Immune Dysregulation by Human Papillomavirus during Head and Neck Cancer Progression

项目总结 人乳头瘤病毒(HPV)是一种高度流行的病原体，与超过5%的人类 癌症，包括约25%的头颈部鳞状细胞癌(HNSCC)。人乳头瘤病毒阳性的发生率 口咽鳞状细胞癌(OPSCC)在过去的二十年里急剧增加，并将 到2020年，可能占美国和全球所有HNSCC的大部分。在目前的治疗方法下，患者 必须处理治疗的深刻后遗症，这需要卫生和社会的大力支持 护理系统。因此，减少发病率和提高治愈率的新疗法对于 HPV阳性HNSCC患者(HPV+HNSCC)。为了开发成功的HNSCC患者治疗方法， 目前迫切需要深入了解HPV是如何抑制宿主免疫反应的。 我们最近的研究表明，虽然促炎趋化因子上调，但相对 新趋化因子CXCL14在HPV+HNSCC中被HPV癌蛋白E7诱导显著下调 CXCL14启动子高甲基化。CXCL14在HPV阳性小鼠HNSCC细胞中的恢复表达 抑制免疫活性同基因小鼠的肿瘤生长，但不能抑制RAG1缺陷小鼠的肿瘤生长。小鼠带有 CXCL14的表达显示肿瘤中CD8+T细胞和自然杀伤(NK)细胞数量显著增加 组织和肿瘤引流淋巴结，免疫抑制免疫细胞减少，包括髓系- 衍生抑制细胞(MDSCs)。因此，我们假设HPV E7驱动器下调了CXCL14的表达 诱导MDSC渗入TME而不能诱导CD8+T细胞和NK细胞发生HNSCC CXCL14在TME中的表达和免疫细胞浸润与临床相关 HNSCC患者的预后及联合放化疗增强肿瘤抑制作用 治疗(CRT)。这一假设将通过4个具体目标进行检验：1)确定CXCL14是否诱导了 CD8+T和NK细胞是HNSCC细胞抗肿瘤免疫应答的必要条件和充分条件 我们的免疫活性同基因小鼠模型移植了工程HPV+小鼠HNSCC细胞。2) 明确HPV癌蛋白E7通过以下途径下调CXCL14表达的机制 启动子高甲基化，使用我们的启动子甲基化报告实验和HPV16 E7突变体。3)考核 CXCL14表达、免疫细胞浸润与HNSCC临床预后的关系 患者，使用来自HNSCC患者的临床标本来开发有用的预后工具。4)定义是否 放化疗改变HPV阳性HNSCC细胞中HPV E7和CXCL14的表达及其机制 使用体外和体内放化疗的标准治疗中，再表达对肿瘤清除的影响 治疗结合Cxcl14在我们免疫活性的小鼠HNSCC模型中的表达。这 拟议的研究将为重新激活HPV和HPV抑制的抗肿瘤免疫反应提供重要线索 目的：开发可用于HNSCC治疗的有效预后和治疗工具。

PROJECT SUMMARY Human papillomaviruses (HPVs) are highly prevalent pathogens causally associated with over 5% of all human cancers, including ~25% of head and neck squamous cell carcinoma (HNSCC). The incidence of HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) has increased steeply in the last two decades, and will likely comprise the majority of all HNSCCs in the US and worldwide by 2020. With current therapies, patients must deal with the profound sequelae of treatment, which requires considerable support from health and social care systems. Therefore, novel therapies that decrease morbidity and increase cure rates are essential for patients with HPV-positive HNSCC (HPV+ HNSCC). To develop successful patient therapies for HNSCC, an in-depth understanding of how HPV suppresses the host immune response is urgently needed. Our recent studies have revealed that, while the proinflammatory chemokines are upregulated, a relatively novel chemokine CXCL14 is significantly downregulated in HPV+ HNSCC by the HPV oncoprotein E7-induced CXCL14 promoter hypermethylation. Restoration of CXCL14 expression in HPV+ mouse HNSCC cells suppresses tumor growth in immunocompetent syngeneic mice, but not in Rag1-deficient mice. Mice with CXCL14 expression showed dramatically increased CD8+ T and natural killer (NK) cell populations in tumor tissues and tumor draining lymph nodes, and decreased immunosuppressive immune cells including myeloid- derived suppressor cells (MDSCs). Thus, we hypothesize that CXCL14 downregulation by HPV E7 drives HNSCC development by inducing MDSC infiltration into the TME and failing to elicit CD8+ T and NK cell responses; and that CXCL14 expression and immune cell infiltration in the TME correlate with clinical outcomes of HNSCC patients and boosts tumor suppression in combination with chemoradiation therapy (CRT). This hypothesis will be tested through 4 specific aims: 1) Determine whether CXCL14-induced CD8+ T and NK cells are necessary and sufficient for the antitumor immune response to HNSCC cells, using our immunocompetent syngeneic mouse models transplanted with engineered HPV+ mouse HNSCC cells. 2) Define the mechanisms by which the HPV oncoprotein E7 downregulates CXCL14 expression through promoter hypermethylation, using our promoter methylation reporter assay and HPV16 E7 mutants. 3) Assess the clinical correlation between CXCL14 expression, immune cell infiltration, and clinical outcomes of HNSCC patients, using clinical specimens from HNSCC patients for developing useful prognostic tools. 4) Define if chemoradiation therapy changes HPV E7 and CXCL14 expression in HPV+ HNSCC cells and how CXCL14 re-expression impacts tumor clearance during standard therapies, using in vitro and in vivo chemoradiation therapy in combination with Cxcl14 expression in our our immunocompetent mouse HNSCC model. This proposed study will provide important clues to reactivate antitumor immune responses suppressed by HPV and to develop effective prognostic and therapeutic tools that may be employed in the treatment of HNSCC.