Molecular Mechanism Of SQSTM1/P62 Enhancing The Resistance Of Cell Apoptosis In Esophageal Cancer Cells

Esophageal squamous cell carcinoma (ESCC) is a common cancer of the digestive system, with an overall five-year survival rate of about 10%. There is an urgent need to discover new therapeutic targets to improve the survival rates of ESCC patients. The previous studies have demonstrated that PKCiota (protein kinase C, iota) is an oncogene and exerts functions in cell apoptosis-resistance and tumor metastasis in ESCC. In the present work, we found the consistent expression of PKCiota and SQSTMl/p62 in the ESCC tissues (P< 0.001).p62 is a multifunctional adapter protein implicated in selective autophagy and multiple signaling pathways. However, the molecular mechanism underlying the role of p62 in esophageal squamous cells remains unclear. In order to characterize the funcution of p62 in ESCC cells, we firstly used fluorescent immunocytochemistry to detect the expression and localization of p62 in esophageal squamous carcinomas KYSE150, KYSE180, KYSE510, KYSE450, Eca-109 and COLO-680N cell lines, and observed that p62 expressed in the cytoplasm of the cell lines. We selected KYSE450 and KYSE180 to performe functional analysis by siRNA-mediated silencing. Under normal culture condition, p62 did not influence cell proliferation, apoptosis, colony formation assays and cell cycle. However, as serum starvation treatment, knockdown of p62 increased cell apoptosis and inhibited the colony formation. Especially, inhibition of p62 expression decreased tumor growth in nude mouse models.We next investigated the molecular mechanism of p62 enhancing the apoptosis resistance in ESCC cells under starvation conditions, and found that both protein and mRNA of SKP2 (S-phase kinase-associated protein 2) were significantly reduced in p62-siRNA transfected KYSE450 and KYSE180 cells detected by the techniques of Western blotting and reverse transcription polymerase chain reaction (RT-PCR). Silencing SKP2 expression markedly induced cell apoptosis. Further analysis showed that knockdown of p62 caused a decrease in SKP2 through regulating PKCiota expression. Co-immunoprecipitation (Co-IP) assays showed that there was an interaction between p62 and PKCiota. Addition of the proteasome inhibitor MG132 prevented the decrease of SKP2 protein level in p62 silenced cells, and polyubiquitin-SKP2 was elevated after p62 knockdown, suggesting that p62 might regulate the expression of SKP2 through the ubiquitin-proteasome pathway under serum starvation. These results indicate that PKCiota-SKP2 signaling pathway might mediate p62-induced apoptosis resistance, which enhances the survival and vitality of esophageal cancer cells.Then we examined the active domain of p62 through constructing three different truncated mutants of the p62 protein. The results showed that PB1 domain of p62 had biological activity. PB1 deletion reduced the colony formation ability of the cells, simultaneously down-regulated the expression of PKCiota. Treatment with an inhibitor of PKCiota, Aurothiomalate (ATM), showed no significant effect on the cell proliferation and apoptosis. However, ATM combined p62-silencing significantly enhanced the percentage of cell apoptosis compare to the non-treated cells. At the molecular level, we found that the expression of p62, PKCiota and SKP2 were declined in the group of ATM combined p62-silencing.In conclusion, p62 connected with PKCiota regulated the expression of SKP2 through the ubiquitin-proteasome pathway, which contributed to the resistance of cell apoptosis and the inhibition of colony formation. The data suggest that p62 may serve as a candidate molecular target for ESCC therapy.