Conditional KrasG12D Mouse Model Research Articles

Deficiency in hormone-sensitive lipase accelerates the development of pancreatic cancer in conditional KrasG12D mice

BackgroundHormone sensitive lipase (HSL) is a neutral lipase that preferentially catalyzes the hydrolysis of diacylglycerol contributing to triacylglycerol breakdown in the adipose tissue. HSL has been implicated to play a role in tumor cachexia, a debilitating syndrome characterized by progressive loss of adipose tissue. Consequently, pharmacological inhibitors of HSL have been proposed for the treatment of cancer-associated cachexia. In the present study we used the conditional KrasG12D (KC) mouse model of pancreatic ductal adenocarcinoma (PDAC) with a deficiency in HSL to determine the impact of HSL suppression on the development of PDAC.MethodsKC;Hsl+/+ and KC;Hsl−/− mice were fed standard rodent chow for 20 weeks. At sacrifice, the incidence of PDAC was determined and inflammation in the mesenteric adipose tissue and pancreas was assessed histologically and by immunofluorescence. To determine statistical significance, ANOVA and two-tailed Student’s t-tests were performed. To compare PDAC incidence, a two-sided Fisher’s exact test was used.ResultsCompared to KC;Hsl+/+ mice, KC;Hsl−/− mice gained similar weight and displayed adipose tissue and pancreatic inflammation. In addition, KC;Hsl−/− mice had reduced levels of plasma insulin and leptin. Importantly, the increased adipose tissue and pancreatic inflammation was associated with a significant increase in PDAC incidence in KC;Hsl−/− mice.ConclusionsHSL deficiency is associated with adipose tissue and pancreatic inflammation and accelerates PDAC development in the KC mouse model.

Metformin prevents obesity-promoted pancreatic cancer development in the conditional KrasG12D mouse model

Metformin prevents obesity-promoted pancreatic cancer development in the conditional KrasG12D mouse model

Metformin Decreases the Incidence of Pancreatic Ductal Adenocarcinoma Promoted by Diet-induced Obesity in the Conditional KrasG12D Mouse Model

Pancreatic ductal adenocarcinoma (PDAC) is a particularly deadly disease. Chronic conditions, including obesity and type-2 diabetes are risk factors, thus making PDAC amenable to preventive strategies. We aimed to characterize the chemo-preventive effects of metformin, a widely used anti-diabetic drug, on PDAC development using the KrasG12D mouse model subjected to a diet high in fats and calories (HFCD). LSL-KrasG12D/+;p48-Cre (KC) mice were given control diet (CD), HFCD, or HFCD with 5 mg/ml metformin in drinking water for 3 or 9 months. After 3 months, metformin prevented HFCD-induced weight gain, hepatic steatosis, depletion of intact acini, formation of advanced PanIN lesions, and stimulation of ERK and mTORC1 in pancreas. In addition to reversing hepatic and pancreatic histopathology, metformin normalized HFCD-induced hyperinsulinemia and hyperleptinemia among the 9-month cohort. Importantly, the HFCD-increased PDAC incidence was completely abrogated by metformin (p < 0.01). The obesogenic diet also induced a marked increase in the expression of TAZ in pancreas, an effect abrogated by metformin. In conclusion, administration of metformin improved the metabolic profile and eliminated the promoting effects of diet-induced obesity on PDAC formation in KC mice. Given the established safety profile of metformin, our findings have a strong translational potential for novel chemo-preventive strategies for PDAC.

Abstract 3163: Diet-induced obesity is associated with increased levels of IL-4 and IL-13, macrophage infiltration, fibrosis, and pancreatic neoplasia in the conditional KrasG12D mouse model

