Expression profiling of colorectal cancer cells reveals inhibition of DNA replication licensing by extracellular calcium

Abstract

Colorectal cancer is one of the most common cancers in industrialised societies. Epidemiological studies, animal experiments, and randomized clinical trials have shown that dietary factors can influence all stages of colorectal carcinogenesis, from initiation through promotion to progression. Calcium is one of the factors with a chemoprophylactic effect in colorectal cancer. The aim of this study was to understand the molecular mechanisms of the anti-tumorigenic effects of extracellular calcium ([Ca²⁺]_o) in colon cancer cells. Gene expression microarray analysis of colon cancer cells treated for 1, 4, and 24h with 2mM [Ca²⁺]_o identified significant changes in expression of 1571 probe sets (ANOVA, p<10^-5). The main biological processes affected by [Ca²⁺]_o were DNA replication, cell division, and regulation of transcription. All factors involved in DNA replication-licensing were significantly downregulated by [Ca²⁺]_o. Furthermore, we show that the calcium-sensing receptor (CaSR), a G protein-coupled receptor is a mediator involved in this process. To test whether these results were physiologically relevant, we fed mice with a standard diet containing low (0.04%), intermediate (0.1%), or high (0.9%) levels of dietary calcium. The main molecules regulating replication licensing were inhibited also in vivo, in the colon of mice fed high calcium diet. We show that among the mechanisms behind the chemopreventive effect of [Ca²⁺]_o is inhibition of replication licensing, a process often deregulated in neoplastic transformation. Our data suggest that dietary calcium is effective in preventing replicative stress, one of the main drivers of cancer and this process is mediated by the calcium-sensing receptor.

Keywords: Calcium; Calcium-sensing receptor; Colorectal cancer; DNA replication licensing; Minichromosome maintenance complex.

Supplementary Fig. 1

Result of the PCA mapping of all transcripts. Principal component one (X-axis) and two (Y-axis) are plotted. These two PCs describe 32.1% of the dataset variance. In PC #1 mainly the 24 h CA treatment condition is separated from all other conditions.

Supplementary Fig. 2

Heat map of the K-Mean clustering of differentially expressed transcripts. FDR < 10³ in ANOVA has been used as the criteria for differentially expression. Clustering has been performed with Euclidean distance measurement, K = 5 groups and 100 runs.

Fig. 1

Hierarchical clustering of the differentially expressed probe-sets. Hierarchical clustering of the 1517 differentially expressed probe-sets identified the time dependent effect of calcium on Caco-2/AQ cells. Cntr refers to control; ca01h, ca04h and ca24h refer to calcium treatment for 1, 4 and 24 h, respectively.

Fig. 2

Regulation of DNA replication licensing by extracellular calcium. A: Schematic presentation of the replication licensing process. B: The expression of the DNA licensing factors is significantly inhibited by 24 hour treatment with 2 mM [Ca²⁺]_o (ca24h). Four replicates were analyzed for each time-point.

Fig. 3

Confirmation of the microarray data in Caco-2/AQ cells. A: qRT-PCR analysis of the expression of CDT1, MCM2, CDC6, and CDC45. Bars represent mean ± SEM. Statistical significance was calculated using ANOVA followed by Tukey's post-test. Asterisks above bars indicate significant changes within groups, *p < 0.05, **p < 0.01, ***p < 0.001. B: Representative Western Blot after treatment of the cells for 24 h with 2 mM [Ca²⁺]_o. C: Representative immunofluorescent images of MCM2, CDC6, and CDC45 (green) after treatment of Caco-2/AQ cells with 2 mM [Ca²⁺]_o for 24 h. Nuclei are stained with DAPI.

Fig. 4

Effect of dietary calcium on the expression of licensing factors in the colon of mice. Effect of dietary calcium on the expression of licensing factors in the colon of mice (n = 10 mice/group) fed for 8 months with AIN76 diet containing 0.04%, 0.1%, or 0.9% calcium. Individual data points are shown ± SEM. Statistical significance was calculated using ANOVA followed by Tukey's post-test. Asterisks indicate significant changes within groups, *p < 0.05, **p < 0.01.

Fig. 5

Expression of licensing factors in HT29 colon cancer cells overexpressing CaSR. Overexpression of CaSR in HT29 colon cancer cells (HT29^CaSR) significantly inhibited the expression of CDT1, MCM2, CDC6, and CDC45 compared with the controls transfected with an empty vector (HT29^Emp). Bars represent mean ± SEM. Statistical significance was calculated using Student's t-test. Asterisks above bars indicate significant changes compared with control cells, *p < 0.05.