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

持续的HPV感染是子宫颈癌的主要原因。虽然HPV感染和肿瘤发生之间的联系是非常明确的，但HPV介导的转化机制尚不完全清楚。STAT3和GLI1是有效的致癌基因，在许多癌症类型中与干细胞的持久性有关，并被发现在靶基因的表达中协同起作用。主要导师的实验室发现了宫颈癌细胞增殖所必需的NF-kB-IL6-STAT3环。二级主管证明STAT3是GLI1高表达和高活性所必需的，GLI1是一种由Hedgehog途径调节的癌基因转录因子。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STAT3/GLI1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。这一目标将通过个别目标来实现：STAT3和GLI1相互依赖维持e6转化宫颈癌细胞的研究。我们将沉默和过表达STAT3和GLI1，并使用这两种蛋白的药理学抑制剂，然后通过qPCR分析它们的转录活性，并研究细胞的致癌特性（增殖、依赖锚定和独立的集落形成、迁移和存活）。在第三位导师的实验室中，将对转染STAT3或GLI1的细胞进行裸鼠皮下异种移植，以分析其体内肿瘤生长情况。2. 分离STAT3/GLI1复合体进行生化分析，包括结构信息和相互作用域的鉴定。从细胞裂解物中纯化或用重组蛋白制备的复合物将用于结构分析以确定相互作用基序并通过诱变进行确认。不能相互作用的突变体将在子宫颈癌细胞中表达，以研究它们在体外和体内对致癌特性的影响。细胞的RNA-seq将识别选择性地需要STAT3/GLI1复合体的靶基因。3. HPV E6诱导的STAT3与GLI1正反馈回路的研究。我们将研究在缺乏另一种转录因子的细胞中，每种转录因子的表达和稳定性的相互调节。确定STAT3和GLI1在宫颈癌中的关键协同作用将为低剂量联合抑制剂的使用提供信息，并指导虚拟筛选阻止复合物形成以减少hpv阳性宫颈癌进展的新化合物。

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.