Abul Elahi

The human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) DNA repair enzyme and its association with lung cancer risk.

OBJECTIVE The human 8-oxoguanine DNA N-glycosylase 1 gene encodes a DNA glycosylase that is involved in the base excision repair of 8-hydroxy-2-deoxyguanine from oxidatively-damaged DNA and expressed in lung tissue. The codon 326 polymorphism in the hOGG1 gene has been suggested to reduce DNA repair enzyme activity based on in vitro functional analysis. The goal of the present study is to determine whether the codon 326 polymorphism was significantly associated with alterations in individual risk for lung cancer. METHODS To determine whether hOGG1 plays a role in risk for lung cancer, we measured the prevalence of the Ser326Cys polymorphism in incident lung cancer patients and matched non-cancer controls. hOGG1 genotyping was performed by PCR-restriction fragment length polymorphism analysis of genomic DNA isolated from 179 Caucasian lung cancer cases and 358 controls individually matched in a 1:2 ratio by race-, sex- and age (+/- 5 years). RESULTS Significantly increased risk for lung cancer was observed for both the hOGG1 326 (odds ratio [OR] = 1.9, 95% confidence interval [CI] = 1.2-2.9) and hOGG1 326 genotypes (OR = 3.8, 95% CI = 1.4-10.6). The increased risk for lung cancer was observed for subjects with both the hOGG1 326 (OR = 1.7, 95% CI = 1.1-2.8) and hOGG1 326 genotypes (OR = 4.9, 95% CI = 1.5-16.1) in ever-smokers. A significant association was found between hOGG1 genotypes and lung cancer risk with a dose-dependent effect with smoking. Significantly increased risk for variant hOGG1 genotypes was observed for all non-small cell lung cancer patients. CONCLUSION These results suggest that the hOGG1 Ser326Cys polymorphism plays an important role in the risk for lung cancer and is linked to exposure to tobacco smoke.

Detection of UGT1A10 polymorphisms and their association with orolaryngeal carcinoma risk

UGT1A10 exhibits glucuronidating activity against metabolites of the tobacco smoke carcinogen, benzo(a)pyrene, and is expressed highly in numerous target tissues for tobacco‐related cancers including the upper aerodigestive tract. The current study was conducted to determine the prevalence of genetic polymorphisms in the UGT1A10‐specific region of the UDP‐glucuronosyltransferase family 1A locus and their relationship with risk for orolaryngeal carcinoma.

Genetic analysis of microsomal epoxide hydrolase gene and its association with lung cancer risk

The human microsomal epoxide hydrolase (EH) gene contains polymorphic alleles, which may be linked to increased risk for tobacco-related lung cancer. The purpose of this study is to screen new polymorphisms and determine whether these polymorphisms can be used to predict individual susceptibility to lung cancer. The polymerase chain reaction–single strand conformation polymorphism (PCR-SSCP) analysis was used to screen for polymorphisms in the coding region of the EH gene. Eleven polymorphisms, including previously reported polymorphisms, were identified and the prevalence of these variants was assessed in at least 50 healthy Caucasians and African–Americans. Among the 11 polymorphisms, the prevalence of the amino acid-changing EH polymorphisms in codons 43, 113 and 139 was examined in 182 Caucasian incident cases with primary lung cancer, as well as in 365 frequency-matched controls to examine the role of EH polymorphisms in lung cancer risk. A significant increase in lung cancer risk was observed for predicted high EH activity genotypes (odds ratio (OR) 2.3, 95% confidence interval (CI) 1.2–4.3) as compared with low EH activity genotypes. This association was more pronounced among patients with lung adenocarcinoma (OR 4.7, 95% CI 1.7–13.1). These results suggest that the EH polymorphism plays an important role in lung cancer risk and is linked to tobacco smoke exposure.

HPP1‐mediated tumor suppression requires activation of STAT1 pathways

HPP1 is a recently discovered gene that is epigenetically silenced in a number of tumor types, suggesting a potential role as a tumor suppressor. However, whether HPP1 has tumor suppressor activity is not clearly known. We have sought to investigate the effects of HPP1 on tumor growth and survival and to identify signaling pathways that mediate HPP1s mechanism of action. Forced expression of HPP1 into HCT116 colon cancer cell lines blocked the ability of HCT116 tumors to grown in vivo in nude mice. In cell culture, ectopic expression of HPP1 induces apoptosis and potently inhibits soft agar colony formation. HPP1 overexpression was also associated with a moderate reduction in in vitro proliferation characterized by an accumulation of cells in the G0/G1 phase of the cell cycle. Microarray analysis revealed that ectopic expression of HPP1 resulted in a dramatic upregulation of STAT1 as well as a large number of associated interferon‐inducible genes. RNA interference‐mediated abrogation of STAT1 reversed HPP1s antiproliferative effects. We conclude that HPP1 demonstrates tumor suppressive and pro‐apoptotic activity, both in vitro and in vivo. Coupled with its inactivation in a number of tumor types, our data provides evidence to support the role of HPP1 as a tumor suppressor gene. Moreover, activation of the STAT1 pathway likely represents the principal mediator of HPP1s tumor suppressive properties.

