Estrogen Receptor Positive Breast Tumors Research Articles

Abstract P3-04-01: Discovery of distinct sub-groups of ER positive breast cancers by DNA repair expression profiling: Implications for patient stratification and personalization of therapy

Abstract Background: The role of DNA repair in estrogen receptor (ER) positive breast cancer is largely unknown. We hypothesized that DNA repair dysregulation could lead to evolution of aggressive phenotypes with increasing resistance to endocrine therapy. Methods: We immuno-profiled 1120 early stage ER positive breast tumours for Pol b, FEN1, APE1, XRCC1, SMUG1, PARP1, ATR, ATM, DNA-PK, BRCA1, CHK1, CHK2, TOPO2, and p53. Multivariate analysis was used to calculate a DNA repair prognostic index after controlling for other validated prognostic factors, confounders and adjuvant therapy. For additional validation, genomic expression as well as array CGH evaluation of key DNA repair genes was performed [discovery cohort (n = 128), external validation in Uppsala (n = 249) and METABRIC cohort (n = 1950)]. Neural network analysis was conducted in 249 tumours to map genetic interactions. Results: DNA repair immuno-profiling stratified ER positive breast tumours into four distinct sub-groups with increasing aggressiveness and endocrine resistance (p = 1.6 × 10 −27). Subgroup 1 had the best breast cancer specific survival, subgroups 2 & 3 have intermediate breast cancer specific survival and sub-group 4 had the worst breast cancer specific survival (Table). Survival analysis HR (95% CI)P ValuesSub-group 2 versus. Sub-group 11.6 (1.1-2.1)0.006Sub-group 3 versus. Sub-group 13.1 (2.3-4.3)1.5 × 10-12Sub-group 4 versus. Sub-group 15.7 (4.0-8.1)9.0 × 10−21 These distinct subgroups manifest progressively increasing tumour size, level of proliferation, de-differentiation, lymphovascular invasion, metastasis, altered apoptosis and increasing level of genomic instability (ps<0.001). At the genomic level DNA repair gene dysregulation remains independently associated with poor outcome and resistance (ps<0.001). Neural network analysis confirms interaction with genes involved in ER response. Conclusion: This is the first and largest study with 20 years long term follow-up data to provide compelling evidence that DNA repair dysregulation impacts ER positive breast cancer pathogenesis and drives evolution of aggressive phenotypes. Our data could transform stratification and the personalization landscape for ER positive tumours. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-01.

Abstract P5-07-02: Mutant GATA3 causes precocious lobuloalveolar development in the mouse mammary gland and is associated with more aggressive human breast cancer

Abstract GATA3 is an essential mediator of mammary gland development and is expressed at high levels in Estrogen Receptor positive tumors. This gene has recently emerged as one of the most frequently mutated genes in Estrogen Receptor positive breast cancer. GATA3 mutant tumors tend to retain and express the wild-type GATA3 allele which, together with the established role for GATA3 in maintaining luminal epithelial differentiation, has led to the suggestion that GATA3 is a haploinsufficient tumor suppressor gene in the mammary gland. We describe a striking contrast between the GATA3 mutation spectrum in breast cancer with that found in a true GATA3 haploinsufficiency syndrome and propose that this is indicative of a strong selective pressure to mutate and retain expression of a substantial portion of the GATA3 coding sequence. We demonstrate that the proteins encoded by GATA3 mutant alleles lack the capacity to bind to canonical GATA sequence motifs and acquire a new recognition specificity governed by the N-terminal zinc finger domain. Transgenic expression of the mutant protein results in a pronounced precocious lobuloalveolar development in 9 week old female mice, suggesting that the mutant gene is not a null allele and can regulate cell proliferation. In addition, we demonstrate that Estrogen Receptor positive GATA3 mutant human breast tumors arise at earlier ages than Estrogen Receptor GATA3 wild-type tumors, which further emphasizes the clinical relevance of these mutations. Based on the estimated mutation prevalence from the TCGA study, approximately 22,000 new cases of GATA3-mutant invasive breast cancer are diagnosed in the US each year. Collectively, our data indicate that GATA3 mutant tumors may comprise a breast cancer subset with distinct biological and clinical behavior and that a detailed understanding of the novel repertoire of target genes may yield useful clinical targets for this population. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-02.

