Annamaria Gentile

Aberrant DNA methylation in non-neoplastic gastric mucosa of H. Pylori infected patients and effect of eradication.

BACKGROUND:Gene promoter methylation is an epigenetic event leading to gene silencing. This mechanism is particularly relevant in cancer since it can interfere with the activity of specific “suppressor” genes.AIM:To evaluate promoter methylation of CDH1, p16, APC, MLH1, and COX2 in patients with H. pylori (Hp) infection before and after eradication.METHODS:Fifty-seven dyspeptic outpatients who had never performed previous endoscopy or Hp testing and treatment underwent clinical interview, endoscopy with three paired gastric biopsy specimens from the antrum, angulus, and corpus, and 13C-urea breath test (UBT). Biopsies were scored for the presence of Hp and intestinal metaplasia (IM). DNA methylation of five tumor-related genes (CDH1, p16, MLH1, APC, and COX2) was evaluated by methylation-specific PCR in each biopsy. Infected patients were given a standard eradicating treatment and, after 1 yr, underwent endoscopy with biopsies and UBT.RESULTS:Hp infection was found in 45 patients. IM was detected in 17 out of 45 (38%) infected patients. Mean number of methylated genes was 0, 1.1 ± 0.9, and 1.6 ± 0.9 among the 12 Hp−/IM−, the 28 Hp+/IM−, and the 17 Hp+/IM+ patients, respectively (P < 0.0001). Specifically, promoter hypermethylation of CDH1, p16, APC, MLH1, and COX2 was found in 68%, 25%, 7%, 0%, and 14% of Hp+/IM− patients and in 71%, 29%, 35%, 12%, and 12% of Hp+/IM+ patients. No significant difference was found among the three groups of patients as far as age, smoking, alcohol, meat and vegetable consumption, and family history of gastric cancer were considered. Twenty-three out of 45 (51%) infected patients underwent the 1-yr follow-up endoscopy: 17 out of 23 (74%) were successfully eradicated. After Hp eradication, CDH1, p16, and APC methylation significantly decreased while COX2 methylation completely disappeared. Conversely, MLH1 methylation did not change significantly in patients with IM.CONCLUSION:Hp infection is associated with promoter methylation of genes which are relevant in the initiation and progression of gastric carcinogenesis. While CDH1 methylation seems to be an early event in Hp gastritis, MLH1 methylation occurs late along with IM. Hp eradication is able to significantly reduce gene methylation thus delaying or reversing Hp-induced gastric carcinogenesis.

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.

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.

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.

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.

Analysis of clock gene-miRNA correlation networks reveals candidate drivers in colorectal cancer

Altered functioning of the biological clock is involved in cancer onset and progression. MicroRNAs (miRNAs) interact with the clock genes modulating the function of genetically encoded molecular clockworks. Collaborative interactions may take place within the coding-noncoding RNA regulatory networks. We aimed to evaluate the cross-talk among miRNAs and clock genes in colorectal cancer (CRC). We performed an integrative analysis of miRNA-miRNA and miRNA-mRNA interactions on high-throughput molecular profiling of matched human CRC tissue and non-tumor mucosa, pinpointing core clock genes and their targeting miRNAs. Data obtained in silico were validated in CRC patients and human colon cancer cell lines. In silico we found severe alterations of clock gene–related coding-noncoding RNA regulatory networks in tumor tissues, which were later corroborated by the analysis of human CRC specimens and experiments performed in vitro. In conclusion, specific miRNAs target and regulate the transcription/translation of clock genes and clock gene-related miRNA-miRNA as well as mRNA-miRNA interactions are altered in colorectal cancer. Exploration of the interplay between specific miRNAs and genes, which are critically involved in the functioning of the biological clock, provides a better understanding of the importance of the miRNA-clock genes axis and its derangement in colorectal cancer.

The expression of leucine-rich repeat gene family members in colorectal cancer.

This study was conducted to evaluate the association of the leucine-rich repeat (LRR) gene family with colorectal cancer (CRC). The expression of members of the LRR gene family were analyzed in 17 CRC specimens and in 59 healthy colorectal tissues by using Human Exon1.0ST microarray, and in 25 CRC specimens and 32 healthy colorectal tissues by U133Plus2.0 microarray. An association was found for 25 genes belonging to the plant-specific (PS) class of LRR genes (P = 0.05 for Exon1.0 ST and P = 0.04 for U133Plus2.0). In both data-sets, in CRC, we found down-regulation of SHOC2 (P < 0.00003) and LRRC28 (P < 0.01) and up-regulation of LRSAM1 (P < 0.000001), while up-regulation of MFHAS1 (P = 0.0005) and down-regulation of WDFY3 (P = 0.026) were found only in the Exon1.0 ST data-set. The PS LLR gene class encodes proteins that activate immune cells and might play a key role in programmed cell death and autophagy. SHOC2 and LRRC28 genes involved in RAS-mediated signaling, which hinders nutrient deprivation-induced autophagy, might be a possible link between the negative control of autophagy and tumorigenesis.

