Theranostics 2020; 10(14):6530-6543. doi:10.7150/thno.44417

Research Paper

Hepatoma cell-intrinsic TLR9 activation induces immune escape through PD-L1 upregulation in hepatocellular carcinoma

Binghai Zhou*1, Jiuliang Yan*1, Lei Guo*1, Bo Zhang*1, Shuang Liu*2, Mincheng Yu1, Zheng Chen1, Kewei Zhang3, Wentao Zhang1, Xiaoqiang Li4, Yongfeng Xu1, Yongsheng Xiao1, Jian Zhou1, Jia Fan1, Mien-Chie Hung5, Hui Li1✉, Qinghai Ye1✉

1. Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion (Fudan University), Ministry of Education, Shanghai, China.
2. Department of Neurosurgery, Zhongshan Hospital, Fudan University, Shanghai, China.
3. Department of General Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
4. Department of Thoracic Surgery, Peking University Shenzhen Hospital, Shenzhen, China.
5. Graduate Institute of Biomedical Sciences and Center for Molecular Medicine, China Medical University, Taichung 404, Taiwan.
* BH-Z, JL-Y, LG, BZ and SL contributed equally to this work.

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Citation:
Zhou B, Yan J, Guo L, Zhang B, Liu S, Yu M, Chen Z, Zhang K, Zhang W, Li X, Xu Y, Xiao Y, Zhou J, Fan J, Hung MC, Li H, Ye Q. Hepatoma cell-intrinsic TLR9 activation induces immune escape through PD-L1 upregulation in hepatocellular carcinoma. Theranostics 2020; 10(14):6530-6543. doi:10.7150/thno.44417. Available from http://www.thno.org/v10p6530.htm

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Abstract

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.

Keywords: PD-L1, Hepatocellular carcinoma, TLR9, PARP1, STAT3