Isolation and characterization of calcium sensing receptor null cells: a highly malignant and drug resistant phenotype of colon cancer.

The expression of calcium sensing receptor (CaSR) in the human colonic crypt epithelium is linked to cellular differentiation while its lack of expression is associated with undifferentiated and invasive colon carcinoma. Human colon carcinoma cell lines contain small subpopulations (10-20%) that do not express CaSR (termed CaSR null cells). Here, we report on the isolation, propagation, maintenance and characterization of CaSR null cells from the CBS and HCT116 human colon carcinoma cell lines. CaSR null cells grew as three-dimensional non-adherent spherical clusters with increased propensity for anchorage independent growth, cellular proliferation and invasion of matrigels. CaSR null cells were highly resistant to fluorouracil and expressed abundant amount of thymidylate synthase and survivin. Molecular profiling by real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blots showed a high level of expression of the previously reported cancer stem cell markers CD133, CD44 and Nanog in CaSR null cells. A significant increase in the expression of epithelial-mesenchymal transitional molecules and transcription factors was also observed. These include N-cadherin, β-catenin, vimentin, fibronectin, Snail1, Snail2, Twist and FOXC2. The expression of the tumor suppressive E-cadherin and miR145, on the other hand, was greatly reduced while expression of the oncogenic microRNAs: miR21, miR135a and miR135b was significantly up-regulated. CaSR null cells possess a myriad of cellular and molecular features that drive and sustain the malignant phenotype. We conclude that CaSR null constitutes a highly malignant and drug resistant phenotype of colon cancer.