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

Siyuan Chang,Yang Si,Zhigang Bai,Jing Wang,Jing Huang,Shan Cheng,Huan Niu,Wei Ding

doi:10.1186/s12935-020-01541-z

Siyuan Chang, Yang Si + Show 6 more

Open Access

https://doi.org/10.1186/s12935-020-01541-z

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Abstract

BackgroundDrug 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.MethodsWe 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.ResultsWe 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.ConclusionsThe 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.

Highlights

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
Maunakea et al previously analyzed the genome-wide distribution of Methyl-CpG binding protein 2 (MeCP2) in colon cancer cell line HCT116 (2013, GSE47678) by Chromatin immunoprecipitation (ChIP)-seq, which helped us to avoid the problem of screening primers to identify the exact target DNA fragments for PCR detections (Additional file 1: Fig. S2).Using HAUS8 gene as a MeCP2 positive control [23], the results identified the binding of MeCP2 to opn exon4 and exon5 in HCT-8 cells at native states (Fig. 6a)
Our results demonstrated that the generation of OPNc (Fig. 5) was a nuclear calcium-controlled event involving the MeCP2 phosphorylation at position serine 421, and was dependent to DNA methylation for binding MeCP2 to the opn alternative splicing exons (Fig. 6)

Summary

Introduction

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. From an increasing number of reports, it strongly indicated that microenvironmental factors secreted from cancer cells exposed to chemotherapy agents directly stimulated and were critically involved in the development of drug resistance through autocrine and paracrine mechanisms [4]. The identification or depletion of such factors was suggested as an important approach to validate potential biomarkers for prognosis, or screening for therapeutic targets when resistance was being developed

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