Abstract Background: Obesity is a well known risk factor for the development of several cancers including pancreatic cancer (PaCa). The formation of an inflammatory microenvironment with a complex communication between (pre)-neoplastic cells and stromal cells, including fibroblasts and inflammatory cells, is thought to promote tumor development during obesity. However, the exact molecular signals that are critical in this process are unknown. Recently, there has been interest in understanding the role of interleukins (IL)-4 and -13 in mediating the crosstalk between several cell types in cancer. E.g. they have been shown to promote the conversion of anti-tumorigenic M1 macrophages to pro-tumorigenic M2 macrophages. Aim: In this study we sought to investigate whether diet-induced obesity in the conditional KrasG12D mouse model of PaCa is associated with enhanced fibrosis and inflammation in the pancreas and to determine a possible role of IL-4 and -13. Methods and Results: Conditional KrasG12D mice and wildtype controls were fed a high fat, high calorie diet (HFCD) or control diet (CD) for 3 months. Mice fed the HFCD gained significantly more weight with a substantial enlargement of the visceral adipose tissue. Mutant mice fed the HFCD had more advanced pancreatic intraepithelial neoplasia lesions (PanINs). Compared to CD, mice fed the HFCD had a significant increase in pancreatic fibrosis (3.0±0.7 fold) and enhanced activation of pancreatic stellate cells (1.9±0.2) as assessed by immunohistochemical staining for fibronectin and α-smooth muscle actin, respectively. Flow cytometry demonstrated increased infiltration of Gr-CD11b+ macrophages (2.0±0.4 fold) into the pancreas in mice fed the HFCD. In addition, compared to the CD the HFCD was associated with increased tissue levels of IL-4 (43±5%) and IL-13 (65±20%) in the pancreas. Conclusion: Our results demonstrate that diet-induced obesity was associated with enhanced pancreatic neoplasia, inflammation, and fibrosis in the conditional KrasG12D mouse model. Our data further show that IL-4 and IL-13 are elevated in the pancreas of obese mice suggesting that these cytokines play a role, possibly in conversion of macrophages to the pro-tumorigenic M2 phenotype thereby promoting inflammation and tumor development. Citation Format: Chiara Birtolo, Guido Eibl, Aune Moro, Xiaoman Jung, Susan Morvaridi, Richard Waldron, Aurelia Lugea, Vay LW Go, Stephen J. Pandol. Diet-induced obesity is associated with increased levels of IL-4 and IL-13, macrophage infiltration, fibrosis, and pancreatic neoplasia in the conditional KrasG12D mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3163. doi:10.1158/1538-7445.AM2015-3163

Abstract IA18: Obesity, inflammation, and pancreatic cancer

Abstract There is epidemiologic evidence that obesity increases the risk of cancers including pancreatic cancer. Several underlying mechanisms, e.g. inflammation and insulin resistance, are proposed. However, the driving mechanisms in pancreatic cancer are poorly understood. We developed a model of diet-induced obesity and pancreatic cancer development in a state-of-the-art mouse model, which resembles important clinical features of human obesity, e.g. weight gain and metabolic disturbances. Offspring of Pdx-1-Cre (or p48-cre) and LSL-KrasG12D crosses were allocated to either a diet high in fats and calories (HFCD; ~4,535 kcal/kg; 40% of calories from fats) or control diet (CD; ~3,725 kcal/kg; 12% of calories from fats) for various time periods. Compared to control animals, mice fed the HFCD significantly gained more weight and developed hyperinsulinemia, hyperglycemia, hyperleptinemia, and elevated levels of IGF-1. Mice fed the HFCD had a substantial increase in visceral fat as measured by micro-CT. The white visceral adipose tissue in mice fed the HFCD showed signs of marked inflammation, which was more pronounced in the peri-pancreatic fat depot. In addition, the pancreas of HFCD-fed animals showed robust signs of inflammation with increased numbers of infiltrating inflammatory cells. Detailed flow cytometry analysis showed a marked elevation of infiltrating natural killer cells, myeloid-derived suppressor cells, T-regulatory cells, and tumor-infiltrating macrophages into the pancreas of mice fed the HFCD. Multiplex analysis demonstrated a significant change in the pancreatic cytokine profile in HFCD-fed mice compared to CD-fed animals. Importantly, the HFCD led to an acceleration of PanIN development in conditional KrasG12D mice. These results demonstrate that a diet high in fats and calories leads to obesity and metabolic disturbances similar to humans and accelerates early pancreatic neoplasia in the conditional KrasG12D mouse model. Based on the notion that obesity is associated with an increase in inflammatory mediators and insulin-resistance and our previous finding that non-steroidal anti-inflammatory drugs inhibit the progression of PanIN lesions in the conditional KrasG12D mouse model, we investigated a potential crosstalk between the PGE2/EP/cAMP and IGF-1/Akt/mTORC1 signaling pathways in pancreatic cancer. In multiple cell lines PGE2 increased intracellular cAMP levels, indicating the activation of Gsα-coupled EP receptors (EP2 and/or EP4). PGE2 dose- and time-dependently increased the phosphorylation of S6 ribosomal protein downstream of mTORC1, suggesting a crosstalk between PGE2/cAMP and mTORC1. In addition, the effect of PGE2 on phospho-S6rp was mimicked by Forskolin, a pharmacological cAMP stimulator. Importantly, PGE2 enhanced the effect of IGF-1 on S6rp phosphorylation, supporting the existence of a positive reinforcement. Interestingly, PGE2 and forskolin had no effect on Akt phosphorylation, suggesting a link downstream of Akt. In the presence of the PKA inhibitor H89, baseline phosphorylation of S6rp and PGE2-activated S6rp phosphorylation were reduced, indicating a role of PKA in the crosstalk. In summary, these data provide evidence of a crosstalk between the PGE2/EP/cAMP and IGF-1/Akt/mTORC1 signaling pathways. Since both pathways are over-activated in obesity-associated cancers, this interaction may be of great importance in elucidating the tumor-promoting effects of obesity in pancreatic cancer. Citation Format: Guido Eibl. Obesity, inflammation, and pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr IA18.