Abstract A132: The HER4 intracellular domain (4ICD) functions as a potent estrogen receptor coactivator mediating estrogen stimulated proliferation of breast tumor cells

Abstract Among women with breast cancer more than 70% present with tumors expressing the estrogen receptor (ER) and will be treated with a therapeutic combination that includes endocrine therapy. Unfortunately, over 50% of these women fail therapy and develop incurable and often lethal metastatic disease. Disengaging ER interactions with essential transcriptional coactivators represents an effective approach to suppress ER driven breast cancer. To this end we have identified the HER4 intracellular domain (4ICD) as a potent estrogen receptor coactivator modulating estrogen induced gene expression and mediating estrogen stimulated proliferation of breast tumor cells. In multiple clinical studies, HER4 expression is significantly associated with ER positive tumors with up to 90% of ER positive breast tumors coexpressing HER4. These clinical observations imply a strong tumor cell selection for HER4 and ER coexpression. Estrogen stimulation of the ER and HER4 positive MCF-7 breast tumor cell line results in proteolytic processing of HER4 to release 4ICD. Significantly, estrogen stimulates nuclear translocation and binding of the 4ICD/ER complex to promoters containing estrogen response elements. Here we performed microarray gene expression analysis comparing MCF-7 cells with endogenous HER4 expression to MCF-7 cells where HER4 was stably suppressed. We found that estrogen significantly stimulated expression (2 fold and above) of 786 genes and 399 (51%) of these genes also required HER4 expression. Furthermore, chromatin immunoprecipitation (ChIP) analysis of two ER-inducible genes, progesterone receptor (PgR) and stromal-derived factor 1 (SDF-1) shows that 4ICD binds to PgR and SDF-1 gene promoters suggesting that it is required for efficient promoter binding of ER to certain ER-mediated genes. These results clearly indicate that 4ICD is an important ER coactivator that enhances ER promoter interactions. Finally, we have evidence that HER4/4ICD is required for estrogen stimulated tumor cell proliferation raising the possibility that disrupting the interaction between 4ICD and ER would represent a novel therapeutic approach to suppress breast tumorigenesis. In the future, we propose to identify additional transcription factors recruited with the ER/4ICD genomic complex. Citation Format: Wen Han, Frank Jones. The HER4 intracellular domain (4ICD) functions as a potent estrogen receptor coactivator mediating estrogen stimulated proliferation of breast tumor cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A132.

Abstract 390: Expression of the actin-binding protein Anillin is a prognostic biomarker for primary ER positive breast cancer.

Abstract Identification of novel prognostic and predictive factors is important for individualizing prognosis and treatment for cancer patients. The present study aimed at examining the potential prognostic value of Anillin (ANLN) expression in breast cancer. ANLN, an actin-binding protein required for cytokinesis, was discovered using the Human Protein Atlas as being differentially expressed in breast cancer and, thus, a potential prognostic biomarker. The purpose of the Human Protein Atlas project (www.proteinatlas.org) is to generate validated antibodies towards the entire human proteome and to use these antibodies for protein profiling in normal tissues and cancer cells. The freely available Human Protein Atlas provides an opportunity to use antibody-based proteomics as a high-throughput strategy to discover novel cancer biomarkers of potential clinical relevance. ANLN has been shown to be up-regulated in several human tumor types including lung, renal and pancreatic tumors. For the purpose of the current study, tissue microarrays with tumor samples from three independent breast cancer cohorts were analyzed by immunohistochemistry (IHC). SiRNA mediated ANLN knockdown of breast cancer cell lines followed by western blot or IHC on cytospin was done for antibody validation. Cell viability assay and cell cycle assay was used to study the function of ANLN knocked breast cancer cells. The majority of breast tumors displayed moderate to strong nuclear positivity in slightly varying fractions, generally below 25%. High nuclear fraction of ANLN was associated with large tumor size, high histological grade, high proliferation and estrogen receptor (ER) negativity. Furthermore, high nuclear expression of ANLN was significantly associated with poor prognosis for patients with ER positive breast tumors in all three cohorts. SiRNA mediated ANLN knockdown was found to induce low cell viability for ER positive breast cancer cells but not for ER negative breast cancer cells. In addition, cell cycle studies showed that ANLN knockdown yielded a shift to G2/M phase, in both ER positive and ER negative breast cancer cells. In conclusion, our results strongly suggest that ANLN can be used as a prognostic biomarker for ER positive breast cancer patients. Citation Format: Kristina Magnusson, Gabriela Gremel, Fredrik Pontén, Karin Jirström. Expression of the actin-binding protein Anillin is a prognostic biomarker for primary ER positive breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 390. doi:10.1158/1538-7445.AM2013-390

Abstract 3462: High-throughput qRT-PCR expression profiling of estrogen receptor positive breast tumors.

