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Kras mutation rate precisely orchestrates ductal derived pancreatic intraepithelial neoplasia and pancreatic cancer.

ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States. Despite the high prevalence of Kras mutations in pancreatic cancer patients, murine models expressing the oncogenic mutant Kras (Kras^mut) in mature pancreatic cells develop PDAC at a low frequency. Independent of cell of origin, a second genetic hit (loss of tumor suppressor TP53 or PTEN) is important for development of PDAC in mice. We hypothesized ectopic expression and elevated levels of oncogenic mutant Kras would promote PanIN arising in pancreatic ducts. To test our hypothesis, the significance of elevating levels of K-Ras and Ras activity has been explored by expression of a CAG driven LGSL-Kras^G12V allele (cKras) in pancreatic ducts, which promotes ectopic Kras expression. We predicted expression of cKras in pancreatic ducts would generate neoplasia and PDAC. To test our hypothesis, we employed tamoxifen dependent CreER^T2 mediated recombination. Hnf1b:CreER^T2;Kras^G12V (cKras^Hnf1b/+) mice received 1 (Low), 5 (Mod) or 10 (High) mg per 20 g body weight to recombine cKras in low (cKras^Low), moderate (cKras^Mod), and high (cKras^High) percentages of pancreatic ducts. Our histologic analysis revealed poorly differentiated aggressive tumors in cKras^High mice. cKras^Mod mice had grades of Pancreatic Intraepithelial Neoplasia (PanIN), recapitulating early and advanced PanIN observed in human PDAC. Proteomics analysis revealed significant differences in PTEN/AKT and MAPK pathways between wild type, cKras^Low, cKras^Mod, and cKras^High mice. In conclusion, in this study, we provide evidence that ectopic expression of oncogenic mutant K-Ras in pancreatic ducts generates early and late PanIN as well as PDAC. This Ras rheostat model provides evidence that AKT signaling is an important early driver of invasive ductal derived PDAC.

SUBMITTER: Singh K

PROVIDER: S-EPMC8172380 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background and aimsDevelopment of pancreatic ductal adenocarcinoma (PDAC) is a multistep process intensively studied; however, precocious diagnosis and effective therapy still remain unsatisfactory. The role for Notch signaling in PDAC has been discussed controversially, as both cancer-promoting and cancer-antagonizing functions have been described. Thus, an improved understanding of the underlying molecular mechanisms is necessary. Here, we focused on RBPJ, the receiving transcription factor in the Notch pathway, examined its expression pattern in PDAC, and characterized its function in mouse models of pancreatic cancer development and in the regeneration process after acute pancreatitis.MethodsConditional transgenic mouse models were used for functional analysis of RBPJ in the adult pancreas, initiation of PDAC precursor lesions, and pancreatic regeneration. Pancreata and primary acinar cells were tested for acinar-to-ductal metaplasia together with immunohistology and comprehensive transcriptional profiling by RNA sequencing.ResultsWe identified reduced RBPJ expression in a subset of human PDAC specimens. Ptf1α-CreERT-driven depletion of RBPJ in transgenic mice revealed that its function is dispensable for the homeostasis and maintenance of adult acinar cells. However, primary RBPJ-deficient acinar cells underwent acinar-to-ductal differentiation in ex vivo. Importantly, oncogenic KRAS expression in the context of RBPJ deficiency facilitated the development of pancreatic intraepithelial neoplasia lesions with massive fibrotic stroma formation. Interestingly, RNA-sequencing data revealed a transcriptional profile associated with the cytokine/chemokine and extracellular matrix changes. In addition, lack of RBPJ delays the course of acute pancreatitis and critically impairs it in the context of KRASG12D expression.ConclusionsOur findings imply that downregulation of RBPJ in PDAC patients derepresses Notch targets and promotes KRAS-mediated pancreatic acinar cells transformation and desmoplasia development.

Project description:Background & aimsActivating mutations in KRAS are detected in most pancreatic ductal adenocarcinomas (PDACs). Expression of an activated form of KRAS (KrasG12D) in pancreata of mice is sufficient to induce formation of pancreatic intraepithelial neoplasia (PanINs)-a precursor of PDAC. Pancreatitis increases formation of PanINs in mice that express KrasG12D by promoting acinar-to-ductal metaplasia (ADM). We investigated the role of the transcription factor Krüppel-like factor 5 (KLF5) in ADM and KRAS-mediated formation of PanINs.MethodsWe performed studies in adult mice with conditional disruption of Klf5 (Klf5fl/fl) and/or expression of KrasG12D (LSL-KrasG12D) via CreERTM recombinase regulated by an acinar cell-specific promoter (Ptf1a). Activation of KrasG12D and loss of KLF5 was achieved by administration of tamoxifen. Pancreatitis was induced in mice by administration of cerulein; pancreatic tissues were collected, analyzed by histology and immunohistochemistry, and transcriptomes were compared between mice that did or did not express KLF5. We performed immunohistochemical analyses of human tissue microarrays, comparing levels of KLF5 among 96 human samples of PDAC. UN-KC-6141 cells (pancreatic cancer cells derived from Pdx1-Cre;LSL-KrasG12D mice) were incubated with inhibitors of different kinases and analyzed in proliferation assays and by immunoblots. Expression of KLF5 was knocked down with small hairpin RNAs or CRISPR/Cas9 strategies; cells were analyzed in proliferation and gene expression assays, and compared with cells expressing control vectors. Cells were subcutaneously injected into flanks of syngeneic mice and tumor growth was assessed.ResultsOf the 96 PDAC samples analyzed, 73% were positive for KLF5 (defined as nuclear staining in more than 5% of tumor cells). Pancreata from Ptf1a-CreERTM;LSL-KrasG12D mice contained ADM and PanIN lesions, which contained high levels of nuclear KLF5 within these structures. In contrast, Ptf1a-CreERTM;LSL-KrasG12D;Klf5fl/fl mice formed fewer PanINs. After cerulein administration, Ptf1a-CreERTM;LSL-KrasG12D mice formed more extensive ADM than Ptf1a-CreERTM;LSL-KrasG12D;Klf5fl/fl mice. Pancreata from Ptf1a-CreERTM;LSL-KrasG12D;Klf5fl/fl mice had increased expression of the tumor suppressor NDRG2 and reduced phosphorylation (activation) of STAT3, compared with Ptf1a-CreERTM;LSL-KrasG12D mice. In UN-KC-6141 cells, PI3K and MEK signaling increased expression of KLF5; a high level of KLF5 increased proliferation. Cells with knockdown of Klf5 had reduced proliferation, compared with control cells, had reduced expression of ductal markers, and formed smaller tumors (71.61 ± 30.79 mm3 vs 121.44 ± 34.90 mm3 from control cells) in flanks of mice.ConclusionLevels of KLF5 are increased in human PDAC samples and in PanINs of Ptf1a-CreERTM;LSL-KrasG12D mice, compared with controls. KLF5 disruption increases expression of NDRG2 and reduces activation of STAT3 and reduces ADM and PanINs formation in mice. Strategies to reduce KLF5 activity might reduce progression of acinar cells from ADM to PanIN and pancreatic tumorigenesis.

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Kras mutation rate precisely orchestrates ductal derived pancreatic intraepithelial neoplasia and pancreatic cancer.

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