Citations

Abstract Background: Triple Negative Breast Cancer (TNBC) affects approximately 15-20% of BC patients, yet accounts for a disproportionately higher rate of BC morbidity and mortality, in part due to lack of targeted therapies. Using well-validated antibodies, Estrogen Receptor Beta (ERβ) protein has been shown to be expressed in approximately 25% of TNBCs and is associated with improved patient outcomes. Using multiple ERβ +/- TNBC cell lines and PDX models, we have demonstrated that ligand-mediated activation of ERβ by estradiol (E2) decreases cell proliferation, invasion, and migration in vitro, as well as primary tumor growth and metastatic spread in vivo. Methods: To determine the mechanisms by which ERβ elicits these anti-cancer effects, we elucidated the ERβ transcriptome and cistrome via Microarray and ChIPseq, respectively, in TNBC cells stably expressing ERβ in a doxycycline-inducible manner. We also performed gene expression and luciferase assays to assess the impact of ERβ on NFκB signaling, followed by ChIP-PCR and ChIPseq to assess how ERβ modifies chromatin architecture near NFκB target genes. Results: Pathway analysis of ERβ-regulated genes identified NFκB signaling as one of the most suppressed pathways in response to E2 treatment. Indeed, numerous NFκB target genes were among the most down-regulated genes following E2 treatment but only in the presence of ERβ expression. Chromatin Immunoprecipitation followed by sequencing (ChIPseq) revealed that ERβ primarily associated with estrogen response elements (EREs), but was also enriched around NFκB binding sites following E2 treatment. In fact, 12% of all ERβ binding sites were enriched for NFκB response elements and ERβ was shown to physically associate with NFκB protein. Using an NFκB reporter construct and qPCR, ERβ was shown to block TNFα-mediated induction of NFκB signaling and NFκB target gene expression. Globally, RNAseq identified 200 genes to be significantly regulated by TNFα in TNBC cells, of which 81 were significantly altered in the presence of E2+TNFα. ChIPseq demonstrated that ligand-mediated activation of ERβ significantly diminished an activating histone mark (H3K27Ac) at many of these NFκB target genes while enhancing a repressive mark (H3K27Me3). These modifications are also associated with recruitment of the histone methyltransferase, EZH2, to enhancer elements of these NFκB target genes. Drug-mediated blockade of HDAC and EZH2 activity reversed suppression of NFκB target gene expression by ERβ. Conclusions: Our data suggest that ERβ may elicit its anti-cancer effects in part via formation of a novel co-repressor complex consisting of ERβ, NFκB, and EZH2. These data are in keeping with prior observations of the importance of NFκB signaling as it relates to TNBC cell proliferation and invasion, and that decreased expression of NFκB target genes is associated with improved outcomes in TNBC patients. Currently, a Mayo Breast SPORE prospective study is underway to investigate the role of estradiol in ERβ expressing TNBC and to further evaluate the cross-talk between ERβ and NFκB signaling in TNBC. Citation Format: Aspros K, Nelson A, Ye Z, Sun Z, Chernukhin I, Carroll J, Ingle J, Goetz M, Hawse J. Estrogen receptor beta elicits anti-cancer effects in triple negative breast cancer through suppression of NFκB signaling [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-03.

Abstract Purpose: Statins may mediate suppression of molecular pathways conferring benefit in cancer. Statins have shown anti-tumor effects in preclinical studies and have been associated with decreased recurrence and improved disease-specific survival. While designed to target cholesterol biosynthesis, statins can also have liver, bone and brain effects. We collected data on statin use in the S0307 adjuvant bisphosphonate trial to test the hypothesis that statin use may decrease risk of recurrence to liver, bone and brain as well as second primary (contralateral) breast cancers, and may act synergistically with bisphosphonates to decrease the risk of recurrence to bone. Patients and Methods: In S0307, 6097 patients diagnosed with Stage I-III breast cancer who had undergone surgery and were receiving adjuvant systemic therapy were randomized to receive zoledronic acid, clodronate, or ibandronate for 3 years. No significant difference was found in disease-free survival (DFS) among the 3 groups, including a sub-analysis of patients > age 55. Statin use was infrequent in younger women in S0307, consequently we analyzed statin use in those > age 55. Cox proportional hazard models were used to determine which variables were independently associated with DFS and to estimate hazard ratios (HR) and 95% confidence intervals (CI). Results: Among women aged ≥ 55 years, 684 (27%) reported taking a statin at baseline and 1,848 did not. Both groups were similar in terms of hormone receptor and HER2 status (p = 0.82). Median age in the statin group was 64.3 versus 61.0 years in the no statin group, mean BMI 31.2 v. 29.5, mean tumor size 2.1cm v. 2.3cm, negative lymph nodes 60% v. 54%, Stage I disease 47% v. 36%, and receipt of chemotherapy 62% v. 71% (all p < 0.01). In the statin group, 122 (17.8%) experienced a DFS event compared to 313 (16.9%) in the no statin group (HR 1.18, CI 0.95-1.46). No difference was observed by statin use in overall recurrence (p=0.28), distant recurrence (p=0.64), or recurrences to the bone (p=0.64), liver (p=0.38) or brain (p=0.65) at initial recurrence. There was no synergy between statin use and specific bisphosphonates. Recurrence and statin useOutcomeGroup 1: On stan at baseline n=684Group 2: No statin at baseline n=1848DFS events122 (17.8%)313 (16.9%)Died without recurrence51 7.5%)97 (5.2%)Recurrence71 (10.4%)216 (11.7%)Contralateral breast cancer9 (1.3%)17 (0.9%)Distant recurrence48 (7%)157 (8.5%)Bone as 1st site of distant recurrence (% distant recurrence)31 (65%)76 (48%)Liver as 1st site of distant recurrence (% distant recurrence)6 (13%)24 (16%)Brain/CNS as 1st site of distant recurrence (% distant recurrence)5 (10%)17 (11%) Conclusions: We found no evidence that statins reduce risk of second primary breast cancers or distant metastases among post-menopausal women with early-stage breast cancer. Despite promising preclinical data, they did not appear to act in synergy with a specific bisphosphonate. Though women in the statin group had less advanced disease at study entry, statin use was not associated with improved DFS. Results are limited by lack of information about type of statin used, adherence, or initiation of statin in control group. Citation Format: Kizub D, Miao J, Stopeck A, Thompson P, Paterson AH, Clemons M, Dees EC, Ingle JN, Falkson CI, Barlow W, Hortobagyi GN, Gralow JR. Statin use, site of recurrence, and survival among post-menopausal women taking bisphosphonates as adjuvant therapy for breast cancer (SWOG S0307) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-17-03.

