Hijacking Hedgehog: identifying targetable pathways co-opted by Human Papillomavirus in cervical cancer

劫持刺猬：确定人乳头瘤病毒在宫颈癌中选择的目标途径

• 批准号: 2745964
• 金额: --
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2745964
• 关键词: Hijacking; Hedgehog; identifying; targetable; pathways

Persistent HPV infection is the leading cause of cervical cancer. While the link between HPV infection and oncogenesis is very well-established, the mechanisms of HPV mediated transformation are not fully understood. STAT3 and GLI1 are potent oncogenes that have been associated with persistence of stem cells in many cancer types and were found to function cooperatively in the expression of target genes. The primary supervisor's lab identified an NF-kB-IL6-STAT3 loop necessary for cervical cancer cells proliferation. The secondary supervisor demonstrated that STAT3 is necessary for high expression and activity of GLI1, an oncogene transcription factor regulated by the Hedgehog pathway. This project will explore if E6 promotes formation of a STAT3/GLI1 complex necessary to maintain oncogenic properties of cervical cancer cells and will develop novel strategies to target its function as proof of concept of a potential therapeutic approach. This aim will be achieved through individual objectives: 1. Determination of STAT3 and GLI1 mutual dependency to sustain E6-transformed cervical cancer cells. We will silence and overexpress STAT3 and GLI1 and use pharmacological inhibitors of both proteins, followed by analysis of their transcriptional activity by qPCR, and by study of oncogenic properties of the cells (proliferation, anchorage-dependent and -independent colony formation, migration and survival). Analysis of in vivo tumour growth of cells with silenced STAT3 or GLI1 will be performed as subcutaneous xenografts of the modified cells in nude mice in the third supervisor's lab. 2. Isolation of the STAT3/GLI1 complex for biochemical analysis, including structural information and identification of interacting domains. The complex purified from cell lysates or made using recombinant proteins will be used for structural analysis to identify the interaction motif and confirmed by mutagenesis. Mutants unable to interact with each other will be expressed in cervical cancer cells to investigate their impact on oncogenic properties, in vitro and in vivo. RNA-seq of cells will identify target genes that selectively require the STAT3/GLI1 complex. 3. Investigation of the positive feedback loop between STAT3 and GLI1 induced by HPV E6. We will investigate the mutual regulation of expression and stability of each transcription factor in cells depleted of the other one. Identification of a key cooperative role of STAT3 and GLI1 in cervical cancer will inform the use of inhibitors in combination at low doses and guide the virtual screening of novel compounds that block complex formation to reduce HPV-positive cervical cancer progression.

HPV感染是导致宫颈癌的主要原因。虽然HPV感染和肿瘤发生之间的联系是非常明确的，但HPV介导的转化机制尚未完全了解。STAT 3和GLI 1是与许多癌症类型中的干细胞持续存在相关的有效癌基因，并且被发现在靶基因的表达中协同发挥作用。主要主管的实验室鉴定了宫颈癌细胞增殖所必需的NF-kB-IL 6-STAT 3环。二级主管证明，STAT 3是必需的高表达和活性的GLI 1，一个致癌基因转录因子调节的刺猬途径。该项目将探索E6是否促进维持宫颈癌细胞致癌特性所必需的STAT 3/GLI 1复合物的形成，并将开发新的策略来靶向其功能，作为潜在治疗方法的概念证明。这一目标将通过个人目标实现：1。确定STAT 3和GLI 1相互依赖性以维持E6转化的宫颈癌细胞。我们将沉默和过表达STAT 3和GLI 1，并使用这两种蛋白质的药理学抑制剂，然后通过qPCR分析其转录活性，并研究细胞的致癌特性（增殖，锚定依赖性和非依赖性集落形成，迁移和存活）。将在第三位主管的实验室中，在裸鼠中进行具有沉默的STAT 3或GLI 1的细胞的体内肿瘤生长分析，作为修饰细胞的皮下异种移植物。2.分离STAT 3/GLI 1复合物用于生化分析，包括结构信息和相互作用结构域的鉴定。从细胞裂解物中纯化或使用重组蛋白制备的复合物将用于结构分析以鉴定相互作用基序并通过突变进行确认。不能相互作用的突变体将在宫颈癌细胞中表达，以研究它们对体外和体内致癌特性的影响。细胞的RNA-seq将识别选择性地需要STAT 3/GLI 1复合物的靶基因。3. HPV E6诱导的STAT 3和GLI 1之间的正反馈回路的研究。我们将调查的相互调节的表达和稳定性的每一个转录因子在细胞耗尽的其他一个。 鉴定STAT 3和GLI 1在宫颈癌中的关键合作作用将为低剂量联合使用抑制剂提供信息，并指导阻断复合物形成以减少HPV阳性宫颈癌进展的新型化合物的虚拟筛选。