Screening of components of the epigenetic machinery functionally associated with chemoresistance and sensitivity to gemcitabine in pancreatic cancer

Abstract

Pancreatic cancer is one of the most lethal neoplasms and only 5% of affected individuals survive more than 5 years after diagnosis, with ductal adenocarcinoma (PDAC) being the most prevalent and aggressive type. Chemotherapy with gemcitabine is the standard treatment protocol for invasive PDAC and is also used as adjuvant therapy after surgical removal of the tumor. However, the clinical benefit of this therapy is extremely limited due to the acquisition of chemoresistance followed by development of metastatic disease. The acquisition of chemoresistance involves different molecular pathways and is associated with other important cellular phenotypic changes (epithelial mesenchymal transition, expression of stem cell markers and stem-like characteristics) that contribute to disease progression. The development of chemoresistance and the resulting establishment of a more aggressive tumor phenotype involve changes in the pattern of gene expression that are maintained after cell division by the action of proteins responsible for the deposition, removal, and reading of chromatin modifications. The importance of epigenetic regulatory mechanisms in cellular homeostasis and the consequences of its deregulation for tumor progression have already been documented in different types of cancer, including PDAC, and is highlighted by the use in the clinic of drugs that act by reversing pro-oncogenic epigenetic marks. However, the contribution of epigenetic regulators to the establishment and maintenance of resistance to gemcitabine is not known yet.Using a functional screening approach, the goal of this proposal is to investigate the existence of epigenetic machinery components that are, not only critical for the survival of PDAC cells, but also for the establishment of gemcitabine resistance. This information will broaden the knowledge about the processes that lead to drug resistance by epigenetic control of gene expression and may also help identify new candidate targets for the treatment of pancreatic cancer. To achieve these goals we will use different cell models from human PDAC, including immortalized lineages and primary cultures derived from patient-derived xenotransplants (PDXs) generated in our laboratory. These cells will be interrogated with a custom library of short hairpin RNAs that target about 300 genes encoding protein components of the epigenetic machinery. Naïve of gemcitabine-resistant tumor cells will be transfected with the library and then maintained in the presence of sub-lethal concentrations of the drug. Analysis of the differential representation of specific shRNAs before and after selection with gemcitabine may reveal genes critical for the survival of PDAC or that sensitize tumor cells to treatment with the drug. The most promising candidate genes will be validated individually in different PDAC lines and in the PDX collection already available for this project. The results will provide original and relevant information about the role played by the epigenetic machinery in the cellular mechanisms of resistance to the main chemotherapeutic agent used to treat PDAC patients. In addition, this project holds the potential to unveil novel promising targets to be explored in combination with gemcitabine in the development of new therapeutic strategies that may increase treatment efficacy and patient survival in pancreatic cancer. (AU)