- Email: [email protected]
316 The Phenotype of Familial Pancreatic Neoplasia
using transcript profiling or known to be important in carcinogenesis using immunohistochemistry and in-situ hybridisation in a cohort of 124 patients with pancreatic cancer to identify potential biomarkers of prognosis and response to operative resection. Results: Aberrant expression of p53, p21WAF1/CIP1, p27KIP1, p16INK4A, cyclin D1, cyclin E, DPC4/Smad4, EGFR, beta-catenin, sfrp4, LMO4, HOXB2, S100P, S100A6, S100A2, RAI3 and CRBP1 were identified in a significant proportion of PC. Multivariate analysis of clinicopathological variables (tumor size, differentiation, subtype, lymph node metastases, perineural invasion and vascular space invasion), treatment parameters (margin involvement, adjuvant therapy, resection type), and aberrant expression of the above candidate biomarkers identified that HOXB2 expression and resection margin involvement by tumor were the only 2 independent prognostic factors. Patients who had absent HOXB2 expression and had clear margins after pancreaticoduodenectomy (48%) had an actual survival of 40% at 3 years and 21% at 5 years, compared to those with HOXB2 expression and/or positive surgical margins who had all died by 28 months. Conclusion: HOXB2 expression may represent a surrogate marker of advanced disease, can potentially be assessed preoperatively using FNAB, and is currently the best predictor of outcome and response to operative resection to be reported. Preoperative assessment of HOXB2 expression has potential clinical utility in predicting response to operative resection for PC to allow better selection of patients for operative intervention.
The Pathogenicity of Citrobacter rodentium (CR) Is Enhanced in Mice Fed Diets Deficient in Selenium (Se) and Vitamin E (VE) Allen Smith, Sebastian Botero, Terez Shea-Donohue Specific nutritional deficiencies can have a significant negative impact on host resistance to infection. We demonstrated that mice fed diets deficient in the antioxidants Se and VE had delayed intestinal clearance of the parasitic nematode, Heligmosomoides polygyrus by interfering with Th2-dependent mechanisms in the small intestine. Citrobacter rodentium is a mouse pathogen that mimics many aspects of enteropathogenic E. coli infection in humans, and is a potent inducer of a Th1 immune response systemically and in the colon. Little is known, however, about the role of antioxidant nutrients on the immune response to this class of pathogens. Aim: To determine the effects of a single or double deficiency in Se and VE on Cr infection in mice. Methods: Three week-old male mice were placed on deficient diets for 5-6 weeks and then infected with Cr. Bacterial load in the feces, colon, and spleen was determined at days 6 or 7, 12, 16, and 20 days post-infection. Colon and spleen weights were also measured. Colonic inflammation was assessed in Geimsa-stained sections. Gene expression in colonic tissue was evaluated by real-time PCR and Cr antigen specific IgM and IgG production by ELISA. Numbers of CD3+, CD4+, CD8+, and B220+ cells in the spleen were determined by flow cytometry. Results: Mice fed a diet deficient in both Se and VE had significantly higher colonic Cr load (p<0.05) at days 12 and 16 post-infection. This was associated with increased tissue damage and cellular infiltration in colonic lamina propria. Colonization of the spleen by Cr was low and variable but consistently higher in mice fed the Se and VE deficient diet compared to infected mice on a sufficient diet. The colon/body weight ratios at day 16 and spleen/body weight ratios at days 12 and 16 postinfection were significantly elevated (p<0.05). Increased spleen size at day 12 days postinfection in mice fed the Se and VE deficient diet correlated with an increase in total cells that was due primarily to cells expressing B220. Gene expression for IFN-γ, TNF-α, and IL-1β in the colon was reduced at day 7, but not day 12, post-infection in mice fed the Se and VE deficient diet. Anti-Cr IgM and IgG levels were not affected by diet. Mice fed diet deficient in Se or VE alone had no effect on the parameters analyzed. Conclusion: A combined dietary deficiency in Se and VE resulted in higher colonic and spleen Cr burden, a delayed Th1-related cytokine response, and greater pathology associated with infection. These data emphasize the importance of antioxidant nutrients in host defense against enteric pathogens.
315 Conditional Deletion of ERBB2/Neu Inhibits Pancreatic Tumorigenesis Martin Majer, Phillip Gray, Xiufen Li, Lyska L. Emerson, Scott K. Kuwada The average 5-year survival for pancreatic adenocarcinoma (ACa) patients is only about 5%. Chemoprevention of pancreatic ACa requires rational targets and new mouse models that accurately recapitulate the PanIN (pancreatic intraductal neoplasia)-ACa sequence provide an excellent system to find druggable targets. Overexpression of ErbB-2/HER-2/Neu occurs early in pancreatic tumorigenesis but its role in this process is untested. AIMS: To determine the role of ErbB-2 in the progression of premalignant pancreatic ductal neoplasia. Methods: PDX1-Cre; LSL(lox-stop-lox)-KRAS-G12D/+ (KRAS-mut) mice conditionally express oncogenic KRAS-G12D in their pancreata and faithfully recapitulate the PanIN-ACa sequence. Knock-in mice expressing floxed Erbb2 alleles were crossed with mice expressing PDX1Cre and LSL-KRAS-G12D alleles. Genotyping was performed by PCR of genomic DNA from tail snips. Confirmation of Cre-mediated recombination of floxed Erbb2 and LSL-KRASG12D alleles was achieved by PCR of genomic DNA isolated from the pancreas, liver and heart. Pancreas sections were examined at 8.5 months by a pathologist blinded to genotype, since this is when PanIN lesions are present. RESULTS: KRAS-mut mice demonstrated pancreatic enlargement and overexpression of ErbB-2 protein in their PanINs as previously reported by others. PDX1-Cre; Erbb2-Lox/Lox (Panc-Erbb2 KO) mice showed LoxP recombination restricted to the pancreas and a 30-fold reduction in Erbb2 mRNA expression in pancreatic tissue by qRT-PCR. Panc-Erbb2 KO mice were healthy throughout life, indicating that Erbb2 is not required for pancreatic development or function. The key finding was that 100% of KRAS- mut mice (n=4) expressed at least PanIN1b or PanIN2 lesions, while all KRAS- mut/Panc-Erbb2 KO mice (n=6) expressed only PanIN1a lesions and had pancreata similar in size to mice lacking KRAS-G12D expression. Thus, conditional homozygous deletion of Erbb2 inhibited PanIN progression caused by mutated KRAS in the pancreas. Homozygous deletion of Erbb2 in the pancreata of KRAS-mut mice did not cause differences in the expression of phospho-MEK1 or phospho-S6Kinase in the PanINs, but greatly decreased the nuclear expression of phospho-Akt, cyclin D1 and Ki-67 in the PanINs. These results suggest that KRAS-G12D is sufficient to activate S6Kinase but that ErbB-2 is required for cyclin D1 expression and cell cycle progression. CONCLUSIONS: ErbB-2 overexpression drives PanIN progression by increasing cyclin D1 expression and cell cycle progression. Drugs that inhibit ErbB-2 may be successful chemopreventive agents in individuals with strong hereditary predispositions for pancreatic neoplasia.
