Normal Pancreatic Ductal Cells Research Articles

Abstract PR01: Downregulation of PTF1A is a crucial and rate-limiting step in pancreatic cancer initiation

Abstract The goal of this study is to understand the endogenous mechanisms limiting the ability of oncogenic KRAS to initiate pancreatic tumorigenesis. Recently, our lab and others discovered that although pancreatic ductal adenocarcinoma (PDAC) shares phenotypic characteristics with normal pancreatic duct cells, it actually originates from mature exocrine acinar cells. In analyzing the initiating events of PDAC, we have identified the acinar cell transcription factor, PTF1A, as a critical factor inhibiting KRAS from reprogramming acinar cells to ductal tumor precursors. Binding sites for PTF1A are found upstream of essentially all acinar differentiation products and, importantly, PTF1A maintains its own expression through a positive autoregulatory loop. Given the central role of PTF1A in regulating acinar-specific gene expression, we wanted to determine if loss of this transcription factor plays a role in pancreatic intraepithelial neoplasia (PanIN) formation. Importantly, we find that PTF1A is downregulated in early (stage 1) PanINs of humans and mouse models, suggesting that loss of PTF1A expression could be a rate-limiting step in pancreatic cancer initiation. To test whether PTF1A loss is a functionally important step in PanIN development, we deleted the Ptf1a gene in mice using an acinar-specific inducible Cre (Ptf1a cKO). These studies confirm that PTF1A downregulation is an essential step in PanIN formation, as Cre-mediated deletion of acinar cell Ptf1a in the presence of oncogenic KRAS (KrasG12D) acutely and dramatically accelerates PanIN formation. Widespread PanINs are observed within two weeks of combined KRAS activation/Ptf1a deletion, by which time KRAS alone has induced few or no PanINs. At six weeks post-recombination, the combination of KRAS/Ptf1a cKO induces >15-fold more PanINs than KRAS alone. Additionally, we have found that Ptf1a heterozygosity sensitizes pancreatic cells to KRAS-mediated PanIN formation, indicating that transformation requires reduction of PTF1A activity below a critical autoregulatory threshold needed to maintain acinar identity. Based on these current studies, we hypothesize the existence of mutual antagonism between acinar differentiation, which is maintained through PTF1A, and transformation driven by KRAS. KRAS normally transforms acinar cells only weakly, but both endogenous and mutant KRAS activity can be increased by inflammation, such as that stimulated by caerulein-induced pancreatitis. As KRAS is potentiated by Ptf1a deletion, we asked whether inflammation would be sufficient to transform Ptf1a-deficient acinar cells even without mutant KRAS. In the absence of pancreatitis, Ptf1a deletion alone has relatively little short-term effect: Ptf1a-deficient acinar cells eventually lose their differentiated phenotype and express ductal markers, but they do not undergo hyperplasia or dysplasia. After subjecting Ptf1a cKO mice to caerulein-induced pancreatitis, however, we observed widespread acinar-to-ductal metaplasia, histological structures that resemble PanINs and stain positively with Alcian blue, and loss of amylase staining throughout the pancreas. Our current data indicate that PTF1A has a critical role in guarding against genetic (oncogenic KRAS) and environmental (pancreatitis) insults, and demonstrate that loss of Ptf1a expression is the critical event of acinar cell transformation. Going forward, we will use gene expression profiling and ChIP-seq analysis to identify PTF1A target genes responsible for inhibiting KRAS, and to characterize the epigenetic processes by which KRAS and inflammation reprogram acinar cells to a PanIN phenotype. This abstract is also presented as Poster A3. Citation Format: Nathan Michael Krah, Mary P. Bronner, Christopher V. Wright, L. Charles Murtaugh. Downregulation of PTF1A is a crucial and rate-limiting step in pancreatic cancer initiation. [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 PR01.

Abstract A11: Ets transcription factor Etv5 regulates ductal morphogenesis and differentiation in association with Sox9 in vitro and increases susceptibility and delays recovery from pancreatitis in vivo

Abstract Introduction: The exocrine pancreas comprises a branched network of ducts that are connected to acini and lined by a monolayered epithelium that derives from the endoderm and is surrounded by mesenchyme. The formation and maturation of pancreatic ductal cells during branching morphogenesis is controlled by a complex hierarchy of transcription factors, which is not fully understood. The Ets-transcription factor Etv5 has been reported to play an important role during the development of organs that undergo branching morphogenesis such as mammary and salivary glands. We therefore aimed to characterize the functional role of Etv5 in pancreatic ductal development and differentiation. Methods: Cells were isolated from wild-type mouse pancreata and lentiviral transduction was used to induce Etv5 overexpression. Cells were grown in 3D organotypic culture and analyzed with time lapsed microscopy for morphology and dynamics in the formation of duct-like cystic structures. Differentiation status of cells grown in 3D was analyzed by immunofluorescence-staining (IF) and qPCR. To asses the role of Etv5 in the normal mouse pancreas and its role in inflammation and regeneration in vivo, Pdx1cre;Etv5-/-; RosaYFP mice were generated and aged up to six months and subjected to a cerulein induced acute pancreatitis protocol. Results: Normal pancreatic ductal cells grown in 3D-organotypic culture form spheroid cysts that resemble pancreatic ductal structures. Etv5 overexpression leads to early and exuberant formation of spheroid cysts within 2 days after seeding. Time-lapse microscopy demonstrated a significantly increased movement of cellular structures along the cyst as well as fusion of cysts into larger tubular structures. mRNA analysis of cysts harvested at day 7 displayed a strong upregulation of Sox9 and Foxa2, important regulators of ductal differentiation. Concurrently, E-cadherin was upregulated significantly whereas N-cadherin was downregulated indicating the terminal differentiation of these cells. IF-staining revealed co-localization of Etv5 and Sox9 in spheroid cysts of Etv5-overexpressing cells. Knockdown of Sox9 in Etv5-overexpressing cells with siRNA partially abrogated the formation of tubular structures and disrupted cyst architecture. Etv5 knockout mice (Pdx1Cre;Etv5-/-) were generated and we performed a cerulein-induced acute pancreatitis model. We found significantly elevated levels of serum amylase in Etv5-/- mice on Days 1 and 3 of the protocol. In a semiquantitative, blinded review of the histology, there was significantly more intense edema, inflammation, vacuolization and necrosis in Etv5-/- mice in all time points, which was also confirmed by quantitative amylase area scoring. Conclusion: Our data suggest a novel role for Etv5 in pancreatic ductal morphogenesis and lumen formation that is at least in part mediated by Sox9. In addition, our data suggests that the loss of Etv5 expression increases susceptibility to pancreatitis and results in persistent pancreatitis with delayed regeneration. Citation Format: Koushik K. Das, Steffen Heeg, Maximilian Reichert, Shigetsugu Takano, Basil S. Bakir, Gregory P. Botta, Christopher Hahn, Andrew D. Rhim, Anil K. Rustgi. Ets transcription factor Etv5 regulates ductal morphogenesis and differentiation in association with Sox9 in vitro and increases susceptibility and delays recovery from pancreatitis in vivo. [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 A11.

MicroRNA-301a-3p promotes pancreatic cancer progression via negative regulation of SMAD4.

Aim to determine the clinicopathological and prognostic role of miR-301a-3p in pancreatic ductal adenocarcinoma(PDAC), to investigate the biological mechanism of miR-301a-3p in vitro and in vivo. By tissue microarray analysis, we studied miR-301a-3p expression in PDAC patients and its clinicopathological correlations as well as prognostic significance. qRT-PCR was used to test miR-301a-3p expression in PDAC tissues and cell lines. Functional experiments including in vitro and in vivo were performed. Significantly higher expression of miR-301a-3p were found in PDAC patients with lymph node metastasis and advanced pathological stages and identified as an independent prognostic factor for worse survival. In PDAC samples and cell lines, miR-301a-3p was significantly up-regulated compared with matched non-tumor tissues and normal pancreatic ductal cells, respectively. Overexpression of miR-301a-3p enhanced PDAC cells colony, invasion and migration abilities in vitro as well as tumorigenicity in vivo. Furthermore, SMAD4 was identified as a target gene of miR-301a-3p by cell as well as mice xenograft experiments. In PDAC tissue microarray, a significantly inverse correlation between miR-301a-3p ISH scores and SMAD4 IHC scores were observed in both tumor and corresponding non-tumor tissues. MiR-301a-3p functions as a novel oncogene in PDAC and the oncogenic activity may involve its inhibition of the target gene SMAD4.

CA 19-9: Biochemical and Clinical Aspects.

CA19-9 (carbohydrate antigen 19-9, also called cancer antigen 19-9 or sialylated Lewis a antigen) is the most commonly used and best validated serum tumor marker for pancreatic cancer diagnosis in symptomatic patients and for monitoring therapy in patients with pancreatic adenocarcinoma. Normally synthesized by normal human pancreatic and biliary ductal cells and by gastric, colon, endometrial and salivary epithelia, CA 19-9 is present in small amounts in serum, and can be over expressed in several benign gastrointestinal disorders. Importantly, it exhibits a dramatic increase in its plasmatic levels during neoplastic disease. However, several critical aspects for its clinical use, such as false negative results in subjects with Lewis (a-b-) genotype and false positive elevation, occasional and transient, in patients with benign diseases, together with its poor positive predictive value (72.3 %), do not make it a good cancer-specific marker and renders it impotent as a screening tool. In the last years a large number of putative biomarkers for pancreatic cancer have been proposed, most of which is lacking of large scale validation. In addition, none of these has showed to possess the requisite sensitivity/specificity to be introduced in clinical use. Therefore, although with important limitations we well-know, CA 19-9 continues being the only pancreatic cancer marker actually in clinical use.

Effects of insulin on human pancreatic cancer progression modeled in vitro.

