L’adénocarcinome pancréatique canalaire est un cancer de mauvais pronostic avec une survie à 5 ans inférieure à 5% et une médiane de survie d’environ 6 mois. Dès les stades précoces de la carcinogenèse pancréatique, la mucine membranaire MUC1, glycoprotéine de haute masse moléculaire, est surexprimée et présente des anomalies de distribution cellulaire, essentiellement une délocalisation membranaire vers le pôle basolatéral et une rétention à l’intérieur de la cellule. Sachant que MUC1 est capable d’interagir avec l’EGFR et de jouer un rôle sur la transduction des signaux, cette séquestration pourrait être à l’origine de signaux oncogéniques et est utilisée par les pathologistes comme indicateur de malignité après ponction sur des lésions pancréatiques. Cependant, les mécanismes permettant cette rétention cytoplasmique ne sont pas connus. Des études antérieures du laboratoire ont montré que le trafic de certaines glycoprotéines vers la membrane apicale des cellules épithéliales était dépendant de la Galectine 4. Dans ce travail, nous nous intéresserons à la Galectine 3, lectine endogène susceptible d’interagir avec les motifs glycanniques des mucines et aussi des récepteurs membranaires. Cette Galectine est surexprimée dans le cancer pancréatique et son expression est corrélée à une forte agressivité tumorale dans d’autres cancers. Le but de ce travail a été dans un premier temps de mettre au point des lignées cellulaires pancréatiques polarisées CAPAN-1 Knocked-down pour la Galectine-3. Dans un second temps d’étudier l’implication de la Galectine-3 : (i) dans le trafic intracellulaire de MUC1 et l’EGFR (ii) dans l’interaction entre MUC1 et l’EGFR (iii) sur les voies de signalisation de l’EGFR.Les résultats montrent que les cellules tumorales pancréatiques CAPAN-1 présentent les anomalies de distribution cellulaire de MUC1 observées dans les tumeurs pancréatiques humaines, notamment la rétention de MUC1 à l’intérieur des cellules. Le silencing de la Galectine-3 entraîne la disparition de cette anomalie de distribution cellulaire, MUC1 retrouvant la distribution membranaire normale. Nous avons montré que l’inhibition de l’expression de la Galectine-3 est associée (i) à une augmentation de la translocation nucléaire de l’EGFR (ii) à une inhibition de l’endoyctose de MUC1 et de l’EGFR en condition de sevrage. Ce phénomène s’accompagne d’une augmentation de l’interaction entre MUC1 et l’EGFR et d’une activation accrue de ce récepteur tyrosine kinase lorsqu’il est soumis à l’EGF : augmentation de la phosphorylation de l’EGFR et augmentation de la phosphorylation des MAPK Erk1 et 2. Mots clés : Galectine, Mucine, EGFR, cancer, endocytose, translocation nucléaire / Pancreatic ductal adenocarcinoma (PDAC) is thought to derive from the epithelial ductal cells. In physiological state, the transmembrane mucin MUC1 is expressed at the apical pole where it protects the cell, senses the environment and modulates signal transduction notably by interacting with EGFR. In tumor cells, the localization of MUC1 is modified. MUC1 expression at the membrane becomes circumferential and accumulates in the cytoplasm. This sequestration is thought to deliver oncogenic signals to the cell, and is used by pathologists as an indicator of malignancy. Since it was previously demonstrated that endogenous lectins, especially galectin-3 (Gal-3) and -4, control the apical targeting of glycoproteins in epithelial cells, our aim was to study the role of Gal-3 in the control of MUC1 expression topography in PDAC, and in the cell invasiveness in vitro. Results/expected results: In control cells, we observed a strong MUC1 labeling in cytoplasm as in pathologic conditions. MUC1 was partially co-localized with M6PR in late endosomes. In KD cells, invalidation of Gal-3 led to a strong decrease of MUC1 cytoplasmic labeling. Gal-3 down-regulation is associated with a decrease of both MUC1 and EGFR protein levels by WB. However, KD Gal-3 cells expressed 2-fold higher levels of phosphoY1173EGFR in response to EGF treatment. Preliminary results showed that KD Gal-3 cells lost their in vitro invasive phenotype.Conclusions: Our data showed that Gal-3 is responsible for MUC1 cytoplasmic retention in pancreatic tumor cells. Silencing of Gal-3 promotes MUC1 localization at the cell membrane, and increases EGF-induced EGFR phosphorylation.keywords : Galectine, Mucine, EGFR, cancer, endocytose, translocation nucléaire

