Thesis presented November 07, 2024
Abstract:
Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a 5-year overall survival rate of only 11%. Its lethality stems from late diagnosis and rapid progression to metastasis. Various large-scale genomic studies have identified multiple potential driver mutations in PDAC, with KRAS (K), TP53 (T), CDKN2A (C), and SMAD4 (S) genes showing the highest mutation frequencies. Notably, KRAS mutations are present in over 90% of PDAC tumors.
In our research project, we aimed to develop a precancerous model that recapitulates PDAC development by introducing the four driver mutations into pancreatic epithelial cells. We extensively characterize this model in both 2D and 3D cultures and assess its suitability for drug testing.
To generate the cellular model, we utilized CRISPR/Cas9 gene editing technology to target human immortalized pancreatic ductal epithelial cells (H6c7) for knocking out three tumor suppressor genes (C-T-S). Additionally, we engineered plasmid-encoded KRAS vectors for both continuous and conditional expression (utilizing TetR systems) of mutant KRAS. To improve the efficiency of mutant KRAS integration, we employed PiggyBac transposase. Simultaneously, we knocked out the KRAS gene to explore cellular pathways influenced by KRAS loss and to identify potential KRAS-complementary targets. Subsequent to each genetic modification, clonal selection was carried out, and genetic mutations were confirmed through biological assays.
For the C-T-S tumor suppressor genes, we successfully generated cell lines with single and triple gene mutations. Notably, the triple gene mutated cells exhibited elevated KRAS expression, leading to increased proliferation, apoptosis, and DNA damage. However, when attempting continuous expression of KRAS mutants using plasmid vectors, we observed induction of senescence in both wild type and triple mutant cells, preventing the establishment of stable clones. To overcome this challenge, we employed PiggyBac (PB) vectors for conditional KRAS expression, which were validated through transfection assays. Subsequently, PB stable cell lines were established and characterized. We further assessed the utility of these cell lines for drug screening, particularly focusing on mutant KRAS degraders and inhibitors. Furthermore, we explored the potential of these cell lines for studying carcinogenesis initiation in both 2D and 3D cultures. In our KRAS knockout experiments, we successfully obtained clones on both wild-type and triple C-T-S mutation backgrounds. We observed that the loss of KRAS affected cellular morphology and proliferation.
To conclude, although p53 and p16 (CDKN2A gene product) have traditionally been recognized as key factors responsible for KRAS oncogene-induced senescence, our initial findings with triple C-T-S mutants indicate that the deletion of these genes alone is not sufficient to initiate carcinogenesis or to counteract senescence induced by KRAS. Consequently, it suggests the involvement of other yet-to-be-identified factors that are crucial for KRAS-induced carcinogenesis. Our data involving KRAS-overexpressing and KRAS knockout cells further supports this notion, indicating that KRAS influences multiple cellular pathways, including those governing cell size, morphology, dynamics, and cell cycle regulation.
Keywords:
pancreatic cancer, carcinogenesis, cell model