Genomic sequencing of pancreatic ductal adenocarcinoma (PDAC) tumours has highlighted the existence of wide genetic diversity alongside frequent mutations in KRAS, TP53 and SMAD4. Within this heterogeneity many components of the epigenetic machinery are mutated, including the histone H3 lysine 4 methyltransferases KMT2C and KMT2D, which are frequently subject to mutation and can identify patients with a more favorable prognosis. In this thesis low expression of KMT2C and KMT2D were shown to also define better outcome groups, with median survivals of 15.9 vs 9.2 months (p = 0.029), and 19.9 vs 11.8 months (p = 0.001) respectively. Experiments across eight human pancreatic cell lines following their depletion suggest that this improved outcome may be due to attenuated cell proliferation, with decreased progression of cells from G0/G1 observed upon KMT2D loss. Whole transcriptome analysis of PDAC cell lines following KMT2C or KMT2D knockdown identified 31 and 124 differentially expressed genes respectively, with 19 common to both. Gene set enrichment analysis revealed a significant downregulation of genes relating to cell-cycle pathways, confirmed by interrogation of the International Cancer Genome Consortium and The Cancer Genome Atlas PDAC data series. Furthermore, these experiments highlighted a potential role for NCAPD3, a subunit of the condensin II complex, as a PDAC outcome predictor across four patient gene expression series. Alongside this, Kmt2d depletion in cells derived from murine models of pancreatic cancer led to an increase in their response to the antimetabolites 5-fluorouracil and gemcitabine. Taken together, the studies herein suggest that lower levels of this methyltransferase may mediate the sensitivity of PDAC patients to particular treatments. Altogether, these data suggest a potential therapeutic benefit in targeting these methyltransferases within PDAC, especially in those patients that demonstrate higher KTM2C/D expression.

No description available.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Metastatic, treatment‐resistant pancreatic ductal adenocarcinoma (PDAC) is a rapidly fatal disease that typically carries a bleak prognosis. Contrast‐enhanced CT is the current standard of care tool for imaging evaluation, and repeat imaging is routinely performed in clinical trials. The availability of these imaging data render them exploitable for further analysis. CT texural analysis (CTTA), a quantitative tool for examining a region of interest on CT and generating statistical parameters based on gray‐level pixel data, is powerful technique that has been studied in other cancers and shown to correlate with features such as tumor grade, stage, and prognosis. However, the application of CTTA to PDAC has not been studied. Given the paucity of diagnostic tests to guide therapy, validated CTTA biomarkers could be immensely useful. Identifying PDAC variants that have a relative deficit in DNA repair might allow these cancers to be treated with targeted cytotoxic regimens sooner. Additionally, identifying prognostic CTTA parameters would be useful in gauging the severity of disease. We sought to perform quantitative textural analysis on CT imaging from a clinical trial cohort of patients with metastatic, treatment‐resistant PDAC. We aimed to correlate CTTA features to molecular profiling results (copy number variations obtained by array CGH) and clinical features (overall survival). Metastatic tumor sites from patients with treatment‐resistant PDAC were biopsied and molecularly profiled. Intrachromosal copy number were assessed by CGH in tumor specimens, and patients were treated based on these individual molecular profiling results. Pre‐biopsy portal‐venous phase and non‐contrast CT scans were obtained for retrospective analysis (n=15). CTTA was performed by drawing regions of interest around the primary pancreas adenocarcinoma and the normal pancreas tissue. CTTA parameters including mean positive pixels, entropy, kurtosis, and skewness were derived using the TexRAD platform at texture filtering densities of 0, 2, 3, 4, 5, and 6 pixels. CTTA values were then compared to intrachromosomal copy number variation (CNV) per tumor and overall survival (OS) post treatment using a Spearman’s rank correlation coefficient. Additional linear regression analysis was performed for positive correlations, and a Kaplan‐Meier statistic was generated for OS using median CTTA entropy. Multivariate analyses for CNV and OS were also performed. CNV were negatively correlated with the kurtosis value of the primary tumor mass using medium texture filtering (p=0.034, n=15). Linear regression revealed a significant negative correlation between kurtosis and CNV (p=0.038). Secondary analysis of the normal pancreas using coarse texture filtering revealed that increasing entropy was associated with decreased OS (p=0.0014, n=12). Using median entropy as a cutoff value (median: 4.165), median OS was greater in the entropy < 4.165 group versus the entropy > 4.165 group (179 days v 43 days; 95% CI 73.137 – 166.87; p=0.004, n=12). This exploratory study with admittedly limited sample size raises interesting questions about the use of CTTA parameters as diagnostic tools and/or biopsy adjuncts in assessing PDAC susceptibility to commercially available cytotoxics. Secondarily, entropy, a potential marker of heterogeneity and inflammation in the normal pancreas, represents an intriguing possibility for gauging prognosis.

Background: The systemic immune-inflammation index based on peripheral neutrophil, lymphocyte, and platelet counts has shown a prognostic impact in several malignancies. The aim of this study was to determine the prognostic role of systemic immune-inflammation index in patients with pancreatic ductal adenocarcinoma undergoing resection. Methods: Consecutive patients who underwent surgical resection at the department of surgery at the Medical University of Vienna between 1995 and 2014 were included into this study. The systemic immune-inflammation index was calculated by the formula platelet*neutrophil/lymphocyte. Optimal cutoffs were determined using Youden's index. Uni-and multivariate analyses were calculated by the Cox proportional hazard regression model for overall survival. Results Three hundred twenty-one patients were included in this study. Clinical data was achieved from a prospective patient database. In univariate survival analysis, elevated systemic immune-inflammation index was found to be significantly associated with shortened patients' overall survival (p = 0.007). In multivariate survival analysis, systemic immune-inflammation index remained an independent prognostic factor for overall survival (p = 0.004). No statistical significance could be found for platelet to lymphocyte ratio and neutrophil to lymphocyte ratio in multivariate analysis. Furthermore, area under the curve analysis showed a higher prognostic significance for systemic immune-inflammation index, compared to platelet to lymphocyte ratio and neutrophil to lymphocyte ratio. Conclusion A high systemic immune-inflammation index is an independent, preoperative available prognostic factor in patients with resectable pancreatic ductal adenocarcinoma and is superior to platelet to lymphocyte ratio and neutrophil to lymphocyte ratio for predicting overall survival in pancreatic ductal adenocarcinoma patients.

