Marie Loh

RUNX3 Is Frequently Inactivated by Dual Mechanisms of Protein Mislocalization and Promoter Hypermethylation in Breast Cancer

A tumor suppressor function has been attributed to RUNX3, a member of the RUNX family of transcription factors. Here, we examined alterations in the expression of three members, RUNX1, RUNX2, and RUNX3, and their interacting partner, CBF-beta, in breast cancer. Among them, RUNX3 was consistently underexpressed in breast cancer cell lines and primary tumors. Fifty percent of the breast cancer cell lines (n = 19) showed hypermethylation at the promoter region and displayed significantly lower levels of RUNX3 mRNA expression (P < 0.0001) and protein (P < 0.001). In primary Singaporean breast cancers, 9 of 44 specimens showed undetectable levels of RUNX3 by immunohistochemistry. In 35 of 44 tumors, however, low levels of RUNX3 protein were present. Remarkably, in each case, protein was mislocalized to the cytoplasm. In primary tumors, hypermethylation of RUNX3 was observed in 23 of 44 cases (52%) and was undetectable in matched adjacent normal breast epithelium. Mislocalization of the protein, with or without methylation, seems to account for RUNX3 inactivation in the vast majority of the tumors. In in vitro and in vivo assays, RUNX3 behaved as a growth suppressor in breast cancer cells. Stable expression of RUNX3 in MDA-MB-231 breast cancer cells led to a more cuboidal phenotype, significantly reduced invasiveness in Matrigel invasion assays, and suppressed tumor formation in immunodeficient mice. This study provides biological and mechanistic insights into RUNX3 as the key member of the family that plays a role in breast cancer. Frequent protein mislocalization and methylation could render RUNX3 a valuable marker for early detection and risk assessment.

MicroRNA-130b regulates the tumour suppressor RUNX3 in gastric cancer

AIM Accumulating evidence indicates that RUNX3 is an important tumour suppressor that is inactivated in many cancer types. This study aimed to assess the role of microRNA (miRNA) in the regulation of RUNX3. METHODS Four bioinformatic algorithms were used to predict miRNA binding to RUNX3. The correlation between candidate miRNAs and RUNX3 expression in cell lines was determined by real-time reverse transcriptase quantitative PCR (RT-qPCR) and Western blot. Candidate miRNAs were tested for functional effects through transfection of miRNA precursors and inhibitors, and monitoring cell viability, apoptosis and Bim expression. miRNA and RUNX3 expression, RUNX3 methylation and RUNX3 protein levels were assessed in gastric tissue by RT-qPCR, Methylight analysis and immunohistochemistry, respectively. RESULTS Bioinformatics, gene and protein expression analysis in eight gastric cell lines identified miR-130b as the top candidate miRNA for RUNX3 binding. Overexpression of miR-130b increased cell viability, reduced cell death and decreased expression of Bim in TGF-beta mediated apoptosis, subsequent to the downregulation of RUNX3 protein expression. In 15 gastric tumours, miR-130b expression was significantly higher compared to matched normal tissue, and was inversely associated with RUNX3 hypermethylation. CONCLUSION Attenuation of RUNX3 protein levels by miRNA may reduce the growth suppressive potential of RUNX3 and contribute to tumourigenesis.

Inflammatory tumour microenvironment is associated with superior survival in hepatocellular carcinoma patients

