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 tumours. 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（Human papillomavirus-related oropharyngeal squamous cell carcinoma, HPV+ OPSCC）是头颈癌中一类独特的疾病实体，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肿瘤中的作用。本研究从109个已发表的候选可变剪接事件中筛选出11个（GIT2、CTNNB1、MKNK2、MRPL33、SIPA1L3、SNHG6、SYCP2、TPRG1、ZHX2、ZNF331及ELOVL1）进行验证，以阐明HPV+相关疾病发生的转录后调控机制。体外（in vitro）实时定量逆转录聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）验证显示，11个候选可变剪接事件中有9个存在差异表达；癌症基因组图谱队列（The Cancer Genome Atlas cohort, TCGA cohort）的计算机模拟（in silico）分析则证实了其中8个的差异表达情况。其中，MRPL33、SIPA1L3、SNHG6、TPRG1、ZHX2及ELOVL1这6个可变剪接事件在两种分析方法中均呈现显著的差异表达。进一步的染色质修饰分析显示，可变剪接事件的表达与组蛋白3赖氨酸27乙酰化（acetylated lysine 27 of histone 3, H3K27ac）的肿瘤特异性分布显著相关。随后使用JQ1对HPV+头颈鳞癌细胞系（UM-SCC-047及UPCI-SCC-090）进行表观遗传干预，不仅可诱导肿瘤特异性可变剪接异构体的表达下调，还能抑制两种细胞系的增殖；其中UPCI-SCC-090细胞系的可变剪接事件表达水平更高，经JQ1处理后其生长抑制效果也更为显著。本研究证实了HPV+ OPSCC中多个新型肿瘤特异性可变剪接事件，并为染色质修饰调控HPV+ OPSCC中的可变剪接过程提供了实验依据，凸显了表观遗传改变在HPV+ OPSCC发生发展中的作用，为可重塑全局转录后调控网络的治疗手段提供了一个独特且尚未被充分探索的靶点。