Introduction: Pancreatic cancer is the 10th most common cause of cancer in the United Kingdom (UK) yet the 5th most common cause of cancer related death. Although excess adiposity, measured as body mass index (BMI), is a risk factor for the development of pancreatic cancer the increase in relative risk is modest. Animal models suggest that the intra-organ deposition of lipids may be more specific to disease risk than anthropometric measurements. There is therefore a need to develop non-invasive methods to quantify intra-pancreatic fat deposition as a potential biomarker for pancreatic cancer predisposition. Cancer Research UK (CRUK) sets out clear guidelines for biomarker discovery and development. Potential biomarkers must go through a process of discovery and assay development followed by qualification. Methods Three streams of research: (i) Stage-one of the PanORAMA project. Assessment of accuracy through comparison of CS-MR and MRS quantified intra-pancreatic fat with histologically quantified intra-pancreatic fat in 12 patients undergoing pancreatic surgery. (ii) Stage-two of the PanORAMA study. Assessment of precision (reproducibility) and comparison with other anthropometric markers of excess adiposity in healthy volunteers (n=15). Refinement of MRS protocols and repeated assessment of precision in healthy volunteers (n=10). (iii) The Breast Risk Reduction Intermittent Dietary Evaluation 2 (BRRIDE-2) trial. Comparison of the effects of Intermittent Energy Restriction (IER) with Daily Energy Restriction (DER) on intra-pancreatic and intra-hepatic fat stores and metabolic markers of disease risk (n=26). Results (i) CS-MR and MRS had agreement with histological assessment of intra-pancreatic fat, but correlations were only moderate to good (rho 0.672 and 0.781 respectively). (ii) CS-MR, and after refinement, MRS, have clinically acceptable precision. This study tested this principle in intra-pancreatic fat in healthy volunteers with a range of intra-pancreatic fat consistent with the literature on the healthy population. (iii) I found no differences in reduction in intra-hepatic or intra-pancreatic fat when comparing IER with DER. Overall, I found that significant reductions (mean: 6.5%) in both of these ectopic fat stores could be achieved with eight-weeks of dietary intervention. Discussion More recent hypotheses on the link between excess adiposity and cancer have focused on the importance of within organ local ectopic fat as an abnormal micro-environment favouring cancer development and progression. Importantly, this hypothesis explains the specificity of epidemiological associations between excess adiposity and cancer risk. The observations that within a given individual, in the presence of short-term weight reduction, there are differential changes in local within organ fats – hepatic fat and pancreatic fat – support the specificity hypothesis. This thesis has put us in position to scale-up and explore the importance of intra-organ fats using non-invasive imaging techniques.

The incidence of cancer worldwide has increased over the years, and gastrointestinal cancers (G.I.) are amongst the most common forms of cancer. Nevertheless, there is still no curative treatments for this group of tumors. Nucleoside analogues are widely used in cancer treatment. The prevailing compounds are Gemcitabine (used for pancreatic cancer and other carcinomas), 5-Fluorouracil (used in breast, colon, and other cancers), Cytarabine and Clofarabine (used in leukemias). Gemcitabine, the current standard of care for various forms of solid tumors, has a limited efficacy against pancreatic cancer. The objective of this project was the development of effective drugs against pancreatic cancer. We focused on a novel class of nucleoside analogues designed to bypass the most common cellular road blocks and resistance mechanisms. After an extensive screen for cell killing activity, two lead molecules were exclusively studied: LCB2151 and LCB2132. These two molecules showed high efficacy in killing human cancer cells from three different human G.I. cell lines: BxPC3 and Capan-2, two pancreatic cell lines representative of K-Ras positive and negative tumors, as well as the liver cell line HepG2. LCB2151 showed high efficacy in killing Gemcitabine-resistant cancer cells, and a low toxicity in normal cells. Interestingly, results show that these prodrugs can efficiently bypass key resistance mechanisms developed by cancer cells. The results obtained in this project are promising and could pave the way for a more effective treatment of pancreatic cancer.

