Animal Models Of Pancreatic Cancer Research Articles

Abstract A75: Meloxicam and nimesulide modulate immunity in pancreatic cancer

Abstract Background: Meloxicam and nimesulide have been studied as potential cancer immunomodulatory agents. Those drugs have been shown to have immune stimulatory effects on the tumor stroma and the microenvironment of several tumors including pancreatic ductal adenocarcinoma. They have several immune effects in vitro and in animal models. We compared the immunomodulatory activity of two preferential Cox-2 inhibitors drugs, meloxicam and nimesulide in advanced pancreatic cancer patient’s peripheral blood mononuclear cells (PBMCs) with good and poor Karnofsky. Materials and Methods: Repository PBMCs from advanced pancreatic patients with Karnofsky < 50% (n=10) and > 80% (n=10) were stimulated with different concentrations of meloxicam and nimesulide in vitro. We performed dose-response curve with concentrations of both drugs ranging from 0.5, 1, 10, 50 and 100 μM and we performed the ELISPOT assays for Interferon-gamma using human anti-CD3 and CMV whole lysate as basal T cell stimulation. Th2 cytokines were measured as well by ELISPOT and Cytokine ELISA. Results: In patients with Karnofsky > 80% both meloxicam and nimesulide were effective in promoting IFN-gamma production after stimulation with both drugs starting at 0.5 μM. The use of meloxicam was associated with significant production of IFN-gamma at 0.5 μM (p<0.001) and 1μM (p<0.0001). Nimesulide significantly induced IFN-gamma secretion at 0.5 μM (p=0.005), 1 μM (p=0.0002) and at 10 μM (p<0.0001). Meloxicam and nimesulide treatment resulted in different responses in PBMCs IFG-gamma production from pancreatic cancer patients that had a Karnofsky <50%. Nimesulide was more effective than meloxicam at promoting IFN-gamma production at 1μM (p<0.005) and 10μM (p<0.0001). Meloxicam did not improve the IFN-gamma secretion at low doses such as 0.5, 1 and 10 μM (n.s.). However, meloxicam at 50 μM and 100 μM was able to induce IFN-gamma (p<0.05 and p<0.0001, respectively). Additionally PBMCs treated with nimesulide in both groups were able to decrease Th2 cytokines such as IL-6, IL-18, TNF-alpha and IL-8 (statistical analysis in progress). Discussion: This preliminary data suggests that the use of meloxicam and nimesulide as putative immunomodulatory drugs may be less effective when pancreatic patients have a poor Karnosky although at high concentration they are effective. Also, the potent induction of IFN-gamma in patients with a good Karnofsky suggest synergy of these drugs in combination with standard of care treatment, immunogenic chemotherapy, anti-stroma strategies and active specific immunotherapy as a combinatorial approach to move forward into animal models of pancreatic cancer and eventually into the clinic. Grant Support: Junior Physician Scientist Fellowship Grant from CICS. Citation Format: Juan Marquez, Erik Ramos, Pedro Alejandro Lucero-Diaz, Fernando Durazo-Bustamante, Gerardo Palacios Diaz De Leon, Rosana Soto-Soto, Sara Carrillo-Marquez, Arturo Suplee-Rivera, Alejandro Camacho-Hernandez, Daniel Camacho-Gil.{Authors}. Meloxicam and nimesulide modulate immunity in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A75.

Animal models of pancreatic cancer and their application in clinical research

Animal models of pancreatic cancer and their application in clinical research

Impaired Synthesis of Stromal Components in Response to Minnelide Improves Vascular Function, Drug Delivery, and Survival in Pancreatic Cancer.

Pancreatic cancer stromal microenvironment is considered to be the major reason for failure of conventional and targeted therapy for this disease. The desmoplastic stroma, comprising mainly collagen and glycosaminoglycans like hyaluronan (HA), is responsible for compression of vasculature in the tumor resulting in impaired drug delivery and poor prognosis. Minnelide, a water-soluble prodrug of triptolide currently in phase I clinical trial, has been very effective in multiple animal models of pancreatic cancer. However, whether Minnelide will have efficacious delivery into the tumor despite the desmoplastic stroma has not been evaluated before. Patient tumor-derived xenografts (PDX) and spontaneous pancreatic cancer mice were treated with 0.42 and 0.21 mg/kg body weight for 30 days. Stromal components were determined by IHC and ELISA-based assays. Vascular functionality and drug delivery to the tumor were assessed following treatment with Minnelide. Our current study shows that treatment with Minnelide resulted in reduction of ECM components like HA and collagen in the pancreatic cancer stroma of both the spontaneous KPC mice as well as in patient tumor xenografts. Furthermore, treatment with Minnelide improved functional vasculature in the tumors resulting in four times more functional vessels in the treated animals compared with untreated animals. Consistent with this observation, Minnelide also resulted in increased drug delivery into the tumor compared with untreated animals. Along with this, Minnelide also decreased viability of the stromal cells along with the tumor cells in pancreatic adenocarcinoma. In conclusion, these results are extremely promising as they indicate that Minnelide, along with having anticancer effects is also able to deplete stroma in pancreatic tumors, which makes it an effective therapy for pancreatic cancer.

An anti-EpCAM antibody EpAb2-6 for the treatment of colon cancer.

