Epigenetic regulation of osteopontin splicing isoform c defines its role as a microenvironmental factor to promote the survival of colon cancer cells from 5-FU treatment

Background: Drug resistance to 5-fluorouracil (5-FU) and recurrence after chemotherapy in colorectal cancer remain a challenge to be resolved for the improvement of patient outcomes. It is recognized that a variety of secretory proteins released from the tumor cells exposed to chemo-drugsinto the tumor microenvironment (TME) contributedto the cell-to-cell communication, and altered the drug sensitivity.One of these important factors is osteopontin (OPN), which exists in several functional forms from alternative splicing and post-translational processing. In colon cancer cells, increased total OPN expression was observed during the progression of tumors, however, the exact role and regulation of the OPN splicing isoforms was not well understood. Methods: We assayed precisely the abundance of majorOPN splicing isoformsunder5-FU treatmentsincolon cancer cell lineswith different sensitivities to 5-FU, and also evaluated the effects of the condition medium from OPNsplicing isoforms overexpressed cells on cell functions. The methods of nuclear calcium reporter assays and ChIP (chromatin immunoprecipitation) assays were used to investigate the molecular mechanism underlining the production of OPN isoforms.Results: We discovered that OPNc was a most increased splicing isoform to a significant abundance following 5-FU treatment of colon cancer cells. OPNc as a secretory protein in the conditioned medium exerted a more potent effect to promote cell survival in 5-FU than other OPN isoforms. The kinetic response of nuclear calcium signalscouldbe used to indicate an immediate effect of the conditioned medium containing OPNc and other isoforms. MeCP2 was identified to regulate the splicing of opn gene, where the phosphorylation of MeCP2 at S421 site, possibly by CAMKII, was required. Conclusions: The results demonstrated that the production of OPNc was highly controlled under epigenetic regulations, where MeCP2 and the activation of nuclear calcium signaling were involved. It was also suggested that OPNc could transmit the stress signal of cells upon chemotherapy in TME and promoted the survival of adjacent colon cancer cells.