Phosphorylation of ETV4 at Ser73 by ERK kinase could block ETV4 ubiquitination degradation in colorectal cancer

https://doi.org/10.1016/j.bbrc.2017.03.163Get rights and content

Highlights

  • ETV4 played an important regulatory role in the migration of embryonic cells and tumor cells.

  • The ETV4 was significantly enhanced by ERK kinase in the colorectal cancer patients, mouse models and CRC cell lines.

  • The ETV4 upregulated by ERK was due to inhibition of its degradation.

  • Phosphorylation of ETV4 at Ser73 could block its binding to COP1 and avoid its ubiquitination degradation.

Abstract

It was reported that Src-mediated and RTK-dependent accumulation of key transcription factor, ETV4, which played an important role in the migration of embryonic cells and tumor cells, were regulated by their common downstream MAPK molecules. However, the detailed mechanism was not completely clear. In the present study, we revealed that ETV4 protein was significantly enhanced by ERK kinase activation in the colorectal cancer (CRC) patients and mouse models as well as in the CRC cell lines. It was further confirmed that the activation of ERK kinase led to the phosphorylation of ETV4 at Ser73 and the ETV4 phosphorylation could block its binding to COP1, thereby stabilized ETV4 via avoiding its ubiquitination degradation. In addition, this effect was not due to altering an E3 ubiquitin ligase, COP1 amount or p-COP1/COP1 ratio. Our results will help understand the mechanism of ETV4 overexpression in CRC patients and provide a clue to search new therapeutic target to treat the related tumors in clinical practice.

Introduction

It was well known that cell migration is a necessary event during the formation of organs in embryonic development and the metastasis of solid tumor cells. E26 transformation-specific (ETS) transcription factors encoded by E26 transformation-specific genes play an important role in this physiological and pathophysiological process. They are characterized by DNA binding domain called ETS domain, which binds to DNA sequences with a 5′-GGA (A/T)-3′ core [1], [2]. Since the first ETS transcription factor was found three decades ago, the majority of family members were found in human tissues and 28 homologs were divided into 12 subfamilies based on their differences in structures and function domains [1]. Among them, polyoma enhancer activator protein (PEA3) subfamily members including ETV1, ETV4 and ETV5 were defined to contribute to the migration of various tumors and embryonic cells. Previous studies demonstrated that PEA3 members functioned in lung branch morphogenesis [3] and kidney development [4]. In neocortical development, PEA3 members activated by FGF/FGFR signals are vital in affecting migratory behaviors on nascent neuronal progeny [5]. Moreover, PEA3 members also functioned as a downstream molecule of FGF/FGFR signaling to lead migration of primary mesenchyme cells [6].

More recent years, it was reported that PEA3 subfamily also played an important role in a variety of solid tumor development. Leading clinical studies showed that PEA3 factors were clearly involved in the invasion and metastasis processes as an oncoprotein in breast cancer, prostate cancer and gastrointestinal neoplasm [7], [8], [9]. Examples included the highly-expressed ETV1 in melanoma and Imatinib-sensitive gastrointestinal stromal tumors (GIST) in which ETV1 was required for the tumor growth and development [10], [11]. Interestingly, persistent activation of KIT signaling not only could upregulate ETV1 expression, but also increase its binding to a target gene, the chemokine receptor-4 (CXCR4) promoter, which promoted GIST liver metastasis [10].

Furthermore, overexpression of ETV4, another key member of PEA3 subfamily, was positively correlated with greater likelihood of malignant behavior and higher recurrence in human breast cancer [8], [12]. ETV4 protein was also highly expressed in colorectal cancer (CRC) which was often correlated with the shorter survival period [13]. Taken together, PEA3 subfamily members not only promoted the migration of embryonic cells and organ development, but also enhanced the metastatic potential of tumor cells, which would contribute to the progress of several solid tumors, as well as aggressive biological behaviors.