Abstract 5353: Leptin regulates cell differentiation and protumorigenic responses in pancreatic stellate cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by marked desmoplasia orchestrated by pancreatic stellate cells (PaSCs). Epidemiological studies indicate that obesity and chronic pancreatitis (from alcoholism) increase the risk of PDAC. We recently showed that a diet high in fats and calories increases blood leptin levels and accelerates the development of desmoplasia and early pancreatic neoplasia in the conditional KrasG12D mouse model of pancreatic carcinogenesis. We hypothesize that environmental risk factors such as obesity and alcohol abuse facilitate PDAC progression by regulating PaSC tumorigenic responses. Here we measured leptin and ethanol effects on cell differentiation into the myofibroblast phenotype, cell proliferation and fibrogenic responses in primary mouse PaSC and mouse and human PaSC lines. Inhibitors of mTOR signaling were used to delineate the role of mTOR on leptin-mediated PaSC responses. We found that leptin receptors are expressed in cultured-activated primary mouse PaSCs and in immortalized mouse and human PaSCs. In quiescent PaSCs isolated from the mouse pancreas, leptin alone or in combination with ethanol markedly accelerated PaSC activation and promoted extracellular matrix (ECM) protein production (collagen and fibronectin) via ERK and Akt/mTORC1 signaling pathways, suggesting that these pathways interact to modulate the activated PaSC phenotype. In activated PaSCs, leptin induced activation of JAK/STAT and mTORC1 signaling pathways, and the mTORC1 inhibitor rapamycin blocked leptin-induced mTORC1 activation and markedly reduced fibronectin production and cell proliferation. Other factors known to regulate cell proliferation and inflammation in obesity and alcohol abuse, such as IGF-1, TNFα, and LPS also transiently activated Akt/mTORC1 signaling in PaSCs. Further, we found that pancreatic intraepithelial neoplasia (PanIN) cells isolated from the KrasG12D mice interact with PaSCs to promote their proliferation and ECM production. Our data support a key role for leptin-induced Akt/mTOR signaling in regulating PaSC pro-tumorigenic responses and indicate that leptin and other inflammatory factors provide the mechanistic underpinnings of environmental risk factor promotion in PDAC. Citation Format: Jun Gong, Raymond Gong, Richard T. Waldron, Aurelia Lugea, Stephen J. Pandol. Leptin regulates cell differentiation and protumorigenic responses in pancreatic stellate cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5353. doi:10.1158/1538-7445.AM2014-5353

High-Fat, High-Calorie Diet Promotes Early Pancreatic Neoplasia in the Conditional KrasG12D Mouse Model

