Human papillomavirus-encoded circular RNA circE7 promotes immune evasion in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a prevalent malignancy with limited success using immunotherapy alone. While immunotherapy using immune checkpoint inhibitors like anti-PD-1/PD-L1 antibodies shows promise, over 80% of patients exhibit drug resistance. Human Papillomavirus (HPV) infection plays a significant role in HNSCC development, and its encoded proteins influence immune responses. High-risk HPVs, such as HPV16, are strongly associated with HNSCC, and the oncogenic proteins E6 and E7 are crucial in tumorigenesis. Circular RNAs (circRNAs), generated from reverse splicing of precursor mRNAs, are involved in various biological processes, including tumor development. HPV can encode circRNAs, with circE7 being one specifically encoded by high-risk HPV16. This study investigates the role of HPV-encoded circE7 in HNSCC immune evasion and its potential impact on immunotherapy response.

The literature review section details previous research on HNSCC, highlighting its prevalence and treatment challenges. It discusses the role of HPV infection and its encoded proteins (E6, E7) in HNSCC development and immune evasion. The review covers the increasing understanding of circRNAs and their role in tumorigenesis, particularly the involvement of HPV-encoded circRNAs. Finally, it highlights the limited existing research on the specific role of circE7 in HNSCC immune evasion and the rationale for further investigation into its function and possible therapeutic implications.

The study used a combination of in vitro and in vivo experiments. Initially, they assessed circE7 expression in HNSCC tissue samples using PCR and in situ hybridization. The correlation between circE7 expression and CD8+ T cell infiltration was analyzed using immunohistochemistry. In vitro experiments involved overexpressing or knocking down circE7 in HNSCC cell lines and co-culturing them with primary human T cells to assess T cell function and activity. The expression of T cell effector molecules (TNF-α, GZMB, IFN-γ) and galectin-9 was evaluated using RT-qPCR, Western blotting, flow cytometry, and ELISA. The molecular mechanism of circE7's action was investigated using RNA pull-down, RNA immunoprecipitation (RIP), and Chromatin immunoprecipitation (ChIP) assays. In vivo experiments utilized HNSCC cell lines (HN30 and SCC090) with manipulated circE7 expression, implanted into nude mice, followed by immunotherapy with PD-1 and TIM-3 monoclonal antibodies. Tumor growth, T cell infiltration, and cytokine expression were monitored. The effects of blocking TIM-3 and PD-1 were investigated using blocking antibodies and lactose. Additional experiments included assessing intracellular acetyl-CoA levels and global/histone acetylation levels. Statistical analysis included Student's t-tests, Pearson correlation tests, and Kaplan-Meier analysis. Specific techniques used included RT-qPCR, Western blotting, flow cytometry, ELISA, immunohistochemistry, immunofluorescence, RNA fluorescence in situ hybridization (FISH), RNA pull-down, RIP, ChIP-qPCR, ChIP-seq, and in vivo imaging.

The study found a significant negative correlation between circE7 expression and CD8+ T cell infiltration in HNSCC tissues. In vitro experiments showed that circE7 suppresses T cell function and activity by downregulating LGALS9, the gene encoding galectin-9. Mechanistically, circE7 binds to ACC1, promoting its dephosphorylation and reducing H3K27 acetylation at the LGALS9 promoter, thereby decreasing galectin-9 expression. Galectin-9 interacts with TIM-3 and PD-1 on T cells, promoting cytotoxic cytokine secretion and inhibiting T cell apoptosis. In vivo experiments demonstrated that combined anti-PD-1 and anti-TIM-3 therapy significantly enhanced the efficacy of HNSCC immunotherapy, especially in HPV16+ HNSCC, reversing the immune evasion promoted by circE7. The study also found that circE7 binds to ACC1, leading to reduced acetyl-CoA levels and decreased H3K27 acetylation at the LGALS9 promoter. This epigenetic modification suppresses galectin-9 expression, resulting in impaired T cell function and immune evasion. Combined treatment with anti-PD-1 and anti-TIM-3 antibodies effectively counteracted the effects of circE7, improving immunotherapy efficacy both in vitro and in vivo. The combination of anti-PD-1 and anti-TIM-3 antibodies resulted in increased T cell infiltration, reduced T cell apoptosis, and increased cytotoxic cytokine production, resulting in decreased tumor size and weight in mouse models.

This study identifies circE7 as a critical mediator of immune evasion in HPV16+ HNSCC. The findings highlight a novel mechanism by which HPV promotes immune escape through epigenetic modification driven by circE7. The negative correlation between circE7 and CD8+ T cell infiltration, along with the in vitro and in vivo data supporting circE7's suppression of T cell function via LGALS9 downregulation, strongly supports this conclusion. The success of combined anti-PD-1 and anti-TIM-3 therapy suggests a promising new therapeutic strategy for HNSCC, especially in patients with high circE7 expression. The mechanistic insights into circE7's function through ACC1 modulation and epigenetic regulation of LGALS9 are significant contributions to the understanding of HPV-related HNSCC.

This research reveals a novel mechanism of immune evasion in HPV16-positive HNSCC driven by the viral-encoded circE7. circE7 suppresses T cell function by downregulating galectin-9 expression through an epigenetic mechanism involving ACC1. The study's findings suggest that targeting both PD-1 and TIM-3 pathways may be a more effective immunotherapy strategy for HPV16-positive HNSCC. Future research should explore the clinical significance of circE7 as a biomarker and therapeutic target.

The study primarily focused on HPV16-positive HNSCC, limiting the generalizability of the findings to other HPV subtypes. The in vivo studies used nude mice, which lack a functional immune system, potentially underestimating the complexity of the immune response in humans. Further investigation is needed to validate the findings in larger cohorts of human patients and to explore the potential efficacy of combined PD-1 and TIM-3 blockade in clinical trials.