Pancreatic ductal adenocarcinoma (PDAC) is characterised by an intense desmoplastic stromal response that can comprise 60 to 80% of tumour volume and has been implicated to be a factor in promoting tumour invasiveness and the poor prognosis associated with this cancer type. It is now well established that pancreatic stellate cells, which are vitamin A storing cells found in the periacinar spaces of the stroma in the normal gland, are primarily responsible for this desmoplastic reaction. Studying the interaction between stellate cells and cancer cells could provide for a better understanding of the disease process. During the evolution of PDAC, the stromal proportion increases from 4% in the normal gland to up to 80%. We hypothesised that there is an optimal proportion of stellate cells and cancer cells that modulates tumour behaviour and we attempted to dissect out this probable ‘tipping point’ for stromal composition upon cancer cell behaviour using a well-established in vitro organotypic culture model of pancreatic cancer. The cancer cell-stromal cell interaction led to extra-cellular matrix contraction and stiffening; and an increase in cancer cell number. The stromal stellate cells conferred a pro-survival and pro-invasive effect on cancer cells which was most pronounced at a stellate cell proportion of 0.66-0.83. The expression of key molecules involved in EMT and metastasis such as E-Cadherin and β-catenin showed a reduction and this was found to be most significant again at a stellate cell proportion of 0.66-0.83. Stellate cells altered the genetic profile of cancer cells leading to differential expression of genes involved in key cellular pathways such as cell-cycle and proliferation, cell movement and death, cell-cell signalling, and inflammatory response. qRT-PCR confirmed the differential expression of the top differentially expressed genes and protein validation by immunofluorescence staining using PIGR as a candidate molecule confirmed the experimental findings in human PDAC specimens. This study demonstrates that the progressive accumulation of desmoplastic stromal cells has a tumour progressive (pro-survival, pro-invasive) effect on cancer cells in addition to stiffening (contraction) of the extracellular matrix (maximum effect when the stromal cell proportion is 60-80%). This is mediated through a number of signalling cascades and molecular targets. Dampening this tumour-promoting interaction between cancer and stromal cells by ‘multi-targeting’ agents may allow traditional chemo- and/or radiotherapy to be effective.

Introduction: Polymeric immunoglobulin receptor (pIgR) traffics Immunoglobulins (IgA and IgM) through epithelial cells in normal mucosae but neither are expressed in the normal pancreas. Recent work has demonstrated pIgR to be upregulated in hepatocellular carcinoma, even though it is not expressed in normal liver cells. High pIgR levels are associated with poor survival and distant metastases for a number of cancers such as nasopharyngeal cancers, lung and oesophageal cancers. Recent work from our laboratory suggested pIgR may be upregulated in pancreatic ductal adenocarcinoma (PDAC). My aim was to assess pIgR's role in PDAC by interrogating human PDAC tissue samples as well using cell biology experimental tools. Methods: pIgR expression was manipulated (siRNA and shRNA) in cell lines to evaluate its subsequent effect on cell behaviour in 2D assays as well as 3D organotypics models. Tissue Microarrays of patients with PDAC were analysed after pIgR, αSMA, E-Cadherin and Picrosirius Red staining to assess their role as a combined bio-marker panel. Results: Cytokines such as interleukin 4 (IL4) and Tumour Necrosis Factor (TNFα) could not modulate pIgR expression in PDAC cell lines despite this effect being seen in other studies using colorectal and nasopharyngeal cancer cell lines. Downregulation in pIgR expression in Capan1 cell line resulted in reduction of cellular proliferation (n= 3, P < 0.05, Friedman test), adhesion (n= 3, P < 0.05, Kruskal-Wallis) and migration (n= 3, P < 0.05, Kruskal-Wallis). In 3D organotypic models, pIgR downregulation resulted in reduced cancer cell invasion (n= 9, P < 0.05, Kruskal- Wallis) and diminished contraction of gels (n= 9, P < 0.05, Kruskal-Wallis). In human PDAC, decreased E-cadherin expression correlates with increased pIgR expression through pancreatic intra-epithelial neoplasia (PanIN) progression. There was no IgA expression in PDAC. pIgR expression had no clinical correlation with routine prognostic measures such as differentiation, lymph node metastasis (n= 88, P=0.5012, Kruskal-Wallis). Even in combination with stromal indices (α-smooth muscle action (SMA) and Picrosirius red), low pIgR scores had no statistically significant impact on prognosis but had a trend towards better survival (n= 88, P=0.2791, Mann-Whitney U test). Conclusion: pIgR may be involved in progression from pre-neoplastic lesions such as PanIN to PDAC. pIgR may have a biological impact on cellular motility and invasion due to yet to be deciphered signalling cascades with marked effect on cellular phenotype. Careful analysis is required to study the impact of pIgR on prognostic impact bearing in mind the histological sub-types of pancreatic cancer.

No description available.

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

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

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24523号 / 医博第4965号 / 新制||医||1065(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 川口 義弥, 教授 藤田 恭之, 教授 波多野 悦朗 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM