Publications from researchers at the Buck Institute for Research on Aging http://www.buckinstitute.org/benzLab © 2011 Buck Institute, All Rights Reserved It is unclear if earlier onset (40years) and greater proliferative capacity confer an equally poor prognosis to endocrinedependent and endocrineindependent breast cancers. Available outcome (distant metastasisfree survival, DMFS) and expression microarray data from 621 adjuvant treatmentnave, nodenegative primary breast cancers were pooled for prognostic evaluation of ageatdiagnosis (40years vs. 40years) and tumor proliferative capacity relative to estrogen receptor status (n=400 ERpositive, n=221 ERnegative). Transcriptome measures of proliferative capacity included a proliferation score (PS) based on a 61gene proliferation signature and the single gene surrogate, FOXM1. KaplanMeier analyses revealed no significant difference in DMFS between ERpositive and ERnegative cases 5years after diagnosis. In contrast, younger age and higher proliferative capacity resulted in significantly more metastatic events cumulated over 15years, but only in ERpositive breast cancers where positive correlations between age and proliferation were observed. While strongly correlated, FOXM1 and PS did not appear equivalent in relation to age and prognosis. The poor prognosis associated with breast cancer arising before age 40 or with higher proliferative capacity pertains only to endocrinedependent (ERpositive) breast cancer, indicating that different biological processes drive the metastatic potential of ERnegative breast cancer. http://www.ncbi.nlm.nih.gov/pubmed/21225456 Fri, 31 Dec 2010 00:00:00 -0800 PURPOSE: We have evaluated the eukaryotic translation initiation factor 4E (eIF4E) as a potential biomarker and therapeutic target in breast cancer. eIF4E facilitates nuclear export and translation of specific, growthstimulatory mRNAs and is frequently overexpressed in cancer. Experimental design: Breast cancer cells were treated with ribavirin, an inhibitor of eIF4E, and effects on cell proliferation and on known mRNA targets of eIF4E were determined. eIF4E expression was assessed, at the mRNA and protein level, in breast cancer cell lines and in skin biopsies from patients with metastatic disease. Additionally, pooled microarray data from 621 adjuvant untreated, node negative breast cancers were analyzed for eIF4E expression levels and correlation with Distant Metastasis Free Survival (DMFS), overall and within each intrinsic breast cancer subtype.RESULTS: At clinically relevant concentrations, ribavirin reduced cell proliferation and suppressed clonogenic potential, correlating with reduced mRNA export and protein expression of important eIF4E targets. This effect was suppressed by knockdown of eIF4E. Although eIF4E expression is elevated in all breast cancer cell lines, variability in ribavirin responsiveness was observed, indicating that other factors contribute to an eIF4Edependent phenotype. Assessment of the prognostic value of high eIF4E mRNA in patient tumors found that significant discrimination between good and poor outcome groups was observed only in luminal B cases, suggesting that a specific molecular profile may predict response to eIF4Etargeted therapy.CONCLUSIONS: Inhibition of eIF4E is a potential breast cancer therapeutic strategy that may be especially promising against specific molecular subtypes and in metastatic as well as primary tumors. http://www.ncbi.nlm.nih.gov/pubmed/21415224 Fri, 31 Dec 2010 00:00:00 -0800 Breast cancer has a long natural history. Established and emerging biologic markers address overall risk but not necessarily timing of recurrence. 346 adjuvant nave breast cancer cases from Guy's Hospital with 23years minimum followup and archival blocks were recut and reassessed for hormonereceptors (HR), HER2receptor and grade. Diseasespecific survival (DSS) was analyzed by recursive partitioning. To validate insights from this analysis, genesignatures (proliferative and HRnegative) were evaluated for their ability to predict early versus late metastatic risk in 683 nodenegative, adjuvant nave breast cancers annotated with expression microarray data. Risk partitioning showed that adjuvant nave nodenegative outcome risk was primarily partitioned by tumor receptor status and grade but not tumor size. HRpositive and HER2negative (HRpos) risk was partitioned by tumor grade low grade cases have very low early risk but a 20 falloff in DSS 10 or more years after diagnosis. Higher grade HRpos cases have risk over 20years. Triplenegative (Tneg) and HER2positive (HER2pos) cases DSS events occurred primarily within the first 5years. Among nodepositive cases, only low grade conferred late risk, suggesting that proliferative gene signatures that identify proliferation would be important for predicting early but not late recurrence. Using pooled data from four publicly available data sets for nodenegative tumors annotated with gene expression and outcome data, we evaluated four prognostic gene signatures: two proliferationbased and two immune functionbased. Tumor proliferative capacity predicted early but not late metastatic risk for HRpos cases. The immune function or HRneg specific signatures predicted only early metastatic risk in Tneg and HER2pos cases. Breast cancer prognostic signatures need to inform both risk and timing of metastatic events and may best be applied within subsets. Current signatures predict for outcome risk within 5years of diagnosis. Predictors of late risk for HR positive disease are needed. http://www.ncbi.nlm.nih.gov/pubmed/21597921 Fri, 31 Dec 2010 00:00:00 -0800 A catalogue of molecular aberrations that cause ovarian cancer is critical for developing and deploying therapies that will improve patients' lives. The Cancer Genome Atlas project has analysed messenger RNA expression, microRNA expression, promoter methylation and DNA copy number in 489 highgrade serous ovarian adenocarcinomas and the DNA sequences of exons from coding genes in 316 of these tumours. Here we report that highgrade serous ovarian cancer is characterized by TP53 mutations in almost all tumours (96) low prevalence but statistically recurrent somatic mutations in nine further genes including NF1, BRCA1, BRCA2, RB1 and CDK12 113 significant focal DNA copy number aberrations and promoter methylation events involving 168 genes. Analyses delineated four ovarian cancer transcriptional subtypes, three microRNA subtypes, four promoter methylation subtypes and a transcriptional signature associated with survival duration, and shed new light on the impact that tumours with BRCA1/2 (BRCA1 or BRCA2) and CCNE1 aberrations have on survival. Pathway analyses suggested that homologous recombination is defective in about half of the tumours analysed, and that NOTCH and FOXM1 signalling are involved in serous ovarian cancer pathophysiology. http://www.ncbi.nlm.nih.gov/pubmed/21720365 Fri, 31 Dec 2010 00:00:00 -0800 ABSTRACT: INTRODUCTION: Various multigene predictors of breast cancer clinical outcome have been commercialized, but proved to be prognostic only for hormone receptor (HR) subsets overexpressing estrogen or progesterone receptors. Hormone receptor negative (HRneg) breast cancers, particularly those lacking HER2/ErbB2 overexpression and known as triplenegative (Tneg) cases, are heterogeneous and generally aggressive breast cancer subsets in need of prognostic subclassification, since most early stage HRneg and Tneg breast cancer patients are cured with conservative treatment yet invariably receive aggressive adjuvant chemotherapy. METHODS: An unbiased search for genes predictive of distant metastatic relapse was undertaken using a training cohort of 199 nodenegative, adjuvant treatment naive HRneg (including 154 Tneg) breast cancer cases curated from three public microarray datasets. Prognostic gene candidates were subsequently validated using a different cohort of 75 nodenegative, adjuvant naive HRneg cases curated from three additional datasets. The HRneg/Tneg gene signature was prognostically compared with eight other previously reported gene signatures, and evaluated for cancer network associations by two commercial pathway analysis programs. RESULTS: A novel set of 14 prognostic gene candidates were identified as outcome predictors: CXCL13, CLIC5, RGS4, RPS28, RFX7, EXOC7, HAPLN1, ZNF3, SSX3, HRBL, PRRG3, ABO, PRTN3, MATN1. A composite HRneg/Tneg gene signature index proved more accurate than any individual candidate gene or other reported multigene predictors in identifying cases likely to remain free of metastatic relapse. Significant positive correlations between the HRneg/Tneg index and three independent immunerelated signatures (STAT1, IFN, and IR) were observed, as were consistent negative associations between the three immunerelated signatures and five other proliferation modulecontaining signatures (MS14, ONCORS, GGI, CSR/wound and NKI70). Network analysis identified 8 genes within the HRneg/Tneg signature as being functionally linked to immune/inflammatory chemokine regulation. CONCLUSIONS: A multigene HRneg/Tneg signature linked to immune/inflammatory cytokine regulation was identified from pooled expression microarray data and shown to be superior to other reported gene signatures in predicting the metastatic outcome of early stage and conservatively managed HRneg and Tneg breast cancer. Further validation of this prognostic signature may lead to new therapeutic insights and spare many newly diagnosed breast cancer patients the need for aggressive adjuvant chemotherapy. http://www.ncbi.nlm.nih.gov/pubmed/20946665 Thu, 30 Sep 2010 00:00:00 -0700 Cancer cells often acquire a constitutively active nuclear factorkappaB (NFkappaB) program to promote survival, proliferation and metastatic potential by mechanisms that remain largely unknown. Extending observations from an immunologic setting, we demonstrate that microRNA146a and microRNA146b (miR146a/b) when expressed in the highly metastatic human breast cancer cell line MDAMB231 function to negatively regulate NFkappaB activity. Lentiviralmediated expression of miR146a/b significantly downregulated interleukin (IL)1 receptorassociated kinase and TNF receptorassociated factor 6, two key adaptor/scaffold proteins in the IL1 and Tolllike receptor signaling pathway, known to positively regulate NFkappaB activity. Impaired NFkappaB activity was evident from reduced phosphorylation of the NFkappaB inhibitor IkappaBalpha, reduced NFkappaB DNAbinding activity and suppressed expression of the NFkappaB target genes IL8, IL6 and matrix metalloproteinase9. Functionally, miR146a/bexpressing MDAMB231 cells showed markedly impaired invasion and migration capacity relative to control cells. These findings implicate miR146a/b as a negative regulator of constitutive NFkappaB activity in a breast cancer setting and suggest that modulating miR146a/b levels has therapeutic potential to suppress breast cancer metastases. http://www.ncbi.nlm.nih.gov/pubmed/18504431 Tue, 31 Aug 2010 00:00:00 -0700 Reactive oxygen species (ROS) are both physiological intermediates in cellular signaling and mediators of oxidative stress. The cysteinespecific redoxsensitivity of proteins can shed light on how ROS are regulated and function, but low sensitivity has limited quantification of the redox state of many fundamental cellular regulators in a cellular context. Here we describe a highly sensitive and reproducible oxidation analysis approach (OxMRM) that combines protein purification, differential alkylation with stable isotopes, and multiple reaction monitoring mass spectrometry that can be applied in a targeted manner to virtually any cysteine or protein. Using this approach, we quantified the sitespecific cysteine oxidation status of endogenous p53 for the first time and found that Cys182 at the dimerization interface of the DNA binding domain is particularly susceptible to diamide oxidation intracellularly. OxMRM enables analysis of sulfinic and sulfonic acid oxidation levels, which we validate by assessing the oxidation of the catalytic Cys215 of protein tyrosine phosphatase1B under numerous oxidant conditions. OxMRM also complements unbiased redox proteomics discovery studies as a verification tool through its high sensitivity, accuracy, precision, and throughput. http://www.ncbi.nlm.nih.gov/pubmed/20233844 Wed, 30 Jun 2010 00:00:00 -0700 The overexpressed ErbB2/HER2 receptor is a clinically validated cancer target whose surface localization and internalization mechanisms remain poorly understood. Downregulation of the overexpressed 185kDa ErbB2 receptor is rapidly (26 hours) induced by the HSP90 chaperone inhibitor geldanamycin (GA), whereas its downregulation and lysosomal degradation are more slowly (24 hours) induced by the proteasome inhibitor bortezomib/PS341. In PS341treated SKBR3 cells, overexpressed ErbB2 coprecipitates with the E3 ubiquitin ligase cCbl and also with the deubiquitinating enzyme USP9x moreover, siRNA downregulation of USP9x enhances PS341induced ErbB2 downregulation. Because polyubiquitin linkages via lysine 48 (K48) or 63 (K63) can differentially address proteins for 26S proteasomal degradation or endosome trafficking to the lysosome, multiple reaction monitoring (MRM)/mass spectrometry (MS) and polyubiquitin linkagespecific antibodies were used to quantitatively track K48linked and K63linked ErbB2 polyubiquitination following either GA or PS341 treatment of SKBR3 cells. MRM/MS revealed that unlike the rapid, modest (4fold to 8fold), and synchronous GA induction of K48 and K63 polyubiquitinated ErbB2, PS341 produces a dramatic (20fold to 40fold) sequential increase in polyubiquitinated ErbB2 consistent with K48 polyubiquitination followed by K63 editing. Fluorescence microscopic imaging confirmed that PS341, but not GA, induces colocalization of K48linked and K63linked polyubiquitin with perinuclear lysosomesequestered ErbB2. Thus, ErbB2 surface overexpression and recycling seem to depend on its polyubiquitination and deubiquitination as well, the contrasting effects of PS341 and GA on ErbB2 receptor localization, polyubiquitination, and degradation point to alternate cytoplasmic trafficking likely regulated by different K48 and K63 polyubiquitin editing mechanisms. http://www.ncbi.nlm.nih.gov/pubmed/20406983 Wed, 31 Mar 2010 00:00:00 -0700 BACKGROUND: Recent declines in invasive breast cancer have been reported in the US, with many studies linking these declines to reductions in the use of combination estrogen/progestin hormone therapy (EPHT). We evaluated the changing use of postmenopausal hormone therapy, mammography screening rates, and the decline in breast cancer incidence specifically for Marin County, California, a population with historically elevated breast cancer incidence rates. METHODS: The Marin Women's Study (MWS) is a communitybased, prospective cohort study launched in 2006 to monitor changes in breast cancer, breast density, and personal and biologic risk factors among women living in Marin County. The MWS enrolled 1,833 women following routine screening mammography between October 2006 and July 2007. Participants completed a selfadministered questionnaire that included items regarding historical hormone therapy regimen (estrogen only, progesterone only, EPHT), age of first and last use, total years of use, and reason(s) for stopping, as well as information regarding complementary hormone use. Questionnaire items were analyzed for 1,083 nonHispanic white participants ages 50 and over. Breast cancer incidence rates were assessed overall and by tumor histology and estrogen receptor (ER) status for the years 19902007 using data from the Northern California Surveillance, Epidemiology and End Results (SEER) cancer registry. RESULTS: Prevalence of EPHT use among nonHispanic white women ages 50 and over declined sharply from 21.2 in 1998 to 6.7 by 200607. Estrogen only use declined from 26.9 in 1998 to 22.4 by 200607. Invasive breast cancer incidence rates declined 33.4 between 2001 and 2004, with drops most pronounced for ER cancers. These rate reductions corresponded to declines of about 50 cases per year, consistent with population attributable fraction estimates for EPHTrelated breast cancer. Selfreported screening mammography rates did not change during this period. Use of alternative or complementary agents did not differ significantly between ever and never hormone users. Of women who reported stopping EPHT in the past 5 years, 60 cited "health risks" or "news reports" as their primary reasons for quitting. CONCLUSION: A dramatic reduction in EPHT use was followed temporally by a significant reduction in invasive and ER breast cancer rates among women living in Marin County, California. http://www.ncbi.nlm.nih.gov/pubmed/20433756 Thu, 31 Dec 2009 00:00:00 -0800 Topotecan (TPT), a highly active anticancer camptothecin drug, would benefit from nanocarriermediated sitespecific and intracellular delivery because of a labile lactone ring whose hydrolysis inactivates the drug, poor cellular uptake resulting from both lactone hydrolysis and a titratable phenol hydroxyl, and the scheduledependency of its efficacy due to its mechanism of action. We have encapsulated topotecan in liposomes using transmembrane gradients of triethylammonium salts of polyphosphate (Pn) or sucroseoctasulfate (SOS). Circulation lifetimes were prolonged, and the rate of drug release in vivo depended on the drug load (T(1/2)=5.4 h vs. 11.2 h for 124 and 260 g TPT/mol PL, respectively) and the nature of intraliposomal drug complexing agent used to stabilize the nanoliposome formulation (T(1/2)=11.2 h vs. 27.3 h for Pn and SOS, respectively). AntiEGFR and antiHER2immunoliposomal formulations dramatically increased uptake of topotecan compared to nontargeted nanoliposomal topotecan and poorly permeable free topotecan in receptoroverexpressing cancer cell lines, with a corresponding increase in cytotoxicity in multiple breast cancer cell lines and improved antitumor activity against HER2overexpressing human breast cancer (BT474) xenografts. We conclude that stabilization of topotecan in nanoliposomes significantly improves the targetability and pharmacokinetic profile of topotecan, allowing for highly active formulations against solid tumors and immunotargeting to canceroverexpressing cell surface receptors. http://www.ncbi.nlm.nih.gov/pubmed/19686789 Mon, 30 Nov 2009 00:00:00 -0800 PURPOSE: Liposome and immunoliposome formulations of two vinca alkaloids, vincristine and vinblastine, were prepared using intraliposomal triethylammonium sucroseoctasulfate and examined for their ability to stabilize the drug for targeted drug delivery in vivo. METHODS: The pharmacokinetics of both the encapsulated drug (vincristine or vinblastine) and liposomal carrier were examined in Sprague Dawley rats, and the in vivo drug release rates determined. AntiHER2 immunoliposomal vincristine was prepared from a human antiHER2/neu scFv and studied for targeted cytotoxic activity in cell culture, and antitumor efficacy in vivo. RESULTS: Nanoliposome formulations of vincristine and vinblastine demonstrated similar pharmacokinetic profiles for the liposomal carrier, but increased clearance for liposome encapsulated vinblastine (t (1/2) = 9.7 h) relative to vincristine (t (1/2) = 18.5 h). Immunoliposome formulations of vincristine targeted to HER2 using an antiHER2 scFv antibody fragment displayed a marked enhancement in cytotoxicity when compared to nontargeted liposomal vincristine control 63 or 253fold for BT474 and SKBR3 breast cancer cells, respectively. Targetspecific activity was also demonstrated in HER2overexpressing human tumor xenografts, where the HER2targeted formulation was significantly more efficacious than either free vincristine or nontargeted liposomal vincristine. CONCLUSIONS: These results demonstrate that active targeting of solid tumors with liposomal formulations of vincristine is possible when the resulting immunoliposomes are sufficiently stabilized. http://www.ncbi.nlm.nih.gov/pubmed/19184019 Tue, 30 Jun 2009 00:00:00 -0700 PURPOSE: Excess histone deacetylase (HDAC) activity can induce hypoacetylation of histone and nonhistone protein substrates, altering gene expression patterns and cell behavior potentially associated with malignant transformation. However, HDAC expression and protein acetylation have not been studied in the context of breast cancer progression. EXPERIMENTAL DESIGN: We assessed expression levels of acetylated histone H4 (acH4), acH4K12, actubulin, HDAC1, HDAC2, and HDAC6 in 22 reduction mammoplasties and in 58 specimens with synchronous normal epithelium, ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) components. Differences among groups were tested for significance using nonparametric tests. RESULTS: From normal epithelium to DCIS, there was a marked reduction in histone acetylation (P 0.0001). Most cases showed similar levels of acetylation in DCIS and IDC, although some showed further reduction of acH4 and acH4K12 from DCIS to IDC. Expression of HDAC1, HDAC2, and HDAC6 was also significantly reduced but by a smaller magnitude. Greater reductions of H4 acetylation and HDAC1 levels were observed from normal to DCIS in estrogen receptornegative compared with estrogen receptorpositive, and in highgrade compared with nonhighgrade tumors. CONCLUSION: Overall, there was a global pattern of hypoacetylation associated with progression from normal to DCIS to IDC. These findings suggest that the reversal of this hypoacetylation in DCIS and IDC could be an early measure of HDAC inhibitor activity. http://www.ncbi.nlm.nih.gov/pubmed/19383825 Thu, 30 Apr 2009 00:00:00 -0700 A systematic study of posttranslational modifications of the estrogen receptor isolated from the MCF7 human breast cancer cell line is reported. Proteolysis with multiple enzymes, mass spectrometry, and tandem mass spectrometry achieved very high sequence coverage for the fulllength 66kDa endogenous protein from estradioltreated cell cultures. Nine phosphorylated serine residues were identified, three of which were previously unreported and none of which were previously observed by mass spectrometry by any other laboratory. Two additional modified serine residues were identified in recombinant protein, one previously reported but not observed here in endogenous protein and the other previously unknown. Although major emphasis was placed on identifying new phosphorylation sites, Nterminal loss of methionine accompanied by amino acetylation and a lysine side chain acetylation (or possibly trimethylation) were also detected. The use of both HPLCESI and MALDI interfaced to different mass analyzers gave higher sequence coverage and identified more sites than could be achieved by either method alone. The estrogen receptor is critical in the development and progression of breast cancer. One previously unreported phosphorylation site identified here was shown to be strongly dependent on estradiol, confirming its potential significance to breast cancer. Greater knowledge of this array of posttranslational modifications of estrogen receptor, particularly phosphorylation, will increase our understanding of the processes that lead to estradiolinduced activation of this protein and may aid the development of therapeutic strategies for management of hormonedependent breast cancer. http://www.ncbi.nlm.nih.gov/pubmed/18984578 Sat, 28 Feb 2009 00:00:00 -0800 Breast cancer metastasis suppressor 1 (BRMS1) is a predominantly nuclear protein that differentially regulates expression of multiple genes, leading to suppression of metastasis without blocking orthotopic tumor growth in multiple human and murine cancer cells of diverse origins. We hypothesized that miR146 may be involved in the ability of BRMS1 to supress metastasis because miR146 expression is altered by BRMS1 and because BRMS1 and miR146 are both associated with decreased signaling through the nuclear factorkappaB pathway. BRMS1 significantly upregulates miR146a by 6 to 60fold in metastatic MDAMB231 and MDAMB435 cells, respectively, and miR146b by 40fold in MDAMB435 as measured by realtime quantitative reverse transcriptionPCR. Transduction of miR146a or miR146b into MDAMB231 downregulated expression of epidermal growth factor receptor, inhibited invasion and migration in vitro, and suppressed experimental lung metastasis by 69 and 84, respectively (mean / SE: empty vector = 39 / 6, miR146a = 12 / 1, miR146b = 6 / 1). These results further support the recent notion that modulating the levels of miR146a or miR146b could have a therapeutic potential to suppress breast cancer metastasis. http://www.ncbi.nlm.nih.gov/pubmed/19190326 Sat, 31 Jan 2009 00:00:00 -0800 It has been over 20 years since the discovery of the human epidermal growth factor receptor 2 (HER2), a