Anna Panza

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.

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.

Altered expression of the clock gene machinery in kidney cancer patients.

BACKGROUND AND AIM Kidney cancer is associated with alteration in the pathways regulated by von Hippel-Lindau protein and hypoxia inducible factor α. Tight interrelationships have been evidenced between hypoxia response pathways and circadian pathways. The dysregulation of the circadian clock circuitry is involved in carcinogenesis. The aim of our study was to evaluate the clock gene machinery in kidney cancer. METHODS mRNA expression levels of the clock genes ARNTL1, ARNTL2, CLOCK, PER1, PER2, PER3, CRY1, CRY2, TIMELESS, TIPIN and CSNK1E and of the clock controlled gene SERPINE1 were evaluated by DNA microarray assays and by qRT-PCR in primary tumor and matched nontumorous tissue collected from a cohort of 11 consecutive kidney cancer patients. RESULTS In kidney tumor tissue, we found down-regulation of PER2 (median=0.658, Q1-Q3=0.562-0.744, P<0.01), TIMELESS (median=0.705, Q1-Q3=0.299-1.330, P=0.04) and TIPIN (median=0.556, Q1-Q3=0.385-1.945, P=0.01), up-regulation of SERPINE1 (median=1.628, Q1-Q3=0.339-4.071, P=0.04), whereas the expression of ARNTL2 (median=0.605, Q1-Q3=0.318-1.738, P=0.74) and CSNK1E (median=0.927, Q1-Q3=0.612-2.321, P=0.33) did not differ. A statistically significant correlation was evidenced between mRNA levels of PER2 and CSNKIE (r=0.791, P<0.01), PER2 and TIPIN (r=0.729, P=0.01), PER2 and SERPINE1 (r=0.704, P=0.01), TIMELESS and TIPIN (r=0.605, P=0.04), TIMELESS and CSNKIE (r=0.637, P=0.03), TIPIN and CSNKIE (r=0.940, P<0.01). CONCLUSION In kidney cancer, the circadian clock circuitry is deregulated and the altered expression of the clock genes might be involved in disease onset and progression.

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.

Loss of connectivity in cancer co-expression networks

Differential gene expression profiling studies have lead to the identification of several disease biomarkers. However, the oncogenic alterations in coding regions can modify the gene functions without affecting their own expression profiles. Moreover, post-translational modifications can modify the activity of the coded protein without altering the expression levels of the coding gene, but eliciting variations to the expression levels of the regulated genes. These considerations motivate the study of the rewiring of networks co-expressed genes as a consequence of the aforementioned alterations in order to complement the informative content of differential expression. We analyzed 339 mRNAomes of five distinct cancer types to find single genes that presented co-expression patterns strongly differentiated between normal and tumor phenotypes. Our analysis of differentially connected genes indicates the loss of connectivity as a common topological trait of cancer networks, and unveils novel candidate cancer genes. Moreover, our integrated approach that combines the differential expression together with the differential connectivity improves the classic enrichment pathway analysis providing novel insights on putative cancer gene biosystems not still fully investigated.

Peroxisome proliferator-activated receptor γ-mediated induction of microRNA-145 opposes tumor phenotype in colorectal cancer

UNLABELLED MicroRNAs (miRNAs) regulate diverse biological processes by inhibiting translation or inducing degradation of target mRNAs. miR-145 is a candidate tumor suppressor in colorectal carcinoma (CRC). Colorectal carcinogenesis involves deregulation of cellular processes controlled by a number of intertwined chief transcription factors, such as PPARγ and SOX9. Since PPAR family members are able to modulate complex miRNAs networks, we hypothesized a role of miRNA-145 in the interaction between PPARγ and SOX9 in colorectal carcinogenesis. To address this issue, we evaluated gene expression in tissue specimens of CRC patients and we took advantage of invitro models represented by CRC derived cell lines (CaCo2, SW480, HCT116, and HT-29), employing PPARγ activation and/or miRNA-145 ectopic overexpression to analyze how their interplay impact the expression of SOX9 and the development of a malignant phenotype. RESULTS PPARγ regulates the expression of miR-145 by directly binding to a PPAR response element (PPRE) in its promoter at -1207/-1194bp from the transcription start site. The binding is essential for miR-145 upregulation by PPARγ upon rosiglitazone treatment. Ectopic expression of miR-145, in turn, regulates SOX9 expression through the binding to specific seed motifs. The PPARγ-miR-145-SOX9 axis overarches cell cycle progression, invasiveness and differentiation of CRC derived cell lines. Together, these results suggest that miR-145 is a novel target of PPARγ, acts as a tumor suppressor in CRC cell lines and is a key regulator of intestinal cell differentiation by directly targeting SOX9, a marker of undifferentiated progenitors in the colonic crypts.

Identification and surveillance of 19 Lynch syndrome families in southern Italy: report of six novel germline mutations and a common founder mutation.

