Detection of Methylation Targets in Advanced Adenoma in African Americans: Discovery of Colorectal Cancer Risk Markers

Colorectal cancer (CRC) arises from epigenetic and genetic alterations in a stepwise histological progression sequence. CpG island methylator phenotype (CIMP) is one of the basic mechanisms in initiation of CRC. The objectives were to; 1) Establish a set of CIMP markers as a methylome signature in high-risk population such as African Americans (AAs) with CRC to be granted a better value for clinical treatment and management. A systematic review was used to identify CIMP and non-CIMP markers that were hypermethylated, and a list of the 16 non-CIMP gene panel was formed based on Illumina Human Methylation 27 array (IHM27) data and review of the literature. 2) Validate the list of cancer candidate methylated genes in AAs and Caucasians by Methylation specific PCR (MSP). The rate of methylation in AAs was significantly higher for GPNMB, CHD5, and ICAM5. 3) Examine the role of GPNMB gene in normal, adenoma and CRC in AA patients to elucidate epigenetic drivers of colon oncogenic transformation. Methylation status of 13 CpG sites in GPNMB gene's promoter was analyzed by pyrosequencing in human CRC cell lines as well as paraffin embedded samples with normal, non-advanced adenoma (NA), advanced adenoma (AD), and cancer tissue. GPNMB expression was analyzed by immunohistochemistry (IHC). The functions of GPNMB were examined by cell proliferation, migration and invasion assays in HCT116 colon cell line that was stably transfected with a GPNMB cDNA expression vector. GPNMB methylation was lower in normal mucosa compared to CRC samples (P<0.001). AD also had a significantly higher GPNMB methylation frequency than normal colon samples (P<0.001). The frequency of GPNMB methylation in NA differs significantly from that in the normal mucosa (P<0.009). GPNMB protein expression decreased in CRC tissues compared with AD and NA colon mucosa. GPNMB overexpression in HCT116 colon cancer cell line decreased cell proliferation (P<0.02), invasion (P0.2). Our data indicate a high methylation profile and a lower GPNMB expression in adenoma and CRC samples. As such, GPNMB might be useful as a biomarker of adenomas with high carcinogenic potential in AAs.