Epithelial cell adhesion molecule (EpCAM) is known to be overexpressed in epithelial cancers associated with enhanced malignant potential, particularly colorectal carcinoma (CRC) and head and neck squamous cell carcinoma (HNSCC). However, it is unknown whether progression of malignance can be directly inhibited by targeting EpCAM. Here, we have generated five novel monoclonal antibodies (mAbs) against EpCAM. One of these anti-EpCAM mAbs, EpAb2-6, was found to induce cancer cell apoptosis in vitro, inhibit tumor growth, and prolong the overall survival of both a pancreatic cancer metastatic mouse model and mice with human colon carcinoma xenografts. EpAb2-6 also increases the therapeutic efficacy of irinotecan, fluorouracil, and leucovorin (IFL) therapy in a colon cancer animal model and gemcitabine therapy in a pancreatic cancer animal model. Furthermore, EpAb2-6, which binds to positions Y95 and D96 of the EGF-II/TY domain of EpCAM, inhibits production of EpICD, thereby decreasing its translocation and subsequent signal activation. Collectively, our results indicate that the novel anti-EpCAM mAb can potentially be used for cancer-targeted therapy.

Abstract B57: Gnetin C, a novel resveratrol dimer, targets pancreatic cancer metabolism

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is aggressively invasive and treatment-resistant malignancy, and is the fourth leading cause of cancer deaths in the United States. The pancreatic cancer cells alter specific metabolic pathways to meet their tremendous energy and biomass demands which contribute to the progression and dissemination of this disease. More importantly, the oncogenic signaling enables cancer cells to reprogram the cellular metabolism that afford growth and proliferative advantages over normal cells and, thus, may contribute to pancreatic cancer pathophysiology. There is an increasing interest to investigate the oncogenic signaling that controls the metabolic reprogramming of cancer cells. Objectives: Mammalian Target of Rapamycin (mTOR), a central downstream target of Akt, is a highly conserved protein kinase, and is frequent activated in PDAs. Earlier studies reported that mTOR, a master regulator of cell growth and proliferation downstream of oncogenic signaling pathways, controls specific aspects of cellular metabolism through the induction of metabolic gene expression. In our earlier microarray studies, we observed differential expression of genes associated with glucose and lipid metabolic pathways, with concomitant increase in mTOR expression. However, the mechanism and critical link between mTOR and the pancreatic cancer metabolism has not been fully understood. Methodology: In the present study, we evaluated the effectiveness of Melinjo (Indonesian name; Gnetum gnemon L.) seed extract (MSE) and gnetin C (GC), a resveratrol dimer found abundantly in MSE in human (Aspc-1 and PANC-1) and mouse (Pan-02) pancreatic cancer cells. MSE has been reported to exert antioxidant, lipase and amylase inhibition, anti-metabolic syndrome effects and anticancer activities. A recent clinical study reported that MSE decreased serum uric acid and increased HDL cholesterol levels in humans, suggesting that MSE may improve lipid metabolism. To test the anti-tumor activities, pancreatic cancer cells were treated with various concentrations of MSE (0 - 400μg/ml) and GC (0 – 100μM) for 48 h. MTS cell proliferation and apoptotic assays were performed to determine the cell growth inhibition and induction of apoptosis. Trans-resveratrol (RES) that is abundant in grapes and wine was used to compare the effects of GC. Real-time PCR was performed to evaluate the effect of a MSE or GC on mTOR and its downstream targets, as well selected glucose and lipid metabolic pathway genes. Western blot was performed to confirm their expression at the protein level. Results and conclusion: Human (ASPC-1 and PANC-1), and mouse (Pan-02) pancreatic cancer cells treated with MSE, GC and RES showed a dose-dependent decrease in cancer cell proliferation. The IC50 values of MSE, GC and RES related to their anti-proliferative effects revealed that GC, the major metabolite of MSE has superior anti-proliferative properties than resveratrol. Further, flow cytometry analysis showed a pre-G1 peak of apoptosis (>10%) with GC. We observed that GC significantly downregulated mTOR complex, namely (mTORC1 and mTOC2). mTORC1 is a sensor of systemic and local levels of nutrients that regulates cancer cell proliferation and survival. mTORC2 is associated with the ribosome and insulin-stimulated oncogenic PI3K signaling. We also found that GC downregulated p-ribosomal protein S6 (p-RPS6), a downstream target of mTOR that plays important role in both neoplastic and inflammatory processes. In addition, GC also downregulated glycogen synthase kinase 3 alpha and beta (GSK3α and β) that is linked to glycogen metabolism and 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), which is associated with lipids and lipoproteins metabolism (cholesterol biosynthesis). Further analysis on key genes of the lipid metabolism is in progress. Given the preliminary evidence that GC has superior anti-proliferative activities and its ability to inhibit mTOR and metabolic/lipid pathway genes can be expected to yield more potent therapies against this deadly disease. The future goal will be to test the potentials of GC on the antitumor efficacy and the effect on metabolic pathway targets in preclinical animal models of pancreatic cancer. Citation Format: Narayanan K. Narayanan, Kazuhiro Kunimasa, Di Tian, Lori Horton, Igor Dolgaev, Adriana Heguy, George Miller, Amit Tiwari, Bhagavathi A. Narayanan. Gnetin C, a novel resveratrol dimer, targets pancreatic cancer metabolism. [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 B57.