Abstract Background: Despite the prevalent treatment options for ERα-positive breast cancer patients, and their initial efficacy for many women, ERα-positive disease still accounts for more breast cancer related deaths than any other subtype. Relapse in these patients is largely due to the development of resistance to anti-estrogen therapies such as tamoxifen. While tamoxifen and its resistance mechanisms have been extensively studied from both the bench and the bedside, relatively little is known about its active metabolite endoxifen. Our group has provided evidence that endoxifen is the most potent and clinically relevant metabolite of tamoxifen, suggesting that its characterization may be crucial to understanding tamoxifen resistance. Methods: We have developed novel endoxifen resistant MCF7 and T47D cell lines through chronic exposure to endoxifen during a period of 12-24 months. Using these models and their respective controls, we compared global gene expression profiles of endoxifen resistant cells to tamoxifen resistant cells and found marked differences between the two models. Additionally, we subjected treatment naïve cells to a genome-wide, CRISPR-mediated knockout screen to identify genes, and their associated pathways, that are likely involved in mediating endoxifen resistance. Results: Analysis of CRISPR guide RNAs enriched or depleted in response to chronic endoxifen treatment revealed that disruption of genes regulated by dexamethasone (Dex), a potent glucocorticoid receptor (GR) agonist, enhanced cells' ability to survive and proliferate in the presence of endoxifen. These data suggest that GR activation may inhibit endoxifen resistance, and that treatment of resistant cells with Dex may restore endoxifen efficacy. Indeed, Dex treatment significantly inhibited the proliferation rates of endoxifen resistant cells by 50-60% with little to no inhibitory effects in endoxifen sensitive models. Further, Dex was shown to synergize with endoxifen in resistant cells to further suppress cell proliferation, implying that Dex treatment could be utilized as an effective therapy for endocrine resistant disease. Conditioned media harvested from cells chronically exposed to Dex also resulted in substantial inhibition of endoxifen resistant cell proliferation rates. To explore potential mechanisms of these effects, we performed RNA-seq on both treatment-naïve and endoxifen resistant cells following Dex treatment. Out of 246 genes significantly regulated by Dex in endoxifen resistant cells, we identified 61 genes that were not differentially regulated in treatment naïve cells. These genes may provide insights into the mechanisms of GR activity specific to endoxifen resistant cells. Conclusions: To our knowledge, we have developed the first models of endoxifen resistance and have demonstrated that global transcriptomic changes that occur during this process are substantially different than those observed in tamoxifen resistant models. We have shown that activation of GR signaling elicits significant growth-inhibitory effects specifically in the setting of endoxifen resistance. These data identify the GR pathway as a potential novel therapeutic target for the treatment of endocrine resistant breast cancer. Citation Format: Jones CJ, Goetz MP, Ingle JN, Hawse JR. Glucocorticoid receptor activation inhibits proliferation of endoxifen resistant breast cancer cells and resensitizes cells to hormonal therapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-05.

Anastrozole is a widely prescribed aromatase inhibitor for the therapy of estrogen receptor positive (ER+) breast cancer. We performed a genome‐wide association study (GWAS) for plasma anastrozole concentrations in 687 postmenopausal women with ER+ breast cancer. The top single‐nucleotide polymorphism (SNP) signal mapped across SLC38A7 (rs11648166, P = 2.3E‐08), which we showed to encode an anastrozole influx transporter. The second most significant signal (rs28845026, P = 5.4E‐08) mapped near ALPPL2 and displayed epistasis with the SLC38A7 signal. Both of these SNPs were cis expression quantitative trait loci (eQTL)s for these genes, and patients homozygous for variant genotypes for both SNPs had the highest drug concentrations, the highest SLC38A7 expression, and the lowest ALPPL2 expression. In summary, our GWAS identified a novel gene encoding an anastrozole transporter, SLC38A7, as well as epistatic interaction between SNPs in that gene and SNPs near ALPPL2 that influenced both the expression of the transporter and anastrozole plasma concentrations.