Abstract The class I phosphatidylinositol 3’ kinases (PI3K) play a major role in proliferation and survival in a wide variety of human cancers, and activation of the PI3K pathway is thought to be an important driver in estrogen receptor positive (ER+) breast cancer. A key factor in successful development of drugs targeting this pathway will be development in appropriate molecular subsets. Important questions relevant to PI3K inhibitor development in ER+ breast cancers are whether these inhibitors will work equally well in luminal A compared to luminal B tumors, and whether gene expression signatures of pathway activation may have additional utility in patient stratification beyond PIK3CA mutation status alone. The goal of this study was to develop a methodology for high throughput profiling of ER+ breast cancers, in order to enable molecular subtyping of patients enrolled in clinical studies. To accomplish this, we developed an analysis platform to measure the relative expression of 90 breast cancer and PI3K pathway specific genes in formalin-fixed paraffin-embedded (FFPE) tissue. The content for this panel consists of genes known to be important for epithelial-mesenchymal biology, proliferation rate, and transcriptional output of the PI3K pathway. The 96 assay panel (including 6 housekeeping genes) was developed on the Fluidigm Biomark microfluidics platform and was extensively validated using well-characterized breast cancer cell lines and FFPE breast cancer samples of known subtypes based on immunohistochemistry for HER2, ER, and PR. All assays showed high levels of inter-and intra-chip reproducibility and were sensitive on standard curves down to 3ng RNA input. Using this method we were able to separate breast cancers into distinct molecular subtypes, as well as identify more proliferative luminal B type tumors. In addition, PIK3CA mutation status, a potential biomarker, was determined using a highly specific and sensitive qRT-PCR mutation assay, in order to allow comparison with the PI3K pathway activation signature. We extended these analyses to a small cohort of patient samples consisting of matched primary and metastatic tumor tissues, and report here the correlation of primary and matched metastatic ER+ breast cancer FFPE tumor samples at both the gene expression and mutational levels. We found that the majority of matched pairs were concordant for both mutation status and gene expression, though a subset did show differences. Future studies will examine the prognostic significance and clinical relevance of this gene signature. Citation Format: Erica B. Schleifman, Rupal M. Desai, Jill Spoerke, Cheryl Victoria Wong, Ilma Abbas, Carol O'Brien, Garret Hampton, Timothy Wilson, Hartmut Koeppen, Rajesh Patel, Teiko Sumiyoshi, Ling Fu, Rachel Tam, Rajiv Raja, Mark Lackner. High-throughput qRT-PCR expression profiling of estrogen receptor positive breast tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3462. doi:10.1158/1538-7445.AM2013-3462

Abstract 3464: CEBPD (C/EBPδ) acts as a tumor suppressor in hormone receptor positive breast cancer cells and may serve as biomarker to predict the need for adjuvant therapy.

Abstract The transcription factor CCAAT/enhancer binding protein delta (CEBPD, C/EBPδ) is expressed in normal breast epithelial cells and down-regulated in breast cancer tissue and cell lines. In the MMTV-c-Neu mouse mammary tumor model, loss of CEBPD leads to increased tumor incidence but reduced tumor metastasis, suggesting dual functions as a tumor suppressor and tumor promoter. To investigate the role of CEBPD in human breast cancer, we analyzed tissue microarrays (TMA) by immunostaining. Analysis of 79 breast tumor samples confirmed overall downregulation of CEBPD in cancer but also revealed that CEBPD was expressed in 50% of estrogen receptor (ER) positive (+) breast tumors and associated with longer disease-specific patient survival (P=0.034, log-rank test). Furthermore, CEBPD was correlated with expression of progesterone receptor (PR; P=0.0007). Results from an independent TMA of 292 tumors from patients that were randomized to no adjuvant treatment or two years of tamoxifen (TAM) treatment confirmed that CEBPD expression was an independent good prognostic marker for ER(+) patients (progression-free survival, Multivariate Cox analysis P=0.041). Within the subgroup with high PR labeling, low or no expression of CEBPD was associated with worst prognosis if untreated and associated with significant response to TAM (P=0.034). On the other hand, patients with high CEBPD had better prognosis but did not further benefit from TAM. These data indicate that CEBPD expression is significantly associated with ER/PR(+) breast cancer and has potential as prognostic and/or predictive biomarker. To investigate the functions of CEBPD we silenced its expression in the ER/PR(+) MCF-7 breast tumor cell line. We found that CEBPD attenuates proliferation and promotes estrogen-dependence. In this cell culture model, CEBPD augmented MCF-7 cell sensitivity to TAM. Furthermore, we found that CEBPD promotes cell-cell adhesion through expression of alpha-catenin. These observations are consistent with a tumor suppressor-like function for CEBPD in ER/PR(+) cancer cells. Currently, we are using ChIP-Seq and mRNA-Seq approaches in MCF-7 cells to identify a CEBPD-dependent gene signature and test its prognostic value in breast cancer gene expression data. Our long-term goal is to contribute to the development of more precise multiparameter predictors of outcome and/or response through the combination of biological assays and analysis of gene expression signatures. Citation Format: Daniel Y. Mendoza-Villanueva, Suryun Kim, H.Raza Ali, Shikha Sharan, Tapasree R. Sarkar, Carlos Caldas, Göran Landberg, Esta Sterneck. CEBPD (C/EBPδ) acts as a tumor suppressor in hormone receptor positive breast cancer cells and may serve as biomarker to predict the need for adjuvant therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3464. doi:10.1158/1538-7445.AM2013-3464

Abstract 509: RNF115/BCA2 E3 ubiquitin ligase promotes breast cancer cell proliferation through targeting p21Waf1/Cip1 for ubiquitin-mediated degradation .