Abstract B119: Triptolide abrogates expression of the Met and epidermal growth factor receptors in pancreatic cancer

Abstract Background: Minnelide, a pro-drug of the Chinese herb triptolide, is currently in Phase I clinical trials against pancreatic cancer. Preclinical studies with Minnelide have demonstrated its efficacy in preventing tumor formation and causing tumor regression in multiple animal models of pancreatic cancer. However, its mechanism of action remains unclear. Pancreatic cancer cells overexpress several receptor tyrosine kinases including the Met and EGF receptors which drive proliferation, migration and invasion of these cells. Here, using human pancreatic cancer cells as well as the KRasG12D; Trp53R172H; Pdx-1 Cre (KPC) animal model, we show the ability of triptolide, or its water soluble pro-drug, Minnelide, to down-regulate the Met and EGF receptors, both in vitro and in vivo. Methods:Pancreatic cancer cells were treated with triptolide for varying times (0-24h) and receptor levels evaluated using western blotting or confocal microscopy. The KPC mouse model recapitulates human disease progression and is therefore the most relevant model for chemotherapeutic drug testing. KPC animals, which spontaneously form pancreatic tumors, were treated with Minnelide for one week and tumors harvested. mRNA expression was analyzed using real time PCR and protein expression assessed by immunohistochemistry. Results: Treatment with triptolide causes an increase in tyrosine phosphorylation at earlier time points (0.5 - 6h), accompanied by an increase in levels of the Met and EGF receptor tyrosine kinases. However, longer treatments (12-24h) resulted in marked decrease in levels of both Met and EGF receptors. Further investigation using tumors from KPC animals treated with Minnelide show a decrease in gene expression of both the Met and EGF receptors compared with tumors from saline injected animals. Furthermore, immunohistochemical analysis of formalin fixed paraffin embedded sections from these tumors show a decrease in levels of the Met receptor. Conclusion: Treatment of cells or KPC pancreatic tumor bearing animals with triptolide or Minnelide leads to decreased expression of the Met and EGF receptor tyrosine kinases. These results lend further insight into the mechanism of action of Minnelide, a pro-drug currently in clinical trials against pancreatic cancer. Citation Format: Veena Sangwan, Kelsey M. Jensen, Vikas Dudeja, Rohit Chugh, Sulagna Banerjee, Selwyn M. Vickers, Morag Park, Ashok K. Saluja. Triptolide abrogates expression of the Met and epidermal growth factor receptors in 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 B119.

Risk factors for pancreatic cancer: underlying mechanisms and potential targets.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths among both men and women in the United States and remains essentially without effective therapies. The 5-year survival of patients with pancreatic cancer is a dismal 6% or less. Environmental risk factors such as smoking, diabetes, obesity, and alcoholism play major roles in the promotion of PDAC. However, currently we have limited understanding of how these risk factors promote the disease. The papers presented in this topic illustrate the latest knowledge regarding the mechanisms of pancreatic cancer induction and promotion by major risk factors such as smoking, pancreatitis, alcohol abuse, obesity and diabetes (Kolodecik et al., 2013), and less studied factors such blood group types (Pelzer et al., 2013) and the genetic mutations (Kong et al., 2013; Reznik et al., 2014; Weiss, 2014). Indeed, an inherited predisposition to PDAC is believed to present in familial cancer syndromes such as the Peutz-Jeghers Syndrome, which is associated with germline mutations in the STK11/LKB1 gene, Familial Atypical Multiple Mole Melanoma syndrome, which results due to germline mutations in the p16/CDKN2A gene, Hereditary Breast-Ovarian Cancer syndrome (BRCA1/2 genes), Hereditary Non-polyposis Colorectal Cancer (mismatch repair genes), and Familial Adenomatous Polyposis syndrome (Reznik et al., 2014). Hereditary causes of pancreatitis, such as the autosomal dominant form caused by germline mutations of the cationic trypsinogen gene, PRSS1, have been indirectly linked to PDAC through early onset chronic pancreatitis with an associated 53-fold increased incidence and approximately 40% of hereditary pancreatitis patients noted to develop pancreatic cancer by age 70 (Weiss, 2014). Finally, more patients with blood group A suffer from PDAC whereas blood group O was less frequent in patients with PDAC (Pelzer et al., 2013). Kras mutations remain the most abundant genetic alteration found in pancreatic cancer patients. Kras mutations may lead to reducing power for ROS detoxification, leading to low ROS levels in pancreatic pre-neoplastic cells and in cancer cells. In adult stem cells and cancer stem cells, low ROS levels have been associated with the formation of a proliferation-permissive intracellular environment and with perseverance of self-renewal capacities. Therefore, it is conceivable that low intracellular ROS levels may contribute significantly to oncogenic Kras-mediated PDAC formation (Kong et al., 2013). Specific gene-based, gene-product, and marker-based testing for the early detection of pancreatic cancer are currently being developed, with the potential for these to be useful as potential therapeutic targets as well. Today, the role of stromal cells is highly appreciated for pancreatic cancer development. Immune cell infiltration into the tumor not only fail to contribute to disease eradication but rather due to exhibiting a Th2-type inflammation and immunosuppression is associated with more rapid disease progression, cachexia induction, and reduced survival. Polarization of macrophages toward M2-type correlates with a poor prognosis after surgery in resected patients. High CD163+ and CD204+ cell counts correlate with metastasis and poor prognosis in PDAC patients (Protti and De Monte, 2013; Tan et al., 2014). Src kinase might serve as a critical mechanistic link between inflammation and cancer, mediating and propagating a cycle between immune and tissue cells that can ultimately lead to the development and progression of cancer (Liu et al., 2013). In addition, there is now compelling evidence that pancreatic stellate cells interact not only with cancer cells themselves, but with several other cell types in the stroma (endothelial cells, immune cells, and possibly neuronal cells) to promote cancer progression. Strategies to target the tumor microenvironment cells are proposed (Wilson et al., 2014). The central role of the mammalian target of rapamycin (mTOR) in mediating a crosstalk between the insulin/IGF-1 and GPCR signaling in pancreatic cancer cells is discussed in depth and strategies, including the use of metformin, to target this signaling pathway in PDAC cells are proposed (Rozengurt, 2014). Finally, a comprehensive review of the latest animal models of pancreatic cancer are discussed, proposing novel tools to study the mechanism of pancreatic cancer initiation and promotion by major risk factors.