313 Glutamine Regulates Autophagy in IEC-18 Cells Toshio Sakiyama, Mark W. Musch, Mark J. Ropeleski, Hirohito Tsubouchi, Eugene B. Chang Background and aims: Autophagy is a major mechanism contributing to protein catabolism during starvation and potentially other conditions of cell stress. Amino acids, particularly leucine, are potent inhibitors of autophagy. It has been hypothesized that the signaling mechanism by amino acids to modulate autophagy is stimulation of mammalian target of rapamycin (mTOR). Glutamine is a major source of fuel for intestinal epithelial cells, but little is known about glutamine's effect on autophagy. The present studies investigate whether glutamine regulates autophagy in IEC-18 rat intestinal epithelial cells under basal and heat stressed conditions. Methods: Formation and accumulation of membrane-bound LC3phospholipid conjugates (LC3-II) on autophagosomal membranes was determined by Western blotting and also localized by confocal microscopy in unstimulated as well as heatstressed cells in growth media with and without glutamine. Activation of the mTOR targets, p70 S6 kinase (p70S6K) and S6 ribosomal protein (S6RP), were measured using phosphospecific antibodies by Western blotting. Results: Glutamine-treated IEC-18 cells demonstrated both LC3-II as well as soluble unlipidated LC3 (LC3-I). Glutamine depletion decreased the amount of LC3-II, suggesting a decreased autophagy during glutamine starvation. Glutamine depletion increased the phosphorylation of p70S6K and S6RP, indicating mTOR activation, which may play a role in decreasing LC3-II levels. When glutamine was present, heat shock decreased LC3-II and increased the phosphorylation of p70S6K and S6RP. After the heat stress, LC3-II increased above its basal level, and phosphorylation of p70S6K and S6RP returned to near basal levels. Under glutamine starvation, however, the change of LC3-II by heat shock was minimal, and increased phosphorylation of p70S6K and S6RP by heat shock was not observed. Conclusions: Autophagy in intestinal epithelial cells appears to be regulated not only by the presence of glutamine, but also by heat stress. Glutamine is important for maintenance of LC3-II levels, a pivotal protein in assembly of the autophagosome, under both basal as well as heat stressed conditions. Glutamine may regulate autophagy by inhibition of mTOR, since the amount of LC3-II was well inversely correlated with mTOR activity assessed by phosphorylation of p70S6K and S6RP. Further studies on the ability of glutamine to modulate mTOR activity may elucidate important interactions of glutamine status and heat-induced signaling changes.
316 The Phenotype of Familial Pancreatic Neoplasia Marcia I. Canto, Michael G. Goggins, Richard D. Schulick, Charles J. Yeo, Elliot K. Fishman, Ihab R. Kamel, John L. Cameron, Alison P. Klein, Ralph H. Hruban Screening with EUS and CT/MRCP can detect pancreatic neoplasia in asymptomatic high risk individuals (HRI). AIM: To determine the clinical, radiologic, and pathologic correlates of familial pancreatic neoplasia in HRI. METHODS: HRI with at least 2 blood relatives with PC, PJS, and/or a BRCA2 mutation were screened with EUS and multi-detector CT(19982005) or EUS with MRI/MRCP (2005-2007) as part of our research studies or clinical screening program. Questionnaires and EUS and CT/MRCP results were recorded at baseline and follow-up. Annual surveillance was suggested. Gross and microscopic pathologic specimens from surgically-treated patients were evaluated by a single expert pathologist. Neoplastic lesions were counted and graded for dysplasia using international consensus classification systems. The density of pancreatic intraepithelial neoplasia (PanIN) was determined (total number of PanIN /total number of duct profiles). RESULTS: 175 asymptomatic HRI (59% women, age 30-76) had screening EUS and either CT or MRCP. To date, follow-up was available on 77%. 19 patients underwent 22 operations for suspected pancreatic neoplasms (10 Whipple, 8 distal, 1 total pancreatectomy) detected at baseline or during surveillance (follow-up time median 5.4 years). HRI presented with cystic masses (n=15, 8 multiple) or solid masses (> 1 cm) or nodules (< 1 cm) (n=7, 2 multiple). 14 (64%) had chronic pancreatitis-like features with EUS. 12/22 resected pancreata contained intraductal papillary mucinous neoplasms (IPMN), multiple in 42%, size 5-20 mm; 2 patients had IPMN had high-grade dysplasia (HGD), the rest had moderate or low-grade dysplasia. 6 patients had incipient IPMN (larger than PanIN with finger-like papillae, size <1 cm), none with HGD, alone or with IPMN. 3 patients had single or multiple pancreatic endocrine neoplasms (PEN)(size 2-15 mm). One patient had an invasive PC without associated IPMN, another had 2 IPMN with multiple PanIN and microinvasive PC. 5 patients had PanIN-3 (1-14 lesions per pancreas), 4 larger lesions visualized by EUS. The median PanIN density for patients with EUS changes of chronic pancreatitis (> 4 features) was significantly greater than that for those that did not meet criteria (14.3% vs. 3.2%, p=.004). PanIN density was
314 The Novel Biomarker (HOXB2) Predicts Response to Pancreatectomy for Pancreatic Cancer Andrew V. Biankin, James G. Kench, Devendra Segara, David A. Skalicky, Sandra A. Biankin, Nam Q. Nguyen, Neil Merrett, Robert L. Sutherland, Susan M. Henshall, Elizabeth A. Musgrove Background: Pancreatectomy is the only therapeutic intervention that offers the chance of long term survival for patients with pancreatic cancer, however, only 10% of patients who undergo pancreatectomy survive > 3 years with prognostic factors only determined after pathological examination of the resected specimen. Identification of a biomarker of response to surgical resection that can be determined preoperatively offers the potential to significantly improve survival and quality of life for patients with PC by improving patient selection for pancreatectomy. Methods: We examined the aberrant expression of over 20 genes identified
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positively correlated with the number of EUS features (p=.0012). All cases with multifocal PanIN were associated with lobulocentric parenchymal atrophy. No patient under 45 years of age had a high grade or malignant neoplasm. CONCLUSIONS: The phenotype of early pancreatic neoplasia includes multifocal PanIN with associated lobulocententric atrophy, single or multiple IPMNs, and/or endocrine neoplasms.
