Mohammad Daremipouran

Distinct Genetic Alterations in Colorectal Cancer

Background 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. Methodology/Principal Findings 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. Conclusions/Significance Chromosome X amplification in male patients and chromosomes 4, 8 and 18 deletions were prominent aberrations in AAs. Some CAN genes were altered at high frequencies in AAs with EXOC4, EPHB6, GNAS, MLL3 and TBX22 as the most frequently deleted genes and HAPLN1, ADAM29, SMAD2 and SMAD4 as the most frequently amplified genes. The observed CIN may play a distinctive role in CRC in AAs.

Toward a comprehensive and systematic methylome signature in colorectal cancers

CpG Island Methylator Phenotype (CIMP) is one of the underlying mechanisms in colorectal cancer (CRC). This study aimed to define a methylome signature in CRC through a methylation microarray analysis and a compilation of promising CIMP markers from the literature. Illumina HumanMethylation27 (IHM27) array data was generated and analyzed based on statistical differences in methylation data (1st approach) or based on overall differences in methylation percentages using lower 95% CI (2nd approach). Pyrosequencing was performed for the validation of nine genes. A meta-analysis was used to identify CIMP and non-CIMP markers that were hypermethylated in CRC but did not yet make it to the CIMP genes’ list. Our 1st approach for array data analysis demonstrated the limitations in selecting genes for further validation, highlighting the need for the 2nd bioinformatics approach to adequately select genes with differential aberrant methylation. A more comprehensive list, which included non-CIMP genes, such as APC, EVL, CD109, PTEN, TWIST1, DCC, PTPRD, SFRP1, ICAM5, RASSF1A, EYA4, 30ST2, LAMA1, KCNQ5, ADHEF1, and TFPI2, was established. Array data are useful to categorize and cluster colonic lesions based on their global methylation profiles; however, its usefulness in identifying robust methylation markers is limited and rely on the data analysis method. We have identified 16 non-CIMP-panel genes for which we provide rationale for inclusion in a more comprehensive characterization of CIMP+ CRCs. The identification of a definitive list for methylome specific genes in CRC will contribute to better clinical management of CRC patients.

Transactivation of the EGFR by AP-1 Is Induced by Helicobacter pylori in Gastric Cancer

BACKGROUND:Helicobacter pylori infection of the gastric mucosa is strongly associated with gastritis, peptic ulcer disease, and gastric cancer. However, the mechanisms by which H. pylori causes cancer are currently unknown. Binding of epidermal growth factor (EGF) to its receptor (EGFR) may be important in the development of gastric cancer. This interaction accelerates cell proliferation and migration, and triggers epithelial cell signaling. In this study, we investigated the effects of H. pylori on EGFR- and AP-1-mediated signal transduction pathways in the AGS gastric epithelial cell line and gastric tissue from humans.METHODS:Cells were treated with H. pylori and cell death was examined at a variety of time points using cell viability and trypan blue exclusion dye assay. To investigate the effects on EGFR regulation, AGS cells were transfected with a full-length and truncated EGFR luciferase (luc) reporter. Tissue microarray containing 44 samples of gastric biopsies from H. pylori-positive patients was analyzed for protein expression level of EGFR by immunohistochemistry.RESULTS:EGFR promoter activity was increased (˜twofold) 3 h after treatment with H. pylori commenced. Using a series of EGFR promoter deletion mutants, we identified a region that was crucial for transactivation of the EGFR by H. pylori. To determine whether AP-1 binding was altered, we transfected AGS cells with an AP-1 luciferase construct and then treated them with H. pylori for up to 6 h. We found that AP-1 activity was induced by H. pylori in gastric cells, while electrophoretic mobility shift assays confirmed that binding of AP-1 to the EGFR promoter site was increased following H. pylori treatment. Binding of c-Jun and c-Fos to the EGFR promoter region −1,062/−900 was induced eight- and six fold, respectively, using ChIP assay. Active EGFR staining was markedly increased in gastric mucosa from infected persons, compared to uninfected controls.CONCLUSIONS:We conclude that exposure of gastric cells to H. pylori induces increased production of EGFR through various signal transduction pathways, including those mediated by the EGFR and AP-1. Distinct effects on EGFR activation may specify the subset of AP-1 target genes that are selected, including those involved in proliferation and apoptosis. This is consistent with EGFR activation that was found in the gastric mucosa of humans infected with H. pylori. Hence, the balance between apoptosis and proliferation in these cells may be altered in response to injury caused by H. pylori infection, leading to an increased risk of cancer.