BackgroundPancreatic adenocarcinoma is one of the most lethal cancers, yet it remains understudied and poorly understood. Hyperinsulinemia has been reported to be a risk factor of pancreatic cancer, and the rapid rise of hyperinsulinemia associated with obesity and type 2 diabetes foreshadows a rise in cancer incidence. However, the actions of insulin at the various stages of pancreatic cancer progression remain poorly defined.MethodsHere, we examined the effects of a range of insulin doses on signalling, proliferation and survival in three human cell models meant to represent three stages in pancreatic cancer progression: primary pancreatic duct cells, the HPDE immortalized pancreatic ductal cell line, and the PANC1 metastatic pancreatic cancer cell line. Cells were treated with a range of insulin doses, and their proliferation/viability were tracked via live cell imaging and XTT assays. Signal transduction was assessed through the AKT and ERK signalling pathways via immunoblotting. Inhibitors of AKT and ERK signalling were used to determine the relative contribution of these pathways to the survival of each cell model.ResultsWhile all three cell types responded to insulin, as indicated by phosphorylation of AKT and ERK, we found that there were stark differences in insulin-dependent proliferation, cell viability and cell survival among the cell types. High concentrations of insulin increased PANC1 and HPDE cell number, but did not alter primary duct cell proliferation in vitro. Cell survival was enhanced by insulin in both primary duct cells and HPDE cells. Moreover, we found that primary cells were more dependent on AKT signalling, while HPDE cells and PANC1 cells were more dependent on RAF/ERK signalling.ConclusionsOur data suggest that excessive insulin signalling may contribute to proliferation and survival in human immortalized pancreatic ductal cells and metastatic pancreatic cancer cells, but not in normal adult human pancreatic ductal cells. These data suggest that signalling pathways involved in cell survival may be rewired during pancreatic cancer progression.

Inhibition of RAC1 GTPase sensitizes pancreatic cancer cells to γ-irradiation.

Radiation therapy is a staple treatment for pancreatic cancer. However, owing to the intrinsic radioresistance of pancreatic cancer cells, radiation therapy often fails to increase survival of pancreatic cancer patients. Radiation impedes cancer cells by inducing DNA damage, which can activate cell cycle checkpoints. Normal cells possess both a G1 and G2 checkpoint. However, cancer cells are often defective in G1 checkpoint due to mutations/alterations in key regulators of this checkpoint. Accordingly, our results show that normal pancreatic ductal cells respond to ionizing radiation (IR) with activation of both checkpoints whereas pancreatic cancer cells respond to IR with G2/M arrest only. Overexpression/hyperactivation of Rac1 GTPase is detected in the majority of pancreatic cancers. Rac1 plays important roles in survival and Ras-mediated transformation. Here, we show that Rac1 also plays a critical role in the response of pancreatic cancer cells to IR. Inhibition of Rac1 using specific inhibitor and dominant negative Rac1 mutant not only abrogates IR-induced G2 checkpoint activation, but also increases radiosensitivity of pancreatic cancer cells through induction of apoptosis. These results implicate Rac1 signaling in the survival of pancreatic cancer cells following IR, raising the possibility that this pathway contributes to the intrinsic radioresistance of pancreatic cancer.

Elevation of MMP-9 and IDO induced by pancreatic cancer cells mediates natural killer cell dysfunction.

BackgroundNatural killer (NK) cells play a key role in non-specific immune response in different cancers, including pancreatic cancer. However the anti-tumor effect of NK cells decreases during pancreatic cancer progression. The regulatory pathways by which NK cells facilitate tumor immune escape are unclear, therefore our purpose was to investigate the roles of the contributory factors.MethodsNK cells isolated from fresh healthy peripheral blood were co-cultured with normal human pancreatic ductal cells hTERT-HPNE and human pancreatic cancer cell lines SW1990 and BxPc-3 in vitro. Then NK cell function was determined by Flow cytometric analysis of surface receptors and cytotoxic granules in NK cells, NK cell apoptosis and cytotoxicity, and Enzyme-linked immunosorbent assay of cytokines. Expression level of MMP-9, IDO and COX-2 in hTERT-HPNE and SW1990 cells were detected by quantitative RT-PCR. Statistical differences between data groups were determined by independent t-tests using SPSS 19.0 software.ResultsOur results showed that NK cell function was significantly downregulated following exposure to pancreatic cancer cells compared to normal pancreatic cells, as demonstrated by lower expressions of activating surface receptors (NKG2D, DNAM-1, NKp30 and NKp46) and cytotoxic granules (Perforin and Granzyme B); decreased secretion of cytokines (TNF-α and IFN-γ); and reduced cytotoxicity against myelogenous leukemia K562 cells. Further investigations revealed that MMP-9 and IDO may be implicated in SW1990 cell-induced NK cell dysfunction by facilitating tumor immune evasion. Blockade by TIMP-1 and/or 1-MT could partially restore NK function.ConclusionsTaken together, elevation of MMP-9 and IDO induced by pancreatic cancer cells mediates NK cell dysfunction. Our findings could contribute to the development of NK cell-based immunotherapy in patients with pancreatic cancer.

Abstract 2787: Modulation of autophagy and oncogenic phenotypes through CYB5A-TRAF6 signaling influence prognosis of resected and metastatic pancreatic cancer