by real time RT-PCR and Western blot analysis; NADPH oxidase-mediated superoxide production, using lucigenin-derived chemiluminescence in cell lysates; and cellular reactive oxygen species (ROS) levels, by FACS analysis using the ROS-sensitive dye DCF. Pharmacologic inhibitors and transfections with siRNAs and antisense oligonucleotides were used to determine the roles of different NADPH oxidase subunits, as well as the Akt/PKB kinase, in NADPH oxidase activation. Results: Knockdown of the p22 subunit (using both siRNA and antisense) showed its critical role in IGF-I-induced Nox4 activation and ROS production in the PaCa cell lines. By contrast, siRNAs against the regulatory p67 and p47 subunits had little effect on NADPH oxidase activity despite the presence of these subunits in PaCa cells. Knockdown of p22, but not p67 and p47, greatly attenuated the anti-apoptotic effect of IGF-I. The p22 siRNA (but not the non-targeting negative control siRNA) markedly decreased Nox4 protein level, suggesting that a complex with p22 stabilizes Nox4 in PaCa cells. We found that the IGF-I-induced NADPH oxidase activation was also prevented by siRNA against Akt1 or the Akt1/2 inhibitor VIII, indicating that it is mediated by Akt. Conclusions: Both Nox4 and p22 subunits are necessary for the IGF-I-induced NADPH oxidase activation and ROS production in PaCa cells. Further, complex formation between these subunits stabilizes Nox4 protein. Knocking down of either Nox4 or p22 stimulates apoptosis in PaCa cells. IGF-I-induced activation of NADPH oxidase is mediated by Akt, which identifies a novel pathway of the pro-survival effect of Akt in PaCa cells, namely, via activating the antiapoptotic NADPH oxidase. The results suggest Nox4 and p22 as promising therapeutic targets to stimulate PaCa cell death.
317 Pancreatic Cancer Biomarkers in Saliva James J. Farrell, Bernhard Zimmerman, Ali Ammar, Hui Zhou, Qing Wei, Wael Ghacham, Brad Henson, David T. Wong Background: Early detection of pancreatic cancer carries the best hope for improved survival in this disease. Saliva offers great advantages for the non-invasive surveillance of human health and detection of disease. Based on our recent technological developments with mRNA markers in saliva, we investigated the role of the salivary transcriptome for the detection of pancreatic cancer. Methods: Saliva was collected from patients with clinically confirmed early pancreatic cancer, chronic pancreatitis, and healthy controls. A transcriptomic discovery study was performed using the Affymetrix All-Exon-Array, profiling pancreatic cancers and healthy control saliva samples. By a novel RT-PCR pre-amplification and real-time PCR, a large number of candidate markers were validated in the initial study cohort. These candidate markers were then validated using a second, independently collected sample set from patients with pancreatic cancers, chronic pancreatitis and healthy controls. T-test and Wilcoxon rank test was used for statistical analysis) Results: Microarray profiling led to the selection of 38 candidate exon/transcripts elevated in pancreatic cancer patients' saliva (n=9) compared with normal controls' saliva (n =11), in the initial study cohort. We confirmed a >4 fold elevation of 22 transcripts in the initial study cohort using RT-PCR. The elevation of 14 of these 22 candidate transcripts was then validated in the second independent cohort comparing pancreatic cancer patients' saliva (n=13) with chronic pancreatitis (n=11) and healthy control saliva (n=33) samples. Conclusions: This is the first report validating salivary mRNA markers for the detection of a systemic non-oral disease: pancreatic cancer. These results augment the value of saliva as a non-invasive diagnostic medium, and particularly its potential utility in the diagnosis of systemic disease. We further aim to use the “salivary exon expression profiling” technology to stratify early and locally advanced pancreatic cancer patients. Additional validation studies with large sample cohorts are necessary to translate this finding into clinical applications, and may lead to the inclusion of saliva for the early detection of pancreatic cancer. Supported by PHS grant R01 DE015970
320 VEGF-D Promotes Tumor Growth and Lymphatic Spread in Hepatocellular Carcinoma Armin Thelen, Arne Scholz, Christoph Benckert, Maik Schröder, Peter Neuhaus, Bertram Wiedenmann, Sven Jonas Lymphatic spread is an important clinical determinant for the prognosis of hepatocellular carcinoma (HCC), but little is known about the control of lymphangiogenesis in HCC. We addressed expression and biological role of the pro-(lymph)angiogenic protein VEGF-D in this tumor entity. Using immunohistochemistry and in situ hybridization on specimens of HCC, cirrhotic and normal liver we found abundant expression of VEGF-D exclusively in the tumor cells. The cognate receptor, VEGFR-3 was detected on blood and lymphatic vessels. VEGF-D expression correlated with pT-stage of the primary, lymph node metastasis and lymphatic invasion. To experimentally address the functional role of VEGF-D for lymphangiogenesis, HCC cell lines were studied. Three out of 4 human HCC cell lines expressed and secreted VEGF-D. We took advantage of the VEGF-D deficient cell line, SK-Hep-1, and stably transfected these cells with VEGF-D cDNA to determine effects on tumor progression In Vivo. Compared to Mock controls, subcutaneous tumors derived from VEGF-D expressing cells were larger and more frequently metastasized to regional lymph nodes. VEGF-D expressing tumors exhibited increased microvessel density and increased abundance of periand intratumoral lymphatics, as assessed by immunostaining for CD31 and for LYVE-1 and/ or podoplanin, respectively. Furthermore, coexpression of the soluble extracellular VEGFR3 domain blocked VEGF-D-induced tumor growth and lymphatic spread via reduction of angiogenesis and lymphangiogenesis. In the orthotopic approach, VEGF-D expression resulted in an increased rate of intra- and extrahepatic as well as lymph node metastasis. In conclusion, our study suggests that expression of VEGF-D is involved in growth and lymphatic spread of HCC. Therefore, VEGF-D might represent a therapeutic target in HCC.
