Deletion Hotspots in AMACR Promoter CpG Island Are cis-Regulatory Elements Controlling the Gene Expression in the Colon

Abstract

Alpha-methylacyl-coenzyme A racemase (AMACR) regulates peroxisomal β-oxidation of phytol-derived, branched-chain fatty acids from red meat and dairy products — suspected risk factors for colon carcinoma (CCa). AMACR was first found overexpressed in prostate cancer but not in benign glands and is now an established diagnostic marker for prostate cancer. Aberrant expression of AMACR was recently reported in Cca; however, little is known about how this gene is abnormally activated in cancer. By using a panel of immunostained-laser-capture-microdissected clinical samples comprising the entire colon adenoma–carcinoma sequence, we show that deregulation of AMACR during colon carcinogenesis involves two nonrandom events, resulting in the mutually exclusive existence of double-deletion at CG3 and CG10 and deletion of CG12-16 in a newly identified CpG island within the core promoter of AMACR. The double-deletion at CG3 and CG10 was found to be a somatic lesion. It existed in histologically normal colonic glands and tubular adenomas with low AMACR expression and was absent in villous adenomas and all CCas expressing variable levels of AMACR. In contrast, deletion of CG12-16 was shown to be a constitutional allele with a frequency of 43% in a general population. Its prevalence reached 89% in moderately differentiated CCas strongly expressing AMACR but only existed at 14% in poorly differentiated CCas expressing little or no AMACR. The DNA sequences housing these deletions were found to be putative cis-regulatory elements for Sp1 at CG3 and CG10, and ZNF202 at CG12-16. Chromatin immunoprecipitation, siRNA knockdown, gel shift assay, ectopic expression, and promoter analyses supported the regulation by Sp1 and ZNF202 of AMACR gene expression in an opposite manner. Our findings identified key in vivo events and novel transcription factors responsible for AMACR regulation in CCas and suggested these AMACR deletions may have diagnostic/prognostic value for colon carcinogenesis.

Author Summary

Men consuming high amounts of red meat and dairy products are at a higher risk of developing colon and prostate cancer. Alpha-methylacyl-coenzyme A racemase (AMACR) is an enzyme that helps to break down fat from these foods to produce energy. An increase in the utilization of energy from fat is a hallmark of many cancers including colon and prostate cancers. Indeed, the AMACR gene was first found to be abnormally active in prostate cancers, and its abnormal expression has become a diagnostic marker for the cancer. However, little is known about how AMACR becomes activated in cancer cells. Here, we show that AMACR is also highly expressed in certain stages of colon cancer, though not all stages. A close examination of the AMACR gene in a panel of normal and progressively malignant colon tissues reveals that deletions of specific sequences in the AMACR gene may trigger its abnormal expression during the evolution of colon cancer. We also identify unique proteins known as “transcription factors” that normally bind to these deleted sequences to maintain normal expression of the gene. Finally, we report a new deletion variant of the AMACR gene in the general population that may influence the course of colon carcinogenesis.