Original ArticlesUSP26 promotes esophageal squamous cell carcinoma metastasis through stabilizing Snail
Introduction
Esophageal carcinoma is a major health problem, being the sixth most common cause of cancer death worldwide [1]. Esophageal squamous cell carcinoma, which is the most common histological subtype of esophageal cancer, is high-incidence in eastern Asia, eastern and southern Africa [[1], [2], [3]]. The five-year survival rate of ESCC patients is between 15% and 25%, and a large proportion of patients have previously metastasized before diagnosis [4,5].
Metastasis is the primary cause of death in cancer patients [6]. Epithelial-mesenchymal transition (EMT) is an essential program in the process of metastasis. EMT is a process that epithelioid cells lose their differentiated epithelial-like properties and gain a mesenchymal-like phenotype [7], and losing the expression of E-cadherin is a hallmark of EMT [8]. EMT-activating transcription factors (EMT-TFs) are vital executors of the EMT process, such as ZEB, TWIST and Snail families which have been reported to induce EMT by down-regulating E-cadherin [9].
Snail is a transcription factor containing C2H2 zinc-finger domain and is first characterized in Drosophila [10,11]. As an EMT-TF, the well-studied function of Snail is inducing EMT progress, and repression of E-cadherin transcription is an important way to activate EMT [12,13]. In many types of human cancers, Snail is found overexpressed and associated with poor prognosis and distant metastasis of cancer patients [14,15]. Snail expression also correlated with metastasis in ESCC patients [16,17]. Thus, Snail protein level must be tightly regulated, and it is important to expound the mechanism of Snail regulation.
Snail is a liable protein and degraded by ubiquitin-proteasome system. There are various E3 ligases mediating its degradation, such as SPSB3, FBXO11, FBXL14 and β-TrCP [[18], [19], [20], [21]]. For example, FBXO11 ubiquitinates Snail which is phosphorylated by PKD1, and β-TrCP or SPSB3 mediated Snail degradation in response to it phosphorylation by GSK3β [18,19,21]. It is well known that the ubiquitination process could be reversed by deubiquitinating enzymes. There are about 100 deubiquitinases, and they are classified in five families including UCHs, USPs, OTUs, Josephins and JAMMs [22]. But the mechanism of deubiquitinases stabilizing Snail is unclear. DUB3 is a deubiquitinating enzyme that has been reported to deubiquitinates and stabilizes Snail, and it is one of cytokine induced DUBs [23,24]. DUB3 is a target of CDK4/6, and phosphorylation by CDK4/6 is essential for DUB3 deubiquitinating and stabilizing Snail [23]. In recent studies, we identified OTUB1 and PSMD14 as deubiquitinases of Snail [25,26]. As an important transcription factor, Snail might be regulated by multi-deubiquitinating enzymes. Other deubiquitinases might participate in the post-translational network of Snail.
In this study, we screened a DUB expression library and found USP26 is a potent deubiquitinase responsible for Snail deubiquitination and stabilization. We also identified that USP26 is a booster of ESCC cell migration and invasion, and it is highly expressed in ESCC samples. Overall, our study demonstrates USP26 is a novel deubiquitinating enzyme of Snail and provides a possible target for the therapy of esophageal cancer metastasis.
Section snippets
Cell culture
The human ESCC cell lines KYSE30, KYSE70, KYSE140, KYSE150, KYSE180, KYSE410 and KYSE510 were munificently given by Dr. Shimada Y. and grown in RPMI/1640 medium (Hyclone, USA) plus with 10% fetal bovine serum (Hyclone, USA). HEK293T cell line was purchased from American Type Culture Collection (ATCC) and grown in DMEM medium (Hyclone, USA) plus with 10% fetal bovine serum.
Plasmids and antibodies
The USP26, USP26 mutant (C304S) and Snail were cloned into pLVX-IRES vector. The USP26 and Snail truncated mutants were
USP26 increases stability of Snail
To find potential deubiquitinating enzymes that increased Snail stability, we screened a DUB cDNA expression library. We co-transfected various deubiquitinases and Snail into the HEK293T cells. In order to enlarge the differences and eliminate the fake positive results, the cells were treated with CHX for two hours before harvest. We found USP26 significantly increased Snail stability (Fig. 1A and Fig. S1). DUB3 also enhanced the stabilization of Snail (Fig. 1A). To further validate USP26
Discussion
Despite the rapid development of antitumor therapy, metastasis is still a great threat to the cancer patients and the understanding of its biological mechanisms remains limited [5]. Metastasis is a complex multistep process that begins with cell detachment from the original sites and finally generated a micro metastatic lesions in the distant organs [28]. Before transferred and colonized to other sites, most epithelial cells detached from epithelial tissues went through a crucial step of
Conflicts of interest
The authors declare that there is no conflict of interest.
Acknowledgements
This study was supported by the National Key R&D Program of China (2016YFC1302100), the CAMS Innovation Fund for Medical Sciences (2016-I2M-1-001), and National Science Foundation of China (81773134). We thank Dr. Shimada Y. (Kyoto University, Kyoto, Japan) for kindly providing the KYSE series ESCC cell lines.
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2020, Archives of Oral BiologyCitation Excerpt :This process is mediated by different epithelial-mesenchymal transition-inducing transcription factors (EMT-TFs), including Twist1/2, SNAI1/2 (also called Snail and Slug, respectively), and ZEB1/2 (Zinc finger E-box binding homeobox 1/2) (Imani, Hosseinifard, Cheng, Wei, & Fu, 2016; van Staalduinen, Baker, Ten Dijke, & van Dam, 2018). The acquisition of mesenchymal markers such as vimentin, the increased expression of EMT-TFs and the reduced expression of epithelial markers can increase the disseminating potential of HNSCC and are consequently related to a poor prognosis (de Morais et al., 2019; Li, Zhou, Zhu, & Liu, 2019; Zang et al., 2017). The EMT-TFs suppress the expression of key epithelial genes involved in the maintenance of epithelial phenotype integrity, such as CDH1 (E-cadherin gene) which encodes the adherens junction protein E-cadherin (de Morais et al., 2019; Zhang, Cheng, Zhou, Wang, & Chen, 2013).
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Present address: Key Laboratory of RNA Biology, Center for Big Data Research in Health, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.