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.

miR-135b- and miR-146b-dependent silencing of calcium-sensing receptor expression in colorectal tumors

“…It is well established that the expression of the CaSR is reduced, or even lost during colorectal tumorigenesis . Recently, we demonstrated that changes in epigenetic patterns, like CaSR promoter 2 hypermethylation and loss of lysine 9 acetylation on histone 3, are among the mechanisms involved in silencing expression of the CaSR in colorectal tumors . However, our data suggested that other causes must also be involved in the loss of CaSR expression in these tumors.…”

“…We conducted microarray analysis for microRNAs in Caco2/AQ and Caco2/15 cells, which are subclones derived from the Caco‐2 cell line to screen for potential microRNAs that might target the CaSR. Caco2/AQ and Caco2/15 cell lines were maintained under standard conditions for 21 days. These cells differentiate spontaneously in cell culture after reaching confluency .…”

“…We performed our experiments in two CRC cell lines: Caco2/AQ, well‐differentiated cells and HT29, a moderately differentiated cell line. Caco2/AQ and HT29 cells were seeded at 30% confluency in regular growth medium in 24‐well plates as described previously . 24 hr after seeding, cells were transfected using DharmaFECT 1 transfection reagent with miR‐135b‐5p and miR‐146b‐5p mimics or inhibitors, or control for 48 hr in regular growth medium without penicillin/streptomycin, after which cells were harvested for RNA and protein analyses.…”

“…Epidemiological studies demonstrate an inverse correlation between calcium intake and the risk of colorectal tumor development . In the colon, the calcium‐sensing receptor (CaSR) is a tumor suppressor that mediates, at least in part, the antiproliferative and prodifferentiating actions of calcium . It is well established that the expression of the CaSR is reduced, or even lost during colorectal tumorigenesis .…”

“…In the colon, the calcium‐sensing receptor (CaSR) is a tumor suppressor that mediates, at least in part, the antiproliferative and prodifferentiating actions of calcium . It is well established that the expression of the CaSR is reduced, or even lost during colorectal tumorigenesis . Recently, we demonstrated that changes in epigenetic patterns, like CaSR promoter 2 hypermethylation and loss of lysine 9 acetylation on histone 3, are among the mechanisms involved in silencing expression of the CaSR in colorectal tumors .…”