Role of Clostridium perfringens Enterotoxin on YAP Activation in Colonic Sessile Serrated Adenoma/Polyps with Dysplasia

Sessile serrated adenoma/polyp with dysplasia (SSA/P-D) is an SSA/P with cellular dysplasia and has a higher risk of progressing to colon carcinogenesis. Previously, we reported that tight junction impairment by Clostridium perfringens enterotoxin (CPE) leads to activation of the transcriptional co-activator yes-associated protein (YAP) in oral squamous cell carcinoma. Here, we investigated whether CPE activates YAP to promote the malignant progression of SSA/P. E-cadherin expression was lower in the 12 cases with SSA/P-D examined than that in normal mucosa, SSA/P, or tubular adenoma (TA). Furthermore, intracellular translocation of claudin-4 (CLDN4) and nuclear translocation of YAP were observed. The CPE gene was detected in DNA extracted from SSA/P-D lesions, but not in SSA/P or TA. Treatment of the rat intestinal epithelial cell line IEC6 with low-dose CPE resulted in intracellular translocation of CLDN4 to the cytoplasmic membrane. Cytoplasmic CLDN4 showed co-precipitation with transcriptional co-activator with PDZ-binding motif, zonula occludens (ZO)-1, large tumor suppressor, and mammalian Ste20-like. Additionally, YAP co-precipitated with ZO-2 under CPE treatment led to decreased YAP phosphorylation and nuclear translocation. YAP activation promoted increase in nuclear TEA domain family member level, expression of cyclin D1, snail, vimentin, CD44, NS and decrease in E-cadherin levels, thereby inducing stemness and epithelial-mesenchymal-transition (EMT). The Hippo complex with the incorporation of CLDN4 increased stability. Upon low-dose CPE treatment, HT29 cells with BRAF(V600E) gene mutation showed increased growth, enhanced invasive potential, stemness, and induced EMT phenotype, whereas HCT116 cells, which carry KRAS(G13D) gene mutation, did not show such changes. In an examination of 10 colorectal cancers, an increase in EMT and stemness was observed in CPE (+) and BRAF mutation (+) cases. These findings suggest that C. perfringens might enhance the malignant transformation of SSA/P-D via YAP activation. Our findings further highlight the importance of controlling intestinal flora using probiotics or antibiotics.