Citations

Abstract Background: Male breast cancer (MBC) is rare, and usually managed by extrapolation from female breast cancer. We report on the characteristics and survival outcomes of MBC patients from Mayo Clinic Rochester (MCR). Methods: Medical records of MBC patients treated at MCR during a 25-year period (1990-2015) were reviewed. Demographic variables, tumor characteristics, recurrences, and overall survival (OS) were collected. Progression free survival (PFS) and OS were estimated by the Kaplan-Meier method. Multivariate Cox-proportional hazard regression was used to identify predictors of OS. Results: One hundred sixty-seven patients were included in the final analysis, with a median follow-up of 58 months after diagnosis. Baseline characteristics are presented in Table 1. Eighty percent of patients with ER-positive tumors received endocrine therapy. Among men with stage I-III disease, approximately 90% underwent mastectomy, and 44% received adjuvant chemotherapy. The 5-year locoregional and distant recurrence rates for patients with stage I-III disease were 4.4% and 21.5%, respectively. The 5-year PFS and OS for patients with stage I-III disease were 65.5% and 80.1%, respectively. In a multivariate analysis assessing predictors of OS in patients with stage I-III disease, older age (HR 1.05; 95% CI: 1.02 – 1.09), stage II (HR 11.06; 95% CI: 3.84 – 31.85) or stage III disease (HR 14.74; 95% CI (3.99 – 54.45), and omission of surgery (HR 45.33; 95% CI: 3.97 – 517.32) were associated with poorer OS, while endocrine therapy (HR 0.21, 95% CI: 0.09 – 0.51) was associated with better OS. ER, PR, HER2 and grade were not independently prognostic. The median OS for stage IV patients was 10 months, though this 11-man cohort was too small to allow assessment of prognostic factors in advanced male breast cancer. Conclusions: MBC remains an understudied condition. Prognostic factors in this stage I-III disease are consistent with those identified in other MBC retrospective cohorts. Prospective studies are needed to better understand the unique clinical features of MBC, and to improve outcomes, particularly for advanced disease. Table 1:Baseline characteristics N=167 Median age at diagnosis (Years)64.4 Ethnicity/Race: Caucasian131 (78.4%)African American4 (2.4%)Other or unknown32 (19.2%) Overall AJCC 7th edition stage: Stage I39 (23.4%)Stage II80 (47.9%)Stage III32 (19.2%)Stage IV11 (6.6%)Unknown5 (3.0%) Grade: 18 (4.8%)247 (28.1%)3101 (60.5%)Unknown12 (7.1%) ER status: Negative8 (4.8%)Positive153 (91.6%)Unknown6 (3.6%) PR status: Negative17 (10.2%)Positive141 (84.4%)Unknown9 (5.4%) HER-2 status: Negative70 (41.9%)Positive12 (7.2%)Unknown85 (50.9%) Citation Format: Yadav S, Leon-Ferre RA, Jimenez RE, Hawse JR, Hieken TJ, Couch FJ, Boughey JC, Ruddy KJ. Clinical characteristics and survival of patients with male breast cancer: The Mayo Clinic experience [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 P6-19-05.

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 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.

Significance Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer and patients exhibit high rates of recurrence and mortality in part due to lack of treatment options beyond standard-of-care chemotherapy regimens. In the subset of TNBCs that express estrogen receptor beta (ERβ), ligand-mediated activation of ERβ elicits potent anticancer effects. We report here the elucidation of the ERβ cistrome and transcriptome in TNBC and identify a mechanism whereby ERβ induces cystatin gene expression resulting in inhibition of canonical TGFβ signaling and a blockade of metastatic phenotypes. These findings suggest that ERβ-targeted therapies represent a treatment option for the subset of women with ERβ-expressing TNBC.

1043 Background: In estrogen receptor (ER) positive MBC, mutations (e.g. ESR1), identified from tumor biopsies or cfDNA, confer resistance. The concordance between mutations observed in tumor and cfDNA and the implications for response to Z-endoxifen, a potent anti-estrogen, are unknown. Methods: We previously conducted a phase I trial of Z-endoxifen in endocrine refractory, ER positive MBC. Seven dose levels were considered ranging from 20 to 160 mg/day followed by expansion cohorts (EC) of 40, 80, and 100 mg/day. Pretreatment blood samples (all pts) and fresh tumor biopsies (EC) were collected prospectively. Tumor and cfDNA were evaluated by targeted NGS. Results: 41 pts (38 evaluable) were enrolled. Prior endocrine therapy included aromatase inhibitors (37/38, 97%), fulvestrant (22/38, 58%) and tamoxifen (26/38, 68%). Substantial endoxifen exposure without DLTs at doses above 80 mg/day led to a halt in dose escalation and opening of the EC. Overall clinical benefit (stable > 6 months [7 pts.] or partial response by RECIST criteria [3 pts.]) rate was 26.3% (95%CI: 13.4-43.1%). cfDNA was obtained from 36 pts and mutations were identified in 13 (36%) including ESR1 [ Y537N or D538G] (5), PIK3CA [ H1047R or E542K] (8), TP53 [ K132R, R248Q, R267Q, or H179Y] (4), AKT ( Q79K) (1), and KRAS ( G12D) (1). In 5 pts with cf ESR1 mutations, 4 had additional cfDNA mutations including PIK3CA (3), and TP53 (1). PFS was shorter in pts with cfDNA mutations relative to those without (median: 61 vs.132 days; log-rank p = 0.021). Discordance was observed between tumor and cfDNA mutations where 3/7 PIK3CA tumor mutations were detected by cfDNA, 1/2 TP53 tumor mutations were detected by cfDNA, and 0/1 AKT tumor mutations were detected by cfDNA. Conversely, 2 pts had cfDNA mutations (either ESR1, TP53 or AKT) undetected in tumor. Conclusions: The absence of cfDNA mutations in patients with endocrine resistant, MBC treated with Z-Endoxifen was associated with significantly longer PFS. Given the discordance between tumor and cfDNA sequence data, future studies must determine which approach maximizes prognosis and prediction of benefit for estrogen-targeted therapy. Clinical trial information: NCT01327781.

