Erin N. Howe

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 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.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 ER + breast cancers, a high ratio of AR:ER (≥2.0) indicated an over four fold increased risk for failure while on tamoxifen (HR = 4.43). 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 (HR = 4.04, 95% CI: 1.68, 9.69; p = 0.002) and disease specific survival (HR = 2.75, 95% CI: 1.11, 6.86; p = 0.03). 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+/AR + breast 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.

Loss of miR-200c: A Marker of Aggressiveness and Chemoresistance in Female Reproductive Cancers

We focus on unique roles of miR-200c in breast, ovarian, and endometrial cancers. Members of the miR-200 family target ZEB1, a transcription factor which represses E-cadherin and other genes involved in polarity. We demonstrate that the double negative feedback loop between miR-200c and ZEB1 is functional in some, but not all cell lines. Restoration of miR-200c to aggressive cancer cells causes a decrease in migration and invasion. These effects are independent of E-cadherin status. Additionally, we observe that restoration of miR-200c to ovarian cancer cells causes a decrease in adhesion to laminin. We have previously reported that reintroduction of miR-200c to aggressive cells that lack miR-200c expression restores sensitivity to paclitaxel. We now prove that this ability is a result of direct targeting of class III beta-tubulin (TUBB3). Introduction of a TUBB3 expression construct lacking the miR-200c target site into cells transfected with miR-200c mimic results in no change in sensitivity to paclitaxel. Lastly, we observe a decrease in proliferation in cells transfected with miR-200c mimic, and cells where ZEB1 is knocked down stably, demonstrating that the ability of miR-200c to enhance sensitivity to paclitaxel is not due to an increased proliferation rate.

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.

Progestin suppression of miR-29 potentiates dedifferentiation of breast cancer cells via KLF4

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.

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.

MicroRNA regulation of epithelial plasticity in cancer

MicroRNAs (miRNAs) are small non-coding RNA molecules capable of regulating multiple target genes through repression of mRNA stability or translation. miRNAs regulate a wide variety of developmental and pathological processes, and many have oncogenic or tumor suppressor activity. Carcinoma cells can undergo changes resembling a partial or transient epithelial-mesenchymal transition (EMT) as part of the metastatic cascade. Numerous miRNAs regulate EMT by targeting genes that control epithelial or mesenchymal characteristics. The involvement of miRNAs in feedback loops with EMT-regulating transcription factors reveals a critical role in governing epithelial plasticity. In this review we summarize miRNAs involved in carcinoma cell plasticity.

Abstract B70: Decreasing ovarian cancer metastases and progression via restoration of microRNAs that target mediators of anoikis- and chemoresistance.

Background: Epithelial ovarian cancer (EOC) accounts for the majority of deaths from gynecological malignancies, with a 5-year survival rate for women with advanced stage disease of only 46%. In EOC metastasis primarily occurs by a mechanism termed direct seeding, which involves shedding of tumor cells from the primary site into the peritoneal cavity. To allow survival in suspension before attachment at metastatic sites, ovarian cancer cells must acquire resistance to anoikis, a form of apoptosis initiated by detachment from native extracellular matrix. A targeted therapy that could simultaneously affect multiple factors critical for ovarian cancer progression such as anoikis resistance, chemoresistance and attachment to metastatic sites in the peritoneal cavity, would be a revolutionary breakthrough for this aggressive malignancy. Due to their ability to inhibit multiple targets involved in tumor survival, microRNAs (miRNAs) are attractive therapeutic candidates. We previously demonstrated that miR-200c is downregulated in ovarian cancer cell lines and restoration of miR-200c increased sensitivity to taxanes in vitro, by targeting TUBB3, a known mediator of chemoresistance. We tested the effect of miR-200c restoration in an intraperitoneal xenograft model of human ovarian cancer and observed dramatically decreased tumor formation. Further, even in established tumors, inducible restoration of miR-200c alone and in combination with paclitaxel, resulted in significantly reduced tumor burden. Hypothesis: Since restoration of miR-200c to multiple anoikis resistant EOC cell lines increased anoikis sensitivity and reduced adherence to biological substrates in vitro, we hypothesized that the decreased intraperitoneal tumor burden observed upon miR-200c restoration resulted from increased anoikis sensitivity. Methods and results: To elucidate mechanisms of anoikis resistance in ovarian cancer and identify targets involved in the ability of miR-200c to reverse anoikis resistance, we performed gene and miRNA expression profiling of attached Hey ovarian cancer cells as compared to the same cells grown under conditions of forced suspension for 24h. We identified miR-193 as downregulated in anoikis resistant cells in suspension. Among numerous genes predicted as miR-200c and/or miR-193 targets, SCLC7A11 (xCT), which encodes the light chain of the cystine/glutamate antiporter system xc, was increased by 10.7 fold in suspended versus attached cells. The xc- system is involved in glutathione (GSH) synthesis, mediating intracellular defense against oxidative stress. Reactive oxygen species (ROS) increased in anoikis-sensitive (OvCA 433) cells in suspension, but not in anoikis-resistant cells (Hey), suggesting that xCT might protect these cells from ROS-induced cell death. Consistent with this hypothesis, xCT mRNA and protein increased in anoikis resistant EOC cells in suspension, and transient xCT knockdown rendered cells anoikis sensitive. Interestingly, the xCT 39UTR contains three predicted binding sites for miR-200c and two sites for miR-193b, and restoration of either miRNA blocked the suspension induced-increase in cXT protein, suggesting that both miRNAs target this gene. Conclusions: Our results indicate that the ability of miR-200c to reduce tumor burden results, at least in part, from its ability to restore anoikis sensitivity by inhibiting a suspension-induced increase in xCT that protects tumor cells from ROS. Thus, miR-200c could be a powerful candidate to block progression of ovarian cancer by enhancing anoikis sensitivity and restoring chemo-sensitivity to already attached tumor. Citation Format: Diana M. Cittelly, Dawn R. Cochrane, Erin N. Howe, Irina Dimitrova, Miriam D. Post, Broaddus R. Russell, Monique A. Spillman, Jennifer K. Richer. Decreasing ovarian cancer metastases and progression via restoration of microRNAs that target mediators of anoikis- and chemoresistance.. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B70.