Abstract 3075: Dysregulated stromal microRNAs promote metastatic progression in colorectal cancer.
Authors: Bullock Marc D. , Pickard Karen , Nielsen Boye , Jenei Veronika , Mellone Max , Mitter Richard , Primrose John , Thomas Gareth , Packham Graham , Mirenzami Alexander H. 
Published: August 2014

Background

MicroRNAs, which are key drivers of colorectal cancer (CRC) tumorogenesis, are also increasingly implicated in metastatic progression. Equally relevant in CRC metastasis is the critical part played by the tumour microenvironment and interactions between epithelial cells and cancer-associated stromal cells such as myofibroblasts.

The present study aims to address whether miRNAs in cancer-associated stroma influence the metastatic cascade by identifying miRNAs dysregulated in CRC specimens of progressive pathological stage and exploring their functional significance.

Methods and Results

RT-PCR-based QuantimiR™ microarray profiling revealed 18 miRNAs significantly differentially expressed in the laser-microdissected stroma of 20 CRC specimens compared with paired normal tissue. The highest scoring miRNA candidates were validated using high-sensitivity Taqman™ qRT-PCR assays. Stromal miR21, miR19a and miR215 expression were capable of distinguishing tumor from normal tissue (p&lt;0.03) and miR192, distinguishing between tumors of different pathological stage (Duke's A vs C p&lt;0.008).

MiR21, an important oncomiR with pleiotropic actions in numerous cancer relevant biological pathways was one of our highest scoring candidates, with a 3.4 mean fold increase in CRC compared with normal tissue (p&lt;0.01). In-situ hybridisation localized miR21 expression to fibroblast type cells in the stroma. Staining was absent in normal tissue and CRC epithelium.

To assess the biological significance of stromal miR21, stable over-expression was induced in cultured fibroblasts. This resulted in up-regulated αSMA expression, implying miR21 supports myofibroblast transdifferentiation. Medium supernatant from miR21 over-expressing fibroblasts protected CRC cells from oxaliplatin induced apoptosis and increased their proliferative and invasive capacity. 3D organotypic co-cultures, developed to model in-vivo circumstances, revealed that ectopic stromal miR21 expression was associated with 3.6 fold increased invasion of CRC epithelium into stroma (p&lt;0.001). Western blotting and immunofluorescence confirm RECK and TIMP3, inhibitors of the matrix remodelling enzymes MMPs, and known miR21 targets, are significantly downregulated in ex-vivo and cultured fibroblasts, with a corresponding increase in MMP2 activity by zymography.

Conclusions

Stromal miRNA expression profiles can distinguish CRC specimens of different pathological stage and could form the basis of a molecular staging approach in CRC. Dysregulation of key oncomiR, miR21 is a stromal phenomenon and supports fibroblast-to-myofibroblast transdifferentiation. Malignant invasion is enhanced by miR21 induced RECK/TIMP3 downregulation and rise in MMP2 activity. This novel mechanism highlights the importance of tumour stroma in CRC progression, and presents an interesting perspective for the development of novel drugs.

Citation Format: Marc D. Bullock, Karen Pickard, Boye Nielsen, Veronika Jenei, Max Mellone, Richard Mitter, John Primrose, Gareth Thomas, Graham Packham, Alexander H. Mirenzami. Dysregulated stromal microRNAs promote metastatic progression in colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3075. doi:10.1158/1538-7445.AM2013-3075