ABSTRACT There is a clinical need to identify new molecular targets for the treatment of osteoporosis, particularly those that simultaneously inhibit bone resorption while stimulating bone formation. We have previously shown in overexpression studies that retinoic acid receptor-related orphan receptor β (Rorβ) suppresses in vitro osteoblast differentiation. In addition, the expression of Rorβ is markedly increased in bone marrow–derived mesenchymal stromal cells with aging in both mice and humans. Here we establish a critical role for Rorβ in regulating bone metabolism using a combination of in vitro and in vivo studies. We used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing to demonstrate that loss of Rorβ in osteoblasts enhances Wnt signaling, specifically through increased recruitment of β-catenin to T-cell factor/lymphoid enhancer factor (Tcf/Lef) DNA binding sites in the promoters of the Wnt target genes Tcf7 and Opg. This resulted in increased osteogenic gene expression and suppressed osteoclast formation through increased osteoprotegerin (OPG) secretion in Rorβ-deficient cells. Consistent with our in vitro data, genetic deletion of Rorβ in both female and male mice resulted in preserved bone mass and microarchitecture with advancing age due to increased bone formation with a concomitant decrease in resorption. The improved skeletal phenotype in the Rorβ–/– mice was also associated with increased bone protein levels of TCF7 and OPG. These data demonstrate that loss of Rorβ has beneficial skeletal effects by increasing bone formation and decreasing bone resorption, at least in part through β-catenin–dependent activation of the Wnt pathway. Thus, inhibition of Rorβ represents a novel approach to potentially prevent or reverse osteoporosis. © 2017 American Society for Bone and Mineral Research.

TIEG knockout (KO) mice exhibit a female‐specific osteopenic phenotype and altered expression of TIEG in humans is associated with osteoporosis. Gene expression profiling studies identified sclerostin as one of the most highly up‐regulated transcripts in the long bones of TIEG KO mice relative to WT littermates suggesting that TIEG may regulate SOST expression. TIEG was shown to substantially suppress SOST promoter activity and the regulatory elements through which TIEG functions were identified using promoter deletion and chromatin immunoprecipitation assays. Knockdown of TIEG in IDG‐SW3 osteocyte cells using shRNA and CRISPR‐Cas9 technology resulted in increased SOST expression and delayed mineralization, mimicking the results obtained from TIEG KO mouse bones. Given that TIEG is an estrogen regulated gene, and as changes in the hormonal milieu affect SOST expression, we performed ovariectomy (OVX) and estrogen replacement therapy (ERT) studies in WT and TIEG KO mice followed by miRNA and mRNA sequencing of cortical and trabecular compartments of femurs. SOST expression levels were considerably higher in cortical bone compared to trabecular bone. In cortical bone, SOST expression was increased following OVX only in WT mice and was suppressed following ERT in both genotypes. In contrast, SOST expression in trabecular bone was decreased following OVX and significantly increased following ERT. Interestingly, a number of miRNAs that are predicted to target sclerostin exhibited inverse expression levels in response to OVX and ERT. These data implicate important roles for TIEG and estrogen‐regulated miRNAs in modulating SOST expression in bone.

Abstract Human induced pluripotent stem cells (hiPSC) hold great promise in diagnostic and therapeutic applications. However, translation of hiPSC technology depends upon a means of assessing hiPSC quality that is quantitative, high-throughput, and can decipher malignant teratocarcinoma clones from normal cell lines. These attributes are lacking in current approaches such as detection of cell surface makers, RNA profiling, and/or teratoma formation assays. The latter remains the gold standard for assessing clone quality in hiPSCs, but is expensive, time-consuming, and incompatible with high-throughput platforms. Herein, we describe a novel method for determining hiPSC quality that exploits pluripotent cells’ documented hypersensitivity to the topoisomerase inhibitor etoposide (CAS No. 33419-42-0). Based on a study of 115 unique hiPSC clones, we established that a half maximal effective concentration (EC50) value of <300 nM following 24 hours of exposure to etoposide demonstrated a positive correlation with RNA profiles and colony morphology metrics associated with high quality hiPSC clones. Moreover, our etoposide sensitivity assay (ESA) detected differences associated with culture maintenance, and successfully distinguished malignant from normal pluripotent clones independent of cellular morphology. Overall, the ESA provides a simple, straightforward method to establish hiPSC quality in a quantitative and functional assay capable of being incorporated into a generalized method for establishing a quality control standard for all types of pluripotent stem cells.