Francesca Tavano

Mirna Expression Profiles Identify Drivers in Colorectal and Pancreatic Cancers

Background and Aim Altered expression of microRNAs (miRNAs) hallmarks many cancer types. The study of the associations of miRNA expression profile and cancer phenotype could help identify the links between deregulation of miRNA expression and oncogenic pathways. Methods Expression profiling of 866 human miRNAs in 19 colorectal and 17 pancreatic cancers and in matched adjacent normal tissues was investigated. Classical paired t-test and random forest analyses were applied to identify miRNAs associated with tissue-specific tumors. Network analysis based on a computational approach to mine associations between cancer types and miRNAs was performed. Results The merge between the two statistical methods used to intersect the miRNAs differentially expressed in colon and pancreatic cancers allowed the identification of cancer-specific miRNA alterations. By miRNA-network analysis, tissue-specific patterns of miRNA deregulation were traced: the driving miRNAs were miR-195, miR-1280, miR-140-3p and miR-1246 in colorectal tumors, and miR-103, miR-23a and miR-15b in pancreatic cancers. Conclusion MiRNA expression profiles may identify cancer-specific signatures and potentially useful biomarkers for the diagnosis of tissue specific cancers. miRNA-network analysis help identify altered miRNA regulatory networks that could play a role in tumor pathogenesis.

Common variation at 2p13.3, 3q29, 7p13 and 17q25.1 associated with susceptibility to pancreatic cancer.

Pancreatic cancer is the fourth leading cause of cancer death in the developed world. Both inherited high-penetrance mutations in BRCA2 (ref. 2), ATM, PALB2 (ref. 4), BRCA1 (ref. 5), STK11 (ref. 6), CDKN2A and mismatch-repair genes and low-penetrance loci are associated with increased risk. To identify new risk loci, we performed a genome-wide association study on 9,925 pancreatic cancer cases and 11,569 controls, including 4,164 newly genotyped cases and 3,792 controls in 9 studies from North America, Central Europe and Australia. We identified three newly associated regions: 17q25.1 (LINC00673, rs11655237, odds ratio (OR) = 1.26, 95% confidence interval (CI) = 1.19–1.34, P = 1.42 × 10−14), 7p13 (SUGCT, rs17688601, OR = 0.88, 95% CI = 0.84–0.92, P = 1.41 × 10−8) and 3q29 (TP63, rs9854771, OR = 0.89, 95% CI = 0.85–0.93, P = 2.35 × 10−8). We detected significant association at 2p13.3 (ETAA1, rs1486134, OR = 1.14, 95% CI = 1.09–1.19, P = 3.36 × 10−9), a region with previous suggestive evidence in Han Chinese. We replicated previously reported associations at 9q34.2 (ABO), 13q22.1 (KLF5), 5p15.33 (TERT and CLPTM1), 13q12.2 (PDX1), 1q32.1 (NR5A2), 7q32.3 (LINC-PINT), 16q23.1 (BCAR1) and 22q12.1 (ZNRF3). Our study identifies new loci associated with pancreatic cancer risk.

Clock Gene Expression Levels and Relationship With Clinical and Pathological Features in Colorectal Cancer Patients

The clock gene machinery controls cellular metabolism, proliferation, and key functions, such as DNA damage recognition and repair. Dysfunction of the circadian clock is involved in tumorigenesis, and altered expression of some clock genes has been found in cancer patients. The aim of this study was to evaluate the expression levels of core clock genes in colorectal cancer (CRC). Quantitative real-time polymerase chain reaction (qPCR) was used to examine ARNTL1, CLOCK, PER1, PER2, PER3, CRY1, CRY2, Timeless (TIM), TIPIN, and CSNK1Ε expression levels in the tumor tissue and matched apparently healthy mucosa of CRC patients. In the tumor tissue of CRC patients, compared to their matched healthy mucosa, expression levels of ARNTL1 (p = .002), PER1 (p = .002), PER2 (p = .011), PER3 (p = .003), and CRY2 (p = .012) were lower, whereas the expression level of TIM (p = .044) was higher. No significant difference was observed in the expression levels of CLOCK (p = .778), CRY1 (p = .600), CSNK1Ε (p = .903), and TIPIN (p = .136). As to the clinical and pathological features, a significant association was found between low CRY1 expression levels in tumor mucosa and age (p = .026), and female sex (p = .005), whereas high CRY1 expression levels in tumor mucosa were associated with cancer location in the distal colon (p = .015). Moreover, high TIM mRNA levels in the tumor mucosa were prevalent whenever proximal lymph nodes were involved (p = .013) and associated with TNM stages III–IV (p = .005) and microsatellite instability (p = .015). Significantly poorer survival rates were evidenced for CRC patients with lower expression in the tumor tissue of PER1 (p = .010), PER3 (p = .010), and CSNKIE (p = .024). In conclusion, abnormal expression levels of core clock genes in CRC tissue may be related to the process of tumorigenesis and exert an influence on host/tumor interactions. (Author correspondence: g.mazzoccoli@tin.it)

Changes in miR-143 and miR-21 expression and clinicopathological correlations in pancreatic cancers.

