A systems approach identifies time-dependent associations of multimorbidities with pancreatic cancer risk.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is usually diagnosed in late adulthood; therefore, many patients suffer or have suffered from other diseases. Identifying disease patterns associated with PDAC risk may enable a better characterization of high-risk patients. METHODS: Multimorbidity patterns (MPs) were assessed from 17 self-reported conditions using hierarchical clustering, principal component, and factor analyses in 1705 PDAC cases and 1084 controls from a European population. Their association with PDAC was evaluated using adjusted logistic regression models. Time since diagnosis of morbidities to PDAC diagnosis/recruitment was stratified into recent (<3 years) and long term (≥3 years). The MPs and PDAC genetic networks were explored with DisGeNET bioinformatics-tool which focuses on gene-diseases associations available in curated databases. RESULTS: Three MPs were observed: gastric (heartburn, acid regurgitation, Helicobacter pylori infection, and ulcer), metabolic syndrome (obesity, type-2 diabetes, hypercholesterolemia, and hypertension), and atopic (nasal allergies, skin allergies, and asthma). Strong associations with PDAC were observed for ≥2 recently diagnosed gastric conditions [odds ratio (OR), 6.13; 95% confidence interval CI 3.01-12.5)] and for ≥3 recently diagnosed metabolic syndrome conditions (OR, 1.61; 95% CI 1.11-2.35). Atopic conditions were negatively associated with PDAC (high adherence score OR for tertile III, 0.45; 95% CI, 0.36-0.55). Combining type-2 diabetes with gastric MP resulted in higher PDAC risk for recent (OR, 7.89; 95% CI 3.9-16.1) and long-term diagnosed conditions (OR, 1.86; 95% CI 1.29-2.67). A common genetic basis between MPs and PDAC was observed in the bioinformatics analysis. CONCLUSIONS: Specific multimorbidities aggregate and associate with PDAC in a time-dependent manner. A better characterization of a high-risk population for PDAC may help in the early diagnosis of this cancer. The common genetic basis between MP and PDAC points to a mechanistic link between these conditions.

S100PBP is regulated by mutated KRAS and plays a tumour suppressor role in pancreatic cancer.

We have previously shown that expression of S100PBP, an S100P binding partner, gradually decreases during progression of pancreatic ductal adenocarcinomas (PDAC). Here, we show that loss of S100PBP leads to oncogenic transformation of pancreatic cells; after deregulation of S100PBP expression, both in silico and in vitro analyses highlighted alterations of genes known to modulate cytoskeleton, cell motility and survival. Overexpression of S100P reduced S100PBP expression, while co-immunoprecipitation indicated the interaction of S100P with S100PBP-p53-ubiquitin protein complex, likely causing S100PBP degradation. The doxycycline-induced KrasG12D activation resulted in decreased S100PBP levels, while low-dose treatment with HDAC inhibitor MS-275 rescued its expression in both human and mouse PDAC cell lines. This indicates KrasG12D as an upstream epigenetic regulator of S100PBP. Finally, analysis of TCGA PanCancer Atlas PDAC datasets demonstrated poor prognosis in patients with high S100P and low S100PBP expression, suggesting that S100PBP is a novel tumour suppressor gene with potential clinical utility.

Antibody combination therapy targeting CD25, CD70 and CD8 reduces islet inflammation and improves glycaemia in diabetic mice.

