Comparison of fluorescence in-situ hybridisation with dual-colour in-situ hybridisation for assessment of HER2 gene amplification of breast cancer in Hong Kong

published_or_final_versio

Forkhead box K2 modulates epirubicin and paclitaxel sensitivity through FOXO3a in breast cancer

published_or_final_versio

OTUB1 inhibits the ubiquitination and degradation of FOXM1 in breast cancer and epirubicin resistance

The forkhead transcription factor FOXM1 has a key role in DNA damage response, and its deregulated overexpression is associated with genotoxic drug resistance in breast cancer. However, little is known about the posttranslational mechanisms by which FOXM1 expression is regulated by genotoxic agents and how they are deregulated in resistant cells. Initial co-immunoprecipitation studies verified previous proteomic analysis finding that the OTUB1 is a novel FOXM1-interacting protein. Western blot analysis showed that both OTUB1 and FOXM1 expression reduced upon genotoxic agent treatment in MCF-7 cells, but remained relatively constant in resistant cells. FOXM1 expression reduced upon OTUB1 depletion by siRNA and increased with OTUB1 overexpression in MCF-7 cells, arguing that OTUB1 positively regulates FOXM1 expression. In agreement, co-immunoprecipitation experiments demonstrated that FOXM1 expression is associated with OTUB1 binding but inversely correlates with conjugation to the protein degradation-associated Lys-48-linked ubiquitin-chains. Overexpression of wild-type (WT) OTUB1, but not the OTUB1(C91S) mutant, disrupted the formation of Lys48-linked ubiquitin-conjugates on FOXM1. Importantly, knockdown of OTUB1 by siRNA resulted in an increase in turnover of FOXM1 in MCF-7 cells treated with the protein synthesis inhibitor cycloheximide, whereas overexpression of WT OTUB1, but not the OTUB1(C91S) mutant, significantly enhances the half-life of FOXM1. In addition, proliferative and clonogenic assays also show that OTUB1 can enhance the proliferative rate and epirubicin resistance through targeting FOXM1, as OTUB1 has little effect on FOXM1-deficient cells. The physiological relevance of the regulation of FOXM1 by OTUB1 is further underscored by the significant correlations between FOXM1 and OTUB1 expression in breast cancer patient samples. Cox-regression survival analysis indicates that OTUB1 overexpression is linked to poorer outcome in particular in patients treated with chemotherapy. Collectively, these data suggest that OTUB1 limits the ubiquitination and degradation of FOXM1 in breast cancer and has a key role in genotoxic agent resistance

BRCA1 positively regulates FOXO3 expression by restricting FOXO3 gene methylation and epigenetic silencing through targeting EZH2 in breast cancer.

BRCA1 mutation or depletion correlates with basal-like phenotype and poor prognosis in breast cancer but the underlying reason remains elusive. RNA and protein analysis of a panel of breast cancer cell lines revealed that BRCA1 deficiency is associated with downregulation of the expression of the pleiotropic tumour suppressor FOXO3. Knockdown of BRCA1 by small interfering RNA (siRNA) resulted in downregulation of FOXO3 expression in the BRCA1-competent MCF-7, whereas expression of BRCA1 restored FOXO3 expression in BRCA1-defective HCC70 and MDA-MB-468 cells, suggesting a role of BRCA1 in the control of FOXO3 expression. Treatment of HCC70 and MDA-MB-468 cells with either the DNA methylation inhibitor 5-aza-2'-deoxycitydine, the N-methyltransferase enhancer of zeste homologue 2 (EZH2) inhibitor GSK126 or EZH2 siRNA induced FOXO3 mRNA and protein expression, but had no effect on the BRCA1-competent MCF-7 cells. Chromatin immunoprecipitation (ChIP) analysis demonstrated that BRCA1, EZH2, DNMT1/3a/b and histone H3 lysine 27 trimethylation (H3K27me3) are recruited to the endogenous FOXO3 promoter, further advocating that these proteins interact to modulate FOXO3 methylation and expression. In addition, ChIP results also revealed that BRCA1 depletion promoted the recruitment of the DNA methyltransferases DNMT1/3a/3b and the enrichment of the EZH2-mediated transcriptional repressive epigenetic marks H3K27me3 on the FOXO3 promoter. Methylated DNA immunoprecipitation assays also confirmed increased CpG methylation of the FOXO3 gene on BRCA1 depletion. Analysis of the global gene methylation profiles of a cohort of 33 familial breast tumours revealed that FOXO3 promoter methylation is significantly associated with BRCA1 mutation. Furthermore, immunohistochemistry further suggested that FOXO3 expression was significantly associated with BRCA1 status in EZH2-positive breast cancer. Consistently, high FOXO3 and EZH2 mRNA levels were significantly associated with good and poor prognosis in breast cancer, respectively. Together, these data suggest that BRCA1 can prevent and reverse FOXO3 suppression via inhibiting EZH2 and, consequently, its ability to recruit the transcriptional repressive H3K27me3 histone marks and the DNA methylases DNMT1/3a/3b, to induce DNA methylation and gene silencing on the FOXO3 promoter

