Dawn R. Cochrane

MicroRNA-200c mitigates invasiveness and restores sensitivity to microtubule-targeting chemotherapeutic agents.

The transcription factor ZEB1 is normally not expressed in epithelial cells. When inappropriately expressed in carcinomas, ZEB1 initiates epithelial to mesenchymal transition due to its ability to repress E-cadherin and other genes involved in polarity. Recently, ZEB1 and ZEB2 have been identified as direct targets of the microRNA-200c family. We find that miR-200c levels are high in well-differentiated endometrial, breast, and ovarian cancer cell lines, but extremely low in poorly differentiated cancer cells. Low or absent miR-200c results in aberrant expression of ZEB1 and consequent repression of E-cadherin. Reinstatement of miR-200c to such cells restores E-cadherin and dramatically reduces migration and invasion. Microarray profiling reveals that in addition to ZEB1 and ZEB2, other mesenchymal genes (such as FN1, NTRK2, and QKI), which are also predicted direct targets of miR-200c, are indeed inhibited by addition of exogenous miR-200c. One such gene, class III β-tubulin (TUBB3), which encodes a tubulin isotype normally found only in neuronal cells, is a direct target of miR-200c. This finding is of particular significance because we show that restoration of miR-200c increases sensitivity to microtubule-targeting agents by 85%. Because expression of TUBB3 is a common mechanism of resistance to microtubule-binding chemotherapeutic agents in many types of solid tumors, the ability of miR-200c to restore chemosensitivity to such agents may be explained by its ability to reduce TUBB3. Because miR-200c is crucial for maintenance of epithelial identity, behavior, and sensitivity to chemotherapy, we propose that it warrants further investigation as a therapeutic strategy for aggressive, drug-resistant cancers. [Mol Cancer Ther 2009;8(5):OF1–12]

Targets of miR-200c mediate suppression of cell motility and anoikis resistance

IntroductionmiR-200c and other members of the miR-200 family promote epithelial identity by directly targeting ZEB1 and ZEB2, which repress E-cadherin and other genes involved in polarity. Loss of miR-200c is often observed in carcinoma cells that have undergone epithelial to mesenchymal transition (EMT). Restoration of miR-200c to such cells leads to a reduction in stem cell-like characteristics, reduced migration and invasion, and increased sensitivity to taxanes. Here we investigate the functional role of novel targets of miR-200c in the aggressive behavior of breast and endometrial cancer cells.MethodsPutative target genes of miR-200c identified by microarray profiling were validated as direct targets using dual luciferase reporter assays. Following restoration of miR-200c to triple negative breast cancer and type 2 endometrial cancer cell lines that had undergone EMT, levels of endogenous target mRNA and respective protein products were measured. Migration and sensitivity to anoikis were determined using wound healing assays or cell-death ELISAs and viability assays respectively.ResultsWe found that restoration of miR-200c suppresses anoikis resistance, a novel function for this influential miRNA. We identified novel targets of miR-200c, including genes encoding fibronectin 1 (FN1), moesin (MSN), neurotrophic tyrosine receptor kinase type 2 (NTRK2 or TrkB), leptin receptor (LEPR), and Rho GTPase activating protein 19 (ARHGAP19). These targets all encode proteins normally expressed in cells of mesenchymal or neuronal origin; however, in carcinoma cells that lack miR-200c they become aberrantly expressed and contribute to the EMT phenotype and aggressive behavior. We showed that these targets are inhibited upon restoration of miR-200c to aggressive breast and endometrial cancer cells. We demonstrated that inhibition of MSN and/or FN1 is sufficient to mediate the ability of miR-200c to suppress cell migration. Lastly, we showed that targeting of TrkB mediates the ability of miR-200c to restore anoikis sensitivity.ConclusionsmiR-200c maintains the epithelial phenotype not only by targeting ZEB1/2, which usually facilitates restoration of E-cadherin expression, but also by actively repressing a program of mesenchymal and neuronal genes involved in cell motility and anoikis resistance.

Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide

IntroductionThe 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 ERu2009+u2009tumors. 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.MethodsWe examined nuclear AR to ER protein ratios in primary breast cancers in relation to response to endocrine therapy. The effects of AR inhibition with enzalutamide were examined in vitro and in preclinical models of ER positive and negative breast cancer that express AR.ResultsIn a cohort of 192 women with ERu2009+u2009breast cancers, a high ratio of AR:ER (≥2.0) indicated an over four fold increased risk for failure while on tamoxifen (HRu2009=u20094.43). The AR:ER ratio had an independent effect on risk for failure above ER % staining alone. AR:ER ratio is also an independent predictor of disease-free survival (HRu2009=u20094.04, 95% CI: 1.68, 9.69; pu2009=u20090.002) and disease specific survival (HRu2009=u20092.75, 95% CI: 1.11, 6.86; pu2009=u20090.03). Both enzalutamide and bicalutamide inhibited 5-alpha-dihydrotestosterone (DHT)-mediated proliferation of breast cancer lines in vitro; however, enzalutamide uniquely inhibited estradiol (E2)-mediated proliferation of ER+/ARu2009+u2009breast cancer cells. In MCF7 xenografts (ER+/AR+) enzalutamide inhibited E2-driven tumor growth as effectively as tamoxifen by decreasing proliferation. Enzalutamide also inhibited DHT- driven tumor growth in both ER positive (MCF7) and negative (MDA-MB-453) xenografts, but did so by increasing apoptosis.ConclusionsAR to ER ratio may influence breast cancer response to traditional endocrine therapy. Enzalutamide elicits different effects on E2-mediated breast cancer cell proliferation than bicalutamide. This preclinical study supports the initiation of clinical studies evaluating enzalutamide for treatment of AR+ tumors regardless of ER status, since it blocks both androgen- and estrogen- mediated tumor growth.

Restoration of miR-200c to Ovarian Cancer Reduces Tumor Burden and Increases Sensitivity to Paclitaxel

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. miRNAs have emerged as potential therapeutic candidates due to their ability to downregulate multiple targets involved in tumor progression and chemoresistance. miRNA-200c (miR-200c) is downregulated 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 class III β-tubulin gene (TUBB3), a tubulin known to mediate chemoresistance. Indeed, we find that patients with tumors having low TUBB3 had significantly prolonged survival (average survival 52.73 ± 4.08 months) as compared with those having high TUBB3 (average survival 42.56 ± 3.19 months). miR-200c also targets TrkB, a mediator of resistance to anoikis. We show that restoration of miR-200c to ovarian cancer cells results in increased anoikis sensitivity and reduced adherence to biologic substrates in vitro. Because 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 before cytotoxic chemotherapy may allow for a better response or lower effective dose. Mol Cancer Ther; 11(12); 2556–65. ©2012 AACR.

The miR-200 and miR-221/222 microRNA Families: Opposing Effects on Epithelial Identity

Carcinogenesis is a complex process during which cells undergo genetic and epigenetic alterations. These changes can lead tumor cells to acquire characteristics that enable movement from the primary site of origin when conditions become unfavorable. Such characteristics include gain of front-rear polarity, increased migration/invasion, and resistance to anoikis, which facilitate tumor survival during metastasis. An epithelial to mesenchymal transition (EMT) constitutes one way that cancer cells can gain traits that promote tumor progression and metastasis. Two microRNA (miRNA) families, the miR-200 and miR-221 families, play crucial opposing roles that affect the differentiation state of breast cancers. These two families are differentially expressed between the luminal A subtype of breast cancer as compared to the less well-differentiated triple negative breast cancers (TNBCs) that exhibit markers indicative of an EMT. The miR-200 family promotes a well-differentiated epithelial phenotype, while high miR-221/222 results in a poorly differentiated, mesenchymal-like phenotype. This review focuses on the mechanisms (specific proven targets) by which these two miRNA families exert opposing effects on cellular plasticity during breast tumorigenesis and metastasis.

miR-200c Targets a NF-κB Up-Regulated TrkB/NTF3 Autocrine Signaling Loop to Enhance Anoikis Sensitivity in Triple Negative Breast Cancer