RAB32 hypermethylation and microsatellite instability in gastric and endometrial adenocarcinomas

The recently described gene, RAB32, is a ras proto‐oncogene family member that encodes an A‐kinase‐anchoring protein. RAB32 has been found to be frequently hypermethylated in microsatellite instability‐high (MSI‐H) colon cancers. We sought to determine the prevalence of RAB32 hypermethylation in gastric and endometrial adenocarcinomas, the 2 other major tumor types in which MSI‐H is common. Moreover, we delineated the association of RAB32 hypermethylation with microsatellite instability (MSI) and hMLH1 hypermethylation. MSI status and hypermethylation of the RAB32 and hMLH1 genes were studied in paired primary normal and tumor tissues from 48 patients with gastric cancer. An additional 80 endometrial cancer patients were studied for RAB32 methylation and MSI status. Thirteen (27%) of 48 gastric cancers demonstrated evidence of RAB32 hypermethylation. MSI status was determined in 46 of the tumors, with 7 (100%) of 7 MSI‐H tumors, 1 (33%) of 3 MSI‐low (MSI‐L) tumors and 4 (11%) of 36 microsatellite‐stable (MSS) tumors found to harbor RAB32 hypermethylation. RAB32 methylation was significantly associated with intestinal type histology and concomitant hMLH1 hypermethylation in gastric cancer. In contrast, RAB32 methylation occurred in only 1 of 80 endometrial cancers, including 20 MSI‐H, 8 MSI‐L and 52 MSS tumors. Hypermethylation of hMLH1 was noted in 16 (20%) of 80 endometrial tumors. We conclude that although RAB32 methylation is rare in endometrial cancers, it is strongly associated with hMLH1 hypermethylation and MSI in gastric adenocarcinomas. Given its similar involvement in colon cancer, RAB32 inactivation may represent a component of the oncogenic pathway of microsatellite‐unstable gastrointestinal adenocarcinomas.

Complementary Strand MicroRNAs Mediate Acquisition of Metastatic Potential in Colonic Adenocarcinoma

BackgroundAltered expression of specific microRNAs (miRNA) is known to occur during colorectal carcinogenesis. However, little is known about the genome-wide alterations in miRNA expression during the neoplastic progression of primary colorectal cancers.MethodsUsing a miRNA array platform, we evaluated the expression of 668 miRNA in primary colonic adenocarcinomas. Biological functions of selected miRNA were evaluated with in vitro invasion assays.ResultsRNA was extracted for miRNA analysis from 65 primary colon cancers. We identified a seven-miRNA expression signature that differentiated stage I and stage IV primary colon cancers. We then demonstrated this signature was able to discriminate between stage II and III primary colon cancers. Six differentially expressed miRNA were downregulated in association with the development of metastases, and all 7 miRNA were complementary strand miRNA. We transfected HCT-116, a highly invasive colon cancer cell line, with corresponding downregulated miRNA and demonstrated that overexpression of three miRNA (miR200c*, miR143*, and miR424*) significantly abrogated invasive potential.ConclusionWe have identified a seven-miRNA signature that is associated with metastatic potential in the primary tumor. Forced overexpression of three downregulated miRNA resulted in attenuation of in vitro invasion, suggesting direct tumor suppressive function and further supporting the biological importance of complementary strand miRNA.

HPV L1 capsid protein detection and progression of anal squamous neoplasia.

The progression of cervical intraepithelial lesions to invasive cancer is associated with corresponding reductions in human papillomavirus (HPV) L1 capsid antigen (L1) expression. We sought to determine whether a similar loss of L1 occurs during anal carcinogenesis using immunohistochemistry on paraffin-embedded sections as well as INNO-LiPA HPV Genotyping (Innogenetics, Gent, Belgium) technology to determine HPV infection status. We analyzed 31 squamous cell carcinomas (SCCs), 26 SCCs in situ (SCC-IS), and 11 normal anal mucosae from 36 patients. High-risk HPV subtypes were detected in all patients. L1 nuclear staining was identified in 38% of SCC-IS; however, there was no detection in normal anal mucosae, SCC, or recurrent SCC. Of those SCC-IS associated with a concomitant invasive SCC, only 15% demonstrated nuclear L1 expression as compared to 62% of isolated SCC-IS (P = .02). Nuclear expression of L1 is lost in the progression of anal SCC-IS to SCC and may serve as a possible prognostic marker of enhanced malignant potential.

