Abstract 175: Carcinoma-associated fibroblasts promote breast cancer cell invasion via IGF-1 and RhoA

Abstract

Abstract In triple-negative breast cancer (TNBC) and other breast cancer subtypes, the presence of a fibrotic focus - composed of carcinoma-associated fibroblasts (CAFs) and collagen fibers correlates with high-grade malignancies and reduced survival. CAFs secrete specific soluble pro-survival factors and extracellular matrix (ECM) molecules that modulate the adhesion of cancer cells, leading to the remodeling of a permissive and supportive environment for tumor progression and dissemination. Hence, the main goal of our study was to determine how CAFs affect breast cancer cell invasion. To study in vitro invasion, we established a 3D co-culture model by seeding human TNBC spheroids (MDA-MB-231 and HCC-1937), alone or in presence of human mammary CAFs, in a collagen I gel. Our findings show that CAFs increase active RhoA-GTP expression in MDA-MB-231 cells and significantly promote their invasion, by inducing cellular scattering and a switch from an elongated to an amœboid mode of migration. In presence of CAFs, the ROCK inhibitor Y27632, as well as the genetic silencing of RhoA, inhibited the rounding of MDA-MB-231 cells and significantly reduced their invasion. We then determined the expression of pro-invasive cytokines in CAFs and we found that IGF-1 expression was increased in CAFs co-cultured with MDA-MB-231 cells. To date, the IGF-1R pathway has been shown to stimulate proliferation and cell survival in TNBC cells in vitro, and several studies have suggested that the presence of stromal IGF-1 could modulate TNBC cell plasticity and drive tumors to metastasize to bone. To assess the role of IGF-1 in CAF-promoted invasion of TNBC cells, we first assayed the effect of recombinant IGF-1 on RhoA activation and the invasion of MDA-MB-231 cells. IGF-1 increased RhoA-GTP expression and promoted invasion in a RhoA-dependent manner. Furthermore, the addition of an IGF-1 blocking antibody to the co-culture of MDA-MB-231 cells and CAFs reduced the expression of RhoA-GTP, and the scattering and invasion of MDA-MB-231. Together, our results suggest that CAFs increase the invasion of MDA-MB-231 by promoting cell scattering and amœboid migration, which is dependent on the IGF-1/RhoA/ROCK pathway. Interestingly, we also found that CAFs as well as recombinant IGF-1, promote multicellular invasion of HCC-1937, in a ROCK-dependent manner, indicating that CAFs, via the IGF-1/RhoA/ROCK pathway, can support different modes of invasion. In conclusion, our results highlight the different roles of CAFs in invasion of TNBC cells. In breast cancer patients, the expression of RhoA and its downstream effector ROCK1 has been associated with cancer outgrowth and dissemination. Our findings may lead to the use of ROCK inhibitors (such as Fasudil), potentially in combination with IGF-1R targeting therapy, in the treatment of invasive breast cancer. Citation Format: Julien Daubriac, Yves Boucher. Carcinoma-associated fibroblasts promote breast cancer cell invasion via IGF-1 and RhoA. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 175. doi:10.1158/1538-7445.AM2014-175