Destruction of pancreatic islets in type 1 diabetes is caused by infiltrating, primed and activated T cells. In a clinical setting this autoimmune process is already in an advanced stage before intervention therapy can be administered. Therefore, an effective intervention needs to reduce islet inflammation and preserve any remaining islet function. In this study we have investigated the role of targeting activated T cells in reversing autoimmune diabetes. A combination therapy consisting of CD25-, CD70- and CD8-specific monoclonal antibodies was administered to non-obese diabetic (NOD) mice with either new-onset diabetes or with advanced diabetes. In NOD mice with new-onset diabetes antibody combination treatment reversed hyperglycaemia and achieved long-term protection from diabetes (blood glucose <13·9 mmol/l) in >50% of mice. In contrast, in the control, untreated group blood glucose levels continued to increase and none of the mice were protected from diabetes (P < 0·0001). Starting therapy early when hyperglycaemia was relatively mild proved critical, as the mice with advanced diabetes showed less efficient control of blood glucose and shorter life span. Histological analysis (insulitis score) showed islet preservation and reduced immune infiltration in all treated groups, compared to their controls. In conclusion, antibody combination therapy that targets CD25, CD70 and CD8 results in decreased islet infiltration and improved blood glucose levels in NOD mice with established diabetes.

Gallbladder disease and pancreatic cancer risk: a multicentric case-control European study.

BACKGROUND AND AIMS: The overall evidence on the association between gallbladder conditions (GBC: gallstones and cholecystectomy) and pancreatic cancer (PC) is inconsistent. To our knowledge, no previous investigations considered the role of tumour characteristics on this association. Thus, we aimed to assess the association between self-reported GBC and PC risk, by focussing on timing to PC diagnosis and tumour features (stage, location, and resection). METHODS: Data derived from a European case-control study conducted between 2009 and 2014 including 1431 PC cases and 1090 controls. We used unconditional logistic regression models to estimate odds ratios (ORs) and corresponding 95% confidence intervals (CIs) adjusted for recognized confounders. RESULTS: Overall, 298 (20.8%) cases and 127 (11.6%) controls reported to have had GBC, corresponding to an OR of 1.70 (95% CI 1.33-2.16). The ORs were 4.84 (95% CI 2.96-7.89) for GBC diagnosed <3 years before PC and 1.06 (95% CI 0.79-1.41) for ≥3 years. The risk was slightly higher for stage I/II (OR = 1.71, 95% CI 1.15-2.55) vs. stage III/IV tumours (OR = 1.23, 95% CI 0.87-1.76); for tumours sited in the head of the pancreas (OR = 1.59, 95% CI 1.13-2.24) vs. tumours located at the body/tail (OR = 1.02, 95% CI 0.62-1.68); and for tumours surgically resected (OR = 1.69, 95% CI 1.14-2.51) vs. non-resected tumours (OR = 1.25, 95% CI 0.88-1.78). The corresponding ORs for GBC diagnosed ≥3 years prior PC were close to unity. CONCLUSION: Our study supports the association between GBC and PC. Given the time-risk pattern observed, however, this relationship may be non-causal and, partly or largely, due to diagnostic attention and/or reverse causation.

Sperm-associated antigen 1 is expressed early in pancreatic tumorigenesis and promotes motility of cancer cells.

Sperm-associated antigen 1 (SPAG1) was recently identified in a rare form of infertility where anti-SPAG1 antibodies derived from the serum of an infertile woman were reported to cause sperm agglutination. Except for its expression and potential role in spermatogenesis, the function of SPAG1 is completely unknown. The unexpected finding of high levels of SPAG1 expression in pancreatic adenocarcinoma compared to normal pancreatic tissue in our previous cDNA array experiments prompted us to look in more detail at the expression and role of this gene in a panel of normal and malignant human tissues as well as in a larger series of pancreatic cancer specimens. We have generated an SPAG1-specific monoclonal antibody and showed high levels of SPAG1 protein in testis and in a large proportion of pancreatic ductal adenocarcinomas (PDAC). In the latter, SPAG1 expression was predominantly cytoplasmic and confined to malignant cells. Furthermore, the extent and intensity of SPAG1 expression was shown to be associated with stage and tumour nodal status, while analysis of precursor lesions, pancreatic intraepithelial neoplasias (PanINs), demonstrated its increased immunoreactivity with increasing PanIN grade, suggesting that SPAG1 is a novel marker of PDAC progression. Immunocytochemical analysis demonstrated colocalization of SPAG1 with microtubules, and their association was confirmed by co-immunoprecipitation; subsequent motility assays further substantiated a potential role of SPAG1 in cancer cell motility. Combined with the finding of its early expression in PDAC development, our data suggest that SPAG1 could contribute to the early spread and poor prognosis of pancreatic adenocarcinoma.