318 Early Vaccination Targeting Mutated KRAS As An Antigen Expressed By Panins Delays Progression to Pancreatic Tumors in a Mouse Model Michel Kafrouni, Todd Armstrong, Elizabeth M. Jaffee Similar to human pancreatic cancer, the PdX1Cre-Kras-p53 pancreatic cancer mouse model develops spontaneous pancreatic adenocarcinoma from precancerous lesions called pancreatic intraepithelial neoplasias (PanINs). The objective of this study was to target these early lesions with an antigen specific vaccine to prevent progression to invasive pancreatic cancer. Methods: The PdX1Cre-Kras-p53 mouse model carries a mutated version of the human Kras gene that is over-expressed in the pancreas. Mutated Kras (a glycine to aspartate change at codon 12) is a known oncogene involved early in the progression of human pancreatic adenocarcinoma. A listeria based vaccine construct that contains this same mutant Kras (LmKras) was used for vaccination. To assess the vaccine's ability to induce immunity, splenocytes isolated from vaccinated mice were evaluated to detect the presence of Kras specific, activated CD8+ T cells using intracellular cytokine staining for interferon-γ. To assess the clinical utility of the vaccine, 4 groups of mice were treated with either: no vaccine, the LmKras vaccine only, regulatory T cells (Tregs) depletion only, or LmKras vaccine and Tregs depletion. Mice were assessed for survival and progression of the PanINs. Results: Vaccination with LmKras produced a robust immune response. 10-12 % of CD8+ T cells exhibited an antigen specific immune response against the Kras peptide after 1 week of vaccination. However, following a single vaccination, this response becomes undetectable after 1 month. Serial monthly vaccination and Tregs depletion provided a statistically significant improvement in survival of the mice when compared to the no vaccine group and the vaccine only group (p=0.034 and 0.004 respectively). One fourth of the mice were alive at 360 days compared to (0-10)% in the other 3 groups. Furthermore, initiation of treatment of mice at a younger age (30-45 days old) showed better survival (25%) then at an older age (0)% at 360 days. Vaccination given concurrently with Tregs depletion also prevented the progression of the PanINs to more advanced histologic stages (PanIN 3 or cancer). Only 1 out of 6 mice that received 2 cycles of vaccine and Tregs depletion had an advanced PanIN stage compared to 4 out of 5 mice that recieved no vaccine. Conclusion: Vaccination with LmKras produces a systemic CD8+ T cell response against Kras. This immune response coupled with Tregs depletion produced improvement in survival and delayed the progression of early PanINs to pancreatic cancer. Vaccination against PanIN expressed antigens can prevent progression to pancreatic adenocarcinoma when given early and with Tregs depletion.
321 Hepatocellular Carcinoma in a Non-Cirrhotic Liver: Presentation of a MedicoSurgical Series of 40 Cases Edwige Morineaux, Anne-Marie Marion-Audibert, Philippe Merle, Sylvie Radenne, Brigitte Bancel, Jacques Baulieux, Fabien Zoulim, Christian Ducerf, Jean Christophe Souquet, Christian Trepo Hepatocellular carcinoma (HCC)occurring in non-cirrhotic liver is a rare entity. This retrospective study aimed to describe the etiology, the pathological features of HCC arising in liver with fibrosis ≤ F2 and to measure the prognostic impact of moderate fibrosis. PATIENTS: Among 648 patients with HCC treated between January 1997 and January 2007, patients were included when they had histologically proven HCC, pathology of the peri-tumoral liver showing fibrosis ≤ F2 (operative specimen, liver biopsy), no radiological or endoscopic signs of cirrhosis. Biological, pathological parameters, treatment and survival were collected. Prognostic factors were evaluated by univariate and multivariate analyses. RESULTS: 40 patients (mean age 60 yr, sex ration 1.2) were included, 6.2% of the whole series. Potential etiologic factors were: alcohol consumption in 16 patients (40%), HBV in 4 (11.1%), HCV in 3 (8.6%), dysmetabolic syndrome in 16 (40%), type 2 diabetes in 6 (15%), obesity in 9 (22.5%) and drugs or toxics in 15 (37.5%). Thus, at least one factor was present in 90% of patients. The tumor was unique in 75 and quite large (mean diameter 13 cm). 31 patients (77.5%) had pathological abnormalities in peri-tumoral liver (activity ≥ 1 and/or fibrosis ≥ 1 and ≤ F2). Only in 9 cases, pathology did not show any abnormality. The tumor was well/very well differentiated in 65%. Vascular invasion was observed in 55%. Therapeutic procedures involved transplantation in 3 cases and hepatectomy in 34. Resection was R0 in 25 (62.5%). 19 patients (47.5%) had early recurrence (<3 yr) related to intrahepatic recurrence or metastatic dissemination. 16 patients underwent additional therapy (1 radiofrequency ablation, 2 transarterial chemoembolization, 12 131 I-lipiodol and 1 chemotherapy). At the end of the follow-up (median 47 months), 17 patients had died, 18 were in remission, 3 had stable recurrence and 2 progressive disease. Disease-free median rate was 24 months. Median survival was 51 months. The 3-year disease-free and overall survival rates were respectively 45 and 56%. Multivariate analysis identified vascular invasion and lymph node metastases as independent factors of poor prognosis. Fibrosis F0, F1 or F2 had no effect of median survival. CONCLUSIONS: Almost all patients without cirrhosis presented risk factors for HCC development. 40% had type 2 diabetes and/or overweight or obesity and/or dyslipidemia underlying the potential role of NAFLD, independently of advanced fibrosis. Recurrence of HCC in this setting is common. However hepatectomy remains a legitimate treatment and a favorable prognosis could be expected in those patients with better liver function.