Abstract 1434: Metformin-induced metabolic changes are k-ras-dependent in animal models of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy, that is very difficult to treat. To date, systemic treatment for PDA, either a single agent or combinations of agents, has shown only modest benefits. Numerous epidemiological studies have reported that metformin (MET), a widely used anti-diabetic drug, provides protective benefits in reducing PDA risk among the diabetic population. Using a stable isotope glucose (GLUC) tracer for dynamic metabolic profiling (SiDMAP), we recently reported that high cholesterol (CHOL) alters the cellular metabolism of MiaPaCa2 (a PDA cell line harboring mutant K-Ras) cells by redirecting glucose-derived acetyl-CoA toward fatty acid (FA) synthesis. This response to MET is depended on the level of intracellular CHOL synthesis. We now performed a SiDMAP study in the LSL-K-RasG12D/+, LSL-Trp53R172H/+, Pdx-1-Cre (KPC) and the LSL-K-RasG12D/+, Pdx-1-Cre (KC) mouse models for PDA, and their wild-type littermates (C57Bl6.129). The mice were treated (or not) with MET (250 mg/kg, i.p. Q5D) and subjected to an Intra Peritoneal GLUC Tolerance Test (IPGTT) pre- and post-MET treatment. The KPC mice with the average tumor volume of 80.83+8.57 mm3 and the KC mice with PanIN lesions (aver. 9 mo old) were put on study. K-Ras mutation, with the presence of the tumor (KPC mice), induced an increase in plasma GLUC production via de novo synthesis by the liver using futile cycling of GLUC derived lactate and pyruvate. MET treatment decreased this flux in mutated (KC) and tumor-bearing animals (KPC), but increased this flux in liver of control mice. MET treatment increased the complete GLUC oxidation into 13CO2 in the pancreas of KC and KPC animals. This indicates that the pancreas in MET-treated animals use less GLUC for RNA, DNA and FA synthesis. For 13CO2 production, we found that tumor growth has to be established; the mutation is not enough to induce changes in this flux. As in plasma, tumor growth increases pancreas lactate production from GLUC. MET treatment dramatically decreased the Warburg effect in the pancreas of mutated (KC) and mutated/tumor-bearing (KPC) mice. Finally, MET decreased GLUC-derived acetyl-CoA delivery to FAS and palmitate in control, mutated and tumor-bearing mice. Immunohistochemical analysis of the tumors from the KPC mice revealed that treatment with MET decreased the staining of phospho-Ser79-ACC by ∼54%, total FAS staining (mainly in the tumor stroma) by ∼43%, and the growth-associated transcription factor HMGA2 by ∼36.7%. These decreases correlated with a decrease in Ki67 staining by almost 80%, indicative of an inhibitory effect of MET on PDA growth. These results suggest that mutant K-Ras is responsible for the metabolic adaptation in both the tumor- (KPC) and non-tumor-bearing animals (KC). Our findings provide a strong rationale for targeting the metabolic changes induced by activated K-RAS in PDA patients which harbor mutations in K-RAS gene in >95% of cases. Citation Format: Mary Jo Cantoria, Laszlo G. Boros, Hitendra Patel, Haiyong Han, Natalia Ignatenko, Emmanuelle J. Meuillet. Metformin-induced metabolic changes are k-ras-dependent in animal models of pancreatic cancer. [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 1434. doi:10.1158/1538-7445.AM2014-1434

Abstract 4321: Effect of pretreatment, dose and route of administration of BPM31510 (Coenyzme Q10 containing proprietary formulation) alone or in combination with gemcitabine improves survival in pancreatic cancer

Abstract In-vitro studies have demonstrated the ability of BPM31510, a Coenzyme Q10 (CoQ10) containing proprietary formulation to effectuate a metabolic switch from glycolysis to mitochondrial OXPHOS resulting in the activation of apoptotic pathways specifically in cancer cells. The effectiveness of BPM31510 in combination with standard of care chemotherapy agents in in-vitro and in-vivo models has been previously established. The current study was to determine the effect of dose, route of administration and combination with gemcitabine on survival in an animal model of pancreatic cancer. BPM31510 administered in three divided doses (50mg/kg vs 75mg/kg body weight daily, intravenous) was associated with dose dependent increase in survival and was significantly better when combined with gemcitabine. Continuous infusion of BPM31510 significantly improved survival rates compared to three divided doses (50mg/kg or 75mg/kg body weight) of BPM31510, with best outcomes at 200mg/kg body weight dose. Sixty day pretreatment with BPM31510 alone followed by combination with gemcitabine was also associated with improved survival outcomes with either gemcitabine or BPM31510 alone. The data suggests that dose and route of administration of BPM31510 alone or in combination with standard of care chemotherapy agents influences and improves survival in animal model of pancreatic cancer. Citation Format: Niven Narain, Lucia Mauro, Assuan Lens, Viatcheslav Akmaev, Rangaprasad Sarangarajan, Joaquin Jimenez. Effect of pretreatment, dose and route of administration of BPM31510 (Coenyzme Q10 containing proprietary formulation) alone or in combination with gemcitabine improves survival in pancreatic cancer. [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 4321. doi:10.1158/1538-7445.AM2014-4321