Periostin promotes invasiveness and resistance of pancreatic cancer cells to hypoxia-induced cell death: role of the beta4 integrin and the PI3k pathway.

Pancreatic ductal adenocarcinoma is a devastating disease, characterized by a rapid progression and poor treatment response. Using gene expression profiling of pancreatic cancer tissues, we previously identified periostin as a potential diagnostic and therapeutic target. In this study, we report the overexpression of periostin in a larger set of pancreatic cancer tissues and show that although the periostin transcript is exclusively expressed in tumour cells, the protein product is only detected in the extracellular matrix adjacent to cancer cells. Using an enzyme-linked immunosorbent assay (ELISA) assay, we show significantly increased levels of periostin in the sera of pancreatic cancer patients compared to non-cancer controls. We demonstrate that periostin promotes the invasiveness of tumour cells by increasing the motility of cells without inducing expression of proteases, and enhances the survival of tumour cells exposed to hypoxic conditions. At the molecular level, we provide evidence that the alpha(6)beta(4) integrin complex acts as the cell receptor of periostin in pancreatic cancer cells and that interaction promotes phosphorylation of focal adhesion kinase (FAK) and protein kinase B (AKT) though activation of the PI3 kinase pathway, but not the RAS/MEK/ERK pathway. These findings suggest an important role of periostin in pancreatic cancer and provide a rationale to study periostin for diagnostic and therapeutic applications.

Risk of pancreatic cancer associated with family history of cancer and other medical conditions by accounting for smoking among relatives.

Background: Family history (FH) of pancreatic cancer (PC) has been associated with an increased risk of PC, but little is known regarding the role of inherited/environmental factors or that of FH of other comorbidities in PC risk. We aimed to address these issues using multiple methodological approaches. Methods: Case-control study including 1431 PC cases and 1090 controls and a reconstructed-cohort study (N = 16 747) made up of their first-degree relatives (FDR). Logistic regression was used to evaluate PC risk associated with FH of cancer, diabetes, allergies, asthma, cystic fibrosis and chronic pancreatitis by relative type and number of affected relatives, by smoking status and other potential effect modifiers, and by tumour stage and location. Familial aggregation of cancer was assessed within the cohort using Cox proportional hazard regression. Results: FH of PC was associated with an increased PC risk [odds ratio (OR) = 2.68; 95% confidence interval (CI): 2.27-4.06] when compared with cancer-free FH, the risk being greater when ≥ 2 FDRs suffered PC (OR = 3.88; 95% CI: 2.96-9.73) and among current smokers (OR = 3.16; 95% CI: 2.56-5.78, interaction FHPC*smoking P-value = 0.04). PC cumulative risk by age 75 was 2.2% among FDRs of cases and 0.7% in those of controls [hazard ratio (HR) = 2.42; 95% CI: 2.16-2.71]. PC risk was significantly associated with FH of cancer (OR = 1.30; 95% CI: 1.13-1.54) and diabetes (OR = 1.24; 95% CI: 1.01-1.52), but not with FH of other diseases. Conclusions: The concordant findings using both approaches strengthen the notion that FH of cancer, PC or diabetes confers a higher PC risk. Smoking notably increases PC risk associated with FH of PC. Further evaluation of these associations should be undertaken to guide PC prevention strategies.

ER stress protein AGR2 precedes and is involved in the regulation of pancreatic cancer initiation.

