Chintan Chheda

Characterization of Mouse Models of Early Pancreatic Lesions Induced by Alcohol and Chronic Pancreatitis.

Objective We describe the first mouse model of pancreatic intraepithelial neoplasia (PanIN) lesions induced by alcohol in the presence and absence of chronic pancreatitis. Methods Pdx1-Cre;LSL-K-ras mice were exposed to Lieber-DeCarli alcohol diet for 6 weeks with cerulein injections. The PanIN lesions and markers of fibrosis, inflammation, histone deacetylation, epithelial-to-mesenchymal transition (EMT), and cancer stemness were measured by immunohistochemistry and Western. Results Exposure of Pdx1-Cre;LSL-K-ras mice to an alcohol diet significantly stimulated fibrosis and slightly but not significantly increased the level of PanIN lesions associated with an increase in tumor-promoting M2 macrophages. Importantly, the alcohol diet did not increase activation of stellate cells. Alcohol diet and cerulein injections resulted in synergistic and additive effects on PanIN lesion and M2 macrophage phenotype induction, respectively. Cerulein pancreatitis caused stellate cell activation, EMT, and cancer stemness in the pancreas. Pancreatitis caused histone deacetylation, which was promoted by the alcohol diet. Pancreatitis increased EMT and cancer stemness markers, which were not further affected by the alcohol diet. Conclusions The results suggest that alcohol has independent effects on promotion of PDAC associated with fibrosis formed through a stellate cell-independent mechanism and that it further promotes early PDAC and M2 macrophage induction in the context of chronic pancreatitis.

HDAC3 mediates smoking-induced pancreatic cancer

Smoking is a major risk factor for developing pancreatic adenocarcinoma (PDAC); however, little is known about the mechanisms involved. Here we employed a genetic animal model of early stages of PDAC that overexpresses oncogenic Kras in the pancreas to investigate the mechanisms of smoking-induced promotion of the disease in vivo. We confirmed the regulation of the interactions between the tumor microenvironment cells using in vitro cellular systems. Aerial exposure to cigarette smoke stimulated development of pancreatic intraepithelial neaoplasia (PanIN) lesions associated with a tumor microenvironment-containing features of human PDAC including fibrosis, activated stellate cells, M2-macrophages and markers of epithelial-mesenchymal transition (EMT). The pro-cancer effects of smoking were prevented by Histone Deacetylase HDAC I/II inhibitor Saha. Smoking decreased histone acetylation associated with recruitment of and phenotypic changes in macrophages; which in turn, stimulated survival and induction of EMT of the pre-cancer and cancer cells. The interaction between the cancer cells and macrophages is mediated by IL-6 produced under the regulation of HDAC3 translocation to the nucleus in the cancer cells. Pharmacological and molecular inhibitions of HDAC3 decreased IL-6 levels in cancer cells. IL-6 stimulated the macrophage phenotype change through regulation of the IL-4 receptor level of the macrophage. This study demonstrates a novel pathway of interaction between cancer cells and tumor promoting macrophages involving HDAC3 and IL-6. It further demonstrates that targeting HDAC3 prevents progression of the disease and could provide a strategy for treating the disease considering that the HDAC inhibitor we used is FDA approved for a different disease.

Cadherin-11 Is a Cell Surface Marker Up-Regulated in Activated Pancreatic Stellate Cells and Is Involved in Pancreatic Cancer Cell Migration

Chronic pancreatitis is a prominent risk factor for the development of pancreatic ductal adenocarcinoma. In both conditions, the activation of myofibroblast-like pancreatic stellate cells (PSCs) plays a predominant role in the formation of desmoplastic reaction through the synthesis of connective tissue and extracellular matrix, inducing local pancreatic fibrosis and an inflammatory response. Yet the signaling events involved in chronic pancreatitis and pancreatic cancer progression and metastasis remain poorly defined. Cadherin-11 (Cad-11, also known as OB cadherin or CDH11) is a cell-to-cell adhesion molecule implicated in many biological functions, including tissue morphogenesis and architecture, extracellular matrix-mediated tissue remodeling, cytoskeletal organization, epithelial-to-mesenchymal transition, and cellular migration. In this study, we show that, in human chronic pancreatitis and pancreatic cancer tissues, Cad-11 expression was significantly increased in PSCs and pancreatic cancer cells. In particular, an increased expression of Cad-11 can be detected on the plasma membrane of activated PSCs isolated from chronic pancreatitis tissues and in pancreatic cancer cells metastasized to the liver. Moreover, knockdown of Cad-11 in cancer cells reduced pancreatic cancer cell migration. Taken together, our data underline the potential role of Cad-11 in PSC activation and pancreatic cancer metastasis.

Essential Role of Lyn in Fibrosis

Fibrotic disorders involve replacement of normal parenchyma with myofibroblasts, which deposit connective tissue, leading to obliteration of the function of the underlying organ. The treatment options are inadequate and reflect the fact that signaling targets in myofibroblasts are unknown. Here we identify the hyperactive Lyn signaling in myofibroblasts of patients with chronic pancreatitis-induced fibrosis. Lyn activation coexpress with markers of activated myofibroblasts, and is increased ~11-fold in chronic pancreatitis compared to normal tissue. Inhibition of Lyn with siRNA or INNO-406 leads to the substantial decrease of migration and proliferation of human chronic pancreatitis myofibroblasts in vitro, while leaving migration and proliferation of normal myofibroblasts only slightly affected. Furthermore, inhibition of Lyn prevents synthesis of procollagen and collagen in myofibroblasts in a mouse model of chronic pancreatitis-induced fibrosis. We conclude that Lyn, as a positive regulator of myofibroblast migration, proliferation, and collagen production, is a key target for preventing fibrosis.