A large number of experiments showed that highly-expressed ETV4 promoted EMT process, namely loss of cell polarity and adhesion and led the epithelial cells to obtain migratory and invasive properties [14], [15]. As a result, these alterations could increase tumor invasion and metastasis ability [9]. Therefore, it was widely accepted that PEA3 subfamily were regarded as potential malignant behavior markers for several solid tumors. Regarding to mechanism of the increased expression of PEA3 members, it was considered that ETV1 overexpression was resulted from ETV1 translocation fused with TMPRSS2 gene which induced by the androgen-dependent in prostate cancer [16]. Whereas, recent studies indicated that overexpression of ETV4 was regulated by activation of receptor tyrosine kinase (RTKs) signaling in multiple kinds of tumors. Indeed, high activity of HER2/Neu signaling was accompanied by high expression of ETV4 in breast cancer, showing higher recurrence and malignant behavior [12]. It was worth mentioning that activation of KIT, also a main member of RTK superfamily, and its downstream MAPK molecules consistently enhanced ETV4 expression in CRC [17].

Several interesting studies indicated that MAPK-dependent phosphorylation could bring about PEA3 factor overexpression attributed to post-transcriptional upregulation and increased protein expression. It was known that evolutionarily conserved E3 ubiquitin ligase Constitutive Photomorphogenic Protein 1 (COP1) could recognize two binding motifs in conserved residues VP in the N-terminal of PEA3 factors and promote their ubiquitination degradation [18], [19]. Recent years, ETS1, a vital transcriptional factor in ETS family was also defined as a target of COP1 and its phosphorylation resulted by Src kinase protected ETS1 from COP1-mediated ubiquitination degradation [20]. Collectively, it raises a possibility that phosphorylation of ETV4 by activation of ERK kinase can not be ubiquitinated degradation, however, the detailed molecular mechanism is still unclear. Therefore, the main purpose of this study is to investigate the mechanism of high expression of ETV4 by the activated ERK kinase in the CRC. Our results demonstrated that ETV4 protein was significantly enhanced by the activation of ERK kinase in the CRC. The activation of ERK kinase could phosphorylate ETV4 at Ser73 and destruct the binding with COP1, thereby inhibiting ETV4 ubiquitination degradation. These results might help understand the mechanism of RTK-dependent accumulation of ETV4 in the CRC patients and provide a clue to searching new therapeutic target to treat the related tumors in clinic.

Section snippets

Cells and reagents

The human colorectal cell lines HCT-116 (p53-/- and p53+/+), RKO and Caco2 were purchased from the America Type Culture Collection (ATCC, USA) and were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin (Life Technologies, USA) at 37 °C in the presence of 5% CO2. The medium was replaced every 24 h. TPA, MG132, CHX and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (USA), U0126 from Cell Signaling Technology (USA), SCH772984

Increased expression of ETV4 in CRC was associated with ERK activation

In CRC mouse models, ETV4 expression was higher in cancer tissues compared with normal colorectal epithelium tissues (Fig. 1A). Then, we collected 13 human CRC tissues and corresponding adjacent normal tissues to further confirm this issue. ETV4 expression in tumor tissues were significantly elevated in 9 out of 13 patients compared with their matched adjacent tissues (Fig. 1B). P-ERK was also increased in these cancer tissues (Fig. 1B). Furthermore, correlation analysis revealed that there was

Discussion

Previous studies indicated that ERK kinase could attribute to post-transcriptional upregulation and increase protein stability of ETV4 that was considered to protect ETV4 from ubiquitination degradation, but the detailed mechanism remains unclear. In the present study, we conducted a series of plasmids of truncated wild-type ETV4 and 3 single residue mutants near to COP1 recognition motifs based on structural prediction and demonstrated that only Ser73 was the potential phosphorylated site

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (81572322 and 31371220), the Beijing Natural Science Foundation (7172021 and 5172008) and the Scientific Research Key Program of Beijing Municipal Commission of Education (KM201610025002).

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