Abstract The E3 ubiquitin ligase Ring Finger Protein 115 (RNF115), also known as Breast Cancer Associated gene 2 (BCA2), has previously been reported to be overexpressed in estrogen receptor (ERα) positive breast tumors and to promote breast cell proliferation; however, the mechanism is unknown. In this study, we demonstrated that silencing of BCA2 by siRNA in two ERα-positive breast cancer cell lines MCF7 and T47D decreases cell proliferation and increases the protein levels of the cyclin-dependent kinases inhibitor p21Waf/Cip1. The protein stability of p21 was negatively regulated by BCA2. BCA2 directly interacts with p21 and promotes p21 ubiquitination and proteasomal degradation. Knockdown of p21 partially rescues the cell growth arrest induced by the BCA2 siRNA. These results suggest that BCA2 promotes ERα-positive breast cancer cell proliferation at least partially through down-regulating the expression of p21. This study was supported by National Nature Science Foundation of China (81072162, 81120108019, and U1132605) . Citation Format: Zehua Wang, Arun Seth, Ceshi Chen. RNF115/BCA2 E3 ubiquitin ligase promotes breast cancer cell proliferation through targeting p21Waf1/Cip1 for ubiquitin-mediated degradation . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 509. doi:10.1158/1538-7445.AM2013-509

Abstract 3452: YM155 induces autophagy-dependent cell death in Tamoxifen-resistant breast cancer cells.

Abstract Typically, patients with estrogen receptor positive (ER-positive) breast tumors will receive Tamoxifen either as a first line treatment or as a second line treatment after surgery. Despite the benefit of using Tamoxifen as hormonal therapy in treating ER-positive breast cancers has been widely demonstrated in patients at early treatment stages, its effectiveness seems to be significantly reduced in patients after five years post-treatment. Interestingly, two thirds of the tumors that become resistant to Tamoxifen continue to express ER. Thus, it is necessary to develop novel strategies that can be applied to target ER-positive Tamoxifen-resistant breast cancer cells. On the other hand, it is also in urgent to develop novel agents that can be used to target triple-negative metastatic aggressive breast cancers, given that current therapeutic selection for this cancer is still limited. Survivin is a member of the inhibitor-of-apoptosis proteins family. It has been shown that expression of survivin is positively correlated to the breast cancer progression and inversely correlated to the survival period of patient after chemotherapeutic/hormonal treatments. YM155 is a small molecule survivin gene suppressant. Although the functionality of YM155 in targeting lymphoma and lung cancer cells has been shown in vitro and in vivo, and the pharmacokinetics and the maximum tolerance dose of YM155 have also been evaluated in patients with non-Hodgkin's lymphoma and non-small cell lung cancer, its effectiveness in targeting ER-positive Tamoxifen resistant breast cancers and triple-negative metastatic aggressive breast cancers has not yet been determined. Furthermore, the detailed molecular mechanism of actions of YM155 is still unclear. In this study, the effectiveness of YM155 in targeting a series of newly-generated MCF7-derivded ER-positive Tamoxifen-resistant breast cancers (TamR7, TamR8, TamC3 and TamC6) and the triple-negative Tamoxifen-resistant MDA-MB-231 breast cancer cells has been determined. MTT cell viability assay revealed that YM155 is equally effective in targeting both the parental MCF7 and MCF7-derivded Tamoxifen-resistant breast cancer cells in vitro. YM155 is also effective in targeting the triple-negative MDA-MB-231 breast cancer cells with an IC50 value in low nanomolar range. Interestingly, Western blot analysis and MTT assay (3MA or Z-VAD-FMK co-treated) revealed that YM155 induces autophagy-dependent cell death, but not caspase-3 dependent apoptosis, in the treated cancer cells, despite it is widely believed that survivin can functionally inhibit apoptosis through caspase-3 inhibition. In addition, YM155 also induces the expression of gamma H2AX, indicating the induction of DNA damage in the treated breast cancer cells. In conclusion, YM155 is effective in targeting various types of Tamoxifen-resistant breast cancers through induction of autophagy-dependent cell death. Citation Format: Chih-Yun Liu, Siao Muk Cheng, Euphemia Leung, Chun Hei Antonio Cheung. YM155 induces autophagy-dependent cell death in Tamoxifen-resistant breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3452. doi:10.1158/1538-7445.AM2013-3452

Selective Estrogen Receptor Modulators Resveratrol, Genistein, Tamoxifen and Pterostilbene down‐regulate the Expression of Estrogen‐Regulated Trefoil Factor 1 (TFF1) Gene in MCF‐7 Breast Cancer Cells