Novel Small Molecule Inhibitors of Protein Kinase D Suppress NF-kappaB Activation and Attenuate the Severity of Rat Cerulein Pancreatitis

Nuclear factor-kappa B (NF-κB) activation is a key early signal regulating inflammatory and cell death responses in acute pancreatitis. Our previous in vitro studies with molecular approaches on AR42J cell showed that protein kinase D (PKD/PKD1) activation was required in NF-κB activation induced by cholecystokinin 8 (CCK) or carbachol (CCh) in pancreatic acinar cells. Recently developed small molecule PKD inhibitors, CID755673 and CRT0066101, provide potentially important pharmacological approaches to further investigate the effect of PKD in pancreatitis therapy. The aim of this study was to explore whether CID755673 and CRT0066101 block NF-κB activation with in vitro and in vivo models of experimental pancreatitis and whether the small molecule PKD inhibitors have therapeutic effects when given before or after the initiation of experimental pancreatitis. Freshly prepared pancreatic acini were incubated with CID755673 or CRT006101, followed by hyperstimulation with CCK or CCh. For in vivo experimental pancreatitis, rats were treated with intraperitoneal injection of CID755673 or CRT0066101 prior to or after administering cerulein or saline. PKD activation and NF-κB-DNA binding activity in nuclear extracts from pancreatic acini and tissue were measured. The effects of PKD inhibitors on pancreatitis responses were evaluated. Our results showed that both CID755673 or CRT0066101 selectively and specifically inhibited PKD without effects on related protein kinase Cs. Inhibition of PKD resulted in significantly attenuation of NF-κB activation in both in vitro and in vivo models of experimental pancreatitis. NF-κB inhibition by CID755673 was associated with decreased inflammatory responses and attenuated severity of the disease, which were indicated by less inflammatory cell infiltration, reduced pancreatic interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), decreased intrapancreatic trypsin activation, and alleviation in pancreatic necrosis, edema and vacuolization. Furthermore, PKD inhibitor CID755673, given after the initiation of pancreatitis in experimental rat model, significantly attenuated the severity of acute pancreatitis. Therapies for acute pancreatitis are limited. Our results indicate that small chemical PKD inhibitors have significant potential as therapeutic interventions by suppressing NF-κB activation.

Abstract 4200: Novel treatment strategy for pancreatic cancer

Background: Solid association exists between high expression level of glycogen synthase kinase 3 beta (GSK-3β) and pancreatic cancer (PaCa) progression in humans. GSK-3β inhibition in a mouse orthotopic model of PaCa induced tumor shrinkage. However, GSK-3β inhibition stimulates pro-metastasis epithelial to mesenchymal transition (EMT). HDAC1/2 is involved in regulation of EMT in PaCa cells. We developed a novel strategy based on the combination of GSK-3β inhibition with inhibition of HDAC1 to prevent cancer cell survival, EMT and metastasis in vitro and in vivo. We designed, synthesized and tested a novel dual-inhibitor CSME-357 which inhibits both the proteins. Methods: Pdx-Cre;LSL-Kras (KC) mice were ip injected with GSK-3β inhibitor TDZD-8 and/or HDAC1/2 inhibitor Saha. Pancreatic intraepithelial neoplasia (PanIN) lesions, fibrosis, and inflammation were measured by IHC. Pancreatic cancer cell lines MIA PaCa-2, BxPC3, AsPC1 were cultured in the presence of HDAC and/or GSK-3β inhibitors or with the novel compound CSME-357 which inhibits both proteins. Cell survival and apoptosis were measured by MTT assay and DNA fragmentation, respectively. EMT and cancer stemness markers, histone acetylation and GSK-3β level were measured by IHC and Western in tissue and cells. Invasion of the cells was measured by invasion assay. Ability of the cells to form metastatic niches in nude mice was measured by injecting PaCa cells in mice tails in the presence or absence of CSME-357 and live mice were analyzed by MRI and by tissue analysis after necropsy. Results: Treatment of KC mice with Saha decreased the level of PanIN lesions, fibrosis, inflammation, and EMT. TDZD-8 potentiated the effect of Saha on PanIN prevention. Saha and TDZD-8 decreased EMT in KC mice. Pharmacological and molecular inhibitions of GSK-3β significantly, dose dependently, and synergistically decreased proliferation and at a lesser extent stimulated apoptosis in PaCa cell lines. GSK-3β inhibition stimulated EMT in PaCa cells; whereas, Saha reversed this effect. CSME-357 significantly and more potently (compared to the combination of two inhibitors) decreased proliferation, invasion and EMT and stimulated apoptosis in PaCa cells. Significance was achieved at 300nM. Toxicity assays showed no to very little toxicity of the novel compound. CSME-357 decreased the ability of cancer cells to form metastatic niches in nude mice. Conclusion: Combination of HDAC1 and GSK-3β inhibitions induced a triple beneficial effect by decreasing proliferation, stimulating apoptosis and inhibiting EMT/metastasis. CSME-357 is a novel compound with promising anti-cancer properties. CSME-357 is a very potent inhibitor for both GSK-3β and HDAC1/2; in addition, CSME-357 prevents cancer cell growth, resistance to apoptosis, EMT, invasion and metastasis. Note: This abstract was not presented at the meeting. Citation Format: Chintan Chheda, Ramachandran Murali, Paul Grippo, Dale Uyeminami, Kent Pinkerton, Stephen Pandol, Mouad Edderkaoui. Novel treatment strategy for pancreatic cancer. [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 4200. doi:10.1158/1538-7445.AM2015-4200