There is epidemiologic evidence that obesity increases the risk of cancers. Several underlying mechanisms, including inflammation and insulin resistance, are proposed. However, the driving mechanisms in pancreatic cancer are poorly understood. The goal of the present study was to develop a model of diet-induced obesity and pancreatic cancer development in a state-of-the-art mouse model, which resembles important clinical features of human obesity, for example, weight gain and metabolic disturbances. Offspring of Pdx-1-Cre and LSL-KrasG12D mice were allocated to either a high-fat, high-calorie diet (HFCD; ∼4,535 kcal/kg; 40% of calories from fats) or control diet (∼3,725 kcal/kg; 12% of calories from fats) for 3 months. Compared with control animals, mice fed with the HFCD significantly gained more weight and developed hyperinsulinemia, hyperglycemia, hyperleptinemia, and elevated levels of insulin-like growth factor I (IGF-I). The pancreas of HFCD-fed animals showed robust signs of inflammation with increased numbers of infiltrating inflammatory cells (macrophages and T cells), elevated levels of several cytokines and chemokines, increased stromal fibrosis, and more advanced PanIN lesions. Our results show that a diet high in fats and calories leads to obesity and metabolic disturbances similar to humans and accelerates early pancreatic neoplasia in the conditional KrasG12D mouse model. This model and findings will provide the basis for more robust studies attempting to unravel the mechanisms underlying the cancer-promoting properties of obesity, as well as to evaluate dietary- and chemopreventive strategies targeting obesity-associated pancreatic cancer development.

Oxidative Stress Induced by Inactivation of TP53INP1 Cooperates with KrasG12D to Initiate and Promote Pancreatic Carcinogenesis in the Murine Pancreas

Tumor protein p53-induced nuclear protein 1 (TP53INP1) is involved in cell stress response. Itsexpression is lost at the pancreatic intraepithelial neoplasia 1b (PanIN1b)/PanIN2 stage of pancreatic carcinogenesis. Our objective was to determine whether TP53INP1 loss of expression contributes to pancreatic cancer formation in a conditional KrasG12D mouse model. We generated Kras-INP1KO mice using LSL-Kras(G12D/+);Pdx1-Cre(+/-) mice (Kras mice) and TP53INP1(-/-) mice. Analysis of pancreases during ageing shows that in the presence of activated Kras, TP53INP1 loss of expression accelerated PanIN formation and increased pancreatic injury and the number of high-grade lesions as compared with what occurs in Kras mice. Moreover, cystic lesions resembling intraductal papillary mucinous neoplasm (IPMN) were observed as early as 2 months of age. Remarkably, TP53INP1 is down-regulated in human IPMN. Activation of the small GTPase Rac1 shows that more oxidative stress is generated in Kras-INP1KO than in Kras mice pancreas despite elevated levels of the Nrf2 antioxidant regulator. We firmly establish the link between Kras-INP1KO pancreatic phenotype and oxidative stresswith rescue of the phenotype by the antioxidant action of N-acetylcysteine. Our data provide invivo functional demonstration that TP53INP1 deficiency accelerates progression of pancreatic cancer, underlining its role in the occurrence of IPMN and highlighting the importance of TP53INP1 in the control of oxidative status during development of pancreatic cancer.

Acceleration of pancreatic cancer development in the conditional KrasG12D mouse model by a high fat and high calorie diet

Acceleration of pancreatic cancer development in the conditional KrasG12D mouse model by a high fat and high calorie diet

Antifibrotic agent, pirfenidone, inhibits pancreatic stellate cells and tumor-stromal interaction in pancreatic cancer

s / Pancreatology 13 (2013) e1–e94 e4 greatly accelerates pancreatic cancer development in the conditional KrasG12Dmouse model. This model and findings will provide the basis for more robust studies attempting to unravel the mechanisms underlying the cancer-promoting properties of obesity.

