SIX3, a tumor suppressor, inhibits astrocytoma tumorigenesis by transcriptional repression of AURKA/B

Zhibin Yu,Jianbo Feng,Yan Zhang,Qing Liu,Xiaoling She,Changhong Liu,Minghua Wu,Guiyuan Li,Chunhua Zhao,Peiyao Li,Qiang Liu,Shuai Chen,Zhiyong Deng,Yingnan Sun,Haijuan Fu,Zeyou Wang

doi:10.1186/s13045-017-0483-2

Abstract

BackgroundSIX homeobox 3 (SIX3) is a member of the sine oculis homeobox transcription factor family. It plays a vital role in the nervous system development. Our previous study showed that the SIX3 gene is hypermethylated, and its expression is decreased in astrocytoma, but the role of SIX3 remains unknown.MethodsChromatin-immunoprecipitation (ChIP) and luciferase reporter assay were used to confirm the binding of SIX3 to the promoter regions of aurora kinase A (AURKA) and aurora kinase B (AURKB). Confocal imaging and co-immunoprecipitation (Co-IP) were used to detect the interaction between AURKA and AURKB. Flow cytometry was performed to assess the effect of SIX3 on cell cycle distribution. Colony formation, EdU incorporation, transwell, and intracranial xenograft assays were performed to demonstrate the effect of SIX3 on the malignant phenotype of astrocytoma cells.ResultsSIX3 is identified as a novel negative transcriptional regulator of AURKA and AURKB, and it decreases the expression of AURKA and AURKB in a dose-dependent manner in astrocytoma cells. Importantly, interactions between AURKA and AURKB stabilize and protect AURKA/B from degradation, and overexpression of SIX3 does not affect these interactions; SIX3 also acts as a tumor suppressor, and it increases p53 activity and expression at the post-translational level by the negative regulation of AURKA or AURKB, reduces the events of numerical centrosomal aberrations and misaligned chromosomes, and significantly inhibits the proliferation, invasion, and tumorigenesis of astrocytoma in vitro and in vivo. Moreover, experiments using primary cultured astrocytoma cells indicate that astrocytoma patients with a low expression of SIX3 and mutant p53 are more sensitive to treatment with aurora kinase inhibitors.ConclusionSIX3 is a novel negative transcriptional regulator and acts as a tumor suppressor that directly represses the transcription of AURKA and AURKB in astrocytoma. For the first time, the functional interaction of AURKA and AURKB has been found, which aids in the protection of their stability, and partially explains their constant high expression and activity in cancers. SIX3 is a potential biomarker that could be used to predict the response of astrocytoma patients to aurora kinase inhibitors.

Highlights

SIX homeobox 3 (SIX3) is a member of the sine oculis homeobox transcription factor family
SIX3 is a transcriptional repressor of aurora kinase A (AURKA) and aurora kinase B (AURKB) In addition to the positive regulatory regions identified by Tanaka [19] (AURKA −124 to −75 bp, TSS = 0) and Kimura [18] (AURKB −104 to −74 bp), we analyzed the promoter regions of AURKA from −2939 to −528 bp and AURKB from −1715 to −105 bp (Fig. 1a), which cover the regulatory regions of the respective genes according to the data from the Encyclopedia of DNA
To confirm whether SIX3 regulates the transcriptional activity of the promoter regions of AURKA and AURKB, SIX3-expressing plasmid (Additional file 1: Figure S1) and AURKA/AURKB luciferase reporters were co-transfected into U251 cells

Summary

Introduction

SIX homeobox 3 (SIX3) is a member of the sine oculis homeobox transcription factor family. It plays a vital role in the nervous system development. Our previous study showed that the SIX3 gene is hypermethylated, and its expression is decreased in astrocytoma, but the role of SIX3 remains unknown. Malignant astrocytomas are the most common and aggressive malignancy of the central nervous system, especially glioblastoma (GBM). Chromosomal instability (CIN) is one of the hallmarks of the cancer, and its causative role in tumorigenesis has been recognized for over a century [2]. Complex mechanisms of CIN have been explored and could provide attractive therapeutic targets in cancer [3]. Mutation and misexpression of these genes are often an early event in tumor initiation, resulting in aneuploidy and heterogeneity of cancer cells [4]

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Discussion

Conclusion