Abstract High-resolution array comparative genomic hybridization in a cohort of 44 radically resected pancreatic cancer patients revealed a correlation between shorter survival and loss of the 18q22.3cytoband. The present study investigated the genes encoded by this cytoband and provided mechanistic insights on their role in the suppression of oncogenic properties. We studied mRNA and protein expression of FBXO15, C18orf55, CYB5A, CPGL and CPGL-B in frozen laser-microdissected tissues from radically resected (N=48) and biopsies from metastatic patients (N=50). Low CYB5A expression correlated with 18q22.3 deletion and shorter survival, both in resected and metastatic patients (i.e. 6.5 vs. 12.7 months, P=0.004). Association with outcome was validated in a tissue-microarray of a second cohort of radically resected patients (N=100): patients with low expression levels of CYB5A had significantly shorter OS, and multivariate analysis confirmed CYB5A prognostic relevance (increased risk of death=2.0, P=0.02). The role of CYB5A was evaluated in 11 pancreatic cancer cell lines, 5 primary cultures, and a normal pancreatic ductal cell line through retrovirus-mediated up-regulation and siRNA. With these analyses we characterized a novel function of CYB5A, autophagy induction, concomitant with reduced proliferation and migration/invasion of pancreatic cancer cells. Marked accumulation of autophagic vacuoles was detected by electron microscopy, while immunofluorescence revealed CYB5A modulation of LC-3. Activation of pro-autophagic pathways was corroborated by gene and kinase arrays showing a significant up-regulation of several ATG-genes, accompanied by down-regulation of BCL-2 and MAPK14. Additionally, inhibition of multiple components of EGFR, Akt and Src signalling favored cancer cell death while preventing potentially deleterious cross-talk between key oncogenic players. Network analysis suggested CYB5A interaction with TRAF6, which was confirmed by TRAF6 down-regulation after CYB5A reconstitution (-69% in SU.86.86-CYB5A+, P=0.005). CYB5A silencing had opposite effects, restoring TRAF6 expression and wound-healing. In vivo studies were performed in genetically and histopathologically characterized patient-derived CYB5A+ orthotopic models (N=6 mice/group), monitored by Firefly and Gaussia-luciferase bioluminescence, MRI and high-frequency-ultrasound. CYB5A induced autophagy, as demonstrated by immunohistochemical analyses of p62 down-regulation, and ATG7/ATG16L2 overexpression, while inhibiting tumor growth/metastasis and increasing survival (57 vs. 44 days, P=0.03). These results define CYB5A as a novel prognostic factor, exerting its tumor-suppressor function via autophagy-induction and TRAF6 modulation, which holds a potential as a novel therapeutic approach in the clinical management of pancreatic cancer. Citation Format: Elisa Giovannetti, Qiuyan Wang, Niccola Funel, Amir Avan, Tonny Lagerweij, Viola Caretti, Ugo Boggi, Yisong Wang, Sara Caponi, Arjan van der Velde, Enrico Vasile, Henk M. Verheul, Thomas Wurdinger, Giuseppe Giaccone. Modulation of autophagy and oncogenic phenotypes through CYB5A-TRAF6 signaling influence prognosis of resected and metastatic 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 2787. doi:10.1158/1538-7445.AM2014-2787

SOX2 promotes dedifferentiation and imparts stem cell-like features to pancreatic cancer cells

SOX2 (Sex-determining region Y (SRY)-Box2) has important functions during embryonic development and is involved in cancer stem cell (CSC) maintenance, in which it impairs cell growth and tumorigenicity. However, the function of SOX2 in pancreatic cancer cells is unclear. The objective of this study was to analyze SOX2 expression in human pancreatic tumors and determine the role of SOX2 in pancreatic cancer cells regulating CSC properties. In this report, we show that SOX2 is not expressed in normal pancreatic acinar or ductal cells. However, ectopic expression of SOX2 is observed in 19.3% of human pancreatic tumors. SOX2 knockdown in pancreatic cancer cells results in cell growth inhibition via cell cycle arrest associated with p21Cip1 and p27Kip1 induction, whereas SOX2 overexpression promotes S-phase entry and cell proliferation associated with cyclin D3 induction. SOX2 expression is associated with increased levels of the pancreatic CSC markers ALDH1, ESA and CD44. Importantly, we show that SOX2 is enriched in the ESA+/CD44+ CSC population from two different patient samples. Moreover, we show that SOX2 directly binds to the Snail, Slug and Twist promoters, leading to a loss of E-Cadherin and ZO-1 expression. Taken together, our findings show that SOX2 is aberrantly expressed in pancreatic cancer and contributes to cell proliferation and stemness/dedifferentiation through the regulation of a set of genes controlling G1/S transition and epithelial-to-mesenchymal transition (EMT) phenotype, suggesting that targeting SOX2-positive cancer cells could be a promising therapeutic strategy.

MicroRNA Array Analysis Finds Elevated Serum miR-1290 Accurately Distinguishes Patients with Low-Stage Pancreatic Cancer from Healthy and Disease Controls

Our goal was to identify circulating micro RNA (miRNA) levels that could distinguish patients with low-stage pancreatic cancer from healthy and disease controls. We measured 735 miRNAs in pancreatic cancer case and control sera by QRTPCR using TaqMan MicroRNA Arrays. After array analysis, we selected 18 miRNA candidates for validation in an independent set of cases and control samples. Of the significantly elevated circulating miRNAs in patients with pancreatic cancer compared with controls, miR-1290 had the best diagnostic performance: receiver operating characteristic (ROC) analysis on miR-1290 serum level yielded curve areas (AUC) of 0.96 [95% confidence interval (CI), 0.91-1.00], 0.81 (0.71-0.91), and 0.80 (0.67-0.93), for subjects with pancreatic cancer (n = 41) relative to healthy controls (n = 19), subjects with chronic pancreatitis (n = 35), and pancreatic neuroendocrine tumors (n = 18), respectively. Serum miR-1290 levels were also significantly higher than healthy controls among patients with intraductal papillary mucinous neoplasm (IPMN; n = 20; AUC = 0.76, 0.61-0.91). Serum miR-1290 levels distinguished patients with low-stage pancreatic cancer from controls better than CA19-9 levels, and like CA19-9, higher miR-1290 levels predicted poorer outcome among patients undergoing pancreaticoduodenectomy. Greater numbers of miR-1290 transcripts were detected by FISH in primary pancreatic cancer and IPMN than normal pancreatic duct cells. miR-1290 influenced in vitro pancreatic cancer cell proliferation and invasive ability. Several other circulating miRNAs distinguished sera of patients with pancreatic cancer from those of healthy controls with AUCs >0.7, including miR-24, miR-134, miR-146a, miR-378, miR-484, miR-628-3p, and miR-1825. The detection of elevated circulating miR-1290 has the potential to improve the early detection of pancreatic cancer.

