Distinct Genetic Alterations in Colorectal Cancer

Colon cancer (CRC) development often includes chromosomal instability (CIN) leading to amplifications and deletions of large DNA segments. Epidemiological, clinical, and cytogenetic studies showed that there are considerable differences between CRC tumors from African Americans (AAs) and Caucasian patients. In this study, we determined genomic copy number aberrations in sporadic CRC tumors from AAs, in order to investigate possible explanations for the observed disparities.We applied genome-wide array comparative genome hybridization (aCGH) using a 105k chip to identify copy number aberrations in samples from 15 AAs. In addition, we did a population comparative analysis with aCGH data in Caucasians as well as with a widely publicized list of colon cancer genes (CAN genes). There was an average of 20 aberrations per patient with more amplifications than deletions. Analysis of DNA copy number of frequently altered chromosomes revealed that deletions occurred primarily in chromosomes 4, 8 and 18. Chromosomal duplications occurred in more than 50% of cases on chromosomes 7, 8, 13, 20 and X. The CIN profile showed some differences when compared to Caucasian alterations. as the most frequently amplified genes. The observed CIN may play a distinctive role in CRC in AAs

Helicobacter pylori-induced gastric cancer is orchestrated by MRCK beta-mediated Siah2 phosphorylation

Background Helicobacter pylori-mediated gastric carcinogenesis is initiated by a plethora of signaling events in the infected gastric epithelial cells (GECs). The E3 ubiquitin ligase seven in absentia homolog 2 (Siah2) is induced in GECs in response to H. pylori infection. Posttranslational modifications of Siah2 orchestrate its function as well as stability. The aim of this study was to evaluate Siah2 phosphorylation status under the influence of H. pylori infection and its impact in gastric cancer progression. Methods H. pylori-infected various GECs, gastric tissues from H. pylori-infected GC patients and H. felis-infected C57BL/6 mice were evaluated for Siah2 phosphorylation by western blotting or immunofluorescence microscopy. Coimmunoprecipitation assay followed by mass spectrometry were performed to identify the kinases interacting with Siah2. Phosphorylation sites of Siah2 were identified by using various plasmid constructs generated by site-directed mutagenesis. Proteasome inhibitor MG132 was used to investigate proteasome degradation events. The importance of Siah2 phosphorylation on tumorigenicity of infected cells were detected by using phosphorylation-null mutant and wild type Siah2 stably-transfected cells followed by clonogenicity assay, cell proliferation assay, anchorage-independent growth and transwell invasion assay. Results Siah2 was phosphorylated in H. pylori-infected GECs as well as in metastatic GC tissues at residues serine(6) (Ser(6)) and threonine(279) (Thr(279)). Phosphorylation of Siah2 was mediated by MRCK beta, a Ser/Thr protein kinase. MRCK beta was consistently expressed in uninfected GECs and noncancer gastric tissues but its level decreased in infected GECs as well as in metastatic tissues which had enhanced Siah2 expression. Infected murine gastric tissues showed similar results. MRCK beta could phosphorylate Siah2 but itself got ubiquitinated from this interaction leading to the proteasomal degradation of MRCK beta and use of proteasomal inhibitor MG132 could rescue MRCK beta from Siah2-mediated degradation. Ser(6) and Thr(279) phosphorylated-Siah2 was more stable and tumorigenic than its non-phosphorylated counterpart as revealed by the proliferation, invasion, migration abilities and anchorage-independent growth of stable-transfected cells. Conclusions Increased level of Ser(6) and Thr(279)-phosphorylated-Siah2 and downregulated MRCK beta were prominent histological characteristics of Helicobacter-infected gastric epithelium and metastatic human GC. MRCK beta-dependent Siah2 phosphorylation stabilized Siah2 which promoted anchorage-independent survival and proliferative potential of GECs. Phospho-null mutants of Siah2 (S6A and T279A) showed abated tumorigenicity.Peer reviewe

Distinct High-Profile Methylated Genes in Colorectal Cancer

Background: Mutations and promoters ’ methylation of a set of candidate cancer genes (CAN genes) are associated with progression of colorectal cancer (CRC). We hypothesized that these genes ’ promoters are inactivated through epigenetic silencing and may show a different profile in high-risk populations. We investigated the status of CAN gene methylation and CHD5 protein expression in African American CRC tissue microarrays (TMA) using immunohistochemical staining. Methodology/Principal Findings: The promoter methylation status of the CAN genes was studied by methylation-specific PCR (MSP) in 51 Iranians (a white population) and 51 African Americans (AA). Microsatellite instability (MSI) was analyzed as well. The differential frequency of methylation for each gene was tested by chi-square analysis between the two groups based on matched age and sex. CHD5 protein expression was evaluated in moderate to well differentiated and poorly differentiated carcinomas compared to matched normal tissue using TMA. In addition, the correlation between these epigenetic biomarkers and various clinicopathological factors, including, age, location, and stage of the disease were analyzed. Seventy-seven and 34 % of tumors were distal in Iranian and African American patients, respectively. In both populations, the percentage of methylation was.65 % for SYNE1, MMP2, APC2, GPNMB, EVL, PTPRD, and STARD8, whereas methylation was,50 % for LGR6, RET, CD109, and RNF. The difference in methylation between the two populations was statistically significant for CHD5, ICAM5 and GPNMB. Thirty-one percent AA tumors showed MSI-H, compared to 28 % i

Impact of BRAF, MLH1 on the incidence of microsatellite instability high colorectal cancer in populations based study