DNA methylome profiling identifies novel methylated genes in African American patients with colorectal neoplasia.

The identification of genes that are differentially methylated in colorectal cancer (CRC) has potential value for both diagnostic and therapeutic interventions specifically in high-risk populations such as African Americans (AAs). However, DNA methylation patterns in CRC, especially in AAs, have not been systematically explored and remain poorly understood. Here, we performed DNA methylome profiling to identify the methylation status of CpG islands within candidate genes involved in critical pathways important in the initiation and development of CRC. We used reduced representation bisulfite sequencing (RRBS) in colorectal cancer and adenoma tissues that were compared with DNA methylome from a healthy AA subject’s colon tissue and peripheral blood DNA. The identified methylation markers were validated in fresh frozen CRC tissues and corresponding normal tissues from AA patients diagnosed with CRC at Howard University Hospital. We identified and validated the methylation status of 355 CpG sites located within 16 gene promoter regions associated with CpG islands. Fifty CpG sites located within CpG islands—in genes ATXN7L1 (2), BMP3 (7), EID3 (15), GAS7 (1), GPR75 (24), and TNFAIP2 (1)—were significantly hypermethylated in tumor vs. normal tissues (P < 0.05). The methylation status of BMP3, EID3, GAS7, and GPR75 was confirmed in an independent, validation cohort. Ingenuity pathway analysis mapped three of these markers (GAS7, BMP3 and GPR) in the insulin and TGF-β1 network—the two key pathways in CRC. In addition to hypermethylated genes, our analysis also revealed that LINE-1 repeat elements were progressively hypomethylated in the normal-adenoma-cancer sequence. We conclude that DNA methylome profiling based on RRBS is an effective method for screening aberrantly methylated genes in CRC. While previous studies focused on the limited identification of hypermethylated genes, ours is the first study to systematically and comprehensively identify novel hypermethylated genes, as well as hypomethylated LINE-1 sequences, which may serve as potential biomarkers for CRC in African Americans. Our discovered biomarkers were intimately linked to the insulin/TGF-B1 pathway, further strengthening the association of diabetic disorders with colon oncogenic transformation.

Folate Status and Risk of Colorectal Polyps in African Americans

Dietary folate status appears to influence risk for colorectal cancer possibly by alterations in DNA methylation and nucleotide precursor pools. Polymorphisms (677C→T and 1298A→C) in methylenetetrahydrofolate reductase (MTHFR), a key enzyme in folate metabolism, determines enzyme activity. The frequency of polymorphisms in the gene varies extensively in different populations. We sought to determine the association between folate status, folate metabolism, DNA methylation, tobacco, alcohol consumption, and the risk of colorectal adenomas in African Americans. Among 58 patients who underwent a clinically indicated colonoscopy, 23 patients with histology confirmed colorectal polyps and 35 patients without were recruited for a case-control study. Blood samples were collected from fasting patients for determination of serum and red blood cell (RBC) folate, homocysteine, vitamin B12, and methylation status. Polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) technique was performed to identify the MTHFR 677 C→T polymorphism and specific PCR was used to analyze adenomatous polyposis coli (APC) gene-promoter sequence methylation. Among 23 cases, 49 polyps (adenomatous, n = 41 and hyperplastic, n= 8) were identified. Twenty-eight (57%) of the polyps were on the left side and 21 (42%) were on the right side of the colon. There was no association between the presence of colon polyps and levels of folate (serum, RBC), vitamin B12, or homocysteine. Forty-eight individuals (84%) were homozygous for 677 CC. Of these individuals, 18 (37.5%) had ≥1 colorectal polyps, whereas 30 (62.5%) had no polyps. Nine individuals were heterozygous for 677 CT, and 4 (44%) of these individuals had colon polyps. Eighty-eight percent of the APC gene-promoter sequences tested using peripheral blood DNA from patients were unmethylated. Among the individuals who showed APC methylation, 66% had polyps; 33% were polyp free using their blood DNA. There was highly significant association between smoking and alcohol consumption with the presence of a colon polyp (P= .0006 and P= .05, respectively). In conclusion, the lack of the 677 TT may be a significant risk factor for colon neoplasm in the African-American population. Smoking and alcohol consumption were found to be risk factors for colon polyps. APC gene-promoter sequence methylation found in peripheral blood may be an indicator of risk for polyp formation and an important screening tool.