The mechanisms of initiation of pancreatic ductal adenocarcinoma (PDAC) are still largely unknown. In the present study, we analysed the role of anterior gradient-2 (AGR2) in the earliest stages of pancreatic neoplasia. Immunohistochemical analysis of chronic pancreatitis (CP) and peritumoral areas in PDAC tissues showed that AGR2 was present in tubular complexes (TC) and early pancreatic intraepithelial neoplasia (PanINs). Moreover, AGR2 was also found in discrete subpopulations of non-transformed cells neighbouring these pre-neoplastic lesions. In primary cells derived from human patient-derived xenograft (PDX) model, flow-cytometry revealed that AGR2 was overexpressed in pancreatic cancer stem cells (CSC) compared with non-stem cancer cells. In LSL-KrasG12D;Pdx1-Cre (KC) mouse model Agr2 induction preceded the formation of pre-neoplastic lesions and their development was largely inhibited by Agr2 deletion in engineered LSL-KrasG12D;Pdx1-Cre; Agr2-/- mice. In vitro, AGR2 expression was stimulated by tunicamycin-induced endoplasmic reticulum (ER) stress in both KRAS wild-type normal pancreas cells, as well as in KRAS mutated pancreatic cancer cells and was essential for ER homoeostasis. The unfolded protein response proteins GRP78, ATF6 and XBP1s were found expressed in CP and PDAC peritumoral tissues, but in contrast to AGR2, their expression was switched off during TC and PanIN formation. Real-time PCR and ELISA analyses showed that ER stress induced a pro-inflammatory phenotype in pancreatic normal, cancer and stellate cells. Moreover, AGR2 expression was inducible by paracrine transfer of ER stress and pro-inflammation between different pancreatic cell types. Our findings demonstrate that AGR2 induced in ER-stressed and inflammatory pre-neoplastic pancreas is a potential marker of cancer progenitor cells with an important functional role in PDAC initiation.

Gene expression profiles of progressive pancreatic endocrine tumours and their liver metastases reveal potential novel markers and therapeutic targets.

The intrinsic nature of tumour behaviour (stable vs progressive) and the presence of liver metastases are key factors in determining the outcome of patients with a pancreatic endocrine tumour (PET). Previous expression profile analyses of PETs were limited to non-homogeneous groups or to primary lesions only. The aim of this study was to investigate the gene expression profiles of a more uniform series of sporadic, non-functioning (NF) PETs with progressive disease and, for the first time, their liver metastases, on the Affymetrix human genome U133A and B GeneChip set. Thirteen NF PET samples (eight primaries and five liver metastases) from ten patients with progressive, metastatic disease, three cell lines (BON, QGP and CM) and four purified islet samples were analysed. The same samples were employed for confirmation of candidate gene expression by means of quantitative RT-PCR, while a further 37 PET and 15 carcinoid samples were analysed by immunohistochemistry. Analysis of genes differentially expressed between islets and primaries and metastases revealed 667 up- and 223 down-regulated genes, most of which have not previously been observed in PETs, and whose gene ontology molecular function has been detailed. Overexpression of bridging integrator 1 (BIN1) and protein Z dependent protease inhibitor (SERPINA10) which may represent useful biomarkers, and of lymphocyte specific protein tyrosine kinase (LCK) and bone marrow stromal cell antigen (BST2) which could be used as therapeutic targets, has been validated. When primary tumours were compared with metastatic lesions, no significantly differentially expressed genes were found, in accord with cluster analysis which revealed a striking similarity between primary and metastatic lesions, with the cell lines clustering separately. We have provided a comprehensive list of differentially expressed genes in a uniform set of aggressive NF PETs. A number of dysregulated genes deserve further in-depth study as potentially promising candidates for new diagnostic and treatment strategies. The analysis of liver metastases revealed a previously unknown high level of similarity with the primary lesions.

Gene expression profiles of pancreatic cancer and stromal desmoplasia.

