Bernard Kwabi-Addo

DNA methylation profile associated with rapid decline in kidney function: findings from the CRIC Study

BACKGROUND Epigenetic mechanisms may be important in the progression of chronic kidney disease (CKD). METHODS We studied the genome-wide DNA methylation pattern associated with rapid loss of kidney function using the Infinium HumanMethylation 450 K BeadChip in 40 Chronic Renal Insufficiency (CRIC) study participants (n = 3939) with the highest and lowest rates of decline in estimated glomerular filtration rate. RESULTS The mean eGFR slope was 2.2 (1.4) and -5.1 (1.2) mL/min/1.73 m(2) in the stable kidney function group and the rapid progression group, respectively. CpG islands in NPHP4, IQSEC1 and TCF3 were hypermethylated to a larger extent in subjects with stable kidney function (P-values of 7.8E-05 to 9.5E-05). These genes are involved in pathways known to promote the epithelial to mesenchymal transition and renal fibrosis. Other CKD-related genes that were differentially methylated are NOS3, NFKBIL2, CLU, NFKBIB, TGFB3 and TGFBI, which are involved in oxidative stress and inflammatory pathways (P-values of 4.5E-03 to 0.046). Pathway analysis using Ingenuity Pathway Analysis showed that gene networks related to cell signaling, carbohydrate metabolism and human behavior are epigenetically regulated in CKD. CONCLUSIONS Epigenetic modifications may be important in determining the rate of loss of kidney function in patients with established CKD.

Genome-wide differentially methylated genes in prostate cancer tissues from African-American and Caucasian men

Increasing evidence suggests that aberrant DNA methylation changes may contribute to prostate cancer (PCa) ethnic disparity. To comprehensively identify DNA methylation alterations in PCa disparity, we used the Illumina 450K methylation platform to interrogate the methylation status of 485,577 CpG sites focusing on gene-associated regions of the human genome. Genomic DNA from African-American (AA; 7 normal and 3 cancers) and Caucasian (Cau; 8 normal and 3 cancers) was used in the analysis. Hierarchical clustering analysis identified probe-sets unique to AA and Cau samples, as well as common to both. We selected 25 promoter-associated novel CpG sites most differentially methylated by race (fold change > 1.5-fold; adjusted P < 0.05) and compared the β-value of these sites provided by the Illumina, Inc. array with quantitative methylation obtained by pyrosequencing in 7 prostate cell lines. We found very good concordance of the methylation levels between β-value and pyrosequencing. Gene expression analysis using qRT-PCR in a subset of 8 genes after treatment with 5-aza-2′-deoxycytidine and/or trichostatin showed up-regulation of gene expression in PCa cells. Quantitative analysis of 4 genes, SNRPN, SHANK2, MST1R, and ABCG5, in matched normal and PCa tissues derived from AA and Cau PCa patients demonstrated differential promoter methylation and concomitant differences in mRNA expression in prostate tissues from AA vs. Cau. Regression analysis in normal and PCa tissues as a function of race showed significantly higher methylation prevalence for SNRPN (P = 0.012), MST1R (P = 0.038), and ABCG5 (P < 0.0002) for AA vs. Cau samples. We selected the ABCG5 and SNRPN genes and verified their biological functions by Western blot analysis and siRNA gene knockout effects on cell proliferation and invasion in 4 PCa cell lines (2 AA and 2 Cau patients-derived lines). Knockdown of either ABCG5 or SNRPN resulted in a significant decrease in both invasion and proliferation in Cau PCa cell lines but we did not observe these remarkable loss-of-function effects in AA PCa cell lines. Our study demonstrates how differential genome-wide DNA methylation levels influence gene expression and biological functions in AA and Cau PCa.

Identification of novel DNA-methylated genes that correlate with human prostate cancer and high-grade prostatic intraepithelial neoplasia.

