title: Calcium-sensing receptor silencing in colorectal cancer is associated with promoter hypermethylation and loss of acetylation on histone 3.
abstract: The calcium-sensing receptor (CaSR) is suggested to mediate the antiproliferative effects of calcium in colon. However, in colorectal cancer (CRC) the expression of the CaSR is silenced and the underlying mechanisms leading to its loss are poorly understood. We investigated whether loss of the CaSR expression in colorectal tumors is caused by DNA hypermethylation and imbalance of transcriptionally permissive/repressive histone alterations. We observed significantly lower CaSR mRNA expression (n = 65, p < 0.001) in colorectal tumors compared with the adjacent mucosa from the same patient. Immunofluorescence staining confirmed downregulation of the CaSR protein also. The CaSR promoter was methylated to a greater extent in tumors compared with adjacent mucosa as determined by bisulfite sequencing (n = 20, p < 0.01) and by pyrosequencing (n = 45, p < 0.001), and methylation correlated inversely with mRNA expression (n = 20, ρ = -0.310, p < 0.05 and n = 45, ρ = -0.588, p < 0.001). Treatments with 5-aza-2'-deoxycytidine (DAC), a DNA methyltransferase inhibitor and/or with two different histone deacetylase inhibitors, trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) restored the expression of CaSR in colon cancer cells. Restored CaSR expression in Coga1A and HT29 cells was functional. Inhibition of lysine-specific demethylase 1 (LSD1) to prevent demethylation of mono- and dimethylated H3K4, increased CaSR expression only marginally. Our data show that hypermethylation of the CaSR promoter and H3K9 deacetylation, but not H3K4me2 demethylation are important factors that cause silencing of the CaSR in colorectal cancer.
authors.affiliation: Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria.
authors.forename: Irfete S
authors.initials: IS
authors.lastname: Fetahu
authors.forename: Julia
authors.initials: J
authors.lastname: Höbaus
authors.forename: Abhishek
authors.initials: A
authors.lastname: Aggarwal
authors.forename: Doris M
authors.initials: DM
authors.lastname: Hummel
authors.forename: Samawansha
authors.initials: S
authors.lastname: Tennakoon
authors.forename: Ildiko
authors.initials: I
authors.lastname: Mesteri
authors.forename: Sabina
authors.initials: S
authors.lastname: Baumgartner-Parzer
authors.forename: Enikő
authors.initials: E
authors.lastname: Kállay
mesh_terms: D000107:Acetylation; D000368:Aged; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D047369:Chromatin Immunoprecipitation; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D019175:DNA Methylation; D005260:Female; D005455:Fluorescent Antibody Technique; D020868:Gene Silencing; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006657:Histones / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008297:Male; D060787:Neoplasm Grading; D009367:Neoplasm Staging; D011379:Prognosis; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D044169:Receptors, Calcium-Sensing / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
chemical_list: C095550:CASR protein, human; D056572:Histone Deacetylase Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; D012333:RNA, Messenger; D044169:Receptors, Calcium-Sensing; C012589:trichostatin A
keywords: DNA methylation; calcium-sensing receptor; colorectal cancer; histone acetylation; histone methylation