tyrosine kinase receptor that is a potent oncoprotein in breast and other cancers and has become an opportune target for therapy. HER2 plays a critical role in normal development, forming homodimers or heterodimers with other HER family members and triggering downstream signaling cascades controlling proliferation, cell survival, and apoptosis. However, amplification of the HER2 gene in cancer cells results in overexpression of HER2 receptors on the cell surface, leading to excessive and dysregulated signaling. HER2driven signaling also upregulates transcription factors that act on the HER2 promoter, increasing its expression. In breast cancer, HER2 is gene amplified in 2025 of primary tumors and is associated with a more aggressive phenotype and poorer prognosis. The key role HER2 plays in tumorigenesis makes it an ideal target for therapy. Trastuzumab, a monoclonal antibody against HER2, inhibits downstream signaling and has proven to be effective against HER2overexpressing metastatic breast cancer both as a single agent and in combination with chemotherapy. Seminal clinical trial data also show that the use of adjuvant trastuzumab in combination with chemotherapy or as a single agent after chemotherapy significantly increases diseasefree and overall survival. Lapatinib, a dual tyrosine kinase inhibitor against HER1 and HER2, has been approved in combination with capecitabine for HER2overexpressing advanced or metastatic breast cancer, which has progressed following previous anthracycline, taxane, and trastuzumab therapy. Other HER2targeting strategies are also under active investigation. http://www.ncbi.nlm.nih.gov/pubmed/18952552 Tue, 30 Sep 2008 00:00:00 -0700 Two paradigms central to geroscience research are that aging is associated with increased oxidative stress and increased cancer risk. Therefore, it could be deduced that cancers arising with ageing will show evidence of increased oxidative stress. Recent studies of gene expression in agecontrolled breast cancer cases indicate that this deduction is false, posing parallax views of these two paradigms, and highlighting the unanswered question: does ageing cause or simply permit cancer development http://www.ncbi.nlm.nih.gov/pubmed/18948997 Tue, 30 Sep 2008 00:00:00 -0700 INTRODUCTION: Oxidative stress can modify estrogen receptor (ER) structure and function, including induction of progesterone receptor (PR), altering the biology and clinical behavior of endocrine responsive (ERpositive) breast cancer. METHODS: To investigate the impact of oxidative stress on estrogen/ERregulated gene expression, RNA was extracted from ERpositive/PRpositive MCF7 breast cancer cells after 72 hours of estrogen deprivation, smallinterfering RNA knockdown of ERalpha, shortterm (8 hours) exposure to various oxidant stresses (diamide, hydrogen peroxide, and menadione), or simultaneous ERalpha knockdown and oxidant stress. RNA samples were analyzed by highthroughput expression microarray (Affymetrix), and significance analysis of microarrays was used to define gene signatures responsive to estrogen/ER regulation and oxidative stress. To explore the association of these signatures with breast cancer biology, microarray data were analyzed from 394 ERpositive primary human breast cancers pooled from three independent studies. In particular, an oxidantsensitive estrogen/ERresponsive gene signature (OxE/ER) was correlated with breast cancer clinical parameters and diseasespecific patient survival (DSS). RESULTS: From 891 estrogen/ERregulated probes, a core set of 75 probes (62 unique genes) responsive to all three oxidants were selected (OxE/ER signature). Ingenuity pathway analysis of this signature highlighted networks involved in development, cancer, and cell motility, with intersecting nodes at growth factors (plateletderived growth factorBB, transforming growth factorbeta), a proinflammatory cytokine (tumor necrosis factor), and matrix metalloproteinase2. Evaluation of the 394 ERpositive primary breast cancers demonstrated that OxE/ER index values correlated negatively with PR mRNA levels (rp = 0.2 P = 0.00011) and positively with tumor grade (rp = 0.2 P = 9.741 x e5), and were significantly higher in ERpositive/PRnegative versus ERpositive/PRpositive breast cancers (ttest, P = 0.0008). Regardless of PR status, the OxE/ER index associated with reduced DSS (n = 201 univariate Cox, P = 0.078) and, using the optimized cutpoint, separated ERpositive cases into two significantly different DSS groups (log rank, P = 0.0009). CONCLUSION: An oxidantsensitive subset of estrogen/ERresponsive breast cancer genes linked to cell growth and invasion pathways was identified and associated with loss of PR and earlier diseasespecific mortality, suggesting that oxidative stress contributes to the development of an aggressive subset of primary ERpositive breast cancers. http://www.ncbi.nlm.nih.gov/pubmed/18631401 Tue, 30 Sep 2008 00:00:00 -0700 In addition to repressing ERBB2 promoter function, histone deacetylase (HDAC) inhibitors induce the accelerated decay of mature ERBB2 transcripts the mechanism mediating this transcript destabilization is unknown but depends on the 3' untranslated region (UTR) of ERBB2 mRNA. Using ERBB2overexpressing human breast cancer cells (SKBR3), the mRNA stability factor HuR was shown to support ERBB2 transcript integrity, bind and endogenously associate with a conserved Urich element within the ERBB2 transcript 3' UTR, coimmunoprecipitate with RNAassociated HDAC activity, and colocalize with HDAC6. HDAC6 also coimmunoprecipitates with HuR in an RNAdependent manner and within 6 hours of exposure to a panHDAC inhibitor dose, that does not significantly alter cytosolic HuR levels or HuR binding to ERBB2 mRNA. Cellular ERBB2 transcript levels decline while remaining physically associated with HDAC6. Knockdown of HDAC6 protein by small interfering RNA partially suppressed the ERBB2 transcript decay induced by either panHDAC or HDAC6selective enzymatic inhibitors. Three novel hydroxamates, ST71, ST17, and ST80 were synthesized and shown to inhibit HDAC6 with 14fold to 31fold greater selectivity over their binding and inhibition of HDAC1. Unlike more potent panHDAC inhibitors, these HDAC6selective inhibitors produced dosedependent growth arrest of ERBB2overexpressing breast cancer cells by accelerating the decay of mature ERBB2 mRNA without repressing ERBB2 promoter function. In sum, these findings point to the therapeutic potential of HuR and HDAC6selective inhibitors, contrasting ERBB2 stability effects induced by HDAC6 enzymatic inhibition and HDAC6 protein knockdown, and show that ERBB2 transcript stability mechanisms include exploitable targets for the development of novel anticancer therapies. http://www.ncbi.nlm.nih.gov/pubmed/18644987 Mon, 30 Jun 2008 00:00:00 -0700 BACKGROUND: Preclinical studies have shown the anticancer potential of HMGCoA reductase enzyme inhibitors (statins), whereas epidemiologic studies remain controversial. Because lipophilic statins show preclinical anticancer activity against hormone receptor estrogen receptor (ER)/progesterone receptor (PR)negative breast cancer models, we explored the hormone receptor phenotype of breast cancers that arise in statin users. METHODS: We did a retrospective cohort analysis via electronic pharmacy records from the Kaiser Permanente Northern California Cancer Registry on 2,141 female patients listed in 2003 as incident cases of breast malignancy. Measures included tumor grade, stage, and receptor phenotype in statin users versus nonusers and controlled for hormone replacement therapy and race. RESULTS: 387 of the 2,141 breast cancer patients used lipophilic statins lovastatin (85), simvastatin, and atorvastatin. Fiftyone women developed ER/PRnegative tumors. The ageadjusted odds ratio (OR) of developing an ER/PR negative tumor was 0.63 (95 confidence interval, 0.430.92 P = 0.02) for statin use or=1 year before breast cancer diagnosis compared with statin use 1 year (including nonuse). Breast cancers in patients with or=1 year of statin use were more likely to be low grade (OR, 1.44) and less invasive stage (OR, 1.42). CONCLUSIONS: Breast cancer patients with exposure to statins have proportionately fewer ER/PRnegative tumors that are of lower grade and stage. Although our data set cannot address whether statins affect the incidence of breast cancer, we show that statin use may influence the phenotype of tumors. This suggests a new potential strategy for breast cancer prevention, that of combining statins with agents that prevent ERpositive cancer (tamoxifen, aromatase inhibitors). http://www.ncbi.nlm.nih.gov/pubmed/18463402 Wed, 30 Apr 2008 00:00:00 -0700 Activated estrogen receptor (ERalpha) plays a critical role in breast cancer development and is a major target for drug treatment. Serine phosphorylation within the Nterminal domain (NTD) contributes to ERalpha activation and may also cause drug resistance. Previous biochemical identification of phosphorylated ERalpha residues was limited to protein artificially overexpressed in transfected cell lines. We report mass spectrometric methods that have allowed the identification of a new site within the NTD of ERalpha isolated from cultured human breast cancer cells. Immunoprecipitation, trypsin digestion, and analysis by nanoLCESIMS/MS (QSTAR, MDS Sciex) and vMALDIMS(n) (Finnigan LTQ, ThermoElectron) identified peptides containing 8 of 14 serine residues within the NTD, one being partially phosphorylated Ser167, known but not previously reported by MS. Chymotrypsin digestion revealed other known sites at Ser102/104/106 and 118. Tandem methods developed for the peptide containing Ser118 and the use of hypothesisdriven experimentsi.e., the assumption that an intact phosphopeptide showing no molecular ion might yield fragment ions including loss of phosphoric acid in vMALDIMS/MSallowed the identification of a novel site at Ser154. Quantitation by selected reaction monitoring demonstrated 6fold and 2.5fold increases in Ser154 phosphorylation in estradiol and EGFtreated cells, respectively, compared to controls, confirmed by immunoblotting with a novel rabbit polyclonal antibody. Thus, the protein isolation and MS strategies described here can facilitate discovery of novel phosphorylation sites within low abundance, clinically important cancer targets like ERalpha, and may thereby contribute to our understanding of the role of phosphorylation in the development of breast cancer. http://www.ncbi.nlm.nih.gov/pubmed/18367407 Wed, 30 Apr 2008 00:00:00 -0700 Breast cancer is a heterogeneous malignancy its agespecific incidence profile rises exponentially until menopause and increases more slowly thereafter, reflecting the superimposition of earlyonset and lateonset breast cancer rates. While earlyonset breast cancers largely represent inherited or early life transforming effects on immature mammary epithelium, lateonset breast cancers likely follow extended exposures to promoting stimuli of susceptible epithelium that has failed to age normally. Among stimuli thought to promote lateonset breast tumorigenesis are the altered extracellular matrix and secreted products of senescent fibroblasts however, the extent to which these senescent influences exist within the aging breast remains unknown. Clinical observations and biomarker studies indicate that lateonset breast cancers grow more slowly and are biologically less aggressive than earlyonset breast cancers, even when controlled for hormone receptor (e.g. estrogen receptor, ER) and growth factor receptor (e.g. HER2) expression, supporting the conclusion that the biology of breast cancer is agedependent. http://www.ncbi.nlm.nih.gov/pubmed/17949989 Fri, 29 Feb 2008 00:00:00 -0800 INTRODUCTION: Age is one of the most important risk factors for human malignancies, including breast cancer in addition, age at diagnosis has been shown to be an independent indicator of breast cancer prognosis. Except for inherited forms of breast cancer, however, there is little genetic or epigenetic understanding of the biological basis linking aging with sporadic breast cancer incidence and its clinical behavior. METHODS: DNA and RNA samples from matched estrogen receptor (ER)positive sporadic breast cancers diagnosed in either younger (age or= 45 years) or older (age or= 70 years) Caucasian women were analyzed by array comparative genomic hybridization and by expression microarrays. Array comparative genomic hybridization data were analyzed using hierarchical clustering and supervised age cohort comparisons. Expression microarray data were analyzed using hierarchical clustering and gene set enrichment analysis differential gene expression was also determined by conditional permutation, and an age signature was derived using prediction analysis of microarrays. RESULTS: Hierarchical clustering of genomewide copynumber changes in 71 ERpositive DNA samples (27 younger women, 44 older women) demonstrated two ageindependent genotypes one with few genomic changes other than 1q gain/16q loss, and another with amplifications and lowlevel gains/losses. Age cohort comparisons showed no significant differences in total or sitespecific genomic breaks and amplicon frequencies. Hierarchical clustering of 5.1 K genes variably expressed in 101 ERpositive RNA samples (53 younger women, 48 older women) identified six transcriptome subtypes with an apparent age bias (P 0.05). Samples with higher expression of a poor outcomeassociated proliferation signature were predominantly (65) younger cases. Supervised analysis identified cancerassociated genes differentially expressed between the cohorts with younger cases expressing more cell cycle genes and more than threefold higher levels of the growth factor amphiregulin (AREG), and with older cases expressing higher levels of four different homeobox (HOX) genes in addition to ER (ESR1). An age signature validated against two other independent breast cancer datasets proved to have 80 accuracy in discerning younger from older ERpositive breast cancer cases with characteristic differences in AREG and ESR1 expression. CONCLUSION: These findings suggest that epigenetic transcriptome changes, more than genotypic variation, account for ageassociated differences in sporadic breast cancer incidence and prognosis. http://www.ncbi.nlm.nih.gov/pubmed/17850661 Mon, 31 Dec 2007 00:00:00 -0800 Targeted delivery of smallmolecule drugs has the potential to enhance selective killing of tumor cells. We have identified previously an internalizing single chain single chain variable fragment (scFv) antibody that targets prostate cancer cells and identified the target antigen as CD166. We report here the development of immunoliposomes using this antiCD166 scFv (H3). We studied the effects of a panel of intracellularly delivered, antiCD166 immunoliposomal smallmolecule drugs on prostate cancer cells. Immunoliposomal formulations of topotecan, vinorelbine, and doxorubicin each showed efficient and targeted uptake by three prostate cancer cell lines (Du145, PC3, and LNCaP). H3immunoliposomal topotecan was the most effective in cytotoxicity assays on all three tumor cell lines, showing improved cytotoxic activity compared with nontargeted liposomal topotecan. Other drugs such as liposomal doxorubicin were highly effective against LNCaP but not PC3 or Du145 cells, despite efficient intracellular delivery. Postinternalization events thus modulate the overall efficacy of intracellularly delivered liposomal drugs, contributing in some cases to the lower than expected activity in a cell linedependent manner. Further studies on intracellular tracking of endocytosed liposomal drugs will help identify and overcome the barriers limiting the potency of liposomal drugs. http://www.ncbi.nlm.nih.gov/pubmed/17938267 Sun, 30 Sep 2007 00:00:00 -0700 MicroRNAs (miRNAs or mirs) are small, noncoding RNAs that bind specific mRNAs and decrease their translation or increase their degradation. miRNAs may modulate the formation and maintenance of tumors by regulating oncogene and tumor suppressor expression. For example, overexpression of a subset of miRNAs has been inversely correlated with certain tumor phenotypes, suggesting a role in tumor suppression. Pairs of oncogenes and the corresponding miRNAs that attenuate their expression have been recently identified. These miRNAs, or "antioncomirs," can act as natural inhibitors of oncogene function, indicating the possibility that they might be developed as novel therapeutics. http://www.ncbi.nlm.nih.gov/pubmed/17827440 Tue, 31 Jul 2007 00:00:00 -0700 INTRODUCTION: We investigated whether mRNA levels of E2F1, a key transcription factor involved in proliferation, differentiation and apoptosis, could be used as a surrogate marker for the determination of breast cancer outcome. METHODS: E2F1 and other proliferation markers were measured by quantitative RTPCR in 317 primary breast cancer patients from the Stiftung Tumorbank Basel. Correlations to one another as well as to the estrogen receptor and ERBB2 status and clinical outcome were investigated. Results were validated and further compared with expressionbased prognostic profiles using The Netherlands Cancer Institute microarray data set reported by Fan and colleagues. RESULTS: E2F1 mRNA expression levels correlated strongly with the expression of other proliferation markers, and low values were mainly found in estrogen receptorpositive and ERBB2negative phenotypes. Patients with low E2F1expressing tumors were associated with favorable outcome (hazard ratio = 4.3 (95 confidence interval = 1.89.9), P = 0.001). These results were consistent in univariate and multivariate Cox analyses, and were successfully validated in The Netherlands Cancer Institute data set. Furthermore, E2F1 expression levels correlated well with the 70gene signature displaying the ability of selecting a common subset of patients at good prognosis. Breast cancer patients' outcome was comparably predictable by E2F1 levels, by the 70gene signature, by the intrinsic subtype gene classification, by the wound response signature and by the recurrence score. CONCLUSION: Assessment of E2F1 at the mRNA level in primary breast cancer is a strong determinant of breast cancer patient outcome. E2F1 expression identified patients at low risk of metastasis irrespective of the estrogen receptor and ERBB2 status, and demonstrated similar prognostic performance to different gene expressionbased predictors. http://www.ncbi.nlm.nih.gov/pubmed/17535433 Sat, 30 Jun 2007 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/17510382 Mon, 30 Apr 2007 00:00:00 -0700 A major challenge to broadening oncology applications for inhibitors of the ubiquitinproteasome system (UPS) is the identification of UPSdependent cancer pathways predictive of tumors responsive to peptidomimetic inhibitors of its 20S core protease activity. To inform clinical studies evaluating UPS inhibitors as breast cancer therapeutics, seven phenotypically diverse human breast cancer cell line models were characterized for their cellular and molecular responses to the clinically approved 20S inhibitor bortezomib (PS341 Velcade), focusing on those overexpressing estrogen receptor (ER) or ERBB2/HER2, because these oncogenic receptor pathways are constitutively activated in approximately 80 of all breast cancers. All models demonstrated dosedependent bortezomib reduction in intracellular 20S activity correlating with cell growth inhibition, and bortezomib IC(50) values (concentrations producing 50 growth inhibition) varied directly with pretreatment 20S activities (r = 0.74 , p 0.05), suggesting that basal 20S activity may serve as a clinical predictor of tumor responsiveness to UPS inhibition. Reduction in 20S activity ( 60) was associated with early (24 h) intracellular relocalization of ER (nucleus to cytoplasm) and ERBB2 (plasma membrane to perinuclear lysosomes), buildup of ubiquitinated and Hsp70associated receptor, degradation and loss of ER and ERBB2 function, and induction of cellular apoptosis. These models were also used to screen a pharmacologic panel of pathwaytargeted anticancer agents 4hydroxy3methoxy5(benzothiazolylthiomethyl)benzylidenecyanoacetamide (AG825), 6(4bromo2chlorophenylamino)7fluoro3methyl3Hbenzoimidazole5carboxylic acid (2hydroxyethoxy)amide (AZD6244/ARRY142886), 2(4morpholinyl)8phenyl4H1benzopyran4one hydrochloride (LY294002), 17Nallylamino17demethoxy geldanamycin (17AAG), and (2E)Nhydroxy34(2hydroxyethyl)2(1Hindol3yl)ethylaminomethylphenyl2propenamide (LAQ824) for those capable of sensitizing to bortezomib. In keeping with the observation that 20S reduction has little effect on mitogenactivated protein kinase kinase 1/2 (MEK1/2) signaling in either ERpositive or ERBB2positive models, only the MEK1/2 inhibitor AZD6244 consistently improved the antitumor activity of bortezomib. http://www.ncbi.nlm.nih.gov/pubmed/17392524 Mon, 30 Apr 2007 00:00:00 -0700 Human ERBB2 presents several SNPs. One of these, Ile655Val, introduces a structural change in the transmembrane region of ERBB2 and has been the focus of debate over its potential role as a susceptibility marker for breast cancer risk. Another SNP, Ala1170Pro, introduces a structural change in the carboxylterminal regulatory domain of the protein, but its clinical and biological importance remains undefined. The aim of this study was to investigate the association of rare alleles of both SNPs and the risk of developing breast cancer, BRCA1 alterations and clinicalpathological features of Caucasian breast cancer patients with familial history of breast/ovarian cancer. The originality of the present paper is that it is the only specifically focusing on the relationship between ERBB2 SNPs and familiarity/BRCA1 characteristics. A consecutive series of 628 patients with first diagnosis of breast cancer and 169 healthy people had DNA analyzed for both SNPs. Genotypic or allelic frequencies of ERBB2 SNPs in breast cancer patients were similar than in controls. The variant allele 655Val was significantly associated with younger age (p=0.009) particularly associated with patient family history of breast cancer (p=0.02). The 655Val allele was also more commonly found in invasive, while the variant 1170Pro in estrogen receptor positive breast cancers. Furthermore, this last SNP seems to be strictly associated with the presence of BRCA1 polymorphisms. In conclusion, these findings point to the existence of an association of ERBB2 allelic variants at both loci with specific breast tumor phenotypes and to the need of deeply investigate different gene SNPs association for risk defining. http://www.ncbi.nlm.nih.gov/pubmed/17452776 Sat, 31 Mar 2007 00:00:00 -0700 BACKGROUND: Signaling pathways that converge on two different transcription factor complexes, NFkappaB and AP1, have been identified in estrogen receptor (ER)positive breast cancers resistant to the antiestrogen, tamoxifen. METHODS: Two cell line models of tamoxifenresistant ERpositive breast cancer, MCF7/HER2 and BT474, showing increased AP1 and NFkappaB DNAbinding and transcriptional activities, were studied to compare tamoxifen effects on NFkappaB and AP1 regulated reporter genes relative to tamoxifensensitive MCF7 cells. The model cell lines were treated with the IKK inhibitor parthenolide (PA) or the proteasome inhibitor bortezomib (PS341), alone and in combination with tamoxifen. Expression microarray data available from 54 UCSF nodenegative ERpositive breast cancer cases with known clinical outcome were used to search for potential genes signifying upregulated NFkappaB and AP1 transcriptional activity in association with tamoxifen resistance. The association of these genes with patient outcome was further evaluated using nodenegative ERpositive breast cancer cases identified from three other published data sets (Rotterdam, n = 209 Amsterdam, n = 68 Basel, n = 108), each having different patient age and adjuvant tamoxifen treatment characteristics. RESULTS: Doses of parthenolide and bortezomib capable of sensitizing the two endocrine resistant breast cancer models to tamoxifen were capable of suppressing NFkappaB and AP1 regulated gene expression in combination with tamoxifen and also increased ER recruitment of the transcriptional corepressor, NCoR. Transcript profiles from the UCSF breast cancer cases revealed three NFkappaB and AP1 upregulated genescyclin D1, uPA and VEGFcapable of dichotomizing nodenegative ERpositive cases into early and late relapsing subsets despite adjuvant tamoxfien therapy and most prognostic for younger age cases. Across the four independent sets of nodenegative ERpositive breast cancer cases (UCSF, Rotterdam, Amsterdam, Basel), high expression of all three NFkappaB and AP1 upregulated genes was associated with earliest metastatic relapse. CONCLUSION: