Hassan Ashktorab

MicroRNA-211 Expression Promotes Colorectal Cancer Cell Growth In Vitro and In Vivo by Targeting Tumor Suppressor CHD5

Background Chromodomain-helicase-DNA-binding protein 5 (CHD5) is a newly identified tumor suppressor that is frequently downregulated in a variety of human cancers. Our previous work revealed that the low expression of CHD5 in colorectal cancer is correlated with CHD5 promoter CpG island hypermethylation. In this study, we investigated the effect of microRNA-211 (miR-211)-regulated CHD5 expression on colorectal tumorigenesis. Methodology/Principal Findings miR-211 was predicted to target CHD5 by TargetScan software analysis. A stably expressing exogenous miR-211 colorectal cancer cell line (HCT-116miR-211) was generated using lentiviral transduction and used as a model for in vitro and in vivo studies. The expression level of miR-211 in HCT-116miR-211 cells was upregulated by 16-fold compared to vector control cells (HCT-116vector). Exogenous miR-211 directly binds to the 3′-untranslated region (3′-UTR) of CHD5 mRNA, resulting in a 50% decrease in CHD5 protein level in HCT-116miR-211 cells. The levels of cell proliferation, tumor growth, and cell migration of HCT-116miR-211 cells were significantly higher than HCT-116vector cells under both in vitro and in vivo conditions, as determined using the methods of MTT, colony formation, flow cytometry, scratch assay, and tumor xenografts, respectively. In addition, we found that enforced expression of miR-211 in HCT-116 cells was able to alter p53 pathway-associated regulatory proteins, such as MDM2, Bcl-2, Bcl-xL, and Bax. Conclusion/Significance Our results demonstrate that CHD5 is a direct target of miR-211 regulation. Enforced expression of miR-211 promotes tumor cell growth at least in part by downregulating the expression level of the CHD5 tumor suppressor. Our results provide a better understanding of the association of between miR-211-regulated CHD5 expression and CHD5 function in colorectal tumorigenesis.

Isorhamnetin inhibits proliferation and invasion and induces apoptosis through the modulation of peroxisome proliferator-activated receptor γ activation pathway in gastric cancer.

Background: PPAR-γ, a nuclear transcription factor, plays a critical role in the development of gastric cancer (GC). Hence, novel agents that can modulate PPAR-γ cascade have a great potential for the treatment of GC. Results: Isorhamnetin (IH) modulates PPAR-γ pathway in GC. Conclusion: IH induces apoptosis through the activation of the PPAR-γ pathway. Significance: The study proposes a novel agent for GC treatment. Gastric cancer (GC) is a lethal malignancy and the second most common cause of cancer-related deaths. Although treatment options such as chemotherapy, radiotherapy, and surgery have led to a decline in the mortality rate due to GC, chemoresistance remains as one of the major causes for poor prognosis and high recurrence rate. In this study, we investigated the potential effects of isorhamnetin (IH), a 3′-O-methylated metabolite of quercetin on the peroxisome proliferator-activated receptor γ (PPAR-γ) signaling cascade using proteomics technology platform, GC cell lines, and xenograft mice model. We observed that IH exerted a strong antiproliferative effect and increased cytotoxicity in combination with chemotherapeutic drugs. IH also inhibited the migratory/invasive properties of GC cells, which could be reversed in the presence of PPAR-γ inhibitor. We found that IH increased PPAR-γ activity and modulated the expression of PPAR-γ regulated genes in GC cells. Also, the increase in PPAR-γ activity was reversed in the presence of PPAR-γ-specific inhibitor and a mutated PPAR-γ dominant negative plasmid, supporting our hypothesis that IH can act as a ligand of PPAR-γ. Using molecular docking analysis, we demonstrate that IH formed interactions with seven polar residues and six nonpolar residues within the ligand-binding pocket of PPAR-γ that are reported to be critical for its activity and could competitively bind to PPAR-γ. IH significantly increased the expression of PPAR-γ in tumor tissues obtained from xenograft model of GC. Overall, our findings clearly indicate that antitumor effects of IH may be mediated through modulation of the PPAR-γ activation pathway in GC.