The human trefoil factor 1 (TFF1) is a small cysteine‐rich secretory protein expressed in the breast tumor in response to estrogen. The selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene provide an effective treatment for estrogen receptor (ER) positive breast cancers. Our studies indicate that these SERMs at pharmacological concentrations modulate the expression of TFF1. The MCF‐7 breast cancer cells were treated with increasing concentrations of estrogen and SERMs such as resveratrol, genistein, tamoxifen and pterostilbene, and its effects were studied using RNA and protein analyses. The cell viability studies using estrogen showed increased cell growth even at low concentrations whereas the SERMs showed a concentration dependent cell growth arrest and death by apoptosis. Reverse Transcription‐Polymerase Chain Reaction (RT‐PCR) and Northern blot analyses indicated decreased levels of transcription of TFF1 mRNA. Western blot analysis showed a corresponding decrease in the expression of the protein. The above results indicate the potential use of these SERMs for the down‐regulation of TFF1 expression and consequently the regression of ER positive breast tumors. This work was supported by science research funds from Arnold and Marie Schwartz College of Pharmacy, Long Island University, NY.

Metastasis Awakening: Targeting dormant cancer

An important challenge for oncologists is to treat overt metastasis, the major source of cancer-related deaths1. Adjuvant therapy should prevent distant recurrences by targeting residual disseminated tumor cells (DTCs) that give origin to metastasis, as well as existing undetected micrometastasis. Whereas in some cases, such as breast cancer, patients can show delayed metastasis after hormonal therapies (for example, in estrogen receptor–positive tumors) or treatment with trastuzumab, for HER2 (also called ERBB2)-positive tumors2, adjuvant therapy is for the most part ineffective in fully blocking metastasis development and improving overall survival.

In silico discovery of mitosis regulation networks associated with early distant metastases in estrogen receptor positive breast cancers.

The aim of this study was to perform comparative analysis of multiple public datasets of gene expression in order to identify common genes as potential prognostic biomarkers. Additionally, the study sought to identify biological processes and pathways that are most significantly associated with early distant metastases (<5 years) in women with estrogen receptor-positive (ER+) breast tumors. Datasets from three published studies were selected for in silico analysis of gene expression profiles of ER+ breast cancer, using time to distant metastasis as the clinical endpoint. A subset of 44 differently expressed genes (DEGs) was found common to all three studies and characterized by mitotic checkpoint genes and pathways that regulate mitotic spindle and chromosome dynamics. DEG promoter regions were enriched with NFY binding sites. Analysis of miRNA target sites identified significant enrichment of miR-192, miR-193B, and miR-16-1 targets. Aberrant mitotic regulation could drive increased genomic instability leading to a progression towards an early onset metastatic phenotype. The relative importance of mitotic instability may reflect the clinical utility of mitotic poisons in metastatic breast cancer, including poisons such as the taxanes, epothilones, and vinca alkaloids.

Abstract P2-10-40: Correlation between expression of the prognostic marker Progranulin (GP88) with Oncotype Dx Recurrence Score in estrogen receptor positive breast tumors

Abstract Background: GP88 (Progranulin, acrogranin) is an 88kDa glycoprotein overexpressed in breast tumors and involved in their proliferation and survival. Biological studies have shown that GP88 in ER+ breast cancer cells was associated with estrogen independence and resistance to anti-estrogen therapies and aromatase inhibitors. In addition, GP88 stimulated migration, invasion and angiogenesis, hallmarks of metastasis. Pathological studies have demonstrated that GP88 was preferentially expressed in invasive ductal carcinoma while lobular carcinoma was mostly negative. No expression was found in normal mammary tissue. Two retrospective studies totaling 530 cases of ER+ formalin-fixed paraffin-embedded invasive ductal carcinoma (IDC) established that high GP88 expression was associated with a 4-fold increase in risk of recurrence and 2.5-fold increase in death when compared to low GP88 scores. Multivariate analysis showed that GP88 remains a predictor of recurrence even when adjusted for prognostic factors such as tumor size, grade, stage, lymph node status and age. High level of GP88 was also found to be elevated in the serum of breast cancer patients when compared to healthy individuals. Since GP88 tumor tissue expression appears as a prognostic factor for ER+ breast cancer, we proposed to investigate whether GP88 tumor tissue expression would correlated to Oncotype Dx® Recurrence Score which is now for early stage ER+ node negative breast cancer patients. Methods: Eight five cases from women ages 37–77 with ER+ invasive mammary carcinoma from three different institutions and with an available Oncotype Dx recurrence score were selected with approval from each site's IRB. GP88 expression was determined by immunohistochemistry (IHC) using the anti-human GP88 6B3 monoclonal antibody (A&amp;G) on a Ventana automated staining platform. For all cases examined, GP88 IHC scores (0, 1+, 2+, 3+) were compared to routine clinicopathologic factors (tumor size, grade, and stage), PR, HER2/neu and Ki67 expression (by image analysis) and to their Oncotype DX® recurrence score (Genomic Health). The associations of GP88 with the parameters described above were assessed by t test or ANOVA. Results: The GP88 tissue expression correlated with Oncotype Dx Recurrence score (p &amp;lt; 0.03) as well as with Ki-67 index (p &amp;lt; 0.004). Age, HER2/neu and PR status did not correlate with GP88 expression. Details of these finding will be presented here. Conclusion: This study shows that GP88, an important tumor aggressiveness indicator, significantly correlates with Oncotype DX score and confirms that GP88 correlates with other tumor clinicopathologic parameters and prognostic markers. Further studies are underway to determine whether GP88 in combination with routine markers, particularly Ki67, could be used instead of Oncotype DX Assay to predict outcome and provide information for the management of ER+ breast cancer patients. This study is supported by grant 02–2010-010 from the Avon Foundation for Women. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-40.

