Introduction: The androgen receptor (AR) is widely expressed in breast cancers and has been proposed as a therapeutic target in estrogen receptor alpha (ER) negative breast cancers that retain AR. However, controversy exists regarding the role of AR, particularly in ER + tumors. Enzalutamide, an AR inhibitor that impairs nuclear localization of AR, was used to elucidate the role of AR in preclinical models of ER positive and negative breast cancer.

Purpose Anti-endocrine therapy remains the most effective treatment for ER+ breast cancer, but development of resistance is a major clinical complication. Effective targeting of mechanisms that control the loss of ER dependency in breast cancer remains elusive. We analyzed breast cancer-associated fibroblasts (CAFs), the largest component of the tumor microenvironment, as a factor contributing to ER expression levels and anti-endocrine resistance. Experimental Design Tissues from ER+ breast cancer patients were analyzed for the presence of CD146 positive (CD146pos) and CD146 negative (CD146neg) fibroblasts. ER dependent proliferation and tamoxifen sensitivity were evaluated in ER+ tumor cells co-cultured with CD146pos or CD146neg fibroblasts. RNAseq was used to develop a high confidence gene signature that predicts for disease recurrence in tamoxifen treated patients with ER+ breast cancer. Results We demonstrate that ER+ breast cancers contain two CAF subtypes defined by CD146 expression. CD146neg CAFs suppress ER expression in ER+ breast cancer cells, decrease tumor cell sensitivity to estrogen, and increase tumor cell resistance to tamoxifen therapy. Conversely, the presence of CD146pos CAFs maintains ER expression in ER+ breast cancer cells and sustains estrogen-dependent proliferation and sensitivity to tamoxifen. Conditioned media from CD146pos CAFs with tamoxifen-resistant breast cancer cells is sufficient to restore tamoxifen sensitivity. Gene expression profiles of patient breast tumors with predominantly CD146neg CAFs correlate with inferior clinical response to tamoxifen and worse patient outcomes. Conclusions Our data suggest that CAF composition contributes to treatment response and patient outcomes in ER+ breast cancer, and should be considered a target for drug development.

BackgroundTumor resistance to the selective estrogen receptor modulator tamoxifen remains a serious clinical problem especially in patients with tumors that also overexpress HER2. We have recently demonstrated that the clinically important isoform of HER2, HERΔ16, promotes therapeutically refractory breast cancer including resistance to endocrine therapy. Likewise additional breast tumor cell models of tamoxifen resistance have been developed that do not involve HER2 overexpression. However, a unifying molecular mechanism of tamoxifen resistance has remained elusive.ResultsHere we analyzed multiple cell models of tamoxifen resistance derived from MCF-7 cells to examine the influence of microRNAs (miRNAs) on tamoxifen resistance. We compared miRNA expression profiles of tamoxifen sensitive MCF-7 cells and tamoxifen resistant MCF-7/HER2Δ16 cells. We observed significant and dramatic downregulation of miR-342 in the MCF-7/HER2Δ16 cell line as well as the HER2 negative but tamoxifen resistant MCF-7 variants TAMR1 and LCC2. Restoring miR-342 expression in the MCF-7/HER2Δ16 and TAMR1 cell lines sensitized these cells to tamoxifen-induced apoptosis with a dramatic reduction in cell growth. Expression of miR-342 was also reduced in a panel of tamoxifen refractory human breast tumors, underscoring the potential clinical importance of miR-342 downregulation. Towards the goal of identifying direct and indirect targets of miR-342 we restored miR-342 expression in MCF-7/HER2Δ16 cells and analyzed changes in global gene expression by microarray. The impact of miR-342 on gene expression in MCF-7/HER2Δ16 cells was not limited to miR-342 in silica predicted targets. Ingenuity Pathways Analysis of the dataset revealed a significant influence of miR-342 on multiple tumor cell cycle regulators.ConclusionsOur findings suggest that miR-342 regulates tamoxifen response in breast tumor cell lines and our clinical data indicates a trend towards reduced miR-342 expression and tamoxifen resistance. In addition, our results suggest that miR-342 regulates expression of genes involved in tamoxifen mediated tumor cell apoptosis and cell cycle progression. Restoring miR-342 expression may represent a novel therapeutic approach to sensitizing and suppressing the growth of tamoxifen refractory breast tumors.