Abstract 5405: Limiting glucose availability with metformin increased reactive oxygen species, decreased cell survival, and promoted apoptosis in human and mouse pancreatic cancer cells.

Abstract Background: Recently, we found that calorie restriction (CR) protected K-ras G12D; Pdx-1Cre mice against the progression of advanced PanIn lesions and pancreatic cancer. Since CR limits glucose availability, the CR mimetic metformin is also potential approach to prevent pancreatic cancer. Recent studies found that metformin reduced the incidence of pancreatic cancer in diabetic patients. However, it is not clear how metformin promotes the anticancer effects. One potential mechanism is by altering reactive oxygen species (ROS) and autophagy. In this in vitro study, we explore the role of ROS and autophagy in mediating the growth-inhibitory effects of metformin by treating pancreatic cancer cells and normal pancreatic ductal cells with metformin. Methods: We acclimated MiaPaca and KP mouse pancreatic tumor cells from K-ras G12D; Pdx-1Cre mice to physiological concentrations of glucose and then treated cells cells with metformin with and without the antioxidant N-acetyl cysteine (NAC) or chloroquine (autophagy inhibitor) for 24h. Cell viability, ROS production, and apoptosis were measured. Results: Metformin decreased viability of MiaPaca and KP cells in a concentration-dependent manner in comparison to untreated cells; metformin did not have any effect in HPNE human pancreatic normal epithelial cells. Pretreating MiaPaca and KP cells with NAC increase survival and decreased apoptosis, which suggests that ROS promotes the growth-inhibitory and apoptotic effect of metformin. Metformin increased ROS formation in MiaPaca and KP cells but not in HPNE cells. Cell survival increased and apoptosis decreased when MiaPaca cells were pretreated with chloroquine. Our findings suggest that ROS and autophagy may mediate the cytotoxic effects of metformin in MiaPaca and KP pancreatic cancer cells. Conclusion: In summary, metformin may be a potential chemoprevention agent against pancreatic cancer without cytotoxic effects in normal pancreatic ductal cells. Citation Format: Guang Yan, Theresa Freeman, Susan Lanza-Jacoby. Limiting glucose availability with metformin increased reactive oxygen species, decreased cell survival, and promoted apoptosis in human and mouse pancreatic cancer cells. [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 5405. doi:10.1158/1538-7445.AM2013-5405

Abstract 1517: Pancreatic stellate cells and chemoresistant pancreatic cancer cells induce EMT gene expression in normal pancreatic ductal cells.

Abstract Pancreatic cancer, one of the deadliest of human cancers, is associated with a collagen-rich pronounced stromal reaction. Previously, we showed that the collagen microenvironment increased the motility of human pancreatic ductal epithelial (HPDE) cells (Ottaviano AJ et al, Cancer Research 2006). Since stellate cells are one of the major contributors to the stromal reaction, we examined the effect of stellate cells on HPDE cells. HPDE cells were labeled with cell tracking dye and then co-cultured stellate cells to allow intimate cell-cell contact. HPDE cells were then isolated 72 hours later using FACS and changes in gene expression analyzed by real time PCR. We found that HPDE cells upregulated expression of epithelial-mesenchymal transition (EMT) markers Zeb1, Snail and vimentin. Stellate cells also increased expression of these EMT markers in HPDE cells expression Kras (HPDE-Kras). However, no change in these EMT markers were seen when the HPDE and stellate cells were prevented from direct cell-cell contact using transwell co-culture system. Treatment with TGF-b type I receptor inhibitor SB431542 blocked the effect of stellate cells on HPDE gene expression. Since ductal cells also encounter malignant cells in vivo, we also examined the effect of co-culture of ductal cells with cancer cells. HPDE cells failed to upregulate EMT markers when they were co-cultured with HPAFII/CD18 cell. However, when HPDE cells were co-cultured with chemo-resistant CD18 cells (CD18-CR), HPDE cells upregulated EMT markers. The effect of CD18-CR cells on HPDE gene expression was only observed when there was intimate cell-cell contact. Also, treatment with SB431542 blocked the effect on HPDE gene expression. These results indicate that the tumor microenvironment may affect the behavior of peri-tumoral non-malignant cells and thereby contribute to tumor progression. Citation Format: Krishan Kumar, Vaibhav Sahai, Kazumi Ebine, Hidayatullah G. Munshi. Pancreatic stellate cells and chemoresistant pancreatic cancer cells induce EMT gene expression in normal pancreatic ductal cells. [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 1517. doi:10.1158/1538-7445.AM2013-1517

Abstract 5291: Non-coding satellite RNAs are highly expressed in cancer adjacent lesions of human pancreatic carcinoma and may promote oncogenesis .

