Background. Chromatin remodeling and activation of transcription are important aspects of gene regulation, but these often go awry in disease progression, including during colon cancer development. Histone modification is an important factor in Cancer and Colorectal Cancer development. In addition, micro-satellite instability (MSI) is implicated in 15% of Colorectal Cancers. We investigated the status of localized histone acetylation and expression of histone deacetylase 2 (HDAC2) in African-Americans (AA) human colon cancer tissue microarrays using immunohistochemical staining. We also investigated the correlation between MSI and localized histone acetylation and HDAC2 expression.
Methods. Specifically, HDAC2 and the acetylation of histones H4K12 and H3K18 were evaluated in colonic adenoma (134 patients) and carcinoma (58 patients) compared to matched normal tissue from patients undergoing elective colonoscopy at Howard University Hospital. In addition, the correlation between expression of these epigenetic biomarkers and various clinicopathological factors, including, age, location and stage of the disease were analyzed. A fluorescence method was used to analyze MSI. Briefly, MSI status of the neoplasms was determined by PCR to amplify five microsatellite markers (Boland, Thibodeau et al. 1998); BAT25, BAT26, NR21, NR22 and NR24) were used to evaluate MSI status.
Results. HDAC2 nuclear expression was detected at high levels in 81.9%, 62.1%, and 53.1% of colorectal adenocarcinomas, adenomas, and normal tissue, respectively (P=0.002). The corresponding nuclear expression levels in well differentiated tumors for H4K12 acetylation were 71.7%, 61%, and 43.6%, respectively (P=0.002). HDAC2 expression was correlated significantly with progression of adenoma to carcinoma (P=0.002). Based on area under curve assessment, the discriminative power was 0.74 for HDAC2 (Receiver Characteristics Curve fitting model; specificity=0.75, sensitivity=0.80), when comparing cancer and non-cancer cases. Our results showed marginal differences between tissue designated non-MSI-High and MSI-High. The mean values of Non-MSI-H and MSI-H for H3K18 are 84.00 and 87.84 respectively. The standard deviations for these two groups are 24.59 and 24.10 respectively. The mean values for Non-MSI-H and MSI-H are 69 and 74 respectively. The standard deviation values for these two groups are 39.85 and 33.02 respectively. The mean values for Non-MSI-H and MSI-H are 88.75 and 81.46 respectively. The standard deviations of Non-MSI-H and MSI-H are 20.310 and 29.880respectively. From the p-values for this data, it can be said that there exists little to no correlation between MSI status and H3K18 and H4K12 acetyation and HDAC2 expression. Our immunohistochemistry (IHC) results show that there was no statistically significant difference in H4K12 and HDAC2 expression and tumor stages. Co-expression of H4K12 and HDAC2 was found in 80%, and 86% patients with Stage II and III disease, respectively. The results also show an increased acetylation of H4K12 and expression of HDAC2 as the progression proceeds from normal to carcinoma tissue. These results suggest that: (1) H4K12 acetylation is involved in the activation of regions of the genome that are oncogenic or that promote proliferation, and (2) HDAC2 deacetylates or inactivates regions that are involved in tumor suppression.
Conclusion. Our MSI results showed minimal differences between tissue designated non-MSI-High and MSI-High. From the statistical analysis of this data, we concluded that there exists little to no correlation between MSI status and H3K18 and H4K12 acetyation and HDAC2 expression. Also from our IHC results we made the conclusion that overall histone modifications of CRC cells are predictive of disease progression from adenoma to carcinoma. The mechanistic basis of such changes are currently unclear but may be related to the altered expression and/or global activities of various histone-modifying enzymes. The variability in the levels of any one modification was not sufficient for predicting outcome. However, in combination, these changes proved to be indicative of the tumor risk in patients with colon adenoma.