The tumor microenvironment is now appreciated to be an active participant in driving the oncogenic behavior of cancer cells. In particular, evidence is mounting that carcinoma-associated fibroblasts (CAFs) in the microenvironment are a critical player 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 importance of CAFs in promoting tumor progression and 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 (KO 3T3s), a cell line that has previously been demonstrated to share many similarities to human CAFs. Interestingly, the addition of media conditioned by KO 3T3s led to robust anoikis inhibition in detached MCF-10A cells, suggesting that factors secreted by CAFs can block anoikis. We validated these findings by demonstrating that factors secreted from patient-derived human CAFs can inhibit anoikis to a similar degree. 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 cell lines and in 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. Surprisingly, this inhibition was independent of signaling through PI(3)K or MAPK and did not involve alterations in Bim protein levels. However, 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. Furthermore, using mass spectrometric analyses, we have identified proteins of the IGFBP family that are differentially secreted by CAFs in a fashion that regulates anoikis induction. 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: Kelsey Weigel, Ana Jakimenko, Brooke Conti, William Kaliney, Matthew M. Champion, Zachary T. Schafer. The regulation of anoikis by carcinoma-associated fibroblasts in breast cancer cells. [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 1634. doi:10.1158/1538-7445.AM2013-1634