Human SHPRH suppresses genomic instability through proliferating cell nuclear antigen polyubiquitination

Differential modifications of proliferating cell nuclear antigen (PCNA) determine DNA repair pathways at stalled replication forks. In yeast, PCNA monoubiquitination by the ubiquitin ligase (E3) yRad18 promotes translesion synthesis (TLS), whereas the lysine-63–linked polyubiquitination of PCNA by yRad5 (E3) promotes the error-free mode of bypass. The yRad5-dependent pathway is important to prevent genomic instability during replication, although its exact molecular mechanism is poorly understood. This mechanism has remained totally elusive in mammals because of the lack of apparent RAD5 homologues. We report that a putative tumor suppressor gene, SHPRH, is a human orthologue of yeast RAD5. SHPRH associates with PCNA, RAD18, and the ubiquitin-conjugating enzyme UBC13 (E2) and promotes methyl methanesulfonate (MMS)–induced PCNA polyubiquitination. The reduction of SHPRH by stable short hairpin RNA increases sensitivity to MMS and enhances genomic instability. Therefore, the yRad5/SHPRH-dependent pathway is a conserved and fundamental DNA repair mechanism that protects the genome from genotoxic stress

Identifying DNA methylation biomarkers for non-endoscopic detection of Barrett’s esophagus

We report a biomarker-based non-endoscopic method for detecting Barrett’s esophagus (BE), based on detecting methylated DNAs retrieved via a swallowable balloon-based esophageal sampling device. BE is the precursor of, and a major recognized risk factor for, developing esophageal adenocarcinoma (EAC). Endoscopy, the current standard for BE detection, is not cost-effective for population screening. We performed genome-wide screening to ascertain regions targeted for recurrent aberrant cytosine methylation in BE, identifying high-frequency methylation within the CCNA1 locus. We tested CCNA1 DNA methylation as a BE biomarker in cytology brushings of the distal esophagus from 173 individuals with or without BE. CCNA1 DNA methylation demonstrated an area under the curve (AUC)=0.95 for discriminating BE-related metaplasia and neoplasia cases versus normal individuals, performing identically to methylation of VIM DNA, an established BE biomarker. When combined, the resulting two biomarker panel was 95% sensitive and 91% specific. These results were replicated in an independent validation cohort of 149 individuals, who were assayed using the same cutoff values for test positivity established in the training population. To progress toward non-endoscopic esophageal screening, we engineered a well-tolerated, swallowable, encapsulated balloon device able to selectively sample the distal esophagus within 5 minutes. In balloon samples from 86 individuals, tests of CCNA1 plus VIM DNA methylation detected BE metaplasia with 90.3% sensitivity and 91.7% specificity. Combining the balloon sampling device with molecular assays of CCNA1 plus VIM DNA methylation enables an efficient, well-tolerated, sensitive, and specific method of screening at-risk populations for BE

The DNMT1-associated lincRNA DACOR1 reprograms genome-wide DNA methylation in colon cancer

Abstract Background DNA methylation is a key epigenetic mark in mammalian organisms that plays key roles in chromatin organization and gene expression. Although DNA methylation in gene promoters is generally associated with gene repression, recent studies demonstrate that DNA methylation in gene bodies and intergenic regions of the genome may result in distinct modes of gene regulation. Furthermore, the molecular mechanisms underlying the establishment and maintenance of DNA methylation in human health and disease remain to be fully elucidated. We recently demonstrated that a subset of long non-coding RNAs (lncRNAs) associates with the major DNA methyltransferase DNMT1 in human colon cancer cells, and the dysregulation of such lncRNAs contribute to aberrant DNA methylation patterns. Results In the current study, we assessed the impact of a key DNMT1-associated lncRNA, DACOR1, on genome-wide DNA methylation using reduced representation bisulfite sequencing (RRBS). Our findings demonstrated that induction of DACOR1 in colon cancer cells restores DNA methylation at thousands of CpG sites throughout the genome including promoters, gene bodies, and intergenic regions. Importantly, these sites overlap with regions of the genome that become hypomethylated in colon tumors. Furthermore, induction of DACOR1 results in repression of FOS and JUN and, consequently, reduced AP-1 transcription factor activity. Conclusion Collectively, our results demonstrate a key role of lncRNAs in regulating DNA methylation in human cells, and the dysregulation of such lncRNAs could emerge as a key mechanism by which DNA methylation patterns become altered in human tumors

Phase I/II study of azacitidine and capecitabine/oxaliplatin (CAPOX) in refractory CIMP-high metastatic colorectal cancer: evaluation of circulating methylated vimentin

International audiencePURPOSE: Hypermethylation of promoter CpG islands (CIMP) has been strongly implicated in chemotherapy resistance and is implicated in the pathogenesis of a subset of colorectal cancers (CRCs) termed CIMP-high.EXPERIMENTAL DESIGN: This phase I/II study in CRC (phase II portion restricted to CIMP-high CRC), treated fluoropyrimidine/oxaliplatin refractory patients with azacitidine (75 mg/m2/day subcutaneously D1-5) and CAPOX (capecitibine and oxaliplatin) every three weeks.RESULTS: Twenty-six patients (pts) were enrolled in this study: 15 pts (12 treated at MTD) in phase I and 11 pts in phase II. No dose limiting toxicities were observed. A total of 14 pts were CIMP-high. No responses were seen. CIMP-high status did not correlate with efficacy endpoints [stable disease (SD) or progression-free survival (PFS)] or baseline vimentin methylation level. Changes in vimentin methylation over time did not correlate with efficacy outcomes. Baseline methylated vimentin correlated with tumor volume (P<0.001) and higher levels of baseline methylation correlated with the obtainment of stable disease (P=0.04).CONCLUSIONS: Azacitidine and CAPOX were well tolerated with high rates of stable disease in CIMP-high pts, but no objective responses. Serum methylated vimentin may be associated with benefit from a regimen including a hypomethylation agent, although this study is not able to separate a potential prognostic or predictive role for the biomarker

A Novel Blood???Based Colorectal Cancer Diagnostic Technology Using Electrical Detection of Colon Cancer Secreted Protein???2

Colorectal cancer (CRC) is the second-leading cause of cancer-related mortality worldwide, which may be effectively reduced by early screening. Colon cancer secreted protein-2 (CCSP-2) is a promising blood marker for CRC. An electric-field effect colorectal sensor (E-FECS), an ion-sensitive field-effect transistor under dual gate operation with nanostructure is developed, to quantify CCSP-2 directly from patient blood samples. The sensing performance of the E-FECS is verified in 7 controls and 7 CRC samples, and it is clinically validated on 30 controls, 30 advanced adenomas, and 81 CRC cases. The concentration of CCSP-2 is significantly higher in plasma samples from CRC and advanced adenoma compared with controls (both P &lt; 0.001). Sensitivity and specificity for CRC versus controls are 44.4% and 86.7%, respectively (AUC of 0.67), and 43.3% and 86.7%, respectively, for advanced adenomas (AUC of 0.67). CCSP 2 detects a greater number of CRC cases than carcinoembryonic antigen does (45.6% vs 24.1%), and the combination of the two markers detects an even greater number of cases (53.2%). The E-FECS system successfully detects CCSP-2 in a wide range of samples including early stage cancers and advanced adenoma. CCSP-2 has potential for use as a blood-based biomarker for CRC