The Role And Molecular Mechanism Of STUB1 In Regulating Anti-tumor Immunity Of Hepatocellular Carcinoma

Background:Liver cancer is the second leading cause of cancer-related death worldwide.Hepatocellular carcinoma(HCC)accounts for more than 90% of primary liver cancers,and the vast majority of patients are diagnosed in the advanced stages of the disease.Molecular targeted drugs are currently the only effective treatment for advanced HCC patients,but the overall survival benefit of patients is still not ideal.Therefore,there is an urgent need to develop more effective and innovative treatments for liver cancer.STUB1(STIP1 is homologous and contains U-box protein 1),a member of the E3 ligase containing U-box domain,mainly involves in the ubiquitination process of protein-related endoplasmic reticulum degradation pathways,and acts as a chaperone molecule involved in the quality control of endoplasmic reticulum by recognizing the unnatural state of the protein.STUB1 is widely involved in tumor development,proliferation,and invasion.However,the effects of STUB1 on tumor microenvironment have not been reported.Currently,the role of STUB1 in liver cancer is unclear.PD-L1(Programmed cell death ligand 1,also known as CD274)is the major inhibitory ligand of PD-1(Programmed cell death protein 1,also known as CD279)on the surface of T cells after antigen activation.After PD-L1 binds to PD-1,it can limit the proliferation and activation of T cells,so that tumor cells can escape the killing and elimination of T cells.Previous studies have reported that STUB1 may be a negative regulator of PD-L1 in melanoma,but the specific regulatory mechanism is unclear.In liver cancer,the effect of STUB1 on PD-L1 and its molecular mechanism have not been reported.Aims:To study the difference of STUB1 expression in HCC cancer and adjacent tissues,analyze the role of STUB1 expression in the long-term prognosis of HCC patients,and verify the potential value of STUB1 as a biomarker in HCC.To clarify the effect of STUB1 on immune cell infiltration in liver cancer microenvironment and its underlying mechanism.To elucidate the effect of STUB1 on the expression of PD-L1 in liver cancer cells and its underlying molecular mechanism.Methods:We used immunohistochemical staining to detect the expression of STUB1 in pathological tissues and adjacent tissues of 70 pairs of HCC patients,and compared the difference in STUB1 expression between HCC tissues and their adjacent tissues.The correlation between STUB1 protein expression level and clinicopathological characteristics and long-term prognosis in 70 cases of HCC was analyzed.STUB1 stable transfection cell lines was constructed.The effects of STUB1 on the proliferation and apoptosis of HCC cells in vitro were investigated by CCK-8 experiments,clone formation experiments,and flow cytometry.To observe the effects of knockdown or overexpression of STUB1 on tumor growth in vivo,we constructed mouse orthotopic liver cancer models using STUB1 stable knockdown or overexpression Hepa 1-6 cells.Next,we analyzed the correlation between STUB1 expression level and tumor-associated macrophages(TAM)?CD8+T cells and other immune cell infiltration levels through the TIMER database.Subsequently,the expression of STUB1 and the infiltration level of TAM(CD163 +)in the tumor tissues of 70 HCC patients were detected by immunohistochemical staining.Then the flow cytometry was used to analyze the infiltration level of M2 TAM in mouse liver carcinoma orthotopic tumors constructed by Hepa 1-6 stable knockdown cells and their normal controls.Through RNAseq and subsequent KEGG and GSEA analysis,we explored the genes and related signaling pathways regulated by STUB1.The effect of STUB1 on the expression of CSF-1 in the cell supernatant was detected by ELISA experiments.the TIMER database was used to predict the correlation between STUB1 m RNA expression level and PD-L1 m RNA expression level in HCC.The effects of STUB1 knockdown on PD-L1 m RNA and protein levels in HCC cells with or without IFN-? stimulation were analyzed by q RT-PCR,western blotting,and flow cytometry.Flow cytometry was used to detect the apoptosis and exhaustion of CD8+T cells in orthotopic tumors constructed by Hepa 1-6 stable knockdown cells and their normal controls.Finally,through western blotting,and co-immunoprecipitation experiments,the regulation of STUB1 on the IFN-?-JAK2-STAT3 signal axis in liver cancer and its direct mechanism were explored.Results:The expression level of STUB1 in HCC was significantly lower than that in adjacent tissues.The expression level of STUB1 in HCC has no significant correlation with clinical pathological parameters such as tumor size,distant metastasis,and TNM staging.However,the survival analysis showed that the overall survival rate of patients with low expression of STUB1 was worse than that of patients with high expression of STUB1.Multivariate Cox regression analysis showed that low expression of STUB1 was an independent risk factor for survival prognosis in patients with HCC.Cell functional experiments showed that STUB1 does not affect the proliferation and apoptosis of HCC cells in vitro,but in vivo experiments have found that knocking down STUB1 in Hepa1-6 cells can promote the growth of HCC in orthotopically bearing mice,and overexpress STUB1 in Hepa1-6 cells can inhibit the growth of HCC in orthotopically bearing mice.By analyzing the correlation between the expression of STUB1 and the degree of TAM infiltration in tissue samples from HCC patients and mouse orthotopic tumors,we found that the expression level of STUB1 in liver cancer was inversely correlated with the infiltration level of M2 TAM in the microenvironment.The results of KEGG and GSEA analysis suggest that STUB1 is mainly involved in the signaling pathway of cytokine-cytokine receptor interaction.ELISA results showed that STUB1 can regulate the secretion of CSF-1 in HCC cells.Using TIMER database analysis,we found that the m RNA expression level of STUB1 in HCC was inversely related to the m RNA expression level of PD-L1.In vitro experiments revealed that the PD-L1 m RNA and protein expression levels regulated by STUB1 depends on the effect of IFN-?.In vivo experiments further found that knockdown of STUB1 in tumor cells can increase the proportion of apoptotic and exhausted CD8+T cells in the microenvironment.Mechanism studies have found that STUB1 can mediate the ubiquitination of IFNGR2,thereby inhibiting the activation of JAK2-STAT3 signaling pathway by IFN-?,and then down-regulating the expression of PD-L1 in tumor cells.Conclusion:1.Compared with the adjacent tissues of HCC,the expression level of STUB1 in HCC cancer tissues decreased significantly.The overall survival of HCC patients with low expression of STUB1 is worse than that of patients with high expression,and low expression of STUB1 is an independent risk factor for prognosis of HCC patients.2.The down-regulation of STUB1 expression in HCC promotes the secretion of CSF-1 and increases the infiltration level of M2 TAM in the tumor microenvironment,which in turn affects the occurrence and development of tumors.3.STUB1 can mediate the ubiquitination of IFNGR2 in HCC.STUB1 expression is down-regulated in HCC,which can activate IFN-?-mediated activation of the JAK2-STAT3 pathway,up-regulate PD-L1 expression on the surface of tumor cells,thereby inhibiting the activity of CD8 + T cells in the tumor microenvironment,and ultimately suppress anti-tumor immunity.