Genotyping of the lactase-phlorizin hydrolase C/T- 13910 polymorphism by means of a new rapid denaturing high-performance liquid chromatography : Based assay in healthy subjects and colorectal cancer patients

Adult-type hypolactasia results from the progressive decline of lactase-phlorizin hydrolase activity in enterocytes after weaning. Lactase nonpersistence may determine a primary lactose intolerance with reduced diary product consumption, which is possibly related to an increased risk of colon cancer. Recently, a genetic variant C/T—13910 upstream of the lactase-phlorizin hydrolase (LCT) gene has been strongly correlated with the lactase persistence/nonpersistence trait in both family and case-control studies. The authors validate a denaturing high-performance liquid chromatography (dHPLC)—based assay versus conventional genotype sequencing in detecting the C/T—13910 polymorphism of LCT and evaluate its prevalence in 2 different Italian geographical areas and in colorectal cancer patients. DNA samples of 157 healthy subjects and 124 colon cancer patients from Apulia and of 97 healthy subjects from Sardinia were evaluated for the C/T—13910 polymorphism by dHPLC, sequencing, and restriction fragment length polymorphism (RFLP). Under optimized conditions, dHPLC was as sensitive as DNA sequencing and detected a new genetic variant (T/C-13913) in 2 individuals that was not identified by RFLP assay. Frequency of lactase nonpersistence genotype (C/C—13910) was similar in healthy subjects from 2 different Italian geographical areas and not increased in patients with colorectal cancer. The results indicate that the dHPLC method may be used as a rapid, noninvasive, and laborsaving screening tool for genotyping C/T—13910 polymorphism, with high success, low cost, and reproducibility. (Journal of Biomolecular Screening 2007:733-739)

Deregulated expression of cryptochrome genes in human colorectal cancer

BackgroundCircadian disruption and deranged molecular clockworks are involved in carcinogenesis. The cryptochrome genes (CRY1 and CRY2) encode circadian proteins important for the functioning of biological oscillators. Their expression in human colorectal cancer (CRC) and in colon cancer cell lines has not been evaluated so far.MethodsWe investigated CRY1 and CRY2 expression in fifty CRCs and in the CaCo2, HCT116, HT29, SW480 cell lines.ResultsCRY1 (p = 0.01) and CRY2 (p < 0.0001) expression was significantly changed in tumour tissue, as confirmed in a large independent CRC dataset. In addition, lower CRY1 mRNA levels were observed in patients in the age range of 62-74 years (p = 0.018), in female patients (p = 0.003) and in cancers located at the transverse colon (p = 0.008). Lower CRY2 levels were also associated with cancer location at the transverse colon (p = 0.007). CRC patients displaying CRY1 (p = 0.042) and CRY2 (p = 0.043) expression levels over the median were hallmarked by a poorer survival rate. Survey of selected colon cancer cell lines evidenced variable levels of cryptochrome genes expression and time-dependent changes in their mRNA levels. Moreover, they showed reduced apoptosis, increased proliferation and different response to 5-fluorouracil and oxaliplatin upon CRY1 and CRY2 ectopic expression. The relationship with p53 status came out as an additional layer of regulation: higher CRY1 and CRY2 protein levels coincided with a wild type p53 as in HCT116 cells and this condition only marginally affected the apoptotic and cell proliferation characteristics of the cells upon CRY ectopic expression. Conversely, lower CRY and CRY2 levels as in HT29 and SW480 cells coincided with a mutated p53 and a more robust apoptosis and proliferation upon CRY transfection. Besides, an heterogeneous pattern of ARNTL, WEE and c-MYC expression hallmarked the chosen colon cancer cell lines and likely influenced their phenotypic changes.ConclusionCryptochrome gene expression is altered in CRC, particularly in elderly subjects, female patients and cancers located at the transverse colon, affecting overall survival. Altered CRY1 and CRY2 expression patterns and the interplay with the genetic landscape in colon cancer cells may underlie phenotypic divergence that could influence disease behavior as well as CRC patients survival and response to chemotherapy.

Multifaceted enrichment analysis of RNA–RNA crosstalk reveals cooperating micro-societies in human colorectal cancer

Alterations in the balance of mRNA and microRNA (miRNA) expression profiles contribute to the onset and development of colorectal cancer. The regulatory functions of individual miRNA-gene pairs are widely acknowledged, but group effects are largely unexplored. We performed an integrative analysis of mRNA–miRNA and miRNA–miRNA interactions using high-throughput mRNA and miRNA expression profiles obtained from matched specimens of human colorectal cancer tissue and adjacent non-tumorous mucosa. This investigation resulted in a hypernetwork-based model, whose functional backbone was fulfilled by tight micro-societies of miRNAs. These proved to modulate several genes that are known to control a set of significantly enriched cancer-enhancer and cancer-protection biological processes, and that an array of upstream regulatory analyses demonstrated to be dependent on miR-145, a cell cycle and MAPK signaling cascade master regulator. In conclusion, we reveal miRNA-gene clusters and gene families with close functional relationships and highlight the role of miR-145 as potent upstream regulator of a complex RNA–RNA crosstalk, which mechanistically modulates several signaling pathways and regulatory circuits that when deranged are relevant to the changes occurring in colorectal carcinogenesis.