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

头颈部鳞状细胞癌（HNSCC）是全球发病率第六大癌症。人乳头瘤病毒（HPV）感染已成为一个被证实的危险因素。hpv引起的HNSCC是一种不同的病理实体，与非hpv相关的HNSCC相比具有不同的分子背景和更好的预后。妊娠X受体（PXR）在恶性肿瘤相关基因的调控中起着关键作用。PXR活性介导其靶基因的转录，可分为内在活性（基础的、与配体无关的）和诱导活性（通过配体结合增强）。PXR活性是PXR表达、其二聚化伙伴类维生素a - x受体(RXR) α的表达、辅助因子（辅激活因子和辅抑制因子）的表达和功能以及翻译后修饰（磷酸化和乙酰化）的最终结果。PXR与良好预后（如食管癌和前列腺癌）或不良预后（如乳腺癌和卵巢癌）相关。HPV E6和E7蛋白已被证明可调节核受体功能（如甲状腺激素受体）。这通常通过与相应受体或其辅因子的物理相互作用发生。此外，HPV调节激酶和乙酰化酶能够翻译后修饰核受体。然而，HPV与PXR的相互作用及其对与HNSCC预后相关的PXR靶基因的影响目前尚未研究。本项目的主要目的是评估HPV和PXR在HNSCC细胞系和原位（肿瘤样本）中的相互作用，从而估计其临床相关性。我们的假设是，高风险hpv通过改变其表达，其伴侣RXR α的表达，通过与辅助因子相互作用或通过影响PXR翻译后修饰来调节PXR活性。不能排除E6/E7抗坏血因子的直接作用。这将导致PXR靶点的基础表达和诱导性的变化，可能影响HNSCC的恶性表型。在使用HNSCC细胞系的先导实验中，我们观察到hpv16e6 /E7过表达增加了PXR的内在活性和诱导性。该项目的具体目标是：1。1 .高危型HPV E6/E7对hnscc细胞系PXR活性的影响（报告基因测定）；2 .潜在分子机制的表征，具体而言，通过E6/E7（染色质免疫沉淀、western blot、qPCR、DuoLink检测）调节PXR、RXR α和辅助因子的表达和功能，以及PXR翻译后修饰。3 .这种相互作用对PXR靶基因的影响的表征（qPCR，增殖-，细胞周期-，凋亡和迁移测定）；在原位分析中对所得结果的验证。

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.