Abstract 1613: Delta-tocotrienol delays the progression of pancreatic intraepithelial neoplasia (PanIN) lesions in a conditional KrasG12D mouse model

Abstract Background: We have shown that natural vitamin E γ-tocotrienol is the most bioactive tocotrienols in inhibiting the cell proliferation and induction of apoptosis in pancreatic cancer cells as well as in xenograft models. Therefore in this study we evaluated the chronic 12-month feeding of γ-tocotrienol on the prevention of the progression of PanIN lesions in a conditional KrasG12D mouse model. Methods: Offspring of LSLKRASG12D x PDX-1-Cre intercrosses (double positive by genotyping) were randomized to three groups: 1) No treatment (NT), 2) Vehicle (ethanol extracted olive oil, 1.0 ml/kg x 2/ day, PO) and 3) γ-tocotrienol (200 mg/kg x 2/day, PO). The treatment was started at the age of 10 weeks and continued for 12 months. The survival of the mice in three groups was plotted by Kaplan-Meir graph. The pancreas of KRASG12D; PDX-1-Cre mice was analyzed for the presence of murine PanINs. The apoptosis, cell cycle and signaling markers were determined by Western blotting and immunostaining. Results: No significant difference in food intake and body weight gain between γ-tocotrienol and control groups was observed during 12 months feeding. γ-tocotrienol treatment significantly increased survival of mice compared to control groups (p&amp;lt;0.025). Mice fed γ-tocotrienol had a significant delay in the progression of mPanIN lesions. The proportion of mPanIN-3 lesion was significantly decreased (P&amp;lt;0.001), whereas the relative numbers of all other lesions combined were slightly increased in the γ-tocotrienol-fed animals. γ-tocotrienol feeding also inhibited the pMEK and pERK signaling and increased p27Kip1 expression in the pancreatic tumor tissues. The apoptosis markers such as caspase-3, PARP1 cleavage, and Bax expression were increased in tumor tissues of mice treated with γ-tocotrienol compared to controls. NF-κB/p65 and the NF-κB transcriptional targets Bcl-XL was down-regulated by γ-tocotrienol treatment. The ratio of the proapoptotic (Bax) and antiapoptotic (Bcl-XL) proteins was increased almost 4-folds in the pancreatic tumor tissues compared to controls. Conclusion: γ-tocotrienol delays the progression of pancreatic cancer precursor lesions in a preclinical animal model hence warrant it's clinical use for the prevention and treatment of pancreatic cancer in high-risk patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1613. doi:1538-7445.AM2012-1613

Delayed Progression of Pancreatic Intraepithelial Neoplasia in a Conditional KrasG12D Mouse Model by a Selective Cyclooxygenase-2 Inhibitor

Pancreatic ductal adenocarcinomas are thought to arise from noninvasive, intraductal precursor lesions called pancreatic intraepithelial neoplasias (PanIN). The study of PanINs holds great promise for the identification of early detection markers and effective cancer-preventing strategies. Cyclooxygenase-2 (COX-2) represents an intriguing target for therapeutic and preventive approaches in various human malignancies. The aim of the present study was to evaluate the efficacy of a selective COX-2 inhibitor to prevent the progression of PanINs in a conditional Kras(G12D) mouse model. Offspring of LSL-KRAS(G12D) x PDX-1-Cre intercrosses were randomly allocated to a diet supplemented with the selective COX-2 inhibitor nimesulide (400 ppm) or a control diet. After 10 months, animals were sacrificed. Successful recombination in the pancreas was evaluated by PCR. The pancreas of KRAS(G12D);PDX-1-Cre mice was analyzed for the presence of murine PanINs. Animals fed the COX-2 inhibitor had significantly fewer PanIN-2 and PanIN-3 lesions than control animals (P < 0.05). Ten percent of all pancreatic ducts in the nimesulide-fed animals showed PanIN-2 or PanIN-3 lesions, whereas 40% of the pancreatic ducts in the control animals had PanIN-2 or PanIN-3 lesions. Intrapancreatic prostaglandin E(2) levels were reduced in nimesulide-fed animals. Immunohistochemistry confirmed COX-2 expression in early and late PanINs. In summary, we found that the selective COX-2 inhibitor nimesulide delays the progression of pancreatic cancer precursor lesions in a preclinical animal model. These data highlight the importance of COX-2 in the development of pancreatic cancer. Inhibition of COX-2 may represent an intriguing strategy to prevent pancreatic cancer in high-risk patients.