We have identified an alternative pathway of tumorigenesis in sporadic colon cancer, involving microsatellite instability due to mismatched repair methylation, which may be driven by mutations in the BRAF gene (V600E). Colorectal cancer (CRC) is the most common cancer in the world, and African Americans show a higher incidence than other populations in the United States. We analyzed sporadic CRCs in Omani (of African origin, N = 61), Iranian (of Caucasian origin, N = 53) and African American (N = 95) patients for microsatellite instability, expression status of mismatched repair genes (hMLH1, hMSH2) and presence of the BRAF (V600E) mutation. In the Omani group, all tumors with BRAF mutations were located in the left side of the colon, and for African Americans, 88% [7] of tumors with BRAF mutations were found in the right side of the colon. In African Americans, 31% of tumors displayed microsatellite instability at two or more markers (MSI-H), while this rate was 26% and 13% for tumors in the Iranian and Omani groups, respectively. A majority of these MSI-H tumors were located in the proximal colon (right side) in African American and Iranian subjects, whereas most were located in the distal colon (left side) in Omani subjects. Defects in hMLH1 gene expression were found in 77% of MSI-H tumors in both African Americans and Iranians and in 38% of tumors in Omanis. BRAF mutations were observed in all subjects: 10% of tumors in African Americans (8/82), 2% of tumors in Iranians (1/53), and 19% of tumors in Omanis (11/59). Our findings suggest that CRC occurs at a younger age in Omani and Iranian patients, and these groups showed a lower occurrence of MSI-H than did African American patients. Our multivariate model suggests an important and significant role of hMLH1 expression and BRAF mutation in MSI-H CRC in these populations. The high occurrence of MSI-H tumors in African Americans may have significant implications for treatment, since patients with MSI-H lesions display a different response to chemotherapeutic agents such as 5-fluorouracil

Identification of Specific Oral and Gut Pathogens in Full Thickness Colon of Colitis Patients: Implications for Colon Motility

Impaired colon motility is one of the leading problems associated with inflammatory bowel disease (IBD). An expanding body of evidence supports the role of microbiome in normal gut function and in progression of IBD. The objective of this work is to determine whether diseased full thickness colon specimens, including the neuromuscular region (critical for colon motility function), contain specific oral and gut pathogens. In addition, we compared the differences in colon microbiome between Caucasians (CA) and African Americans (AA). Thirty-nine human full thickness colon (diseased colon and adjacent healthy colon) specimens were collected from Crohn's Colitis (CC) or Ulcerative Colitis (UC) patients while they underwent elective colon surgeries. We isolated and analyzed bacterial ribosomal RNA (rRNA) from colon specimens by amplicon sequencing of the 16S rRNA gene region. The microbiome proportions were quantified into Operational Taxonomic Units (OTUs) by analysis with Quantitative Insights Into Microbial ecology (QIIME) platform. Two hundred twenty-eight different bacterial species were identified by QIIME analysis. However, we could only decipher the species name of fifty-three bacteria. Our results show that proportion of non-detrimental bacteria in CC or UC colon samples were altered compared to adjacent healthy colon specimens. We further show, for the first time in full thickness colon specimens, that microbiome of CC and UC diseased specimens is dominated by putative oral pathogens belonging to the Phyla Firmicutes (Streptococcus, Staphylococcus, Peptostreptococcus), and Fusobacteria (Fusobacterium). In addition, we have identified patterns of differences in microbiome levels between CA and AA specimens with potential implications for health disparities research. Overall, our results suggest a significant association between oral and gut microbes in the modulation of colon motility in colitis patients

NOD2 Mutations and Anti-Saccharomyces cerevisiae Antibodies Are Risk Factors for Crohn's Disease in African Americans

NOD2 mutations and anti-Saccharomyces cerevisiae antibodies (ASCA) are associated with Crohn’s disease (CD), ileal involvement and complicated disease behavior in whites. ASCA and the three common NOD2 mutations have not been assessed in African American (AA) adults with CD

Case-Control Study of Vitamin D, dickkopf homolog 1 (DKK1) Gene Methylation, VDR Gene Polymorphism and the Risk of Colon Adenoma in African Americans

There are sparse data on genetic, epigenetic and vitamin D exposure in African Americans (AA) with colon polyp. Consequently, we evaluated serum 25(OH) D levels, vitamin D receptor (VDR) polymorphisms and the methylation status of the tumor suppressor gene dickkopf homolog 1 (DKK1) as risk factors for colon polyp in this population.The case-control study consisted of 93 patients with colon polyp (cases) and 187 healthy individuals (controls) at Howard University Hospital. Serum levels of 25(OH)D (including D3, D2, and total) were measured by liquid chromatography-mass spectrometry. DNA analysis focused on 49 single nucleotide polymorphisms (SNPs) in the VDR gene. Promoter methylation analysis of DKK1 was also performed. The resulting data were processed in unadjusted and multivariable logistic regression analyses.Cases and controls differed in vitamin D status (D(3)<50 nmol/L: Median of 35.5 in cases vs. 36.8 in controls nmol/L; P = 0.05). Low levels of 25(OH)D(3) (<50 nmol/L) were observed in 86% of cases and 68% of controls and it was associated with higher risks of colon polyp (odds ratio of 2.7, 95% confidence interval 1.3-3.4). The SNP analysis showed no association between 46 VDR polymorphisms and colon polyp. The promoter of the DKK1 gene was unmethylated in 96% of the samples.We found an inverse association between serum 25(OH)D(3) and colon polyp in AAs. VDR SNPs and DKK1 methylation were not associated with colon polyp. Vitamin D levels may in part explain the higher incidence of polyp in AAs

Contribution of higher risk genes and European admixture to Crohnʼs disease in African Americans:

African Americans (AA) are an admixed population of West African (WA) and European ancestry (EA). Crohn's disease (CD) susceptibility genes have not been established. We therefore evaluated the contribution of European admixture and major established risk genes to AA CD