La mucine MUC4 est un acteur important de la cancérogenèse pancréatique. Elle favorise la progression tumorale et son expression est associée à un mauvais pronostic. Il a également été montré l’implication de la mucine MUC4 dans la résistance aux chimiothérapies. L’ensemble de ces données souligne l’intérêt de la mucine MUC4 comme cible thérapeutique. De plus, sa néoexpression dès les stades précoces de la cancérogenèse pancréatique lui confère un rôle potentiel de marqueur précoce de la carcinogenèse. Les mécanismes moléculaires responsables de l’induction précoce de l’expression de MUC4 sont toutefois encore peu connus.L’étude de la régulation de l’expression de la mucine MUC4 contribuerait donc mieux à comprendre son rôle dans la cancérogenèse. Ainsi, il a été montré que le gène MUC4 est régulé in vitro par des mécanismes transcriptionnels et par des mécanismes épigénétiques de méthylation de l’ADN et des modifications post-traductionnelles des histones. En revanche, la régulation post-transcriptionelle de l’expression de MUC4 notamment par les miARN est peu connue. Nos travaux ont pour but d’identifier les miARN dérégulés dans le cancer du pancréas et/ou ciblant potentiellement MUC4, de déterminer le(s) miARN inhibant l’expression de la protéine oncogénique MUC4 et l’impact de l’administration de ce(s) miARN dans la cancérogenèse pancréatique, et d’identifier les miARN impliqués dans la chimiorésistance médiée par MUC4 dans le cancer du pancréas.Dans un premier temps, nous avons dressé le profil d’expression des miARN dans des lignées cellulaires pancréatiques humaines normales et cancéreuses par puces miARN. Nous avons pu mettre en évidence une signature d’expression de miARN qui a permis de valider nos modèles cellulaires. Puis, à l’aide des bases de données TargetScan, Microcosm et MiRanda, nous avons identifié les miARN ciblant potentiellement MUC4. L’analyse par PCR quantitative a permis de montrer que seuls le miR-145 et miR-219-1-3p étaient sous-exprimés dans l’ensemble des lignées cancéreuses étudiées. Finalement, uniquement miR-219-1-3p est capable d’inhiber l’expression protéique de MUC4 ; c’est pourquoi nous nous sommes intéressés à son rôle dans le cancer du pancréas.Nous avons observé une perte d’expression du miR-219-1-3p dans des tissus de patients atteints d’adénocarcinome pancréatique. Par deux approches complémentaires de surexpression (transitoire ou stable) ou d’inhibition de miR-219-1-3p, nous avons montré qu’il était capable de réprimer l’expression de MUC4 au niveau protéique en se fixant directement sur son 3’-UTR. Nous avons observé une inhibition de la migration et de la prolifération cellulaires associées à une diminution de l’expression de la cycline D1 et de l’activation des voies de signalisation Akt et Erk. In vivo, la croissance tumorale est fortement ralentie après l’injection intratumorale de miR-219-1-3p. Grâce au modèle murin Pdx1-Cre;LSL-KrasG12D de lésions PanIN, nous avons pu mettre en évidence que la perte d’expression du miR-219-1-3p intervient précocement dès les stades PanIN-1/2 de la cancérogenèse pancréatique et qu’il existait une corrélation inverse entre l’expression de miR-219-1-3p et celle de la mucine Muc4.Par ailleurs, nous avons observé que le traitement des cellules cancéreuses pancréatiques humaines par la gemcitabine induit une forte surexpression du miR-219-1-3p qui laisse penser à un rôle potentiel de ce miARN dans la sensibilité des cellules à la chimiothérapie. Cependant, cette surexpression n’a montré aucun effet sur la survie