BACKGROUND & AIMS Hepatocellular carcinoma (HCC) is an aggressive malignancy with few treatment options. As the status of the tumour immune microenvironment can affect progression of established tumours, we evaluated potential immune mechanisms associated with survival in HCC. METHODS Immune gene expression profiles were analyzed in tumour and non-tumour liver tissues from resected HCC patients using quantitative PCR and immunohistochemistry. Tumour-infiltrating leukocytes (TILs) were isolated to verify the expression of immune genes and to identify proliferating TILs. These parameters were analyzed statistically in relation with patient survival and tumour phenotype (apoptosis and proliferation). RESULTS The immune microenvironment within tumours was found to be heterogeneous, although globally more inert compared to the adjacent non-tumour liver tissue. Univariate analysis in 61 patients identified a group of innate immune genes whose expression within tumours is positively associated with patient survival. TNF, IL6 and CCL2 are the most significant genes, with TNF being an independent predictor of survival in multivariate analysis. The gene set includes macrophage and NK-associated molecules such as TLR4, TLR3, CCR2, NCR3. Most of these molecules are expressed by TILs. Importantly, proliferating immune cells, predominantly NK and T cells, are present in tumours of patients with longer survival, and exclusively in areas devoid of proliferating tumour cells. NK and CD8(+) T cell densities are correlated positively with tumour apoptosis, and negatively with tumour proliferation. CONCLUSIONS Hence, an inflammatory immune microenvironment within HCC tumours could be an important means to control tumour progression via TIL activation and proliferation.

Epigenome-wide association of DNA methylation markers in peripheral blood from Indian Asians and Europeans with incident type 2 diabetes: a nested case-control study.

BACKGROUND Indian Asians, who make up a quarter of the worlds population, are at high risk of developing type 2 diabetes. We investigated whether DNA methylation is associated with future type 2 diabetes incidence in Indian Asians and whether differences in methylation patterns between Indian Asians and Europeans are associated with, and could be used to predict, differences in the magnitude of risk of developing type 2 diabetes. METHODS We did a nested case-control study of DNA methylation in Indian Asians and Europeans with incident type 2 diabetes who were identified from the 8-year follow-up of 25 372 participants in the London Life Sciences Prospective Population (LOLIPOP) study. Patients were recruited between May 1, 2002, and Sept 12, 2008. We did epigenome-wide association analysis using samples from Indian Asians with incident type 2 diabetes and age-matched and sex-matched Indian Asian controls, followed by replication testing of top-ranking signals in Europeans. For both discovery and replication, DNA methylation was measured in the baseline blood sample, which was collected before the onset of type 2 diabetes. Epigenome-wide significance was set at p<1 × 10(-7). We compared methylation levels between Indian Asian and European controls without type 2 diabetes at baseline to estimate the potential contribution of DNA methylation to increased risk of future type 2 diabetes incidence among Indian Asians. FINDINGS 1608 (11·9%) of 13 535 Indian Asians and 306 (4·3%) of 7066 Europeans developed type 2 diabetes over a mean of 8·5 years (SD 1·8) of follow-up. The age-adjusted and sex-adjusted incidence of type 2 diabetes was 3·1 times (95% CI 2·8-3·6; p<0·0001) higher among Indian Asians than among Europeans, and remained 2·5 times (2·1-2·9; p<0·0001) higher after adjustment for adiposity, physical activity, family history of type 2 diabetes, and baseline glycaemic measures. The mean absolute difference in methylation level between type 2 diabetes cases and controls ranged from 0·5% (SD 0·1) to 1·1% (0·2). Methylation markers at five loci were associated with future type 2 diabetes incidence; the relative risk per 1% increase in methylation was 1·09 (95% CI 1·07-1·11; p=1·3 × 10(-17)) for ABCG1, 0·94 (0·92-0·95; p=4·2 × 10(-11)) for PHOSPHO1, 0·94 (0·92-0·96; p=1·4 × 10(-9)) for SOCS3, 1·07 (1·04-1·09; p=2·1 × 10(-10)) for SREBF1, and 0·92 (0·90-0·94; p=1·2 × 10(-17)) for TXNIP. A methylation score combining results for the five loci was associated with future type 2 diabetes incidence (relative risk quartile 4 vs quartile 1 3·51, 95% CI 2·79-4·42; p=1·3 × 10(-26)), and was independent of established risk factors. Methylation score was higher among Indian Asians than Europeans (p=1 × 10(-34)). INTERPRETATION DNA methylation might provide new insights into the pathways underlying type 2 diabetes and offer new opportunities for risk stratification and prevention of type 2 diabetes among Indian Asians. FUNDING The European Union, the UK National Institute for Health Research, the Wellcome Trust, the UK Medical Research Council, Action on Hearing Loss, the UK Biotechnology and Biological Sciences Research Council, the Oak Foundation, the Economic and Social Research Council, Helmholtz Zentrum Munchen, the German Research Center for Environmental Health, the German Federal Ministry of Education and Research, the German Center for Diabetes Research, the Munich Center for Health Sciences, the Ministry of Science and Research of the State of North Rhine-Westphalia, and the German Federal Ministry of Health.