Abstract It was believed that most satellite repeats in pericentric heterochromatin region were silenced and rarely transcribed into RNAs. However, a few reports suggest that non-coding satellite RNAs may play a significant role in cell division, heterochromatin remodeling or DNA damage such as other well-researched non-coding RNAs. In fact, a next generation digital gene expression method recently revealed that human satellite II (HSATII) transcripts are highly and specifically upregulated in human pancreatic tumors. To further explore these findings, we examined the precise localization of HSATII transcripts in human clinical pancreatic tissues by in situ hybridization using highly sensitive locked nucleic acid-modified oligonucleotide probes. Unexpectedly, HSATII RNAs were not detected in pancreatic ductal cancerous lesions, in almost all cases examined. Instead, HSATII RNAs were clearly expressed in non-cancerous adjacent acinar cells, surrounding carcinoma tissues. On the other hand, none of normal or inflammatory pancreatic tissues derived from the ones without cancer expressed HSATII RNAs. While, HSATII transcript expression in normal pancreatic ductal cells could not be induced in vitro even under stress conditions which mimic the tumor-adjacent environments, the cells with ectopic expression of HSATII transcripts showed the increase foci of gH2AX, which may reflect the increased genomic instability. These results suggest that the expression of HSATII may not be phenotypic changes in cancers but may directly contribute to the oncogenesis in the cells in precancerous lesions. Citation Format: Takahiro Kishikawa. Non-coding satellite RNAs are highly expressed in cancer adjacent lesions of human pancreatic carcinoma and may promote oncogenesis . [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 5291. doi:10.1158/1538-7445.AM2013-5291

Abstract 5207: Targeting prion protein as a potential oncogene in pancreatic cancer.

Abstract Background: Human prion protein (PrP) has been found to be up-regulated in gastric, breast, pancreatic cancers and its expression in cancer cells contributes to cancer progression and resistance to various cancer therapies. Current literature suggests that overexpression of PrP induces inherent resistance to cancer therapeutics, while repression of PrP induces sensitization to cancer therapeutics. Our previous studies using expression microarray have revealed that PrP is not detected in normal human pancreatic ductal cells but is expressed in human pancreatic ductal adenocarcinoma (PDAC) cell lines and 40% of pancreatic cancer specimens. Further, expression of PrP confers a worse prognosis in PDAC patients. The poor prognosis of pancreatic cancer demonstrates needs for better target and drug development. Our recent studies also found that PrP is universally expressed in fetal pancreatic precursor ductal cells, and co-expresses with pancreatic stem cell markers, such as Pdx1 and Notch1, suggesting that PrP is a marker of pancreatic stem cells and may cross-talk with Notch1 during development and pancreatic tumorigenesis. However, the role for PrP in the establishment or progression of cancer is unknown, and whether PrP cross-talks with oncogenes such as Notch in these processes need to be elucidated. Methods: We applied immunoprecipitation (Abcam, Anti-Prion protein antibody 8H4) and western blotting to investigate the binding partners for PrP. Knockdown and over-expression of PRNP gene were carried out on various pancreatic cell lines, followed by in vitro proliferation assay and apoptotic activity analysis of these cells. Results: Pancreatic cancer cell lines express different levels of PrP, e.g. BxPC3 and Pan02.03 have higher PrP expression when compared to Capan-1. Western blot analysis revealed that PrP can bind with Notch1 protein in both BxPC-3 and CAPAN-1 cell lines. Moreover, down-regulation of PrP led to decreased cell proliferation and accelerated apoptosis, accompanied by down-regulation of cleaved/activated Notch1 (ICN1) and Notch2 (ICN2) expression in BxPC3 cells, but had lesser effect on CAPAN-1 cells. In comparison, over-expression of PrP significantly increased the proliferation rate of CAPAN-1 cells and dramatically upregulated the expression of ICN1. In conjunction with our previous findings that PrP exists as a pro-prion that binds filamin A in pancreatic cancer cells, these findings suggest that PrP may mediate pancreatic cancer cell progression through regulation of Notch activity and other cell mechanic and signaling integrators. This hypothesis is being further tested by xenograft analysis. Elucidating the mechanisms for PrP mediated Notch activation in PDAC will further our long-term goal of understanding the role of oncogenic PrP in conferring pancreatic cancer aggressiveness, and potentially will lead to novel therapeutic options for treatment of pancreatic cancer. Citation Format: Yiwei Wang, Lan Zhou, Wei Xin. Targeting prion protein as a potential oncogene in pancreatic cancer. [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 5207. doi:10.1158/1538-7445.AM2013-5207

Abstract 1143: Unraveling the role of CYB5A in pancreatic ductal adenocarcinoma (PDAC): correlation with clinical outcome and functional characterization in the modulation of autophagy and oncogenic phenotypes.