FOXM1 Promotes the Warburg Effect and Pancreatic Cancer Progression via Transactivation of LDHA Expression

The transcription factor Forkhead box protein M1 (FOXM1) plays critical roles in cancer development and progression. However, the regulatory role and underlying mechanisms of FOXM1 in cancer metabolism are unknown. In this study, we characterized the regulation of aerobic glycolysis by FOXM1 and its impact on pancreatic cancer metabolism. The effect of altered expression of FOXM1 on expression of glycolytic enzymes and tumor development and progression was examined using animal models of pancreatic cancer. Also, the underlying mechanisms of altered pancreatic cancer glycolysis were analyzed using in vitro molecular biology. The clinical relevance of aberrant metabolism caused by dysregulated FOXM1 signaling was determined using pancreatic tumor and normal pancreatic tissue specimens. We found that FOXM1 did not markedly change the expression of most glycolytic enzymes except for phosphoglycerate kinase 1 (PGK-1) and lactate dehydrogenase A (LDHA). FOXM1 and LDHA were overexpressed concomitantly in pancreatic tumors and cancer cell lines. Increased expression of FOXM1 upregulated the expression of LDHA at both the mRNA and protein level and elevated LDH activity, lactate production, and glucose utilization, whereas reduced expression of FOXM1 did the opposite. Further studies demonstrated that FOXM1 bound directly to the LDHA promoter region and regulated the expression of the LDHA gene at the transcriptional level. Also, elevated FOXM1-LDHA signaling increased the pancreatic cancer cell growth and metastasis. Dysregulated expression and activation of FOXM1 play important roles in aerobic glycolysis and tumorigenesis in patients with pancreatic cancer via transcriptional regulation of LDHA expression.

FOXM1c Promotes Pancreatic Cancer Epithelial-to-Mesenchymal Transition and Metastasis via Upregulation of Expression of the Urokinase Plasminogen Activator System

The transcription factor Forkhead box M1 (FOXM1) plays important roles in the formation of several human tumors, including pancreatic cancer. However, the molecular mechanisms by which FOXM1 promotes pancreatic tumor epithelial-to-mesenchymal transition (EMT) and metastasis are unknown. The effect of altered expression of FOXM1 and urokinase-type plasminogen activator receptor (uPAR) on EMT and metastasis was examined using animal models of pancreatic cancer. Also, the underlying mechanisms of altered pancreatic cancer invasion and metastasis were analyzed using in vitro molecular biology assays. Finally, the clinical relevance of dysregulated FOXM1/uPAR signaling was investigated using pancreatic tumor and normal pancreatic tissue specimens. Pancreatic tumor specimens and cell lines predominantly overexpressed the FOXM1 isoform FOXM1c. FOXM1c overexpression promoted EMT in and migration, invasion, and metastasis of pancreatic cancer cells, whereas downregulation of FOXM1 expression inhibited these processes. The level of FOXM1 expression correlated directly with that of uPAR expression in pancreatic cancer cell lines and tumor specimens. Moreover, FOXM1c overexpression upregulated uPAR expression in pancreatic cancer cells, whereas inhibition of FOXM1 expression suppressed uPAR expression. Furthermore, transfection of FOXM1c into pancreatic cancer cells directly activated the uPAR promoter, whereas inhibition of FOXM1 expression by FOXM1 siRNA suppressed its activation in these cells. Finally, we identified an FOXM1-binding site in the uPAR promoter and demonstrated that FOXM1 protein bound directly to it. Deletion mutation of this site significantly attenuated uPAR promoter activity. Our findings demonstrated that FOXM1c contributes to pancreatic cancer development and progression by enhancing uPAR gene transcription, and thus, tumor EMT and metastasis.

Abstract B27: Tripterine induced cell cycle arrest and apoptosis is associated with modulation of PI3K-AKT-mTOR pathway in human pancreatic cancer cell

