Myles Brown Research Articles

Abstract 966: LSD1 functions as a global androgen receptor coactivator and is a therapeutic target in prostate cancer

Abstract Androgen receptor (AR) plays a pivotal role in prostate cancer (PCa) and remains critical for more aggressive castration-resistant PCa (CRPC) through transactivation of multiple genes. Although CRPC is generally responsive to further CYP17A1 inhibition (abiraterone) or more potent AR antagonist (enzalutamide) treatments, patients treated with these agents still relapse within 1-2 years, and high levels of AR and AR regulated genes in many of these relapsed tumors indicate that AR activity has again been restored. Therefore, there remains a need for further novel AR targeted therapies. Initially identified as a component in the REST complex that functions as a transcriptional suppressor, Lysine Specific Demethylase 1 (LSD1) removes the methyl group from histone 3 lysine 4 (H3K4me1 and 2), and turns off the marks of active transcription. Our recent genome-wide integrated analysis of LSD1 and AR chromatin bindings and gene expression profiles indicates that LSD1 is broadly associated with AR regulated enhancers, and LSD1 functions as a coactivator on AR-stimulated genes through demethylating novel histone or nonhistone substrates in addition to H3K4me. LSD1 is also associated with FOXA1 at AR regulated enhancer sites, and an LSD1-FOXA1 interaction enhances binding of both proteins at these sites. These results clearly demonstrated LSD1 as a therapeutic target in prostate cancer. We then accessed if LSD1 enzymatic activity is required for its global AR coactivator function. AR transcription activity is significantly impaired in cells overexpressing a catalytic-deficient LSD1 mutant. In addition, this mutant also decreased FOXA1 binding on AR-regulated enhancers. We then selected of a panel of LSD1 inhibitors (a few of them have been used in clinical trials for lung cancer and leukemia) to assess the effects on AR activity and PCa cell growth. Inhibition of LSD1 consistently impaired AR activity through abrogating acetylated H3K27 at AR-regulated enhancers. LSD1 inhibitor also decreased FOXA1 binding prior to androgen stimulation. While most of inhibitors showed relatively low efficacy in PCa cells (>50μM), LSD1-C12 is highly potent and specific at μM range to dramatically decrease AR activity and PCa cell growth. Overall, our study suggests that the AR coactivator function of LSD1 depends on its enzymatic activity and targeting LSD1 with more specific and potent inhibitors is a promising therapeutic approach to treat PCa. Furthermore, understanding the mechanism of LSD1 coactivator function will potentially advance PCa therapy by targeting LSD1. Citation Format: Shuai Gao, Yanfei Gao, Hansen He, Myles Brown, Steve Balk, Changmeng Cai. LSD1 functions as a global androgen receptor coactivator and is a therapeutic target in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 966. doi:10.1158/1538-7445.AM2015-966

Abstract LB-016: Estrogen receptor stability is modulated by calpain activity in breast cancer cells

Abstract Breast cancer is a disease that affects thousands of women worldwide, and despite a number of treatment options, death results from metastasis and disease resistance. Two-thirds of all breast cancer cases involve dysregulation of estrogen receptor (ER) signaling; thus, a number of treatment strategies employ using compounds that affect its activity. Included in these drugs is the selective ER modulator (SERM) tamoxifen, which binds ER, leading to its association with DNA and the recruitment of corepressor proteins that inhibit ER signaling. While therapies involving tamoxifen are useful in controlling the disease, resistance inevitably occurs, rendering these treatments ineffective. Thus, other compounds are used such as selective ER down regulators (SERDs), which leads to ubiquitin-mediated ER degradation and complete loss of its signaling. Because resistance to these compounds also occurs, other methods for regulating ER signaling must be explored. In this study, we report loss in ER protein expression in response to treatment with doxorubicin and MG132. We found that treatment of the human breast cancer cell lines MCF7 and T47D with doxorubicin plus MG132 leads to ER protein expression loss as demonstrated by western blotting. In contrast, no ER protein expression loss was observed by treatment with either drug alone. RNA-Seq analysis of cells treated with this drug combination demonstrated loss in downstream ER targets, confirming suppressed ER signaling. As MG132 inhibits both the proteasome and calpain activity, we next sought to determine which of these protease activities was involved. Cells were treated in the presence of the proteasome inhibitors bortezomib and lactacystin, and no effect on ER expression was found with doxorubicin treatment, suggesting that the proteasome does not play a role in the ER loss. In contrast, treatment with doxorubicin plus the calpain-specific inhibitors PD 150606 and EST led to ER protein expression loss, confirming that calpain activity is involved in ER protein regulation. Next, to determine which calpains were specifically involved in the loss of ER protein, we knocked down several calpain proteins found to be expressed in MCF7 cells including calpains 1, 7, 8, 9, and 13 and the regulatory subunit CAPNS1. While knockdown of calpains 1, 7, 8, 9, and 13 had no effect on ER protein expression in the presence of doxorubicin, knockdown of CAPNS1 led to a loss in ER that was similar to treatment with calpain inhibitors in the presence of doxorubicin. In conclusion, this study identifies a new pathway by which ER stability is affected i.e., DNA damage induction plus calpain inhibition, suggesting a novel chemotherapeutic treatment paradigm that may overcome resistance in patients with breast cancer. Thus, future studies will focus on identifying the proteins and mechanisms directly responsible for ER loss as well as analyzing preclinical in vivo mouse models. Citation Format: Shannon T. Bailey, Thomas Westerling, Myles Brown. Estrogen receptor stability is modulated by calpain activity in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-016. doi:10.1158/1538-7445.AM2015-LB-016

