Osteopontin alters DNA methylation through up-regulating DNMT1 and sensitizes CD133+/CD44+ cancer stem cells to 5 azacytidine in hepatocellular carcinoma

Xiaomei Gao,Yuanyuan Sheng,Jing Yang,Sijia Yan ,Jie Wei ,Chaoqun Wang,Rui Zhang,Ying Zhu,Ze Zhang,Kaili Zhang,Haoting Sun,Xuan Wang,Xinxin Yu,Yu Zhang,Qin Luo,Yan Zheng,Peng Qiao,Yue Zhao,Qiongzhu Dong,Lun–Xiu Qin

doi:10.1186/s13046-018-0832-1

Abstract

BackgroundIn hepatocellular carcinoma (HCC), CD133+/CD44+ cells are one subgroup with high stemness and responsible for metastatic relapse and resistance to treatment. Our previous studies have demonstrated that osteopontin (OPN) plays critical roles in HCC metastasis. We further investigated the molecular mechanism underlying the role of OPN in regulating the stemness of HCC epigenetically and explored possible targeting strategy.MethodsCD133+/CD44+ subgroup sorting from HCC cell lines and HCC tissues was used to investigate the effects of OPN knockdown on stemness. iTRAQ and MedIP-sequencing were applied to detect the protein profile and epigenetic modification of CD133+/CD44+ subgroup with or without OPN knockdown. The antitumor effects of 5 Azacytidine were examined in cultured HCC cells and patient derived xenograft (PDX) models.ResultsOPN was accumulated in CD133+/CD44+ subgroup of HCC cells. Knocking down OPN significantly inhibited the sphere formation and stemness-related genes expression, and delayed tumor initiation of CD133+/CD44+ subgroup of HCC cells. Employing MedIP-sequencing, dot blot and iTRAQ analyses of CD133+/CD44+ SCR and CD133+/CD44+ shOPN cells, we found that OPN knockdown leaded to reduction in DNA methylation with particular enrichment in CGI. Meanwhile, DNA (cytosine-5)-methyltransferase 1 (DNMT1), the main methylation maintainer, was downregulated via proteomics analysis, which mediated OPN altering DNA methylation. Furthermore, DNMT1 upregulation could partially rescue the properties of CD133+/CD44+ shOPN cells. Both in vitro and in vivo assays showed that CD133+/CD44+ cells with high OPN levels were more sensitive to DNA methylation inhibitor, 5 Azacytidine (5 Aza). The above findings were validated in HCC primary cells, a more clinically relevant model.ConclusionsOPN induces methylome reprogramming to enhance the stemness of CD133+/CD44+ subgroup and provides the therapeutic benefits to DNMT1 targeting treatment in HCC.

Highlights

In hepatocellular carcinoma (HCC), CD133+/CD44+ cells are one subgroup with high stemness and responsible for metastatic relapse and resistance to treatment
Our findings reveal that OPN-DNA (cytosine-5)-methyltransferase 1 (DNMT1) axis plays important roles in the regulation of stemness through modulating DNA methylation of CD133+/CD44+ cells
OPN knockdown impaired the properties of CD133+/CD44+ cells. (A-B) OPN rescuing reversed the number of spheres and the expression of genes in Huh7, NS, no significance. (C) shOPN in CD133+/CD44+ cells from Hep3B reduced the number and size of spheres, 100x. (D) CD133 and CD44 were down-regulated in CD133 +/CD44+ shOPN from Hep3B. (E) OPN knockdown in CD133+/CD44+ cells from Hep3B inhibited genes expression. (F) shOPN in Hep3B CD133 +/CD44+ cells decreased the potential of migration on gelatin, 100x and 400x. (G-H) OPN rescuing reversed the number of spheres and the expression of genes in Hep3B

Summary

Introduction

In hepatocellular carcinoma (HCC), CD133+/CD44+ cells are one subgroup with high stemness and responsible for metastatic relapse and resistance to treatment. Our previous studies have demonstrated that osteopontin (OPN) plays critical roles in HCC metastasis. We further investigated the molecular mechanism underlying the role of OPN in regulating the stemness of HCC epigenetically and explored possible targeting strategy. Many studies indicate that tumor heterogeneity is responsible for tumor metastasis, relapse and drug resistance of cancers including hepatocellular carcinoma (HCC) [2]. Cancer stem cells are conceited as the cells within the tumors possessing the capacities of self-renewal and heterogeneous lineages differentiation [3]. It is convinced that targeting cancer stem-cells is a promising approach for cancer treatment based on a detailed understanding of this unique sub-group biological features distinct from its parental counterparts [7]. Activated signal pathways and proteins in this subgroup could all be potential therapeutic targets [8, 9]

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Discussion

Conclusion