Abstract Pancreatic cancer is the most common cancer among men and women; the fourth leading cause of cancer death in the United States and the fifth leading cause of cancer death worldwide. This disease has a poor prognosis with a 5-year overall survival rate of less than 20%. Multiple mechanisms have been postulated for the development of benign and malignant pancreatic diseases. However, the nature and origin of the precursor cells for pancreatic cancer have not yet been delineated. Based on several molecular mechanism(s) proposed for pancreatic cancer, the phosphoinositide 3-kinase (PI3K)/Akt pathway is found to be constitutively activated and the mammalian target of rapamycin (mTOR) kinase is reported to be an important mediator for its signaling. The phosphoinositide 3-kinase-AKT-mammalian target of rapamycin (PI3K-AKT-mTOR) pathway is a frequently hyper activated pathway in cancer and is important for tumor cell growth and survival. The development of targeted therapies against mTOR pathway led to the approval of drugs including everolimus and temsirolimus, for the treatment pancreatic and other cancer types. However, the effectiveness and response among high risk patients still remains unclear. Epidemiologic and laboratory studies suggest that plant-derived bioactive food components reverse or prevent the development and progression of early-stage disease before it becomes aggressive and malignant. Lately, naturally occurring non-toxic dietary compounds are increasingly used as a novel strategy for the prevention of more aggressive cancers. Previous reports from our laboratory suggest that prolonged exposure of cancer cells to natural agents may effectively modulate mTOR signaling and promote anti-proliferative effects. In the present study, we evaluated the effectiveness of celastrol in human (AsPC-1) and mouse (Pan-02) pancreatic cancer cells. Celastrol is a plant extract isolated from the root extract of Tripterygium Wilfordi (Thunder of God vine -TGV) and Celastrus Regelii, is also known as tripterine, a multiple bioactive natural product. Recent studies suggest that celastrol may exhibit antioxidant, anti-inflammatory, anticancer, and insecticidal activities. However, a significant lack of information on the bioavailability and the effect on specific molecular targets of celastrol is unknown and is therefore limiting its use for future clinical trials. our preliminary findings on the oral administration of celastrol (10mg/KgBW) and TGV separately to C57BL/6 black mice showed no toxicity or animal death (n=24). Subsequent analysis of serum samples showed bioavailability of celastrol with a retention time between 12 to 15 minutes. Human pancreatic cancer cells AsPC-1, and mouse pancreatic cancer cell Pan-02, treated with celastrol showed a significant decrease in cell proliferation (>4 fold) after 24h with a dose of 20ug/ml (confirmed by MTS assay). In order to determine whether the cell growth inhibition is associated with an effect on cell cycle, we conducted flow cytometry analysis for DNA content. Pan-02 cells treated with celastrol at an optimum dose of 10ug/ml showed cell cycle arrest at the G1 phase. Further analysis of the flow cytometry data showed a pre-G1 peak of apoptosis (>10%). Based on the findings on celastrol inducing cell cycle arrest and apoptosis, we extended our studies to determine the effect on the downstream targets of PI3K-AKT-mTOR pathways. RT-PCR analysis for AKT using specific primers with RNA isolated from pancreatic cancer cells showed a decrease in the mRNA (>2 fold). Further studies are in progress to confirm a specific dose dependent effect celastrol and TGV on the downstream targets of the PI3K-AKT-mTOR and cell cycle regulatory proteins including cyclin D1 in pancreatic cancer cells. The future goal will be to test the efficacy of celastrol and TGV in preclinical animal models of pancreatic cancer. Overall, our findings suggest the potential molecular targets of celastrol in pancreatic cancer cells. Citation Format: Di Tian, Bhagavathi Narayanan, Amitabha Mazumder, George Miller, Narayanan K. Narayanan. Tripterine induced cell cycle arrest and apoptosis is associated with modulation of PI3K-AKT-mTOR pathway in human pancreatic cancer cell. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr B27.

Loss of ATM increases pancreatic cancer formation via induction of EMT

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer deaths in the developed world, with a 5 year survival rate as low as 5%. In recent years, genetically engineered animal models of pancreatic cancer, which recapitulate the human disease, have provided useful means of studying both the morphological and molecular features associated with PDAC. The most commonly studied model to date has been the expression of active Kras under the control of an endogenous, pancreas specific promoter, which leads to a spectrum of changes in the pancreas recapitulating development of human PDAC. Interestingly, PDAC develops much more rapidly when further oncogenes such as SMAD4 or p53 are deleted. In the current study, we address the role of Ataxia Telangiectasia Mutated (ATM) gene in PDAC and describe a loss of ATM protein with advanced UICC stage in primary human tumor specimens. We also found that mutations in the ATM gene are more abundant in large collectives of human pancreatic cancer. Subsequently, we show that loss of ATM enhanced the metastatic potential of the Kras mouse model and decreased survival of mice to 36 and 38 weeks for homozygous and heterozygous ATM deletion, respectively. These mice developed a greater number of pre-malignant pancreatic intraepithelial neoplasias (mPanINs) with a significant increase in proliferation and α-smooth muscle actin positive cells, indicative of activated pancreatic stellate cells (PSCs). This was followed by an increase in large, dysplastic papillary lesions and a significant amount of surrounding fibrosis compared to controls. Our results furthermore establish that the loss of ATM in this mouse model promoted EMT via an increased response to BMP4-signalling. Moreover, early EMT upon ATM loss was associated with increased invasive properties. Taken together, our data suggests that there may be an intimate link between ATM activity and pancreatic cancer formation, one of the major signalling pathways altered in PDAC and targeting this axis may provide therapeutic potential in the future.

Relevance of Animal Models of Pancreatic Cancer and Pancreatitis to Human Disease

h m m f o t T a p c r p Our understanding of the pathogenesis of pancreatic cancer and pancreatitis has advanced so greatly in ecent years that it would hardly be an exaggeration to efer to the present time as the Pancreatic Renaissance. nfortunately, the tangible benefits to patients have not een commensurate with the theoretical advances. In eeking to explain this disparity, most investigators view ith suspicion the relevance of the models thus far used. or novel therapies to emerge, it is necessary that the nimal models recapitulate the human disease with preision. This realization has engendered a vigorous search or better models, especially genetically engineered modls. The key to progress lies in a sound understanding of he strengths, weaknesses, and applicability of the models n the investigator’s repertoire.

Abstract 2730: Novel animal models of pancreatic cancer induced by smoking and chronic pancreatitis.