Maintenance of S-nitrosothiol homeostasis plays an important role in growth suppression of estrogen receptor-positive breast tumors

IntroductionProtein denitrosylation by thioredoxin reductase (TrxR) is key for maintaining S-nitrosothiol (SNO) homeostasis, although its role in tumor progression is unknown. Therefore, the present study aimed to assess the role of altered SNO homeostasis in breast cancer cells.MethodsThe impairment of SNO homeostasis in breast cancer cells was achieved with the highly specific TrxR inhibitor auranofin and/or exposure to S-nitroso-L-cysteine. S-nitrosylated proteins were detected using the biotin switch assay. Estrogen receptor (ER) alpha knockdown was achieved using RNA silencing technologies and subcellular localization of ERα was analyzed by confocal microscopy. The Oncomine database was explored for TrxR1 (TXNRD1) expression in breast tumors and TrxR1, ER and p53 expression was analyzed by immunohistochemistry in a panel of breast tumors.ResultsThe impairment of SNO homeostasis enhanced cell proliferation and survival of ER+ MCF-7 cells, but not of MDA-MB-231 (ER-, mut p53) or BT-474 (ER+, mut p53) cells. This enhanced cell growth and survival was associated with Akt, Erk1/2 phosphorylation, and augmented cyclin D1 expression and was abolished by the ER antagonist fulvestrant or the p53 specific inhibitor pifithrin-α. The specific silencing of ERα expression in MCF-7 cells also abrogated the growth effect of TrxR inhibition. Estrogenic deprivation in MCF-7 cells potentiated the pro-proliferative effect of impaired SNO homeostasis. Moreover, the subcellular distribution of ERα was altered, with a predominant nuclear localization associated with phosphorylation at Thr311 in those cells with impaired SNO homeostasis. The impairment of SNO homeostasis also expanded a cancer stem cell-like subpopulation in MCF-7 cells, as indicated by the increase of percentage of CD44+ cells and the augmented capability to form mammospheres in vitro. Notably, ER+ status in breast tumors was significantly associated with lower TXNDR1 mRNA expression and immunohistochemical studies confirmed this association, particularly when p53 abnormalities were absent.ConclusionThe ER status in breast cancer may dictate tumor response to different nitrosative environments. Impairment of SNO homeostasis confers survival advantages to ER+ breast tumors, and these molecular mechanisms may also participate in the development of resistance against hormonal therapies that arise in this type of mammary tumors.

Exome sequencing identifies somatic point mutations associated with acquired endocrine resistance in breast cancer cell lines

Background Endocrine therapy is an effective treatment of estrogen receptor-positive (ER+) breast tumors, significantly reducing mortality. However, approximately 30% of patients receiving adjuvant endocrine therapy will experience recurrence within a 15-year period. The mechanisms of endocrine resistance are poorly understood. Understanding the underlying genetic diversity of breast cancers responding differently to endocrine therapy is important for the development of more optimal and individualized treatments strategies. Materials and methods In the current study, a panel of isogenic MCF-7-derived human breast cancer cell lines [1-3] that are resistant to tamoxifen only, or to both tamoxifen and fulvestrant, respectively, were analyzed for mutations through exome sequencing and compared with the exome of the parental cell line. In addition, global gene expression levels for the same panel of cell lines were generated. Detected variations were integrated with gene expression profiles and analyzed in the context of prior knowledge of drug action and genes associated with resistance to endocrine therapies as identified by extensive literature curation. Results and conclusion A small panel of somatic point mutations potentially associated with acquired endocrine resistance were identified. Future experimental validation will reveal which of the detected mutations that are causatively involved in resistance to endocrine therapy.

Estrogen receptor-α recruits P-TEFb to overcome transcriptional pausing in intron 1 of the MYB gene.