cellulaire après traitement. Nous avons par la suite mis en évidence un profil d’expression différentiel des miARN entre les cellules invalidées pour MUC4 et les cellules contrôles. [...] / The MUC4 mucin is an important actor of pancreatic tumorigenesis as it contributes to tumor progression and its expression correlates with a poor prognosis. It has also been shown that MUC4 is involved in resistance of cells to chemotherapies. In this context, MUC4 is a potential therapeutic target in pancreatic cancer. MUC4 neoexpression at early stages of carcinogenesis confers to this mucin a potential interest as an early marker. Molecular mechanisms responsible for MUC4 induction of expression are not well defined. Thus, studying MUC4 gene expression regulation would contribute to better understand its role in tumorigenesis. MUC4 gene is regulated at the transcriptional level and epigenetically by DNA methylation and histone modifications mechanisms. However, MUC4 post-transcriptional regulation notably by miRNA is largely unknown. Our work aimed at (i) identifying miRNA dysregulated in pancreatic cancer and/or potentially targeting MUC4, (ii) determining miRNA(s) inhibiting MUC4 expression and its (their) impact on pancreatic carcinogenesis and finally (iii) identifying miRNAs involved in chemoresistance mediated by MUC4 in pancreatic cancer.First, using miRNA microarrays we established the miRNA expression profile of normal and cancerous pancreatic cell lines. We showed a cancer-specific miRNA signature which allows us to validate our cellular models. Then, performing in silico studies with TargetScan, Microcosm and MiRanda databases led us to identify miRNA potentially targeting MUC4. Analysis by qRT-PCR showed that miR-145 and miR-219-1-3p were downregulated in human pancreatic cancer cell lines. Finally, only miR-219-1-3p inhibited MUC4 expression thus we focused on its role in pancreatic cancer.We observed a loss of miR-219-1-3p expression in pancreatic cancer tissues. Complementary approaches overexpressing (transiently or stably) and inhibiting miR-219-1-3p expression, led us to show that miR-219-1-3p represses MUC4 protein expression by interacting directly with its 3’-UTR. We observed a decrease of cell migration and cell proliferation which was associated with an inhibition of cyclin D1 expression and an inhibition of Akt and Erk activation. Tumor growth in scid mice was strongly slowed down following miR-219-1-3p intratumoral injection. In the Pdx1-Cre;LSL-KrasG12D mouse model of PanIN, loss of miR-219-1-3p expression was an early event as soon as PanIN1/2 and miR-219-1-3p expression was conversely correlated to Muc4 expression.While the strong induction of miR-219-1-3p following gemcitabine treatment of pancreatic cancer cells suggests a potential role of this miRNA in sensitivity of cells,miR-219-1-3p has no effect on survival rate of cells treated with gemcitabine. We then established the miRNA expression profile of MUC4 knocked-down (MUC4-KD) cells and control cells (Mock) and showed a dysregulation of miRNA expression in MUC4-KD compared to Mock cells.To conclude, our results indicate that miR-219-1-3p, downregulated in pancreatic cancer, negatively regulates MUC4 expression, alters cancer cell biological properties and has an antimoral effect in vivo. Altogether, these results propose miR-219-1-3p as tumor suppressor in pancreatic cancer. Loss of MUC4 leads to an aberrant miRNA expression profile suggesting a potential role of miRNA as markers of chemoresistance in pancreatic cancer.