Altogether, these findings implicate increased NFkappaB and AP1 transcriptional responses with tamoxifen resistant breast cancer and early metastatic relapse, especially in younger patients. These findings also suggest that agents capable of preventing NFkappaB and AP1 gene activation may prove useful in restoring the endocrine responsiveness of such highrisk ERpositive breast cancers. http://www.ncbi.nlm.nih.gov/pubmed/17407600 Sat, 31 Mar 2007 00:00:00 -0700 Redoxdependent modifications of sulfhydryl groups within the two Cys4 zinc fingers of the estrogen receptor DNAbinding domain (ERDBD) result in structural damage and loss of ER DNAbinding function, which parallels the situation observed in many ERpositive breast cancers. Quantitation of the redox status of cysteinyl thiols within ERDBD employed cysteinespecific oxidants to induce varying degrees of oxidation in recombinant ER, followed by differential alkylation with the stable isotopic labeling reagents 12C2iodoacetic acid and 13C2bromoacetic acid. Subsequent proteolysis with LysC/AspN generated diagnostic peptides of which the Cterminal peptide of the second zinc finger is most strongly detected by mass spectrometry (MS) and serves as a suitable marker of ERDBD redox status. Data were collected from two different MALDIMS instruments: a timeofflight and a linear ion trap (vMALDILIT). An analogous but larger synthetic peptide treated with three isotopic variants of the alkylating reagent modeled isotopic overlaps that might complicate the relative quantitation of cysteine oxidation. Despite the isotopic overlaps, excellent relative quantitation was achieved from MS data obtained from both instruments. This was also true of tandem MS/MS data from the vMALDILIT, which should facilitate selected reaction monitoring. Relative quantitation by MS also closely matched data from immunochemical methods. http://www.ncbi.nlm.nih.gov/pubmed/17373775 Sat, 31 Mar 2007 00:00:00 -0700 Deregulation of microRNAs (miRNAs) is emerging as a major aspect of cancer etiology because their capacity to direct the translation and stability of targeted transcripts can dramatically influence cellular physiology. To explore the potential of exogenously applied miRNAs to suppress oncogenic proteins, the ERBB oncogene family was chosen with a bioinformatics search identifying targeting seed sequences for miR125a and miR125b within the 3'untranslated regions of both ERBB2 and ERBB3. Using the human breast cancer cell line SKBR3 as a model for ERBB2 and ERBB3 dependence, infection of these cells with retroviral constructs expressing either miR125a or miR125b resulted in suppression of ERBB2 and ERBB3 at both the transcript and protein level. Luciferase constructs containing the 3' 3'untranslated regions of ERBB2 and ERBB3 demonstrated approximately 35 less activity in miR125a and miR125bexpressing cells relative to controls. Additionally, phosphorylation of ERK1/2 and AKT was suppressed in SKBR3 cells overexpressing either miR125a or miR125b. Consistent with suppression of both ERBB2 and ERBB3 signaling, miR125aor miR125boverexpressing SKBR3 cells were impaired in their anchoragedependent growth and exhibited reduced migration and invasion capacities. Parallel studies performed on MCF10A cells demonstrated that miR125a or miR125b overexpression produced only marginal influences on the growth and migration of these nontransformed human mammary epithelial cells. These results illustrate the feasibility of using miRNAs as a therapeutic strategy to suppress oncogene expression and function. http://www.ncbi.nlm.nih.gov/pubmed/17110380 Sun, 31 Dec 2006 00:00:00 -0800 BACKGROUND: It is unclear whether hormone replacement therapy (HRT), in addition to increasing risk for breast cancer, affects the type of breast cancer diagnosed. We conducted this investigation to assess whether the type of hormone used (none, estrogen, progesterone, or combined) and duration of use influences subsequent breast cancer histology. METHODS: We performed a retrospective cohort analysis among women listed as incident cases of breast malignancy in the Kaiser Permanente Northern California Cancer Registry during 2003 (n = 2830). Type and duration of hormone used (none, estrogen, progesterone, or combined) before breast cancer diagnosis was obtained from electronic pharmacy records. The association between type and duration of hormone use with characteristics of subsequent breast cancers was examined. RESULTS: Among women aged 50 years (n = 1701), any use of estrogen, progesterone, or combination therapy was not associated with an increased risk of estrogen receptor (ER)positive disease. However, 6 months' use of combined HRT increased the odds of ERpositive tumors (odds ratio, 1.65 95 confidence interval, 1.072.5 P = .02). Estrogen HRT patients were more likely than nonusers to present with lowgrade (P = .05), and earlystage tumors (P = .03). This trend was not seen in combined HRT users. CONCLUSIONS: Shortduration HRT did not increase the likelihood of ERpositive breast cancer. However, prolonged duration of combined HRT, but not estrogen or progesterone alone, resulted in a marked increase in ERpositive disease. Our findings suggest that the effect of combined HRT on breast cancer incidence or progression is not immediate and that longterm use is more likely to affect breast cancer histology. http://www.ncbi.nlm.nih.gov/pubmed/17103262 Sun, 31 Dec 2006 00:00:00 -0800 Hormonedependent breast cancers that overexpress the ligandbinding nuclear transcription factor, estrogen receptor (ER), represent the most common form of breast epithelial malignancy. Exposure of breast epithelial cells to a redoxcycling and arylating quinone induces mitogenactivated protein kinase phosphorylation of the cytoskeletal filament protein, cytokeratin8, along with thiol arylation of H3 nuclear histones. Exogenous or endogenous quinones can also induce ligandindependent nuclear translocation and phosphorylation of ER with excess exposure, these quinones can arylate ER zinc fingers, impairing ER DNAbinding and altering ERinducible gene expression. Immunoaffinity enrichment for low abundance proteins such as ER, coupled with modern mass spectrometry techniques, promises to improve understanding of the proteinmodifications produced by endogenous and exogenous quinone exposure and their role in the development or progression of epithelial malignancies such as breast cancer. http://www.ncbi.nlm.nih.gov/pubmed/17145690 Thu, 30 Nov 2006 00:00:00 -0800 Genospheres are cationic lipidnucleic acid nanoparticles prepared by the assembly of the lipids and nucleic acids from an aqueous/organic liquid monophase that independently dissolves the components, where the resultant particles are homogeneously sized (70110 nm), with efficiently incorporated and protected DNA. In the present study, we demonstrate pHdependent modulation of the Genosphere surface charge using pHtitratable lipids. By incorporation of the lipids with titratable anionic or imidazole headgroups, Genospheres with neutral or anionic surface charge at neutral pH were produced and compared for cellular uptake and transfection of a reporter gene (luciferase) in culture of breast cancer cells. The extent of particlecell association was also studied by fluorescent microscopy and quantified by cytofluorometery. The effects of Genosphere surface modification with poly(ethylene glycol) (molecular weight 2000) at low (0.5 mol ) and high (5 mol ) grafting densities, as well as the effects of HER2receptordirected targeting by an internalizable antiHER2 scFv F5, linked via PEG spacer, were also studied. Inclusion in the Genosphere formulation of pHtitratable lipids CHEMS (cholesteryl hemisuccinate), CHIM (1(3(cholesteryloxycarbonylamino)propyl)imidazole), or DSGG (1,2distearoylsnglycero3hemiglutarate) rendered the particles surfacecharge neutral or slightly anionic at neutral pH, and cationic at mildly acidic pH, as shown by zetapotential measurements. In HER2targeted systems, transfection activity and target specificity with HER2overexpressing SKBR3 breast cancer cells were dependent on Genosphere surface charge and PEGylation. The highest target specificity correlated with low cationic charge at neutral pH, while incorporation of 5 mol PEGlipid had only minor effects on Genospherecell association, internalization, and transfection activity. The implications of this work for potential in vivo applications are discussed. http://www.ncbi.nlm.nih.gov/pubmed/17140260 Thu, 30 Nov 2006 00:00:00 -0800 Statins are cholesterollowering drugs with pleiotropic activities including inhibition of isoprenylation reactions and reduction of signals driving cell proliferation and survival responses. The objectives of this study were to examine the effects of statins on breast cancer cells, both in vitro and in vivo, and to begin to determine their mechanism of action. We evaluated the effects of statins on breast cancer cell growth, phosphoprotein signaling intermediates, survival/apoptosis regulators, cell cycle regulators, and activated transcription factors. We also examined the in vivo effect of statin administration in a mouse ErbB2() breast cancer model. Only lipophilic statins had direct anticancer activity in vitro. Breast cancer cells with activated Ras or ErbB2 pathways seemed to be more sensitive than those overexpressing estrogen receptor, and this correlated with endogenous levels of activated nuclear factor kappaB (NFkappaB). Key intermediates regulating cell survival by NFkappaB activation, as well as cell proliferation by the mitogen activated protein kinase cascade, were among the earliest phosphoproteins influenced by statin treatment. These early effects were followed by declines in activator protein1 and NFkappaB activation and concordant changes in other mediators of proliferation and apoptosis. In vivo results showed that oral dosing of statins significantly inhibited the growth of a mouse mammary carcinoma. Lipophilic statins can exert direct anticancer activity in vitro by reducing proliferation and survival signals in susceptible breast cancer phenotypes. Tumor growth inhibition in vivo using a clinically relevant statin dose also seems to be associated with reduced tumor cell proliferation and survival. These findings provide supporting rationale for future statin trials in breast cancer patients. http://www.ncbi.nlm.nih.gov/pubmed/16951186 Thu, 31 Aug 2006 00:00:00 -0700 Analysis of a collection of human breast cancers (n = 150), enriched in ERBB2positive cases (n = 57) and involving tumor genotyping relative to populationmatched blood genotyping (n = 749) for a common ERBB2 single nucleotide polymorphism Ala(G)1170Pro(C), revealed that ERBB2 amplification in breast cancer is invariably monoallelic. Analysis of paired breast cancer and blood samples from informative (G1170C heterozygotic) ERBB2positive (n = 12) and ERBB2negative (n = 17) cases not only confirmed monoallelic amplification and ERBB2 transcriptional overexpression but also revealed that most low ERBB2 expressing breast cancers (12/17) exhibit unbalanced allelic transcription, showing 3fold to nearly 5,000fold preferential expression from one of two inherited alleles. To explore cisacting transcriptional mechanisms potentially selected during ERBB2 amplification, levels of four different ERBB2 transcript variants (5.2, 4.7, 2.1, and 1.4 kb) were correlated with total (4.6 kb) ERBB2 mRNA levels in ERBB2positive (n = 14) versus ERBB2negative (n = 43) primary breast cancers. Relative expression of only the 2.1 kb extracellular domainencoding splice variant and a 4.7 kb mRNA variant that uses an alternative start site were significantly increased in association with ERBB2positivity, implicating altered promoter usage and selective transcript regulation within the ERBB2 amplicon. Altogether, these findings provide new mechanistic insights into the development of ERBB2positive breast cancer and strong rationale for delineating candidate cisacting regulatory elements that may link allelespecific ERBB2 transcription in premalignant breast epithelia with subsequent development of breast cancers bearing monoallelic ERBB2 amplicons. http://www.ncbi.nlm.nih.gov/pubmed/16883574 Thu, 31 Aug 2006 00:00:00 -0700 BACKGROUND: Recent studies indicate that microRNAs (miRNAs) are mechanistically involved in the development of various human malignancies, suggesting that they represent a promising new class of cancer biomarkers. However, previously reported methods for measuring miRNA expression consume large amounts of tissue, prohibiting highthroughput miRNA profiling from typically small clinical samples such as excision or core needle biopsies of breast or prostate cancer. Here we describe a novel combination of linear amplification and labeling of miRNA for highly sensitive expression microarray profiling requiring only picogram quantities of purified microRNA. RESULTS: Comparison of microarray and qRTPCR measured miRNA levels from two different prostate cancer cell lines showed concordance between the two platforms (Pearson correlation R2 = 0.81) and extension of the amplification, labeling and microarray platform was successfully demonstrated using clinical core and excision biopsy samples from breast and prostate cancer patients. Unsupervised clustering analysis of the prostate biopsy microarrays separated advanced and metastatic prostate cancers from pooled normal prostatic samples and from a nonmalignant precursor lesion. Unsupervised clustering of the breast cancer microarrays significantly distinguished ErbB2positive/ERnegative, ErbB2positive/ERpositive, and ErbB2negative/ERpositive breast cancer phenotypes (Fisher exact test, p = 0.03) as well, supervised analysis of these microarray profiles identified distinct miRNA subsets distinguishing ErbB2positive from ErbB2negative and ERpositive from ERnegative breast cancers, independent of other clinically important parameters (patient age tumor size, node status and proliferation index). CONCLUSION: In sum, these findings demonstrate that optimized highthroughput microRNA expression profiling offers novel biomarker identification from typically small clinical samples such as breast and prostate cancer biopsies. http://www.ncbi.nlm.nih.gov/pubmed/16784538 Thu, 31 Aug 2006 00:00:00 -0700 A whole cell highthroughput screening assay was developed and tested against 2,000 structurally and functionally diverse druglike small molecules to identify lead compounds capable of cell permeability and selective silencing of ErbB2 transcription. Screening employed reporter sublines clonally selected from ErbB2negative MCF7 breast cancer cells after stable genomic integration of the ErbB2 proximal promoter driving a luciferase reporter antiErbB2 activities (50 inhibitory concentration values) were compared to inhibition of control MCF7 sublines bearing integrated reporters driven by either a mutated ErbB2 promoter or the cyclin D1 promoter. Of the seven resulting lead compounds, four emerged from the National Cancer Institute (NCI)/ Developmental Therapeutics Program (DTP) Structural Diversity Set (NSC131547, NSC176328, NSC259968, and NSC321237) three others emerged from a panel of anticancer compounds with known mechanistic actions and included a minor groove DNAbinding antibiotic (NSC58514, chromomycin A3), a hydroxamic acid inhibitor of histone deacetylases (NSC709238, trichostatin A), and a tripeptide aldehyde proteasome inhibitor (MG132). For optimization, 58 scaffold analogs of the four NCI/DTP structural leads and nine functional analogs of the mechanistic leads were secondarily screened to identify seven compounds with comparable or superior activity relative to the leads, including an approved anticancer drug, PS341 (bortezomib). PS341 activity was validated against cultured ErbB2positive breast cancer cell lines (SKBr3 and BT474) and a trastuzumabresistant ErbB2positive breast cancer xenograft model (B585), in which PS341 antitumor activity correlated with selective downregulation of ErbB2 mRNA and protein levels, confirming the ErbB2 silencing potential of proteasome inhibitors. http://www.ncbi.nlm.nih.gov/pubmed/16834533 Fri, 30 Jun 2006 00:00:00 -0700 We describe evidence for a novel mechanism of monoclonal antibody (MAb)directed nanoparticle (immunoliposome) targeting to solid tumors in vivo. Longcirculating immunoliposomes targeted to HER2 (ErbB2, Neu) were prepared by the conjugation of antiHER2 MAb fragments (Fab' or single chain Fv) to liposomegrafted polyethylene glycol chains. MAb fragment conjugation did not affect the biodistribution or longcirculating properties of i.v.administered liposomes. However, antibodydirected targeting also did not increase the tumor localization of immunoliposomes, as both targeted and nontargeted liposomes achieved similarly high levels (78 injected dose/g tumor tissue) of tumor tissue accumulation in HER2overexpressing breast cancer xenografts (BT474). Studies using colloidal goldlabeled liposomes showed the accumulation of antiHER2 immunoliposomes within cancer cells, whereas matched nontargeted liposomes were located predominantly in extracellular stroma or within macrophages. A similar pattern of stromal accumulation without cancer cell internalization was observed for antiHER2 immunoliposomes in nonHER2overexpressing breast cancer xenografts (MCF7). Flow cytometry of disaggregated tumors posttreatment with either liposomes or immunoliposomes showed up to 6fold greater intracellular uptake in cancer cells due to targeting. Thus, in contrast to nontargeted liposomes, antiHER2 immunoliposomes achieved intracellular drug delivery via MAbmediated endocytosis, and this, rather than increased uptake in tumor tissue, was correlated with superior antitumor activity. Immunoliposomes capable of selective internalization in cancer cells in vivo may provide new opportunities for drug delivery. http://www.ncbi.nlm.nih.gov/pubmed/16818648 Fri, 30 Jun 2006 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/16717179 Sun, 30 Apr 2006 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/16648516 Sun, 30 Apr 2006 00:00:00 -0700 We describe the assembly of a cationic lipidnucleic acid nanoparticle from a liquid monophase containing water and a water miscible organic solvent where both lipid and DNA components are separately soluble prior to their combination. Upon removal of the organic solvent, stable and homogenously sized (70100 nm) lipidnucleic acid nanoparticles (Genospheres) were formed. The low accessibility (15) of the nanoparticleencapsulated DNA to a DNA intercalating dye indicated wellprotected nucleic acids and high DNA incorporation efficiencies. It was demonstrated that Genospheres could be stably stored under a variety of conditions including a lyophilized state where no appreciable increase in particle size or DNA accessibility was observed following reconstitution. Finally, Genospheres were made targetspecific by insertion of an antibodylipopolymer (antiHER2 scFv (F5)PEGDSPE) conjugate into the particle. The target specificity (100fold) in HER2 overexpressing SKBR3 breast cancer cells was dependent on the degree of PEGylation, where the incorporation of high amounts of PEGlipid on the particle surface (up to 5 mol) had only a minor effect on the transfection activity of the targeted Genospheres. In summary, this work describes a novel, readily scalable method for preparing highly stable immunotargeted nucleic acid delivery vehicles capable of achieving a high degree of specific transfection activity. http://www.ncbi.nlm.nih.gov/pubmed/16341056 Tue, 28 Feb 2006 00:00:00 -0800 Improved understanding of the molecular mechanisms by which smallmolecule inhibitors of histone deacetylases (HDAC) induce programs, such as cellular differentiation and apoptosis, would undoubtedly assist their clinical development as anticancer agents. As modulators of gene transcript levels, HDAC inhibitors (HDACi) typically affect only 5 to 10 of actively transcribed genes with approximately as many mRNA transcripts being upregulated as downregulated. Using microRNA (miRNA) array analysis, we report rapid alteration of miRNA levels in response to the potent hydroxamic acid HDACi LAQ824 in the breast cancer cell line SKBr3. Within 5 hours of exposure to a proapoptotic dose of LAQ824, significant changes were measured in 40 of the 60 different miRNA species expressed in SKBr3 cells with 22 miRNA species downregulated and 5 miRNAs upregulated. To explore a potential functional link between HDACi induced mRNA upregulation and miRNA downregulation, antisense experiments were done against miR27a and miR27b, both abundantly expressed and downregulated in SKBr3 cells by LAQ824. Correlating a set of genes previously determined by cDNA array analysis to be rapidly upregulated by LAQ824 in SKBr3 with a database of potential 3' untranslated region miRNA binding elements, two genes containing putative miR27 anchor elements were identified as transcriptionally upregulated following miR27 antisense transfection, ZBTB10/RINZF, a Sp1 repressor, and RYBP/DEDAF, an apoptotic facilitator. These findings emphasize the importance of posttranscriptional mRNA regulation by HDACi in addition to their established effects on promoterdriven gene expression. http://www.ncbi.nlm.nih.gov/pubmed/16452179 Tue, 31 Jan 2006 00:00:00 -0800 The Ets transcription factor, ESX, exhibits a unique pattern of epithelialrestricted expression and transactivates genes involved in epithelial differentiation and cancer. The aim of this study was to determine the underlying genetic basis for epithelialspecific expression of ESX. We have identified a 30bp ESX enhancer sequence (EES) approximately 3 kb upstream of the proximal promoter. This region displays enhancer activity in an epithelialspecific manner and deletion of this region abrogates ESX gene transcription. An EES binding protein complex (EBC) was identified through electrophoretic mobility shift assays whose degree of EES binding correlated well with endogenous ESX levels in epithelial cells and was regulated by epithelial differentiation. Understanding the regulation of this element will lend insight into mechanisms of epithelial differentiation and the etiology of breast cancer and may provide novel targets for cancer therapeutic intervention. http://www.ncbi.nlm.nih.gov/pubmed/16307850 Tue, 31 Jan 2006 00:00:00 -0800 Estrogen receptor (ER, alpha isoform) is a 67 kDa zinc finger transcription factor that plays a fundamental role in both normal reproductive gland development and breast carcinogenesis, and also represents a critical molecular target for breast cancer therapy. We are investigating the structural consequences of chemical exposures thought to modify essential zinc finger cysteine residues in human ER. The current study employs mass spectrometry to probe ER zinc finger structural changes induced by a redoxreactive vitamin K3 analog, menadione a commonly used cysteine alkylator, iodoacetic acid and a thiol alkylating fluorophore, monobromobimane. Although they are slower to react, the sterically bulkier reagents, monobromobimane and menadione, effectively alkylate the most susceptible ER zinc finger cysteine sulfhydryl groups. Menadione arylation results first in Michael addition of the hydroquinone followed by rapid oxidation to the corresponding quinone, evidenced by a 2 Da mass loss per cysteine residue. Mass spectrometric analysis performed under MALDI conditions reveals both hydroquinone and quinone forms of arylated menadione, whereas only the quinone product is detectable under ESI conditions. Tandem mass spectrometry of a synthetic peptide encompassing the Cterminal half of the structurally more labile second zinc finger of ER (ZnF2B) demonstrates that the two nucleophilic thiols in ZnF2B (Cys237, Cys240) are not chemically equivalent in their reactivity to bromobimane or menadione, consistent with their unequal positioning near basic amino acids that affect thiol pKa, thereby rendering Cys240 more reactive than Cys237. These findings demonstrate important differential susceptibility of ER zinc finger cysteine residues to thiol reactions. http://www.ncbi.nlm.nih.gov/pubmed/16246571 Mon, 31 Oct 2005 00:00:00 -0800 The vitamin K analog menadione (K3), capable of both redox cycling and arylating nucleophilic substrates by Michael addition, has been extensively studied as a model stressinducing quinone in both cell culture and animal model systems. Exposure of keratin 8 (k8) expressing human breast cancer cells (MCF7, T47D, SKBr3) to K3 (50100 microM) induced rapid, sustained, and sitespecific k8 serine phosphorylation (pSer73) dependent on signaling by a single mitogen activated protein kinase (MAPK) pathway, MEK1/2. Normal nuclear morphology and k8 immunofluorescence coupled with the lack of DNA laddering