Abstract 3587: Carcinoma-associated fibroblast-mediated regulation of anoikis in breast cancer cells

Abstract

Abstract There is accumulating evidence that carcinoma-associated fibroblasts (CAFs) in the microenvironment are critical players in the promotion of tumor progression and metastasis. However, the precise mechanisms utilized by CAFs to enhance malignancy are only beginning to be understood. Given the fact that cancer cells lack normal extracellular matrix (ECM) attachment after breaking through the basement membrane, we hypothesized that CAFs may be actively involved in the inhibition of anoikis (ECM-detachment-induced cell death). To investigate this question, we utilized NIH-3T3 mouse fibroblasts lacking the caveolin-1 gene (Cav -/-), a cell line that has previously been demonstrated to share many similarities to human CAFs. Interestingly, the addition of media conditioned by Cav-/- and patient-derived human CAFs led to robust anoikis inhibition in detached MCF-10A cells, suggesting that factors secreted by CAFs can block anoikis. Moreover, the ability of CAF-conditioned media to block anoikis was not limited to MCF-10A cells as we obtained similar results in other non-tumorigenic and breast cancer cell lines. To examine if the observed differences in anoikis induction caused by secreted factors from CAFs manifest in a more physiologically relevant context, we employed a 3-dimensional (3D) cell culture model of mammary acinus development. MCF-10A acini exposed to secreted factors from CAFs were more prone to have filled lumen suggesting that factors secreted by CAFs can promote the survival of ECM-detached cells in the luminal space. In addition, when investigating the mechanism by which CAFs block anoikis, we discovered that the release of mitochondrial cytochrome c into the cytosol was robustly inhibited by CAF-conditioned media in MCF-10A cells. Additionally, we discovered that the degradation of Mcl-1 was strongly inhibited by CAF-conditioned media suggesting that Mcl-1 was not properly targeted to the proteasome. Stability of Mcl-1 is triggered by activation of ILK and p-ERK and inhibition of Gsk-3β. Furthermore, using proteomic analyses, we have identified proteins of the IGFBP family that are differentially secreted by CAFs in a fashion that is both necessary for anoikis inhibition by CAFs and sufficient to inhibit anoikis on their own. Lastly, we found that tumor formation in immunocompromised mice was reduced when cancer cells were co-injected with IGFBP-deficient CAFs rather than normal CAFs. In aggregate, these data identify a novel mechanism by which CAFs contribute to tumorigenesis (the inhibition of anoikis) and suggest that targeting factors secreted from CAFs could be a novel therapeutic approach to eliminate ECM-detached cells through the selective induction of anoikis. Citation Format: Zachary T. Schafer. Carcinoma-associated fibroblast-mediated regulation of anoikis in breast cancer cells. [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 3587. doi:10.1158/1538-7445.AM2014-3587