Identification of novel mutations by exome sequencing in African American colorectal cancer patients

The purpose of this study was to identify genome‐wide single nucleotide variants and mutations in African American patients with colorectal cancer (CRC). There is a need of such studies in African Americans, because they display a higher incidence of aggressive CRC tumors.

Association of Cumulative Ultraviolet Radiation Exposure with Prostate Cancer Risk in a Case-control Study of African-American Men

It is well established that exposure to ultraviolet (UV) radiation has beneficial effects in reducing prostate cancer risk. To determine if there is a correlation between UV exposure and prostate cancer risk, we assessed sun exposure in a case-control study of 182 African-American men aged 40 years and older residing in the Metropolitan Washington, DC area. Using data on cumulative exposure per year and adult sunbathing scores derived from a validated questionnaire, analysis revealed significant difference in cumulative sun exposure between cases and controls (p=0.003). Additionally, the outdoor and recreation UV exposures were significantly higher in controls when compared to cases (p=0.003; p=0.03 in age-matched cases and controls). Although the results of conditional logistic regression analysis indicate that there was no association between total UV exposure and risk of prostate cancer after adjusting for age (OR=2.04, 95% CI 0.54-7.70, p=0.29), outdoor UV exposure was associated with decreased prostate cancer risk (OR= 0.31, 95% CI 0.14-0.65, p=0.002). Furthermore, a trend for reduced prostate cancer risk was found among men with early life high sun exposure during childhood ages 0-5 years (OR=0.17, 95% CI 0.03-0.74, p=0.02) and 6-11 years (OR= 0.28, 95% CI 0.07-1.05, p=0.06). Interestingly, this inverse association between prostate cancer risk and early life high sun exposure intensity was also observed among young men at ages 12-17 years although not statistically significant (OR=0.41, 95% CI 0.09-1.95, p=0.26). These findings indicate that UV exposure earlier in life may affect susceptibility to prostate cancer.

T2090 CAN1 Gene Methylation Profile in African Americans with Colon Cancer and Adenoma, New Candidate Genes

SOCS1, CD109, RET, and CGB were 100%, 92%, 92%, 92%, 83%, 77%, 75%, 70%, 58%, 50%, 42%, 38%, 23%, 15%, 17%, 8%, and 4%, respectively. GPNMB, APC2, EVL, PTPRD and CHD5 are frequently silenced in more than 70% cases of cancer, however, the level of GPNMB, APC2, OST, HCAD and TCF21 methylation were identical in the adenoma compared to cancer. There were no statistically significant differences between right and left sided tumors for studied gene methylation. Conclusion: Our data indicated a high methylation profile of GPNMB, APC2, OST, HCAD and TCF21 as potential markers in adenoma as part of the early phase of colon carcinogenesis while EVL, PTPRD, LGR6, and CHD5 methylation profile represent the late phase of CRC. A higher methylation rate in CDH5, EVL, LGR6 and lower methylation rate in CD109, APC2 in AA than in Caucasians (Schuebel et al 2007) is shown in this study.