Abstract 1865: TRIM24 acts as a transcriptional co-activator of the androgen receptor during prostate cancer progression

Abstract The steroid hormone androgen mediates a wide range of developmental, physiological and malignant responses by acting as an agonist for the androgen receptor (AR). AR in turn functions as a nuclear receptor transcription factor and executes specific gene expression programs in an androgen-dependent manner. Since the growth of prostate cancer cells initially depends on androgen, cancer therapy uses hormone-deprivation approaches to reduce the levels of androgen. While prostate cancer (PC) initially responds to androgen-ablation, most tumors progress to a castration resistant (CR) state insensitive to treatment. Both the androgen-dependent and the CRPC state depend on AR, which is probably activated by alternative molecular pathways in CRPC in response to low androgen levels. Here, we assessed the role of the transcriptional co-activator TRIM24 in mediating AR-dependent gene expression and growth in our CRPC cell model LNCaP-abl. We observed that knock-down of TRIM24 impairs CRPC cell growth, while TRIM24 and AR physically interact in these cells in the absence of androgen. When we identified TRIM24-dependent gene expression programs by combining genome-wide binding analyses with microarray studies, we found that TRIM24 and AR co-regulated many genes in CRPC cells. TRIM24- and AR-co-activated genes often function in mitosis and their expression is higher in patients with an increased chance of prostate cancer recurrence. These findings are corroborated by IHC-analyses of prostate cancer patients, where higher TRIM24 protein levels correlated with a greater chance of biochemical recurrence, higher Gleason scores and larger tumor sizes. Importantly, TRIM24 staining was positively correlated with AR staining. This supports a model where TRIM24 acts as an important activator of AR in the castration-resistant setting and therefore may harbor therapeutic potential. Citation Format: Anna C. Groner, Jonas de Tribolet-Hardy, Jean-Philippe Theurillat, Peter J. Wild, Myles Brown. TRIM24 acts as a transcriptional co-activator of the androgen receptor during prostate cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1865. doi:10.1158/1538-7445.AM2015-1865

Abstract 1849: Androgen receptor transcriptionally represses genes mediating DNA synthesis and repair in prostate cancer

Abstract In the U.S., prostate cancer (PCa) ranked second for cancer related deaths for men. It has been well established that androgen receptor (AR) plays an essential role in the development of primary PCa and its activity is restored in castration resistant prostate cancers (CRPC). While AR has been extensively characterized as a transcriptional activator, it can also function by direct or indirect mechanisms to suppress gene expression. Despite the critical role AR plays in PCa, mechanisms by which it functions as a transcriptional repressor are poorly understood. Using VCaP and VCaP derived CRPC cell lines, we have reported that androgen-repressed genes were functionally enriched for DNA synthesis and repair, while AR stimulated genes were associated with lipid/sterol synthesis and other aspects of cellular metabolism. Significantly, a subset of the genes that were androgen-repressed in VCaP cells were consistently increased in CRPC clinical samples, and this 53-gene subset was also highly enriched for genes mediating DNA synthesis and repair. Using AR ChIP-seq and transcriptome profiling in VCaP cells, we found that the majority of this AR-suppressed gene subset (39/53) have one or more AR binding sites within the gene locus. The expression level of these direct AR-suppressed genes is associated with poor survival of disease in clinical cohorts. Interestingly, although the suppression activity of AR on this subset of genes is direct, in other PCa cell lines such as LNCaP, there is a secondary mechanism that allows AR to indirectly stimulate the expression of these genes. Importantly, the direct AR mediated repression of these genes is enhanced in PCa cells expressing higher levels of AR, and overexpression of AR in LNCaP cells resulted in increased and more persistent AR binding to these genes, and also enhanced the direct AR repression of this gene subset. Mechanistically, we found that the indirect activation of these DNA synthesis genes by AR is mediated through transcriptionally stimulating lipid synthesis, which could subsequently drive the G1/S cell cycle progression and RB1 phosphorylation. Indeed, this indirect stimulatory effect of AR on these genes could be suppressed by cyclin dependent kinase inhibition or by preventing the AR stimulation of mTOR (rapamycin) or sterol synthesis (statins). Taken together, these findings indicate that the ability of AR to repress this gene set mediating DNA synthesis may persist in CRPC, and may be exploited therapeutically by combination therapies that stimulate AR while repressing its metabolic functions. Citation Format: Yanfei Gao, Shuai Gao, Housheng He, Xiaming Liu, Sen Chen, Fen Ma, X. Shirley Liu, Myles Brown, Steven P. Balk, Shaoyong Chen, Changmeng Cai. Androgen receptor transcriptionally represses genes mediating DNA synthesis and repair in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1849. doi:10.1158/1538-7445.AM2015-1849

Abstract P3-05-14: Modeling chemoendocrine therapy for ER+/p53wt luminal breast cancer