(A) Gastric Secretion The present study was performed to investigate the canine post-pancreatic duct ligation GIP secretion in response to fat ingestion using a meat meal mixed with unhydrolyzed or hydrolyzed whipping cream, and to determine whether GIP plays a role in the production of hyperacid secretion in the pancreatic duct ligated dogs. Four mongrel female dogs were prepared with Heidenhain pouch (HP) and gastric fistula (GF), and daily acid secretion from the HP was measured before and after pancreatic duct ligation (PDL). HP acid output, serum immunoreactive gastrin (IR-Ga) and serum immunoreactive gastric inhibitory polypeptide (IR-GIP) concentrations during five hours following oral ingestion of a meat meal alone, a meat meal mixed with 125g of unhydrolyzed cream and meat meal mixed with 125g of hydrolyzed cream were measured before and after PDL. Twenty four hour HP acid outputs increased significantly in each of the four dogs after PDL. Five hour HP acid outputs in response to a meat meal alone and a meat meal plus unhydrolyzed cream were modestly increased, while those in response to a meat meal plus hydrolyzed cream were rather reduced after PDL. Serum IR-Ga responses to all stimulants were lowered after PDL and those to meat meal plus hydrolyzed cream lowered most markedly. Serum IR-GIP responses to a meat meal alone were significantly increased, while those to a meat meal plus unhydrolyzed and hydrolyzed cream were reduced. The results of the present study demonstrate serum IR-GIP in response to a meat meal is increased by PDL in dogs, suggesting augmented acid juice passing into the intestinal lumen is responsible for the increased GIP response. It is indicated that hypo-secretion of GIP is not the cause of hypersecretion of gastric acid in the PDL dogs. (B) Glucose Metabolism. Functional alteration in glucose homeostasis especially concerning the early onset of diabetes after PDL was studied in dogs. Intravenous (i.v.) and intragastric glucose tolerance tests were performed at two to ten weeks and two weeks after PDL respectively. Serum glucose, IRI, and IR-GIP in response to a meat meal with and without unhydrolyzed or hydrolyzed fat were estimated at six weeks after PDL. Significantly impaired glucose tolerance and early phase IRI secretion after i.v. glucose were shown at two to ten weeks after PDL. Intragastric glucose load revealed delayed pattern of serum glucose and IRI (no evidence of glucose intolerance or diminished IRI secretion), indicating decreased gastric motility after PDL. Serum IR-GIP response to intragastric glucose load was not attenuated by the operation but showed a similar pattern to IRI response. Serum IRI responses to meat meals with and without unhydrolyzed or hydrolyzed cream were impaired after PDL. It is indicated that dogs after PDL show early onset (two to ten weeks) of diabetes, i.e. blunted early phase insulin secretion, 2 the mechanism of GIP secretion as an insulinotropic enterohormone remains intact after PDL if sufficient stimulants are given. / Surgery, Department of / Medicine, Faculty of / Graduate

Glicose e oxigênio desempenham um importante papel na regulação do metabolismo celular. Dada a importância de ambos no metabolismo - o primeiro como fonte de carbono preferencial da maior parte das células, e o segundo como aceptor final de elétrons na cadeia respiratória, em diversos organismos desenvolveram-se métodos adequados para detectar sua presença de modo a ajustar o metabolismo em função de sua disponibilidade. Neste trabalho foi realizado o estudo da expressão, no nível transcricional, dos genes envolvidos nas vias metabólicas primárias e genes envolvidos em morte celular, em células humanas, com o intuito de determinar as alterações no metabolismo energético em resposta a condições de hipóxia e anóxia, por meio da técnica de microarrays de cDNA. Utilizamos, inicialmente, células normais de fibroblasto humano ASl98 e células de fibroblasto humano MRC-5 imortalizadas por transfecção por SV40, e por fim células provenientes de ilhotas pancreáticas humanas, para a elaboração de um protocolo de cultura celular em que as mesmas crescessem aderidas a microcarregadores Cytodex I. Numa segunda etapa, células de ilhotas pancreáticas humanas foram cultivadas em suspensão, aderidas aos microcarregadores, num biorreator, sendo então realizada a análise do perfil transcricional dos genes escolhidos, frente às condições de baixa tensão de oxigênio. É apresentada a análise da expressão gênica de aproximadamente 160 genes na qual foram