or other features of apoptosis indicated that K3induced cytotoxicity, evident within 4 h of treatment and delayed but not prevented by MEK1/2 inhibition, was due to a form of stressactivated cell death known as oncosis. Independent of MAPK signaling was the progressive appearance of K3induced cellular fluorescence, principally nuclear in origin and suggested by in vitro fluorimetry to have been caused by K3 thiol arylation. Imaging by UV transillumination of protein gels containing nuclear extracts from K3treated cells revealed a prominent 17kDa band shown to be histone H3 by immunoblotting and mass spectrometry (MS). K3 arylation of histones in vitro followed by electrospray ionizationtandem MS analyses identified the unique Cys110 residue within H3, exposed only in the open chromatin of transcriptionally active genes, as a K3 arylation target. These findings delineate new pathways associated with K3induced stress and suggest a potentially novel role for H3 Cys110 as a nuclear stress sensor. http://www.ncbi.nlm.nih.gov/pubmed/15939799 Wed, 31 Aug 2005 00:00:00 -0700 To investigate the dysregulating effect of excess oxidative stress on mammary gland development, mammary anlage from newborn female mice with normal (/) or absent (null, /) manganese superoxide dismutase (SOD2) were excised and implanted under the renal capsule of normal host female nude mice with/without concurrent estrogen supplementation. After 30 days the transplanted glands were excised for wholemount, microscopic and immunohistochemical evaluation. In contrast to the normal growth and maturation of transplanted SOD2/ glands, SOD2/ glands showed arrested development, reduced ductal outgrowth and branching, and absent lumen. These hypomorphic SOD2/ ducts contained hyperplastic epithelium with increased Ki67 labelling, loss of Ecadherin expression, and disorganized p63 and cytokeratin (K)14 expressing basal and myoepithelial components. Estrogen treatment failed to upregulate progesterone receptor or normalize development. These findings suggest that excess oxidative stress from loss of SOD2 function can arrest mammary gland maturation and induce hyperplastic epithelium with early premalignant features. http://www.ncbi.nlm.nih.gov/pubmed/16085231 Sun, 31 Jul 2005 00:00:00 -0700 Antihormones (notably tamoxifen), chemotherapy and modern radiotherapeutic approaches are invaluable in the management of breast cancer, and collectively have contributed substantially to the improved survival in this disease. Moreover, there is promise that these successes will continue with the emergence of other endocrine agents (for example, aromatase inhibitors and pure antioestrogens). However, de novo and acquired resistance comprises a significant problem with all treatment approaches examined to date. This Workshop aimed to evaluate the contribution made by growth factor signalling pathways in the various resistant states, primarily focusing on resistance to antihormonal strategies and spanning experimental models and, where possible, clinical breast cancer data. The successes and limitations of therapeutic targeting of these pathways with various signal transduction inhibitors (STIs) were evaluated in model systems and from emerging clinical trials (including epidermal growth factor receptor inhibitors such as gefitinib). It was concluded that growth factor signalling is an important contributor in the development of endocrine resistance in breast cancer and that use of STIs provides a promising therapeutic strategy for this disease. However, the cancer cell is clearly able to harness alternative growth factor signalling pathways for growth and cell survival in the presence of STI monotherapy and, as a consequence, the efficacy of STIs is likely to be limited by the acquisition of resistance. A number of strategies were proposed from studies in model systems that appeared to enhance antitumour actions of existing STI monotherapy, notably including combination therapies targeting multiple pathways. With the increased availability of diverse STIs and improved drug delivery, there is much hope that the more complex therapeutic strategies proposed may ultimately be achievable in clinical practice. http://www.ncbi.nlm.nih.gov/pubmed/16113086 Sun, 31 Jul 2005 00:00:00 -0700 Endocrine therapy with an estrogen receptor (ER)targeted antiestrogen, such as tamoxifen, or estrogen ablation by aromatase inhibitors is clinically indicated for the management of all forms of ERpositive breast cancer. However, 3050 of ERpositive breast cancer cases fail to benefit clinically from endocrine therapy alone, and recent molecular evidence suggests that 'crosstalk' pathways originating from activated receptor tyrosine kinases and/or other proliferative and survival signals may be contributing to this endocrine resistance. Molecular identification and validation of candidate ER crosstalking pathways will likely lead to clinically important prognostic markers and targets for the application of novel therapeutics in combination with standard endocrine agents. This review focuses on a critical survival and proliferation pathway involving activation of nuclear factorkappaB (NFkappaB), a family of ubiquitously expressed transcription factors that for nearly two decades have been known to be critical regulators of mammalian immune and inflammatory responses, and more recently have been associated with chemotherapy resistance. With the demonstration that activation of NFkappaB is absolutely required for normal mammary gland development, NFkappaB involvment in human breast cancers was initially explored and linked to the development of hormoneindependent (ERnegative) breast cancer. Newer clinical evidence now implicates NFkappaB activation, particularly DNAbinding by the p50 subunit of NFkappaB, as a potential prognostic marker capable of identifying a highrisk subset of ERpositive, primary breast cancers destined for early relapse despite adjuvant endocrine therapy with tamoxifen. Furthermore, initial preclinical studies suggest that treatment strategies designed to prevent or interrupt activation of NFkappaB in cellline models of these more aggressive, ERpositive breast cancers can restore their sensitivity to such standard endocrine agents as tamoxifen. http://www.ncbi.nlm.nih.gov/pubmed/16113098 Sun, 31 Jul 2005 00:00:00 -0700 ErbB2overexpressing human cancers represent potentially sensitive targets for therapy by candidate histone deacetylase (HDAC) inhibitors as we have shown that HDAC inhibitors can selectively reduce ErbB2 expression by repressing the ErbB2 promoter and accelerating the decay of cytoplasmic ErbB2 transcripts. To extend these in vitro findings and enhance the in vivo pharmacodynamic properties of HDAC inhibitors, we stably encapsulated a potent hydroxamatebased HDAC inhibitor (LAQ824) within longcirculating liposomes (LsLAQ824) and immunoliposomes (ILsLAQ824) bearing 10,000 LAQ824 molecules per nanovesicle. Liposomal LAQ824 exhibits prolonged in vivo stability and, unlike free LAQ824, circulates with a halflife of 10.8 hours following a single i.v. injection. Three weekly i.v. injections of 20 to 25 mg/kg LsLAQ824 in nude mice with ErbB2 overexpressing BT474 breast tumor xenografts significantly impairs tumor growth, and administration of ErbB2targeted ILsLAQ824 may further improve this antitumor activity. Studies of tumorbearing mice 24 hours after single treatment indicate that: (a) 10 of injected liposomal LAQ824 is still circulating (whereas free LAQ824 is undetectable in the blood after 15 minutes) and (b) tumor uptake of LsLAQ824 and ILsLAQ824 is 3 injected drug per gram of tumor, producing levels of acetylated tumor histones that are 5 to 10fold increased over those following free LAQ824 or saline treatments and resulting in concordantly reduced levels of tumor ErbB2 mRNA. These preclinical results support the clinical evaluation of HDAC inhibitors against ErbB2overexpressing malignancies, and further indicate that encapsulation into targeted and nontargeted liposomes substantially improves the in vivo pharmacokinetics, tumor uptake, and antitumor properties of hydroxamatebased HDAC inhibitors. http://www.ncbi.nlm.nih.gov/pubmed/15867240 Sat, 30 Apr 2005 00:00:00 -0700 Acetylation is a key posttranslational modification of many proteins responsible for regulating critical intracellular pathways. Although histones are the most thoroughly studied of acetylated protein substrates, histone acetyltransferases (HATs) and deacetylases (HDACs) are also responsible for modifying the activity of diverse types of nonhistone proteins, including transcription factors and signal transduction mediators. HDACs have emerged as uncredentialed molecular targets for the development of enzymatic inhibitors to treat human cancer, and six structurally distinct drug classes have been identified with in vivo bioavailability and intracellular capability to inhibit many of the known mammalian members representing the two general types of NADindependent yeast HDACs, Rpd3 (HDACs 1, 2, 3, 8) and Hda1 (HDACs 4, 5, 6, 7, 9a, 9b, 10). Initial clinical trials indicate that HDAC inhibitors from several different structural classes are very well tolerated and exhibit clinical activity against a variety of human malignancies however, the molecular basis for their anticancer selectivity remains largely unknown. HDAC inhibitors have also shown preclinical promise when combined with other therapeutic agents, and innovative drug delivery strategies, including liposome encapsulation, may further enhance their clinical development and anticancer potential. An improved understanding of the mechanistic role of specific HDACs in human tumorigenesis, as well as the identification of more specific HDAC inhibitors, will likely accelerate the clinical development and broaden the future scope and utility of HDAC inhibitors for cancer treatment. http://www.ncbi.nlm.nih.gov/pubmed/15822187 Thu, 31 Mar 2005 00:00:00 -0800 Activation of nuclear factorkappaB (NFkappaB) has been linked to the development of hormoneindependent, estrogen receptor (ER)negative human breast cancers. To explore the possibility that activated NFkappaB marks a subset of clinically more aggressive ERpositive breast cancers, NFkappaB DNAbinding was measured in ERpositive breast cancer cell lines and primary breast cancer extracts by electrophoretic mobility shift assay and ELISAbased quantification of specific NFkappaB p50 and p65 DNAbinding subunits. Oxidant (menadione 100 microMx30 min) activation of NFkappaB was prevented by pretreatment with various NFkappaB inhibitors, including the specific IkappaB kinase (IKK) inhibitor, parthenolide (PA), which was found to sensitize MCF7/HER2 and BT474 but not MCF7 cells to the antiestrogen tamoxifen. Early stage primary breast cancers selected a priori for lower ER content (2187 fmol/mg n=59) and known clinical outcome showed two to fourfold increased p50 and p65 NFkappaB DNAbinding over a second set of primary breast cancers with higher ER content (100 fmol/mg n=22). Breast cancers destined to relapse (13/59) showed significantly higher NFkappaB p50 (but not p65) DNAbinding over those not destined to relapse (46/59 p=0.04). NFkappaB p50 DNAbinding correlated positively with several prognostic biomarkers however, only NFkappaB p50 DNAbinding (p=0.04), Activator Protein1 DNAbinding (AP1 por=0.01) and urokinasetype plasminogen activator expression (uPA p=0.0014) showed significant associations with metastatic relapse and diseasefree patient survival. These clinical findings indicate that highrisk ERpositive breast cancers may be prognostically identified by increased NFkappaB p50 DNAbinding, and support preclinical models suggesting that therapeutic inhibition of NFkappaB activation may improve the endocrine responsiveness of highrisk ERpositive breast cancers. http://www.ncbi.nlm.nih.gov/pubmed/15743683 Mon, 28 Feb 2005 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/15743664 Mon, 28 Feb 2005 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/15770520 Mon, 28 Feb 2005 00:00:00 -0800 Nanoscale drug delivery systems including liposomes, polymers, and other nanoparticles provide potential solutions for improved cancer therapeutics. Of these drug delivery systems, liposomebased agents, particularly liposomal anthracyclines, have had the greatest impact in oncology to date. Current liposomal drugs evolved from a number of design strategies for improved biodistribution over free drugs. Reticuloendothelial systemtargeted formulations significantly reduce systemic exposure to high peak levels of free drug, but do not facilitate targeting to tumors. Passive or physiologic targeting of drugs to tumors is achievable using longcirculating liposomes, including pure lipid systems as well as surfacemodified formulations designed to resist recognition and uptake by reticuloendothelial system cells. The latter, represented by pegylated or STEALTH liposomes, circulate for days as stable constructs and slowly extravasate in neoangiogenic vessels in tumors, providing a degree of passive targeting to tumor tissue. Future liposome therapeutics are building on these validated designs as well as on pharmacologic insights into their mechanisms of delivery. For example, camptothecin analogues, antiangiogenesis agents, and antisense oligonucleotides each represent rational candidates for delivery in highly stabilized and longcirculating liposomes. For such agents, pegylated liposome delivery offers improved chemical stability of encapsulated drug, enhanced accumulation in tumors, and prolonged drug exposure. True molecular targeting can be achieved using liposomes linked to ligands such as monoclonal antibody fragments directed against cancerassociated antigens. Immunoliposomes combine antibodymediated tumor recognition with liposomal delivery and, when designed for target cell internalization, provide intracellular drug release. Recent advances in immunoliposome design include rapid selection of phage antibodyderived scFv for targeting, and methods for conjugation of ligands to existing US Food and Drug Administrationapproved liposomal drugs such as pegylated liposomal doxorubicin (Doxil/Caelxy PLD). An immunoliposome consisting of novel antiHER2 scFv F5 conjugated to PLD, currently in development, selectively binds to and internalizes in HER2overexpressing tumor cells. The modular organization of immunoliposome technology enables a combinatorial approach in which a repertoire of monoclonal antibody fragments can be used in conjunction with a series of liposomal drugs to yield a new generation of molecularly targeted agents. http://www.ncbi.nlm.nih.gov/pubmed/15717745 Mon, 31 Jan 2005 00:00:00 -0800 Targeting HER2(ErbB2/neu) overexpressing tumor cells to selectively deliver anticancer agents and thereby reduce host toxicity represents a rational and emerging strategy for the treatment of breast and other epithelial cancers. The extracellular domain of the HER2 receptor tyrosine kinase is readily accessible to systemically administered antibodybased therapeutics, including growthinhibiting monclonals such as rhuMAbHER2 (trastuzmab/Herceptin) as well as antiHER2 immunotoxins, antibodydependent enzyme prodrug therapy (ADEPT), and immune cell recruiting bispecific antibodies. In addition to summarizing recent advances in these antibodybased strategies, this review focuses on preclinical advances in the development of antiHER2 immunoliposomes (ILs) as a platform technology for targeted drug delivery. Extensive in vitro and in vivo testing including efficacy and tumor uptake studies in multiple human tumor xenograft models now provide conclusive evidence for the superior therapeutic efficacy of antiHER2 ILsdoxorubicin (dox) over free dox or liposomal (Ls)dox, and even over combinations of dox and Lsdox with rhuMAbHER2. As antiHER2 ILsdox approaches clinical testing in patients with advanced HER2 overexpressing breast cancer, future applications of this novel targeting strategy will also broaden to include intracellular delivery of other anticancer agents as well as therapeutic nucleic acids (oligonucleotides, genes). http://www.ncbi.nlm.nih.gov/pubmed/15687597 Mon, 31 Jan 2005 00:00:00 -0800 HYPOTHESIS: Some risk factors associated with breast cancer may be more predictive of estrogen receptor (ER) positive than ERnegative tumors. DESIGN: Survey of patients enrolled in a study of breast cancer risk factors. SETTING: Community population in a northern California county. PATIENTS: A total of 234 individuals diagnosed as having breast cancer between July 1, 1997, and June 30, 1999, reporting Marin County, California, residence and participating in a questionnaire regarding exposure to breast cancer risk factors. MAIN OUTCOME MEASURE: Diagnosis of ERpositive vs ERnegative breast cancer. RESULTS: Comparison between ERpositive and ERnegative cases showed several factors predictive of ERpositive tumors. In a multivariate model, years of hormone therapy use remained the most significant predictor of ERpositive disease. CONCLUSIONS: Patients diagnosed as having ERpositive breast cancer were more likely to have undergone hormone therapy. The excess of ERpositive breast cancers reported in Marin County could, therefore, in part, be related to hormone therapy. http://www.ncbi.nlm.nih.gov/pubmed/15655207 Fri, 31 Dec 2004 00:00:00 -0800 The continued evolution of targeted liposomal therapeutics has resulted in new agents with remarkable antitumour efficacy and relatively mild toxicity profiles. A careful selection of the ligand is necessary to reduce immunogenicity, retain extended circulation lifetimes, target tumourspecific cell surface epitopes, and induce internalisation and subsequent release of the therapeutic substance from the liposome. Methods for assembling targeted liposomes, including a novel micellar insertion technology, for incorporation of targeting molecules that efficiently transforms a nontargeted liposomal therapeutic to a targeted one, greatly assist the translation of targeted liposome technology into the clinic. Targeting strategies with liposomes directed at solid tumours and vascular targets are discussed. The authors believe the development of ligandtargeted liposomes is now in the advanced stage and offers unique and important advantages among other targeted therapies. AntiHER2 immunoliposomal doxorubicin is awaiting Phase I clinical trials, the results of which should provide new insights into the promise of ligandtargeted liposomal therapies. http://www.ncbi.nlm.nih.gov/pubmed/15268628 Wed, 30 Jun 2004 00:00:00 -0700 Elevated and more rapidly increasing breast cancer incidence rates have been described for Marin County, California (CA), a homogeneous, high socioeconomic status population for which yearly surveillance is facilitated by its status as a county. The present study evaluates the histology and hormonal phenotype of the excess breast cancer cases occurring in white, nonHispanic women living in Marin County between 1992 and 2000 and compares them with patterns occurring in the rest of the San Francisco Bay Area (SFBA) and other urban parts of CA. Incidence data for invasive breast cancer histological subtypes and estrogen receptor (ER) and progesterone receptor (PR) status were obtained from the 19922000 Surveillance, Epidemiology, and End Results program. Expected numbers for Marin County were computed based on agespecific rates for five other SFBA counties. Incidence rates were ageadjusted to the 2000 United States standard. Marin County breast cancer diagnoses during 19922000 compared with other SFBA and other urban CA Surveillance, Epidemiology, and End Results county rates for white, nonHispanic women consisted of a disproportionate increase in ER/PR tumors. The observed absolute excess (versus expected) numbers of Marin County ER/PR lobular and nonlobular (predominantly ductal) cases were similar however, the relative increase appeared greatest for lobular breast cancer. The progressive increase in breast cancer incidence rates observed in Marin County over the past decade is occurring in women with high prevalence of risk factors predisposing toward excess development of ER/PR breast cancer. http://www.ncbi.nlm.nih.gov/pubmed/14693747 Sun, 30 Nov 2003 00:00:00 -0800 Oxidantinduced structural modifications within the cysteinerich DNAbinding domain (DBD) of the overexpressed estrogen receptor (ER) likely contribute to its loss of DNAbinding function and altered transcriptional activity during human breast cancer development. Using recombinant ER protein as a model, procedures to detect such endogenously produced structural changes in the two Cys(4)type zinc fingers within the DBD of ER extracted from breast cancer cells are being developed. Unfortunately, ex vivo oxidation of these ERDBD cysteine residues can occur during routine ER purification and preparation procedures. Also, cysteine residues readily undergo thioldisulfide exchange reactions that can result in artificial oxidation and incorrect disulfide bond assignments. These problems can be circumvented by an initial irreversible alkylation of all free thiols followed by reduction of any disulfides and treatment with a second alkylating agent, prior to proteolysis and highperformance liquid chromatography mass spectrometry analysis of peptides in the doubly alkylated ER digest, to differentiate between the originally free and the disulfidebonded cysteine residues. Although the use of chemically identical but isotopically different alkylating agents was more effective than the use of chemically different alkylating agents, subsequent problems were encountered with incomplete alkylation of particular Cys residues in the native ER protein. To overcome this limitation, the initial alkylation was accompanied by denaturation and the second alkylation was carried out during the proteolytic digestion. These improved analytical strategies should facilitate the monitoring of structurally altered endogenous ER produced within oxidantstressed human breast cancer cells. http://www.ncbi.nlm.nih.gov/pubmed/12895466 Thu, 31 Jul 2003 00:00:00 -0700 This review and study of nearly 4,000 primary breast cancers evaluates the hypothesis that human aging not only increases breast cancer incidence but also alters breast cancer biology. Clinically validated biomarkers were chosen as surrogate measures of genetic instability and tumor growth, invasiveness and metastatic potential. Our results support the premise that breast cancer clinical behavior and biology are significantly affected by patient age, but call into question key aspects of the current canceraging paradigm. http://www.ncbi.nlm.nih.gov/pubmed/12820531 Sat, 31 May 2003 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/12611465 Fri, 28 Feb 2003 00:00:00 -0800 The antitumor activity of histone deacetylase (HDAC) inhibitors has been linked to gene expression induced by acetylation of histone and nonhistone proteins but the molecular basis for their antitumor selectivity remains largely unknown. With development of a genomically integrated, ErbB2 promoterreporting breast cancer cell screen, ErbB2 promoter inhibiting activity was observed by the HDAC inhibitors trichostatin A (TSA) and sodium butyrate. Paradoxically, these agents stimulated the episomal form of this ErbB2 promoterreporter introduced by transient transfection. Transcriptional runoff assays in ErbB2 amplified and overexpressing breast cancer cells confirmed that within 5 h, TSA exposure profoundly inhibits ErbB2 transcript synthesis from the amplified oncogene yet preserves transcription from single copy genes such as the epithelialspecific Ets family member, ESX. Northern analyses of ErbB2overexpressing breast cancer lines (SKBR3, BT474, and MDA453) showed that within 24 h of submicromolar treatment by TSA, ESX transcript levels increase while ErbB2 transcript levels rapidly decline, with no TSA effect apparent on the open chromatin configuration of either gene as monitored by DNase I hypersensitivity. Actinomycin D studies confirmed that in addition to inhibiting ErbB2 transcript synthesis, TSA selectively destabilizes mature ErbB2 transcripts enhancing their decay. Whereas TSA markedly reduced ErbB2 protein levels in these overexpressing cell lines, TSA treatment of MCF/HER218 cells engineered to overexpress the ErbB2 receptor under control of a heterologous promoter increased their expression of ErbB2 protein. These findings suggest that further studies are warranted to determine whether ErbB2positive human cancers represent unusually sensitive clinical targets for HDAC inhibitor therapy. http://www.ncbi.nlm.nih.gov/pubmed/12477051 Sat, 30 Nov 2002 00:00:00 -0800 Breast cancer incidence increases with age but this relationship has not been fully explored with regard to expression of