Objectives Despite advances in clinical management of pancreatic cancer (PC), there is still room for improvement in early detection, diagnosis, and treatment strategies. The role of microRNAs (miRNAs) in tumor biology might pinpoint an alteration in expression of miRNAs as new diagnostic/prognostic biomarkers. Methods Expression levels of miR-143 and miR-21 and correlations with clinicopathological features were analyzed in 26 matched pairs of tumor and adjacent noncancerous tissue samples collected from patients with PCs, including 18 pancreatic ductal adenocarcinomas (PDACs) and 8 adenocarcinomas of Vater’s papilla (PVACs). Results Compared to normal tissues, miR-143 was up-regulated in both PDAC and PVAC tumor samples (P = 0.0028 and P = 0.039, respectively). Conversely, alterations in miR-21 expression were significantly different in PDAC versus PVAC samples (P = 0.0049). Tumor levels of miR-21 were associated with preoperative serum levels of CA 19-9 (r = 0.63, P = 0.0022), whereas miR-143 expression was negatively correlated to lymph node spreading (r = −0.64; P = 0.0004). Correlation between miR-143 and miR-21 expression levels in patients with PDAC was observed (r = 0.53, P = 0.023). Conclusions Deregulation of miR-143 and miR-21 may reflect histological features and biological behavior of different PCs. Association data with clinical parameters might indicate a prognostic significance for miR-143 and miR-21 in PCs.

Expression of the Antiapoptotic Protein BAG3 Is a Feature of Pancreatic Adenocarcinoma and Its Overexpression Is Associated With Poorer Survival

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers, being the fourth leading cause of cancer-related deaths. Long-term survival reaching 15% is achieved in less than 5% of patients who undergo surgery, and median survival is only 6 months in those with inoperable lesions. A deeper understanding of PDAC biologic characteristics as well as novel prognostic markers are therefore required to improve outcomes. Herein we report that BAG3, a protein with recognized anti-apoptotic activity, was expressed in 346 PDACs analyzed, but was not expressed in the surrounding nonneoplastic tissue. In a cohort of 66 patients who underwent radical resection (R0), survival was significantly shorter in patients with high BAG3 expression (median, 12 months) than in those with low BAG3 expression (median, 23 months) (P = 0.001). Furthermore, we report that BAG3 expression in PDAC-derived cell lines protects from apoptosis and confers resistance to gemcitabine, offering a partial explanation for the survival data. Our results indicate that BAG3 has a relevant role in PDAC biology, and suggest that BAG3 expression level might be a potential marker for prediction of patient outcome.

Chemokine receptor CXCR4: Role in gastrointestinal cancer

Chemokines (CK)s, small proinflammatory chemoattractant cytokines that bind to specific G-protein coupled seven-span transmembrane receptors, are major regulators of cell trafficking and adhesion. The CXCL12 [stromal cell-derived factor-1 (SDF-1)] binds primarily to CXC receptor 4 (CXCR4; CD184). The binding of CXCL12 to CXCR4 induces intracellular signaling through several divergent pathways initiating signals related to chemotaxis, cell survival and/or proliferation, increase in intracellular calcium, and gene transcription. CXCR4 is expressed on multiple cell types including lymphocytes, hematopoietic stem cells, endothelial and epithelial cells, and cancer cells. One of the most intriguing and perhaps important roles that CKs and the CK receptors have is in regulating metastasis. Here, CK receptors may potentially facilitate tumor dissemination at each of the key steps of metastasis, including adherence of tumor cells to endothelium, extravasation from blood vessels, metastatic colonization, angiogenesis, proliferation, and protection from the host response via activation of key survival pathways such as ERK/MAPK, PI-3K/Akt/mTOR, or Jak/STAT, etc. In addition, it is increasingly recognized that CKs play an important role in facilitating communication between cancer cells and non-neoplatic cells in the tumor microenvironment (TME), including endothelial cells and fibroblasts, promoting the infiltration, activation of neutrophils, and tumor-associated macrophages within the TME. In this review, we mainly focus on the roles of chemokines CXCL12 and its cognate receptors CXCR4 as they pertain to cancer progression. In particular, we summarizes our current understanding regarding the contribution of CXCR4 and SDF-1 to gastrointestinal tumor behavior and its role in local progression, dissemination, and immune evasion of tumor cells. Also, describes recent therapeutic approaches that target these receptors or their ligands.