Gene expression studies were undertaken in normal pancreas and pancreatic adenocarcinomas to determine new candidate genes that can potentially be used as markers of the disease. The characteristic desmoplastic stromal reaction of pancreatic adenocarcinoma greatly hampers expression studies in this tumour type, and usually necessitates time-consuming tissue microdissection for enrichment of the tumour cell population. We show that fine needle aspiration of cancer provides a fast and efficient way of obtaining samples highly enriched in tumour cells with sufficient yields of RNA. Using Atlas cancer cDNA arrays with 588 cancer-related genes, we describe gene expression profiles of normal pancreas, bulk pancreatic tumour tissues and pancreatic tumour aspirates containing more than 95% tumour cells. Analysis of bulk tissue specimens revealed differentially expressed genes belonging predominantly to the stromal component of the tumour. This contrasted with the results obtained from tumour-cell enriched samples. Several genes already described in pancreatic cancer (caspase 8, TIMP1, CD9, IL-13) were also differentially expressed in our study. Furthermore, we found dysregulated expression of genes not previously associated with pancreatic adenocarcinoma, such as Rac 1, GLG1, NEDD5, RPL-13a, RPS9 and members of the Wnt5A gene family. In summary, we present a panel of genes newly identified in the pathogenesis of pancreatic adenocarcinoma and demonstrate that fine needle aspirates of the tumour mass are a convenient source of material for gene expression studies in tumours accompanied by desmoplastic reactions.

Comparative analysis of the DRADA A-to-I RNA editing gene from mammals, pufferfish and zebrafish.

The DRADA gene in mammals encodes an A-to-I RNA editase, an adenosine deaminase that acts on pre-mRNAs to produce site specific inosines. DRADA has been shown to deaminate specific adenosine residues in a subset of glutamate and serotonin receptors, and this editing results in proteins of altered sequences and functional properties. DRADA thus plays a role in creating protein diversity. To study the evolutionary significance of this gene, we have characterized the genomic structure of DRADA from Fugu rubripes, and compared the protein sequences of DRADA from mammals, pufferfish and zebrafish. The DRADA gene from Fugu is three-fold compacted with respect to the human gene, and contains a novel intron within the large second coding exon. DRADA cDNAs were isolated from zebrafish and a second pufferfish, Tetraodon fluviatilis. Comparisons among fish, and between fish and mammals, of the protein sequences show that the catalytic domains are highly conserved for each gene, while the RNA binding domains vary within a single protein in their levels of conservation. Conservation within the Z DNA binding domain has also been assessed. Different levels of conservation among domains of different functional roles may reflect differences in editase substrate specificity and/or substrate sequence conservation.

Analysis of pufferfish homologues of the AT-rich human APP gene.

Mutations in the beta-amyloid precursor protein (APP) gene are associated with some forms of Familial Alzheimer's Disease. The human APP gene is large, the 19 exons span approximately 300 kb, and AT-rich, at 40% GC. We have examined the genomic structure and cDNA sequence of the APP gene in the pufferfish Fugu rubripes and Tetraodon fluviatilis, respectively. In contrast to human, the Fugu APP gene spans less than 10 kb of DNA, with the introns compacted 48-fold on average. Two axons, alternatively processed in humans, are absent in both pufferfish. APP is the largest, most AT-rich gene examined in Fugu and is also the most highly compressed. The genomic sequences spanning the human and the Fugu APP genes were analysed with a set of exon and gene prediction programs. Results show that these are highly reliable for the Fugu gene with lower false positive and false negative rates than are seen in the analysis of the human gene. Comparative analysis of Fugu sequences homologous to very AT-rich regions in the human genome may, therefore, be advantageous in gene-finding efforts, both for their highly reduced sizes and their reliable gene predictions.

Pancreatic cancer cells overexpress gelsolin family-capping proteins, which contribute to their cell motility.

