UNVEILING THE ROLE OF CD81/TSPAN28 IN COLON CANCER: INSIGHTS INTO PROLIFERATION, CELL CYCLE PROGRESSION, AND MOTILITY

Background: Cluster of Differentiation 81 (CD81), also known as TSPAN28, is a transmembrane protein belonging to the tetraspanin (TSPAN) family, which interacts with various proteins at the cell membrane. CD81 has pleiotropic biological functions, including regulating cell growth, differentiation, and motility. Its role in cancer is complex, acting as either a tumor promoter or suppressor depending on the cellular context. However, its contribution to colon cancer remains unclear. Aim: This study aims to investigate the molecular mechanisms by which CD81 influences colon cancer cell behaviour in vitro. Methods: CD81 knockdown (CD81KD) was achieved in DLD1 colon cancer cells using shRNA in pSiren retroviral vectors. Cell proliferation was monitored via real-time imaging using the IncuCyte ZOOM system. Cell motility was assessed through wound healing assays, while tumorigenic potential was evaluated using an anchorage-independent growth assay. The effects of CD81 on the expression of key cell cycle regulators, E2F1, CCND1, and CDC6, were analysed using RT-qPCR. Results: CD81KD DLD1 cancer cells demonstrated significantly reduced cell proliferation (p = 0.049) and cell motility (p < 0.001) compared to control cells. Additionally, CD81KD impaired the ability of DLD1 cells to form anchorage-independent colonies in soft agar (p=0.002). Finally, CD81KD in DLD1 cells reduced the expression of the cell cycle genes E2F1 & CDC6 (p<0.001). Conclusions: Our findings indicate that CD81 plays a crucial role in colon cancer progression by enhancing cell proliferation and motility. The observed downregulation of E2F1 and CDC6 following CD81KD suggests a potential mechanism by which CD81 influences cell cycle progression. Further studies are required to elucidate the full scope of CD81-mediated signalling and its therapeutic implications in colon cancer.