To identify microRNAs (miRNAs) associated with estrogen receptor (ESR1) status, we profiled luminal A, ESR1+ breast cancer cell lines versus triple negative (TN), which lack ERα, progesterone receptor and Her2/neu. Although two-thirds of the differentially expressed miRNAs are higher in ESR1+ breast cancer cells, some miRNAs, such as miR-222/221 and miR-29a, are dramatically higher in ESR1− cells (~100 and 16 fold higher, respectively). MiR-222/221 (which target ESR1 itself) and miR-29a are predicted to target the 3' UTR of Dicer1. Addition of these miRNAs to ESR1+ cells reduces Dicer protein, whereas antagonizing miR-222 in ESR1− cells increases Dicer protein. We demonstrate via luciferase reporter assays, that these miRNA directly target the Dicer1 3’UTR. In contrast, miR-200c, which promotes an epithelial phenotype, is 58 fold higher in the more well-differentiated ERα+ cells and restoration of miR-200c to ERα− cells causes increased Dicer protein, resulting in increased levels of other mature miRNAs typically low in ESR1− cells. Together our findings explain why Dicer is low in ERα negative breast cancers, since such cells express high miR-221/222 and miR-29a levels (which repress Dicer) and low miR-200c (which positively affect Dicer levels). Furthermore, we find that miR-7, which is more abundant in ERα cells and is estrogen regulated, targets growth factor receptors and signaling intermediates such as EGFR, IGF1R and IRS-2. In summary, miRNAs differentially expressed in ERα+ versus ERα− breast cancers actively control some of the most distinguishing characteristics of the luminal A and TN subtypes, such as ESR1 itself, Dicer and growth factor receptor levels.

Tamoxifen is the most commonly prescribed therapy for patients with estrogen receptor (ER)α-positive breast tumors. Tumor resistance to tamoxifen remains a serious clinical problem especially in patients with tumors that also overexpress human epidermal growth factor receptor 2 (HER2). Current preclinical models of HER2 overexpression fail to recapitulate the clinical spectrum of endocrine resistance associated with HER2/ER-positive tumors. Here, we show that ectopic expression of a clinically important oncogenic isoform of HER2, HER2Δ16, which is expressed in >30% of ER-positive breast tumors, promotes tamoxifen resistance and estrogen independence of MCF-7 xenografts. MCF-7/HER2Δ16 cells evade tamoxifen through upregulation of BCL-2, whereas mediated suppression of BCL-2 expression or treatment of MCF-7/HER2Δ16 cells with the BCL-2 family pharmacological inhibitor ABT-737 restores tamoxifen sensitivity. Tamoxifen-resistant MCF-7/HER2Δ16 cells upregulate BCL-2 protein levels in response to suppressed ERα signaling mediated by estrogen withdrawal, tamoxifen treatment or fulvestrant treatment. In addition, HER2Δ16 expression results in suppression of BCL-2-targeting microRNAs miR-15a and miR-16. Reintroduction of miR-15a/16 reduced tamoxifen-induced BCL-2 expression and sensitized MCF-7/HER2Δ16 to tamoxifen. Conversely, inhibition of miR-15a/16 in tamoxifen-sensitive cells activated BCL-2 expression and promoted tamoxifen resistance. Our results suggest that HER2Δ16 expression promotes endocrine-resistant HER2/ERα-positive breast tumors and in contrast to wild-type HER2, preclinical models of HER2Δ16 overexpression recapitulate multiple phenotypes of endocrine-resistant human breast tumors. The mechanism of HER2Δ16 therapeutic evasion, involving tamoxifen-induced upregulation of BCL-2 and suppression of miR-15a/16, provides a template for unique therapeutic interventions combining tamoxifen with modulation of microRNAs and/or ABT-737-mediated BCL-2 inhibition and apoptosis.

