Determining the mechanism for YAP1 activation by HPV E7 in oropharyngeal carcinoma

确定口咽癌中 HPV E7 激活 YAP1 的机制

• 批准号: 10606110
• 负责人: WILLIAM JAMES BLAKELY
• 金额: $ 6.84万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10606110
• 关键词: 3-Dimensional; Automobile Driving; Basal Cell; Binding; Biotin; Carcinoma; Cell Differentiation process; Cell Line; Cell Nucleus; Cell model; Cycloheximide; Cytoplasm; DNA biosynthesis; Data; Differentiated Gene; Epithelial; Exclusion; Gene Expression; Genetic Transcription; Goals; Growth; HPV E7; HPV-High Risk; Head and Neck Squamous Cell Carcinoma; Human; Human Activities; Human Papillomavirus; Human papilloma virus infection; Immunofluorescence Microscopy; Immunoprecipitation; Knock-out; Knowledge; LATS1 gene; Label; Longevity; Malignant Epithelial Cell; Malignant Neoplasms; Measures; Mediating; Modeling; Mutate; Nuclear; Nuclear Translocation; Oncogenic; Oncoproteins; Oral; Organoids; Oropharyngeal; Oropharyngeal Squamous Cell Carcinoma; Pathway interactions; Phosphorylation; Phosphorylation Inhibition; Phosphotransferases; Protein Binding Domain; Protein Dephosphorylation; Proteins; RB1 gene; Regulation; Repression; Serine; Signal Pathway; Signal Transduction; Somatic Mutation; Stratified Squamous Epithelium; Streptavidin; Testing; Transcript; Transcription Coactivator; Tumor Suppressor Proteins; Viral; Virus; Virus Replication; cancer cell; carcinogenesis; cell growth; cell transformation; experimental study; high risk; keratinocyte; knock-down; knockout gene; malignant mouth neoplasm; malignant oropharynx neoplasm; mutant; overexpression; penis foreskin; prevent; retinoblastoma tumor suppressor; tumorigenesis; tumorigenic; two-dimensional

PROJECT SUMMARY Human papillomavirus (HPV) is a driver of carcinogenesis in oropharyngeal squamous cell carcinoma (OPSCC). HPV infects epithelial keratinocytes and alters host cell signaling to suit viral replication. HPV simultaneously promotes basal cell growth and represses cell differentiation programming causing increased basal cell retention within stratified squamous epithelia and tumorigenesis from high-risk HPVs. The HPV protein E7 is a major driving oncogenic factor responsible for promoting these effects. While it has been documented that high-risk E7 causes degradation of host retinoblastoma tumor suppressor RB1, leading to increased cell growth and DNA replication, RB1 degradation does not solely cause E7 tumorigenic activity. E7 activity is more tumorigenic than RB1 loss alone. In addition to growth stimulation, HPV requires repression of cell differentiation. Our lab has documented E7 facilitates degradation of an additional tumor suppressor, PTPN14. PTPN14 degradation by E7 represses cell differentiation and induces translocation of YAP1, an oncogenic transcription coactivator. The mechanism connecting PTPN14 to YAP1 regulation is unknown. My preliminary data indicates PTPN14 degradation by E7 suppresses YAP1 inhibition. Phosphorylation of YAP1 at S127 coincides with YAP1 exclusion from the nucleus. YAP1 inhibition is induced by the Hippo kinase cascade. I have shown PTPN14 loss is transformative and E7-mediated degradation promotes dephosphorylation of YAP1 at S127. I have also shown PTPN14 induces YAP1 phosphorylation at S127. My results show these effects in immortalized human foreskin keratinocytes and my goal is to apply my findings to oral keratinocyte models. The specific aims of this proposal are to 1) determine if E7-mediated PTPN14 degradation promotes YAP1 dephosphorylation, stability and nuclear translocation in HPV positive OPSCC and 2) determine the mechanism of YAP1 phosphorylation by PTPN14. In Aim 1 I will establish effects of PTPN14 degradation in an oral keratinocyte cell model, quantifying phosphorylation levels of YAP1 and YAP1 stability by cycloheximide-chase experiments. I will test rescue of YAP1 inhibition on E7 knockdown and also perform rescue experiments with an E7-binding mutant of PTPN14 to reestablish normal YAP1 regulation in HPV positive OPSCC cells. YAP1 localization in three-dimensional organoid cultures of OPSCC cells will also be analyzed by immunofluorescence microscopy. In Aim 2 I will determine how PTPN14 promotes YAP1 S127 phosphorylation and subsequent nuclear exclusion in oral keratinocytes. PTPN14 mutants containing single deletions of function domains will be expressed with YAP1 phosphorylation quantified by immunoblot. I will determine interactors of PTPN14 important for its ability to induce YAP1 phosphorylation by performing BioID biotin labelling of proteins under conditions of phosphorylated YAP1 and dephosphorylated YAP1.

项目总结 人乳头瘤病毒(HPV)是口咽鳞状细胞癌的致癌因素 (OPSCC)。HPV感染上皮角质形成细胞并改变宿主细胞信号以适应病毒复制。人类乳头瘤病毒 同时促进基底细胞生长和抑制细胞分化编程，导致 复层鳞状上皮中的基底细胞滞留与高危HPV的肿瘤发生。人乳头瘤病毒 E7蛋白是促进这些效应的主要致癌因子。虽然它一直是 研究证明，高危E7会导致宿主视网膜母细胞瘤肿瘤抑制因子RB1的降解，导致 增加细胞生长和DNA复制，RB1的降解并不是唯一导致E7致瘤活性的原因。E7 活性比单独失去RB1更容易致癌。除了生长刺激外，HPV还需要抑制 细胞分化。我们的实验室已经证明E7可以促进另一种肿瘤抑制因子的降解， PTPN14.E7降解PTPN14抑制细胞分化并诱导YAP1、AND易位 致癌转录辅活化子。PTPN14与YAP1调控相关的机制尚不清楚。 我的初步数据表明，E7降解PTPN14会抑制YAP1的抑制。磷酸化 S127的YAP1与YAP1的核排除相一致。河马对YAP1的抑制作用 激酶级联反应。我已经证明了PTPN14的缺失是变革性的，E7介导的降解促进了 YAP1在S127的去磷酸化。我还证明了PTPN14在S127处诱导YAP1磷酸化。我的 结果显示在永生化的人类包皮角质形成细胞中有这些效应，我的目标是将我的发现应用于 口腔角质形成细胞模型。该提案的具体目的是：1)确定E7介导的PTPN14 降解促进HPV阳性患者YAP1去磷酸化、稳定性和核转位 OPSCC和2)确定PTPN14磷酸化YAP1的机制。在目标1中，我将确立 口腔角质形成细胞模型中PTPN14降解对YAP1磷酸化水平的影响 环己酰亚胺追逐实验确定YAP1的稳定性。我将测试YAP1对E7基因敲除的抑制作用 并对PTPN14的E7结合突变体进行救援实验，以重建正常的YAP1 HPV阳性OPSCC细胞的调控。YAP1在OPSCC三维类器官培养中的定位 细胞也将通过免疫荧光显微镜进行分析。在目标2中，我将确定PTPN14如何 促进口腔角质形成细胞中YAP1 S127的磷酸化和随后的核排斥。PTPN14 含有单一功能结构域缺失的突变体将通过YAP1磷酸化来表达 用免疫印迹法定量。我将确定PTPN14的相互作用因子对其诱导YAP1的能力至关重要 在磷酸化的YAP1和YAP1的条件下进行生物素标记的蛋白质的磷酸化 去磷酸化的YAP1。