The function of KRAS4B isoform in pancreatic cancer.

4147 Background: Pancreatic ductal adenocarcinoma (PDAC) remains a significant challenge in the field of oncology, demanding the creation of models that faithfully replicate the human condition to aid in early diagnosis and the discovery of effective treatments. To address this need, we have developed the genetically engineered pig PDAC model. The model closely recapitulates the pathophysiological aspects of the human disease, including disease progression, histopathological subtypes of tumors, and the occurrence of metastasis. Alternative splicing variants have been proven to be novel targets in PDAC. KRAS undergoes mutations in PDAC more often than any other oncogene. The distinction between the two KRAS isoforms arises from the alternative splicing of exon 4 within the KRAS locus, resulting in the expression of major KRAS4A and minor KRAS4B splice isoforms, with KRAS4B is understudied, and functions in cancer are unknown. Based on this strong rationale, we initiated a study to functionally validate the role of the KRAS4B isoform in PDAC. Methods: qPCR was carried out to determine the expression level of KRAS4A and KRAS 4B in PDAC tumors and cell lines as well as in different tissues. To investigate the function of KRAS4B, we generated two pancreatic cancer cell lines harboring the genetic signature of Pdx Cre /Kras G12D /p53 R167H mutation (KPC) or Pdx Cre /Kras G12D mutation (KC). Using CRISPR/Cas9 technology to knockout KRAS4B isoform. Additionally, we designed a vector for investigating the overexpression of KRAS4B. Results: At the molecular level, we revealed a differential expression of KRAS isoforms 4A and 4B in PDAC tumors and cell lines. Notably, the KRAS4B isoform was exclusively expressed in the advanced stages of PDAC. At the cellular level, we successfully disrupted the KRAS4B isoform in both cell lines. The overexpression vector of KRAS4B has been successfully constructed for further analysis. Subsequent functional assays will encompass analyses of mRNA and protein expression levels, along with assessments of key cellular behaviors, including proliferation, invasion, migration, and morphological alterations. Moreover, we will discern changes in tumor subtypes by examining the distinct expression patterns of epithelial and mesenchymal markers. Conclusions: Therefore, we engineered KRAS4B knockout and overexpression vectors to investigate its role in PDAC, utilizing our cell lines derived from our genetically engineered pig model. This approach offers a robust platform that enhances the bridge between translational and preclinical research in studying PDAC.