Interplay between SOX9, β-catenin and PPARγ activation in colorectal cancer

Colorectal carcinogenesis relies on loss of homeostasic mechanisms regulating cell proliferation, differentiation and survival. These cell processes have been reported to be influenced independently by transcription factors activated downstream of the Wnt pathway, such as SOX9 and β-catenin, and by the nuclear receptor PPARγ. The purpose of this study was to explore the expression levels and functional link between SOX9, β-catenin and PPARγ in the pathogenesis of colorectal cancer (CRC). We evaluated SOX9, β-catenin and PPARγ expression levels on human CRC specimens by qPCR and immunoblot detection. We tested the hypothesis that PPARγ activation might affect SOX9 and β-catenin expression using four colon cancer cell lines (CaCo2, SW480, HCT116, and HT29 cells). In CRC tissues SOX9 resulted up-regulated at both mRNA and protein levels when compared to matched normal mucosa, β-catenin resulted up-regulated at protein levels, while PPARG mRNA and PPARγ protein levels were down-regulated. A significant relationship was observed between high PPARG and SOX9 expression levels in the tumor tissue and female gender (p=0.005 and p=0.04, respectively), and between high SOX9 expression in the tumor tissue and age (p=0.04) and microsatellite instability (MSI), in particular with MSI-H (p=0.0002). Moreover, treatment with the synthetic PPARγ ligand rosiglitazone induced different changes of SOX9 and β-catenin expression and subcellular localization in the colon cancer cell lines examined. In conclusion, SOX9, β-catenin and PPARγ expression levels are deregulated in the CRC tissue, and in colon cancer cell lines ligand-dependent PPARγ activation unevenly influences SOX9 and β-catenin expression and subcellular localization, suggesting a variable mechanistic role in colon carcinogenesis.

Genetic susceptibility to pancreatic cancer and its functional characterisation: The PANcreatic Disease ReseArch (PANDoRA) consortium

Pancreatic cancer is the fourth leading cause of cancer deaths in the European Union and in the USA, but little is known about its genetic susceptibility. The PANcreatic Disease ReseArch (PANDoRA) consortium was established to unite the efforts of different research groups; its aim is to create a large bio-database to uncover new genetic factors for pancreatic cancer risk, response to treatment, and patient survival. So far 2220 cases of pancreatic adenocarcinoma, a smaller number of cases of endocrine pancreatic tumours (n=86), chronic pancreatitis (n=272) and 3847 healthy controls have been collected. As a collective effort of the consortium, SNPs associated with pancreatic adenocarcinoma risk from a genome-wide association study performed in Caucasians were replicated. The possibility that the same genetic polymorphisms may influence patient survival as well was also addressed. This collective effort is particularly important for pancreatic cancer because it is a relatively rare disease for which little is known about aetiopathogenesis and risk factors. The recruitment of additional collaborators and partner institutions is continuously on-going.

TERT gene harbors multiple variants associated with pancreatic cancer susceptibility

A small number of common susceptibility loci have been identified for pancreatic cancer, one of which is marked by rs401681 in the TERT–CLPTM1L gene region on chromosome 5p15.33. Because this region is characterized by low linkage disequilibrium, we sought to identify whether additional single nucleotide polymorphisms (SNPs) could be related to pancreatic cancer risk, independently of rs401681. We performed an in‐depth analysis of genetic variability of the telomerase reverse transcriptase (TERT) and the telomerase RNA component (TERC) genes, in 5,550 subjects with pancreatic cancer and 7,585 controls from the PANcreatic Disease ReseArch (PANDoRA) and the PanScan consortia. We identified a significant association between a variant in TERT and pancreatic cancer risk (rs2853677, odds ratio = 0.85; 95% confidence interval = 0.80–0.90, p = 8.3 × 10−8). Additional analysis adjusting rs2853677 for rs401681 indicated that the two SNPs are independently associated with pancreatic cancer risk, as suggested by the low linkage disequilibrium between them (r2 = 0.07, D′ = 0.28). Three additional SNPs in TERT reached statistical significance after correction for multiple testing: rs2736100 (p = 3.0 × 10−5), rs4583925 (p = 4.0 × 10−5) and rs2735948 (p = 5.0 × 10−5). In conclusion, we confirmed that the TERT locus is associated with pancreatic cancer risk, possibly through several independent variants.

Neuroimmune interactions in patients with inflammatory bowel diseases: Disease activity and clinical behavior based on Substance P serum levels

BACKGROUND AND AIM The neuropeptide Substance P, plays a key role in modulating neuroimmune interactions in patients with inflammatory bowel diseases. We analyzed Substance P serum levels in patients with ulcerative colitis and Crohns disease, to detail the involvement of the neuropeptide in the pathophysiology of these disorders. METHODS Serum samples were collected from 61 patients with ulcerative colitis (24 with active and 37 with inactive disease), 66 patients with Crohns disease (29 with active and 37 with inactive disease) and 45 healthy subjects, enrolled into the study. Neuropetide serum levels were measured by means of an ELISA/EIA. Associations with disease activity and patients clinical features were also taken into account. RESULTS Compared to controls, Substance P serum levels were significantly increased in both patients with ulcerative colitis and Crohns disease, (p<0.001). In patients with ulcerative colitis, levels paralleled disease activity (p=0.014), and the amount of the neuropeptide was considerably decreased during clinical and endoscopic remission of the disease, (p=0.025). Conversely, median Substance P levels did not differ between patients with active and inactive Crohns disease. However, levels of the neuropeptide were more often elevated in patients with inactive and stricturing/fistulizing Crohns disease, (p=0.002). CONCLUSIONS Data underline that Substance P might exerts important immunomodulatory functions in inflammatory bowel disease. This study suggests a potential role for Substance P serum levels in monitoring intestinal inflammation in patients with inflammatory bowel disease.