Abstract Background ER+ breast cancers are predominantly p53 wildtype (wt). Despite this genotype, these cancers are relatively resistant to chemotherapy-induced apoptosis in the presence of estrogen. Thus, current clinical practice is to administer hormonal therapy and chemotherapy sequentially rather than concurrently. Using the ER+/p53wt cell line MCF-7, we previously demonstrated that ER bound by either the ER agonist estradiol or partial antagonist tamoxifen inhibits the expression of a set of proapoptotic p53 target genes, whereas the full ER antagonist fulvestrant, a selective ER downregulator (SERD), removes the ER-mediated suppression of these genes, sensitizing breast cancer cells to p53-mediated cell death .We hypothesize that effective chemoendocrine therapy requires complete ER antagonism resulting in ER downregulation, increased p53 activity, and ultimately apoptosis. Methods: To model mechanisms of tumor sensitivity and resistance to therapy in vivo, therapeutic experiments were performed in immunodeficient mice bearing ER+/p53wt tumor xenografts derived from a 61-year-old African-American female with grade 2 invasive ductal carcinoma. The mice were randomly assigned to the following treatment groups: A) fulvestrant B) tamoxifen, C) doxorubicin, D) fulvestrant plus doxorubicin, E) tamoxifen plus doxorubicin, and F) control. Tumors were measured weekly for 42 days to determine treatment effects on growth. In addition, RNA was harvested from tumors at an early time point for RNA sequencing to determine genes regulated by the treatments. Results: Combination therapy comprising doxorubicin plus fulvestrant resulted in tumor regression compared with single-agent or doxorubicin plus tamoxifen treatment, which inhibited the growth of tumors but did not lead to their regression. Conclusions: Current paradigms for the treatment of ER+ breast cancer either in the adjuvant or advanced setting have involved the sequential use of endocrine therapy and chemotherapy. This protocol is partially based on evidence of potential antagonism between tamoxifen and chemotherapy as observed in early studies. Our published work in vitro and the preclinical study presented here suggest that complete ER antagonism with SERDs such as fulvestrant is required to overcome the ability of ER to block p53-mediated apoptosis. Our results suggest treatments involving fulvestrant concurrent with chemotherapies involving p53 activation should be considered for the treatment of patients with ER+/p53wt breast cancer. Citation Format: Shiliang A Cao, Elgene Lim, Myles Brown, Shannon T Bailey. Modeling chemoendocrine therapy for ER+/p53wt luminal breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-14.

Abstract PD6-2: FoxA1 gene amplification in ER+ breast cancer mediates endocrine resistance by increasing IL-8

Abstract Background: ER transcriptional programming is associated with fundamental changes when endocrine resistance develops. The Forkhead transcription factor, FoxA1, is a pioneer factor for ER-DNA binding. We hypothesize that FoxA1 plays a critical role in ER transcriptional reprogramming in endocrine resistance by augmenting itself and the specific downstream effectors. Methods: Next generation sequencing was applied to characterize a panel of endocrine-resistant (Endo-R) cell models. Genomic PCR amplification and FISH assays were developed to measure FoxA1 copy number gain (CNG). Q-RT-PCR, Western blots, IHC, ELISA, and cytokine arrays were used to determine the levels of FoxA1 and IL-8 in cell culture and in vivo xenograft tumors. Effects of gene knockdown (ER, FoxA1, or IL-8) or inducible FoxA1 overexpression on ER and growth factor receptor (GFR) downstream signaling were determined by cell growth and Western blots. ER and FoxA1 binding at the IL-8 gene locus was measured by ChIP-qPCR. ChIP-seq analysis was integrated with RNA-seq data. Kaplan-Meier analysis evaluated the predictive role of FoxA1in ER+ breast tumors. Results: Exome-seq revealed that FoxA1 is the most highly amplified gene in TamR vs. P cells from two independent MCF7 models. Genomic PCR and FISH also indicate FoxA1 CNG in Endo-R models of ZR75-1 and BT474. Increased FoxA1 expression was found in multiple Endo-R cells and in MCF7L Endo-R xenograft tumors. Cytokines, especially IL-8, are more highly expressed in multiple Endo-R cell models, similar to our previous microarray data from MCF7 Endo-R xenograft tumors. FoxA1 forced overexpression significantly induced IL-8 expression in MCF7L-P cells. It also activated multiple GFR downstream signaling pathways, and conferred endocrine resistance. Conversely, knockdown of either FoxA1 or ER significantly decreased IL-8 levels in TamR cells, and inhibited cell growth in both P and TamR cells. Knockdown of IL-8 in TamR cells substantially inhibited GFR downstream signaling, and was more cytotoxic than in P cells. A novel FoxA1-binding site (10 kb at 5’UTR of IL-8) recruited more FoxA1 and p300 in MCF7L-TamR than -P cells. ChIP-seq shows a general enhancement of FoxA1 binding around the genes (within 20 kb) that are differentially expressed in TamR vs. P cells. We identified a FoxA1 CNG-associated gene signature from TCGA breast tumors that predicts worse relapse-free survival (RFS) in Tam-treated ER+ tumors (from Loi et al). Meta-analysis showed that FoxA1 mRNA levels in the top 25th percentile predict worse RFS in ER+ patients treated with Tam (N=615), but not in systemically untreated patients (N=500). FoxA1 CNG and overexpression in clinical specimens by using our newly developed FISH and IHC assays are currently being investigated. Conclusions: FoxA1 gene amplification was enriched in two independent MCF7 Tam-R cell models. Clonal selection of FoxA1 gene amplification may occur and lead to endocrine resistance. High levels of FoxA1 may mediate endocrine resistance by directly inducing IL-8. The data suggest that IL-8 signaling is a component of a cytokine loop controlled by the FoxA1/ER transcriptional reprogramming, which might be exploited in therapeutics to overcome endocrine resistance. Citation Format: Xiaoyong Fu, Rinath Jeselsohn, Emporia F Hollingsworth, Dolores Lopez-Terrada, Chad J Creighton, Agostina Nardone, Martin Shea, Laura M Heiser, Pavana Anur, Nicholas Wang, Catie Grasso, Paul Spellman, Carolina Gutierrez, Mothaffar F Rimawi, Susan G Hilsenbeck, Joe W Gray, Myles Brown, C K Osborne, Rachel Schiff. FoxA1 gene amplification in ER+ breast cancer mediates endocrine resistance by increasing IL-8 [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD6-2.