Polymorphisms of the insertion / deletion ACE and M235T AGT genes and hypertension: surprising new findings and meta-analysis of data.

BackgroundEssential hypertension is a common, polygenic, complex disorder resulting from interaction of several genes with each other and with environmental factors such as obesity, dietary salt intake, and alcohol consumption. Since the underlying genetic pathways remain elusive, currently most studies focus on the genes coding for proteins that regulate blood pressure as their physiological role makes them prime suspects.The present study examines how polymorphisms of the insertion/deletion (I/D) ACE and M235T AGT genes account for presence and severity of hypertension, and embeds the data in a meta-analysis of relevant studies.MethodsThe I/D polymorphisms of the ACE and M235T polymorphisms of the AGT genes were determined by RFLP (restriction fragment length polymorphism) and restriction analysis in 638 hypertensive patients and 720 normotensive local blood donors in Weisswasser, Germany. Severity of hypertension was estimated by the number of antihypertensive drugs used.ResultsNo difference was observed in the allele frequencies and genotype distributions of ACE gene polymorphisms between the two groups, whereas AGT TT homozygotes were more frequent in controls (4.6% vs. 2.7%, P = .08). This became significant (p = 0.035) in women only. AGT TT genotype was associated with a 48% decrease in the risk of having hypertension (odds ratio: 0.52; 95% CI, 0.28 to 0.96), and this risk decreased more significantly in women (odds ratio: 0.28; 95% CI, 0.1 to 0.78). The meta-analysis showed a pooled odds ratio for hypertension of 1.21 (TT vs. MM, 95% CI: 1.11 to 1.32) in Caucasians. No correlation was found between severity of hypertension and a specific genotype.ConclusionThe ACE I/D polymorphism does not contribute to the presence and severity of essential hypertension, while the AGT M235T TT genotype confers a significantly decreased risk for the development of hypertension in the population studied here. This contrasts to the findings of meta-analyses, whereby the T allele is associated with increased risk for hypertension.

Meta-analysis of genetic polymorphisms and gastric cancer risk: Variability in associations according to race

The goal of this study was to consolidate information on genetic risk factors for gastric cancer. An additional aim was to investigate the influence of race on these genetic risk associations. Relevant studies were identified from PubMed and references of retrieved articles. Meta-analysis techniques were used to summarise associations between genetic polymorphisms and gastric cancer. A total of 203 relevant studies were identified, assessing 225 polymorphisms across 95 genes. Subgroup analysis indicated that Chinese, Japanese and Korean data were consistent and could be pooled. However, 6 of 13 polymorphisms (ACE I/D, CCND1 870G>A, CDH1 -160C>A, IL1B -511C>T, IL4 -590C>T, IL10 -592A>C) displayed conflicting effects between Asian and Caucasian populations, three of which (ACE I/D, CCND1 870G>A, IL1B -511C>T) had significantly different odds ratios between the two racial groups. In total, 37 polymorphisms across 27 genes were found to be significantly associated with gastric cancer in Asians, and 12 polymorphisms across 11 genes in Caucasians. Consolidated panels of polymorphisms associated with gastric cancer risk were identified in Asians and Caucasians. The results caution against the assumption that genetic risk factors are consistent between races.