Metabolic syndrome and colorectal cancer: Is hyperinsulinemia/insulin receptor-mediated angiogenesis a critical process?

OBJECTIVE Components of metabolic syndrome (MS) have been individually linked to colorectal cancer risk and prognosis; however, an understanding of the dominant mechanisms is lacking. MATERIALS AND METHODS Twenty-one patients (10 MS; 11 non-MS) with resectable colorectal cancer were prospectively enrolled. Patients were classified for MS by the World Health Organization criteria and tested for circulating vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1), fasting insulin, and tumor expression of IGF-1 receptor (IGF-1R), insulin-receptor (IR) and receptor for advanced glycation end-products (RAGE). Circulating markers were re-tested 6 months after surgery. RESULTS The MS group had significantly higher baseline and post-operative fasting insulin levels (p < 0.001 and 0.003). No differences were observed in circulating IL-6, VEGF, IGF-1 and free IGF-1. By immunohistochemistry (IHC), IGF-1R expression was significantly higher in tumor vs. normal tissues (p < 0.001) while IR expression showed no difference. Interestingly, 64% of tumors demonstrated high IR positivity in the vessels within or surrounding the tumor stroma, but not in the vessels away from the tumor. By reverse transcription polymerase chain reaction (RT-PCR), tumor IGF-1R over-expression (80%) was confirmed, but there was no difference between MS and non-MS patients. Tumor RAGE over-expression was found in 67% of patients and was equally distributed between the two groups. CONCLUSIONS Hyperinsulinemia was the only significant factor distinguishing patients with colorectal cancer who have MS. The preferential over-expression of IR in the peri-tumoral microvessels suggests that hyperinsulinemia might contribute to colorectal cancer growth by enhancing angiogenesis.

DNA Methylation Profiling across the Spectrum of HPV-Associated Anal Squamous Neoplasia

Background Changes in host tumor genome DNA methylation patterns are among the molecular alterations associated with HPV-related carcinogenesis. However, there is little known about the epigenetic changes associated specifically with the development of anal squamous cell cancer (SCC). We sought to characterize broad methylation profiles across the spectrum of anal squamous neoplasia. Methodology/Principal Findings Twenty-nine formalin-fixed paraffin embedded samples from 24 patients were evaluated and included adjacent histologically normal anal mucosa (NM; n = 3), SCC-in situ (SCC-IS; n = 11) and invasive SCC (n = 15). Thirteen women and 11 men with a median age of 44 years (range 26–81) were included in the study. Using the SFP10 LiPA HPV-typing system, HPV was detected in at least one tissue from all patients with 93% (27/29) being positive for high-risk HPV types and 14 (93%) of 15 invasive SCC tissues testing positive for HPV 16. Bisulfite-modified DNA was interrogated for methylation at 1,505 CpG loci representing 807 genes using the Illumina GoldenGate Methylation Array. When comparing the progression from normal anal mucosa and SCC-IS to invasive SCC, 22 CpG loci representing 20 genes demonstrated significant differential methylation (p<0.01). The majority of differentially methylated gene targets occurred at or close to specific chromosomal locations such as previously described HPV methylation “hotspots” and viral integration sites. Conclusions We have identified a panel of differentially methlylated CpG loci across the spectrum of HPV-associated squamous neoplasia of the anus. To our knowledge, this is the first reported application of large-scale high throughput methylation analysis for the study of anal neoplasia. Our findings support further investigations into the role of host-genome methylation in HPV-associated anal carcinogenesis with implications towards enhanced diagnosis and screening strategies.

The interplay between histone deacetylases and c-Myc in the transcriptional suppression of HPP1 in colon cancer

HPP1 (hyperplastic polyposis protein 1), a tumor suppressor gene, is downregulated by promoter hypermethylation in a number of tumor types including colon cancer. c-Myc is also known to play a role in the suppression of HPP1 expression via binding to a promoter region cognate E-box site. The contribution of histone deacetylation as an additional epigenetic mechanism and its potential interplay with c-Myc in the transcriptional regulation of HPP1 are unknown. We have shown that the treatment of the HPP1-non-expressing colon cancer cell lines, HCT116 and DLD-1 with HDAC inhibitors results in re-expression of HPP1. RNAi-mediated knockdown of c-Myc as well as of HDAC2 and HDAC3 in HCT116 and of HDAC1 and HDAC3 in DLD-1 also resulted in significant re-expression of HPP1. Co-immunoprecipitation (IP), chromatin IP (ChIP), and sequential ChIP experiments demonstrated binding of c-Myc to the HPP1 promoter with recruitment of and direct interaction with HDAC3. In summary, we have demonstrated that c-Myc contributes to the epigenetic regulation of HPP1 via the dominant recruitment of HDAC3. Our findings may lead to a greater biologic understanding for the application of targeted use of HDAC inhibitors for anti-cancer therapy.