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

Abstract

<jats:title>Abstract</jats:title> <jats:p>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-drugs into the tumor microenvironment (TME) contributed to 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 major OPN splicing isoforms under 5-FU treatments in colon cancer cell lines with different sensitivities to 5-FU, and also evaluated the effects of the condition medium from OPN splicing 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 signals could be used to indicate an immediate effect of the conditioned medium containing OPNc and other isoforms. Methyl-CpG binding protein 2 (MeCP2) was identified to regulate the splicing of opn gene, where the phosphorylation of MeCP2 at S421 site, possibly by calmodulin dependent protein kinase II (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.</jats:p>

Recent Updates on Mechanisms of Resistance to 5-Fluorouracil and Reversal Strategies in Colon Cancer Treatment

“…Interestingly, demethylation in vitro exerted reinforced 5-FU sensitivity in MSI phenotypic cells via targeting pleiomorphic adenoma gene 1 (PLAG1). Although most studies have reported negative correlations between gene hyper-/hypo-methylation and poor patient prognosis through resistance, such as in BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) facilitated by DNA methyltransferase 1 (DNMT1) enzyme [178]; miR-26b via P-gp down-regulation [179]; DNMT3A and DNMT3B overexpression [180]; CCNE1, cyclin D1 binding protein 1 (CCNDBP1), paraoxonase 3 (PON3), as well as DEAD-box helicase 43 (DDX43) and cell adhesion molecule L1-like I (CHL1) via the mitogen-activated protein kinase (MAPK) apoptosis and PI3K/AKT proliferation pathways, respectively [181]; and osteopontin splicing isoform c via methyl-CpG binding protein 2 (MeCP2) [182], there are studies that had reported otherwise; WNT5A [183]; and NME/NM23 nucleoside diphosphate kinase 2 (NME2) [184]. Histone modifications are another common cause for epigenetic alterations and can be observed in the histone methylation H3K9me9 dysregulation and H3K27 PCAF-mediated histone acetylation of p53 [66,185].…”