Increased copy-number and not DNA hypomethylation causes overexpression of the candidate proto-oncogene CYP24A1 in colorectal cancer

In colorectal cancer (CRC) the vitamin D catabolizing enzyme 1,25-dihydroxyvitamin D 24-hydroxylase (CYP24A1) is overexpressed with a potentially significant, positive impact on the catabolism of 1,25-dihydroxyvitamin D3 (1,25-D3). However, the underlying mechanism of CYP24A1 overexpression is poorly understood. In the present study, we investigated possible causes including hypomethylation of the CYP24A1 promoter, amplification of the CYP24A1 gene locus (20q13.2), and altered expression of CYP24A1-specific transcription factors. We quantified CYP24A1 gene copy-number, performed bisulfite sequencing of the CYP24A1 promoter to assess DNA methylation, and measured mRNA expression of CYP24A1, 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1), vitamin D receptor (VDR) and retinoid X receptor (RXR). We found that 77 (60%) out of 127 colorectal tumors showed increased CYP24A1 gene copy-number and that more than 6 copies of CYP24A1 correlated positively with CYP24A1 mRNA expression suggestive of a causal relationship. No differences in CYP24A1 promoter methylation were found between tumor tissue and adjacent mucosa from the same patient or between tissues with high or low mRNA expression, thus excluding DNA hypomethylation as a possible cause of CYP24A1 overexpression in CRC. Furthermore, mRNA expression of several factors involved in replication licensing positively correlated with CYP24A1 mRNA expression, raising the possibility that CYP24A1 overexpression might favor increased proliferation in tumors by suppressing local 1,25-D3 levels. We conclude that high copy-number gain is a key determinant of CYP24A1 overexpression in CRC. Other postulated causes of CYP24A1 overexpression including promoter hypomethylation and enhanced VDR and/or RXR expression do not appear to be involved. What’s new? Recently, it has been suggested that the association between colorectal cancer and reduced levels of circulating vitamin D may be related to overexpression of the vitamin D-catabolizing enzyme, CYP24A1 in the tumor. In this search for a mechanistic explanation, increased CYP24A1 gene copy number was associated with the enzyme’s overexpression in 60 percent of colorectal tumors, and expression was correlated strongly with proliferation markers. The findings suggest that CYP24A1 overexpression is likely to deplete tumor calcitriol (1,25-dihydroxyvitamin D3) levels, possibly increasing the proliferative potential of the tumors.