verificados um comportamento de indução daqueles envolvidos no metabolismo de lipídios e alguns na morte celular e um comportamento inicial de indução, e posterior inibição, do metabolismo primário como um todo. Em vista dos dados obtidos é de interesse ressaltar que essas células deveriam ser mantidas em saturações de oxigênio acima de 5% para evitar o efeito deletério observado na baixa concentração de oxigênio sobre a viabilidade celular, em termos da indução de alguns genes envolvidos na morte celular e da repressão geral dos relacionados ao metabolismo energético. Também foi verificado que, em saturações de oxigênio de até 10%, as células adotaram um padrão transcricional que indicou uma resposta ao estresse por falta de oxigênio, este por sua vez reflete-se na viabilidade celular, característica crucial para o sucesso do transplante clínico de ilhotas. / Glucose and oxygen have important roles on the regulation of cellular metabolism. Due to their importance in metabolism, the former as the preferential carbon source and the later as the final electron acceptor of the respiratory chain, many organisms have developed suitable processes to detect their presence in order to adjust the cellular metabolism to their availability. In this work, we have studied, in human cells and at the transcriptional level, the expression of genes involved in primary metabolism pathways and some of those related to cell death, aiming to resolve alterations in the energetic metabolism as a response to hypoxic and anoxic conditions, by means of cDNA microarrays. We initially used AS198 human fibroblastic normal cells and MRC-5 human fibroblastic cells immortalized by SV40, and later on cells from human pancreatic islets, to develop a cell culture protocol in which they would grow on the surface of Cytodex 1 microcarriers. As a second step, cells from human pancreatic islets were cultured on microcarriers in suspension inside a bioreactor. This culture was then used to carry out the transcriptional profile analysis of selected genes in response to low levels of oxygen. This work presents the analysis of gene expression of approximately 160 genes that can be divided into two distinct groups. The first group, the expression of which is induced, comprises genes involved in lipid metabolism and some of those related to cell death. The expression of the second group, consisting of diverse genes of the primary metabolism, suffers an initial induction followed by repression. Given the data acquired it is interesting to note that the human pancreatic islets should be maintained under at least 5% dissolved oxygen to avoid the deleterious effects on cell viability observed at lower oxygen concentrations, resulting in the induction of some genes involved in cell death and the repression of those related to energetic metabolism. It was also verified that, under oxygen saturation of at least 10%, these cells adopted a transcriptional profile that indicated a response to the stress created by the lack of oxygen, which would in turn reflect on cell viability, a crucial characteristic for success in clinical islet transplantation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21991号 / 医博第4505号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武田 俊一, 教授 坂井 義治, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / Background: Carcinoembryonic antigen (CEA) in the pancreatic cystic fluid is the most important biomarker for differentiating mucinous from non-mucinous pancreatic cystic lesions (PCLs). However, recent studies have shown that glucose levels in pancreatic cystic fluid can discriminate mucinous from non-mucinous cysts. Aims: To perform a meta-analysis to determine the utility of intracystic fluid glucose of pancreatic mucinous cysts compared with intracystic CEA. Methods: We conducted a systematic review of the literature in the PubMed, OVID Medline, and Cochrane databases. This meta-analysis considers studies published up to October 2020. Results: Six studies comprising 506 patients were selected; 61.2% of the population was female. Of the 480 PCLs, 287 (59.7%) were mucinous. Pooled sensitivity and specificity of cystic fluid glucose levels for mucinous PCLs were 91% and 85%, respectively. The positive likelihood ratio (PLR) and negative likelihood ratio (NLR) were 6.33 and 0.11, respectively. Pooled diagnostic odds ratio (DOR) was 60.94. The pooled area under the summary receiver operating characteristic (SROC) curve was 0.959. Pooled sensitivity and specificity of pancreatic cystic fluid CEA levels were 61% and 93%. The PLR and NLR were 8.51 and 0.40, respectively. Pooled DOR was 23.52, and the pooled area under the SROC curve was 0.861. Conclusion: Glucose has become a useful method and appears to be better than CEA for differentiating between mucinous PCLs and non-mucinous PCLs. We suggest that the analysis of glucose in PCLs be routinely performed for the differential diagnosis of these lesions. / Revisión por pares

