Abstract 1136: Gene amplification and epigenetic modifications: Mechanisms behind unbalanced expression of the proto-oncogene CYP24A1 in colorectal tumors
Authors: Julia Hoebaus, Thomas Nittke, Doris Hummel, Teresa Manhardt, Enikö Kallay
Journal: Cancer Research (2011)

Abstract There is an inverse association between colorectal cancer and pre-diagnostic Vitamin D levels (25-OHD3), underlining the importance of Vitamin D in the prevention of colon cancer. 1α,25(OH)2D3 (1,25D3) is the most active form of Vitamin D and exhibits anti-proliferative, pro-differentiating, anti-angiogenic functions. Both, calcitriol and the calcitriol precursor 25(OH)D3 can be catabolized by the 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1). CYP24A1 is expressed at very low basal levels in the colon but is strongly induced by 1,25D3. This induction is due to a complex formation of the 1,25D3 and the Vitamin D Receptor and binding of this complex to Vitamin D Responsive Elements (VDRE) located within the CYP24A1 promoter region. This feedback loop shortens the biological half-life of 1,25D3. A high expression of the Vitamin D catabolizing enzyme CYP24A1 implies faster degradation of 1,25D3, thereby restricting its anti-tumorigenic functions. A genomic copy number gain and an overexpression of CYP24A1 on mRNA level have been observed in different malignancies, such as colon and breast. Our aim is to test whether the observed overexpression of CYP24A1 is due to gene amplification and/or epigenetic modulations. In human colon cancer cell lines we found an increase in genomic copy number of CYP24A1 compared to human placenta using real time-PCR. In human colorectal cancer patients (n=89) we observed a gain of CYP24A1 copy number in approximately 40% of the tumors compared to their respective adjacent mucosa indicating a role of CYP24A1 gene amplification in tumorigenesis. Further, on mRNA level we could show a trend toward increased CYP24A1 expression in tumors compared with their adjacent mucosa using reverse transcription qRT-PCR. However, the effect of copy number gain can be impaired by epigenetic regulation. Aberrant DNA methylation is a characteristic of various tumors. The promoter region of the CYP24A1 gene contains several CpG islands. Therefore we have examined if there are any differences in the methylation status of the CYP24A1 promoter between the tumor tissue and the adjacent “normal” mucosa. First we have compared the methylation status in 4 cell lines. Bisulfit-sequencing of the genomic DNA of the highly differentiated colon cancer cell line Caco2Aq showed heavy methylation of the CYP24A1 promoter including the VDREs located within this promoter area. In comparison, the colon cell lines Coga1A, LS174, and Coga13 (derived from less differentiated tumors) appeared largely unmethylated. Using methylation specific PCR we found that human colorectal tumors are often less methylated than their adjacent mucosa. In conclusion, our data indicate that both gene amplification and DNA-hypomethylation could be mechanisms leading to the overexpression of CYP24A1 in colorectal tumor patients. 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 1136. doi:10.1158/1538-7445.AM2011-1136