MicroRNA 135a Suppresses Lymph Node Metastasis through Down-Regulation of ROCK1 in Early Gastric Cancer

MicroRNAs (miRNAs) play a critical role in gastric cancer progression and metastasis. This study investigated the role of miRNA-135a in early gastric cancer (EGC) including lymph node (LN) metastasis. We examined the correlation between miRNA-135a expression and clinical outcomes in 59 patients who underwent surgery for EGC. Using gastric cancer cell lines, we performed functional and target gene analyses. miRNA-135a expression was down-regulated in 33.9% of patients. These patients showed a significantly more advanced stage (TNM stage≥IB, 35.0% vs. 12.8%, pu200a=u200a0.045) and higher rate of LN metastasis (30.0% vs. 5.1%, pu200a=u200a0.014) than those with up-regulation of miRNA-135a expression. In a multivariate analysis, down-regulation of miRNA-135a was an independent risk factor for LN metastasis (adjusted odds ratio, 8.04; 95% confidence interval, 1.08–59.81; pu200a=u200a0.042). Functional analyses using gastric cancer cell lines showed that miRNA-135a suppressed cell viability, epithelial-mesenchymal transition, cell invasion, and migration. ROCK1 was a target of miRNA-135a and its expression was inversely correlated to that of miRNA-135a. ROCK1 expression was significantly increased in EGC patients with LN metastasis than in those without LN metastasis. Our results confirm the tumor-suppressive role of miRNA-135a, and demonstrate its role in LN metastasis in EGC. miRNA-135a and its target gene ROCK1 may be novel therapeutic and prognostic targets for EGC.

Genomic aberrations in an African American colorectal cancer cohort reveals a MSI-specific profile and chromosome X amplification in male patients.

Objective DNA aberrations that cause colorectal cancer (CRC) occur in multiple steps that involve microsatellite instability (MSI) and chromosomal instability (CIN). Herein, we studied CRCs from AA patients for their CIN and MSI status. Experimental Design Array CGH was performed on 30 AA colon tumors. The MSI status was established. The CGH data from AA were compared to published lists of 41 TSG and oncogenes in Caucasians and 68 cancer genes, proposed via systematic sequencing for somatic mutations in colon and breast tumors. The patient-by-patient CGH profiles were organized into a maximum parsimony cladogram to give insights into the tumors aberrations lineage. Results The CGH analysis revealed that CIN was independent of age, gender, stage or location. However, both the number and nature of aberrations seem to depend on the MSI status. MSI-H tumors clustered together in the cladogram. The chromosomes with the highest rates of CGH aberrations were 3, 5, 7, 8, 20 and X. Chromosome X was primarily amplified in male patients. A comparison with Caucasians revealed an overall similar aberration profile with few exceptions for the following genes; THRB, RAF1, LPL, DCC, XIST, PCNT, STS and genes on the 20q12-q13 cytoband. Among the 68 CAN genes, all showed some level of alteration in our cohort. Conclusion Chromosome X amplification in male patients with CRC merits follow-up. The observed CIN may play a distinctive role in CRC in AAs. The clustering of MSI-H tumors in global CGH data analysis suggests that chromosomal aberrations are not random.

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

Background 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. Methods 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. Results Cases and controls differed in vitamin D status (D3<50 nmol/L: Median of 35.5 in cases vs. 36.8 in controls nmol/L; Pu200a=u200a0.05). Low levels of 25(OH)D3 (<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. Conclusion We found an inverse association between serum 25(OH)D3 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.

Gastrokine 1 regulates NF-κB signaling pathway and cytokine expression in gastric cancers.