Background:Prostate cancer (PCa) harbors a myriad of genomic and epigenetic defects. Cytosine methylation of CpG-rich promoter DNA is an important mechanism of epigenetic gene inactivation in PCa. There is considerable amount of data to suggest that DNA methylation-based biomarkers may be useful for the early detection and diagnosis of PCa. In addition, candidate gene-based studies have shown an association between specific gene methylation and alterations and clinicopathologic indicators of poor prognosis in PCa.Methods:To more comprehensively identify DNA methylation alterations in PCa initiation and progression, we examined the methylation status of 485 577 CpG sites from regions with a broad spectrum of CpG densities, interrogating both gene-associated and non-associated regions using the recently developed Illumina 450K methylation platform.Results:In all, we selected 33 promoter-associated novel CpG sites that were differentially methylated in high-grade prostatic intraepithelial neoplasia and PCa in comparison with benign prostate tissue samples (false discovery rate-adjusted P-value <0.05; β-value ⩾0.2; fold change >1.5). Of the 33 genes, hierarchical clustering analysis demonstrated BNC1, FZD1, RPL39L, SYN2, LMX1B, CXXC5, ZNF783 and CYB5R2 as top candidate novel genes that are frequently methylated and whose methylation was associated with inactivation of gene expression in PCa cell lines. Pathway analysis of the genes with altered methylation patterns identified the involvement of a cancer-related network of genes whose activity may be regulated by TP53, MYC, TNF, IL1 and 6, IFN-γ and FOS in prostate pathogenesis.Conclusion:Our genome-wide methylation profile shows epigenetic dysregulation of important regulatory signals in prostate carcinogenesis.

The Role of Epigenetics

Genetic variations among individuals and populations are providing insights into how information in the DNA sequence is executed at the cellular level. However, phenotypic differences between individuals cannot be entirely explained by genetic differences. Epigenetic events such as DNA-methylation changes have increasingly been found to be associated with health disparities. Social and environmental determinants, such as diet, stress, infection, and exposure to toxins can cause epigenetic changes. The epigenome of the developing fetus is especially sensitive to maternal nutrition, exposure to environmental toxins and psychological stress, and this can result in differences in health outcomes later in life. The emerging epigenetic model of health disparities points to social determinants and environmental exposures as key to addressing racial differences in disease burdens.

Abstract 388: Epigenome-wide profiling identified significant differences in DNA methylation between African-American and European-American men with prostate cancer

Background: DNA methylation is a dynamic epigenetic mark that is essential for mammalian organismal development. For human prostate cancer (PCa), abundant evidence has accumulated to suggest that somatic epigenetic alterations may appear early during cancer development, as well as more commonly and consistently, than genetic changes. Several cancer-associated genes have been reported to show significant differences in DNA methylation pattern in prostate tissues from African-American (AA) and European-American men, but the genome-wide pattern and extent of these differences are largely unknown. The current study investigated genome-wide DNA methylation differences in AA and EA prostate tissue samples with the aim of assessing the variation of genome-wide patterns of DNA methylation. Methods: To comprehensively examine the DNA methylation pattern in AA and EA samples, we used illumina 450 K methylation platform (Infunium Chip) to conduct genome-wide large scale analysis of DNA methylation changes in 7 normal and 3 PCa tissue samples from AA versus 8 normal and 3 PCa tissue samples from EA. This chip interrogates >485,000 CpG methylation sites per sample at single-nucleotide resolution and assigns each site an average beta value for quantitative methylation level. Results: Pathway analysis of the genes with altered methylation patterns identified top canonical pathways for the involvement of cancer related network for genes involved in axonal guidance, antigen presentation, androgen signaling and protein ubiquitination pathways in prostate cancer tissues compared with normal prostate tissues obtained from AA men. On the other hand, the top canonical pathways identified in prostate cancer tissues compared with normal prostate tissues obtained from EA men are genes involved in epithelial-mesenchymal transition, p53 signaling, nucleotide sugars metabolism and germ cell-sertoli cell junction signaling pathways. Conclusion: Using this quantitative sequencing-based approach, our work uncovers significant global DNA methylation alterations in AA versus EA prostate tissues and provides a mechanistic explanation for the disease disparity. Citation Format: Bernard Kwabi-Addo, Songping Wang, Joseph Devaney. Epigenome-wide profiling identified significant differences in DNA methylation between African-American and European-American men with prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 388. doi:10.1158/1538-7445.AM2014-388

Abstract 1974: Functional characterization of Basonuclin 1 (BNC1): a novel tumor suppressor gene commonly downregulated in human prostate cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Prostate cancer (PCa) harbors a myriad of aberrant genomic and epigenetic alterations. Epigenetic inactivation of genes in PCa is largely based on transcriptional silencing by aberrant CpG methylation of CpG rich promoter regions. To better understand the role of aberrant methylation changes and to identify biological pathways likely to be affected in PCa, we used a genome-wide methylation array from Illumina (Infunium) to identify novel methylated genes associated to PCa. We identified several candidate genes including BNC1 as novel genes which are frequently methylated and whose methylation closely related to inactivation of gene expression in prostate cancer cell lines. Basonuclin1 is a zinc finger transcription factor that interacts with a subset of promoters of genes transcribed by both RNA polymerase-I and -II and has roles in maintaining ribosomal biogenesis and the proliferative potential of immature epithelial cells. We found that the expression of BNC1 negatively correlated with the degree of methylation at the CpG island in prostate tumor samples compared to benign prostate tissues. The forced-expression of BNC1 significantly decreased prostate cancer cell proliferation whereas the siRNA mediated BNC1 knock-down increased prostate cancer cell proliferation. Overall, our data suggest a potential tumor suppressor role for BNC1 that includes regulation of cell proliferation and cell-cell interaction in prostate carcinogenesis. Citation Format: Bernard Kwabi-Addo, Songping Wang, Paulett Furbert-Harris, Srinivasan Yegnasubramanian, Joseph Devaney. Functional characterization of Basonuclin 1 (BNC1): a novel tumor suppressor gene commonly downregulated in human prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1974. doi:10.1158/1538-7445.AM2013-1974