Abstract Background and Aims: Smoking and pancreatitis are two major risk factors for pancreatic cancer. We developed novel mouse models of pancreatic cancer induced by smoking and chronic pancreatitis. We used two pancreatic cancer mouse models induced by overexpression of Kras with two different degrees of disease complexity. Methods: Wild type (WT), EL-Kras and Pdx1-Cre;LSL-Kras transgenic mice were subjected to a major cigarette smoke compound nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at doses close to those accumulated with smoking (100mg/kg) or exposed to tobacco smoke (80mg/m3) in tobacco smoke chambers for five days a week during 6 weeks. Standard model of chronic pancreatitis induced by cerulein injections (50μg/kg) for 3 weeks was applied to WT and Pdx1-Cre;LSL-Kras mice. Mice were then sacrificed and pancreatic tissue analyzed. Apoptosis was assessed by TUNEL. Autophagy was assessed by measuring autophagic markers LC3-II and p62. Pancreatic lesions, proliferation, fibrosis and inflammation were analyzed by immunohistochemistry. Epithelial to mesenchymal transition (EMT) and expression of stem cell markers were analyzed by Western. Results: NNK and exposure to tobacco smoke significantly increased the number and stage of pancreatic neoplastic (PanIN) lesions in Kras transgenic mice. Smoking stimulated fibrosis and stellate cells activation but had no significant effect on inflammation. EMT markers (decrease in E-Cadherin and increase in vimentin protein levels) were stimulated by NNK and tobacco smoke exposure in both WT and transgenic mice. NNK decreased apoptosis and LC3-II level in Kras mice. In addition, smoking stimulated expression of stem cell markers sox2 and CD133. Cerulein stimulated fibrosis, stellate cells activation, inflammation, and slightly stimulated EMT in WT and transgenic mice. Combination of smoking and cerulein had additive effect on stimulation of PanIN lesion formation, fibrosis, EMT and stem cell markers induction. Conclusion: The results indicate that both tobacco smoke and chronic cerulein pancreatitis promote pancreatic cancer progression in both EL-Kras and Pdx1-Cre;LSL-Kras mice models. Smoking acts through enhancing fibrosis and EMT; whereas pancreatitis stimulates mainly inflammation and fibrosis. Smoking and pancreatitis have additive effect on fibrosis, EMT and acquiring of stem cell characteristics. Citation Format: Shiping Xu, Paul J. Grippo, Oxana M. Khayrutdinova, Samuel W. French, Stephen J. Pandol, Anna S. Gukovskaya, Mouad Edderkaoui. Novel animal models of pancreatic cancer induced by smoking and chronic pancreatitis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2730. doi:10.1158/1538-7445.AM2013-2730

The Flavonoid Quercetin Inhibits Pancreatic Cancer Growth In Vitro and In Vivo

The flavonoid quercetin holds promise as an antitumor agent in several preclinical animal models. However, the efficacy of oral administration of quercetin in a pancreatic cancer mouse model is unknown. The antiproliferative effects of quercetin alone or in combination with gemcitabine were tested in 2 human pancreatic cancer cell lines using cell count and MTT assays. Apoptosis was evaluated by flow cytometry. Tumor growth in vivo was investigated in an orthotopic pancreatic cancer animal model using bioluminescence. Quercetin was administered orally in the diet. Quercetin inhibited the growth of pancreatic cancer cell lines, which was caused by an induction of apoptosis. In addition, dietary supplementation of quercetin attenuated the growth of orthotopically transplanted pancreatic xenografts. The combination of gemcitabine and quercetin had no additional effect compared with quercetin alone. In vivo quercetin caused significant apoptosis and reduced tumor cell proliferation. Our data provide evidence that oral administration of quercetin was capable of inhibiting growth of orthotopic pancreatic tumors in a nude mouse model. These data suggest a possible benefit of quercetin in patients with pancreatic cancer.