Gastrokine 1 (GKN1) plays an important role in the gastric mucosal defense mechanism and also acts as a functional gastric tumor suppressor. In this study, we examined the effect of GKN1 on the expression of inflammatory mediators, including NF‐κB, COX‐2, and cytokines in GKN1‐transfected AGS cells and shGKN1‐transfected HFE‐145 cells. Lymphocyte migration and cell viability were also analyzed after treatment with GKN1 and inflammatory cytokines in AGS cells by transwell chemotaxis and an MTT assay, respectively. In GKN1‐transfected AGS cells, we observed inactivation and reduced expression of NF‐κB and COX‐2, whereas shGKN1‐transfected HFE‐145 cells showed activation and increased expression of NF‐κB and COX‐2. GKN1 expression induced production of inflammatory cytokines including IL‐8 and ‐17A, but decreased expression of IL‐6 and ‐10. We also found IL‐17A expression in 9 (13.6%) out of 166 gastric cancer tissues and its expression was closely associated with GKN1 expression. GKN1 also acted as a chemoattractant for the migration of Jurkat T cells and peripheral B lymphocytes in the transwell assay. In addition, GKN1 significantly reduced cell viability in both AGS and HFE‐145 cells. These data suggest that the GKN1 gene may inhibit progression of gastric epithelial cells to cancer cells by regulating NF‐κB signaling pathway and cytokine expression. J. Cell. Biochem. 114: 1800–1809, 2013.

Gastric Helicobacter pylori infection associates with an increased risk of colorectal polyps in African Americans.

BackgroundGastric Helicobacter pylori (H. pylori) infection and colorectal polyps are more prevalent in African Americans than in the general population. We aimed to investigate whether gastric H. pylori infection is associated with colorectal polyps in African Americans.MethodsMedical records of African Americans, 40xa0years and older (nu2009=u20091256) who underwent bidirectional gastrointestinal endoscopy on the same day were reviewed. H. pylori status was assessed by immunohistochemistry on gastric specimens. Colorectal polyps were confirmed by histological examination of colorectal biopsies. A subset of serum samples from healthy and polyp-bearing patients (nu2009=u2009163) were analyzed by ELISA for anti-H. pylori and anti-CagA antibodies. The crude and adjusted effect of H. pylori on the risk of colorectal adenoma and polyp were computed by logistic regression models.ResultsThe prevalence of colorectal polyps and adenomas were 456 (36%) and 300 (24%) respectively. Colorectal polyps were more prevalent in gastric H. pylori infected than non-infected subjects [43% vs. 34%; Odds Ratio (OR) (95% CI): 1.5 (1.2-1.9), Pu2009=u20090.001]. Patients with H. pylori-associated chronic active gastritis were at high risk to have adenomas [Unadjusted OR (95% CI): 1.3 (1.0-1.8); Pu2009=u20090.04]. There was no difference in histopathology, size, or location of polyps with respect to H. pylori status. Gastric H. pylori infection, age, male gender and high risk clinical presentations were independent risk factors for colorectal polyps. Serological testing also revealed a higher prevalence of H. pylori and its toxin Cag-A in polyp patients vs. non polyp patients’ sera, although in a non-statistically significant manner.ConclusionsThis study showed that current gastric H. pylori infection is associated with an increased risk of colorectal polyps in African Americans. Patients with H. pylori induced gastritis may benefit from early screening colonoscopy as a preventative measure for colorectal cancer.

Racial Disparity in Gastrointestinal Cancer Risk

Cancer from the gastrointestinal tract and its associated excretory organs will occur in more than 300,000 Americans in 2017, with colorectal cancer responsible for >40% of that burden; there will be more than 150,000 deaths from this group of cancers in the same time period. Disparities among subgroups related to the incidence and mortality of these cancers exist. The epidemiology and risk factors associated with each cancer bear out differences for racial groups in the United States. Esophageal adenocarcinoma is more frequent in non-Hispanic whites, whereas esophageal squamous cell carcinoma with risk factors of tobacco and alcohol is more frequent among blacks. Liver cancer has been most frequent among Asian/Pacific Islanders, chiefly due to hepatitis B vertical transmission, but other racial groups show increasing rates due to hepatitis C and emergence of cirrhosis from non-alcoholic fatty liver disease. Gastric cancer incidence remains highest among Asian/Pacific Islanders likely due to gene-environment interaction. In addition to esophageal squamous cell carcinoma, cancers of the small bowel, pancreas, and colorectum show the highest rates among blacks, where the explanations for the disparity are not as obvious and are likely multifactorial, including socioeconomic and health care access, treatment, and prevention (vaccination and screening) differences, dietary and composition of the gut microbiome, as well as biologic and genetic influences. Cognizance of these disparities in gastrointestinal cancer risk, as well as approaches that apply precision medicine methods to populations with the increased risk, may reduce the observed disparities for digestive cancers.