The MYB proto-oncogene is expressed in most estrogen receptor-positive (ERα+) breast tumors and cell lines. Expression of MYB is controlled, in breast cancer and other cell types, by a transcriptional pausing mechanism involving an attenuation site located ∼1.7 kb downstream from the transcription start site. In breast cancer cells, ligand-bound ERα binds close to, and drives transcription beyond this attenuation site, allowing synthesis of complete transcripts. However, little is known, in general, about the factors involved in relieving transcriptional attenuation, or specifically how ERα coordinates such factors to promote transcriptional elongation. Using cyclin dependent kinase 9 (CDK9) inhibitors, reporter gene assays and measurements of total and intronic MYB transcription, we show that functionally active CDK9 is required for estrogen-dependent transcriptional elongation. We further show by ChIP and co-immunoprecipitation studies that the P-TEFb complex (CDK9/CyclinT1) is recruited to the attenuation region by ligand-bound ERα, resulting in increased RNA polymerase II Ser-2 phosphorylation. These data provide new insights into MYB regulation, and given the critical roles of MYB in tumorigenesis, suggest targeting MYB elongation as potential therapeutic strategy.

Estrogen Receptor Ligands for Targeting Breast Tumours: A Brief Outlook on Radioiodination Strategies

The design and development of radiolabelled estradiol derivatives has been an important area of research due to their recognized value in breast cancer management. The estrogen receptor (ER) is a relevant biomarker in the diagnosis, prognosis and prediction of the therapeutic response in estrogen receptor positive breast tumours. Hence, many radioligands based on estradiol derivatives have been proposed for targeted functional ER imaging. The main focus of this review is to survey the current knowledge on estradiol-based radioiodinated receptor ligands synthesis for breast tumour functional imaging. The main preclinical and clinical achievements in the field will also be briefly presented to make the manuscript more comprehensive.

Novel estradiol based metal complexes of Tc-99m

Aiming to contribute to the design of technetium imaging agents for estrogen receptor (ER) positive breast tumors, we have synthesized and evaluated the novel organometallic estradiol complexes (fac-[M(CO)3(κ3-10)]+ and fac-[M(CO)3(κ3-12) M=Re/99mTc) using two different bifunctional tridentate ligands (4 and 8). The rhenium complexes (13 and 14) were fully characterized by IR, 1H NMR, 13C NMR, mass spectrometry and elemental analyses. The 99mTc complexes (15 and 16) were obtained with high radiochemical purity and exhibited high in vitro radiochemical stability. To get a first insight into the relevance of these complexes for targeting ER positive tumors, ER binding affinity assays and cellular internalization studies in an ER expressing cell line, MCF-7, have also been performed suggesting a non ER mediated uptake.

Histone Deacetylase 6 (HDAC6) Deacetylates Survivin for Its Nuclear Export in Breast Cancer

Survivin is an oncogenic protein that is highly expressed in breast cancer and has a dual function that is dependent on its subcellular localization. In the cytosol, survivin blocks programmed cell death by inactivating caspase proteins; however, in the nucleus it facilitates cell division by regulating chromosomal movement and cytokinesis. In prior work, we showed that survivin is acetylated by CREB-binding protein (CBP), which restricts its localization to the nuclear compartment and thereby inhibits its anti-apoptotic function. Here, we identify histone deacetylase 6 (HDAC6) as responsible for abrogating CBP-mediated survivin acetylation in the estrogen receptor (ER)-positive breast cancer cell line, MCF-7. HDAC6 directly binds survivin, an interaction that is enhanced by CBP. In quiescent breast cancer cells in culture and in malignant tissue sections from ER+ breast tumors, HDAC6 localizes to a perinuclear region of the cell, undergoing transport to the nucleus following CBP activation where it then deacetylates survivin. Genetically modified mouse embryonic fibroblasts that lack mhdac6 localize survivin predominantly to the nuclear compartment, whereas wild-type mouse embryonic fibroblasts localize survivin to distinct cytoplasmic structures. Together, these data imply that HDAC6 deacetylates survivin to regulate its nuclear export, a feature that may provide a novel target for patients with ER+ breast cancer.

Comparative Effects of Sesame Seed Lignan and Flaxseed Lignan in Reducing the Growth of Human Breast Tumors (MCF-7) at High Levels of Circulating Estrogen in Athymic Mice

Flaxseed (FS) has a breast tumor-reducing effect, possibly because of its high content of secoisolariciresinol diglucoside (SDG) lignan. Sesame seed (SS) is rich in the lignan sesamin (SES) but is non-protective. Both lignans are metabolized to estrogen-like enterodiol and enterolactone. The objective of this study was to differentiate the effects of SDG and SES on established human estrogen receptor-positive breast tumors (MCF-7) in athymic mice with high serum estrogen to help explain the different effects of FS and SS. Mice were fed for 8 wk the basal diet (BD, control) or BD supplemented with 1 g/kg SDG or SES. SES reduced palpable tumor size by 23% compared to control, whereas SDG did not differ from SES or control. Both treatments reduced tumor cell proliferation, but only SES increased apoptosis. SDG and SES reduced human epidermal growth factor receptor 2 and endothelial growth factor receptor expressions, but only SES reduced downstream pMAPK. Neither treatment affected IGF-1R, vascular endothelial growth factor receptor-2, Akt, pAkt, or MAPK of the growth factor signaling pathway. Thus, at high serum estrogen levels, SDG may not account for the tumor reducing effect of FS. SES was more effective than SDG in reducing breast tumor growth, but its effect may have been lost when consumed as a component of SS.