319 Role of Different Subunits of NADPH Oxidase in IGF-I-Induced ROS Production and Pro-Survival Effect in Pancreatic Cancer Cells Mouad Edderkaoui, Claudia J. Nitsche, Guido Eibl, Anna S. Gukovskaya Background & Aims: We have recently shown that NADPH oxidase, in particular its Nox4 isoform, mediates the protection of pancreatic cancer (PaCa) cells from death by growth factors such as IGF-I. NADPH oxidase is a multi-subunit enzyme; its subunit composition and the roles of its catalytic and regulatory subunits in non-phagocytic cells remain largely unknown. Here we investigate the composition of NADPH oxidase and the mechanism of its activation by IGF-I in PaCa cells. Methods: We measured the expression of catalytic and regulatory NADPH oxidase subunits in human pancreatic MIA PaCa-2 and PANC-1 cells
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The Pathogenicity of Citrobacter rodentium (CR) Is Enhanced in Mice Fed Diets Deficient in Selenium (Se) and Vitamin E (VE) Allen Smith, Sebastian Botero, Terez Shea-Donohue Specific nutritional deficiencies can have a significant negative impact on host resistance to infection. We demonstrated that mice fed diets deficient in the antioxidants Se and VE had delayed intestinal clearance of the parasitic nematode, Heligmosomoides polygyrus by interfering with Th2-dependent mechanisms in the small intestine. Citrobacter rodentium is a mouse pathogen that mimics many aspects of enteropathogenic E. coli infection in humans, and is a potent inducer of a Th1 immune response systemically and in the colon. Little is known, however, about the role of antioxidant nutrients on the immune response to this class of pathogens. Aim: To determine the effects of a single or double deficiency in Se and VE on Cr infection in mice. Methods: Three week-old male mice were placed on deficient diets for 5-6 weeks and then infected with Cr. Bacterial load in the feces, colon, and spleen was determined at days 6 or 7, 12, 16, and 20 days post-infection. Colon and spleen weights were also measured. Colonic inflammation was assessed in Geimsa-stained sections. Gene expression in colonic tissue was evaluated by real-time PCR and Cr antigen specific IgM and IgG production by ELISA. Numbers of CD3+, CD4+, CD8+, and B220+ cells in the spleen were determined by flow cytometry. Results: Mice fed a diet deficient in both Se and VE had significantly higher colonic Cr load (p<0.05) at days 12 and 16 post-infection. This was associated with increased tissue damage and cellular infiltration in colonic lamina propria. Colonization of the spleen by Cr was low and variable but consistently higher in mice fed the Se and VE deficient diet compared to infected mice on a sufficient diet. The colon/body weight ratios at day 16 and spleen/body weight ratios at days 12 and 16 postinfection were significantly elevated (p<0.05). Increased spleen size at day 12 days postinfection in mice fed the Se and VE deficient diet correlated with an increase in total cells that was due primarily to cells expressing B220. Gene expression for IFN-γ, TNF-α, and IL-1β in the colon was reduced at day 7, but not day 12, post-infection in mice fed the Se and VE deficient diet. Anti-Cr IgM and IgG levels were not affected by diet. Mice fed diet deficient in Se or VE alone had no effect on the parameters analyzed. Conclusion: A combined dietary deficiency in Se and VE resulted in higher colonic and spleen Cr burden, a delayed Th1-related cytokine response, and greater pathology associated with infection. These data emphasize the importance of antioxidant nutrients in host defense against enteric pathogens.
315 Conditional Deletion of ERBB2/Neu Inhibits Pancreatic Tumorigenesis Martin Majer, Phillip Gray, Xiufen Li, Lyska L. Emerson, Scott K. Kuwada The average 5-year survival for pancreatic adenocarcinoma (ACa) patients is only about 5%. Chemoprevention of pancreatic ACa requires rational targets and new mouse models that accurately recapitulate the PanIN (pancreatic intraductal neoplasia)-ACa sequence provide an excellent system to find druggable targets. Overexpression of ErbB-2/HER-2/Neu occurs early in pancreatic tumorigenesis but its role in this process is untested. AIMS: To determine the role of ErbB-2 in the progression of premalignant pancreatic ductal neoplasia. Methods: PDX1-Cre; LSL(lox-stop-lox)-KRAS-G12D/+ (KRAS-mut) mice conditionally express oncogenic KRAS-G12D in their pancreata and faithfully recapitulate the PanIN-ACa sequence. Knock-in mice expressing floxed Erbb2 alleles were crossed with mice expressing PDX1Cre and LSL-KRAS-G12D alleles. Genotyping was performed by PCR of genomic DNA from tail snips. Confirmation of Cre-mediated recombination of floxed Erbb2 and LSL-KRASG12D alleles was achieved by PCR of genomic DNA isolated from the pancreas, liver and heart. Pancreas sections were examined at 8.5 months by a pathologist blinded to genotype, since this is when PanIN lesions are present. RESULTS: KRAS-mut mice demonstrated pancreatic enlargement and overexpression of ErbB-2 protein in their PanINs as previously reported by others. PDX1-Cre; Erbb2-Lox/Lox (Panc-Erbb2 KO) mice showed LoxP recombination restricted to the pancreas and a 30-fold reduction in Erbb2 mRNA expression in pancreatic tissue by qRT-PCR. Panc-Erbb2 KO mice were healthy throughout life, indicating that Erbb2 is not required for pancreatic development or function. The key finding was that 100% of KRAS- mut mice (n=4) expressed at least PanIN1b or PanIN2 lesions, while all KRAS- mut/Panc-Erbb2 KO mice (n=6) expressed only PanIN1a lesions and had pancreata similar in size to mice lacking KRAS-G12D expression. Thus, conditional homozygous deletion of Erbb2 inhibited PanIN progression caused by mutated KRAS in the pancreas. Homozygous deletion of Erbb2 in the pancreata of KRAS-mut mice did not cause differences in the expression of phospho-MEK1 or phospho-S6Kinase in the PanINs, but greatly decreased the nuclear expression of phospho-Akt, cyclin D1 and Ki-67 in the PanINs. These results suggest that KRAS-G12D is sufficient to activate S6Kinase but that ErbB-2 is required for cyclin D1 expression and cell cycle progression. CONCLUSIONS: ErbB-2 overexpression drives PanIN progression by increasing cyclin D1 expression and cell cycle progression. Drugs that inhibit ErbB-2 may be successful chemopreventive agents in individuals with strong hereditary predispositions for pancreatic neoplasia.
