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BCLAF1 binds SPOP to stabilize PD-L1 and promotes the development and immune escape of hepatocellular carcinoma.

ABSTRACT: Interaction between programmed death-1 (PD-1) ligand 1 (PD-L1) on tumor cells and PD-1 on T cells allows tumor cells to evade T cell-mediated immune surveillance. Strategies targeting PD-1/PD-L1 have shown clinical benefits in a variety of cancers. However, limited response rates in hepatocellular carcinoma (HCC) have prompted us to investigate the molecular regulation of PD-L1. Here, we identify B cell lymphoma-2-associated transcription factor 1 (BCLAF1) as a key PD-L1 regulator in HCC. Specifically, BCLAF1 interacts with SPOP, an E3 ligase that mediates the ubiquitination and degradation of PD-L1, thereby competitively inhibiting SPOP-PD-L1 interaction and subsequent ubiquitination and degradation of PD-L1. Furthermore, we determined an SPOP-binding consensus (SBC) motif mediating the BCLAF1-SPOP interaction on BCLAF1 protein and mutation of BCLAF1-SBC motif disrupts the regulation of the SPOP-PD-L1 axis. In addition, BCLAF1 expression was positively correlated with PD-L1 expression and negatively correlated with biomarkers of T cell activation, including CD3 and CD8, as well as with the level of immune cell infiltration in HCC tissues. Besides, BCLAF1 depletion leads to a significant reduction of PD-L1 expression in vitro, and this reduction of PD-L1 promoted T cell-mediated cytotoxicity. Notably, overexpression of BCLAF1 sensitized tumor cells to checkpoint therapy in an in vitro HCC cells-Jurkat cells co-culture model, whereas BCLAF1-SBC mutant decreased tumor cell sensitivity to checkpoint therapy, suggesting that BCLAF1 and its SBC motif serve as a novel therapeutic target for enhancing anti-tumor immunity in HCC.

SUBMITTER: Yu Z

PROVIDER: S-EPMC10858942 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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BCLAF1 binds SPOP to stabilize PD-L1 and promotes the development and immune escape of hepatocellular carcinoma.

Yu Zongdong Z Wu Xiang X Zhu Jie J Yan Huan H Li Yuxuan Y Zhang Hui H Zhong Yeling Y Lin Man M Ye Ganghui G Li Xinming X Jin Jiabei J Li Kailang K Wang Jie J Zhuang Hui H Lin Ting T He Jian J Lu Changjiang C Xu Zeping Z Zhang Xie X Li Hong H Jin Xiaofeng X

Cellular and molecular life sciences : CMLS 20240210 1

Interaction between programmed death-1 (PD-1) ligand 1 (PD-L1) on tumor cells and PD-1 on T cells allows tumor cells to evade T cell-mediated immune surveillance. Strategies targeting PD-1/PD-L1 have shown clinical benefits in a variety of cancers. However, limited response rates in hepatocellular carcinoma (HCC) have prompted us to investigate the molecular regulation of PD-L1. Here, we identify B cell lymphoma-2-associated transcription factor 1 (BCLAF1) as a key PD-L1 regulator in HCC. Specif ...[more]

PMID: 38340178

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Project description:BackgroundHypoxia is a hallmark of cancer, and is closely intertwined with tumor immune evasion. Circular RNAs (circRNAs) have been implicated in tumor response to immune checkpoint blockades. However, hypoxia-associated circRNAs that orchestrate the association between hypoxia and response to immunotherapy remain poorly understood. Here, we aimed to determine the roles of hypoxia-associated circRNAs in immune escape of hepatocellular carcinoma (HCC) cells.MethodsDifferentially expressed hypoxia-associated circRNAs were determined using high-throughput sequencing technology. HCC patients treated with PD-1 blockade were enrolled to assess the clinical significance of circPRDM4. RT-qPCR, western blotting, flow cytometry, T cell-mediated tumor cell killing assay, and enzyme linked immunosorbent assay were used to investigate the roles of circPRDM4 in immune escape of HCC cells in vitro. Patient-derived xenograft mouse models and adoptive human tumor infiltrating lymphocyte-CD8+ T cell transfer were adopted to evaluate the effects of circPRDM4 in vivo. RNA pull-down, mass spectrometry, RNA immunoprecipitation, chromatin immunoprecipitation, chromatin isolation by RNA purification, dual-luciferase reporter assays, dot blotting, DNA in situ hybridization, and immunoprecipitation were utilized to examine the interaction between circPRDM4, HIF-1α, and CD274 promoter.ResultsWe identified circPRDM4 as a hypoxia-associated circRNA in HCC. circPRDM4 was upregulated in responders to PD-1 blockade and associated with therapeutic efficacy. In vitro and in vivo experiments showed that circPRDM4 induced PD-L1 expression and promoted CD8+ T cell-mediated immune escape under hypoxic conditions. Mechanistically, circPRDM4 acted as a scaffold to recruit HIF-1α onto CD274 promoter, and cemented their interaction, ultimately promoting the HIF-1α-mediated transactivation of PD-L1.ConclusionsThese findings illustrated that circPRDM4 promoted immune escape of HCC cells by facilitating the recruitment of HIF-1α onto the promoter of CD274 under hypoxia, thereby inhibiting CD8+ T cell infiltration in the tumor microenvironment. This work may provide a novel prognostic biomarker and therapeutic candidate for HCC immunotherapy.

Project description:Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor with poor prognosis, and currently effective therapeutic strategies are still limited. Although temozolomide (TMZ) is commonly used for GBM therapy and its mechanism was well characterized, while its side effects were required comprehensive investigation. In the present study, we revealed that TMZ-challenged GBM cells strongly suppressed pro-inflammatory cytokines expression in activated periphery blood mononuclear cells (PBMC), which depended on enhanced transcription of CD274 (encoding PD-L1), but not other immune checkpoints, such as CD276, HVEM and galectin-9. Moreover, abundance of membranous PD-L1 was also increased in TMZ-treated GBM cells. When PD-L1 expression was knocked down by short hairpin RNA (shRNA), inhibitory effect of TMZ-treated GBM cells on PBMC became weakened, suggesting that PD-L1 was crucial for immune inhibition capacity of TMZ-treated GBM cells. Additionally, actinomycin D reduced PD-L1 expression in GBM cells after TMZ challenge, indicating that PD-L1 induction occurred at transcriptional level. The immunoblotting results demonstrated that STAT3 signaling was involved in TMZ-mediated PD-L1 induction, and attenuated expression of PD-L1 was observed using STAT3 inhibitor VI or STAT3 shRNA. Finally, the animal study showed that combination of TMZ and PD-1 antibody therapy strongly inhibited tumor growth and achieved the improved survival rate of GBM mice. Accordingly, this study revealed the classical chemotherapy drug TMZ promoted GBM cells immune escape, even TMZ combine with PD-1 antibody treatment not further improve survival ratio of recurrent GBM patients compared with traditional therapy methods, while our animal study provided evidence that combination of TMZ and PD-1 antibody was a promising way to treat GBM, these contradictory results indicate improving the PD-1 antibody delivery efficiency can exert strong combinational therapy outcomes.

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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BCLAF1 binds SPOP to stabilize PD-L1 and promotes the development and immune escape of hepatocellular carcinoma.

Publications

BCLAF1 binds SPOP to stabilize PD-L1 and promotes the development and immune escape of hepatocellular carcinoma.

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