Epigenetic role of GRHL2 in HPV-associated oral cancer

描述（由申请人提供）：我们的长期研究目标是发现新的生物标志物和有效的治疗口腔癌的方法。目前的项目将通过表观遗传机制研究grainyhead -样2 （GRHL2）在介导“高风险”人乳头瘤病毒（HPV）口腔癌发生中的作用。GRHL2通过多个靶基因调控角质形成细胞的命运，包括人类端粒酶逆转录酶（hTERT）基因、叉头盒蛋白1 （FoxM1）和表皮分化复合体（EDC）基因。我们最近发表的数据显示，GRHL2是角化细胞增殖和分化的主要调节因子。GRHL2通过表观遗传机制引发这些影响，包括改变靶基因启动子的DNA甲基化和组蛋白修饰。我们发现GRHL2在含有“高风险”HPV和HPV+口腔鳞状细胞癌（oscc）的人口腔角化细胞（HOKs）中被显著诱导。此外，在富含癌症干细胞（CSCs）的OSCC肿瘤球体中检测到高水平的GRHL2。当GRHL2在OSCCs中被敲低时，体外肿瘤球体的自我更新和体内的致瘤性均显著受损。因此，HPV可能通过诱导FoxM1癌基因导致GRHL2的失调，从而促进其致癌活性，并增强CSC的自我更新。近年来的研究强调了非编码rna在多能性调控和CSC自我更新中的重要性。GRHL2上调miR-21，一种与HPV和CSC表型相关的“肿瘤因子”。因此，GRHL2对非编码rna的差异调控可能是OSCCs中CSC表型增强的机制基础。为了研究HPV在口腔癌变中的病理生物学作用，我们构建了能够表达病毒致癌基因的感染性HPV16病毒粒子。这些新的HPV病毒粒子将使我们能够在控制良好的实验中测试HPV感染对原发性NHOK的影响。在当前的项目中，我们将通过改变其靶基因的表观遗传调控和增强CSC自我更新来验证GRHL2是hpv相关细胞畸变所必需的假设，从而维持口腔癌表型。这一新假设将通过研究GRHL2在hpv相关细胞畸变中的病理生理作用来验证；确定GRHL2调节上皮细胞增殖和分化的表观遗传机制，以及HPV如何改变GRHL2相关的表观基因组谱；并研究GRHL2是否通过调节非编码rna来决定hpv相关口腔癌的CSC表型。本项目将阐明GRHL2介导“高危”HPV口腔癌变的表观遗传机制，并验证GRHL2通过调控非编码rna在CSC更新中的作用，为发现新的诊断标志物和治疗靶点提供依据。

DESCRIPTION (provided by applicant): Long-term goal of our research is to discover novel biomarkers and effective therapies against oral cancer. The current project will investigate the role of Grainyhead-like 2 (GRHL2) in mediating oral carcinogenesis by "high risk" human papillomavirus (HPV) through epigenetic mechanisms. GRHL2 regulates keratinocyte cell fate through multiple target genes, including human telomerase reverse transcriptase (hTERT) gene, forkhead box protein 1 (FoxM1), and epidermal differentiation complex (EDC) genes. Our recent published data revealed that GRHL2 is a master regulator of keratinocyte proliferation and differentiation. GRHL2 elicits these effects through epigenetic mechanisms that involve altered DNA methylation and histone modifications at target gene promoters. We found that GRHL2 is notably induced in human oral keratinocytes (HOKs) harboring "high risk" HPV and HPV+ oral squamous cell carcinomas (OSCCs). Furthermore, high level of GRHL2 was detected in OSCC tumor spheroids, which are enriched with cancer stem cells (CSCs). When GRHL2 was knocked down in OSCCs, self-renewal of tumor spheroids in vitro and tumorigenicity in vivo were significantly impaired. Thus, HPV may cause deregulation of GRHL2 to promote its oncogenic activity by inducing the FoxM1 oncogene and enhance CSC self-renewal. Recent studies underscore the importance of non-coding RNAs in regulation of pluripotency and CSC self-renewal. GRHL2 upregulates miR-21, an "oncomir" associated with HPV and CSC phenotype. Thus, differential regulation of non-coding RNAs by GRHL2 may underlie the mechanism of enhancing CSC phenotype in OSCCs. To investigate the pathobiology of HPV in oral carcinogenesis, we have constructed infectious HPV16 virions capable of expressing the viral oncogenes. These novel HPV virions will allow us to test the effects of HPV infection in primary NHOK in well-controlled experiments. In the current project, we will test the hypothesis that GRHL2 is necessary for HPV-associated cellular aberrations by altered epigenetic regulation of its target genes and by enhancing CSC self-renewal, resulting in the maintenance of oral cancer phenotype. This novel hypothesis will be tested by investigating the pathophysiologic role of GRHL2 in HPV-associated cellular aberrations; determining the epigenetic mechanisms by which GRHL2 modulates epithelial proliferation and differentiation, and how GRHL2-associated epigenomic profile is altered by HPV; and investigating whether GRHL2 determines CSC phenotype in HPV-associated oral cancer by regulation of non-coding RNAs. This project will delineate the epigenetic mechanisms by which GRHL2 mediates pathobiology of "high risk" HPV in oral carcinogenesis, and will validate the role of GRHL2 in CSC renewal through regulation of non-coding RNAs, which may be used for discovery of novel diagnostic marker and therapeutic targets.