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  • FFA expression was also noted to be upregulated in eight

    2021-11-26

    FFA4 expression was also noted to be upregulated in eight human CRC cell lines. Compared to two normal colon cell lines with relative one-fold expression of FFA4, CRC cell lines HCT116 (3.5-fold higher), Colo205 (3-fold), Caco-2 (2.2-fold), HT-29 (2.3-fold), RKO (2.8-fold), DLD-1 (2.9-fold), SW480 (3.2-fold), and SW620 (2.2-fold) all expressed significantly higher levels of FFA4 protein [51]. Since the HCT116 and SW480 lines had highest FFA4 expression, they were studied further and noted to lack expression of FFA1 mRNA, allowing for use of GW9508 as a selective FFA4 agonist in these cells. Agonism of FFA4 with GW9508 resulted in enhanced mRNA and protein expression of CRC proangiogenic factors including VEGF, IL-8, and COX-2, and this effect was completely blocked in Aminoallyl-dUTP treated with FFA4 shRNA [51]. Importantly, reintroduction of FFA4 into the knockdown models was sufficient to restore proangiogenic gene expression, demonstrating that the observed effects were mediated via FFA4. Conditioned media from GW9508-treated CRC cell lines stimulated growth and endothelial branching of human umbilical cord vein endothelial cells (HUVEC) and this response was lost with conditioned media retrieved from HCT116 and SW480 that expressed FFA4 shRNA [51]. The effects of FFA4-mediated proangiogenic gene expression were further characterized and shown to result from FFA4-induced activation of PI3K/AKT-NF-κB signaling. This was evidenced by rapid (within 5–10 min) increases in phosphorylation of IκBα and AKT upon GW9508 stimulation, which were blocked by the PI3K inhibitor LY294002. Additionally, increased phosphorylation of IκBα and AKT was not observed upon GW9508 stimulation in the FFA4 knockdown model of HCT 116 and SW480 cells. Pretreatment with either LY294002 or NF-κB inhibitor BAY 11-7082 suppressed the GW9508 induced proangiogenic gene expression noted earlier. Finally, RNA interference of AKT and IκBα eliminated FFA4-mediated proangiogenic gene expression. The proposed CRC signaling pathway is shown in Fig. 2, however, the mechanism of signal transduction (i.e., G protein or β-arrestin-2) between FFA4 and PI3K was not investigated. Based on previous studies in adipocytes that show a Gαq/11-dependency of FFA4-signaling to PI3K, it is tempting to speculate that this is the mechanism occurring to link the two proteins in CRC. GW9508 stimulation of FFA4 in SW480 and HCT116 cells also significantly increased chemotactic capacity of the tumor cells in an in vitro transwell chemotaxis assay induced by serum, but failed to promote cell migration upon FFA4 knockdown. Migration was restored upon overexpression of FFA4 in shRNA knockdown cells. Migration of epithelial cells is dependent on loss of cell polarity and adhesion and subsequent gain of mesenchymal function, an effect termed epithelial-mesenchymal transition (EMT), which is characterized biochemically by decreases in the epithelial tumor-suppressing adhesion protein E-cadherin and correlated increases in mesenchymal markers such as vimentin, fibronectin, and N-cadherin. Importantly, treatment of HCT116 and SW480 cells with GW9508 facilitated a significant and time-dependent decrease in E-cadherin that correlated temporally with increased expression of both N-cadherin and vimentin, signifying that FFA4 agonism modulates cell migration via EMT (Fig. 2). To assess the effects of FFA4 on angiogenesis and tumor growth in vivo, HCT116 cells expressing scrambled shRNA were used in a nude mouse xenograft paradigm and animals were treated intraperitoneally with 10 mg/kg/day of GW9508. Tumors in these mice grew significantly faster from day six than control tumors treated with vehicle, and were four-fold larger at the 30-day period [51]. Additionally, mice treated with GW9508 had higher expression of proangiogenic markers that included VEGF, IL-8, and COX-2, as well as its synthetic byproduct PGE2. Importantly, tumors generated from HCT116 cells expressing shRNA for FFA4 grew at the same rate and size in both mice treated with IP injections of GW9508 and vehicle, and lacked upregulation of VEGF, IL-8, and COX-2, demonstrating a specific role for FFA4-mediated angiogenesis in the tumor growth. Upon analyzing whether FFA4 expression correlated with angiogenesis and migration capability of CRC cell lines, it was found that higher FFA4 expression yielded greater proangiogenic gene expression and chemotactic activity. In summary, this work revealed increased FFA4 expression in human CRC tissue, correlating positively with advanced tumor staging, and signaled through the PI3K/AKT-NF-κB pathway to promote proangiogenic gene expression in CRC cell lines. Additionally, higher FFA4 expression induced proangiogenic gene expression and migration capability in CRC cell lines in vivo.