Interaction between the human papilloma virus and the pregnane X receptor in head and neck squamous cell carcinoma. Impact on the multidrug resistance and malignant phenotype

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Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer by incidence worldwide. The infection with human papilloma viruses (HPV) has become a validated risk factor. HPV-caused HNSCC represents a different pathological entity with a differential molecular background and better prognosis than not-HPV-associated HNSCC. The pregnane X receptor (PXR) plays a key role in the regulation of genes associated with cancer malignancy. PXR activity mediates the transcription of its target genes and can be divided into intrinsic activity (basal, ligand-independent) and inducibility (enhanced by ligand binding). PXR activity is the net result of PXR expression, the expression of its dimerization partner retinoid-X-receptor (RXR) alpha, the expression and function of cofactors (coactivators and corepressors) and posttranslational modifications (phosphorylation and acetylation). PXR has been associated with either a good (e.g. esophagusandprostate cancer) or poor prognosis (breast- and ovarian cancer). HPV E6 and E7 proteins have been shown to modulate nuclear receptor function (e.g. the thyroid hormone receptor). This usually takes place through the physical interaction with the corresponding receptor or with its cofactors. Additionally, HPV modulates kinases and acetylases capable of posttranslationally modifying nuclear receptors. However, the interaction between HPV and PXR and its impact on PXR target genes associated with HNSCC prognosis has not been investigated so far. The main aim of this project is to evaluate the interaction between HPV and PXR in HNSCC cell lines and in situ (tumor samples) and thus to estimate its clinical relevance. Our hypothesis is that high risk HPVs modulate PXR activity through modifying its expression, the expression of its partner RXR alpha, by interacting with cofactors or by affecting PXR posttranslational modifications. A direct role of E6/E7 ascofactors cannot be ruled out. This would lead to changes in basal expression and inducibility of PXR targets potentially affecting the malignant phenotype of HNSCC. In a pilot experiment using a HNSCC cell line weobserved that HPV16 E6/E7 overexpression increases both PXR intrinsic activity and inducibility. The specific objectives of the project are: 1. the characterization of the effect of high risk HPV E6/E7 on PXR activity inHNSCC cell lines (reporter gene assay), 2. the characterization of the underlying molecular mechanisms, concretely, the modulation of the expression and function of PXR, RXR alpha and cofactors as well as PXRposttranslational modifications by E6/E7 (chromatin immunoprecipitation, western blot, qPCR, DuoLink assays), 3. the characterization of the impact of this interaction on PXR target genes (qPCR, proliferation-, cell cycle-, apoptosis and migration assays) and 4. the validation of the obtained results in an in situ analysis.