Abstract 1529: Cross-talk between carcinoma-associated fibroblasts and invasive breast cancer cells: Role of RhoA / ROCK signaling

Abstract

Abstract The high density of collagen and carcinoma-associated fibroblasts (CAFs) correlates with metastasis and reduced survival in breast cancer. CAFs have been shown to contribute to tumor progression by promoting notably cancer cell survival, migration and invasion. Furthermore, in breast cancer patients, the expression of RhoA and its downstream effector ROCK1 has been associated with cancer outgrowth and dissemination. RhoA has been shown to promote the motility and invasion of breast cancer cells. Hence, the main goal of our study is to determine how CAFs affect breast cancer cell survival and invasion, and the role of RhoA/ROCK signaling in these two processes. To study in vitro invasion, we grew MDA-MB-231 cells expressing GFP on a low adhesive substrate (poly-HEMA) in order to produce spheroids, which we then seeded alone or with human mammary CAFs in a collagen I gel. We measured RhoA activation with a pull-down assay. Our findings show that CAFs activate RhoA in MDA-MB-231 cells and promote their invasion, by inducing cellular scattering and a switch from an elongated to an amoeboid mode of migration. In presence of CAFs, the ROCK inhibitor Y27632 inhibited the rounding of cancer cells and significantly reduced their invasion, confirming the role of ROCK in the CAF-promoted invasiveness of cancer cells. Then to determine the role of RhoA signaling in cancer cells in response to CAFs, we used an shRNA construct to downregulate the expression of RhoA. Interestingly, RhoA downregulation prevented cellular aggregation and the formation of spheroids on poly-HEMA, and induced anoikis in MDA-MB-231 cells, thus suggesting that RhoA plays a role in cell-cell adhesion, which can promote the survival of cells with relatively poor matrix adhesion. These results further suggest that the high levels of active-RhoA induced by CAFs – in rounded cancer cells – maybe necessary for the survival of cancer cells with an amoeboid migration phenotype. To determine if CAFs could affect other pathways that would improve the survival of rounded cancer cells, we used a pathway-focused RT-PCR array to analyze apoptosis signaling. Our preliminary results show that CAFs can enhance the expression of several pro-survival genes in cancer cells, including Akt, which has been recently reported to promote anoikis resistance in melanoma cells via RhoA. In conclusion, our results suggest that CAFs-dependent activation of RhoA/ROCK signaling promotes anoikis resistance and amoeboid migration in invasive breast cancer cells. Since the ROCK inhibitor Fasudil has been approved for the treatment of cardiovascular diseases in Japan, our findings may lead to the use of this drug or other ROCK inhibitors in the treatment of invasive breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1529. doi:1538-7445.AM2012-1529