Abstract S1-01: TransCONFIRM: The correlative analysis of breast tumors from patients with advanced hormone receptor positive disease identifies a genetic signature associated with decreased benefit from single agent fulvestrant

Abstract Introduction: Several multi-gene expression based assays have been developed to assess the prognosis and predict response to endocrine treatments in early stage hormone receptor positive (HR+) breast cancer. Although a significant number of patients with metastatic ER+ disease will not respond to endocrine treatments, molecular assays to predict response in this setting are limited. In addition, tissue specimens of metastatic lesions for molecular studies are not always available. In this study we sought to identify a molecular profile in the primary tumors of patients who developed disease recurrence that could predict response to endocrine treatment in metastatic disease. Methods: We used the primary breast tumor samples from a subgroup of patients participating in the randomized phase III CONFIRM trial, which compared 500mg versus 250mg of fulvestrant in post-menopausal women with HR+ advanced breast cancer. Formalin- fixed paraffin embedded tumors were collected from 130 of the participants and were centrally reviewed for ER, PR, HER2 and Ki67. RNA was sufficient for gene expression profiling in 112 of the cases using the NuGEN Ovation FFPE WTA System and Affymetrix HTA 2.0 GeneChip. The majority of the patients in this analysis developed metastatic disease during adjuvant endocrine treatment (N=55) or had de-novo metastatic disease (N=39) versus relapse after adjuvant treatment (N=18). The association between gene expression and progression free survival (PFS) was investigated using a multivariate Cox proportional hazard model adjusting for statistically significant clinicopatholgical factors. In addition we performed pathway-level analysis and evaluated the PAM50 subtype predictor and Risk of Relapse (ROR) score. Results: The median PFS was 8 months in our cohort. HER2 level by immunohistochemistry above 1+, high PR level, defined as Allred score of above 6, and Ki67 of above 50% were significantly associated with PFS and were included in the multivariate model. Dose of fulvestrant was not associated with PFS in this cohort. We identified a signature of 25 genes that is inversely associated with PFS on fulvestrant treatment (FDR 20%). When compared to other published datasets of breast cancer tumors, these genes are enriched in tumors with poor outcome and triple negative cancers. Pathway analysis revealed an association between activation of the EGFR pathway and decreased PFS (P=0.01). PAM50 subtypes varied with the luminal subtype being the most common (65%) and were generally concordant with the clinical subtype. However, we did not detect a significant trend between PAM50 subtype or ROR score and PFS or overall survival. Conclusions: In this cohort of patients with early and de-novo metastatic disease we identified a gene signature in the primary tumors that is associated with decreased response to fulvestrant treatment in metastatic disease. This signature warrants further validation to determine it’s predictive value and potential to assist in treatment decision making for patients with HR+ metastatic disease. Citation Format: Rinath M Jeselsohn, William T Barry, Jin Zhao, Gilles Buchwalter, Cristina Guarducci, Ilenia Migliaccio, Chiara Biagioni, Martina Bonechi, Naomi Laing, Yuri Rukazenkov, Eric P Winer, Myles Brown, Angelo Di Leo, Luca Malorni. TransCONFIRM: The correlative analysis of breast tumors from patients with advanced hormone receptor positive disease identifies a genetic signature associated with decreased benefit from single agent fulvestrant [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S1-01.

Abstract 5238: Integrative analysis of functional long noncoding RNAs during prostate cancer progression

Abstract Prostate cancer progression from an androgen-dependent state to an androgen-independent phase, which is also referred to castration-resistant prostate cancer (CRPC), not only results in cancer refractory to androgen deprivation therapy but also marks its lethal prognosis. However, it remains largely unknown that how CRPC develops. As a result, ideal diagnosis and therapeutic tools are still highly in demand and under investigation. Recently, many studies indicate that long non-coding RNA (lncRNA) can be extensively functional rather than just transcriptional noise and lncRNAs are also widely involved in the pathogenesis of different types of cancers. To identify and characterize functional lncRNAs during prostate cancer progression, we firstly developed an integrative computational strategy for predicting lncRNAs that may be functionally important for tumorigenesis or tumor suppression via analysis of lncRNA expression profiles, clinical information and somatic genomic alteration profiles of tumor samples. By applying this approach, we predicted a cohort of lncRNAs that may be clinically important for prostate tumorigenesis and experimentally confirmed two functional lncRNAs - PCAT6 and PCAT7 in prostate cancer cell line model. Work from others and our lab have also shown that both androgen receptor (AR) and polycomb group protein enhancer of zest 2 (EZH2) play pivotal roles during prostate cancer progression to CRPC. In contrast to protein coding genes, little is known about AR or EZH2 regulated lncRNAs during this process. To this purpose, we also performed RNAseq in prostate cancer cell lines and combined ChIPseq data to identify androgen-regulated and AR/EZH2-regulated lncRNAs. These regulated lncRNAs are also intersected with above clinically significant lncRNA list to narrow down interesting hits for follow-up mechanistic studies. Our integrative approach has successfully identified a cohort of potentially functional lncRNAs which hold great potentials to be novel prognostic biomarkers and/or therapeutic targets for treatment of advanced prostate cancer. Citation Format: Teng Fei, Yiwen Chen, Zhou Du, Xiaole Shirley Liu, Myles Brown. Integrative analysis of functional long noncoding RNAs during prostate cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5238. doi:10.1158/1538-7445.AM2014-5238

