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

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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.