Anoikis is apoptosis initiated upon cell detachment from the native extracellular matrix. Since survival upon detachment from basement membrane is required for metastasis, the ability to resist anoikis contributes to the metastatic potential of breast tumors. miR-200c, a potent repressor of epithelial to mesenchymal transition, is expressed in luminal breast cancers, but is lost in more aggressive basal-like, or triple negative breast cancers (TNBC). We previously demonstrated that miR-200c restores anoikis sensitivity to TNBC cells by directly targeting the neurotrophic receptor tyrosine kinase, TrkB. In this study, we identify a TrkB ligand, neurotrophin 3 (NTF3), as capable of activating TrkB to induce anoikis resistance, and show that NTF3 is also a direct target of miR-200c. We present the first evidence that anoikis resistant TNBC cells up-regulate both TrkB and NTF3 when suspended, and show that this up-regulation is necessary for survival in suspension. We further demonstrate that NF-κB activity increases 6 fold in suspended TNBC cells, and identify RelA and NF-κB1 as the transcription factors responsible for suspension-induced up-regulation of TrkB and NTF3. Consequently, inhibition of NF-κB activity represses anoikis resistance. Taken together, our findings define a critical mechanism for transcriptional and post-transcriptional control of suspension-induced up-regulation of TrkB and NTF3 in anoikis resistant breast cancer cells.

A TDO2-AhR Signaling Axis Facilitates Anoikis Resistance and Metastasis in Triple-Negative Breast Cancer

The ability of a cancer cell to develop resistance to anoikis, a programmed cell death process triggered by substratum detachment, is a critical step in the metastatic cascade. Triple-negative breast cancers (TNBC) exhibit higher rates of metastasis after diagnosis, relative to estrogen-positive breast cancers, but while TNBC cells are relatively more resistant to anoikis, the mechanisms involved are unclear. Through gene expression and metabolomic profiling of TNBC cells in forced suspension culture, we identified a molecular pathway critical for anchorage-independent cell survival. TNBC cells in suspension upregulated multiple genes in the kynurenine pathway of tryptophan catabolism, including the enzyme tryptophan 2,3-dioxygenase (TDO2), in an NF-κB-dependent manner. Kynurenine production mediated by TDO2 in TNBC cells was sufficient to activate aryl hydrocarbon receptor (AhR), an endogenous kynurenine receptor. Notably, pharmacologic inhibition or genetic attenuation of TDO2 or AhR increased cellular sensitivity to anoikis, and also reduced proliferation, migration, and invasion of TNBC cells. In vivo, TDO2 inhibitor-treated TNBC cells inhibited colonization of the lung, suggesting that TDO2 enhanced metastatic capacity. In clinical specimens of TNBC, elevated expression of TDO2 was associated with increased disease grade, estrogen receptor-negative status, and shorter overall survival. Our results define an NF-κB-regulated signaling axis that promotes anoikis resistance, suggest functional connections with inflammatory modulation by the kynurenine pathway, and highlight TDO2 as an attractive target for treatment of this aggressive breast cancer subtype.

The role of miRNAs in progesterone action

Small non-coding RNAs termed microRNAs (miRNAs) are mediators of post-transcriptional gene silencing and are involved in all aspects of cell biology. Progesterone receptors (PR) are intimately involved in the normal physiology and diseases of hormone responsive tissues including the uterus and the breast. Recent evidence suggests that hormone regulated miRNAs play a substantial role in hormone receptor mediated gene regulation. However, relatively little is known regarding miRNAs regulated by PR or that target PR as compared to those regulated by or targeting estrogen receptors (ER). We summarize the state of current knowledge regarding miRNAs and PR action. We also delineate how progesterone regulated miRNAs might provide an additional level of control and fine tuning of gene regulation by hormone receptors and also facilitate cell- and tissue-specific gene regulation PR.

Metformin-Induced Killing of Triple-Negative Breast Cancer Cells Is Mediated by Reduction in Fatty Acid Synthase via miRNA-193b

The anti-diabetic drug metformin (1,1-dimethylbiguanide hydrochloride) reduces both the incidence and mortality of several types of cancer. Metformin has been shown to selectively kill cancer stem cells, and triple-negative breast cancer (TNBC) cell lines are more sensitive to the effects of metforminxa0as compared to luminal breast cancer. However, the mechanism underlying the enhanced susceptibility of TNBC to metformin has not been elucidated. Expression profiling of metformin-treated TNBC lines revealed fatty acid synthase (FASN) as one of the genes most significantly downregulated following 24xa0h of treatment, and a decrease in FASN protein was also observed. Since FASN is critical for de novo fatty acid synthesis and is important for the survival of TNBC, we hypothesized that FASN downregulation facilitates metformin-induced apoptosis. Profiling studies also exposed a rapid metformin-induced increase in miR-193 family members, and miR-193b directly targets the FASN 3′UTR. Addition of exogenous miR-193b mimic to untreated TNBC cells decreased FASN protein expression and increased apoptosis of TNBC cells, while spontaneously immortalized, non-transformed breast epithelial cells remained unaffected. Conversely, antagonizing miR-193 activity impaired the ability of metformin to decrease FASN and cause cell death. Further, the metformin-stimulated increase in miR-193 resulted in reduced mammosphere formation by TNBC lines. These studies provide mechanistic insight into metformin-induced killing of TNBC.

Abstract 4756: Elucidating the role of AR in breast cancer .

Background: In breast cancers, the androgen receptor (AR) is more widely expressed than estrogen receptor alpha (ER) or progesterone receptor (PR), and AR has recently emerged as a useful marker for refinement of breast cancer subtype classification. Approximately 77% of invasive breast cancer tumors are positive for AR and among the subtypes, 88% of ER+, 59% of HER2+, and 32% of triple negative breast cancers (ER-/PR-/HER2-) are positive for AR protein expression by IHC. AR expression has been associated with resistance to current endocrine therapies (tamoxifen and aromatase inhibitors) in cell line and preclinical models, and clinical studies. Hypothesis: We hypothesized that ER+ breast cancers that become resistant to traditional endocrine therapies might do so by switching from estrogen to androgen-dependence and that AR holds potential as a therapeutic target in AR+ breast cancers, whether ER+ or ER-. Methods: We quantified the percentage of cells positive for nuclear AR compared to the percentage of cells positive for ER in a cohort of tamoxifen treated patients with clinical outcome data. We also examined the effect of dihydrotestosterone (DHT) and estradiol (E2) in the presence of enzalutamide (formerly MDV3100), a novel AR inhibitor, in in vitro and in vivo models of ER+, ER-, and Her2+ breast cancer that retain AR. Results: Our clinical data indicate that a high ratio of AR to ER protein is indicative of a shorter time to relapse in patients treated with tamoxifen and a lack of response to neo-adjuvant AI treatment. In ER+ models, bicalutamide and enzalutamide both inhibit DHT-mediated proliferation, while enzalutamide uniquely inhibited E2-mediated proliferation. In xenograft tumor studies, enzalutamide significantly reduced estrogen- and androgen-mediated growth of ER+/AR+ xenograft tumors. Remarkably, enzalutamide demonstrated an anti-proliferative effect comparable to tamoxifen on tumors in E2 treated mice and inhibited the expression of classic E2-regulated genes such as SDF-1. Enzalutamide opposed DHT-stimulated proliferation of ER-/AR+ MDA-MB-453 tumors in vivo and caused decreased nuclear AR localization and increased tumor cell apoptosis. In Her2+ breast cancer cell lines, enzalutamide enhanced the response to trastuzumab and decreased the amount of both phosphorylated and total Her3. Conclusions: Enzalutamide demonstrated significant anti-tumor activity in preclinical models of breast cancers that express AR, regardless of ER status. In Her2+ breast cancer, inhibition of AR with enzalutamide may enhance response to Her2-directed therapy or overcome resistance to such agents by reducing levels of Her3. The AR:ER ratio may be a new predictor of response to traditional E2/ER directed endocrine therapy and may indicate that patients who relapse while on tamoxifen or AIs might be good candidates for AR-directed therapy. Citation Format: Nicholas C. D9Amato, Haihua Gu, Dawn R. Cochrane, Sebastian Bernales, Britta M. Jacobsen, Paul Jedlicka, Kathleen C. Torkko, Susan M. Edgerton, Ann D. Thor, Anthony D. Elias, Andrew A. Protter, Jennifer K. Richer. Elucidating the role of AR in breast cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4756. doi:10.1158/1538-7445.AM2013-4756