Expression of the Receptor for Advanced Glycation End Products (RAGE) and is upregulated in a several cancers. Based on published studies, we hypothesized that RAGE, when overexpressed in pancreatic cancer cells, will promote cell proliferation and migration. To study the role of RAGE in pancreatic cancer, we selected the human pancreatic cancer cell-line PANC-1, and stably transfected the cells with full length RAGE to generate model cell-lines that overexpress RAGE. We obtained two cell-lines PANC-1 FLR2 and PANC-1 FLR3 and examined the influence of RAGE on cellular properties. A significant increase in proliferation but a reduction in migratory abilities of PANC-1 FLR2 and PANC-1 FLR3 cells was observed. The increase in proliferation and reduction in migration was reverted upon knockdown of RAGE in PANC-1 FLR2 cells with siRNA specific for RAGE. The reduction in migration was supported by the reduced levels of vimentin and several integrins in RAGE transfected cells. Furthermore, we observed a downregulation in FAK, AKT, ERK1/2 and NF-κB activity. Growing evidence supports that RAGE is essential for pancreatic cancer progression. It has also been shown that RAGE facilitates pancreatic tumor cell survival by enhancing autophagy and inhibiting apoptosis. The goal of our study was to determine the effect of RAGE inhibition during gemcitabine chemotherapy on the growth of pancreatic tumor. Hence, we investigated the effect of RAGE inhibitors and their combination with gemcitabine in an orthotopic mouse model of pancreatic cancer using mouse pancreatic cancer cell-line KPC 5508. We used two RAGE inhibitors, an anti-RAGE monoclonal antibody (IgG2A11) and a small molecule RAGE inhibitor (FPS-ZM1). We observed a significant reduction in tumor weights of the mice treated with the combination of IgG2A11 and gemcitabine as compared to gemcitabine alone treated mice. The reduction in tumor growth was accompanied with increase in p62 levels (marker of autophagy) and increase in levels of cleaved PARP (marker of apoptosis). We also observed reduction in HMGB1 and phosphorylation levels of ERK1/2 in tumors from the group treated with the combination as compared to the gemcitabine alone treated group. / North Dakota State University. College of Health Professions / NIH Grant # P20 GM109024 from the National Institute of General Medicine

Spleen tyrosine kinase (SYK) regulates mitogenic signaling, inflammatory responses and cell fate in a number of diverse cell types. KRAS is a proto-oncogene that controls cell growth and proliferation through several mitogenic pathways. In pancreatic cancer, KRAS is frequently mutated, resulting in constitutive activation in 90% of pancreatic cancer cell lines. We previously showed that SYK is highly expressed in a subset of KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cell lines. We demonstrated that SYK kinase inhibition with PRT062607 (SYKi) causes decreased cell proliferation of PDAC cell lines. Furthermore, combined SYKi and MEK inhibitor (MEKi) treatment promotes additive effects on suppression of PDAC cell proliferation and clonogenic growth. Mechanistically, SYK activates the mTORC1 kinase complex as shown by reduced phosphorylation of ribosomal S6 protein and its upstream kinase p70 S6 kinase (p70S6K) following SYKi treatment in PDAC cell lines. SYK-mediated mTORC1 activation occurs independently of MEK/ERK and PI3K/AKT effector signaling pathways. The mTORC1 complex suppresses lysosome biogenesis and macroautophagy (autophagy). Consequently, mTORC1 suppression via SYK inhibition or shRNA-mediated depletion causes accumulation of autolysosomes. These effects are mediated by the enhanced nuclear localization of MITF, a key transcriptional regulator of genes involved in lysosome biogenesis and autophagy pathway activation. In summary, SYK positively regulates mTORC1 activation in a subset of PDAC cell lines to suppress hyperactivation of autophagy. These findings open new avenues for further exploration of SYK as a critical regulator of the autophagy pathway in KRAS/mTORC1-dependent PDAC, and how this may be exploited for therapeutic benefit.