Abstract 1289: Breast cancer predisposition in multiple endocrine neoplasia type 1

Abstract Recent genetic and animal studies have suggested that the Multiple Endocrine Neoplasia type 1 (MEN1) gene is involved in breast carcinogenesis. In addition, the MEN1 gene product menin is a regulator of estrogen receptor alpha function in vitro. In this retrospective study in two independent cohorts we aimed to investigate the prevalence of breast cancer in female MEN1 patients and to characterize MEN1-related breast tumors. The Dutch MEN1 cohort contains data from 190 female MEN1 patients from 1990-2011. The second cohort from the U.S. has data from 68 female patients from 2004-2013. Observations were matched with demographic and cancer incidence and prevalence information from the general Dutch and U.S. populations. Breast cancer tissues from MEN1 patients were analyzed immunohistochemically and loss of heterozygosity (LOH) at the MEN1 gene locus was assessed. Females with MEN1 were found to be at increased risk to develop breast cancer (relative risks 2.83 and 3.20). Eighty percent of MEN1-related breast tumors showed loss of expression of menin and in a subset also loss of heterozygosity at the MEN1 gene locus was found. MEN1 is a breast cancer susceptibility syndrome. Further research will determine the relevance of the MEN1 gene for patients with non-familial breast cancer. Intensified breast cancer screening should be considered for female MEN1 patients. Citation Format: Koen Dreijerink, Elfi Conemans, Chantal Cornelissen, Marja Van Blokland, Michael Mannstadt, Carla Pieterman, Wouter De Herder, Bas Havekes, Eric Fliers, Ad Hermus, Menno Vriens, Marc Timmers, Elsken Van der Wall, Myles Brown, Rob Van der Luijt, Paul van Diest, Gerlof Valk. Breast cancer predisposition in multiple endocrine neoplasia type 1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1289. doi:10.1158/1538-7445.AM2014-1289

Abstract LB-275: Adult body size and physical activity in relation to risk of breast cancer defined by tumor androgen receptor status

Abstract Background: In contrast to the well-established role of estrogens in breast cancer risk and survival, the role of androgens in breast carcinogenesis is unclear. Previous studies have shown that, in postmenopausal women, higher body mass index (BMI) and being less physically active are associated with an increased risk of invasive breast cancer, and the associations generally are stronger for estrogen receptor (ER) and/or progesterone receptor (PR) positive tumors. Although the androgen pathway also may play a mechanistic role with these breast cancer risk factors, no prior studies have specifically evaluated these associations by tumor androgen receptor (AR) status. Observing differential associations by AR status would provide etiologic insights into the contribution of the androgen pathway in breast carcinogenesis. Methods: We evaluated the associations of adult body size and physical activity with risk of incident invasive breast cancer by AR status in the Nurses’ Health Study. We used validated questionnaires to assess height, weight, and physical activity. AR status was determined using immunohistochemistry on sections from tumor tissue microarrays (TMAs); ER and PR status were determined using TMAs or medical record pathology reports. We applied a constrained competing risk survival model to evaluate these associations by AR status while controlling for ER/PR status and established breast cancer risk factors. Results: A total of 2,198 cases (1,701 AR+; 497 AR- tumors) were documented during 26 years of follow-up of 96,966 eligible women. After adjusting for ER/PR status and other breast cancer risk factors, higher BMI was associated with an increased risk of both AR+ and AR- tumors. The multivariable hazard ratios (MV HRs) and 95% confidence intervals (95%CIs) for every 5 kg/m2 increase in BMI were 1.05 (0.99, 1.10) for AR+ and 1.10 (1.00, 1.22) for AR- tumors (p-value for heterogeneity=0.46). Further, women engaged in higher amounts of total physical activity including brisk walking were at a lower risk of developing breast cancer. The MV HRs (95%CIs) per approximately 5 hours of brisk walking per week were 0.87 (0.72, 1.05) for AR+ and 0.69 (0.46, 1.04) for AR- tumors (p-value for heterogeneity=0.32). We further examined these associations by the combinations of ER/PR/AR status and observed that only BMI associations differed significantly across these subtypes (p-value for heterogeneity=0.03). The MV HRs (95%CIs) for 5 kg/m2 increase in BMI were 1.15 (1.08, 1.23) for ER+PR+AR+, 1.23 (1.04, 1.45) for ER+PR+AR-, 0.88 (0.75, 1.03) for ER+PR-AR+, 1.08 (0.92, 1.28) for ER-PR-AR+, 1.19 (1.01, 1.39) for ER-PR-AR-, 0.92 (0.70,1.22) for ER+PR-AR-. Conclusion: In postmenopausal women, higher adult body size was associated with an increased risk of both AR+ and AR- tumors while physical activity, including brisk walking, was associated with a reduced risk of both subtypes. The strongest associations observed with the ER+PR+AR- and ER+PR+AR+ tumors (i.e., subtypes with the most active estrogen signaling) supports the important role of estrogen signaling in the postmenopausal BMI/breast cancer relationship. Further, the positive association observed between BMI and ER-PR-AR- tumors suggests pathways other than sex steroids (e.g., the insulin pathway) also might play an etiologic role and more studies are warranted to confirm and extend these findings. Citation Format: Xuehong Zhang, A. Heather Eliassen, Rulla Tamimi, Aditi Hazra, Andrew H. Beck, Myles Brown, Laura C. Collins, Bernard Rosner, Susan E. Hankinson. Adult body size and physical activity in relation to risk of breast cancer defined by tumor androgen receptor status. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-275. doi:10.1158/1538-7445.AM2014-LB-275