313 Glutamine Regulates Autophagy in IEC-18 Cells Toshio Sakiyama, Mark W. Musch, Mark J. Ropeleski, Hirohito Tsubouchi, Eugene B. Chang Background and aims: Autophagy is a major mechanism contributing to protein catabolism during starvation and potentially other conditions of cell stress. Amino acids, particularly leucine, are potent inhibitors of autophagy. It has been hypothesized that the signaling mechanism by amino acids to modulate autophagy is stimulation of mammalian target of rapamycin (mTOR). Glutamine is a major source of fuel for intestinal epithelial cells, but little is known about glutamine's effect on autophagy. The present studies investigate whether glutamine regulates autophagy in IEC-18 rat intestinal epithelial cells under basal and heat stressed conditions. Methods: Formation and accumulation of membrane-bound LC3phospholipid conjugates (LC3-II) on autophagosomal membranes was determined by Western blotting and also localized by confocal microscopy in unstimulated as well as heatstressed cells in growth media with and without glutamine. Activation of the mTOR targets, p70 S6 kinase (p70S6K) and S6 ribosomal protein (S6RP), were measured using phosphospecific antibodies by Western blotting. Results: Glutamine-treated IEC-18 cells demonstrated both LC3-II as well as soluble unlipidated LC3 (LC3-I). Glutamine depletion decreased the amount of LC3-II, suggesting a decreased autophagy during glutamine starvation. Glutamine depletion increased the phosphorylation of p70S6K and S6RP, indicating mTOR activation, which may play a role in decreasing LC3-II levels. When glutamine was present, heat shock decreased LC3-II and increased the phosphorylation of p70S6K and S6RP. After the heat stress, LC3-II increased above its basal level, and phosphorylation of p70S6K and S6RP returned to near basal levels. Under glutamine starvation, however, the change of LC3-II by heat shock was minimal, and increased phosphorylation of p70S6K and S6RP by heat shock was not observed. Conclusions: Autophagy in intestinal epithelial cells appears to be regulated not only by the presence of glutamine, but also by heat stress. Glutamine is important for maintenance of LC3-II levels, a pivotal protein in assembly of the autophagosome, under both basal as well as heat stressed conditions. Glutamine may regulate autophagy by inhibition of mTOR, since the amount of LC3-II was well inversely correlated with mTOR activity assessed by phosphorylation of p70S6K and S6RP. Further studies on the ability of glutamine to modulate mTOR activity may elucidate important interactions of glutamine status and heat-induced signaling changes.
316 The Phenotype of Familial Pancreatic Neoplasia Marcia I. Canto, Michael G. Goggins, Richard D. Schulick, Charles J. Yeo, Elliot K. Fishman, Ihab R. Kamel, John L. Cameron, Alison P. Klein, Ralph H. Hruban Screening with EUS and CT/MRCP can detect pancreatic neoplasia in asymptomatic high risk individuals (HRI). AIM: To determine the clinical, radiologic, and pathologic correlates of familial pancreatic neoplasia in HRI. METHODS: HRI with at least 2 blood relatives with PC, PJS, and/or a BRCA2 mutation were screened with EUS and multi-detector CT(19982005) or EUS with MRI/MRCP (2005-2007) as part of our research studies or clinical screening program. Questionnaires and EUS and CT/MRCP results were recorded at baseline and follow-up. Annual surveillance was suggested. Gross and microscopic pathologic specimens from surgically-treated patients were evaluated by a single expert pathologist. Neoplastic lesions were counted and graded for dysplasia using international consensus classification systems. The density of pancreatic intraepithelial neoplasia (PanIN) was determined (total number of PanIN /total number of duct profiles). RESULTS: 175 asymptomatic HRI (59% women, age 30-76) had screening EUS and either CT or MRCP. To date, follow-up was available on 77%. 19 patients underwent 22 operations for suspected pancreatic neoplasms (10 Whipple, 8 distal, 1 total pancreatectomy) detected at baseline or during surveillance (follow-up time median 5.4 years). HRI presented with cystic masses (n=15, 8 multiple) or solid masses (> 1 cm) or nodules (< 1 cm) (n=7, 2 multiple). 14 (64%) had chronic pancreatitis-like features with EUS. 12/22 resected pancreata contained intraductal papillary mucinous neoplasms (IPMN), multiple in 42%, size 5-20 mm; 2 patients had IPMN had high-grade dysplasia (HGD), the rest had moderate or low-grade dysplasia. 6 patients had incipient IPMN (larger than PanIN with finger-like papillae, size <1 cm), none with HGD, alone or with IPMN. 3 patients had single or multiple pancreatic endocrine neoplasms (PEN)(size 2-15 mm). One patient had an invasive PC without associated IPMN, another had 2 IPMN with multiple PanIN and microinvasive PC. 5 patients had PanIN-3 (1-14 lesions per pancreas), 4 larger lesions visualized by EUS. The median PanIN density for patients with EUS changes of chronic pancreatitis (> 4 features) was significantly greater than that for those that did not meet criteria (14.3% vs. 3.2%, p=.004). PanIN density was
314 The Novel Biomarker (HOXB2) Predicts Response to Pancreatectomy for Pancreatic Cancer Andrew V. Biankin, James G. Kench, Devendra Segara, David A. Skalicky, Sandra A. Biankin, Nam Q. Nguyen, Neil Merrett, Robert L. Sutherland, Susan M. Henshall, Elizabeth A. Musgrove Background: Pancreatectomy is the only therapeutic intervention that offers the chance of long term survival for patients with pancreatic cancer, however, only 10% of patients who undergo pancreatectomy survive > 3 years with prognostic factors only determined after pathological examination of the resected specimen. Identification of a biomarker of response to surgical resection that can be determined preoperatively offers the potential to significantly improve survival and quality of life for patients with PC by improving patient selection for pancreatectomy. Methods: We examined the aberrant expression of over 20 genes identified
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positively correlated with the number of EUS features (p=.0012). All cases with multifocal PanIN were associated with lobulocentric parenchymal atrophy. No patient under 45 years of age had a high grade or malignant neoplasm. CONCLUSIONS: The phenotype of early pancreatic neoplasia includes multifocal PanIN with associated lobulocententric atrophy, single or multiple IPMNs, and/or endocrine neoplasms.
