Preferential Activation of the Hedgehog Pathway by Epigenetic Modulations in HPV Negative HNSCC Identified with Meta-Pathway Analysis

Head and neck squamous cell carcinoma (HNSCC) is largely divided into two groups based on their etiology, human papillomavirus (HPV)-positive and –negative. Global DNA methylation changes are known to drive oncogene and tumor suppressor expression in primary HNSCC of both types. However, significant heterogeneity in DNA methylation within the groups results in different transcriptional profiles and clinical outcomes. We applied a meta-pathway analysis to link gene expression changes to DNA methylation in distinguishing HNSCC subtypes. This approach isolated specific epigenetic changes controlling expression in HPV− HNSCC that distinguish it from HPV+ HNSCC. Analysis of genes identified Hedgehog pathway activation specific to HPV− HNSCC. We confirmed that GLI1, the primary Hedgehog target, showed higher expression in tumors compared to normal samples with HPV− tumors having the highest GLI1 expression, suggesting that increased expression of GLI1 is a potential driver in HPV− HNSCC. Our algorithm for integration of DNA methylation and gene expression can infer biologically significant molecular pathways that may be exploited as therapeutics targets. Our results suggest that therapeutics targeting the Hedgehog pathway may be of benefit in HPV− HNSCC. Similar integrative analysis of high-throughput coupled DNA methylation and expression datasets may yield novel insights into deregulated pathways in other cancers.头颈部鳞状细胞癌（head and neck squamous cell carcinoma, HNSCC）根据病因学特征可大体分为两大亚型：人乳头瘤病毒（human papillomavirus, HPV）阳性亚型与HPV阴性亚型。已有研究证实，两种亚型的原发性HNSCC中，全基因组DNA甲基化改变均可调控癌基因与抑癌基因的表达。然而，同一亚型内部的DNA甲基化存在显著异质性，进而导致不同的转录组特征与临床结局。本研究采用元通路分析方法，将基因表达变化与DNA甲基化水平相关联，以区分HNSCC的不同亚型。该方法筛选出了调控HPV阴性亚型肿瘤基因表达的特异性表观遗传改变，这些改变可有效区分HPV阳性与阴性亚型的HNSCC。对筛选得到的基因进行分析后，发现刺猬信号通路（Hedgehog pathway）的激活特异性存在于HPV阴性亚型的HNSCC中。本研究进一步证实，作为刺猬信号通路的核心下游靶点，GLI1在肿瘤组织中的表达水平显著高于正常组织；其中HPV阴性亚型肿瘤的GLI1表达水平最高，这提示GLI1表达上调可能是HPV阴性亚型HNSCC的潜在驱动因素。本研究开发的整合DNA甲基化与基因表达数据的算法，可推断出具有重要生物学意义的分子通路，这些通路有望成为潜在治疗靶点。研究结果表明，靶向刺猬信号通路的治疗手段或许可使HPV阴性亚型的HNSCC患者获益。类似的针对高通量配对DNA甲基化与基因表达数据集的整合分析，或可为其他癌症中失调通路的研究提供全新的科学见解。