Kruppel-like Factor 5, Increased in Pancreatic Ductal Adenocarcinoma, Promotes Proliferation, Acinar-to-Ductal Metaplasia, Pancreatic Intraepithelial Neoplasia, and Tumor Growth in Mice.
ABSTRACT: BACKGROUND & AIMS:Activating 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. METHODS:We 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. RESULTS:Of 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. CONCLUSION:Levels 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:<h4>Background</h4>Pancreatic cancer is currently the third leading cause of cancer deaths in the United States and it is predicted to become the second by the year 2030. High-mobility group A1 protein (HMGA1) is an oncogenic transcription factor, localised and active in cell nuclei, that is linked to tumour progression in many human cancers, including pancreatic cancer. Overexpression of HMGA1 renders cancer cells resistant to chemotherapy. Although the Ptf1a-Cre; LSL-KrasG12D transgenic mouse is perhaps the most widely utilised animal model for human pancreatic cancer, expression levels of HMGA1 in pancreata from this mouse model have not been characterised.<h4>Methods</h4>Quantitative immunohistochemical analysis was used to determine nuclear HMGA1 levels in pancreatic tissue sections from Ptf1a-Cre; LSL-KrasG12D mice aged 5, 11, and 15 months. The H Score method was used for quantitative analysis.<h4>Results</h4>The HMGA1 levels were significantly elevated in pancreatic intraepithelial neoplasia (PanIN) epithelia compared with untransformed acinar tissues or fibroinflammatory stroma.<h4>Conclusions</h4>The PanINs have long been regarded as precancerous precursors to pancreatic adenocarcinoma. Significantly elevated HMGA1 levels observed in the nuclei of PanINs in Ptf1a-Cre; LSL-KrasG12D mice validate this animal model for investigating the role that HMGA1 plays in cancer progression and testing therapeutic approaches targeting HMGA1 in human cancers.
Project description:Pancreatic cancer (PC) is an almost uniformly lethal disease with inflammation playing an important role in its progression. Sustained stimulation of purinergic receptor P2X7 drives induction of NLRP inflammasome activation. To understand the role of P2X7 receptor and inflammasome, we performed transcriptomic analysis of p48Cre/+-LSL-KrasG12D/+ mice pancreatic tumors by next generation sequencing. Results showed that P2X7R's key inflammasome components, IL-1? and caspase-1 are highly expressed (p < 0.05) in pancreatic tumors. Hence, to target P2X7R, we tested effects of two P2X7R antagonists, A438079 and AZ10606120, on pancreatic intraepithelial neoplasms (PanINs) and their progression to PC in p48Cre/+-LSL-KrasG12D/+ mice. Following dose optimization studies, for chemoprevention efficacy, six-week-old p48Cre/+-LSL-KrasG12D/+ mice (24-36/group) were fed modified AIN-76A diets containing 0, 50 or 100 ppm A438079 and AZ10606120 for 38 weeks. Pancreata were collected, weighed, and evaluated for PanINs and PDAC. Control diet-fed male mice showed 50% PDAC incidence. Dietary A438079 and AZ10606120 showed 60% PDAC incidence. A marginal increase of PanIN 3 (carcinoma in-situ) was observed in drug-treated mice. Importantly, the carcinoma spread in untreated mice was 24% compared to 43-53% in treatment groups. Reduced survival rates were observed in mice exposed to P2X7R inhibitors. Both drugs showed a decrease in caspase-3, caspase-1, p21 and Cdc25c. Dietary A438079 showed modest inhibition of P2X7R, NLRP3, and IL-33, whereas AZ10606120 had no effects. In summary, targeting the P2X7R pathway by A438079 and AZ10606120 failed to show chemopreventive effects against PC and slightly enhanced PanIN progression to PDAC. Hence, caution is needed while treating high-risk individuals with P2X7R inhibitors.