by real time RT-PCR and Western blot analysis; NADPH oxidase-mediated superoxide production, using lucigenin-derived chemiluminescence in cell lysates; and cellular reactive oxygen species (ROS) levels, by FACS analysis using the ROS-sensitive dye DCF. Pharmacologic inhibitors and transfections with siRNAs and antisense oligonucleotides were used to determine the roles of different NADPH oxidase subunits, as well as the Akt/PKB kinase, in NADPH oxidase activation. Results: Knockdown of the p22 subunit (using both siRNA and antisense) showed its critical role in IGF-I-induced Nox4 activation and ROS production in the PaCa cell lines. By contrast, siRNAs against the regulatory p67 and p47 subunits had little effect on NADPH oxidase activity despite the presence of these subunits in PaCa cells. Knockdown of p22, but not p67 and p47, greatly attenuated the anti-apoptotic effect of IGF-I. The p22 siRNA (but not the non-targeting negative control siRNA) markedly decreased Nox4 protein level, suggesting that a complex with p22 stabilizes Nox4 in PaCa cells. We found that the IGF-I-induced NADPH oxidase activation was also prevented by siRNA against Akt1 or the Akt1/2 inhibitor VIII, indicating that it is mediated by Akt. Conclusions: Both Nox4 and p22 subunits are necessary for the IGF-I-induced NADPH oxidase activation and ROS production in PaCa cells. Further, complex formation between these subunits stabilizes Nox4 protein. Knocking down of either Nox4 or p22 stimulates apoptosis in PaCa cells. IGF-I-induced activation of NADPH oxidase is mediated by Akt, which identifies a novel pathway of the pro-survival effect of Akt in PaCa cells, namely, via activating the antiapoptotic NADPH oxidase. The results suggest Nox4 and p22 as promising therapeutic targets to stimulate PaCa cell death.
317 Pancreatic Cancer Biomarkers in Saliva James J. Farrell, Bernhard Zimmerman, Ali Ammar, Hui Zhou, Qing Wei, Wael Ghacham, Brad Henson, David T. Wong Background: Early detection of pancreatic cancer carries the best hope for improved survival in this disease. Saliva offers great advantages for the non-invasive surveillance of human health and detection of disease. Based on our recent technological developments with mRNA markers in saliva, we investigated the role of the salivary transcriptome for the detection of pancreatic cancer. Methods: Saliva was collected from patients with clinically confirmed early pancreatic cancer, chronic pancreatitis, and healthy controls. A transcriptomic discovery study was performed using the Affymetrix All-Exon-Array, profiling pancreatic cancers and healthy control saliva samples. By a novel RT-PCR pre-amplification and real-time PCR, a large number of candidate markers were validated in the initial study cohort. These candidate markers were then validated using a second, independently collected sample set from patients with pancreatic cancers, chronic pancreatitis and healthy controls. T-test and Wilcoxon rank test was used for statistical analysis) Results: Microarray profiling led to the selection of 38 candidate exon/transcripts elevated in pancreatic cancer patients' saliva (n=9) compared with normal controls' saliva (n =11), in the initial study cohort. We confirmed a >4 fold elevation of 22 transcripts in the initial study cohort using RT-PCR. The elevation of 14 of these 22 candidate transcripts was then validated in the second independent cohort comparing pancreatic cancer patients' saliva (n=13) with chronic pancreatitis (n=11) and healthy control saliva (n=33) samples. Conclusions: This is the first report validating salivary mRNA markers for the detection of a systemic non-oral disease: pancreatic cancer. These results augment the value of saliva as a non-invasive diagnostic medium, and particularly its potential utility in the diagnosis of systemic disease. We further aim to use the “salivary exon expression profiling” technology to stratify early and locally advanced pancreatic cancer patients. Additional validation studies with large sample cohorts are necessary to translate this finding into clinical applications, and may lead to the inclusion of saliva for the early detection of pancreatic cancer. Supported by PHS grant R01 DE015970
320 VEGF-D Promotes Tumor Growth and Lymphatic Spread in Hepatocellular Carcinoma Armin Thelen, Arne Scholz, Christoph Benckert, Maik Schröder, Peter Neuhaus, Bertram Wiedenmann, Sven Jonas Lymphatic spread is an important clinical determinant for the prognosis of hepatocellular carcinoma (HCC), but little is known about the control of lymphangiogenesis in HCC. We addressed expression and biological role of the pro-(lymph)angiogenic protein VEGF-D in this tumor entity. Using immunohistochemistry and in situ hybridization on specimens of HCC, cirrhotic and normal liver we found abundant expression of VEGF-D exclusively in the tumor cells. The cognate receptor, VEGFR-3 was detected on blood and lymphatic vessels. VEGF-D expression correlated with pT-stage of the primary, lymph node metastasis and lymphatic invasion. To experimentally address the functional role of VEGF-D for lymphangiogenesis, HCC cell lines were studied. Three out of 4 human HCC cell lines expressed and secreted VEGF-D. We took advantage of the VEGF-D deficient cell line, SK-Hep-1, and stably transfected these cells with VEGF-D cDNA to determine effects on tumor progression In Vivo. Compared to Mock controls, subcutaneous tumors derived from VEGF-D expressing cells were larger and more frequently metastasized to regional lymph nodes. VEGF-D expressing tumors exhibited increased microvessel density and increased abundance of periand intratumoral lymphatics, as assessed by immunostaining for CD31 and for LYVE-1 and/ or podoplanin, respectively. Furthermore, coexpression of the soluble extracellular VEGFR3 domain blocked VEGF-D-induced tumor growth and lymphatic spread via reduction of angiogenesis and lymphangiogenesis. In the orthotopic approach, VEGF-D expression resulted in an increased rate of intra- and extrahepatic as well as lymph node metastasis. In conclusion, our study suggests that expression of VEGF-D is involved in growth and lymphatic spread of HCC. Therefore, VEGF-D might represent a therapeutic target in HCC.
