The calcium-sensing receptor suppresses epithelial-to-mesenchymal transition and stem cell- like phenotype in the colon

Abstract

Background: The calcium sensing receptor (CaSR), a calcium-binding G protein-coupled receptor is expressed also in tissues not directly involved in calcium homeostasis like the colon. We have previously reported that CaSR expression is down-regulated in colorectal cancer (CRC) and that loss of CaSR provides growth advantage to transformed cells. However, detailed mechanisms underlying these processes are largely unknown.

Methods and results: In a cohort of 111 CRC patients, we found significant inverse correlation between CaSR expression and markers of epithelial-to-mesenchymal transition (EMT), a process involved in tumor development in CRC. The colon of CaSR/PTH double-knockout, as well as the intestine-specific CaSR knockout mice showed significantly increased expression of markers involved in the EMT process. In vitro, stable expression of the CaSR (HT29(CaSR)) gave a more epithelial-like morphology to HT29 colon cancer cells with increased levels of E-Cadherin compared with control cells (HT29(EMP)). The HT29(CaSR) cells had reduced invasive potential, which was attributed to the inhibition of the Wnt/β-catenin pathway as measured by a decrease in nuclear translocation of β-catenin and transcriptional regulation of genes like GSK-3β and Cyclin D1. Expression of a spectrum of different mesenchymal markers was significantly down-regulated in HT29(CaSR) cells. The CaSR was able to block upregulation of mesenchymal markers even in an EMT-inducing environment. Moreover, overexpression of the CaSR led to down-regulation of stem cell-like phenotype.

Conclusions: The results from this study demonstrate that the CaSR inhibits epithelial-to-mesenchymal transition and the acquisition of a stem cell-like phenotype in the colon of mice lacking the CaSR as well as colorectal cancer cells, identifying the CaSR as a key molecule in preventing tumor progression. Our results support the rationale to develop new strategies either preventing CaSR loss or reversing its silencing.

Figure 1

Effect of global CaSR knock-down in vivo on genes modulating EMT/stemness in the colon. Colons sampled from CaSR ^−/−/PTH ^−/− (DKO) and CaSR ^+/+/PTH ^−/− (control) mice were investigated for mRNA expression of the mesenchymal markers αSma (A), Fsp1(B), Snail2 (C), Twist2 (D), Vimentin (E), Zeb1 (F), and the pluripotency markers Nanog (G) and Stella (H). Dots indicate individual data points, and the line represents median. Statistical significance was calculated using t test. n = 9 animals/group, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

Effect of intestine-specific CaSR knock-down in vivo on EMT-associated genes in the colon. Colons sampled from CaSR ^−/− (CaSR^int-KO) and CaSR ^+/+ (CaSR^WT) mice were investigated for protein expression of mesenchymal markers αSma, N-cad, Snai1, Snai2 and Vimentin. Representative images in black and white for the markers are shown in addition to the merged images (red or white channels for the indicated markers and blue for DAPI). n = 5 animals/group. Scale bar: 50 μm.

Figure 3

Impact of the CaSR on the invasive potential of HT29 colon cancer cells. (A) HT29^EMP cells have an elongated, spindle-like morphology whereas HT29^CaSR cells attain a more compact, epithelial-like morphology of well adherent cells. (B) Expression of the epithelial marker E-Cadherin is significantly upregulated in HT29^CaSR cells compared with HT29^EMP cells. (C-D) Invasion and migration was assessed using a 3D spheroid forming assay. The area of the spheroid invading the surrounding matrix is presented as the invasive index. The number of daughter spheroids migrated from the parental spheroid was counted. Bars represent means ± SEM of three independent experiments. Statistical significance was calculated using t tests. **p < 0.01, ***p < 0.001.

Figure 4

Ectopic CaSR prevents nuclear β-catenin translocation in HT29 colon cancer cells. (A) HT29 cells overexpressing the CaSR (HT29^CaSR) show reduced β-catenin nuclear translocation as assessed by western blot and (B) by quantification of β-catenin signal normalized to house-keeping genes (Lamin C: nuclear fraction and α-Tubulin: cytosolic fraction). (C-F) HT29^CaSR cells show increased mRNA expression of GSK-3β, of the differentiation markers CDX2 and Villin, and reduced levels of the proliferation marker Cyclin D1 compared with HT29^EMP cells. Data represent mean ± SEM of three independent experiments. Statistical significance was calculated using t test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Induction of CaSR expression/function suppresses EMT in HT29 colon cancer cells. Expression of CaSR and E-Cadherin are upregulated in HT29^EMP cells treated with 1 μM NPS R-568 or in HT29^CaSR cells whereas expression of αSMA and Vimentin is downregulated. The merged images (red or white channels for the indicated markers and blue for DAPI) are shown. Scale bar: 20 μm.

Figure 6

Ectopic CaSR prevents induction of mRNA expression of EMT markers in HT29 colon cancer cells. HT29^CaSR cells (grey bars) show downregulation in mRNA expression of mesenchymal markers αSMA, FOXC2, SNAI1, TWIST2, Vimentin and Zeb1 compared with HT29^EMP cells (white bars). Treatment with EMT promoting cocktail, further induced mesenchymal transition in HT29^EMP cells (white striped bars), which was blocked by ectopic expression of CaSR (HT29^CaSR, grey striped bars). Data represent mean ± SEM of three independent experiments. Statistical significance was determined by ANOVA followed by Tukey’s post-test. Asterisks above bars indicate all significant changes. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

Ectopic CaSR prevents induction of protein expression of EMT markers in HT29 colon cancer cells. Treatment with the EMT promoting cocktail induced protein expression of mesenchymal markers, αSMA and Vimentin only in HT29^EMP cells, which was blocked by ectopic expression of CaSR (HT29^CaSR). In HT29^CaSR cells, the upregulated E-Cadherin expression was downregulated upon treatment with the EMT promoting cocktail but remained higher than in HT29^EMP cells. The merged images (red or white channels for the indicated markers and blue for DAPI) are shown. Scale bar: 20 μm.

Figure 8

Overexpression of CaSR blocks acquisition of stem cell-like characteristics. HT29 cells overexpressing the CaSR (HT29^CaSR) or empty transfected cells (HT29^EMP) were grown in stem cell media. (A) Ectopic expression of CaSR blocked acquisition of the pluripotency markers Nanog, Oct3/4, Stella, and FOXC2. (B) Treatment with NPS R-568 or ovexpression of CaSR diminished expression of pluripotency-associated markers Sox2, Nanog, Oct4 and CD44. (C) Extreme limiting dilution assay revealed a significantly lower sphere forming ability of stem-like cells in HT29^CaSR cells. Data represent mean ± SEM of 3-5 independent experiments. Scale bar: 50 μm. Statistical significance was calculated using t test. *p < 0.05, **p < 0.01.