Project description:Obesity is a risk factor for pancreatic ductal adenocarcinoma (PDAC), but it is not clear how obesity contributes to pancreatic carcinogenesis. The oncogenic form of KRAS is expressed during early stages of PDAC development and is detected in almost all of these tumors. However, there is evidence that mutant KRAS requires an additional stimulus to activate its full oncogenic activity and that this stimulus involves the inflammatory response. We investigated whether the inflammation induced by a high-fat diet, and the accompanying up-regulation of cyclooxygenase-2 (COX2), increases Kras activity during pancreatic carcinogenesis in mice.We studied mice with acinar cell-specific expression of KrasG12D (LSL-Kras/Ela-CreERT mice) alone or crossed with COX2 conditional knockout mice (COXKO/LSL-Kras/Ela-CreERT). We also studied LSL-Kras/PDX1-Cre mice. All mice were fed isocaloric diets with different amounts of fat, and a COX2 inhibitor was administered to some LSL-Kras/Ela-CreERT mice. Pancreata were collected from mice and analyzed for Kras activity, levels of phosphorylated extracellular-regulated kinase, inflammation, fibrosis, pancreatic intraepithelial neoplasia (PanIN), and PDACs.Pancreatic tissues from LSL-Kras/Ela-CreERT mice fed high-fat diets (HFDs) had increased Kras activity, fibrotic stroma, and numbers of PanINs and PDACs than LSL-Kras/Ela-CreERT mice fed control diets; the mice fed the HFDs also had shorter survival times than mice fed control diets. Administration of a COX2 inhibitor to LSL-Kras/Ela-CreERT mice prevented these effects of HFDs. We also observed a significant reduction in survival times of mice fed HFDs. COXKO/LSL-Kras/Ela-CreERT mice fed HFDs had no evidence for increased numbers of PanIN lesions, inflammation, or fibrosis, as opposed to the increases observed in LSL-Kras/Ela-CreERT mice fed HFDs.In mice, an HFD can activate oncogenic Kras via COX2, leading to pancreatic inflammation and fibrosis and development of PanINs and PDAC. This mechanism might be involved in the association between risk for PDAC and HFDs.
Project description:Gastrin signaling mediated through cholecystokinin-2 receptor (CCK2R) and its downstream molecules is altered in pancreatic cancer. CCK2R antagonists, YF476 (netazepide) and JNJ-26070109, were tested systematically for their effect on pancreatic intraepithelial neoplasia (PanIN) progression to pancreatic ductal adenocarcinoma (PDAC) in KrasG12D mice. After dose selection using wild-type mice, six-week-old p48Cre/+ -LSL-KrasG12D (22-24 per group) genetically engineered mice (GEM) were fed AIN-76A diets containing 0, 250, or 500 ppm JNJ-26070109 or YF-476 for 38 weeks. At termination, pancreata were collected, weighed, and evaluated for PanINs and PDAC. Results demonstrated that control-diet-fed mice showed 69% (males) and 33% (females) incidence of PDAC. Administration of low and high dose JNJ-26070109 inhibited the incidence of PDAC by 88% and 71% (P?<?.004) in male mice and by 100% and 24% (P?>?.05) in female mice, respectively. Low and high dose YF476 inhibited the incidence of PDAC by 74% (P?<?.02) and 69% (P?<?.02) in male mice and by 45% and 33% (P?>?.05) in female mice, respectively. Further, transcriptome analysis showed downregulation of Cldn1, Sstr1, Apod, Gkn1, Siglech, Cyp2c44, Bnc1, Fmo2, 623169, Kcne4, Slc27a6, Cma1, Rho GTPase activating protein 18, and Gpr85 genes in JNJ-26070109-treated mice compared with untreated mice. YF476-treated mouse pancreas showed downregulation of Riks, Zpbp, Ntf3, Lrrn4, Aass, Skint3, Kcnb1, Dgkb, Ddx60, and Aspn gene expressions compared with untreated mouse pancreas. Overall, JNJ-26070109 showed better chemopreventive efficacy than YF476. However, caution is recommended when selecting doses, as the agents appeared to exhibit gender-specific effects.
