Role of miR-26b in carcinoma-associated fibroblasts and effect on migration and invasion of breast cancer epithelial cells

Abstract

BackgroundIn recent years there has been an increasing awareness of the role of the microenvironment surrounding breast cancer epithelium, particularly the carcinoma-associated fibroblast (CAF), in modulating the behaviour of breast tumours. MicroRNAs, a family of small non-coding RNAs that are key in the post-transcriptional regulation of mRNAs, have a role in controlling the behaviour of cancers and have been studied extensively in breast cancer epithelial cells. We hypothesised that miRNAs have important roles in the behaviour of breast CAFs and, in turn, affect the behaviour of malignant breast epithelial cells. MethodsWe used laser capture microdissected normal fibroblast and CAFs from clinical samples, and a tissue co-culture model, to examine expression of miRNAs in breast CAFs. Deregulated miRNAs that were identified with this screening strategy were further assessed in a larger set of paired laser capture microdissected normal fibroblasts and CAFs. We assessed functional effects on fibroblasts by overexpressing, or knocking down miRNAs (or both) in immortalised fibroblast cell lines and using various growth, migration, and invasion assays. Functional effects of fibroblasts with reduced miRNA on breast cancer epithelial cells were examined in co-cultures. To identify potential miRNA targets and pathways downstream, we used mass spectrometry and in-silico analysis. The clinical relevance of these targets was examined by interrogating publicly available datasets. FindingsSix miRNAs were consistently downregulated in CAFs compared with normal fibroblasts with a fold change of more that ten in both the tissue co-culture model and in patient samples. Of these miRNAs, miR-26b was downregulated in 12 of 14 cases of microdissected matched normal fibroblasts and CAFs from clinical formalin fixed paraffin embedded samples (Wilcoxon signed rank test, p=0·04), and consistently in a further four of four cases of matched primary cultures of normal fibroblasts and CAFs. Reduction of the level of miR-26b in immortalised breast fibroblasts showed a small decrease in proliferation but a very notable increase in migration (p<0·01) and invasion of fibroblasts (p=0·03). There was no difference in the growth of cancer epithelial cells when they were co-cultured with fibroblasts with low miR-26b compared with controls. However, these fibroblasts increased the migration (p<0·01) and invasion (p<0·01) of breast cancer epithelial cells. We identified numerous proteins that were differentially expressed between miR-26b knockdown and control fibroblasts. Pathway analyses of the differentially expressed proteins (p<0·05) were enriched for components of the glycolytic pathway and the cytoskeleton regulation by RHO GTPase pathway. COL12A1 was identified as a target of miR-26b. Increased stromal mRNA expression of COL12A1 was significantly associated with increased recurrence of breast cancer on multivariate analysis in publically available datasets (hazard ratio 17, 95% CI 1·9–159; p=0·01). InterpretationWe have shown that downregulation of miR-26b in breast cancer fibroblasts increases the migration and invasion of breast cancer epithelial cells, and we have identified molecular changes that are downstream of miR-26b. These findings add to the growing body of evidence that epithelial-stromal interactions are important in modulating the behaviour of breast cancer. FundingUK Medical Research Council, Pathological Society of Great Britain and Ireland, Breast Cancer Campaign, National Institute of Health Research.