Background: Colorectal cancer (CRC) arises from epigenetic and genetic alterations in a stepwise histological progression sequence. Aim: We examined the role of glycoprotein non-metastatic melanoma protein B (GPNMB) gene in normal, adenoma and CRC in African American (AA) patients to elucidate epigenetic drivers of colon oncogenic transformation.

Patients and Methods: Methylation status of 13 CpG sites (chr7: 23287345-23287426) in GPNMB gene's promoter, selected from IHM27 array methylation profiling, was analyzed by pyrosequencing in human CRC cell lines (HCT116, SW480, and HT29) as well as paraffin embedded samples with normal (n=20), non-advanced adenoma (NA; n=20), advanced adenoma (AD; n=48), and cancer tissue (n=20). Furthermore, GPNMB expression was analyzed by immunohistochemistry (IHC). Tumor suppressor 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. In addition, correlations between the methylation status of GPNMB and various clinicopathological features (age, location and sex) were analyzed.

Results: GPNMB methylation was lower in normal mucosa compared to CRC samples (4/20 [20%] vs. 19/20 [95%]; P<0.001). AD also had a significantly higher GPNMB methylation frequency than normal colon samples (45/48 [94%] vs 4/20 [20%]; P<0.001). GPNMB was more frequently methylated in AD than in matched normal mucosa from three patients (3/3 [100%] vs 1/3 [33.3%]; P<0.001). Finally, the frequency of GPNMB methylation in NA differs significantly from that in the normal mucosa (16/21 [76%] vs 4/20 [20%]; P0.5). Gender, location, and age were independent of GPNMB methylation. However, there was statistically significant correlation of higher methylation at advanced stages and lower methylation at stage 1 CRCs (P<0.05). In agreement with these findings, GPNMB protein expression decreased in CRC tissues compared with AD and NA colon mucosa, p<.05. GPNMB expression silencing through promoter methylation was evident in AD and CRC. GPNMB overexpression in HCT116 colon cancer cell line decreased cell proliferation [(24h, P=0.026), (48h, P<0.001), and (72h, P=0.007)], invasion (p<.05) and migration compared to the mock-transfected cells (p>.05).

Conclusion: Our data indicate a high methylation profile leading to 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 African Americans.

Citation Format: Hamed Rahi, Tahmineh Haidary, Hassan Brim, Edward L. Lee, Babak Shokrani, H Zhifeng, Peter M. Siegel, Hassan Ashktorab. GPNMB methylation: a new marker of potentially colonic adenoma in African Americans. [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 554. doi:10.1158/1538-7445.AM2014-554