Project description:BACKGROUND & AIMS:Obesity is a risk factor for pancreatic cancer. In mice, a high-fat diet (HFD) and expression of oncogenic KRAS lead to development of invasive pancreatic ductal adenocarcinoma (PDAC) by unknown mechanisms. We investigated how oncogenic KRAS regulates the expression of fibroblast growth factor 21, FGF21, a metabolic regulator that prevents obesity, and the effects of recombinant human FGF21 (rhFGF21) on pancreatic tumorigenesis. METHODS:We performed immunohistochemical analyses of FGF21 levels in human pancreatic tissue arrays, comprising 59 PDAC specimens and 45 nontumor tissues. We also studied mice with tamoxifen-inducible expression of oncogenic KRAS in acinar cells (KrasG12D/+ mice) and fElasCreERT mice (controls). KrasG12D/+ mice were placed on an HFD or regular chow diet (control) and given injections of rhFGF21 or vehicle; pancreata were collected and analyzed by histology, immunoblots, quantitative polymerase chain reaction, and immunohistochemistry. We measured markers of inflammation in the pancreas, liver, and adipose tissue. Activity of RAS was measured based on the amount of bound guanosine triphosphate. RESULTS:Pancreatic tissues of mice expressed high levels of FGF21 compared with liver tissues. FGF21 and its receptor proteins were expressed by acinar cells. Acinar cells that expressed KrasG12D/+ had significantly lower expression of Fgf21 messenger RNA compared with acinar cells from control mice, partly due to down-regulation of PPARG expression-a transcription factor that activates Fgf21 transcription. Pancreata from KrasG12D/+ mice on a control diet and given injections of rhFGF21 had reduced pancreatic inflammation, infiltration by immune cells, and acinar-to-ductal metaplasia compared with mice given injections of vehicle. HFD-fed KrasG12D/+ mice given injections of vehicle accumulated abdominal fat, developed extensive inflammation, pancreatic cysts, and high-grade pancreatic intraepithelial neoplasias (PanINs); half the mice developed PDAC with liver metastases. HFD-fed KrasG12D/+ mice given injections of rhFGF21 had reduced accumulation of abdominal fat and pancreatic triglycerides, fewer pancreatic cysts, reduced systemic and pancreatic markers of inflammation, fewer PanINs, and longer survival-only approximately 12% of the mice developed PDACs, and none of the mice had metastases. Pancreata from HFD-fed KrasG12D/+ mice given injections of rhFGF21 had lower levels of active RAS than from mice given vehicle. CONCLUSIONS:Normal acinar cells from mice and humans express high levels of FGF21. In mice, acinar expression of oncogenic KRAS significantly reduces FGF21 expression. When these mice are placed on an HFD, they develop extensive inflammation, pancreatic cysts, PanINs, and PDACs, which are reduced by injection of FGF21. FGF21 also reduces the guanosine triphosphate binding capacity of RAS. FGF21 might be used in the prevention or treatment of pancreatic cancer.
Project description:To investigate the role of SHP2 (Ptpn11) in pancreatic carcinogenesis, murine pancreatic whole tissue RNA samples of 9 week old mice with the genotypes Ptf1a-Cre;LSL-KrasG12D (ID-labels Kxxx) and Ptf1a-Cre;LSL-KrasG12D;Ptpn11fl/fl (ID-labels Mxxxx) were analyzed by microarray.
Project description:Preclinical studies suggest that diets rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs) may be beneficial for prevention of pancreatic cancer. Nutritional intervention studies are often complex, and there is no clear evidence, without potential confounding factors, on whether conversion of n-6 PUFAs to n-3 PUFAs in pancreatic tissues would provide protection. Experiments were designed using n-3 fatty acid desaturase (Fat-1) transgenic mice, which can convert n-6 PUFA to n-3 FAs endogenously, to determine the impact of n-3 PUFAs on pancreatic intraepithelial neoplasms (PanINs) and their progression to pancreatic ductal adenocarcinoma (PDAC). Six-week-old female p48(Cre/+)-LSL-Kras(G12D/+) and compound Fat-1-p48(Cre/+)-LSL-Kras(G12D/+) mice were fed (AIN-76A) diets containing 10% safflower oil for 35 weeks. Pancreata were evaluated histopathologically for PanINs and PDAC. Results showed a dramatic reduction in incidence of PDAC (84%; P < .02) in Fat-1-p48(Cre/+)-LSL-Kras(G12D/+) mice compared to p48(Cre/+)-LSL-Kras(G12D/+) mice. Importantly, significant reductions of pancreatic ducts with carcinoma (90%; P < .0001) and PanIN 3 (~50%; P < .001) lesions were observed in the compound transgenic mice. The levels of n-3 PUFA were much higher (>85%; P < .05-0.01) in pancreas of compound transgenic mice than in those of p48(Cre/+)-LSL-Kras(G12D/+) mice. Molecular analysis of the pancreas showed a significant down-regulation of proliferating cell nuclear antigen, cyclooxygenase-2, 5-lipoxygenase (5-LOX), 5-LOX-activating protein, Bcl-2, and cyclin D1 expression levels in Fat-1-p48(Cre/+)-LSL-Kras(G12D/+) mice compared to p48(Cre/+)-LSL-Kras(G12D/+) mice. These data highlight the promise of dietary n-3 FAs for chemoprevention of pancreatic cancer in high-risk individuals.
