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MAPK signaling is required for dedifferentiation of acinar cells and development of pancreatic intraepithelial neoplasia in mice.

ABSTRACT: Kras signaling via mitogen-activated protein kinase (MAPK) is highly up-regulated in pancreatic cancer cells. We investigated whether MAPK signaling is required for the initiation and maintenance of pancreatic carcinogenesis in mice.We studied the formation and maintenance of pancreatic intraepithelial neoplasia (PanINs) in p48Cre; TetO-KrasG12D; Rosa26(rtTa-IRES-EGFP) (iKras*) mice and LSL-KrasG12D mice bred with p48Cre mice (KC). Mice were given oral PD325901, a small-molecule inhibitor of MEK1 and MEK2 (factors in the MAPK signaling pathway), along with injections of cerulein to induce pancreatitis. Other mice were given PD325901 only after PanINs developed. Pancreatic tissues were collected and evaluated using histologic, immunohistochemical, immunofluorescence, and electron microscopy analyses. Acinar cells were isolated from the tissues and the effects of MEK1 and 2 inhibitors were assessed.PD325901 prevented PanIN formation, but not pancreatitis, in iKras* and KC mice. In iKras* or KC mice given PD325901 at 5 weeks after PanINs developed, PanINs regressed and acinar tissue regenerated. The regression occurred through differentiation of the PanIN cells to acini, accompanied by re-expression of the acinar transcription factor Mist1.In iKras* and KC mice, MAPK signaling is required for the initiation and maintenance of pancreatic cancer precursor lesions. MAPK signaling promotes formation of PanINs by enabling dedifferentiation of acinar cells into duct-like cells that are susceptible to transformation.

SUBMITTER: Collins MA

PROVIDER: S-EPMC4037403 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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MAPK signaling is required for dedifferentiation of acinar cells and development of pancreatic intraepithelial neoplasia in mice.

Collins Meredith A MA Yan Wei W Sebolt-Leopold Judith S JS Pasca di Magliano Marina M

Gastroenterology 20131206 3

<h4>Background & aims</h4>Kras signaling via mitogen-activated protein kinase (MAPK) is highly up-regulated in pancreatic cancer cells. We investigated whether MAPK signaling is required for the initiation and maintenance of pancreatic carcinogenesis in mice.<h4>Methods</h4>We studied the formation and maintenance of pancreatic intraepithelial neoplasia (PanINs) in p48Cre; TetO-KrasG12D; Rosa26(rtTa-IRES-EGFP) (iKras*) mice and LSL-KrasG12D mice bred with p48Cre mice (KC). Mice were given oral P ...[more]

PMID: 24315826

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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.

Project description:Background & aimsPancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy that invades surrounding structures and metastasizes rapidly. Although inflammation is associated with tumor formation and progression, little is known about the mechanisms of this connection. We investigate the effects of interleukin (IL) 22 in the development of pancreatic tumors in mice.MethodsWe performed studies with Pdx1-Cre;LSL-KrasG12D;Trp53+/-;Rosa26EYFP/+ (PKCY) mice, which develop pancreatic tumors, and PKCY mice with disruption of IL22 (PKCY Il22-/-mice). Pancreata were collected at different stages of tumor development and analyzed by immunohistochemistry, immunoblotting, real-time polymerase chain reaction, and flow cytometry. Some mice were given cerulean to induce pancreatitis. Pancreatic cancer cell lines (PD2560) were orthotopically injected into C57BL/6 mice or Il22-/-mice, and tumor development was monitored. Pancreatic cells were injected into the tail veins of mice, and lung metastases were quantified. Acini were collected from C57BL/6 mice and resected human pancreata and were cultured. Cell lines and acini cultures were incubated with IL22 and pharmacologic inhibitors, and protein levels were knocked down with small hairpin RNAs. We performed immunohistochemical analyses of 26 PDACs and 5 nonneoplastic pancreas specimens.ResultsWe observed increased expression of IL22 and the IL22 receptor (IL22R) in the pancreas compared with other tissues in mice; IL22 increased with pancreatitis and tumorigenesis. Flow cytometry indicated that the IL22 was produced primarily by T-helper 22 cells. PKCY Il22-/-mice did not develop precancerous lesions or pancreatic tumors. The addition of IL22 to cultured acinar cells increased their expression of markers of ductal metaplasia; these effects of IL22 were prevented with inhibitors of Janus kinase signaling to signal transducer and activator of transcription (STAT) (ruxolitinib) or mitogen-activated protein kinase kinase (MEK) (trametinib) and with STAT3 knockdown. Pancreatic cells injected into Il22-/- mice formed smaller tumors than those injected into C57BL/6. Incubation of IL22R-expressing PDAC cells with IL22 promoted spheroid formation and invasive activity, resulting in increased expression of stem-associated transcription factors (GATA4, SOX2, SOX17, and NANOG), and increased markers of the epithelial-mesenchymal transition (CDH1, SNAI2, TWIST1, and beta catenin); ruxolitinib blocked these effects. Human PDAC tissues had higher levels of IL22, phosphorylated STAT3, and markers of the epithelial-mesenchymal transition than nonneoplastic tissues. An increased level of STAT3 in IL22R-positive cells was associated with shorter survival times of patients.ConclusionsWe found levels of IL22 to be increased during pancreatitis and pancreatic tumor development and to be required for tumor development and progression in mice. IL22 promotes acinar to ductal metaplasia, stem cell features, and increased expression of markers of the epithelial-mesenchymal transition; inhibitors of STAT3 block these effects. Increased expression of IL22 by PDACs is associated with reduced survival times.

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MAPK signaling is required for dedifferentiation of acinar cells and development of pancreatic intraepithelial neoplasia in mice.

Publications

MAPK signaling is required for dedifferentiation of acinar cells and development of pancreatic intraepithelial neoplasia in mice.

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