Comprehensive profiling of DNA methylation in colorectal cancer reveals subgroups with distinct clinicopathological and molecular features

BackgroundMost previous studies of the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) have been conducted on a relatively small numbers of CpG sites. In the present study we performed comprehensive DNA methylation profiling of CRC with the aim of characterizing CIMP subgroups.MethodsDNA methylation at 1,505 CpG sites in 807 cancer-related genes was evaluated using the Illumina GoldenGate® methylation array in 28 normal colonic mucosa and 91 consecutive CRC samples. Methylation data was analyzed using unsupervised hierarchical clustering. CIMP subgroups were compared for various clinicopathological and molecular features including patient age, tumor site, microsatellite instability (MSI), methylation at a consensus panel of CpG islands and mutations in BRAF and KRAS.ResultsA total of 202 CpG sites were differentially methylated between tumor and normal tissue. Unsupervised hierarchical clustering of methylation data from these sites revealed the existence of three CRC subgroups referred to as CIMP-low (CIMP-L, 21% of cases), CIMP-mid (CIMP-M, 14%) and CIMP-high (CIMP-H, 65%). In comparison to CIMP-L tumors, CIMP-H tumors were more often located in the proximal colon and showed more frequent mutation of KRAS and BRAF (P < 0.001).ConclusionsComprehensive DNA methylation profiling identified three CRC subgroups with distinctive clinicopathological and molecular features. This study suggests that both KRAS and BRAF mutations are involved with the CIMP-H pathway of CRC rather than with distinct CIMP subgroups.

Genome-wide association analysis identifies novel blood pressure loci and offers biological insights into cardiovascular risk.

Elevated blood pressure is the leading heritable risk factor for cardiovascular disease worldwide. We report genetic association of blood pressure (systolic, diastolic, pulse pressure) among UK Biobank participants of European ancestry with independent replication in other cohorts, and robust validation of 107 independent loci. We also identify new independent variants at 11 previously reported blood pressure loci. In combination with results from a range of in silico functional analyses and wet bench experiments, our findings highlight new biological pathways for blood pressure regulation enriched for genes expressed in vascular tissues and identify potential therapeutic targets for hypertension. Results from genetic risk score models raise the possibility of a precision medicine approach through early lifestyle intervention to offset the impact of blood pressure–raising genetic variants on future cardiovascular disease risk.

The South Asian genome.

The genetic sequence variation of people from the Indian subcontinent who comprise one-quarter of the worlds population, is not well described. We carried out whole genome sequencing of 168 South Asians, along with whole-exome sequencing of 147 South Asians to provide deeper characterisation of coding regions. We identify 12,962,155 autosomal sequence variants, including 2,946,861 new SNPs and 312,738 novel indels. This catalogue of SNPs and indels amongst South Asians provides the first comprehensive map of genetic variation in this major human population, and reveals evidence for selective pressures on genes involved in skin biology, metabolism, infection and immunity. Our results will accelerate the search for the genetic variants underlying susceptibility to disorders such as type-2 diabetes and cardiovascular disease which are highly prevalent amongst South Asians.

A coherent approach for analysis of the Illumina HumanMethylation450 BeadChip improves data quality and performance in epigenome-wide association studies

DNA methylation plays a fundamental role in the regulation of the genome, but the optimal strategy for analysis of genome-wide DNA methylation data remains to be determined. We developed a comprehensive analysis pipeline for epigenome-wide association studies (EWAS) using the Illumina Infinium HumanMethylation450 BeadChip, based on 2,687 individuals, with 36 samples measured in duplicate. We propose new approaches to quality control, data normalisation and batch correction through control-probe adjustment and establish a null hypothesis for EWAS using permutation testing. Our analysis pipeline outperforms existing approaches, enabling accurate identification of methylation quantitative trait loci for hypothesis driven follow-up experiments.