The combined effect of IL-6 and hypoxia increases KLK4 gene expression in colon cancer cells via STAT-3 activation

Abstract

The aggressive phenotype of colorectal cancer (CRC) is largely driven by interactions within the tumor microenvironment, specifically the co-occurrence of inflammation and hypoxic stress. While the pro-metastatic enzyme Kallikrein-related Peptidase 4 (KLK4) is known to contribute to dissemination, the precise molecular mechanism by which IL-6 and hypoxia converge to regulate KLK4 expression and subsequent metastatic potential remains to be elucidated. This study investigated the influence of the IL-6 cytokine on KLK4 gene expression and metastatic potential in the HT-29 colon cancer cell line under both normal and hypoxic conditions. Healthy non-cancerous endothelial cells (HUVEC) served as a comparative control. Expression was assessed via RealTime PCR (mRNA) and Western blot (protein), while metastatic potential was determined by the scratch assay. Our findings demonstrate a substantial and marked upregulation in KLK4 gene and protein expression in HT-29 cells over a 48-hour period in response to IL-6, hypoxia, and the combined treatments. This increase in KLK4 was found to be associated with simultaneous upregulation of STAT-3 and p-STAT-3 proteins, strongly suggesting that the STAT-3 signaling pathway mediates this induction. The effects observed were tumor-specific: the non-cancerous HUVEC line showed only transient KLK4 changes and decreased proliferation in individual treatments. In sharp contrast, the combined IL-6 and hypoxia treatments significantly enhanced proliferative activity and metastatic potential in HT-29 cells. Western blot analysis collectively indicates that the augmented KLK4 expression in CRC cells is likely mediated through IL-6 and hypoxia-induced STAT-3 activation. These findings establish KLK4 as a potential downstream effector of the IL-6/STAT-3 pathway, offering a novel therapeutic target for mitigating metastatic potential in colon cancer.