Abstract Loss of 18q22.3 was reported to be a prognostic factor in PDAC. This study aimed at evaluating whether the genes of this cytoband were associated with PDAC outcome and provides mechanistic insights on their role in the suppression of oncogenic properties. The expression of FBXO15, C18orf55, CYB5A, CPGL and CPGL-B was evaluated in cells (11 PDAC cell lines, 5 primary cultures, and a normal pancreatic ductal cell line) and in 48 stage IIB PDAC specimens, isolated by laser-microdissection after surgical resection. Low CYB5A expression correlated with 18q22.3 deletion and shorter survival (16.3 vs. 29.5 months, P=0.01). Association with outcome was validated in a tissue-microarray of a second cohort radically resected patients (N=100): patients with CYB5A protein expression levels below median value had significantly shorter OS, and multivariate analysis confirmed CYB5A prognostic relevance (increased risk of death, 2.0, P=0.02). CYB5A expression in primary cultures correlated with expression in their respective tissues, and CYB5A retrovirus-mediated up-regulation of both PDAC-2 primary cells and Su86.86 cells suppressed proliferation and migration/invasion, while enhancing apoptosis and autophagy induction. Marked accumulation of autophagic vacuoles was detected by electron microscopy, while immunofluorescence revealed CYB5A colocalization with LC-3. Activation of pro-autophagic pathways was corroborated by PCR arrays showing a significant up-regulation of several ATG-genes, accompanied by down-regulation of BCL-2 and MAPK14. The phosphorylation status of MAPK14 was also inhibited, together with phospho-EGFR and the pro-invasive kinases phospho-Src/STAT6, as revealed by 144-kinase peptide substrate arrays. Network analysis suggested that this down-regulation was caused by CYB5A interaction with the NF-κB activator TRAF6, whose expression was significantly reduced after reconstitution of CYB5A in a genetic and histopathologic characterized patient-derived orthotopic mouse model. Furthermore, CYB5A upregulation increased survival, while decreasing primary tumor dimension and metastatic spread, as longitudinally monitored with Firefly- and Gaussia-luciferase. We thus identified CYB5A as a novel prognostic factor that modulates autophagy and oncogenic phenotypes, holding a potential as a novel therapeutic approach in the clinical management of PDAC patients. Citation Format: Elisa Giovannetti, Qiuyan Wang, Amir Avan, Niccola Funel, Elena Glavani, Tonny Lagerweij, Davide Chiasserini, Jih-Hsiang Lee, Viola Caretti, Matilde Masini, Ugo Boggi, Yisong Wang, Enrico Vasile, Godefridus J. Peters, Thomas Wurdinger, Giuseppe Giaccone. Unraveling the role of CYB5A in pancreatic ductal adenocarcinoma (PDAC): correlation with clinical outcome and functional characterization in the modulation of autophagy and oncogenic phenotypes. [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 1143. doi:10.1158/1538-7445.AM2013-1143

PH Regulatory Transporters in Pancreatic Ductal Adenocarcinoma (PDAC)

PDAC is among the most aggressive human malignacies, with a 5‐ year survival rate of 1–4%. Elucidation of specific molecular alterations in PDAC is urgently needed to devise new treatment strategies. Malignant tumors with rapid progression such as PDAC often undergo extensive metabolic changes resulting in increased acid production. We and others have shown in other cancers that this leads to the upregulation of acid extruding transporters, which in turn favors further cancer progression. The aim of our studies is to elucidate the regulation and roles of pH regulatory ion transporters in PDAC. We used qPCR to screen a panel of 4 human PDAC cell lines (AsPC‐1, BxPC‐3, Panc‐1, MIAPaCa‐2) against a normal human pancreatic ductal cell line (HPDE). The Na+/H+ exchanger 2 (NHE2) was upregulated in 3 of 4 cell lines, whereas NHE1 was upregulated only in BxPC3 cells; the V‐type H+‐ATPase subunit a3 was upregulated in all PDAC cell lines; all cell lines except BxPC3 exhibited upregulation of either AE2 or AE3 Cl−/HCO3−exchangers, and also the Na+‐HCO3− transporters SLC4A7 and –A8 tended to be upregulated in PDAC; surprisingly, the H+/lactate cotransporter MCT1 was downregulated in all PDAC cell lines and a similar trend was seen for MCT4. In conclusion, PDAC cell lines exhibit dramatic changes in the mRNA expression level of pH‐regulatory transporters. Ongoing experiments are addressing the functional relevance of this in PDAC.Funding: Marie Curie Actions (ITN IonTraC)

Mass spectrometric analysis reveals O-methylation of pyruvate kinase from pancreatic cancer cells

Pyruvate kinase (PK) is an important glycolytic enzyme that catalyzes the dephosphorylation of phosphoenolpyruvate to pyruvate. Human PK isozyme M2 (PKM2), a splice variant of M1, is overexpressed in many cancer cells, and PKM2 has been investigated as a potential tumor marker for diagnostic assays and as a target for cancer therapy. To facilitate identification and characterization of PK, we studied the enzyme from pancreatic cancer cells and normal pancreatic duct cells by electrophoresis and mass spectrometry, and identified multiple O-methylated residues from PK. These findings advance our knowledge of the biochemical properties of PK and will be important in understanding its biological function in cells.

Does This Patient Have Pancreatic-Biliary Cancer?

