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Disulfiram combined with copper induces immunosuppression via PD-L1 stabilization in hepatocellular carcinoma.

ABSTRACT: As a potential antitumor drug and chemotherapeutic sensitizer, disulfiram combined with Copper (DSF/Cu2+) does not exert considerable antitumor effects on an immunocompetent hepatocellular carcinoma (HCC) model. In this article, we will explore the mechanism underlying the resistance to DSF in HCC. We analyzed the antitumor effect of DSF/Cu2+ in vivo studies. Tumor and immune cells collected from mice were analyzed by flow cytometry. Then, we analyzed the transcriptional changes in liver cancer cells after DSF/Cu2+ treatment by transcriptional expression profiling. The expression of PD-L1 was detected by real-time PCR, Western blotting and flow cytometry. The expression of PARP1 and GSK3? was knocked down by small interfering RNAs (siRNAs). A subcutaneous Hepa1-6 tumor model was used for single-drug or combined-drug studies. Tissue chips (268 samples of liver cancer tissue) were used to analyze the relationship among PARP1, p-GSK3? and PD-L1. We found that DSF/Cu2+ failed to inhibit HCC tumor growth in C57BL/6 mice. DSF/Cu2+ upregulated PD-L1 expression by inhibiting PARP1 activity and enhancing GSK3? phosphorylation at Ser9 and ultimately inhibited T cell infiltration. The combination of DSF/Cu2+ and an anti-PD-1 antibody produced an additive effect that slowed HCC growth in mice. In addition, we observed negative associations between PARP1 and p-GSK3? (Ser9) or PD-L1 expression in tumor tissue samples from HCC patients. Through in vitro and in vivo studies, we found that DSF/Cu2+ could restrain GSK3? activity by inhibiting PARP1, leading to the upregulation of PD-L1 expression. Combination therapy with DSF/Cu2+ and an anti-PD-1 antibody showed much better antitumor efficacy than monotherapy.

SUBMITTER: Zhou B

PROVIDER: S-EPMC6895448 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Disulfiram combined with copper induces immunosuppression via PD-L1 stabilization in hepatocellular carcinoma.

Zhou Binghai B Guo Lei L Zhang Bo B Liu Shuang S Zhang Kewei K Yan Jiuliang J Zhang Wentao W Yu Mincheng M Chen Zheng Z Xu Yongfeng Y Xiao Yongsheng Y Zhou Jian J Fan Jia J Li Hui H Ye Qinghai Q

American journal of cancer research 20191101 11

As a potential antitumor drug and chemotherapeutic sensitizer, disulfiram combined with Copper (DSF/Cu<sup>2+</sup>) does not exert considerable antitumor effects on an immunocompetent hepatocellular carcinoma (HCC) model. In this article, we will explore the mechanism underlying the resistance to DSF in HCC. We analyzed the antitumor effect of DSF/Cu<sup>2+</sup> in vivo studies. Tumor and immune cells collected from mice were analyzed by flow cytometry. Then, we analyzed the transcriptional ch ...[more]

PMID: 31815045

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Project description:BackgroundImmune checkpoint inhibitor therapy targeting antiprogrammed cell death-1 (anti-PD-1) or its ligand (anti-PD-L1) is effective in the treatment of some hepatocellular carcinomas (HCC). Hence, further identification of biological targets related to PD-L1 regulation in HCC is beneficial to improve the clinical efficacy of immunotherapy. Some HCC cells express lysine-specific demethylase 1A (KDM1A), which is implicated in the reduced survival time of patients. Here, we studied whether the level of PD-L1 and the immunosuppression are regulated by KDM1A and its miRNA in HCC cells.MethodsIn the present study, we studied clinical data from The Cancer Genome Atlas (TCGA) database. We performed qPCR and western blotting assays to measure the expression level of genes of interest. PD-L1 expression was also analyzed by FACS. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 was used to generate gene knockout cells to investigate the relationships of genes of interest. We also developed a reporter gene assay (RGA) to explore the changes in T cell-induced antitumor immunity relative to PD-L1 expression in HCC cells. The binding between proteins and promoters or miRNAs and their target genes was explored by luciferase reporter assays.ResultsThe results showed that PD-L1 and KDM1A were increased in HCC patients and cells, and KDM1A promoted the expression of PD-L1 in HCC cells. Our findings showed that the enhancement of PD-L1 expression was not attributed to mitochondrial dysfunction caused by increases in KDM1A in HCC cells. Furthermore, we observed a lower level of MEF2D methylation in HCC cells than in normal human liver cells. Demethylated MEF2D could bind to the promoter of PD-L1 and activate its expression, while KDM1A interacted with MEF2D and acted as a demethylase to reduce its methylation. Moreover, a new miRNA, miR-329-3p, targeting KDM1A was found to regulate the PD-L1 expression profile in HCC cells. In the xenograft model, the tumors treated with miR-329-3p showed growth inhibition.ConclusionsMechanistically, miR-329-3p inhibits tumor cellular immunosuppression and reinforces the response of tumor cells to T cell-induced cytotoxic effect by targeting KDM1A mRNA and downregulating its expression, which contributed to MEF2D demethylation and activation of PD-L1 expression.

Project description:A TLR9 agonist in combination with a PD-1 inhibitor produced powerful antitumor responses in a clinical trial despite TLR9 agonists as monotherapies failing to generate systemic antitumor immune responses due to immunosuppressive effects. However, the mechanism involved in the improved response induced by their combination remains unknown. Methods: Subcutaneous and orthotopic Hepa1-6 tumor model was used for single-drug and combined-drug treatment. We used TLR9 agonist stimulation or lentiviral vectors to overexpress TLR9 and activate TLR9 signaling. We next investigated the crosstalk between PARP1 autoPARylation and ubiquitination and between STAT3 PARylation and phosphorylation mediated by TLR9. Tissue chips were used to analyze the relationships among TLR9, PARP1, p-STAT3 and PD-L1 expression. Results: In this study, we found that the TLR9 agonist in combination with anti-PD-1 therapy or anti-PD-L1 therapy yielded an additive effect that inhibited HCC growth in mice. Mechanistically, we found that TLR9 promoted PD-L1 transcription by enhancing STAT3 Tyr705 phosphorylation. Then, we observed that TLR9 negatively regulated PARP1 expression, which mediated a decrease in STAT3 PARylation and an increase in STAT3 Tyr705 phosphorylation. Moreover, we found that TLR9 enhanced PARP1 autoPARylation by inhibiting PARG expression, which then promoted the RNF146-mediated ubiquitination and subsequent degradation of PARP1. Finally, we observed positive associations between TLR9 and p-STAT3 (Tyr705) or PD-L1 expression and negative associations between TLR9 and PARP1 in HCC patient samples. Conclusions: We showed that hepatoma cell-intrinsic TLR9 activation regulated the crosstalk between PARP1 autoPARylation and ubiquitination and between STAT3 PARylation and phosphorylation, which together upregulated PD-L1 expression and finally induces immune escape. Therefore, combination therapy with a TLR9 agonist and an anti-PD-1 antibody or anti-PD-L1 had much better antitumor efficacy than either monotherapy in HCC.

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Disulfiram combined with copper induces immunosuppression via PD-L1 stabilization in hepatocellular carcinoma.

Publications

Disulfiram combined with copper induces immunosuppression via PD-L1 stabilization in hepatocellular carcinoma.

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