Previous research demonstrated that EIF3H is involved in multiple cancers. Overexpression of EIF3H promotes cancer cell growth [
14], results in a malignant phenotype [
46], and serves as a prognostic marker of recurrence and metastasis [
12,
47]. However, little is known about the role of this molecule in ESCC. Thus, this study aimed to elucidate the function of EIF3H and the molecular mechanism underlying the functional relevance in ESCC.
Validated by the expression difference in human ESCC clinical samples and their adjacent tissues, we observed that EIF3H is highly expressed in ESCC. Additionally, an oncogenic role for EIF3H in tumor progression and metastasis in this research is convincingly demonstrated in vitro and in vivo, which is in alignment with previous studies [
15,
48,
49]. EIF3H depletion in ESCC cells significantly decreased the ability of cell proliferation and mobility, examined by CCK8 assay, colony formation assay and transwell assay in vitro and xenograft and tail-vein lung metastatic mouse models in vivo. In this study, we verified that EIF3H is involved in different processes of tumor tumorigenesis and progression. The increase of proliferation rate is significantly higher in EIF3H-overexpression HET1A cells than that of EIF3H-overexpression KYSE150 and KYSE510 cells but is in nearly the same proportion of EIF3H knockdown KYSE150 and KYSE510 cells. In clinical samples, EIF3H levels in primary tumors are significantly higher than their adjacent normal samples. Taken together, EIF3H might be more essential in the process of tumorigenesis. Besides, it also takes part in EMT process and the knockdown of EIF3H suppressed tumor metastasis in vivo. Whether EIF3H expression levels rise continuously during tumor progression? We still need more clinical samples and in vivo evidence to prove this hypothesis.
Besides, the mechanism of EIF3H mediated cell growth and migration also needs further study. EIF3H is one of the 13 subunits of the elongation initiation factor EIF3 and it connected with EIF3F and other subunits to form the functional octamer core in translational regulation and control [
9]. EIF3H also belongs to the JAMM family of deubiquitinating enzymes for its putative nonconserved MPN domain. The uncharacterized deubiquitinating enzyme ability of EIF3H was verified by structural modelling analysis based on its noncanonical metalloprotease motif and in vitro deubiquitination analysis using tetra-ubiquitin cleavage assays biochemically [
50]. The mis-regulation of EIF3H might disrupt this function leading to the cause of human diseases. To address this issue, we focused on the functional partners of EIF3H. We performed a co-IP assay combined with the mass spectrometry analysis and identified that an EMT-associated transcription factor, Snail could interact with EIF3H. In vivo co-IP assays in EIF3H knockdown cells were also carried out to confirm the specificity of the interaction between Snail and EIF3H, but there is no interaction with other EIF3 subunits (EIF3A and EIF3I). Additionally, EIF3H is demonstrated to be responsible for Snail deubiquitination and stability in ESCC cell lines. EIF3H significantly decreased the poly-ubiquitin level of ubiquitinated Snail in an in vitro ubiquitination assay. Therefore, we identified EIF3H as a potential deubiquitinating enzyme of Snail.
It has been reported that Snail plays an eminent role in the tumorigenesis, growth and metastasis of epithelial tumors [
21,
51,
52], and one of its main mechanisms involved is the induction of EMT [
53]. EMT is a cellular process accompanied by the loss of epithelial phenotypes and the gain of mesenchymal features. EMT is integral in cell development, stemness and contributes pathologically to cancer progression [
54]. Mounting evidences also suggest that the acquisition of cancer invasiveness is correlated to EMT [
55,
56] and tumor metastasis is the major cause of mortality in cancer patients [
57]. EMT is featured by loss of E-cadherin expression and gain of N-cadherin and Vimentin [
58]. Snail is a well-known transcriptional repressor of E-cadherin during EMT, and it is also shown to activate the expression of invasion-associated genes and the migratory phenotype [
59]. A recent publication in lung adenocarcinoma demonstrated that EIF3H overexpression could also induce EMT signaling pathway [
16]. Our results exhibited that ectopic expression of EIF3H promoted ESCC metastasis with the increase of Snail, N-Cadherin and Vimentin, and decrease of E-cadherin, while knockdown of EIF3H exerted an opposite effect. Knockdown of Snail strikingly reversed the promoting effects of ectopic expression of EIF3H. The IHC staining of the lung metastatic nodules in tail-vein injection mice model indicated that knockdown of EIF3H correlated with Snail downregulation and it mediated EMT switch in vivo. Clinically, the levels of EIF3H were positively correlated with Snail expression. Therefore, our study demonstrates a critical EIF3H-Snail signaling axis in EMT process and tumor metastasis in ESCC. Snail expression is regulated tightly during development, and posttranslational regulation of Snail has been a frontier issue in recent years. Protein ubiquitination is a reversible posttranslational modification that regulates a broad range of biological processes [
60]. The balance between ubiquitination and deubiquitination contributes to ubiquitin homeostasis, which disruption leading to tumor initiation and progression [
61]. It is quite common that several deubiquitinating enzymes, working like isozymes, regulate the same substrate under different circumstances, especially in the regulation of vital transcription factors. The stability and ubiquitination of p53 are modulated by diverse deubiquitinating enzymes under different cancer types and stimuli, such as USP42, USP49, PHD3, OTUD1, OTUD5, ATXN3 [
62‐
67]. The protein level of Snail is also reported to be precisely regulated by ubiquitination and deubiquitination [
32]. Some deubiquitinating enzymes were reported to regulate its stability, such as DUB3, PSMD14, OTUB1 and USP26 [
27,
36,
38,
40]. Here, we also identified Snail as a novel substrate of EIF3H. EIF3H interacts with and deubiquitinates Snail to prolong its half-life. How these enzymes collaborate with each other to control Snail expression precisely remains still unknown and needs further investigation. Meanwhile, as a potential deubiquitinating enzyme of Snail, EIF3H might be an efficient target for ESCC therapeutic treatment. Specific small molecular inhibitors targeting EIF3H would be our next research direction.
In summary, we demonstrated that EIF3H is responsible for tumorigenesis, tumor growth and metastasis of ESCC through stabilizing Snail, thus promoting the EMT phenotype in ESCC cells. We revealed the novel modulating mechanism of the Snail stability by EIF3H in ESCC. Our study also has further implications in the development of potential new therapeutic strategy for ESCC.