ICAD deficiency in human colon cancer and predisposition to colon tumorigenesis: linkage to apoptosis resistance and genomic instability.

We previously showed that DNA fragmentation factor, which comprises a caspase-3-activated DNase (CAD) and its inhibitor (ICAD), may influence the rate of cell death by generating PARP-1-activating DNA breaks. Here we tested the hypothesis that ICAD-deficient colon epithelial cells exhibiting resistance to death stimuli may accumulate additional genetic modifications, leading to a tumorigenic phenotype. We show that ICAD deficiency may be associated with colon malignancy in humans. Indeed, an examination of ICAD expression using immunohistochemistry in an array of both colon cancer and normal tissues revealed that ICAD expression levels were severely compromised in the cancerous tissues. Upon DNA damage caused by a low dose of irradiation, ICAD cells acquire a tumorigenic phenotype. Colon epithelial cells derived from ICAD mice showed a significant resistance to death induced by the colon carcinogen dimethylhydrazine in vitro and in mice. Such resistance was associated with a decrease in PARP-1 activation. In an animal model of dimethylhydrazine-induced colon tumorigenesis, ICAD−/− mice developed significantly higher numbers of tumors with markedly larger sizes than the wild-type counterparts. Interestingly, the phenotype of the ICAD−/− mice was not associated with a significant increase in the precancerous aberrant crypt foci suggesting a potential link to tumor progression rather than initiation. More importantly, ICAD deficiency was associated with severe genomic instability as assessed by array comparative genomic hybridization. Such genomic instability consisted most prominently of amplifications but with sizable deletions as compared to the wild-type counterparts affecting several cancer-related genes including RAF-1, GSN, LMO3, and Fzd6 independently of p53. Altogether, our results present a viable case for the involvement of ICAD deficiency in colon carcinogenesis and show that apoptosis and genomic instability may comprise the means by which such deficiency may contribute to the process of increasing susceptibility to carcinogen-induced tumorigenesis.

Targeted Exome Sequencing Outcome Variations of Colorectal Tumors within and across Two Sequencing Platforms

BACKGROUND AND AIMnNext generation sequencing (NGS) has quickly the tool of choice for genome and exome data generation. The multitude of sequencing platforms as well as the variabilities within each platform need to be assessed. In this paper we used two platforms (ION TORRENT AND ILLUMINA) to assess single nucleotides variants in colorectal cancer (CRC) specimens.nnnMETHODSnCRC specimens (n = 13) collected from 6 CRC (cancer and matched normal) patients were used to establish the mutational profile using ION TORRENT AND ILLUMINA sequencing platforms. We analyzed a set of samples from Formalin Fixed Paraffin Embedded and FF (FF) samples on both platforms to assess the effect of sample nature (FFPE vs. FF) on sequencing outcome and to evaluate the similarity/differences of SNVs across the two platforms. In addition, duplicates of FF samples were sequenced on each platform to assess variability within platform.nnnRESULTSnThe comparison of FF replicates to each other gave a concordance of 77% (± 15.3%) in Ion Torrent and 70% (± 3.7%) in Illumina. FFPE vs. FF replicates gave a concordance of 40% (± 32%) in Ion Torrent and 49% (± 19%) in Illumina. For the cross platform concordance were FFPE compared to FF (Average of 75% (± 9.8%) for FFPE samples and 67% (± 32%) for FF and 70% (± 26.8%) overall average).nnnCONCLUSIONnOur data show a significant variability within and across platforms. Also the number of detected variants depend on the nature of the specimen; FF vs. FFPE. Validation of NGS discovered mutations is a must to rule-out false positive mutants. This validation might either be performed through a second NGS platform or through Sanger sequencing.