Project description:Pancreatic cancer is the one of most common causes of cancer deaths and has the worst prognosis. Clinical observational studies suggest that statins may reduce the risk of pancreatic cancer. The chemopreventive efficacy of the statin atorvastatin (Lipitor(®)) and the role of the phosphatidyl-inositol 3-kinase (PI3/AKT) signaling pathway were evaluated for the progression of pancreatic intraepithelial neoplasms (PanINs) to pancreatic ductal adenocarcinoma (PDAC) in conditional p48(Cre/+) -LSL-Kras(G12D/+) transgenic mice. Six-week-old male p48(Cre/+) -LSL-Kras(G12D/+) (20/group) mice were fed AIN-76A diets containing 0, 200 and 400 ppm atorvastatin for 35 weeks. At termination, pancreata were evaluated histopathologically for PanINs and PDAC, and for various PI3/AKT signaling markers, and inflammatory cytokines, by immunohistochemistry/immunohistoflourscence, ELISA, Western blotting and/or reverse transcription-PCR methods. Control diet-fed mice showed 85% incidence of PDAC; whereas, mice fed with atorvastatin showed PDAC incidence of 65 and 35%, respectively (p < 0.0001). Similarly, significant suppression of PanIN-3 (22.6%) was observed in mice fed 400 ppm atorvastatin. Importantly, pancreata from atorvastatin-treated mice were ?68% free from ductal lesions. Furthermore, pancreas of mice administered with atorvastatin had significantly reduced expressions levels of PCNA, p2X7, p-ERK, RhoA, cyclin D1, survivin, Akt, pAKT, ?-catenin, cyclin E, cdK2 and caveolin-1. Also, atorvastatin-treated mice had shown dose-dependent suppression of inflammatory cytokines and a significant increase in tunnel-positive cells, p21 and PARP expression levels in pancreas. Atorvastatin significantly delays the progression of PanIN-1 and -2 lesions to PanIN-3 and PDAC by modulating PI3/AKT signal molecules in a preclinical model, suggesting potential clinical benefits of statins for high-risk pancreatic cancer patients.
Project description:Pancreatic cancer (PC) is acquired postnatally; to mimic this scenario, we developed an inducible KrasG12D; Ptf1a-CreER™ (iKC) mouse model, in which Kras is activated postnatally at week 16 upon tamoxifen (TAM) administration. Upon TAM treatment, iKC mice develop pancreatic intraepithelial neoplasia (PanIN) lesions and PC with metastasis at the fourth and fortieth weeks, respectively, and exhibited acinar-to-ductal metaplasia (ADM) and transdifferentiation. Kras activation upregulated the transcription factors Ncoa3, p-cJun and FoxM1, which in turn upregulated expression of transmembrane mucins (Muc1, Muc4 and Muc16) and secretory mucin (Muc5Ac). Interestingly, knockdown of KrasG12D in multiple PC cell lines resulted in downregulation of MUC1, MUC4, MUC5AC and MUC16. In addition, iKC mice exhibited ADM and transdifferentiation. Our results show that the iKC mouse more closely mimics human PC development and can be used to investigate pancreatic ductal adenocarcinoma (PDAC) biomarkers, early onset of PDAC, and ADM. The iKC model can also be used for preclinical strategies such as targeting mucin axis alone or in combination with neo-adjuvant, immunotherapeutic approaches and to monitor chemotherapy response.
Project description:Nuclear Protein 1 (Nupr1) is a major actor of the cell stress response required for KrasG12D-driven formation of pancreatic intraepithelial neoplastic (PanINs) lesions in mice. We investigated the impact of Nupr1-depletion on the development and biology of murin pancreatic adenocarcinomas (PDAC) in the Pdx1-cre;LSL-KrasG12D;Ink4a/Arffl/fl (KIC) mice. We found that only one half of Nupr1-deficient mice developed PDAC. This is related to increased caspase 3 activity and low IER3 expression in Nupr1-deficient;KIC in the pancreas. Moreover, when Nupr1-deficient;KIC mice do develop PDAC, tumors present with impaired epithelial-to-mesenchymal transition (EMT). Transcriptoma analysis revealed that Nupr1-deficient and Nupr1wt;KIC PDACs presented enrichment of gene signatures of the human classical- and quasi-mesenchymal (QM)-PDAC respectively. Moreover, Nupr1-deficient;KIC PDACs shared with human classical-PDACs overexpression of Kras-activation genes. In addition, cells derived from Nupr1-deficient;KIC PDACs formed fewer microspheres in vitro compared to Nupr1wt;KIC cells, indicative of stemness impairment in the absence of Nupr1. Finally, we found that Nupr1-deficient;KIC cells were more sensitive to some anticancer drugs than their Nupr1wt counterpart. Hence, this study establishes the pivotal role of Nupr1 in PDAC progression after PanIN and in PDAC EMT in vivo, with an impact in PDAC cell stemness. As a consequence, according to absence or presence of Nupr1, KIC mice develop tumors that phenocopy human classical- or QM-PDAC, respectively, thus becoming attractive models for preclinical drug trials. We investigated the impact of the homozygous deletion of the Nupr1 gene on pancreatic adenocarcinoma development and biology in the Pdx1-cre;LSL-KrasG12D;Ink4a/Arffl/fl (KIC) mouse model.