Abstract 3033: NKX2-5, a potential tumor suppressor gene in prostate cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Prostate cancer (PCa) is a common malignancy and a leading cause of cancer deaths among men in the United States. Abundant evidence has accumulated to suggest that epigenetic DNA methylation changes may appear earlier during PCa development than genetic changes, as well as more commonly and consistently suggesting that DNA methylated genes can be explored as DNA-based biomarker for PCa disease detection. Recently, we have identified NKX2-5 as a novel that is hypermethylated in prostate cancer. However, there is little information about the biological significance of this gene in prostate carcinogenesis. We hypothesize that NKX2-5 is a potential tumor suppressor gene that is frequently inactivated in prostate cancer. Methods: We carried out gain-and-loss functional studies of NKX2-5 in prostate cancer cell lines and validated expression at the RNA transcript level using quantitative RT-PCR. Protein expression was analyzed by western blotting and cell cycle analysis investigated by flow cytometer. Results: Over-expression of NKX2-5 was detrimental to prostate cancer cell proliferation as evidenced by significant inhibition of prostate cancer cell proliferation in comparison to control (vector only transfection) and this was due to cell arrest in Go/G1 phase and increase apoptosis. In contract, successful knockdown of NKX2-5 by shRNA transfection increased prostate cancer cell proliferation. Western blot analysis demonstrated that NKX2-5 plays a key regulatory role in the expression of several genes including p53, PTEN, Histone H1 and the androgen receptor. Conclusion: Our observation suggests that NKX2-5, a member of the homeobox gene family of plays an important tumor suppressor activity in prostate carcinogenesis. Because this gene plays important role in several signal transduction pathways, this gene can be exploited as potential biomarker for the early detection of prostate cancer and could be an attractive target to explore for drug investigation or gene therapies of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3033. doi:10.1158/1538-7445.AM2011-3033

Epigenetic Biomarkers and Racial Differences in Cancer

Abstract The reason for the persistent cancer disparities in incidence and mortality among different ethnic and racial groups is not fully known. The risk factors for cancer disparities are multifactorial and arise from a combination of socioeconomic determinant, environmental, and genetic factors. Epigenetic changes, in particular DNA methylation, start early in life even at the prenatal stage and increase as a function of age among different racial and ethnic groups. Thus aberrant epigenetic events offer potential mechanism for perpetuating the effects of early life exposures setting the stage for future disease outcomes including cancer. Few studies showed significant differences in epigenetic events in different racial and ethnic groups. These epigenetic changes can be associated with cancer initiation, progression, aggressiveness, and disease outcomes. Therefore, concerted efforts are needed to understand the causes of aberrant epigenetic changes in cancer disparities to reduce cancer health disparities.

Introduction: The African Diaspora and Disease-Specific Disparities

This introductory chapter looks at the disparities that are associated with the incidence and mortality rates for specific diseases such as cardiovascular disease, cancer, stroke, chronic obstructive pulmonary disease, unintentional injuries, pneumonia and influenza, diabetes, suicide, kidney diseases, chronic liver disease, cirrhosis, and HIV/AIDS, which are major public/health concerns in the USA. This chapter then discusses the major causes and the risk factors associated with these diseases that disproportionally affect African Americans as compared to other ethnic/racial groups in the USA.

Behavior and Health Disparities

The roles of diet, physical activity, tobacco use, and alcohol consumption are widely recognized as determinants for poor health outcomes. However, some of the choices are themselves determined by availability such that heavy marketing of unhealthy foods, alcohol, and cigarettes, especially targeting children, is commonplace in low-income neighborhoods along with limited access to stores selling healthy foods. Many of the behavioral factors are complicated by psychological and emotional issues, such as depression. Behavioral intervention is one option to reduce health disparities.