Novel impacts of Fam3C (the family with sequence similarity 3C) in cancer-associated-adipocyte (CAA) within the tumor microenvironment of breast cancer

Abstract

Adipocyte is one of the major constituents of tumor stromal cells in breast cancer. Nevertheless, the roles of cancer-associated adipocytes (CAA) within the tumor microenvironment remain still elusive. In this study, we identified the Fam3C as one of the most differentially regulated genes in CAA compared to that in benign adipocytes by analyzing mRNA-deep sequencing with isolated adipocytes from subcutaneous adipose tissues of MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mice during tumor progression. Interestingly, the protein levels of Fam3C were significantly increased in differentiated 3T3-L1 adipocytes at 24-48 hours after co-cultured with MET1/EO771 breast cancer cells with a trans-well system. This was also confirmed by immunostaining for Fam3C with mammary tumor tissues of MMTV-PyMT during tumor progression. The protein level of Fam3C was significantly increased in CAA regions of tumor tissues at early stage of mammary tumor progression in vivo. Gain-of function studies by utilizing adenoviral-Fam3C transduction into 3T3-L1 adipocytes revealed that Fam3C overexpressing adipocytes increased cell survival compared to adeno-GFP transduced adipocytes. Furthermore, we established the Fam3C knock-out (Fam3C KO) 3T3-L1 preadipocytes by utilizing CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system. We showed that adipogenesis was slightly faster in Fam3C KO cells compared to control cells with a significant increase of cell cycle arrest when Fam3C KO cells were differentiated into adipocytes. We also found that TGFb signaling pathways contributed to the levels of Fam3C within the tumor microenvironment through mediating the crosstalk between adipocytes and cancer cells. TGFb neutralizing antibody treatment into co-culture system of adipocytes and cancer cells, ameliorated the modulation of Fam3C levels in CAA in vitro. Collectively, our findings demonstrate that a crosstalk between cancer cells and adipocytes through a TGFb-Fam3C axis contributes to cell survival of both CAA and cancer cells via cell cycle regulation. These results strongly suggest that CAA provide a tumor prone niche within the tumor microenvironment by supporting cancer cell survival.