The female hormone progesterone (P4) promotes the expansion of stem-like cancer cells in estrogen receptor (ER) and progesterone receptor (PR) positive breast tumors. The expanded tumor cells lose expression of ER and PR, express the tumor-initiating marker CD44, the progenitor marker cytokeratin 5 (CK5), and are more resistant to standard endocrine and chemotherapies. The mechanisms underlying this hormone-stimulated reprogramming have remained largely unknown. In the present study, we investigated the role of microRNAs in progestin-mediated expansion of this dedifferentiated tumor cell population. We demonstrate that P4 rapidly downregulates miR-29 family members, particularly in the CD44+ cell population. Downregulation of miR-29 members potentiates the expansion of CK5+ and CD44+ cells in response to progestins, and results in increased stem-like properties in vitro and in vivo. We demonstrate that miR-29 directly targets Krüppel-like factor 4 (KLF4), a transcription factor required for the reprogramming of differentiated cells to pluripotent stem cells, and for the maintenance of breast cancer stem cells. These results reveal a novel mechanism whereby progestins increase the stem cell-like population in hormone-responsive breast cancers, by decreasing miR-29 to augment PR-mediated upregulation of KLF4. Elucidating the mechanisms whereby hormones mediate the expansion of stem-like cells furthers our understanding of the progression of hormone responsive breast cancers.

A therapeutic intervention that could decrease tumor burden and increase sensitivity to chemotherapy would have a significant impact on the high morbidity rate associated with ovarian cancer. MicroRNAs (miRNAs) have emerged as potential therapeutic candidates due to their ability to down regulate multiple targets involved in tumor progression and chemoresistance. MiR-200c is down regulated in ovarian cancer cell lines and stage III ovarian tumors, and low miR-200c correlates with poor prognosis. MiR-200c increases sensitivity to taxanes in vitro, by targeting TUBB3, a tubulin known to mediate chemoresistance. Indeed, we find that patients with tumors with low TUBB3 had significantly prolonged survival (average survival 52.73 ± 4.08 months) compared to those with high TUBB3 (average survival 42.56 ±3.19 months). MiR-200c also targets TrkB, a mediator of resistance to anoikis. We demonstrate that restoration of miR-200c to ovarian cancer cells results in increased anoikis sensitivity and reduced adherence to biological substrates in vitro. Since both chemo- and anoikis-resistance are critical steps in the progression of ovarian cancer, we sought to determine how restoration of miR-200c affects tumor burden and chemosensitivity in an in vivo preclinical model of ovarian cancer. Restoration of miR-200c in an intraperitoneal xenograft model of human ovarian cancer, results in decreased tumor formation and tumor burden. Furthermore, even in established tumors, restoration of miR-200c, alone or in combination with paclitaxel, results in significantly decreased tumor burden. Our study suggests that restoration of miR-200c immediately prior to cytotoxic chemotherapy may allow for a better response or lower effective dose.

Epithelial ovarian cancer (EOC) has one of the highest death to incidence ratios among all cancers. High grade serous ovarian carcinoma (HGSOC) is the most common and deadliest EOC histotype due to the lack of therapeutic options following debulking surgery and platinum/taxane‐based chemotherapies. For recurrent chemosensitive HGSOC, poly(ADP)‐ribose polymerase inhibitors (PARPi; olaparib, rucaparib, or niraparib) represent an emerging treatment strategy. While PARPi are most effective in homologous recombination DNA repair‐deficient (HRD) HGSOCs, recent studies have observed a significant benefit in non‐HRD HGSOCs. However, all HGSOC patients are likely to acquire resistance. Therefore, there is an urgent clinical need to understand PARPi resistance and to introduce novel combinatorial therapies to manage PARPi resistance and extend HGSOC disease‐free intervals. In a panel of HGSOC cell lines, we established matched olaparib sensitive and resistant cells. Transcriptome analysis of the matched olaparib‐sensitive vs ‐resistant cells revealed activation of the Wnt signaling pathway and consequently increased TCF transcriptional activity in PARPi‐resistant cells. Forced activation of canonical Wnt signaling in several PARPi‐sensitive cells via WNT3A reduced olaparib and rucaparib sensitivity. PARPi resistant cells were sensitive to inhibition of Wnt signaling using the FDA‐approved compound, pyrvinium pamoate, which has been shown to promote downregulation of β‐catenin. In both an HGSOC cell line and a patient‐derived xenograft model, we observed that combining pyrvinium pamoate with olaparib resulted in a significant decrease in tumor burden. This study demonstrates that Wnt signaling can mediate PARPi resistance in HGSOC and provides a clinical rationale for combining PARP and Wnt inhibitors.