estrogen receptor (ER) and ERinducible genes (PR, pS2, Bcl2, cathepsin D), or the agedependence of oxidant stress markers that also affect ERinducible gene expression. In this threepart study, we first correlated age at diagnosis with expression of breast cancer markers ER, PR, pS2, Bcl2, and cathepsin D, quantitated by enzyme immunoassays from a European collective of approximately 3000 cryobanked primary breast cancers and approximately 300 adjacent nonmalignant breast tissues. Results were then compared with ER and PR data reported to the SEER registry for 83,541 US cancers diagnosed during 19921997. Lastly, a homogeneous subset of 70 ERpositive tumors preselected from the European collective was blindly analyzed for agespecific changes in the DNAbinding content of redoxsensitive transcriprtion factors, AP1 and Sp1, and the oxidant stressactivated protein kinase, phosphorylated(P)Erk5. Increases in breast tumor ER from patients aged 30 to 80 years mirrored 10fold lower increases in nonmalignant breast tissue ER content up to age 60, rising faster thereafter and reaching a near 25fold differential between malignant and nonmalignant breast tissue by age 80. ERinducible markers PR, pS2, Bcl2, and cathepsin D were overexpressed in tumors relative to nonmalignant breast tissue but, unlike ER, did not increase with patient age. While SEER data demonstrated that the increase in US breast cancer incidence rates after age 50 is confined to ERpositive tumors in patients of all ethnic subsets, these patients also showed a striking increase in the proportion of higherrisk ERpositive/PRnegative breast cancers arising after age 50. Mechanistically essential for ERinducible PR expression, Sp1 DNAbinding function (but not Sp1 content) was lost with age in ERpositive tumors and this functional defect correlated with increased tumor content of the oxidant stress marker, PErk5. Altogether these findings support two hypotheses: (i) dysregulated ER expression underlies the agespecific increase in breast cancer incidence after age 50 and (ii) oxidative stress and loss of Sp1 DNAbinding may contribute to an increasing incidence in higherrisk ERpositive/PRnegative breast cancers with aging. http://www.ncbi.nlm.nih.gov/pubmed/12462383 Sat, 30 Nov 2002 00:00:00 -0800 To explore the hypothesis that aging not only increases breast cancer incidence but also alters breast cancer biology, we correlated patient age and diagnosis with tumor histology, stage and biomarkers independently determined from two different tumor archives: an American collection of approximately 800 paraffinembedded and immunohistochemically analyzed primary breast cancers, and an European collection of approximately 3000 cryobanked primary breast cancers analyzed by ligandbinding and enzyme immunoassay (EIA). The prognostic biomarkers chosen for comparison represented surrogate measures of tumor: (i). proliferation, growth and genetic instability (mitotic and apoptotic indices, Ki67/MIB1positivity, nuclear grade, p53positivity), (ii). endocrinedependence (estrogen receptor (ER), progesterone receptors (PR), pS2, Bcl2), (iii). growth factor receptordependence (ErbB2, EGFR/ErbB1), and (iv). angiogenic, invasive and proteolytic potential (uPA, PAI1, Cathepsin D, VEGF). No biomarker reflecting tumor angiogenic, invasive or proteolytic potential showed a significant correlation with patient age at diagnosis. In contrast, significant inverse correlations (r0.1 P or =0.05) were observed for all measures of tumor growth and genetic instability as well as growth factor receptor overexpression (ErbB2 or EGFR positivity). Only one marker of endocrinedependence, ER expression, showed a significant positive correlation with patient age at diagnosis. In summary, these findings support the hypothesis that breast cancer biology is significantly affected by patient age. In particular, breast tumors arising in older patients have slower growth rates, are more likely to be ERpositive, and are less likely to be p53positive, EGFRpositive or ErbB2positive. http://www.ncbi.nlm.nih.gov/pubmed/12200028 Wed, 31 Jul 2002 00:00:00 -0700 Many targeted cancer therapies require endocytosis of the targeting molecule and delivery of the therapeutic agent to the interior of the tumor cell. To generate single chain Fv (scFv) antibodies capable of triggering receptormediated endocytosis, we previously developed a method to directly select phage antibodies for internalization by recovering infectious phage from the cytoplasm of the target cell. Using this methodology, we reported the selection of a panel of scFv that were internalized into breast cancer cells from a nonimmune phage library. For this work, an immunotherapeutic was generated from one of these scFv (F5), which bound to ErbB2 (HER2/neu). The F5 scFv was reengineered with a Cterminal cysteine, expressed at high levels in Escherichia coli, and coupled to sterically stabilized liposomes. F5 antiErbB2 immunoliposomes were immunoreactive as determined by surface plasmon resonance (SPR) and were avidly internalized by ErbB2expressing tumor cell lines in proportion to the levels of ErbB2 expression. F5scFv targeted liposomes containing doxorubicin had antitumor activity and produced significant reduction in tumor size in xenografted mice compared to nontargeted liposomes containing doxorubicin. This strategy should be applicable to generate immunotherapeutics for other malignancies by selecting phage antibodies for internalization into other tumor types and using the scFv to target liposomes or other nanoparticles. http://www.ncbi.nlm.nih.gov/pubmed/12183061 Wed, 31 Jul 2002 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/12078526 Fri, 31 May 2002 00:00:00 -0700 Various members of the Ets multigene family exhibit diverse roles in development, cell differentiation, tissuespecific gene expression and human malignancy. In the search for Ets factors involved in mammary gland development and malignancy, ESX was found to be upregulated in a subset of breast tumours and cell lines. We report the transcriptional regulation of ESX in epithelial breast cancer cells. Transient reporter assays using the ESX promoter show that ESX transcription is regulated by ErbB receptor signalling. In cell lines and in 45 primary ductal breast cancers we show that ESX transcript expression significantly correlates with ErbB2 transcript levels. Moreover, expression of ErbB2 in cells upregulates ESX promoter activity while inhibition of ErbB2 or its downstream signaling pathways decrease both ESX promoter activity and endogenous ESX protein levels. These results indicate that the ESX promoter represents a transcriptional target of ErbB2, and ESX expression may represent a downstream mediator of ErbB2 signaling and ErbB2induced gene expression. http://www.ncbi.nlm.nih.gov/pubmed/12032832 Tue, 30 Apr 2002 00:00:00 -0700 PURPOSE: AntiHER2 immunoliposomes combine the tumortargeting of certain antiHER2 monoclonal antibodies (MAbs) with the pharmacokinetic and drug delivery capabilities of sterically stabilized liposomes. We previously showed that antiHER2 immunoliposomes bind efficiently to and internalize in HER2overexpressing cells in vitro, resulting in intracellular drug delivery. EXPERIMENTAL DESIGN: Here we describe the pharmacokinetics and therapeutic efficacy of antiHER2 immunoliposomes containing doxorubicin (dox) in a series of animal models. RESULTS: Immunoliposomes displayed long circulation that was identical to that of sterically stabilized liposomes in single and multipledose studies in normal rats. AntiHER2 immunoliposomedox produced marked therapeutic results in four different HER2overexpressing tumor xenograft models, including growth inhibition, regression, and cures. These results demonstrated that encapsulation of dox in antiHER2 immunoliposomes greatly increased its therapeutic index, both by increasing antitumor efficacy and by reducing systemic toxicity. Immunoliposomedox was significantly superior to all other treatment conditions tested, including free dox, liposomal dox, and antiHER2 MAb (trastuzumab). When compared with liposomal dox in eight separate therapy studies in HER2overexpressing models, immunoliposome delivery produced significantly superior antitumor efficacy in each study (P 0.0001 to 0.04). AntiHER2 immunoliposomedox containing either recombinant human MAb HER2Fab' or scFv C6.5 yielded comparable therapeutic efficacy. Cure rates for immunoliposomedox reached 50 (11 of 21) with optimized immunoliposomes and Matrigelfree tumors and overall was 16 (18 of 115) versus no cures (0 of 124) with free dox or liposomal dox. Finally, antiHER2 immunoliposomedox was also superior to combinations consisting of free MAb plus free dox or free MAb plus liposomal dox. CONCLUSIONS: AntiHER2 immunoliposomes produced enhanced antitumor efficacy via targeted delivery. http://www.ncbi.nlm.nih.gov/pubmed/11948130 Sun, 31 Mar 2002 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/11804185 Mon, 31 Dec 2001 00:00:00 -0800 We have generated antiHER2 (ErbB2) immunoliposomes (ILs), consisting of long circulating liposomes linked to antiHER2 monoclonal antibody (MAb) fragments, to provide targeted drug delivery to HER2overexpressing cells. Immunoliposomes were constructed using a modular strategy in which components were optimized for internalization and intracellular drug delivery. Parameters included choice of antibody construct, antibody density, antibody conjugation procedure, and choice of liposome construct. AntiHER2 immunoliposomes bound efficiently to and internalized in HER2overexpressing cells in vitro as determined by fluorescence microscopy, electron microscopy, and quantitative analysis of fluorescent probe delivery. Delivery via ILs in HER2overexpressing cells yielded drug uptake that was up to 700fold greater than with nontargeted sterically stabilized liposomes. In vivo, antiHER2 ILs showed extremely long circulation as stable constructs in normal adult rats after a single i.v. dose, with pharmacokinetics that were indistinguishable from sterically stabilized liposomes. Repeat administrations revealed no increase in clearance, further confirming that ILs retain the long circulation and nonimmunogenicity of sterically stabilized liposomes. In five different HER2overexpressing xenograft models, antiHER2 ILs loaded with doxorubicin (dox) showed potent anticancer activity, including tumor inhibition, regressions, and cures (pathologic complete responses). ILs were significantly superior vs. all other treatment conditions tested: free dox, liposomal dox, free MAb (trastuzumab), and combinations of doxMAb or liposomal doxMAb. For example, ILs produced significantly superior antitumor effects vs. nontargeted liposomes (P values from 0.0001 to 0.04 in eight separate experiments). In a nonHER2overexpressing xenograft model (MCF7), ILs and nontargeted liposomal dox produced equivalent antitumor effects. Detailed studies of tumor localization indicated a novel mechanism of drug delivery for antiHER2 ILs. Immunotargeting did not increase tumor tissue levels of ILs vs. liposomes, as both achieved very high tumor localization (7.08.5 of injected dose/g tissue) in xenograft tumors. However, histologic studies using colloidalgold labeled ILs demonstrated efficient intracellular delivery in tumor cells, while nontargeted liposomes accumulated within stroma, either extracellularly or within macrophages. In the MCF7 xenograft model lacking HER2overexpression, no difference in tumor cell uptake was seen, with both ILs and nontargeted liposomes accumulating within stroma. Thus, antiHER2 ILs, but not nontargeted liposomes, achieve intracellular drug delivery via receptormediated endocytosis, and this mechanism is associated with superior antitumor activity. Based on these results, antiHER2 immunoliposomes have been developed toward clinical trials. Reengineering of construct design for clinical use has been achieved, including: new antiHER2 scFv F5 generated by screening of a phage antibody library for internalizing antiHER2 phage antibodies modifications of the scFv expression construct to support large scale production and clinical use and development of methods for largescale conjugation of antibody fragments with liposomes. We developed a scalable twostep protocol for linkage of scFv to preformed and drugloaded liposomes. Our final, optimized antiHER2 ILsdox construct consists of F5 conjugated to derivatized PEGPE linker and incorporated into commercially available liposomal doxorubicin (Doxil). Finally, further studies of the mechanism of action of antiHER2 ILsdox suggest that this strategy may provide optimal delivery of anthracyclinebased chemotherapy to HER2overexpressing cancer cells in the clinic, while circumventing the cardiotoxicity associated with trastuzumabanthracycline. We conclude that antiHER2 immunoliposomes represent a promising technology for tumortargeted drug delivery, and that this strategy may also be applicable to other receptor targets and/or using other delivered agents. http://www.ncbi.nlm.nih.gov/pubmed/11489487 Tue, 31 Jul 2001 00:00:00 -0700 Overexpression of the HER2 (neu/cerbB2) oncogene frequently coincides with an aggressive clinical course of certain human adenocarcinomas. Expression and secretion of aberrant HER2 splice variants has been reported in various cell lines and tissues and can interfere with the oncogenic HER2 activity. Here we demonstrate, using two different approaches, that expression of a truncated 100 kDa HER2 variant which encodes the extracellular domain of HER2 (HERECD) inhibits growth factormediated tumour cell proliferation. A HER2ECD cDNA encoding the truncated variant was overexpressed in MCF7 breast cancer cells. HER2ECD overexpression decreased spontaneous proliferation of MCF7 cells as well as heregulinmediated soft agar colony formation. Concomitantly, heregulininduced phosphorylation of HER4 as well as downstream activation of p44/p42 MAPkinases was decreased. To confirm these data, ribozymes were targeted to the 3'untranslated region of the 2.3 kb HER2ECD mRNA which is spontaneously expressed in MKN7 gastric cancer cells. HER2ECDtargeted ribozymes downregulated HER2ECD expression and enhanced EGFmediated soft agar colony formation of MKN7 cells. In parallel, EGFinduced activation of p44/p42 MAPkinases and activation of cFos expression were increased in ribozymetransfected MKN7 cells. Finally, in RTPCR we found a trend towards a progressive loss of 2.3 kb HER2ECD mRNA expression in more advanced gastric tumours. These data show that the HER2ECD variant inhibits growth factormediated tumour cell proliferation suggesting an important role during the progression of human cancer. http://www.ncbi.nlm.nih.gov/pubmed/11360194 Mon, 30 Apr 2001 00:00:00 -0700 Two internalizing monovalent single chain antibody fragments (scFv), C6.5 and F5, that recognize distinct ErbB2 extracellular domain (ECD) epitopes, and their bivalent forms dbC6.5 and F5(scFv')(2), were compared to the growthinhibiting antiErbB2 antibody Herceptin/trastuzumab, in either its bivalent (Her) or monovalent (4D5Fab') form, for their abilities to induce biological responses in the ErbB2overexpressing breast cancer cells, SkBr3. Assays compared internalization by receptormediated endocytosis, effects on cell cycling and culture growth, and interference with intracellular MAPK and PI3K signaling pathways. We found no correlation between ErbB2 epitope affinity or valency on degree of antibodyinduced endocytosis, since all the scFv were able to internalize better than Her. Unlike Her, neither the monovalent or bivalent forms of the internalizing scFv had any sustained effect on cell growth. Basal levels of MAPK and PI3K signaling in SkBr3 cells were not inhibited by up to 8 h scFv treatment, while decreased MAPK and PI3K signals were noted within 8 h of Her treatment. In summary, antibodyinduced ErbB2mediated endocytosis is not a surrogate marker for resultant biological response, as it shows no correlation with cell cycle, culture proliferation, or intracellular kinase signal induction by internalizing antibodies. Thus, the enhanced endocytotic property of scFv like C6.6 and F5 in conjunction with their absence of any growth or signaling impact on ErbB2overexpressing cells favors their choice as ErbB2 targeting moieties for intracellular delivery of novel cancer therapeutics. http://www.ncbi.nlm.nih.gov/pubmed/11162510 Wed, 31 Jan 2001 00:00:00 -0800 Evaluating the chromatinized erbB2 gene in nuclei from breast cancer cells expressing varying levels of ErbB2 transcripts, we identified a nucleasesensitive site within a 0.22 kb region of maximum enhancer activity centered over a conserved 28 bp polypurine(GGA)polypyrimidine(TCC) mirrorrepeat and an adjacent essential Ets binding site (EBS). Promoter footprinting with nuclear extracts reveals an intense Ets hypersensitivity site at the EBS whose degree of intensity correlates with the level of cellular ErbB2 expression. In vitro mapping assays show that the supercoiled erbB2 promoter forms an internal triplex structure (HrDNA) at the mirrorrepeat element. Mutations preventing HrDNA formation can enhance erbB2 promoter activity in human breast cancer cells, a result consistent with previous demonstration that EtserbB2 promoter complexes cannot form when the mirrorrepeat is engaged in triplex binding, and new results suggesting that Ets binding induces severe promoter bending that may restrict local triplex formation. In addition to previously described erbB2regulating breast cancer Ets factors (PEA3, ESX/Elf3), Elf1 is now shown to be another endogenously expressed Ets candidate capable of binding to and upregulating the erbB2 promoter. Given current strategies to transcriptionally inhibit ErbB2 overexpression, including development of novel erbB2 promotertargeted therapeutics, an EBStargeted approach is presented using chimeric Ets proteins that strongly repress erbB2 promoter activity. http://www.ncbi.nlm.nih.gov/pubmed/11175365 Wed, 31 Jan 2001 00:00:00 -0800 PURPOSE: We hypothesize that phosphorylated ErbB2 (PErbB2, identified by a novel antibody PN2A) may provide either more significant or additional prognostic marker data for breast cancer patients. This study was designed to compare the incidence and prognostic value of ErbB2 (HER2/neu) and PErbB2 immunoexpression in archival breast cancer samples. MATERIALS AND METHODS: Eight hundred sixteen invasive breast cancers with a median of 16.3 years of followup were immunostained for ErbB2 (using antibody CB11) and PErbB2 (using antibody PN2A). ErbB2 and PErbB2 data were compared with clinical, histologic, immunohistochemical, and outcome variables. RESULTS: Of 816 primary breast cancers, 307 (38) were positive for ErbB2 and 37 (12 of ErbB2 positive and 5 of the study population) expressed PErbB2. PErbB2 was not detected in ErbB2negative cases (n = 509). ErbB2 immunohistochemical data were bimodal patients with or = 80 cellular expression had the shortest diseasefree and diseasespecific survival. PErbB2 was associated with a higher percentage of ErbB2positive cells, a higher number of positive lymph nodes, and cellular proliferation. ErbB2 and PErbB2 were indicators of poor prognosis in nodepositive patients in both univariate and multivariate analyses. We found that either PErbB2 expression or high ( or = 80) ErbB2 expression provided the most significant prognostic value in nodepositive cases by multivariate analyses. There were too few PErbB2positive cases and events in the nodenegative patient group to allow statistical analysis of PErbB2 in that subgroup. CONCLUSION: PN2A immunostaining identified a subset (approximately 12 of ErbB2positive breast cancers) with activation (phosphorylation) of the receptor ErbB2. PErbB2 expression was strongly associated with higher levels of ErbB2 expression ( or = 80), although it was not a surrogate. Identification of cases with a high percentage of invasive breast cancer cells expressing ErbB2 or determination of receptor activation via PErbB2 may provide additional prognostic value in nodepositive breast cancers. http://www.ncbi.nlm.nih.gov/pubmed/10986055 Sat, 30 Sep 2000 00:00:00 -0700 Liposomeencapsulated anticancer drugs reveal their potential for increased therapeutic efficacy and decreased nonspecific toxicities due to their ability to enhance the delivery of chemotherapeutic agents to solid tumors. Advances in liposome technology have resulted in the development of ligandtargeted liposomes capable of selectively increasing the efficacy of carried agents against receptorbearing tumor cells. Receptors for vitamins and growth factors have become attractive targets for liganddirected liposomal therapies due to their high expression levels on various forms of cancer and their ability to internalize after binding to the liposomes conjugated to receptors' natural ligands (vitamins) or synthetic agonists (receptorspecific antibodies and synthetic peptides). This chapter summarizes various strategies and advances in targeting liposomes to vitamin and growth factor receptors in vitro and in vivo with special emphasis on two extensively studied liposometargeting systems utilizing folate receptor and HER2/neu growth factor receptor. http://www.ncbi.nlm.nih.gov/pubmed/11037627 Sat, 30 Sep 2000 00:00:00 -0700 Three DNA binding polyamides () were synthesized that bind with high affinity (K(a) = 8.7. 10(9) m(1) to 1.4. 