BACKGROUND: Previously, proteomic methods were applied to characterise differentially expressed proteins in microdissected pancreatic ductal adenocarcinoma cells. AIMS: To report that CapG and a related protein, gelsolin, which have established roles in cell motility, are overexpressed in metastatic pancreatic cancer; and to describe their pattern of expression in pancreatic cancer tissue and their effect on cell motility in pancreatic cancer cell lines. METHODS: CapG was identified by mass spectrometry and immunoblotting. CapG and gelsolin expression was assessed by immunohistochemical analysis on a pancreatic cancer tissue microarray and correlated with clinical and pathological parameters. CapG and gelsolin levels were reduced using RNA interface in Suit-2, Panc-1 and MiaPaCa-2 cells. Cell motility was assessed using modified Boyden chamber or wound-healing assays. RESULTS: Multiple isoforms of CapG were detected in pancreatic cancer tissue and cell lines. Immunohistochemical analysis of benign (n = 44 patients) and malignant (n = 69) pancreatic ductal cells showed significantly higher CapG staining intensity in nuclear (p<0.001) and cytoplasmic (p<0.001) compartments of malignant cells. Similarly, gelsolin immunostaining of benign (n = 24 patients) and malignant (n = 68 patients) pancreatic ductal cells showed higher expression in both compartments (both p<0.001). High nuclear CapG was associated with increased tumour size (p = 0.001). High nuclear gelsolin was associated with reduced survival (p = 0.01). Reduction of CapG or gelsolin expression in cell lines by RNAi was accompanied by significantly impaired motility. CONCLUSIONS: Up regulation of these actin-capping proteins in pancreatic cancer and their ability to modulate cell motility in vitro suggest their potentially important role in pancreatic cancer cell motility and consequently dissemination.

Pancreatic cancer genetics.

Cancer is a multi-stage process resulting from accumulation of genetic changes in the somatic DNA of normal cells. Although in the majority of cases the changes occur only in the cancer cells there is a small proportion of cancers where a germline mutation confers an increased risk for cancer. Cancer susceptibility genes have effects that range from high to low penetrance with a corresponding high to lower likelihood for cancer in the carriers. Pancreatic cancer-prone families have been identified and some of the germline mutations responsible elucidated. Germline mutations in the BRCA2, CDKN2A/p16, hMSH2, hMLH1, hPMS1, hPMS2, LKB1/STK1, and PRSS1 genes have been associated with increased risk for pancreatic cancer. The concept of screening high-risk groups for pancreatic cancer is emerging, preferably in specialised centres with a multidisciplinary team approach.

Laser-assisted microdissection: applications in molecular pathology.

Tissue microdissection is potentially one of the most useful techniques in molecular pathology. Laser-assisted microdissection has been developed to procure precisely the cells of interest in a tissue specimen, in a rapid and practical manner. Together with multiplex molecular approaches, it is now feasible to study genetic alterations and isolate genes and proteins in defined cell populations from complex normal and diseased tissues. The fundamental advantage of this technique is the possibility of capturing single cells from which high-quality DNA and mRNA can be isolated for analysis of sequence and quantitation of expression. Moreover, the integration of laser-assisted microdissection and proteomic analysis could identify novel protein markers for disease. The advent of laser-assisted microdissection is likely to have a profound impact on molecular pathology.

Tetraodon fluviatilis, a new puffer fish model for genome studies.

The puffer fish Fugu rubripes rubripes was recently introduced by S. Brenner et al. (1993, Nature 366: 265-268) as a new model for genomic studies. Due to difficulties in obtaining material from this Japanese marine puffer, we have started work on Tetraodon fluviatilis, a small, freshwater puffer fish that can be kept and bred in an aquarium. It was originally described by E. Hinegardner (1968, Am. Nat. 102(928) 517-523) as the teleost with the smallest amount of DNA per cell (0.4 pg, 380 Mb). To estimate the extent of divergence between T. fluviatilis and F. r. rubripes, part of the mitochondrial cytochrome b (cyt b) gene from both fishes was cloned and sequenced. A comparison of these two sequences indicated that F.r. rubripes and T. fluviatilis diverged approximately 18-30 million years ago, and phylogenetic analysis placed both fishes at the base of the Perciformes lineage. To facilitate and extend further the use of the puffer fish as a model for genome studies, we have constructed and characterized a T. fluviatilis cDNA library.