MicroRNAs Regulate Endometrial and Ovarian Cancer Sensitivity to Anoikis and Chemotherapy.

Abstract

Background and objective: MicroRNA-200c directly targets and represses ZEB1 and ZEB2. These transcription factors cause epithelial to mesenchymal transition (EMT) by repressing E-cadherin and other key determinates of apical-basal polarity. We find that miR-200c is high in well-differentiated breast, endometrial and ovarian cancer cells and low grade tumors, but is extremely low in de-differentiated carcinomas that have undergone EMT. We postulated that miR-200c maintains the well-differentiated epithelial phenotype by directly inhibiting translation of additional targets that play a role in multiple steps in the metastatic cascade including loss of polarity, migration/invasion, resistance to anoikis and drug resistance. Using luciferase 3'UTR reporter assays with mutational analysis, we have identified many direct targets of miR-200c. These targets, including fibronectin 1 (FN1), moesin (MSN), class III beta-tubulin (TUBB3), and neurotrophic tyrosine receptor kinase type 2 (NTRK2 or TrkB), are all typically expressed in non-epithelial cells, such as fibroblasts or neurons, but can become expressed in highly de-differentiated endometrial, ovarian and breast cancer cells that have undergone EMT. Restoration of miR-200c to TNBC cells leads to a decrease in the mRNA and protein levels of these targets. We have made the novel discovery that breast and gynecological cancers that have undergone EMT are resistant to anoikis (death by detachment) and that restoration of miR-200c restores anoikis sensitivity to these cells. Anoikis resistance is a relatively understudied step in the metastatic cascade. Interestingly, both TrkB and its ligand NTF3 (as measured by western blot and ELISA) increase when TNBC cells are forced to grow in suspension thereby setting up an inappropriate autocrine loop that enables anoikis resistance. Both the TrkB receptor and the ligand, NTF3, are repressed when miR-200c is restored to tumor cells, leading to anoikis sensitivity. When exogenous TrkB lacking its 3'UTR (such that it cannot be targeted by miR-200c) is added, miR-200c no longer reverses anoikis resistance. Thus we conclude that the inappropriate expression of TrkB and NTF3 (which are normally expressed in neurons) in tumor cells sets up an autocrine loop resulting in anoikis resistance, but this process can be reversed by restoration of miR-200c. We will also discuss how miR-200c enhance chemosensitivity via targeting of class III beta-tubulin and demonstrate that miR-200c reduces tumor burden in an in vivo model of ovarian cancer. Funding source: DOD Breast Cancer Program Idea Award BC084162 and Susan G. Komen Foundation Award KG090415 and the Colorado Cancer League to JKR. (poster)