10(10) m(1)) to two 7base pair sequences overlapping the Ets DNA binding site (EBS GAGGAA) within the regulatory region of the HER2/neu proximal promoter. As measured by electrophoretic mobility shift assay, polyamides binding to flanking elements upstream () or downstream (2 and 3) of the EBS were one to two orders of magnitude more effective than the natural product distamycin at inhibiting formation of complexes between the purified EBS protein, epithelial restricted with serine box (ESX), and the HER2/neu promoter probe. One polyamide, 2, completely blocked EtsDNA complex formation at 10 nm ligand concentration, whereas formation of activator protein2DNA complexes was unaffected at the activator protein2 binding site immediately upstream of the HER2/neu EBS, even at 100 nm ligand concentration. At equilibrium, polyamide 1 was equally effective at inhibiting Ets/DNA binding when added before or after in vitro formation of proteinpromoter complexes, demonstrating its utility to disrupt endogenous Etsmediated HER2/neu preinitiation complexes. Polyamide 2, the most potent inhibitor of EtsDNA complex formation by electrophoretic mobility shift assay, was also the most effective inhibitor of HER2/neu promoterdriven transcription measured in a cellfree system using nuclear extract from an ESX and HER2/neuoverexpressing human breast cancer cell line, SKBR3. http://www.ncbi.nlm.nih.gov/pubmed/10818092 Thu, 31 Aug 2000 00:00:00 -0700 For approximately onethird of estrogen receptor (ER)positive breast cancer patients, extracted tumor ER is unable to bind to its cognate DNA estrogen response element (ERE), an effect that is partly reversible by the thiolreducing agent dithiothreitol (DTT). Fulllength (67 kDa) ER or its 11 kDa recombinant DNAbinding domain (ERDBD) is also susceptible to loss of structure and function by the action of oxidants such as diamide and hydrogen peroxide however, prior DNA binding by ER or ERDBD protects against this oxidant induced loss of function. The ERDBD contains two (Cys)(4)liganded zinc finger motifs that cooperate to stabilize a rigid DNAbinding recognition helix and a flexible helixsupported dimerization loop, respectively. Comparisons between synthetic peptide analogues of each zinc finger and recombinant ERDBD in the presence of zinc by electrophoretic mobility shift assay, circular dichroism, and mass spectrometry confirm that cooperativity between these zinc fingers is required for both ERDBD structure (alphahelicity) and function (dimeric DNA binding). Rapid proteolytic digestion of monomeric, nonDNAbound ERDBD followed by HPLCMS analysis of the resulting peptides demonstrates that zinc inhibits thiol oxidation of the DNAbinding finger, but not the finger supporting the flexible dimerization loop, which remains sensitive to internal disulfide formation. These findings indicate that the loss of ER DNAbinding function in extracts from some primary breast tumors and in ER or ERDBD exposed to thiolreacting oxidants results from this asymmetric zinc finger susceptibility to disulfide formation that prevents dimerization. Although ERDBD contains several strategically located methionine residues, they are less susceptible to oxidation than the thiol groups and, thus, afford no protection against cysteine oxidation and consequent loss of ER DNAbinding function. http://www.ncbi.nlm.nih.gov/pubmed/10913246 Mon, 31 Jul 2000 00:00:00 -0700 Abnormal cell signal transduction arising from protein tyrosine kinases has been implicated in the initiation and progression of a variety of human cancers. Over the past 2 decades pharmaceutical and university laboratories have been involved in a tremendous effort to develop compounds that can selectively modulate these abnormal signalling pathways. Targeting receptor tyrosine kinases, especially the epidermal growth factor receptor subfamily, has been at the forefront of this effort as a result of strong clinical data correlating overexpression of these receptors with more aggressive cancers. There are a variety of strategies under development for inhibiting the kinase activity of these receptors, targeting both the extracellular and intracellular domains. Antibodybased approaches, immunotoxins and ligandbinding cytotoxic agents use the extracellular domain for targeted tumour therapy. Small molecule inhibitors target the intracellular catalytic region by interfering with ATP binding, while nonphosphorylatable peptides are aimed at the intracellular substrate binding region. Compounds that inhibit subsequent downstream signals from the receptor by interrupting intracellular protein recognition sequences are also being investigated. In the past 5 years enormous progress has been made in developing tyrosine kinase inhibitor compounds with sufficient potency, bioavailability and selectivity against this subfamily of receptor tyrosine kinases. The antiHER2 monoclonal antibody, trastuzumab, for patients with metastatic breast cancer is the first of these inhibitor compounds to gain FDA approval. However, preclinical and clinical trials are ongoing with a variety of other monoclonal antibodies, immunotoxins, and small molecule quinazoline and pyrimidinebased inhibitors. Although their cytotoxic and cytostatic potential has been proven, they are not likely to replace standard chemotherapy regimens as singleagent, firstline therapeutics. Instead, their promising additive and synergistic antitumour effects in combination with standard chemotherapeutics suggest that these novel agents will find their greatest utility and efficacy in conjunction with existing anticancer agents. http://www.ncbi.nlm.nih.gov/pubmed/10804033 Fri, 30 Jun 2000 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/10667243 Mon, 31 Jan 2000 00:00:00 -0800 We have previously reported that Heregulin (HRG)/neu differentiation factor (NDF) induced growth arrest and cellular differentiation in breast cancer cells overexpressing erbB2 receptor. To elucidate the cellular mechanisms underlying the growth inhibition by HRG, we developed an in vitro model by transfection of HRG cDNA into the erbB2 overexpressing breast cancer cell line, SKBr3. We showed that the enforced expression of HRG in SKBr3 cells induces dramatic morphological changes and pronounced inhibition of anchoragedependent and independent growth. Most SKBr3/HRGtransfected (SK/HRG) cells exhibited about 15fold increase in size and persisted as multinucleated cells with extended flattened vacuolefilled cytoplasm with reduced cell attachment. The growth suppression of SK/HRG cells was accompanied by a reduction in S phase, the presence of a G2M cell cycle delay, and an increase in DNA aneuploid components. In addition, DNA fragmentation assays showed that HRG induced apoptosis of SKBr3 cells. In contrast, while HRG treatment of other erbB2 overexpressing breast cancer cell lines led to growth arrest and cell detachment, it did not induce apoptotic features. Thus, this study demonstrates that while growth arrest and cell detachment are general effects of HRG towards erbB2 overexpressing cells, the ability of HRG to induce apoptosis is a phenomenon confined to selective cells including SKBr3 cells. http://www.ncbi.nlm.nih.gov/pubmed/10536169 Tue, 30 Nov 1999 00:00:00 -0800 The HER2/neu protooncogene is overexpressed in 25 to 30 of patients with breast cancer. Trastuzumab (Herceptin Genentech, San Francisco, CA), a recombinant humanized monoclonal antibody with high affinity for the HER2 protein, inhibits the growth of breast cancer cells overexpressing HER2. In this phase II study the efficacy and toxicity of weekly administration of trastuzumab was evaluated in 46 patients with metastatic breast cancer whose tumors overexpressed HER2. A loading dose of 250 mg trastuzumab was administered intravenously, which was followed by 10 weekly doses of 100 mg each. Upon completion of this treatment period, patients with no disease progression could receive a weekly maintenance dose of 100 mg. Patients in this trial had extensive metastatic disease, and most had received prior anticancer therapy. Ninety percent of patients achieved adequate serum levels of trastuzumab. Toxicity was minimal, and no antibodies against trastuzumab could be detected. Objective responses were observed in 5 of the 43 evaluable patients, which included 1 complete remission and 4 partial remissions, for an overall response rate of 11.6. Responses were seen in mediastinum, lymph nodes, liver, and chest wall lesions. Minor responses (seen in 2 patients) and stable disease (14 patients) lasted for a median of 5.1 months. These results demonstrate that trastuzumab is well tolerated and clinically active in patients with HER2overexpressing metastatic breast cancers who have received extensive prior therapy. The regression of human cancer through the targeting of putative growth factor receptors such as HER2 warrants further evaluation of trastuzumab in the treatment of breast cancer. http://www.ncbi.nlm.nih.gov/pubmed/10482197 Tue, 31 Aug 1999 00:00:00 -0700 The Ets gene family has a complex evolutionary history with many family members known to regulate genetic programs essential for differentiation and development, and some known for their involvement in human tumorigenesis. To understand the biological properties associated with a recently described epitheliumrestricted Ets factor ESX, an 11 kb fragment from the 1q32.2 genomically localized human gene was cloned and analysed. Upstream of the ESX promoter region in this genomic fragment lies the terminal exon of a newly identified gene that encodes a ubiquitinconjugating enzyme variant, UEV1. Tissues expressing ESX produce a primary 2.2 kb transcript along with a 4.1 kb secondary transcript arising by alternate poly(A) site selection and uniquely recognized by a genomic probe from the 3' terminal region of the 11 kb clone. Endogenous expression of ESX results in a 42 kDa nuclear protein having fivefold greater affinity for the chromatinnuclear matrix compartment as compared to other endogenous transcription factors like AP2 and the homologous Ets factor, ELF1. Exon mapping of the modular structure inferred from ESX cDNA and construction of GAL4(DBD)ESX expression constructs were used to identify a transactivating domain encoded by exon 4 having comparable potency to the acidic transactivation domain of the viral transcription factor, VP16. This exon 4encoded 31 amino acid domain in ESX was shown by mutation and deletion analysis to possess a 13 residue acidic transactivation core which, based on modeling and circular dichroism analysis, is predicted to form an amphipathic alphahelical secondary structure. Using recombinant GSTESX (exon 4) fusion proteins in an in vitro pulldown assay, this ESX transactivation domain was shown to bind specifically to one component of the general transcription machinery, TATAbinding protein (TBP). Transient transfection experiments confirmed the ability of this TBPbinding transactivation domain in ESX to squelch heterologous promoters independent of any promoter binding as efficiently as the transactivation domain from VP16. http://www.ncbi.nlm.nih.gov/pubmed/10391676 Wed, 30 Jun 1999 00:00:00 -0700 Fulllength (67 kDa) immunoreactive estrogen receptor (ER) extracted from a third of untreated ERpositive primary breast tumors appears unable to bind to its cognate estrogen response element (ERE). We have observed partial reversibility of this ER DNAbinding defect upon treatment of these tumor extracts with excess thiol reducing agent (DTT), suggesting that ER DNAbinding is subject to redox modulation as is reported for other zincfinger proteins and transcriptional activators. Treatment of recombinant ER DNAbinding domain (ERDBD) or ERenriched extracts from CHO(ER) and MCF7 cells with thiolreacting oxidants (diamide, iodosobenzoate, H2O2) or alkylator (iodoacetamide) produces a dosedependent loss in ER DNAbinding capacity. Thiolspecific oxidative loss in ER DNAbinding is fully reversible by DTT reduction, unlike the defect caused by thiolspecific alkylation. Circular dichroism spectrometry shows that both forms of treatment substantially modify ER secondary structure, inducing loss of alphahelical content within the ERDBD that is reversible after thiol oxidation but not after thiol alkylation. Oxidant (H2O2, menadione) exposure of cultured CHO(ER) or MCF7 cells impairs the ability of endogenous ER to bind DNA and transactivate an ERresponsive reporter gene (EREtkCAT), demonstrating that extracellular redox stress can modulate intracellular ER function. Since these thiolspecific oxidant and alkylator treatments have no significant effect on either recombinant ER ligandbinding or intracellular immunoreactive ER content, our findings suggest that DNAbinding and transactivation are the most sensitive intracellular ER functions impaired by oxidant stress in some ERpositive human breast tumors. http://www.ncbi.nlm.nih.gov/pubmed/10022773 Fri, 30 Apr 1999 00:00:00 -0700 Human breast tumors that are initially responsive to tamoxifen (TAM) eventually relapse during treatment. Estrogen receptor (ER) expression and function are often preserved in these tumors, and clinical evidence suggests that this relapse may be related to TAM's known agonistic properties. ER can interact with the activator protein1 (AP1) transcription factor complex through proteinprotein interactions that are independent of ER DNA binding and, in certain ERpositive cells, this may allow TAM to exert an agonist response on AP1regulated genes. We, therefore, assessed both AP1 DNA binding and the known AP1 activating enzyme, cJun NH2terminal kinase (JNK), in a panel of 30 ERpositive primary human breast tumors with acquired TAM resistance, as compared to a matched panel of 27 untreated control ERpositive breast tumors and a separate control set of 14 primary tumors, which included 7 ERpositive tumors that were growtharrested by 3 months of preoperative TAM. AP1 DNA binding activity was measured from cryopreserved tumor extracts using a labeled oligonucleotide probe containing a consensus AP1 response element by electrophoretic mobility shift assay. JNK was first extracted from the tumor lysates by incubation over a Sepharosebound cJun(189) fusion protein, and its activity was then measured by chemiluminescent Western blot by detection of the phosphorylated product using a phosphoJun(Ser63)specific primary antibody. The set of control ERpositive breast tumors growth arrested by TAM showed no significant difference from untreated control tumors in their AP1 DNA binding and JNK activities. In contrast, there was a significant (P 0.001) increase in mean AP1 DNA binding activity for the panel of ERpositive TAMresistant (TAMR) tumors as compared to its matched control panel of untreated tumors. Mean JNK activity in the TAMR tumors was also significantly higher than that found in the untreated tumors (P = 0.038). Overall, there was no significant correlation between JNK activity and AP1 DNA binding however, regression analysis showed that, for any given level of JNK activity, the TAMR tumors possessed a 3.5fold increase in AP1 DNA binding activity as compared to the untreated tumors. These findings indicate that, when compared to untreated ERpositive primary breast tumors, TAMR tumors demonstrate significantly increased levels of AP1 DNA binding and JNK activity, consistent with experimental models suggesting that TAMstimulated ERpositive tumor growth may be mediated by enhanced AP1 transcriptional activity. These observations support the need for further evaluation of these markers in breast tumors as predictors of TAM resistance. http://www.ncbi.nlm.nih.gov/pubmed/10037172 Wed, 31 Mar 1999 00:00:00 -0800 To study mammary gland expression of the epitheliumrestricted Ets factor, ESX, mouse cDNA and genomic sequences were cloned and a approximately 350 bp proximal promoter region with 80 mousehuman homology was identified that mediates ESX induction by serum, heregulin (HRG), or epidermal growth factor (EGF). ESX mRNA expression progressively increases during embryonic mouse development from day 7 and is detectable in virgin mammary glands it shows little if any change during pregnancy, then declines to barely detectable levels after 3 days of lactation. Similarly, cultured HC11 cells from midpregnant mouse mammary epithelium show an increase in ESX expression upon reaching lactogenic competency (in the presence of EGF or HRG), with a decline to barely detectable levels upon exposure to lactogenic hormones that induce milk protein (betacasein) expression. In contrast, involuting mouse and rat mammary glands show maximal ESX expression. High ESX levels are also seen in the involuting ventral prostate gland of rats. These findings, including the persistence of upregulated ESX in fully regressed mammary glands, suggest that ESX expression can be induced by soluble growth factors and is maximally upregulated in those partially committed epithelial cells destined to survive both the apoptotic and remodeling phases of glandular involution. http://www.ncbi.nlm.nih.gov/pubmed/9806763 Sat, 31 Oct 1998 00:00:00 -0800 Polyamines are known to inhibit sequence specific DNAbinding activity of several zincfinger transcription factors, including estrogen receptor (ER) binding to its cognate estrogen response element (ERE). The mechanism accounting for this disruption of proteinDNA interaction is unknown, although polyamine induction of DNA conformational changes has been suggested. To determine if polyamines can directly impair ER action, we compared the effects of putrescine (Putr), spermidine (Spd), and spermine (Spm) on ER DNAbinding (ERERE complex formation), ER ligandbinding (estradiol), ER structure (circular dichroism and sucrose gradient sedimentation), and the capacity of ER to transactivate an EREtkCAT reporter in transient transfection assays. Polyamine concentrations causing 50 inhibition of ERERE formation (IC50 values) were found to be 1 mM for Putr, 4 mM for Spd, and 3 mM for Spm. This loss of ER DNAbinding was associated with a direct and irreversible effect on the ER DNAbinding domain (ERDBD). Additionally, polyamines were observed to inhibit ER ligandbinding with IC50 values of 10 mM for Putr, 2 mM for Spd, and 0.1 mM for Spm and this correlated with a measureable change in higherorder ER structure (5S to 3.5S sedimentation) and inhibition of intracellular ER transactivation. These findings suggest that in ERpositive human breast tumors with increased polyamine (especially Spm) content, ER structure and function may be directly altered by tightion polyamine complexing that results in loss of ERmediated gene regulation. http://www.ncbi.nlm.nih.gov/pubmed/9598871 Fri, 31 Jul 1998 00:00:00 -0700 AntiHER2 immunoliposomes (ILs) have been constructed by conjugation of Fab' fragments of recombinant humanized monoclonal antibody rhuMAbHER2 to small sterically stabilized unilamellar liposomes, to create a targeted drug delivery vehicle for the treatment of HER2 (cerbB2, neu)overexpressing cancers. Parameters affecting in vitro binding and internalization of ILs include liposome composition, Fab' linkage site and Fab' density. AntiHER2 ILs have been constructed to optimize intracellular drug delivery. Doxorubicin (dox)loaded ILs are highly stable and exhibit prolonged circulation in rats. In nude mice bearing HER2overexpressing tumor xenografts, antiHER2 ILs administered i.v. resulted in efficient tumor localization, with penetration of the ILs throughout the tumor mass and accumulation within tumor cells. In contrast, nontargeted liposomes resulted in extracellular tumor accumulation only. In multiple HER2overexpressing human breast tumor xenograft models, treatment with doxloaded antiHER2 ILs produces significantly increased antitumor cytotoxicity as compared to free dox or doxloaded nontargeted liposomes and significantly less systemic toxicity than free dox. To explore further the intracellular delivery advantages of ILs, antiHER2 ILs bearing cationic lipids are being developed for nucleic acid delivery. These cationic immunoliposomes mediate efficient and specific transfection of target cells with reporter genes, as well as intracellular delivery of labeled oligonucleotides. Thus, antiHER2 ILs represent an efficient and feasible strategy to achieve targeted intracellular delivery of therapeutic agents. http://www.ncbi.nlm.nih.gov/pubmed/9459205 Sat, 31 Jan 1998 00:00:00 -0800 BACKGROUND: Compounds that either inhibit or induce an estrogen response in vivo are important as potential drugs and biochemical tools. Nonsteroidal stilbene analogs such as tamoxifen are known to function as both estrogen agonists and antagonists depending upon the analog structure. This family of compounds is amenable to parallelmanifold synthesis because stilbene analogs are easily synthesized using a singlestep olefination reaction. RESULTS: We have prepared a small 23component hydroxystilbene library using a solid phase synthesis approach. The library was screened for estrogenic and antiestrogenic activity using a cellbased bioassay that measures estrogen receptormediated transcription of a reporter gene. Three of the analogs proved to have dosedependent estrogenic activity with EC50 values between 5 microM and 15 microM. Further characterization of the hydroxystilbenemediated estrogenic activity suggests that the agonist activity results from direct binding to the steroid site on the estrogen receptor with IC50 values of 110 microM. CONCLUSIONS: The results of this study show that classic olefination chemistry can be adapted to a solidphase format for parallel synthesis of analog libraries. Although yields varied for the individual analogs, sufficient quantity of pure material was obtained directly from the resin for structural characterization and biological evaluation. This study further validates solidphase organic synthesis as a useful approach for rapid parallelmanifold library synthesis to augment both lead compound discovery and optimization. http://www.ncbi.nlm.nih.gov/pubmed/9383402 Wed, 31 Dec 1997 00:00:00 -0800 HER2/Neu is overexpressed in 2530 of all human breast cancers as a result of both gene amplification and enhanced transcription. Transcriptional upregulation of HER2/neu leads to a 68fold increased abundance of its mRNA per gene copy and likely results from the elevated activity of transcription factors acting on the HER2/neu promoter. Here we report that transcripts of PEA3, an ETS transcription factor implicated in oncogenesis, were increased in 93 of HER2/Neuoverexpressing human breast tumor samples. Analyses to uncover the molecular basis for elevated PEA3 transcripts in HER2/Neupositive breast tumors revealed that the HER2/Neu receptor tyrosine kinase initiated an intracellular signaling cascade resulting in increased PEA3 transcriptional activity transcriptionallyactivated PEA3 stimulated HER2/neu and PEA3 gene transcription by binding to sites in the promoters of these genes. PEA3 also activates transcription of genes encoding matrixdegrading proteinases, enzymes required for tumor cell migration and invasion. These findings implicate PEA3 in the initiation and progression of HER2/Neu positive breast cancer, and suggest that PEA3 and signaling proteins affecting its regulation are appropriate therapeutic targets. http://www.ncbi.nlm.nih.gov/pubmed/9380403 Fri, 31 Oct 1997 00:00:00 -0800 Preliminary studies have suggested that measuring the ability of immunoreactive 67kDa estrogen receptor (ER) to bind DNA and form in vitro complexes with its cognate estrogen response element (ERE) might serve to identify breast tumors most likely to respond to antiestrogens like tamoxifen. Data from two different surveys of untreated primary breast tumors confirmed that only 67 (74 of 111) of ERpositive tumors express a receptor capable of forming ERERE complexes by gelshift assay, with tumors of lower ER content having significantly reduced ER DNAbinding frequency (56) relative to those of higher ER content (82 P = 0.007). In contrast to these untreated tumors, a panel of 41 receptorpositive breast tumors excised after acquiring clinical resistance to tamoxifen during either primary (n = 26) or adjuvant therapy (n = 15) showed a significantly greater ER DNAbinding frequency, with nearly 90 capable of forming ERERE complexes (P 0.02). To assess experimentally whether ER DNAbinding function is altered during the development of antiestrogen resistance, nude mouse MCF7 tumor xenografts were analyzed before and after the acquisition of in vivo resistance to either tamoxifen or a pure steroidal antiestrogen, ICI 182,780. Tamoxifenresistant MCF7 tumors retained full expression of 67kDa DNAbinding ER, and despite a markedly reduced ER content in the ICI 182,780treated tumors, the expressed ER in these antiestrogenresistant tumors exhibited full ability to form ERERE complexes. These findings indicate that breast tumors with acquired antiestrogen resistance continue to express ER of normal size and DNAbinding ability and suggest that the failure of antiestrogens to arrest tumor growth during emergence of clinical resistance results from an altered generegulatory mechanism(s) other than ERERE complex formation. http://www.ncbi.nlm.nih.gov/pubmed/9288779 Sun, 31 Aug 1997 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/9217932 Thu, 31 Jul 1997 00:00:00 -0700 The 30 known members of the Ets multigene family of transcriptional regulators are increasingly being recognized for their involvement in early embryonic development and late tissue maturation, directing stagespecific and tissuerestricted programs of target gene expression. Identifiable primarily by their 85 amino acid ETS DNAbinding domain and dispersed across all metazoan lineages into distinct subfamilies, Ets genes also produce malignancies in humans and other vertebrates when overexpressed or rearranged into chimeras retaining the ETS domain, suggesting that their oncogenic potential is determined by the program of target genes they regulate. Searching for Ets factors that regulate expression of the HER2/neu (cerbB2) oncogene in human breast cancer, we identified a new epitheliumrestricted Ets encoding an ETS domain homologous to the Drosophila E74/human Elf1 subfamily, an aminoterminal region (Aregion or Pointed domain) homologous to the distantly related Ets1 subfamily, and a serinerich box homologous to the transactivating domain of the lymphocyterestricted High Mobility Group (HMG) protein, SOX4. Recombinant protein encoded by ESX (for epithelialrestricted with serine box) exhibits Etslike DNA binding specificity in electrophoretic mobility shift assays and, in transient transfection assays, transactivates Etsresponsive promoter elements including that found in the HER2/neu oncogene. ESX is located at chromosome 1q32 in a region known to be amplified in 50 of early breast cancers, is heregulininducible and overexpressed in HER2/neu activated breast cancer cells. Tissue hybridization suggests that ESX becomes overexpressed at an early stage of human breast cancer development known as ductal carcinoma in situ (DCIS). http://www.ncbi.nlm.nih.gov/pubmed/9129154 Wed, 30 Apr 1997 00:00:00 -0700 Liposomes (70100 nm) of 1palmitoyl2oleoylphosphatidylcholine, cholesterol, and poly(ethylene glycol) (PEG)modified phosphatidylethanolamine (PEGDSPE) were conjugated to Fab' fragments of a humanized recombinant MAb against the extracellular domain of HER2/neu to create sterically stabilized immunoliposomes (antiHER2 SL) as a drug carrier targeting HER2overexpressing cancers. Conjugation employed maleimideterminated membraneanchored spacers of two kinds: a short spacer, providing attachment of Fab' close to the liposome bilayer, or a long spacer, with Fab' attachment at the distal terminus of the PEG chain. Confocal microscopy and spectrofluorometry of HER2overexpressing breast cancer cells incubated with fluorescently labeled antiHER2 SL prepared with either spacer showed binding of liposomes (800023000 vesicles/cell) followed by endocytosis (rate constant ke = 0.0120.033 min1) via the coatedpit pathway, evidenced by intracellular acidification and colocalization with transferrin. Uptake of antiHER2 immunoliposomes