318 Early Vaccination Targeting Mutated KRAS As An Antigen Expressed By Panins Delays Progression to Pancreatic Tumors in a Mouse Model Michel Kafrouni, Todd Armstrong, Elizabeth M. Jaffee Similar to human pancreatic cancer, the PdX1Cre-Kras-p53 pancreatic cancer mouse model develops spontaneous pancreatic adenocarcinoma from precancerous lesions called pancreatic intraepithelial neoplasias (PanINs). The objective of this study was to target these early lesions with an antigen specific vaccine to prevent progression to invasive pancreatic cancer. Methods: The PdX1Cre-Kras-p53 mouse model carries a mutated version of the human Kras gene that is over-expressed in the pancreas. Mutated Kras (a glycine to aspartate change at codon 12) is a known oncogene involved early in the progression of human pancreatic adenocarcinoma. A listeria based vaccine construct that contains this same mutant Kras (LmKras) was used for vaccination. To assess the vaccine's ability to induce immunity, splenocytes isolated from vaccinated mice were evaluated to detect the presence of Kras specific, activated CD8+ T cells using intracellular cytokine staining for interferon-γ. To assess the clinical utility of the vaccine, 4 groups of mice were treated with either: no vaccine, the LmKras vaccine only, regulatory T cells (Tregs) depletion only, or LmKras vaccine and Tregs depletion. Mice were assessed for survival and progression of the PanINs. Results: Vaccination with LmKras produced a robust immune response. 10-12 % of CD8+ T cells exhibited an antigen specific immune response against the Kras peptide after 1 week of vaccination. However, following a single vaccination, this response becomes undetectable after 1 month. Serial monthly vaccination and Tregs depletion provided a statistically significant improvement in survival of the mice when compared to the no vaccine group and the vaccine only group (p=0.034 and 0.004 respectively). One fourth of the mice were alive at 360 days compared to (0-10)% in the other 3 groups. Furthermore, initiation of treatment of mice at a younger age (30-45 days old) showed better survival (25%) then at an older age (0)% at 360 days. Vaccination given concurrently with Tregs depletion also prevented the progression of the PanINs to more advanced histologic stages (PanIN 3 or cancer). Only 1 out of 6 mice that received 2 cycles of vaccine and Tregs depletion had an advanced PanIN stage compared to 4 out of 5 mice that recieved no vaccine. Conclusion: Vaccination with LmKras produces a systemic CD8+ T cell response against Kras. This immune response coupled with Tregs depletion produced improvement in survival and delayed the progression of early PanINs to pancreatic cancer. Vaccination against PanIN expressed antigens can prevent progression to pancreatic adenocarcinoma when given early and with Tregs depletion.
321 Hepatocellular Carcinoma in a Non-Cirrhotic Liver: Presentation of a MedicoSurgical Series of 40 Cases Edwige Morineaux, Anne-Marie Marion-Audibert, Philippe Merle, Sylvie Radenne, Brigitte Bancel, Jacques Baulieux, Fabien Zoulim, Christian Ducerf, Jean Christophe Souquet, Christian Trepo Hepatocellular carcinoma (HCC)occurring in non-cirrhotic liver is a rare entity. This retrospective study aimed to describe the etiology, the pathological features of HCC arising in liver with fibrosis ≤ F2 and to measure the prognostic impact of moderate fibrosis. PATIENTS: Among 648 patients with HCC treated between January 1997 and January 2007, patients were included when they had histologically proven HCC, pathology of the peri-tumoral liver showing fibrosis ≤ F2 (operative specimen, liver biopsy), no radiological or endoscopic signs of cirrhosis. Biological, pathological parameters, treatment and survival were collected. Prognostic factors were evaluated by univariate and multivariate analyses. RESULTS: 40 patients (mean age 60 yr, sex ration 1.2) were included, 6.2% of the whole series. Potential etiologic factors were: alcohol consumption in 16 patients (40%), HBV in 4 (11.1%), HCV in 3 (8.6%), dysmetabolic syndrome in 16 (40%), type 2 diabetes in 6 (15%), obesity in 9 (22.5%) and drugs or toxics in 15 (37.5%). Thus, at least one factor was present in 90% of patients. The tumor was unique in 75 and quite large (mean diameter 13 cm). 31 patients (77.5%) had pathological abnormalities in peri-tumoral liver (activity ≥ 1 and/or fibrosis ≥ 1 and ≤ F2). Only in 9 cases, pathology did not show any abnormality. The tumor was well/very well differentiated in 65%. Vascular invasion was observed in 55%. Therapeutic procedures involved transplantation in 3 cases and hepatectomy in 34. Resection was R0 in 25 (62.5%). 19 patients (47.5%) had early recurrence (<3 yr) related to intrahepatic recurrence or metastatic dissemination. 16 patients underwent additional therapy (1 radiofrequency ablation, 2 transarterial chemoembolization, 12 131 I-lipiodol and 1 chemotherapy). At the end of the follow-up (median 47 months), 17 patients had died, 18 were in remission, 3 had stable recurrence and 2 progressive disease. Disease-free median rate was 24 months. Median survival was 51 months. The 3-year disease-free and overall survival rates were respectively 45 and 56%. Multivariate analysis identified vascular invasion and lymph node metastases as independent factors of poor prognosis. Fibrosis F0, F1 or F2 had no effect of median survival. CONCLUSIONS: Almost all patients without cirrhosis presented risk factors for HCC development. 40% had type 2 diabetes and/or overweight or obesity and/or dyslipidemia underlying the potential role of NAFLD, independently of advanced fibrosis. Recurrence of HCC in this setting is common. However hepatectomy remains a legitimate treatment and a favorable prognosis could be expected in those patients with better liver function.
319 Role of Different Subunits of NADPH Oxidase in IGF-I-Induced ROS Production and Pro-Survival Effect in Pancreatic Cancer Cells Mouad Edderkaoui, Claudia J. Nitsche, Guido Eibl, Anna S. Gukovskaya Background & Aims: We have recently shown that NADPH oxidase, in particular its Nox4 isoform, mediates the protection of pancreatic cancer (PaCa) cells from death by growth factors such as IGF-I. NADPH oxidase is a multi-subunit enzyme; its subunit composition and the roles of its catalytic and regulatory subunits in non-phagocytic cells remain largely unknown. Here we investigate the composition of NADPH oxidase and the mechanism of its activation by IGF-I in PaCa cells. Methods: We measured the expression of catalytic and regulatory NADPH oxidase subunits in human pancreatic MIA PaCa-2 and PANC-1 cells
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