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Contribution à l'étude des mécanismes moléculaires de chimiorésistance des cellules cancéreuses pancréatiques

Abstract : Pancreatic cancer (PC) is the fourth leading cause of death by cancer in Western countries with a survival rate of less than 5% at five years. Its incidence has been increasing for the last forty years, and, in the absence of improvement in the detection and treatment, PC will become the second leading cause of death by cancer worldwide in 2020. Accordingly, we recently conducted the first gene therapy clinical trial for patients diagnosed with advanced PC, during which patients received using endoscopic ultrasound an intratumoral injection of a non viral vector encoding DCK::UMK and SSTR2 genes. Patients were also treated by gemcitabine. In addition to the feasibility and safety of this protocol, gene therapy results in a clinical benefit in selected patients. We also highlighted two major elements: this beneficial effect (1) is not correlated with the dose administered and (2) reaches a "therapeutic plateau" beyond which the cancer cells are resistant to treatment. These considerations suggest that the intratumoral gene transfer is limiting and that additional mechanisms of drug resistance remain to be identified and defeated to offer new therapeutic perspectives. Therefore, we first investigated the molecular mechanisms that may be involved in the primo resistance of cancer cells to gemcitabine. We highlighted the importance of cytidine deaminase (CDA) in cohort of patients that poorly responded to gemcitabine treatment. This enzyme, which catalyzes the irreversible hydrolytic deamination of CTP in UTP, is also overexpressed in PC as compared to adjacent tissue. In experimental models, the invalidation of CDA results in the profound inhibition of cell proliferation and massive induction of apoptosis, even in the absence of gemcitabine. Using an unbiased proteomic approach, we found that several proteins involved in mitochondrial metabolism are strongly altered in response to CDA targeting. Finally, combining the invalidation of the CDA and gemcitabine treatment results in a rarely achieved inhibition of cancer cell proliferation and tumor progression in vivo. We next investigated new delivery vectors/virus for the gene therapy of pancreatic cancer. We selected oncolytic viruses derived from HSV-1, in which the viral ribonucleotide reductase is mutated, and the expression of the viral protein gamma 34.5 is under the control of the proto-oncogene B-myb promoter (Myb34.5). We first identified a highly favorable molecular context for the replication of Myb34.5 in human pancreatic cancer cells as they over-express not only B-myb, but also cellular ribonucleotide reductase RRM1 and RRM2 whereas expression of ENT1 is decreased. In cancer cell lines, Myb34.5 replicates athigh rate, strongly inhibits cell proliferation and provokes cancer cell death by apoptosis. This effect is increased by the co-treatment with gemcitabine. Finally, Myb34.5 causes the regression of experimental tumors, when combined with gemcitabine. This thesis work opens several interesting prospects: we have identified new therapeutic targets to alleviate the chemoresistance of PC cells and demonstrated the efficacy of oncolytic viruses derived from HSV-1 for the treatment of experimental pancreatic tumors. Ultimately, this work may stem for new combinations of therapeutic genes, delivered or not by oncolytic viruses, to defeat the "therapeutic plateau" observed in PC experimental models and during the Thergap clinical trial and to specifically eradicate pancreatic tumor cells with high efficacy.
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Marion Gayral. Contribution à l'étude des mécanismes moléculaires de chimiorésistance des cellules cancéreuses pancréatiques. Cancer. Université Paul Sabatier - Toulouse III, 2015. Français. ⟨NNT : 2015TOU30108⟩. ⟨tel-02928980⟩

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