Abstract IA15: Epigenetics of hormone dependence

Abstract A detailed understanding of interplay between genetics and epigenetics in the progression and hormone dependence of distinct cancer subtypes is critical to the development of new approaches to prevention and therapy. Steroid hormones and their receptors play essential roles in the development and progression of both breast and prostate cancers. It remains unknown what role an epigenetically defined cell of origin plays in determining hormone responsiveness. In addition whether acquired resistance to endocrine therapy is the result of clonal evolution or the presence of hormone independent tumor-initiating cells, or both remains to be determined. Gain of function mutations and gene amplification of the androgen receptor (AR) in prostate cancer and the estrogen receptor (ER) in breast cancer in a subset of hormone refractory cases supports clonal evolution as the dominant mechanism of resistance in in these patients. We have investigated how the pathways downstream of AR and ER are activated in cases where they are not genetically altered and whether these depend on mutations in other epigenetic regulators including other transcription factors involved in steroid receptor networks and chromatin modifying enzymes involved in transcriptional regulation mediated by these receptors. Renewed success in targeting the steroid receptors themselves and promising advances in inhibiting the activity of the chromatin modifying enzymes provides new opportunities for the treatment of patients with breast and prostate cancer. Citation Format: Myles Brown. Epigenetics of hormone dependence. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr IA15.

Abstract A46: LSD1 globally mediates epigenetic modifications on androgen receptor-dependent enhancers

Abstract Androgen receptor (AR) belongs to nuclear receptor superfamily and plays pivotal roles in development of prostate and prostate cancer. The lignded AR binds to AR responsive elements (ARE) on the enhancers and mediates the transcription activity. AR binding on chromatin appears to depend on the binding of pioneer factor FOXA1, which recognizes the active sites marked with high level of mono- or di-methylation of Histon 3 lysine 4 (H3K4me1-2) and then make the site accessible for AR binding. However, the in depth mechanism how FOXA1 opens the enhancer and whether this results in further chromatin modification remains unclear. Through ChIP-seq studies coupled with gene expression array, we have found LSD1 (lysine specific demethylase 1) together with its binding partner CoRest were recruited by FOXA1 to the AR-dependent enhancer and its histone demethylation functions were then activated by liganded AR. This results in the loss of methylation marks and activation of gene transcription but not affecting AR binding or AR-mediated nucleosome dynamics. In contrast to this activator function, LSD1 can also formed corepressor complex to suppress the transcription of a subset of genes. This comprehensive study clearly suggested distinct roles of LSD1 as a coactivator versus a corepressor in different protein complexes therefore to mediate different cell functions. Citation Format: Changmeng Cai, Housheng Hansen He, Myles Brown, Steven P. Balk. LSD1 globally mediates epigenetic modifications on androgen receptor-dependent enhancers. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A46.

Abstract 2313: Differences in estrogen receptor signaling in normal mammary epithelial cells and ER-positive primary breast tumors and metastases.

Abstract Background: The estrogen receptor (ER) is expressed in ≈70% of sporadic breast cancer and activates genes driving cell proliferation and tumorigenesis. We have previously performed genome-wide analysis of ER binding sites in MCF-7 breast cancer cells and identified distinct mechanisms of tumorigenic ER signaling. Using EpCAM and CD49f as markers to enrich for ER-positive (ER+) cells obtained from primary non-malignant breast tissue, we seek to elucidate differences in ER signaling between normal and primary and metastatic ER+ breast cancer cells. Methods: Viable breast epithelial cells were obtained from patients undergoing reduction mammoplasties, and ER+ breast cancer cells from primary tumors and metastatic effusions. Following dissociation into single cells, EpCAM+ cell subpopulations were isolated and stimulated with estradiol. Gene expression microarray analysis, chromatin immunoprecipitation and DNA sequencing (ChIP-seq) on transcription factors and histone modifications as well as DNAse I hypersensitivity assays (DHS) were performed, and compared to MCF-7 breast cancer cells. Results: Triplicates of normal, ER+ breast cancers, and metastatic ER+ cancer were analyzed. Gene expression profiles revealed differences in estradiol regulated genes between primary normal, breast tumor, and metastatic ER+ breast cancer cells. Genes that promote cell cycling and cell proliferation were downregulated in non-malignant tissue but were upregulated in breast cancer cells. Our ChIP-seq results showed differential binding of ER between normal and ER+ breast cancer with little common overlap, and motif analysis of these binding sites demonstrated the enrichment of ERE motifs in common sites, TCF12 motifs in unique normal sites, and FOXA1 motifs in unique breast cancer sites. Analyses of the distribution of histone modifications and DHS regions demonstrated distinct patterns at shared, normal, and breast cancer ER binding sites, suggesting functionality and further validating differential ER binding. Conclusions: There are contrasting differences in ER signaling between normal mammary and ER+ breast cancer cells, with estrogen appearing to have anti-proliferative effects in normal luminal cells compared to pro-proliferative effects in BC. ER ChIP-seq has identified unique motifs, distribution of histone modifications, and DHS regions specific to unique normal, cancer and shared ER binding sites. Our studies point to TCF12 as a potential ER pioneer cofactor in non-malignant breast tissue and provide more data in support of FOXA1 as an important ER coregulator in ER+ breast cancer. Our data provides evidence for key alterations in ER-signaling during tumorigenesis and could lead to the identification of novel strategies to target breast cancer specific ER signaling. Citation Format: David Chi, Housheng He, Tony Yeung, Rinath Jeselsohn, Stuart Schnitt, Judy Garber, Andrea Richardson, Elgene Lim, Myles Brown. Differences in estrogen receptor signaling in normal mammary epithelial cells and ER-positive primary breast tumors and metastases. [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 2313. doi:10.1158/1538-7445.AM2013-2313

Abstract 3562: Genome-wide analysis of the vitamin D receptor (VDR) binding sites reveals vitamin D role modulating autophagy and metabolism in luminal-like breast cancer cells.