Abstract 3033: NKX2-5, a potential tumor suppressor gene in prostate cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Prostate cancer (PCa) is a common malignancy and a leading cause of cancer deaths among men in the United States. Abundant evidence has accumulated to suggest that epigenetic DNA methylation changes may appear earlier during PCa development than genetic changes, as well as more commonly and consistently suggesting that DNA methylated genes can be explored as DNA-based biomarker for PCa disease detection. Recently, we have identified NKX2-5 as a novel that is hypermethylated in prostate cancer. However, there is little information about the biological significance of this gene in prostate carcinogenesis. We hypothesize that NKX2-5 is a potential tumor suppressor gene that is frequently inactivated in prostate cancer. Methods: We carried out gain-and-loss functional studies of NKX2-5 in prostate cancer cell lines and validated expression at the RNA transcript level using quantitative RT-PCR. Protein expression was analyzed by western blotting and cell cycle analysis investigated by flow cytometer. Results: Over-expression of NKX2-5 was detrimental to prostate cancer cell proliferation as evidenced by significant inhibition of prostate cancer cell proliferation in comparison to control (vector only transfection) and this was due to cell arrest in Go/G1 phase and increase apoptosis. In contract, successful knockdown of NKX2-5 by shRNA transfection increased prostate cancer cell proliferation. Western blot analysis demonstrated that NKX2-5 plays a key regulatory role in the expression of several genes including p53, PTEN, Histone H1 and the androgen receptor. Conclusion: Our observation suggests that NKX2-5, a member of the homeobox gene family of plays an important tumor suppressor activity in prostate carcinogenesis. Because this gene plays important role in several signal transduction pathways, this gene can be exploited as potential biomarker for the early detection of prostate cancer and could be an attractive target to explore for drug investigation or gene therapies of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3033. doi:10.1158/1538-7445.AM2011-3033

Abstract 2578: Trust in health information source and colorectal cancer screening uptake among U.S. adults

Background: In order to promote positive health behavior of the population, public health outreach is often carried out in various forms to reach the target stakeholders. However, the effectiveness of these outreach programs is unclear. Aim: To determine up-to-date adherence to colorectal cancer (CRC) screening guidelines among US adults according to the trust they have in various sources of health information. Methods: We used the 2007 Health Information National Trends Survey (HINTS) and identified 4342 respondents (weighted population size = 82,811,829) who were at least 50 years old. Respondents expressed the degree of trust they have in various sources of health related information. We considered a source as trusted when it is trusted “a lot” or had “some trust” and regarded as not trusted if it was trusted “a little” or “not at all”. We defined being current with CRC screening as the use of fecal occult blood testing (FOBT) within 1 year, sigmoidoscopy within 5 years, or colonoscopy within 10 years. We used logistic regression models to calculate odds ratios (OR) and 95% confidence intervals (CI). Survey weights were used in all analyses. Results: Approximately 94.3% of respondents trusted their doctors, 59.3% trusted family or friends, 50.2% trusted newsmagazines, 32.7% trusted radio, 66.2% trusted internet, 40.6% trusted television, 68.5% trusted government sources, 44.3% trusted charity organization and 34.5% trusted religious organizations. When compared with those who did not trust the source of health information, trust in radio (64.7% vs 60.7%; OR = 1.24; 95%CI: 1.01-1.52), internet (64.0% vs 58.3%; OR = 1.26; 95%CI: 1.04-1.53), television (64.4% vs 60.9%; OR = 1.23; 95%CI: 1.02-1.48), and government sources (64.0% vs 58.2%; OR = 1.32; 95%CI: 1.09-1.60) were associated with being up-to-date with colorectal cancer screening whereas trust in doctors (63.2% vs 49.2%; OR = 1.53; 95%CI: 0.92-2.55), family or friends (61.5% vs 63.1%; OR = 0.98; 95%CI: 0.84-1.15), newsmagazines (64.0% vs 60.8%; OR = 1.14; 95%CI: 0.98-1.32), charity organizations (62.0% vs 62.1%; OR = 1.04; 95%CI: 0.86-1.24) and religious organizations (59.7% vs 63.3%; OR = 0.90; 95%CI: 0.74-1.09) were not. Conclusion: Even with modest trust in the source of health information, outreach modalities which engage people repeatedly and those that they can readily access over and over again may exert substantial influence on the uptake of colorectal cancer screening. Multiple outreach methods should be used to promote colorectal cancer screening. Citation Format: Adeyinka O. Laiyemo, Hamidat Segunmaru, Jessica Rogers, Mohammad Daremipouran, Clinton Burnside, Florencia Gonzalez, Cherie Spencer, Carla D. Williams. Trust in health information source and colorectal cancer screening uptake among U.S. adults. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2578.