Ectopic over-expression of miR-429 induces mesenchymal-to-epithelial transition (MET) and increased drug sensitivity in metastasizing ovarian cancer cells

Highlights

• A primary cell line was established from cells isolated from the peritoneal fluid of an advanced stage ovarian cancer patient.

• Over-expression of miR-429 induces mesenchymal-to-epithelial transition while increasing sensitivity to cisplatin.

• Targeted delivery of miR-429 in combination with platinum-based therapy may reduce ovarian cancer metastasis and recurrence.

Abstract

Objective

We recently determined that the ectopic over-expression of miR-429 and other members of the miR-200 family of microRNAs in ovarian cancer (OC) mesenchymal-like cell lines induces mesenchymal-to-epithelial transition (MET) with a concomitant increase in sensitivity to platinum drugs. We sought to determine if metastasizing OC cells isolated from an OC patient could also be induced by miR-429 to undergo MET and become sensitized to established first-line platinum-based therapies.

Methods

We established and characterized a new primary cell line (OCI-984) from free-floating OC cells isolated from the ascites fluid of an advanced stage OC patient. miR-429 was ectopically over-expressed in these cells.

Results

The over-expression of miR-429 in OCI-984 cells induced morphological, functional and molecular changes consistent with MET and a concomitant significant increase in the sensitivity of the converted cells to cisplatin.

Conclusions

Our findings indicate that the miR-200 family of microRNAs, and miR-429 in particular, play a critical role in the functioning of OC metastasizing cells and that targeted delivery of miR-429, and perhaps other miR-200 family members, in combination with platinum-based chemotherapies may be an effective strategy in reducing OC metastasis and tumor recurrence.

Introduction

Epithelial ovarian cancer (OC) is the most lethal of all gynecological cancers, the fourth leading cause of cancer-related deaths in women in the United States and the fifth most common malignancy in women in developed countries [1]. OC can be effectively treated by debulking surgery when the malignancy is confined to the ovary, but most OCs remain undetected until more advanced metastatic stages of the disease when treatment requires the administration of chemotherapy [2]. Although the majority of OCs responds favorably to first-line platinum-based chemotherapies, mesenchymal-like metastasizing cells are typically resistant to these chemotherapies and can thus lead to cancer recurrence in many OC patients [3], [4], [5], [6]. For this reason, considerable effort is currently being focused on the development of effective therapeutic strategies to target OC metastasizing cells.

Two basic approaches are currently being taken to address the challenge of OC metastasis. One is to identify drugs that could be effective at growth inhibition or killing of metastasizing mesenchymal-like OC cells directly. Metastasizing OC cells display a variety of mesenchymal and stem cell-like characteristics (e.g., quiescence, efficient DNA repair, high expression of ABC transporters, etc.) that make them generally resistant to classical antitumor regimens [4], [5], [6]. High-throughput screening [7], [8] and other drug discovery strategies [9] have recently identified a variety of compounds with potential for targeting metastasizing mesenchymal/stem cell-like cells directly. Some of these agents are currently in clinical trials [10].

A second possible strategy for the treatment of OC metastasis is to induce metastasizing mesenchymal-like cells to differentiate into cancer epithelial-like cells that are more responsive to existing chemotherapies [11], [12]. One class of regulatory molecules that has shown considerable promise in inducing such mesenchymal-to-epithelial transitions (MET) is microRNAs (miRNAs) [13].

MicroRNAs are small (~ 22 nucleotide) non-encoding RNAs that regulate gene expression at the transcriptional and/or post-transcriptional levels [14], [15]. Our laboratory has previously shown that the exogenous over-expression of miR-429 and other miR-200 family microRNAs in mesenchymal-like OC cell lines induces MET, resulting in an increased expression of epithelial cell molecular and phenotypic markers and a concomitant increased sensitivity to cisplatin [16], [17]. In an effort to test the potential clinical utility of exogenous expression of miR-429 to induce MET in OC metastasizing cells, we established a primary OC cell line from free floating OC cells isolated from the peritoneal fluid of an advanced stage OC patient. These cells display many of the mesenchymal/stem cell-like characteristics of previously characterized metastasizing cells [5], [6], [18], [19]. We show here that the exogenous over-expression of miR-429 in these cells results in MET with a significant increase in sensitivity to cisplatin. Our results indicate that the treatment of metastasizing OC cells with miR-429 may be an effective strategy to induce MET and sensitize these cells to platinum-based therapies.