Abstract CN06-01: Low-dose azacytidine and epigenetic regulation of self-renewal.

Abstract Breast cancer is the most commonly diagnosed cancer among women worldwide and the second leading cause of cancer death. Breast cancer patients do not die from their primary tumor, but rather from distant metastasis. Metastases are maintained by self-renewing, cancer stem cells (CSCs) and these cells are believed to be primarily responsible for the tumor recurrence that occurs during failed therapy. CSCs are difficult to target with conventional cancer therapies; however we show that self renewing cells can be successfully inhibited using epigenetic approaches. Epigenetics (heritable changes in gene function that do not involve alterations in DNA sequence) play a major role in the inactivation and silencing of genes in cancer, leading to a loss of gene function that provides a selective advantage to cancer cells. This silencing is associated with promoter, CpG island, DNA hypermethylation. These promoters are largely protected from DNA methylation in all normal cells. DNA methylation can be reversed via the use of epigenetic, de-methylating agents such as decitabine (5-aza-2'-deoxycytidine, DAC) and azacitidine (5-azacitidine, AZA); however, the mechanisms underlying the clinical efficacies of these drugs is not clear. Historically, DAC and AZA were administered at high micromolar doses and were considered too toxic to be effective as anti-cancer agents. After reassessment in hematologic malignancies (MDS/AML), these agents were found to be effective and less toxic at lower doses, suggesting that dose de-escalation is key for these de-methylating agents. Using cell lines and primary tissue from breast cancer patients, we show that transient, low nanomolar doses of DAC and AZA, which are not immediately cytotoxic, can impart a long term, anti-tumorigenic response, mediated in part, via decreases in self-renewal, and invasiveness and increases in apoptosis and differentiation. In our ex vivo-mouse model, breast cancer cell lines are treated daily for 3 days in culture with 100 nM DAC, or 500 nM AZA. The drugs are then withdrawn and the cells are permitted to recover in culture for 7 days. At day 10, equal numbers of viable cells are harvested and subcutaneously injected into NOD/SCID mice to generate xenografts. Analysis of tumor xenografts showed that low doses of AZA lead to an increased latency, slower tumor growth and a partial regression of some tumors. The tumor xenograft responses are not indicative of tumor de-bulking, but instead suggest a blunting of self-renewal and/or decrease in tumorigenicity of cancer stem cell populations. Moreover, estrogen receptor positive and HER2+ cancer cell lines were more sensitive to azacytidine than triple negative tumors. Similar, but more dramatic results were observed for primary tumors from metastatic breast cancer patients that were orthotopically transplanted into immunodeficient mice. Pre-established, estrogen receptor positive breast tumors in mice, showed diminished growth in response to systemic, AZA administered subcutaneously, five days a week for three weeks. These data represent a potentially a major step for personalization of epigenetic therapy for breast cancer and are currently under further investigation in a phase I/II trial of azacytidine and entinostat in metastatic breast cancer. In a cell culture model for self renewal, pleural effusions from women with metastatic breast cancer were cultured in suspension to generate mammospheres. Serial passage of mammospheres treated with 500nM AZA led to a progressive decrease in both the size and number of spheres while, with each increased passage, mock treated spheres increased in size and number. Likewise, flow analysis revealed alterations in stem cell markers CD44, CD24 and aldehyde dehydrogenase. These data suggest that treatment with AZA targets self-renewing or stem-like cells in primary patient samples. Agilent gene expression data were assessed by Metacore analyses (GeneGo Inc. St. Joseph, MI) for day 3 of AZA treatment, and day 7 and day 10 of post-treatment. Azacytidine led to sustained alterations in gene expression in several key, anti-tumor pathways in both breast cancer cell lines and primary samples. Many of the anti-tumorigenic pathways identified, are critical for self-renewal, mitosis, invasion, and migration, and suggest that self-renewing tumor cell subpopulations may be among those most heavily affected by these changes. The data obtained from our research will prove to be imperative for understanding how epigenetic therapy strategies work to target cancer stem cells, for the development of biomarkers to personalize the therapies, for the design of new drugs for epigenetic therapy, and, most importantly, for the design of our next immediate clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr CN06-01.