Purpose: Introduction: CA 19-9 (serum carbohydrate antigen 19-9) is a glycosphingolipid that was first isolated in 1979 by Koprowski as a monoclonal antibody to cultured cell from human colonic cancer. Since then, it has been used as a tumor marker for pancreatic and biliary cancers. However, high levels of CA 19-9 are occasionally found in benign diseases of the liver and biliary tract with a value usually <5000 U/m. Case: This is a 69-year-old morbidly obese male patient who presented with a 2 day history of painless jaundice with no history of viral hepatitis or liver disease and his family history was significant for pancreatic carcinoma. Physical examination revealed an obese male with scleral icterus and jaundice. Laboratory studies showed aspartate aminotransferase: 196 U/L; alanine aminotransferase: 15 U/L; alkaline phosphatase: 1045 U/L; total bilirubin: 12 mg/dL; carcinoembryonic antigen: 3.2 ng/mL; CA 19-9: 38,310 U/mL. Ultrasonography and computerized tomography of the abdomen were of poor quality due to body habits. Magnetic resonance imaging of the abdomen showed intrahepatic biliary dilatation, a dilated common bile duct of 11 mm and no pancreatic mass. Endoscopic ultrasound was then performed and showed stones in gall bladder as well as an impacted stone in the ampula and normal pancreatic head. Endoscopic retrograde cholangiopancreaticography was then attempted but with failure to cannulate the bile due to body habitus. After 2 days, his symptoms started to improve. A repeat CA 19-9 level obtained 1 week later was 36 U/mL. Discussion: CA 19-9 is produced in normal pancreatic and biliary ductal cells with upper limit of normal 37-40 U/mL, and significant elevations above 1,000 U/mL being 99% specific for the diagnosis of pancreatic cancer. Extreme elevations of serum CA 19-9 levels with benign biliary tract diseases have been reported in the literature. Several hypothesis have been postulated to explain the degree of elevation: 1) Inflammation resulting in increased proliferation of the epithelial cells leading to increased production of CA 19-9; 2) Biliary tract obstruction leading to accumulation of CA 19-9 in the biliary lumen; 3) Increased biliary pressure leading to irritation of bile duct cells and increased production of CA 19-9; 4) Reflux of CA 19-9 into the circulation by obstruction. Tumor markers alone should never use to establish the diagnosis of malignancy.Figure: EUS: Stone in ampulla.

The GCTM-5 Epitope Associated with the Mucin-Like Glycoprotein FCGBP Marks Progenitor Cells in Tissues of Endodermal Origin

Monoclonal antibodies against cell surface markers are powerful tools in the study of tissue regeneration, repair, and neoplasia, but there is a paucity of specific reagents to identify stem and progenitor cells in tissues of endodermal origin. The epitope defined by the GCTM-5 monoclonal antibody is a putative marker of hepatic progenitors. We sought to analyze further the distribution of the GCTM-5 antigen in normal tissues and disease states and to characterize the antigen biochemically. The GCTM-5 epitope was specifically expressed on tissues derived from the definitive endoderm, in particular the fetal gut, liver, and pancreas. Antibody reactivity was detected in subpopulations of normal adult biliary and pancreatic duct cells, and GCTM-5-positive cells isolated from the nonparenchymal fraction of adult liver expressed markers of progenitor cells. The GCTM-5-positive cell populations in liver and pancreas expanded greatly in numbers in disease states such as biliary atresia, cirrhosis, and pancreatitis. Neoplasms arising in these tissues also expressed the GCTM-5 antigen, with pancreatic adenocarcinoma in particular showing strong and consistent reactivity. The GCTM-5 epitope was also strongly displayed on cells undergoing intestinal metaplasia in Barrett's esophagus, a precursor to esophageal carcinoma. Biochemical, mass spectrometry, and immunochemical studies revealed that the GCTM-5 epitope is associated with the mucin-like glycoprotein FCGBP. The GCTM-5 epitope on the mucin-like glycoprotein FCGBP is a cell surface marker for the study of normal differentiation lineages, regeneration, and disease progression in tissues of endodermal origin.

CDK-4 Inhibitor P276 Sensitizes Pancreatic Cancer Cells to Gemcitabine-Induced Apoptosis

Despite advances in molecular pathogenesis, pancreatic cancer remains a major unsolved health problem. It is a rapidly invasive, metastatic tumor that is resistant to standard therapies. The phosphatidylinositol-3-kinase/Akt and mTOR signaling pathways are frequently dysregulated in pancreatic cancer. Gemcitabine is the mainstay treatment for metastatic pancreatic cancer. P276 is a novel CDK inhibitor that induces G(2)/M arrest and inhibits tumor growth in vivo models. Here, we determined that P276 sensitizes pancreatic cancer cells to gemcitabine-induced apoptosis, a mechanism-mediated through inhibition of Akt-mTOR signaling. In vitro, the combination of P276 and gemcitabine resulted in a dose- and time-dependent inhibition of proliferation and colony formation of pancreatic cancer cells but not with normal pancreatic ductal cells. This combination also induced apoptosis, as seen by activated caspase-3 and increased Bax/Bcl2 ratio. Gene profiling studies showed that this combination downregulated Akt-mTOR signaling pathway, which was confirmed by Western blot analyses. There was also a downregulation of VEGF and interleukin-8 expression suggesting effects on angiogenesis pathway. In vivo, intraperitoneal administration of the P276-Gem combination significantly suppressed the growth of pancreatic cancer tumor xenografts. There was a reduction in CD31-positive blood vessels and reduced VEGF expression, again suggesting an effect on angiogenesis. Taken together, these data suggest that P276-Gem combination is a novel potent therapeutic agent that can target the Akt-mTOR signaling pathway to inhibit both tumor growth and angiogenesis.