The role of BQ323636.1, a novel splice variant of NCOR2, in modulation of oxidative stress in breast cancer

Poster Session B: no. B3

Detection of hypermethylated DNA or cyclooxygenase-2 messenger rna in fecal samples of patients with colorectal cancer or polyps

BACKGROUND: Detection of fecal DNA is a promising approach to colorectal cancer screening. However, the sensitivity of current fecal DNA tests for colorectal polyps is low. We evaluated the feasibility of detecting aberrantly methylated DNA or cyclooxygenase-2 (COX-2) mRNA in feces of patients with colorectal cancer or polyps. METHODS: Fecal samples were collected prior to colonoscopy from 20 patients with colorectal cancer, 30 patients with colorectal polyps, and 30 subjects with normal colonic examination. Presence of hypermethylated DNA in 7 tumor-related genes (APC, ATM, hMLH1, sFRP2, HLTF, MGMT, and GSTP1) in stool was analyzed by methylation-specific PCR. COX-2 mRNA in fecal samples was detected by RT-PCR. RESULTS: With the use of this panel of methylation markers, the sensitivity of detecting colorectal cancer and adenoma was 75% (95% CI 50.9-91.3%) and 68% (95% CI 46.5-85.1%), respectively. Three normal subjects also had methylated DNA detected in stool, which gives a specificity of 90% (95% CI 73.5-97.9%). The mean number of genes methylated in DNA from the stool of patients with colorectal cancer and adenoma was 1.4 and 0.9, respectively. In contrast, COX-2 mRNA was detected in the stool samples of 10 (50%) cancer patients and one (4%) patient with advanced adenoma only. Two (6.7%) stool samples from normal subjects also had COX-2 mRNA detected. CONCLUSION: Detection of aberrantly methylated DNA in fecal samples is more sensitive than COX-2 mRNA for detection of colorectal cancer and adenoma. © 2007 by Am. Coll. of Gastroenterology.link_to_subscribed_fulltex

Dual-utility NLS Drives RNF169-dependent DNA Damage Responses

Poster Presentation: no. P2Loading of 53BP1 and RAP80 at DNA double-strand breaks (DSBs) drives cell cycle checkpoint activation but is counterproductive to high-fidelity DNA repair. RNF169 maintains the balance by limiting the deposition of DNA damage mediator proteins at the damaged chromatin. We report here that this is accomplished, in part, by a predicted NLS that not only shuttles RNF169 into the nucleus, but also promotes its stability by mediating a direct interaction with the ubiquitin specific protease USP7. Guided by the crystal structure of USP7 in complex with the RNF169 NLS, we uncoupled USP7 binding from its nuclear import function, and showed that perturbing the USP7-RNF169 complex destabilized RNF169, compromised high-fidelity DSB repair, and hyper-sensitized cells to PARP inhibition. Finally, expression of USP7 and RNF169 positively correlated in breast cancer specimens. Collectively, our findings uncover an NLS-mediated bipartite mechanism that supports the nuclear function of a DSB response protein

Quantitative detection of promoter hypermethylation in multiple genes in the serum of patients with colorectal cancer

