The Molecular Mechanism Of Heat Shock Protein Gp96 Promoting Hepatocarcinogenesis

Liver cancer is the sixth most common cancer in incidence and the second most common cause of cancer mortality worldwide. It is intrinsically resistant to cytotoxic chemotherapies due to potent anti-apoptotic activity and unique regenerative characteristics. Multiple signaling pathways and complex signaling networks are involved in hepatocarcinogenesis and malignant progression, including the NF-κB, mutant p53, JAK-STAT, and Raf/MAPK/ERK pathways. Uncovering the potential specific targets and blocking anti-apoptotic pathways may be a more efficient way to develop liver cancer treatments.As one of the most abundant chaperones in the endoplasmic reticulum (ER), heat shock protein (HSP) gp96 associates with client nascent polypeptides and guides their maturation and assembly into large multimeric protein complexes in the secretory pathway. But elevated expression of gp96 is observed in liver tumor issues and correlates with tumor phenotypes and disease progression. In this study, we investigated the roles and mechanism of gp96 on liver tumor cell growth and apoptosis. Ultimately, our results may evaluate whether gp96 can act as a new therapeutic target for liver cancer treatment.To investigate if gp96 affects hepatocarcinoma apoptosis, we generated two stable gp96 knockdown cell lines, SK-Hep-l-gp96 KD and HepG2-gp96 KD using a shRNA lentiviral expression system. Knockdown of gp96 led to significantly decreased cell proliferation and induced accumulation of cells in G0/G1 phase compared to mock cells. Notably, gp96 depletion resulted in increased cell apoptosis and susceptibility to chemotherapeutic drug, indicating that heat shock protein gp96 act as a potential role in regulating liver tumor cell growth and apoptosis.P53 protein was identified as a gp96 client protein by profiling apoptosis-related proteins in gp96-knockdown liver cancer cells. Overexpression and knockdown studies both demonstrated that gp96 decreases p53 protein levels. Gp96 down-regulates p53 at the posttranscriptional level as no significant change in mRNA levels were observed in gp96-knockdown cells. Then HCT116 p53 wild-type (+/+) and null (-/-) isogenic colorectal cancer cell lines were used to further explore the role of p53 in gp96-mediated apoptosis and cell cycle regulation. The results indicate that the effects of gp96 on p53 and its downstream molecules p21 and caspase-3, as well as its effect on cell apoptosis and cell-cycle arrest, were largely abolished in the p53 null cells, which demonstrated that gp96 inhibits cell apoptosis and cell-cycle G1 arrest in a p53-dependent manner. We further provide evidence that gp96 interacts with both p53 and Mdm2 by Co-IP and GST pull down assays. Gp96 may act as a scaffolding protein in which M-terminal domain interact with p53 and N-terminal domain interact with Mdm2 respectly, which enhances Mdm2-mediated p53 ubiquitination and degradation. Moreover, targeting gp96 with siRNA induced cell apoptosis and led to the suppression of liver tumor growth in vivo.In conclusion. First, this study represents an effort to address the underlying mechanism of altered p53 levels in liver cancer. Second, our results demonstrate that gp96 may function as a scaffolding protein to increase Mdm2 E3 ligase activity for p53 ubiquitination and degradation. Moreover, targeting gp96 with siRNA induces cell apoptosis and led to the suppression of liver tumor growth in vivo. Given that gp96 expression is elevated in liver cancer cells but not in normal hepatocytes, our study therefore supports the notion that the inhibition of gp96 might provide a novel therapeutic approach for liver cancer.