Deregulated stromal microRNA-21 and promotion of metastatic progression in colorectal cancer

Abstract

BackgroundMicroRNAs (miRNAs) are increasingly implicated in the acquisition of metastatic capabilities. The aim of the present study was to determine the identity and function of deregulated miRNAs in colorectal cancer (CRC) associated stroma, and to study epigenetic mechanisms promoting CRC progression in the tumour microenvironment. MethodsRT-PCR-based profiling of 20 laser capture microdissected colonic tissue specimens was used to identify stromal miRNAs differentially expressed in CRC compared with paired normal tissue. Candidate miRNAs were evaluated by in-situ hybridisation to determine cells of origin, and assessed for biological significance with in-vitro and three-dimensional organotypic functional assays. Findings18 miRNAs were differentially expressed between CRC stroma and paired normal colonic stroma including miR-21, a known oncomiR in CRC. In-situ hybridisation showed that miR-21 was expressed exclusively in cancer-associated fibroblasts. Ectopic miR-21 expression in fibroblasts were associated with upregulated α-smooth mucle actin expression, implying that miR-21 supports myofibroblast transdifferentiation, a key prometastatic stromal event. Compared with controls, conditioned medium from miR-21 overexpressing fibroblasts protected CRC cells from oxaliplatin-induced apoptosis and increased their proliferative capacity. Furthermore, three-dimensional organotypic co-cultures, developed to model in-vivo circumstances, showed that ectopic stromal miR-21 expression in fibroblasts was associated with a 2·5-fold increased invasion of CRC epithelium into stroma compared with controls (p<0·001). RECK, an inhibitor of the matrix-remodelling enzyme MMP2, is targeted by miR-21, and was significantly downregulated by ectopic miR-21 in both cultured and ex-vivo human colorectal fibroblasts. Inhibition of MMP2 activity abrogated the invasion-promoting effects of ectopic stromal-miR-21. InterpretationDeregulation of miR-21 is a stromal phenomenon and supports CRC progression through myofibroblast transdifferentiation, resistance to oxaliplatin cytotoxicity, and promotion of tumour cell invasion. Stromal miR-21 induced invasion is mediated by RECK downregulation and a reciprocal rise in MMP2 activity. This mechanism highlights the importance of miRNAs in the tumour microenvironment during CRC progression, and presents a novel perspective for the development of targeted therapeutic agents. To our knowledge, this study is the first functional demonstration of the pathological effect of deregulated stromal miRNAs in cancer. Further experiments are now required to validate these findings in vivo. FundingUK Medical Research Council, Cancer Research UK.