OBJECTIVES: While promoter hypermethylation is a common molecular alteration of human colorectal cancer that could be detected in the bloodstream, we tested the feasibility of quantitative detection of aberrant DNA methylation in multiple genes in the serum samples of colorectal cancer patients. METHODS: The pre-therapeutic serum samples of 49 colorectal cancer patients and 41 age-matched controls with normal colonoscopy were examined. The presence of methylated DNA in APC (adenomatous polyposis coli), hMLH1 (human MutL homolog 1), and HLTF (helicase-like transcription factor) was detected by quantitative methylation-specific PCR (MethyLight). RESULTS: There was a significant difference in the concentration of methylated serum DNA between cancer patients and controls for HLTF (p= 0.015) and hMLH1 (p= 0.0001) genes, but not for APC gene (p= 0.21). In total, 28 patients with colorectal cancer and 4 controls had methylated DNA detected in at least one marker, which gave a sensitivity of 57% and specificity of 90%. All patients with methylation in two methylation markers had advanced (stage III/IV) cancer (p= 0.006) and patients with methylation in at least one marker tended to have a lower probability of survival (p= 0.08). CONCLUSION: The quantitative detection of aberrant DNA methylation in serum may be a promising high-throughput approach for the noninvasive screening and monitoring of colorectal cancer. © 2005 by Am. Coll. of Gastroenterology Published by Blackwell Publishing.link_to_subscribed_fulltex

H. pylori genotypes and cytokine gene polymorphisms influence the development of gastric intestinal metaplasia in a chinese population

BACKGROUND: Cytokine gene polymorphisms and Helicobacter pylori (HP) genotypes have been linked to gastric cancer development in Western countries. We determined the role of host cytokine polymorphisms and bacterial virulent factors in the development of gastric intestinal metaplasia (IM) in a Chinese population with a high background gastric cancer incidence. METHODS: Three hundred two HP-infected noncancer individuals living in Shandong province of China with available DNA were studied. Polymorphisms in different loci of inflammatory cytokines Interleukin IL-1B, IL-1RN, Interleukin IL-8, IL-10, IL-18, tumor necrosis factor-A (TNF-A), and Transforming growth factor (TGF-B), were determined by allelic discriminating TaqMan polymerase chain reaction (PCR) or a variable number of tandem repeats. Presence of HP virulence factors in cagA, vacA, and babA2 were determined by PCR. Baseline gastric biopsies were assessed for the presence of IM. RESULTS: Among HP-infected subjects, carriers of the IL-1B-511 T allele were associated with a modestly greater prevalence of IM (adjusted OR 2.0, 95% CI 1.0-3.7). There was no association between the presence of IM and polymorphisms in other inflammatory cytokines. Although most subjects from this region harbored the virulent HP strains, carriage of the vacA m1 strain was associated with a significantly higher prevalence of IM (adjusted OR 1.8, 1.1-3.0). The presence of both host (IL-1B-511 T) and HP (vacA m1) genotypes further increased the risk of IM (OR 5.7, 2.0-16) when compared with individuals with the low-risk genotype. CONCLUSION: The carriage of proinflammatory IL-1B-511 and HP vacA m1 genotypes was associated with the development of gastric IM in the Chinese. © 2006 by Am. Coll. of Gastroenterology.link_to_subscribed_fulltex

Effects of Helicobacter pylori eradication on methylation status of E-cadherin gene in noncancerous stomach

Purpose: Promoter hypermethylation of E-cadherin plays an important role on gastric cancer development. Whereas E-cadherin methylation was frequently detected in the stomach of Helicobacter pylori - infected individuals, we tested whether eradication of H. pylori alters the methylation status of the noncancerous gastric epithelium. Experimental Design: Endoscopic biopsies were taken from the antrum and corpus of H. pylori - infected subjects without gastric cancer. Presence of methylated E-cadherin sequences in the gastric specimens was detected by methylation-specific PCR. Bisulfite DNA sequencing was done to determine the topographical distribution and changes in methylation profiles with H. pylori eradication. Results: Among the 28 H. pylori - infected subjects (median age, 44.5 years), 15 (53.6%) had E-cadherin methylation detected in stomach at baseline. Discordant methylation patterns between the antrum and corpus were noted in six patients. One year after successful H. pylori eradication, there was a significant reduction in the methylation density of the promoter region and exon 1 of the E-cadherin gene as detected by bisulfite DNA sequencing (P < 0.001). Conclusion: Promoter methylation in E-cadherin was frequently detected in the stomach of H. pylori - infected individuals. Eradication of H. pylori might possibly reduce the methylation density in E-cadherin gene and the chance of subsequent neoplastic transformation. © 2006 American Association for Cancer Research.link_to_subscribed_fulltex