Golgi Membrane Protein 1 Regulates The Mechanism Of Anti-tumor Immune Response By Influencing Autophagy

Objective:As a worldwide public health concern,cancer is the major cause of human death with a rising morbidity and mortality.Recently,immunotherapy has made a breakthrough for cancer treatment.In consideration of the limitations and side effects of immunotherapy,there is an urgent need to find some reasonable biomarkers and therapeutic targets to assess therapeutic rationality according to individualized principles,and continuously optimize therapeutic options.Golgi membrane protein 1(GOLM1)is located in the cis and medial Golgi apparatus,our previous studies have confirmed that serum GOLM1 could be used as a serological biomarker for early screening and diagnosis of various types of tumor,and the serum GOLM1 level in patients with benign liver diseases or hepatocellular carcinoma is significantly higher than that of healthy people.On this basis,this study aims to verify the expression of GOLM1 and immune changes in tumor microenvironment,and clarify the mechanism of GOLM1-mediated anti-tumor immune response as well as provide theoretical possibilities for the translational development and application of GOLM1.Methods:To evaluate the expression of GOLM1 in tumor tissues and its association with clinic-pathologic features,we collected 161 patients with primary hepatocellular carcinoma and 100 control liver samples.Combining with the data of immunohistochemical staining score of GOLM1 and CD8 in tumor tissue chips,the pathological data and follow-up information,statistical analyses were carried out.To prove that GOLM1 deficiency could result in immune-dependent inhibition of tumor growth,we monitored tumor growth of immunocompetent BALB/c or C57BL/6 mice subcutaneously inoculated WT,Golml-/-,Golm1-/-or Golml-/-H22 hepatoma cells and MCA205 fibrosarcoma cells.To explore whether GOLM1 deficiency could mediate local immune response,mouse subcutaneous tumor models were constructed,and the percentages of immune cell component and intracellular cytokines were observed by flow cytometry,immunohistochemical assays and enzyme-linked immune spot assay respectively.The differential expression of genes in GOLM1 deficient tumors was analyzed by RNA-seq and verified by qPCR,and the association between GOLM1 deletion and interferon pathway was verified by the tumor growth of Ifnar--mice.To study the increased immunogenicity mediated by GOLM1 deficiency,cells were treated with anthracyclines,and cell apoptosis,calreticulin exposure and HMGB1 or ATP release were detected through flow cytometry,Western blot,ELISA and chemiluminiscence assay.Furthermore,we constructed the knockout of Atg5 and the overexpression of ATPase CD39 to prove whether the promotion of tumor immune response mediated by GOLM1 deficiency was related to the autophagy-induced ATP release.To explore the correlation between GOLM1 inhibition and autophagy-related pathways,we made RFP-GFP-LC3 overexpression cell lines,and the changes of autophagy flux and expression of key proteins in AKT/mTOR related autophagy pathway was detected by immunofluorescence assay and Western blot.Results:(1)By immunohistochemical detection of hepatocellular carcinoma tissues and control liver tissues,we found that GOLM1 was highly expressed in tumor tissues,and the expression of CD8 showed a negative correlation with GOLM1 level.Combined with clinical information,our analysis showed that high expression of GOLM1 was related to malignant differentiation degree and poor overall survival rate.(2)Tumor growth curves indicated that GOLM1 deficiency had a significant inhibitory effect on tumor growth in immunocompetent mice,rather than in immunodeficient mice.There was a remarkable increase of the proportion of CD4-T cells,CD8-T cells,MHC-IIhiCD11c-dendritic cells,MHC-IIhiF4/80-macrophages,CD1 lb-Ly6ChiLy6G-myeloid cells,and the IFNy-secreting CD4-and CD8-T cells in GOLM1 deficient tumors.(3)Bioinformatic analysis suggested that GOLM1 deficiency was associated with interferon pathway,and deletion of Golml caused the overexpression of interferon related genes,such as Ifnar,Ccl4 and Cxclll.Besides,GOLM1 deficiency could not inhibit tumor growth in Ifnar--mice.(4)Under the treatment of anthracyclines,GOLM1 deficient cells showed more early apoptosis.Moreover,the expression of Cleaved-Caspase8 and Cleaved-PARP increased,c-FLIP decreased,and the releases of HMGB1 and ATP were upregulated in GOLM1 deficeint cells.Knockout of Atg5 might block the increase of ATP release which was induced by GOLM1 deficiency,and overexpression of ATPase CD39 could promote the growth of GOLM1 deficiency tumor.(5)In the condition of starving,autophagy of Golm1--cells increased obviously,the expression of Beclinl and ATG7 increased,SQSTM1/p62 decreased,and the ratio of LC3B-II/LC3B-I increased.Inhibition of GOLM1 affected AKT/mTOR pathway,characterized as enhanced phosphorylation of AKT and mTOR,and increased expression of ULK1,ATG13 and FIP200.Conclusion:GOLM1 is not only a novel biomarker for early diagnosis and prognostic assessment of hepatocellular carcinoma,but also a contributor to inhibit immune-dependent tumor growth.The therapeutic efficacy of cancer treatments relies on tumor cell immunogenicity to trigger antitumor immune response.GOLM1 deficiency promotes autophagy through AKT-mTOR pathway and manifests an increase of autophagy-induced extracellular ATP release,which further enhances tumor cell immunogenicity and benefits for anti-tumor immune response.Therefore,our founding uncovered that GOLM1 could be a potential biomarker and immune checkpoint blocker target for tumor immunotherapy.