Diverse Roles of G‐Protein‐Coupled Receptor 40 (GPR40/FFA1) and GPR120 (FFA4) in the Regulation of Cell Growth and Motile Activities in Renal Cell Carcinoma (RCC)

Abstract

Renal cell carcinoma (RCC) is the 16th most common cancer worldwide with roughly 400,000 new cases diagnosed annually and approximately half of those leading to mortality. Even though the 5-year survival rate for early-stage RCC is 80%-90%, it is only 5% for the patients with metastasis. Unfortunately, anticancer drugs that are used to treat RCC are proven to be useful for only a small number of patients. Therefore, it is crucial to discover the novel molecular pathways underlying RCC to develop better therapies. The G protein-coupled receptors GPR40 and GPR120, also referred to as free fatty acid receptor 1 (FFA1) and 4 (FFA4), respectively, are activated by medium- to long-chain free fatty acids. Emerging evidence suggests that GPR40 and GPR120 are implicated in the proliferation, invasiveness, and apoptosis of various tumor cells, however, the precise mechanisms of GPCRs signaling events leading to the modifications in cancer cell growth and migration are still scarce, and the roles of these receptors in RCC are unstudied to date. Here, for the first time, we reveal the effects of GPR40 and GPR120 agonism on cell proliferation and migration in metastatic tumor-derived renal cancer cells. The present study used in vitro techniques including cell proliferation assay, wound-healing assay, and transwell migration assay to investigate the functions of GPR40 and GPR120 on the proliferative and migration capacities of the RCC cell line. The human renal cancer cell line ACHN was obtained from the American Type Culture Collection (ATCC). Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was performed to confirm the presence of GPR40 and GPR120 transcripts. Cell proliferation was assessed by plating 50,000 cells per well, followed by treatment with GPR40 and GPR120 agonists and antagonists as noted for 6 days. Cells were counted every 24 hours. For the wound-healing assay, 100,000 cells were plated on a 48-well plate and a scratch-made through the center. The time to wound healing was assessed at 0, 24, 36, and 48 hours. For migration assays, cells were plated in the top chamber of a transwell plate and cells migrated was counted by crystal violet staining after 16 hr. Using selective GPR40 and GPR120 agonists and antagonists, our results demonstrate that GPR40 enhanced and GPR120 suppressed cell proliferation, suggesting that the opposite effects of GPR120 and GPR40 are involved in the cell growth activity. The wound-healing assay revealed that both GPR40 and GPR120 agonism markedly covered the wounded area compared to vehicle-controls, an effect that is blocked by a selective GPR40 and GPR120 antagonist, respectively. In transwell migration assay, GPR40 negatively and GPR120 positively affect the ability of single cells to respond to the chemoattractant; further confirming their involvement in the acquisition of migratory properties in RCC cell. Our present results suggest that GPR40/120-mediated signaling may be involved in the regulation of renal cancer tumorigenesis and progression on the cellular level, and mechanistic studies are underway to assess the cellular cascades that are influenced by GPR40 and GPR120.