Abstract 4921: Selective promoter methylation of the cell differentiation marker, intestinal alkaline phosphatase (iALP), in microsatellite unstable colon cancer

Abstract

Tumorigenesis is characterized by excessive cell growth, resistance to apoptosis and aberrant cellular differentiation. In particular, MSI colorectal cancer is frequently characterized by poorly differentiated histology. Histone Deacetylase inhibitors (HDACi) are a novel class of cancer therapeutics that induce growth arrest, apoptosis and differentiation of a variety of tumour types, including colon cancer cells. Differentiation inducing agents have been used effectively to treat hematopoietic malignancies, prompting us to examine the effects of HDACi treatment on differentiation of colon cancer cells, and to compare the effects induced in MSS and MSI colon cancers. Remarkably, while HDACi treatment robustly and consistently induced differentiation induction as measured by alkaline phosphatise activity and iALP gene expression in multiple MSS colon cancer cell lines, approximately 70% of MSI colon cancer cell lines were highly resistant to HDACi-induced ALP induction. Examination of the coding sequence of the iALP gene failed to identify any significant repeat elements suggesting mutation is unlikely to be responsible for lack of iALP induction. Furthermore, transient transfection of an iALP promoter reporter construct demonstrated robust induction in response to butyrate treatment in both MSS and MSI lines, suggesting loss of a key regulatory factor in MSI cell lines is not likely to be responsible. Conversely, examination of iALP promoter methylation by bisulphite conversion and direct sequencing demonstrated multiple CpG sites, including a Sp1/Sp3 binding site, to be methylated in 4 of 5 MSI lines examined, while no methylation was observed in MSS lines. These findings were confirmed using the sequenom assay. ALP promoter methylation was independent of CIMP status. The importance of Sp1/Sp3 in butyrate-induced iALP induction was demonstrated by the ability of the Sp1/Sp3 inhibitor, mithramycin, to attenuate butyrate-induced ALP activity. Finally, consistent with methylation being a key determinant of iALP expression in MSI lines, azacytidine treatment was able to re-induce iALP expression, and butyrate-induction of ALP expression was markedly enhanced in isogenic HCT116 cells lacking the DNA methyltransferases Dnmt1 and Dnmt3b (DKO), compared to parental cells. These findings demonstrate selective promoter methylation of iALP in MSI colon cancer cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4921.