Abstract The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D3), regulates gene expression through the vitamin D receptor (VDR). Even though 1,25D3 is still best known as a regulator of bodily calcium homeostasis, 1,25D3 has a plethora of physiological actions which include immune and nervous system modulation, cellular proliferation and differentiation. Epidemiological and model studies have revealed the cancer-protective properties of 1,25D3 in colon, prostate and notably in breast. We are actively investigating the anti-proliferative properties of 1,25D3 in a panel of human breast cancer cell lines. We found that 1,25D3 induces autophagy in several luminal-like breast cancer cell models, but not in basal-like or mesenchymal-like models. We performed VDR and signaling partner RXR whole-genome ChIP-seq analysis to uncover all of the direct 1,25D3 target genes involved in regulation of metabolism and autophagy. Our studies suggest that energy regulation as an important aspect of 1,25D3 anti-proliferative properties in luminal-like breast cancer cells. Citation Format: Luz E. Tavera, Thomas Westerling, Myles Brown. Genome-wide analysis of the vitamin D receptor (VDR) binding sites reveals vitamin D role modulating autophagy and metabolism in luminal-like 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 3562. doi:10.1158/1538-7445.AM2013-3562

Abstract B12: LSD1 mediates global AR transcription suppression in prostate cancer cells

Abstract This project is to study the molecular basis of LSD1-dependent AR suppression in prostate cancer cells. Androgen receptor (AR) is highly expressed in prostate cancer (PCa) and plays a pivotal role in tumor growth. Patients generally respond to androgen deprivation therapy (ADT). However, the tumors invariably relapse, and these relapsed tumors (called castration-resistant prostate cancer, CRPC) are generally more aggressive and relatively resistant to current AR antagonist treatments. Significantly, these relapsed tumors express increased levels of AR mRNA and express multiple AR regulated genes, indicating that AR transcriptional activity has been restored. Despite the critical role AR plays in PCa development and progression to CRPC, the mechanisms that contribute to the consistent substantial increases in AR mRNA in CRPC are poorly understood. Using a VCaP xenograft model, we initially detected a rapid and substantial upregulation of AR mRNA upon castration, suggesting a negative feedback loop regulating AR mRNA levels, which may make a significant contribution to increasing AR mRNA in CRPC (Cancer Research 2009, 69:6027). In a recent published study (Cancer Cell 2011, 20:457), we reported that the agonist liganded AR binds to an enhancer in the second intron of the AR gene, and that increased AR gene expression and progression to CRPC is associated with increased H3K4 methylation and increased recruitment of other transcription factors to this site. Significantly, in contrast to AR function as a transcriptional activator on previously studied androgen responsive elements (AREs), we found that the agonist liganded AR functions as a direct transcriptional repressor at this site. The repression is due to AR recruitment of an H3K4 demethylase, LSD1. Interestingly, AR binding to this site and repression of AR gene expression occurs at slightly higher androgen levels than those required for classical androgen regulated genes, consistent with a negative feedback loop to regulate AR signaling. Pathway analyses of additional AR repressed genes showed marked enrichment for genes mediating DNA synthesis and cell cycle progression, while AR stimulated genes were associated with lipid/protein synthesis and cellular metabolism. Significantly, a set of these androgen repressed genes that are associated with proliferation are overexpressed in CRPC. Taken together, these results indicate that the agonist liganded AR functions as a transcriptional repressor on a subset of genes that enhance proliferation. We propose that ADT is effective because it initially suppresses all AR functions, but that the partial restoration of androgen levels and AR activity in CRPC cells may provide a strong growth advantage by stimulating cellular metabolism without downregulation of AR repressed genes that enhance cellular proliferation. Combining the expression array data and ChIP-seq analysis on AR in VCaP and VCaP-derived castration-resistant VCS2 cells, we have identified gene subsets with AR direct binding on the gene locus. Interestingly, while AR binding is highly enriched (∼2fold) in AR-activated gene subset, the binding is less enriched (1.4fold) in AR-suppressed gene subset. The lower enrichment could mean that fewer genes in the AR –repressed group are directly regulated by AR, but could also be in part technical and reflect somewhat weaker binding of AR to AR-repressed genes. To further elucidate the molecular basis for AR suppression function, we focused on the role of LSD1 in the global AR transcriptional repression as previous results showed that expressions of a number of AR-suppressed genes are dependent on LSD1 activity. LSD1 was initially identified in corepressor complexes and shown to function by demethylating mono- and dimethylated H3K4. However, it was subsequently shown to also function as a coactivator through demethylation of repressive mono- and dimethylated H3K9 when associated with AR. Our data indicate that the association with AR does not determine the coactivator versus corepressor function of LSD1, and that it is instead determined by properties of the element to which it is being recruited. Through genome wide ChIP-seq analysis of LSD1 in VCaP and LNCaP cells, we found that LSD1 binding is significantly enriched for both AR-activated and –repressed (both ∼1.5fold) genes, suggesting that both AR activation and repression are dependent on LSD1 activity. Furthermore, we searched the transcription factor-binding motif on LSD1 binding sites. While E2F1 is highly enriched for LSD1 binding in both activation and repression loci, the ZBTB (zinc finger and BTB domain containing) transcription repressor binding motif was only significantly enriched in repression loci. Among this family, PLZF and LRF are highly expressed in prostate cancer cells. Therefore, the current work has focused on these two factors and we propose that LSD1 mediates AR gene suppression through interaction with ZBTB transcription repressors. Citation Format: Changmeng Cai, Housheng He, Sen Chen, X. Shirley Liu, Myles Brown, Steven P. Balk. LSD1 mediates global AR transcription suppression in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B12.