A multicellular signal transduction network of AGE/RAGE signaling

Advanced glycation end products (AGEs) are heterogeneous glycated products of proteins, lipids and nucleotides. The major receptor for AGEs, known as receptor for advanced glycation end products (RAGE or AGER), is a multi-ligand transmembrane receptor of immunoglobulin superfamily. It has an extracellular region, a transmembrane domain and a short cytoplasmic domain. Extracellular region of RAGE consists of one V type (critical for ligand binding) and two C type immunoglobulin domains (Schmidt et al. 1994a, b). Although the short cytoplasmic tail of 43 amino acid residues is found to be important for the signaling events mediated by RAGE, it does not have any known domain or motif (Neeper et al. 1992). The other cell surface receptors for AGEs include dolichyl-diphosphooligosaccharide-protein glycosyltransferase (AGE-R1) (Li et al. 1996), protein kinase C substrate, 80KH phosphoprotein (AGE-R2) (Goh et al. 1996), galectin-3 (AGE-R3) (Vlassara et al. 1995), and class A macrophage scavenger receptors type I and II. RAGE is also considered as a pattern recognition receptor due to its ability to bind different AGEs. RAGE has numerous extracellular ligands in addition to AGEs, which include extracellular high mobility group box-1 (HMGB1), S100 family of calcium binding proteins and amyloid-beta peptide (Fritz 2011). RAGE is expressed in diverse tissues such as lung, heart, kidney, brain, and skeletal muscle and in a variety of cells including endothelial cells, macrophages/monocytes, neutrophils, and lymphocytes (Brett et al. 1993; Ding and Keller 2005; Neeper et al. 1992). RAGE has been implicated in the pathogenesis of diverse diseases such as diabetes, cardiovascular disorders, arthritis, cancers and neurological disorders (Yan et al. 2009). Interactions of AGEs with their receptors alter cell function through the generation of free radicals (Schmidt et al. 1994a, b). In diabetes, interaction of AGEs with RAGE induces oxidative stress and inflammatory reactions thereby resulting in vascular damage and related complications (Yamagishi 2011). RAGE also plays an important role in the progression of atherosclerosis through oxidative stress and proinflammatory responses (Sun et al. 2009). Expression of RAGE in synovial tissue, T cells, B cells and macrophages of arthritic patients implies its significance in inflammatory joint disorders (Drinda et al. 2005). Overexpression of RAGE has also been reported in various types of cancers such as pancreatic, gastric, breast, lung cancers and lymphoma (Logsdon et al. 2007). Knockdown of RAGE expression was shown to inhibit ductal neoplasia in an animal model of pancreatic cancer (DiNorcia et al. 2012). A recent study by Liang et al. reported that the inactivation of RAGE in colorectal cancer cells reduced angiogenesis (Liang et al. 2011). The signaling events mediated by RAGE are complex due to the diversity of its ligands and their diverse effects mediated in different cell types. AGE/RAGE signaling in endothelial cells is reported to modulate oxidative stress, inflammation, apoptosis, autophagy, endothelial-mesenchymal-transition, endothelial permeability and dysfunction (Toma et al. 2009; Xu et al. 2010; Li et al. 2011; Xie et al. 2011; Ma et al. 2010; Del Turco et al. 2011). In smooth muscle cells, AGE/RAGE interaction leads to generation of reactive oxygen species, autophagy, proliferation and calcification (Yoon et al. 2009; Hu et al. 2012; Yuan et al. 2011; Tanikawa et al. 2009). AGE/RAGE signaling is reported to mediate proliferation in lymphocytes (Takahashi et al. 2010). In fibroblasts, it induces migration, inflammation and apoptosis (Liu et al. 2010; Shimoda et al. 2011). A diverse array of molecules and signaling modules were identified to be activated by RAGE depending on the intensity and duration of RAGE ligation. Specific signaling modules such as ERK1/2 (Lander et al. 1997), p38 MAPK (Lander et al. 1997), CDC42/RAC (Bondeva et al. 2011), SAPK/JNK (Hu et al. 2012) and NF-κB (Liu et al. 2010) have been shown to be triggered by AGE/RAGE interaction in different cell types. Currently, there are no resources, which contain RAGE signaling pathway data for visualization and analysis. Therefore, we have gathered signaling pathway reactions induced by AGE/RAGE interaction in diverse cell types and tissues from literature. We have also cataloged genes transcriptionally regulated by AGE/RAGE system in humans along with their transcriptional regulators. We have provided the AGE/RAGE signaling pathway data to scientific community through NetPath (http://www.netpath.org), a resource of signaling pathways developed by us (Kandasamy et al. 2010).

Human Correlates of Provocative Questions in Pancreatic Pathology

Studies of cell lines and of animal models of pancreatic cancer have raised a number of provocative questions about the nature and origins of human pancreatic cancer and have provided several leads into exciting new approaches for the treatment of this deadly cancer. In addition, clinicians with little or no contact with human pathology have challenged the way that pancreatic pathology is practiced, suggesting that "genetic signals" may be more accurate than today's multimodal approach to diagnoses. In this review, we consider 8 provocative issues in pancreas pathology, with an emphasis on "the evidence derived from man."

American Pancreatic Association

American Pancreatic Association

Effect of BPM 31510 on survival outcomes when used in combination with gemcitabine in a preclinical model of pancreatic cancer.

e14593 Background: BPM 31510 is a novel small molecule that targets the metabolic machinery of the cancer microenvironment to create a hallmark shift from lactate dependency towards mitochondrial oxidative phosphorylation, reversing the Warburg effect. Effector downstream signaling results in re-capitulation of BCL-2 mediated apoptosis and disruption in tumor vasculature by modulation of VEGF. This study reports on the therapeutic benefit associated with BPM 31510 used in combination with gemcitabine in animal model of pancreatic cancer. Methods: The NGS mouse devoid of innate and adaptive immune system were implanted with human pancreatic cancer cells, Mia PaCa-2. The animals were randomized (n=30/group) to the following dose regiment cohorts: 50 mg/kg body weight (BW) once or twice daily ± gemcitabine (150 mg/kg BW once/week) for 3 weeks followed by one week rest. A group of animals received 75 mg/kg BW thrice daily ± gemcitabine (150 mg/kg BW once/week) for 3 weeks followed by one week rest. Untreated or group treated with gemcitabine alone with corresponding treatment regimen served as controls. The primary objective was to evaluate the difference in overall survival of the animals when treated with gemcitabine alone compared to combination regimens with BPM 31510. Results: All tumor bearing untreated animals (control arm) expired by Day 21 of the study. In contrast, the once daily BPM 31510 dose regimen was associated with significant increase in duration of overall survival (p<0.009) with all animals expiring by Day 142 compared to gemcitabine alone. The combination of BPM 31510 with gemcitabine was associated with significantly increase in overall survival compared to BPM 31510 (p=0.002) or gemcitabine (p = 7.3E-8) alone. This was associated with significant reduction in tumor size and weight (p<0.001). Increased frequency of BPM 31510 dosing alone or in combination with gemcitabine was associated with significant increases in survival rates. Conclusions: The data herein suggest that BPM 31510 may be a viable agent for combination therapy in pancreatic cancer via employment of a novel metabolic pathways.