by breast cancer cells with low HER2 expression, or after preincubation of cells with free antiHER2 Fab', was less than 0.2 and 4.3, respectively, of the uptake by HER2overexpressing cells. Increasing PEGDSPE content (up to 5.7 mol ) in antiHER2SL prepared with the short spacer decreased liposomecell binding affinity 60100fold, while ke decreased only 2fold however, when Fab' fragments were conjugated via a PEG spacer, both binding affinity and ke were unaffected by PEGDSPE content. Cell binding and internalization of antiHER2 immunoliposomes increased at higher surface density of conjugated Fab' fragments, reaching plateaus at approximately 40 Fab'/liposome for binding and approximately 1015 Fab'/liposome for internalization. Uptake of antiHER2 immunoliposomes correlated with the cell surface density of HER2 and significantly (p 0.005) correlated with the antiproliferative effect of the targeting antibody but not with the total level of cellular HER2 expression. The results obtained were used to optimize in vivo preclinical studies of antiHER2 SL loaded with antineoplastic drugs. http://www.ncbi.nlm.nih.gov/pubmed/8993319 Fri, 31 Jan 1997 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/8775733 Sat, 30 Nov 1996 00:00:00 -0800 Preclinical and clinical studies have pointed to the antitumor potential of the naturally occurring polyphenolic binaphthyl dialdehyde, gossypol, as well as its purified (,) enantiomers. To explore further the antitumor properties of this multifunctional agent, we synthesized several reactive derivatives including the (,) enantiomers of gossypolone and four different gossypol Schiff's bases (AR1, AR2, AR3, AR4). The biological activities of these new agents were screened by measuring their in vitro antiproliferative activity against malignant (MCF7, MCF7/adr) or immortalized (HBL100) human breast epithelial cell lines. Racemic gossypolone showed relatively uniform antiproliferative activity against all of the breast epithelial cell lines with 3 to 5fold less activity than ()gossypol against MCF7 and MCF7/adr cells. Of interest, the relative antitumor potency of purified gossypolone enantiomers was reverse that of gossypol enantiomers, since ()gossypolone showed up to 3fold greater inhibition of MCF7 culture growth than ()gossypolone. Of the Schiff's base derivatives only AR3 with its isopropyl amine substituent demonstrated cytotoxic activity comparable to that of ()gossypol derivatives with ethyl, propyl, or butyl amine substituents (AR1, AR2, AR4) had little growth inhibitory activity at culture concentrations up to 25 microM. AR3 activity was greatest against HBL100 and MCF7 cells MCF7 IC50 values: AR3 = 0.9 microM, ()gossypol = 2.3 microM unlike ()gossypol, however, AR3 showed substantially reduced activity against the multidrugresistant subline, MCF7/adr. These structureactivity comparisons suggest that isolation of (,)enantiomers of AR3 and additional chemical modifications including the synthesis of an isopropyl amine Schiff's base of gossypolone will likely yield a newer generation of gossypol analogues with enhanced anticancer potential. http://www.ncbi.nlm.nih.gov/pubmed/8729944 Mon, 30 Sep 1996 00:00:00 -0700 Mechanisms regulating the intracellular level of endogenous U6 small nuclear RNA were studied by transient transfection of ectopic U6 gene constructs into immortalized normal and malignant human cell lines. Transfection and expression of a modified U6 gene containing native promoter, capping, and termination sequences but lacking all highly conserved internal spliceosome sequences produced dosedependent effects on endogenous U6 gene expression. At low transfection doses, no significant changes in endogenous U6 RNA levels or halflife were noted. However, as the dose of the transfected gene and its expression increased, native U6 RNA levels dramatically decreased in association with an apparent decrease in U6 RNA halflife. Downregulation of native U6 RNA levels was transient, with recovery noted within 4896 h in conjunction with declining expression of the ectopic gene. These modulatory effects appeared specific to endogenous U6 transcripts, because no changes were noted in 7sk, U1, U3, or 5S RNA levels or halflives. Transfection with an unmodified U6 gene did not alter total U6 transcript levels but did produce a similar dosedependent decrease in U6 RNA halflife. These studies suggest a hitherto unrecognized U6specific intracellular regulatory mechanism, through which overaccumulation of U6 small nuclear RNA is prevented. http://www.ncbi.nlm.nih.gov/pubmed/8631843 Sun, 30 Jun 1996 00:00:00 -0700 PURPOSE: Breast cancer frequently overexpresses the product of the HER2 protooncogene, a 185kd growth factor receptor (p185HER2). The recombinant humanized monoclonal antibody (rhuMAb) HER2 has high affinity for p185HER2 and inhibits the growth of breast cancer cells that overexpress HER2. We evaluated the efficacy and toxicity of weekly intravenous administration of rhuMAb HER2 in patients with HER2overexpressing metastatic breast cancer. PATIENTS AND METHODS: We treated 46 patients with metastatic breast carcinomas that overexpressed HER2. Patients received a loading dose of 250 mg of intravenous rhuMAb HER2, then 10 weekly doses of 100 mg each. Patients with no disease progression at the completion of this treatment period were offered a maintenance phase of 100 mg/wk. RESULTS: Study patients had extensive metastatic disease, and most had received extensive prior anticancer therapy. Adequate pharmacokinetic levels of rhuMAb HER2 were obtained in 90 of the patients. Toxicity was minimal and no antibodies against rhuMAb HER2 were detected in any patients. Objective responses were seen in five of 43 assessable patients, and included one complete remission and four partial remissions (overall response rate, 11.6 95 confidence interval, 4.36 to 25.9). Responses were observed in liver, mediastinum, lymph nodes, and chest wall lesions. Minor responses, seen in two patients, and stable disease, which occurred in 14 patients, lasted for a median of 5.1 months. CONCLUSION: rhuMAb HER2 is well tolerated and clinically active in patients with HER2overexpressing metastatic breast cancers that had received extensive prior therapy. This is evidence that targeting growth factor receptors can cause regression of human cancer and justifies further evaluation of this agent. http://www.ncbi.nlm.nih.gov/pubmed/8622019 Fri, 31 May 1996 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/8588906 Thu, 29 Feb 1996 00:00:00 -0800 Amplification of the ERBB2 (HER2/neu) gene is accompanied by overexpression of its cell surface receptor product, p185HER2. Heterogeneity has been observed for both the gene copy number and the level of overexpression of its protein product. To better understand their relationship, correlation between the level of cellular expression of p185HER2 and ERBB2 gene amplification was studied in four human breast cancer cell lines (BT474, SKBR3, MDA453, and MCF7) and in a primary human breast tumor sample. The relative expression of p185HER2 was measured by immunofluorescence by using flow and/or image cytometry while correlated DNA analysis was performed on the same cells by fluorescence in situ hybridization to determine ERBB2 gene and chromosome 17 copy numbers. Marked heterogeneity was observed in both protein expression and ERBB2 copy number. Despite this heterogeneity, and in accordance with previous studies, the average levels of p185HER2 expression correlated well with average ERBB2 gene copy numbers in the four lines examined (r = 0.99). When the relationship between copy number and protein expression was studied on a cellbycell basis, p185HER2 expression correlated with both the absolute number of ERBB2 gene copies/cell (r = 0.590.63) and chromosome 17 copy number (r = 0.450.61). It is of interest that there was weak or no correlation between p185HER2 protein expression and the ERBB2 copy number:chromosome 17 copy number ratio (r = 0.00.25). In more than onehalf of cells expressing a high level of p185HER2, the chromosome 17 copy number was high (two or three times the average copy number), whereas 2 of an unselected population had a high chromosome 17 copy number. Bromodeoxyuridine incorporation indicated that the Sphaselabeling index was homogeneous across various p185HER2expressing subpopulations in the SKBR3 cell line. Analysis of the primary breast tumor sample showed results similar to the cell lines, supporting the strong possibility of a mechanistic link among p185HER2 overexpression, ERBB2 amplification, and high chromosome 17 copy number. http://www.ncbi.nlm.nih.gov/pubmed/7585609 Thu, 30 Nov 1995 00:00:00 -0800 Breast cancer is the most frequent cancer of women in developed countries. It is not surprising, therefore, that major research efforts have been made to improve early detection and treatment strategies directed at this malignancy. In this keynote article, we outline future perspectives on breast cancer genetics, risk factors and prognostic factors, focusing on novel developments relating to the estrogen receptor and the family of ErbB growth factor receptors. http://www.ncbi.nlm.nih.gov/pubmed/7607570 Mon, 31 Jul 1995 00:00:00 -0700 Gossypol is present in antiinflammatory poultices made from the medicinal tree Thespesia populnea. Isolated human neutrophils exposed to 320 microM gossypol for 1590 min were assayed in vitro for superoxide production and surface expression of Mac1 (CD11b/CD18). Gossypol increased superoxide production in a time and concentrationdependent fashion consistent with a moderate, delayed respiratory burst. Surface Mac1 expression was increased within 15 min by 35 microM gossypol, resulting in a 14fold increase over controls and a threefold greater increase over that produced by PMA. Staurosporine failed to block gossypol induction of superoxide and Mac1, while EDTA inhibited induction of Mac1 only, implicating a calciumdependent mechanism. Gossypol increased intracellular calcium to peak levels, but in a delayed fashion as compared to FMLP. These findings demonstrate that gossypol is a highly potent stimulant of Mac1 expression and suggest at least two protein kinase Cindependent pathways of neutrophil activation. The resultant exhaustion of neutrophils may account for the antiinflammatory properties of plants containing gossypol. http://www.ncbi.nlm.nih.gov/pubmed/7843790 Tue, 28 Feb 1995 00:00:00 -0800 We have identified a 28bp homopurine/homopyrimidine sequence capable of triple helix (triplex) formation with GTrich oligodeoxyribonucleotides (oligos) within the critical proximal promoter of the HER2/neu/cerbB2 (HER2) protooncogene. To investigate the possible therapeutic potential of triplexforming oligos in HER2 overexpressing breast cancers, we have studied the ability of triplex formation to compete with and to inhibit the binding of a transcription factor to its consensus sequence at an adjacent site. Competition binding assays demonstrate that a triplexforming oligo can inhibit transcription factor binding in a sequencespecific manner. Moreover, we find that the addition of both nucleotide and nonnucleotide 'tails' to triplexforming oligos do not confer any enhancement of binding affinity, but provide additional inhibition of transcription factor binding, potentially by steric hindrance. http://www.ncbi.nlm.nih.gov/pubmed/7958975 Wed, 30 Nov 1994 00:00:00 -0800 We used antisense RNA in a protocol designed to reduce estrogen receptor (ER) content in human breast cancer cells and observed paradoxical increases in ER levels. ER protein activity was measured using a highly sensitive reporter gene assay that relies on the ability of functional ER to bind a consensus estrogen response element (ERE) and drive the production of chloramphenicol acetyltransferase (CAT). Upon transient transfection of ERpositive cell lines with three different vectors containing the fulllength ER cDNA cloned in an antisense orientation, we observed unexpected increases in ERdriven CAT activity. To further investigate this phenomenon, expression from the antisense ER vectors was studied in an ERnegative breast tumor cell line, MDAMB453. ER activity was observed in these ERnegative cells upon transient transfection with each of three antisense ER vectors, but not from control vectors. Expression of ER from antisense constructs was 30100times less efficient than ER expression from isogenic sense constructs. The paradoxical ER activity was consistent with expected ER behavior in that it exhibited characteristic binding to the natural ligand, 17 betaestradiol (E2), and it was inhibited by the antiestrogens, 4hydroxytamoxifen (OHT) and ICI 164384 (ICI). Control vectors containing a truncated antisense ER cDNA produced no ER activity. Although the mechanism for this ER expression has not been determined, it appears likely that it is due to transcription off the opposite strand of the antisense construct.(ABSTRACT TRUNCATED AT 250 WORDS) http://www.ncbi.nlm.nih.gov/pubmed/7958984 Wed, 30 Nov 1994 00:00:00 -0800 BACKGROUND: The prognostic significance of Cathepsin D and optimal methodologies to measure Cathepsin D in breast cancers are controversial. PATIENTS AND METHODS: Quantitative (immunoradiometric) and semiquantitative (immunohistochemical) assays for Cathepsin D expression were compared using 25 breast carcinomas. Immunohistochemical Cathepsin D results were derived using 3 different antiCathepsin D antibodies and significant associations between immunohistochemical and radiometric Cathepsin D data were observed for each reagent. Immunohistochemical analysis of Cathepsin D expression was performed on nearly 500 fixedembedded archival breast cancers with longterm patient followup using 2 antiCathepsin D antibodies (CDR211/23, IC11). RESULTS: The immunohistochemical reagents recognized generally overlapping subsets of Cathepsin D positive tumors (correlation coefficient 0.54 p = 0.00016). Correlations between Cathepsin D data and clinical, histologic or biologic features differed for each antibody. For the nodenegative patient subset, Cathepsin D immunopositivity correlated with erbB2 and stressresponse protein 27 overexpression but not survival. Cathepsin D positivity was associated with subsequent distant metastasis and estrogen receptor positivity in node positive patients. Univariate analysis of all patients suggested that Cathepsin D immunopositivity may be predictive of a reduced metastasisfree but not overall survival. Multivariate analysis, however, failed to confirm an independent prognostic value for Cathepsin D in breast cancer patients. CONCLUSIONS: These data do not confirm an independent prognostic significance for Cathepsin D using immunohistochemical methods on breast cancers. http://www.ncbi.nlm.nih.gov/pubmed/8075029 Fri, 30 Sep 1994 00:00:00 -0700 Triplex formation with RNA oligonucleotides and doublestranded (ds) DNA may provide a means of controlling gene expression from specific promoters and/or creating more selective DNA cleaving agents. We report the development of a novel technique, called triplex blotting, designed to detect RNA species capable of triplex formation with radiolabeled dsDNA probes within a background of total cellular RNA. Triplex blotting offers a new approach for screening potential RNA sequences for triplex formation with dsDNA targets, for comparing relative binding affinities of various triplexforming RNAs and for confirming the specificity of triplex formation of a DNA target probe within total cellular RNA. In addition, the technique allows for repeated probing of the same filter while varying critical hybridization conditions such as pH, temperature or ionic strength. http://www.ncbi.nlm.nih.gov/pubmed/7521186 Fri, 30 Sep 1994 00:00:00 -0700 Reactive oxygen is an important regulator of vascular cell biology however, the mechanisms involved in transducing signals from oxidants in endothelial cells are poorly defined. Because protein phosphorylation is a major mechanism for signal transduction, cultured aortic endothelial cells were exposed to nonlethal concentrations of H2O2 to examine oxidantsensitive changes in phosphorylation state. Addition of H2O2 increases the phosphorylation of the heat shock protein 27 (HSP27) within 2 min. This response is maximal by 20 min and remains constant for more than 45 min. Levels of intracellular free Ca2 in endothelial cells did not change following addition of 100 microM H2O2, nor did the ability of the cells to respond to bradykinin. H2O2induced phosphorylations were either not affected or were slightly increased in cells pretreated with PKC inhibitors (H8, staurosporin, or calphostin c). Twodimensional analysis of phosphoproteins from homogenates of 32Plabeled cells revealed that phorbol myristate acetate (PMA) did not cause the same degree of HSP27 phosphorylation as H2O2. Simultaneous addition of 10 eta M PMA and 50 microM H2O2 decreased the oxidantstimulated phosphorylation of the most acidic HSP27 isoform. These data suggest that signal transduction for H2O2sensitive endothelial cell responses are not only independent of PKC, but may also be suppressed by the action of the kinase. http://www.ncbi.nlm.nih.gov/pubmed/8070680 Wed, 31 Aug 1994 00:00:00 -0700 Antisense and triplex oligonucleotides continue to demonstrate potential as mediators of genespecific repression of protein synthesis. However, inefficient and heterogeneous cellular uptake, intracellular sequestration, and rapid intracellular and extracellular degradation represent obstacles to their eventual clinical utility. Efficient cellular delivery of targeted ribozymes can present similar problems. In this report we describe a system for circumventing these obstacles and producing large quantities of short, sequencespecific RNA oligonucleotides for use in these gene regulation strategies. The oligonucleotides are generated from a vector containing promoter, capping, and termination sequences from the human small nuclear U6 gene, surrounding a synthetic sequence incorporating the oligonucleotide of interest. In vivo, these oligonucleotides are produced constitutively and without cell type specificity in levels up to 5 x 10(6) copies per cell, reach steadystate levels of expression within 9 hours posttransfection, and are still readily detectable 7 days posttransfection. In addition, these oligonucleotides are retained in the nucleus, obtain a 5' gammamonomethyl phosphate cap, and have an intracellular halflife of approximately one hour. This expression vector provides a novel and efficient method of intracellular delivery of antisense or triplex RNA oligonucleotides (and/or ribozymes) for gene regulation, as well as a costeffective means of comparing the biological activity arising from a variety of different potential oligonucleotide sequences. http://www.ncbi.nlm.nih.gov/pubmed/8052538 Wed, 31 Aug 1994 00:00:00 -0700 Promoter elements accounting for HER2 (cerbB2/neu) overexpression were searched for in several human breast cancer cell lines (MDA453, BT474, ZR751, MCF7) known to express constitutively a 30fold range in HER2 transcripts per gene copy. HER2 overexpressing cells showed a single prominent DNase I hypersensitive site near a conserved and hitherto unrecognized ets response element (GAGGAA), located 38 bases downstream from the CAAT box and directly 5' of the TATA box in the human HER2 promoter. Transient transfection of HER2 promoter constructs (0.125, 0.5, and 2.0 kilobase pairs (kb)) demonstrated that the most proximal promoter region (0.125 kb) was capable of conferring up to 30fold enhanced activity in HER2overexpressing cell lines relative to low HER2expressing control lines. Sitedirected mutagenesis of the ets response element (GAGGAAGAGAGA) caused a or = 60 reduction in promoter activity affecting at least 0.5 kb of upstream HER2 regulatory sequence. Gelshift assays with nuclear extracts and oligonucleotide sequences spanning the 0.125kb promoter region detected an ETSimmunoreactive complex, present most abundantly in cells overexpressing HER2, whose highaffinity binding depended on the GAGGAA response element. Methylation interference confirmed the ETSspecific pattern of protein binding by this complex to guanine bases in the ets response element. UV crosslinking and immunoprecipitation implicate a approximately 60kDa ETS protein, and candidate ETS genes expressed in these breast cancer cells include GABP alpha, elk1, elf1, and PEA3. http://www.ncbi.nlm.nih.gov/pubmed/7914192 Wed, 31 Aug 1994 00:00:00 -0700 Evidence to date indicates that structurally abnormal estrogen receptor (variant ER) can be detected in some human breast tumors. Based on in vitro ability to bind DNA sequences containing the cognate estrogen response element (ERE), these variant receptors may be categorized into DNAbinding ER (Type1 variants) and nonDNAbinding ER (Type2 variants). To look for Type2 variants of normal size (67 kDa ER) that lack the ability to form immunoreactive ERERE complexes, a panel of 40 cryopreserved primary breast tumors were extracted and analyzed by enzyme immunoassay (EREIA), gelshift, and Western blot techniques. For the 33 tumor extracts containing or = 10 fmol/mg ER (by EREIA), the amount of 67 kDa ER detectable by D75 antiER monoclonal antibody under fully denatured and reduced assay conditions (Western blotting) did not correlate well with the presence or intensity of D75 immunoreactive ERERE bands seen under native conditions by gelshift assay. Overall, 30 (10 of 33) of these extracts containing 67 kDa ER failed to produce immunoreactive ERERE complexes, with this frequency varying from over 40 in tumor samples with lower ER content (1049 fmol/mg) to 11 in tumor samples with the highest ER content ( 100 fmol/mg). These results indicate that Type2 variant receptors characterized as nonDNAbinding 67 kDa ER may be present in a significant fraction of ERpositive primary breast tumors preliminary evidence suggests that further study of abnormalities in ER tertiary or quaternary structure, such as those produced by intracellular oxidation of ER thiol groups, is warranted. http://www.ncbi.nlm.nih.gov/pubmed/8219255 Tue, 30 Nov 1993 00:00:00 -0800 BACKGROUND: Tumor labeling index has emerged as a strong predictor of the clinical course of women with breast cancer. This study investigated whether labeling index of primary tumors correlates with labeling indices of concurrent regional node metastases. METHODS: With appropriate written consent, preoperative in vivo infusion of the thymidine analogue 5bromodeoxyuridine (BrdUrd) was used to label 109 human breast cancers. Labeled Sphase cells were identified immunohistochemically with an antibody specific to DNAincorporated BrdUrd. Labeling index was the fraction of labeled nuclei in 2000 tumor nuclei. For 30 women, there was sufficient cancer in axillary lymph nodes to compare labeling indices in primary breast cancer and regional lymph node metastases. RESULTS: The 30 women were from 25 to 82 years of age. Tumors were from 1 to 12 cm in size and there were from 1 to 26 positive nodes. Tumor labeling index ranged from 0.1 to 34, (mean, 11.1 median, 10.3) and axillary lymph node metastasis labeling index ranged from 0.1 to 27.7 (mean, 10.8 median, 10.0). There was strong correlation between primary tumor labeling index and regional lymph node metastases labeling index (r = 0.82, with 95 confidence interval 0.650.91). The correlation persisted within subgroups according to age, tumor size, number of positive nodes, and hormone receptor status. Primary tumor and lymph node metastases labeling indices also had statistically similar relationships with age, level of hormone receptors, tumor size, and number of positive nodes. CONCLUSIONS: Primary tumor and regional node labeling indices correlate strongly the relationship is not influenced by age, level of hormone receptors, tumor size, or number of positive nodes. http://www.ncbi.nlm.nih.gov/pubmed/8508357 Wed, 30 Jun 1993 00:00:00 -0700 Cytogeneticists first proposed that the karyotypic abnormalities identified on chromosomes 1, 3, 6, 11, 13, 16, 17, and 18 supported a genetic basis for breast cancer. Such abnormal banding patterns, however, may represent either lossoffunction or gainoffunction molecular events. RFLP analyses have since confirmed that 2060 of primary and spontaneous human breast tumors exhibit allelic losses on these same chromosomes, although the exact genes involved at these chromosomal sites remain largely unknown. Knowledge gained about the Rb1 and p53 tumor suppressor genes at 13q14 and 17p13 in breast and other human tumors supports the paradigm that for any chromosomal locus, allelic loss associated with a mutation in the remaining tumor allele signifies an involved tumor suppressor gene. Given this paradigm, there are nearly a dozen putative breast tumor suppressor genes under active investigation, with most investigators now focusing on various chromosome 17 loci. Among the known protooncogenes found activated in breast cancer, amplification of cerbB2 at 17q21 is the most widely studied and clinically significant gainoffunction event uncovered to date, occurring in about 20 of all primary breast tumors. The involvement of this overexpressed membrane receptor has engendered interest in related tyrosine kinase receptors, such as EGFR, IR, and IGFIR, as well as their respective ligands, which may be overexpressed in a greater fraction of tumors, contributing to the autocrine and paracrine regulation of breast cancer growth and metastasis. New attention is being given to the potentially oncogenic function of structurally altered nuclear transactivating steroid hormone receptors, such as ER, whose overexpression has long been used to determine endocrine therapy and prognosis for individual breast cancer patients. While cmyc was one of the first known protooncogenes to be found amplified and overexpressed in human breast cancers, the actual incidence and clinical significance of its activation remain disputed and in need of further study. Lastly, we can expect greater clarification about the importance of various 11q13 genes found coamplified in nearly 20 of primary breast cancers, and pursuit into the intriguing possibility that a cyclinencoding gene represents the overexpressed locus of real interest in this amplicon. Virtually all of these important genetic abnormalities identified thus far are associated with but not restricted to human breast cancers. The absence of identifiable molecular defects relating to the tissue specificity of this malignancy must be considered a substantial gap in our basic understanding of breast carcinogenesis.