Abstract IA8: Mechanisms regulating distinct AR transcriptional programs in PCa

Abstract Androgen receptor (AR) plays a central role in prostate cancer (PCa), and androgen deprivation is still the standard systemic therapy used for metastatic disease, but patients invariably relapse with a more aggressive form of PCa (castration resistant/recurrent PCa, CRPC). Studies in clinical samples and xenograft models indicate that increased AR gene expression plays a major role in the progression to CRPC. Previous studies showed that the agonist-liganded AR could suppress AR gene expression, so that AR mRNA levels increase acutely after castration, but the molecular basis for this negative regulation was not known. We have found that the agonist-liganded AR represses AR gene transcription through binding to a conserved site in the second intron of the AR gene (AR binding site 2, ARBS2). In the absence of androgen, ARBS2 is associated with FOXA1, OCT1, GATA2, and H3K4 mono- and dimethylation that are further increased in cells adapted to androgen deprivation, consistent with this site functioning as an enhancer that contributes to increased AR gene expression in CRPC. Significantly, the agonist liganded AR recruits lysine specific demethylase 1 (LSD1) to ARBS2, which then represses AR gene transcription through demethylation of the enhancer associated H3K4me1,2 marks. In contrast to this LSD1 mediated H3K4 demethylation and transcriptional repression, LSD1 associated with AR stimulated genes functions as a coactivator through demethylation of repressive H3K9me1,2 marks. The molecular basis for these distinct functions of LSD1 on AR stimulated versus AR repressed may be related to histone phosphorylation, but this or other mechanisms remain to be established and are under investigation. The rapid androgen mediated downregulation of the androgen synthetic enzymes AKR1C3 and HSD17B6 is similarly LSD1 dependent, indicating that the agonist liganded AR directly mediates a physiological intracellular negative feedback loop to regulate AR activity. Further analysis of gene expression in androgen starved versus androgen stimulated PCa cells revealed LSD1 dependent androgen repression of multiple genes mediating DNA synthesis and cell cycle progression, versus androgen stimulated expression of genes mediating synthesis of lipids, amino acids, and other metabolic processes. This profile is consistent with AR function in normal prostate epithelium to drive terminal differentiation and synthesis of seminal fluid, and may provide a molecular basis for the biphasic response to androgen stimulation whereby PCa cells proliferate in response to low levels of androgen but are growth arrested at high concentrations. Significantly, expression of AR repressed genes is generally increased in CRPC, indicating that an unintended consequence of androgen deprivation may be to increase expression of a subset of genes driving tumor growth. Taken together, these findings elucidate a mechanism that contributes to increased AR gene expression and restored AR activity in CRPC, and identify a suppressor element and transcriptional repressor function for the agonist liganded AR. The distinct mechanisms of AR and LSD1 action on enhancer versus suppressor elements may make it possible to selectively augment AR transcriptional repressor function and thereby prevent or delay the emergence of CRPC. Citation Format: Changmeng Cai, Housheng He, Myles Brown, Steven Balk. Mechanisms regulating distinct AR transcriptional programs in PCa [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr IA8.

Abstract B23: Identification of TMPRSS2:ERG-dependent regulators of AR function and differentiation status in prostate cancer

Abstract Oncogenic translocations involve the chromosomal rearrangement and fusion of two genes, which can result in either deregulated gene expression or the generation of a chimeric protein with unique oncogenic properties. Among these, fusions of the androgen-activated gene TMPRSS2 to the ETS transcription factor ERG occur in 40%–80% of prostate cancers and result in elevated expression of the ERG gene. Although a number of studies demonstrate the importance of ERG as a driver for prostate cancer development, the molecular mechanisms of ERG-mediated oncogenesis remain elusive. A deeper understanding of how ERG disrupts normal prostate biology may ultimately assist in the development of therapies that selectively target TMPRSS2:ERG -positive prostate cancer. We provide evidence that a primary role of ERG in prostate cancer is to specifically disrupt the normal regulation of androgen receptor (AR) signaling, resulting in a selective attenuation of AR's ability to promote prostate epithelial cell differentiation. We are testing the hypothesis that ERG proteins require recruitment of additional cofactors to mediate these AR regulatory functions, and that some of these protein partners may be valid therapeutic targets. Towards this end, we are working to elucidate the full network of ERG-interacting proteins, and validating which of these interactors are necessary and/or sufficient for the oncogenic and de-differentiation functions of ERG in prostate cancer. Citation Format: Zineb Mounir, Raymond Pagliarini, Fallon Lin, Markus Schirle, Justin Klekota, Yan Feng, Anke Hartung, Aron Jaffe, Gilles Buchwalter, Myles Brown. Identification of TMPRSS2:ERG-dependent regulators of AR function and differentiation status in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B23.

Thematic Minireview Series on Nuclear Receptors in Biology and Diseases

Thematic Minireview Series on Nuclear Receptors in Biology and Diseases