Chromatin structure regulates Cancer Specific Alternative Splicing Events in Primary HPV related Oropharyngeal squamous cell carcinoma

Human papillomavirus-related oropharyngeal squamous cell carcinoma (HPV+ OPSCC) represents a unique disease entity within head and neck cancer with rising incidence. Previous work has shown that alternative splicing events (ASEs) are prevalent in HPV+ OPSCC, but further validation is needed to understand the regulation of this process and its role in these tumors. In this study, eleven ASEs (<i>GIT2, CTNNB1, MKNK2, MRPL33, SIPA1L3, SNHG6, SYCP2, TPRG1, ZHX2, ZNF331</i>, and <i>ELOVL1)</i> were selected for validation from 109 previously published candidate ASEs to elucidate the post-transcriptional mechanisms of oncogenesis in HPV+ disease. <i>In vitro</i> qRT-PCR confirmed differential expression of 9 of 11 ASE candidates, and <i>in silico</i> analysis within the TCGA cohort confirmed 8 of 11 candidates. Six ASEs (<i>MRPL33, SIPA1L3, SNHG6, TPRG1, ZHX2</i>, and <i>ELOVL1)</i> showed significant differential expression across both methods. Further evaluation of chromatin modification revealed that ASEs strongly correlated with cancer-specific distribution of acetylated lysine 27 of histone 3 (H3K27ac). Subsequent epigenetic treatment of HPV+ HNSCC cell lines (UM-SCC-047 and UPCI-SCC-090) with JQ1 not only induced downregulation of cancer-specific ASE isoforms, but also growth inhibition in both cell lines. The UPCI-SCC-090 cell line, with greater ASE expression, also showed more significant growth inhibition after JQ1 treatment. This study confirms several novel cancer-specific ASEs in HPV+OPSCC and provides evidence for the role of chromatin modifications in regulation of alternative splicing in HPV+OPSCC. This highlights the role of epigenetic changes in the oncogenesis of HPV+OPSCC, which represents a unique, unexplored target for therapeutics that can alter the global post-transcriptional landscape.

人乳头瘤病毒相关口咽鳞状细胞癌（HPV+ OPSCC）是头颈癌中一类独特的病种，其发病率呈逐年上升趋势。既往研究表明，可变剪接事件（alternative splicing events, ASEs）在HPV+ OPSCC中广泛存在，但目前仍需进一步验证以阐明该过程的调控机制及其在这类肿瘤中的作用。本研究从已发表的109个候选ASE中筛选出11个（GIT2、CTNNB1、MKNK2、MRPL33、SIPA1L3、SNHG6、SYCP2、TPRG1、ZHX2、ZNF331及ELOVL1）进行验证，以阐明HPV相关肿瘤发生的转录后调控机制。体外（in vitro）qRT-PCR实验证实，11个候选ASE中有9个呈现差异表达；针对癌症基因组图谱（The Cancer Genome Atlas, TCGA）队列的in silico分析则验证了其中8个候选ASE。其中6个ASE（MRPL33、SIPA1L3、SNHG6、TPRG1、ZHX2及ELOVL1）在两种实验方法中均呈现显著差异表达。进一步的染色质修饰分析显示，ASE的表达与肿瘤特异性的组蛋白3赖氨酸27乙酰化（H3K27ac）分布模式显著相关。后续使用JQ1对HPV阳性头颈鳞状细胞癌（HPV+ HNSCC）细胞系UM-SCC-047和UPCI-SCC-090进行表观遗传处理后，不仅可下调肿瘤特异性的ASE异构体表达，还能抑制两种细胞系的增殖。其中ASE表达水平更高的UPCI-SCC-090细胞系，在JQ1处理后呈现出更为显著的增殖抑制效果。本研究证实了HPV+ OPSCC中多个全新的肿瘤特异性ASE，并为染色质修饰调控HPV+ OPSCC可变剪接过程提供了实验依据。该研究凸显了表观遗传改变在HPV+ OPSCC肿瘤发生中的作用，同时也为可重塑全局转录后调控网络的治疗手段提供了全新的、尚未被探索的药物靶点。