(ABSTRACT TRUNCATED AT 400 WORDS) http://www.ncbi.nlm.nih.gov/pubmed/1363356 Wed, 31 Mar 1993 00:00:00 -0800 Since the poor prognosis associated with HER2 amplified breast cancers might be explained by a mechanistic association between p185HER2 overexpression and therapeutic resistance, we assessed the chemoendocrine sensitivity of estrogen receptor (ER) containing MCF7 breast cancer cells transfected with fulllength HER2 cDNA. Of the 36 isolated MCF/HER2 subclones, 7 were found to overexpress p185HER2 surface receptor at levels 3 to 45fold greater than parental or control transfected cells (MCF/neo). The overexpressing transfectants possessed increased inositol1,4,5triphosphate3'kinase activity comparable to enzyme activity in the endogenously HER2 amplified breast cancer cell lines SKBr3 and BT474. The antip185HER2 monoclonal antibody and receptorspecific partial agonist, muMAb4D5 (4D5), known to inhibit growth of SKBr3 and BT474 cells, produced no significant growth inhibitory effect on any of the transfectants including the 45fold overexpressing MCF/HER218 cells which were studied in greater detail. MCF/HER218 cells contained at least partially functioning exogenous receptor since 4D5 (3 micrograms/ml) specifically stimulated phosphorylation of p185HER2 and its coprecipitating ptyr56 substrate within 5 min, and this was followed at 1 h by a transient induction of cmyc but not cfos mRNA. ER content and the in vitro sensitivity of MCF/HER218 cells to 5fluorouracil and adriamycin were identical to those of control transfectants and parental cells. However, these highly overexpressing transfectants had acquired low level (2 to 4fold) resistance to cisplatin and were no longer sensitive to the antiestrogen tamoxifen (TAM). To compare the hormonedependent tumorigenicity of the HER2 transfectants, MCF/HER218 and control cells (MCF, MCF/neo3) were implanted into ovariectomized athymic nude mice. No tumors were produced in the absence of estradiol (E2) administration. In E2 supplemented mice, MCF/HER218 tumors grew most rapidly. When E2 treatment was stopped and daily TAM injections were initiated, MCF7 and MCF/neo3 tumor growth ceased immediately, while MCF/HER218 tumors continued to show an accelerated growth rate lasting weeks. This pattern of hormonedependent, TAMresistant growth exhibited by the MCF/HER218 tumors in nude mice supports the possibility that p185HER2 overexpression in human breast cancers may be linked to therapeutic resistance. http://www.ncbi.nlm.nih.gov/pubmed/8095168 Wed, 31 Mar 1993 00:00:00 -0800 A partially agonistic monoclonal antibody, 4D5, known to bind to the extracellular domain of p185HER2 and shown to inhibit long term growth of p185HER2overexpressing breast cancer cells, was used to study signal transduction and phosphotyrosyl protein substrates associated with this receptor. Normal breast epithelial cells and breast carcinoma cells expressing low levels of p185HER2 were not affected by 4D5. HER2/neuoverexpressing breast cancer cells (BT474 and SKBr3) exposed to 4D5 exhibited rapid phosphorylation of both p185HER2 and an associated 56kDa phosphotyrosyl protein (ptyr56). Paralleling the 4D5 stimulated phosphorylation of p185HER2 and ptyr56 was a 510fold induction of cfos mRNA and phosphatidylinositol 4kinase activity and a 2fold induction of inositol 1,4,5trisphosphate 3'kinase activity. The increased phosphatidylinositol 4kinase activity immunoprecipitated with p185HER2 and also coeluted with ptyr56 from an antiphosphotyrosine immunoaffinity column. These results indicate that short term (less than 6 h) 4D5 activation of p185HER2 in overexpressing breast cancer cells produces agonisticlike signaling typical of homologous tyrosine kinase growth factor receptors such as epidermal growth factor receptor. The data also suggest that ptyr56 represents a novel phosphorylated substrate associated with 4D5stimulated p185HER2. http://www.ncbi.nlm.nih.gov/pubmed/1677643 Sat, 31 Aug 1991 00:00:00 -0700 The likelihood a breast cancer will respond to antiestrogen therapy depends on the tumor content of immunoreactive or ligandbinding estrogen receptor (ER). To investigate the failure of many ERpositive breast cancers to respond to antiestrogen therapy, we examined by gelshift assay the ability of tumor ER to bind its cognate estrogen response element (ERE). Analysis of 38 primary breast cancers showed that some tumors containing abundant immunoreactive ER failed to demonstrate DNA binding ER. In many other ERpositive tumors, the fraction of DNA binding ER was low and consisted primarily of truncated receptor forms, which on Western analysis were revealed to be 50 kD homodimers and 6750 kD ER heterodimers. The use of protease inhibitors during tumor extraction and the demonstration of nuclearlocalizing ER and EREbinding COUP (chicken ovalbumin upstream promoter) protein in these tumors indicated that the truncated forms of ER were likely present in vivo. The presence of intact DNA binding ER correlated with higher tumor content of immunoreactive sex steroid receptors (ER and/or PR), standard predictors of tumor responsiveness to antiestrogen, suggesting that loss or truncation of DNA binding ER may be an important prognostic parameter accounting for some forms of clinical resistance to antiestrogen therapy. http://www.ncbi.nlm.nih.gov/pubmed/1864980 Sat, 31 Aug 1991 00:00:00 -0700 Isomers (, ) of the antitumor agent gossypol (G) were studied for their ability to reduce tumor ATP and blood flow in rats bearing subcutaneously implanted pancreatic tumors. A 50 reduction in tumor ATP/Pi within ih of a single injection of G was associated with a 60 decline in tumor blood flow. To determine if these changes in tumor physiology could be due to a direct drug effect on tumor endothelium, G isomers were compared for their ability to alter protein (125IBSA) permeability and metabolic (32P) labelling of cultured endothelial cells. Treatments for ih produced no endothelial cell leakage, but 24h exposures to either G (5 microM) or G (50 microM) produced complete permeability of the monolayers to 125IBSA. In contrast, 0.5I.Oh exposures to G (4 microM) produced 2 to 3fold increases in phosphorylated 27 kDa heatshock protein, hsp27. Hsp27 phosphoprotein isoforms were differentially labelled following G and G exposures with the phosphorylation profile of G appearing most similar to that of oxyradical producing agents known to induce hsp27 and injure endothelial cells. We postulate that the tumor ischemic effects of G are mediated by endothelial response to oxyradical production in a mechanism similar to that of tissue ischemiareperfusion injury. http://www.ncbi.nlm.nih.gov/pubmed/1875793 Sat, 31 Aug 1991 00:00:00 -0700 Several cell lines resistant to alkylating agents possess increased activity of glutathioneStransferase (GST) drug detoxifying enzymes. Inhibition of certain enzymes of the glutathione redox system may affect cellular sensitivity to alkylators. We report that the (.)enantiomer of gossypol is a potent and selective inhibitor of GST alpha and GST pi isozymes, and that in combination with buthionine sulfoximine (BSO), causes the enhanced modulation of alkylator resistance in two drug resistant cell lines with increased GST activity. The use of ()gossypol alone had no effect on the 25fold resistance of MCF7 Adr and Walker resistant cells to chlorambucil, melphalan and BCNU. Cellular depletion of glutathione with BSO resulted in a 24fold modulation of cell sensitivity to these alkylators. However, the combination of ()gossypol with BSO resulted in a markedly greater modulation of alkylator sensitivity than with either inhibitor alone. Therefore, the complementary inhibition of glutathione and GST by BSO and ()gossypol, respectively, produced a synergistic modulation of alkylator cytotoxicity in these drug resistant cell lines. The favorable clinical pharmacokinetics of ()gossypol suggest its further evaluation for use in combination with BSO and alkylating agents in clinical trials. http://www.ncbi.nlm.nih.gov/pubmed/2004358 Sun, 31 Mar 1991 00:00:00 -0800 The early effects of in vivo platinumrhodamine (PtR) chemotherapy on tumor highenergy phosphorous metabolism was investigated using phosphorus31 (31P) magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), and histologic examination in a subcutaneously implanted hamster melanoma model. PtR was chosen because of its potential antimitochondrial and antineoplastic properties. All melanomas were clearly observed on both T1 and T2weighted images (T1WI and T2WI), with viable tumor regions generally characterized by low to intermediate intensity on T1WI and high intensity on T2WI. Necrotic regions were more variable in appearance, depending on the amount of cystic fluid and hemorrhage. No changes were detected on either T1WI or T2WI within 90 minutes of a tumoristatic dose of PtR (40 mg/kg) by visual examination, but slight differences were seen on calculations of relative signal intensities. However, this same dose of PtR caused a 50 drop in tumor ATP and phosphocreatine content (relative to Pi) measured by 31P MRS within 90 minutes of drug injection. Magnetic resonance spectroscopy appears to offer a sensitive means of detecting the earliest biochemical effects of chemotherapeutic agents that are known to affect tumor bioenergetics. http://www.ncbi.nlm.nih.gov/pubmed/2079405 Sun, 31 Mar 1991 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/2576998 Fri, 31 Aug 1990 00:00:00 -0700 Racemic gossypol has been shown to have antitumor properties that may be due to its ability to uncouple tumor mitochondria or to its inhibitory effects on a variety of nonmitochondrial enzymes. We have studied the antimitochondrial and enzymeinhibiting properties of gossypol in human carcinoma cell lines of breast (MCF7, T47D), ovarian (OVCAR3) colon (HCT8), and pancreatic (MiaPaCa) origin by comparing the effects of its purified () and ()enantiomers. ()Gossypol shows up to 10fold greater antiproliferative activity than ()gossypol in the cancer cell lines and in normal hematopoietic stem cells grown in vitro, with IC50 values ranging from 1.5 to 4.0 microM for the cancer cells and from 10 to 20 microM for the human marrow stem cells. As well, multidrugresistant MCF/Adr cells appear more resistant to ()gossypol than their parental cell line. Electron microscopy indicates that the earliest ultrastructural change in tumor cells exposed to a cytotoxic (10 microM) concentration of ()gossypol is the selective destruction of their mitochondria. Consistent with this observation, 31P magnetic resonance spectroscopy detects pronounced changes in tumor cell high energy phosphate metabolism within 24 hr of ()gossypol treatment, manifest by 1.6 to greater than 50fold differential reductions in the intracellular ratios of ATP/Pi, relative to ()gossypoltreated cell lines the magnitude of these antimitochondrial effects correlates with the antiproliferative activity of ()gossypol. Northern blot RNA analyses suggest that treatment with a 510 microM dose of ()gossypol induces a transient increase in the expression of heat shock gene products, particularly hsp70 transcripts. The mean 5fold increase in ()gossypolinduced hsp70 mRNA appears coincident with a comparable heatstimulated increase in transcript levels, as compared with control or ()gossypoltreated cells. The enzymeinhibiting properties of gossypol enantiomers were compared in cellfree assays measuring glutathioneStransferasealpha, mu, and pi activities, calmodulin stimulation of cyclic nucleotide phosphodiesterase, and protein kinase C activity. Both enantiomers are near equivalent antagonists of calmodulin stimulation and protein kinase C activity, exceeding the potency of known inhibitors such as phenothiazines by as much as 50fold. In contrast, ()gossypol is a 3fold more potent inhibitor of glutathioneStransferasealpha and pi isozyme activity, resulting in IC50 values of 1.6 and 7.0 microM, respectively, for these two isozymes.(ABSTRACT TRUNCATED AT 400 WORDS) http://www.ncbi.nlm.nih.gov/pubmed/2193225 Tue, 31 Jul 1990 00:00:00 -0700 A multicenter phase I pharmacokinetic study of a new triphenylethylene antiestrogen, toremifene, was examined in 70 patients with advanced breast cancer. Patients were randomized to receive single daily oral doses of either 10, 20, 40, 60, 200, or 400 mg for 8 weeks. Plasma toremifene and its major metabolites. Ndesmethyltoremifene and 4hydroxytoremifene, were determined weekly during therapy and at 0, 7, 14, and 21 days after the discontinuation of therapy. The time to reach steadystate plasma concentrations was between 1 and 5 weeks, with steadystate being achieved earlier (12 weeks) at daily doses of 200 and 400 mg. The time to peak concentration following oral doses of toremifene ranged from 1.5 to 4.5 h. The terminal halflife of elimination was 5.0, 6.0, and 5.0 days for toremifene, desmethyltoremifene, and 4hydroxytoremifene, respectively. Plasma concentrations of 4hydroxytoremifene were detectable only at high doses (200 and 400 mg/day) of toremifene. The results of this phase I pharmacokinetic study show that toremifene has metabolic and kinetic patterns that are similar to those previously reported with tamoxifen. http://www.ncbi.nlm.nih.gov/pubmed/2136809 Wed, 31 Jan 1990 00:00:00 -0800 31PMagnetic resonance spectroscopy has been used to assess the changes in the levels of watersoluble phosphate pools in T47D breast carcinoma cells induced by the antimitochondrial drugs, gossypol and 6aminonicotinamide. A decrease in the NTP/Pi ratio occurred after treatment with gossypol. No change in the NTP/Pi ratio occurred on treatment with 6aminonicotinamide however, a substantial accumulation of 6phosphogluconate was observed. Pretreatment of T47D cells with gossypol prevented the accumulation of 6phosphogluconate. This facile and noninvasive approach suggests that the oxidative part of the pentosephosphate shuttle is an important source of reducing equivalents in T47D cells. This pathway may prove to be a useful target for sitedirected drug attack in carcinoma cell lines that require large quantities of NADP for the synthesis of fatty acids and steroids. http://www.ncbi.nlm.nih.gov/pubmed/2530981 Thu, 30 Nov 1989 00:00:00 -0800 Shortterm cultures of normal human mammary epithelial cells were used to determine the extent to which cmyc, cHaras1, and cerbB2 protooncogenes were expressed in proliferating normal cells. This level of expression was compared with that of primary tumor cells, malignant effusion cells, or permanently established breast cancer cell lines. Pure preparations of epithelial organoids from seven different reduction mammoplasty tissue samples yielded proliferating normal epithelial cells upon shortterm tissue culture. In every sample, protooncogene transcript levels increased upon shortterm culture of the epithelial cells. These levels often exceeded by 10fold the levels measured in uncultured organoids from the same tissue. In four of the seven cultured normal breast samples, at least one of the protooncogenes increased its expression to a level equaling or exceeding that found in a proliferating breast cancer cell line, MCF7. One effusion metastasis sample and two primary ductal adenocarcinomas were also examined for protooncogene expression. The effusion metastasis sample expressed high levels of cerbB2 messenger RNA, in accord with its amplified gene copy number otherwise, the levels of protooncogene transcripts were low in unprocessed tumor and uncultured organoids, but they increased with proliferation of the tumor cells in culture. These results indicate that the variable expression of these protooncogenes observed in breast biopsy specimens needs to be controlled for cellular growth rate or proliferation index. Furthermore, these findings suggest that dysregulated protooncogene expression, rather than overexpression per se, needs to be evaluated as a possible mechanism contributing to the development of human breast cancer. http://www.ncbi.nlm.nih.gov/pubmed/2572702 Tue, 31 Oct 1989 00:00:00 -0800 Oncogene amplification has been found in a variety of human cancers and may have prognostic importance. Therefore, techniques which facilitate detection of gene amplification could have wide applicability. We have devised a sensitive, rapid, and nonradioactive procedure for detecting alterations in gene copy number based on the polymerase chain reaction (PCR). In this technique, called differential PCR, a target gene and a singlecopy reference gene are coamplified by PCR in the same reaction vessel. The level of target gene amplification is reflected in the ratio between the two resulting PCRproduct bands. We show that this method can detect as low as twofold amplification of specific target genes. Furthermore, amplification of neu and the epidermal growth factor receptor gene could be detected in as few as 100 breast carcinoma cells or in single sections of formalinfixed, embedded material. http://www.ncbi.nlm.nih.gov/pubmed/2780051 Sat, 30 Sep 1989 00:00:00 -0700 Triphenylethylene compounds, such as tamoxifen, have shown chemosensitizing activity independent of estrogen receptor status in doxorubicinresistant cells. We examined the chemosensitizing activity of a new triphenylethylene, toremifene, and its major metabolites in a doxorubicinresistant human breast cell line, MCF7/DOX. In addition, we examined the chemosensitizing activity of unbound plasma toremifene and its metabolites isolated from patients treated with toremifene doses of 20 to 400 mg/d. MCF7/DOX cells were exposed to ultrafiltrate plasma specimens in the absence and presence of doxorubicin. These latter studies were singleblinded. Toremifene and its major metabolites were capable of sensitizing multidrugresistant cells to doxorubicin. The degree of chemosensitizing activity in vitro correlated with the plasma concentrations of toremifene and its metabolites (P less than .05). Plasma samples isolated from patients receiving highdose toremifene (400 mg/d) had the greatest chemosensitizing activity. We present evidence that toremifene and its metabolites can sensitize resistant MCF7/DOX cells to doxorubicin, that this effect is concentrationdependent, and that sensitizing activity can be detected at clinically achieved concentrations. http://www.ncbi.nlm.nih.gov/pubmed/2527971 Thu, 31 Aug 1989 00:00:00 -0700 Amplification and overexpression of the cmyc protooncogene have been observed in 22 to 32 of primary human breast cancers, yet the exact role of this gene in mammary tumor progression is unclear. We sought to elucidate this role by overexpressing cloned myc genes in the human breast carcinoma cell line MCF7. We found that augmented myc RNA levels were associated with slower in vitro growth rates, but that estrogen receptor levels, the requirement for estrogen for in vivo growth in castrated athymic nude mice, and sensitivity to the antiestrogen, tamoxifen were not altered. Furthermore, chemosensitivity to the cytotoxic agent, Adriamycin, was not affected. Lastly, overexpression of a transfected myc gene did not suppress endogenous myc levels unlike the findings in lymphoma cells. Thus our data suggest that the effect of augmented myc expression in human breast cancer cells is complex and may not induce more malignant patterns of growth as has been suggested for other human cancers. http://www.ncbi.nlm.nih.gov/pubmed/2668848 Thu, 31 Aug 1989 00:00:00 -0700 To test the hypothesis that ras activation is involved in the final stages of breast cancer progression, we analyzed tumor DNA derived from 60 different patients and extracted from 40 invasive primary breast tumors, seven lymph node and skin metastases, nine metastatic effusions, and five established breast cancer cell lines. The polymerase chain reaction technique was used to amplify DNA fragments containing Kirsten(Ki), Harvey(Ha), and Nras codons 12, 13, and 61 which were then probed on slotblots with labeled synthetic oligomers to detect nonconservative single base mutations. Activating mutations were found in one of 40 primary tumors (Kiras codon 13), zero of seven lymph node and skin metastases, one of nine metastatic effusions (Kiras codon 12), and two of five cell lines (Kiras codons 12 and 13). These results indicate that activating ras mutations are rarely involved in either the initiation or metastatic progression of human breast cancer. http://www.ncbi.nlm.nih.gov/pubmed/2642738 Tue, 31 Jan 1989 00:00:00 -0800 A modification of the polymerase chain reaction technique (PCR) technique, a primermediated enzymatic amplification of specific target sequences in genomic DNA, was used to introduce point mutations into copies of human ras oncogene sequences, and to amplify these mutated copies approximately 10(6)fold. Of the two flanking oligomers used to amplify the DNA, one contained a single base mismatch with the targeted gene segment, either codon 12 or 61 from the Kirsten ras oncogene. Doublestranded fragments harboring any point mutation can be generated using the appropriate oligomer and constitute greater than 99.999 of the PCRamplified fragments. The amplified DNA fragments are readily slotblotted to nylon membranes to serve as positive hybridization controls in the search for gene mutations in human tissue specimens. The synthesis of single base mismatched DNA fragments was also used to demonstrate that oncogene mutations can be detected from mixed DNA populations as might be present in primary tumor specimens, even when the mutation of interest is present in only 5 of the amplified sample DNA. http://www.ncbi.nlm.nih.gov/pubmed/3061761 Tue, 31 Jan 1989 00:00:00 -0800 This article reviews the absorption, bioavailability, distribution, metabolism, and elimination patterns of the agents most commonly used for the treatment of breast cancer. Although the majority of the pharmacokinetic studies reviewed have been examined in nonbreast cancer patients, the kinetic findings are not significantly altered by disease state. The anthracyclines, antimetabolites, alkylating agents, antioestrogens and mitotic inhibitors are among the classes of agents used to treat breast cancer. Although extensively examined, cancer pharmacokinetic research has resulted in very few clinically relevant findings in breast cancer. http://www.ncbi.nlm.nih.gov/pubmed/3052986 Wed, 30 Nov 1988 00:00:00 -0800 A patient with acquired chronic oral and vaginal candidiasis resistant to topical and parenteral therapy was found to have impaired cell mediated immunity to Candida antigen and loss of skin test response to tuberculin (Mantoux). Treatment with Candidaactive transfer factor produced clinical remission lasting 1 year and restitution of in vitro and in vivo immune parameters. Relapse occurred while receiving a second lot of transfer factor from the same donor. Subsequent treatment with levamisole was associated with onset of agranulocytosis. http://www.ncbi.nlm.nih.gov/pubmed/889704 Fri, 30 Sep 1977 00:00:00 -0700