Lynch syndrome (LS), or hereditary non-polyposis colorectal cancer (HNPCC), is an autosomal dominant condition responsible for early onset cancer mostly in the colonrectum and endometrium as well as in other organ sites. Lynch syndrome is caused by germline mutations in mismatch repair genes, prevalently in hMSH2, hMLH1, and less frequently in hMSH6 and hPMS2. Twenty-nine non-related index cases with colorectal cancer (CRC) were collected from a region in southeast Italy (Apulia). Among this set of patients, fifteen fulfilled the Amsterdam criteria II. The presence of tumor microsatellite instability (MSI) was assessed in all index cases and 19 (15 AC+/4 AC−) were classified as MSI-H. Mutation analysis performed on all patients, identified 15 pathogenic mutations in hMLH1 and 4 in hMSH2. 4/15 mutations in hMLH1 and 2/4 hMSH2 mutations have not been previously reported. Three previously reported mutations were further investigated for the possibility of a common founder effect. Genetic counseling was offered to all probands and extended to 183 relatives after molecular testing and 85 (46%) mutation carriers were identified. Eighty mutation carriers underwent an accurate clinical and instrumental surveillance protocol. Our results confirm that the identification of LS patients based exclusively on family history may miss patients carrying germline mutations in the MMR genes. Moreover, our results demonstrated that molecular screening and subsequent instrumental surveillance are very effective in identifying CRCs at earlier stages and reducing the number of deaths from secondary cancers in HNPCC patients.

MUTYH-associated polyposis (MAP): evidence for the origin of the common European mutations p.Tyr179Cys and p.Gly396Asp by founder events

MUTYH-associated polyposis (MAP) is an autosomal recessive adenomatous polyposis caused by biallelic germline mutations of the base-excision-repair gene MUTYH. In MAP patients of European origin, the combined allele frequency of the mutations p.Tyr179Cys and p.Gly396Asp ranges between 50 and 82%, while these mutations have not been identified in Far Eastern Asian populations, supporting the hypothesis that a founder effect has occurred at some point in European history. To investigate the natural history of the two common European MUTYH alleles, we genotyped six gene-flanking microsatellite markers in 80 unrelated Italian and German MAP patients segregating one or both mutations and calculated their age in generations (g) by using DMLE+2.2 software. Three distinct common haplotypes, one for p.Tyr179Cys and two for p.Gly396Asp, were identified. Estimated mutation ages were 305 g (95% CS: 271–418) for p.Tyr179Cys and 350 g (95% CS: 313–435) for p.Gly396Asp. These results provide evidence for strong founder effects and suggest that the p.Tyr179Cys and p.Gly396Asp mutations derive from ancestors who lived between 5–8 thousand years and 6–9 thousand years B.C., respectively.

Molecular pathways undergoing dramatic transcriptomic changes during tumor development in the human colon

BackgroundThe malignant transformation of precancerous colorectal lesions involves progressive alterations at both the molecular and morphologic levels, the latter consisting of increases in size and in the degree of cellular atypia. Analyzing preinvasive tumors of different sizes can therefore shed light on the sequence of these alterations.MethodsWe used a molecular pathway-based approach to analyze transcriptomic profiles of 59 colorectal tumors representing early and late preinvasive stages and the invasive stage of tumorigenesis. Random set analysis was used to identify biological pathways enriched for genes differentially regulated in tumors (compared with 59 samples of normal mucosa).ResultsOf the 880 canonical pathways we investigated, 112 displayed significant tumor-related upregulation or downregulation at one or more stages of tumorigenesis. This allowed us to distinguish between pathways whose dysregulation is probably necessary throughout tumorigenesis and those whose involvement specifically drives progression from one stage to the next. We were also able to pinpoint specific changes within each gene set that seem to play key roles at each transition. The early preinvasive stage was characterized by cell-cycle checkpoint activation triggered by DNA replication stress and dramatic downregulation of basic transmembrane signaling processes that maintain epithelial/stromal homeostasis in the normal mucosa. In late preinvasive lesions, there was also downregulation of signal transduction pathways (e.g., those mediated by G proteins and nuclear hormone receptors) involved in cell differentiation and upregulation of pathways governing nuclear envelope dynamics and the G2>M transition in the cell cycle. The main features of the invasive stage were activation of the G1>S transition in the cell cycle, upregulated expression of tumor-promoting microenvironmental factors, and profound dysregulation of metabolic pathways (e.g., increased aerobic glycolysis, downregulation of pathways that metabolize drugs and xenobiotics).ConclusionsOur analysis revealed specific pathways whose dysregulation might play a role in each transition of the transformation process. This is the first study in which such an approach has been used to gain further insights into colorectal tumorigenesis. Therefore, these data provide a launchpad for further exploration of the molecular characterization of colorectal tumorigenesis using systems biology approaches.