ETV5 overexpression promotes progression of esophageal squamous cell carcinoma by upregulating SKA1 and TRPV2

Esophageal squamous cell carcinoma (ESCC) is notorious for the rapid progression especially early tumor metastasis due to the unclear mechanism. Recently, ETV5 attracts much attention for its potential role as an oncogenic transcription factor involved in multiple cancers. However, no one reported the mechanism behind the association between ETV5 expression and esophageal squamous cell carcinoma progression. In this study, we found that ETV5 was upregulated in ESCC both from online database and our ESCC tissues and ETV5 was associated with tumor staging and prognosis. Knockdown of ETV5 or its downstream genes SKA1 and TRPV2 significantly suppress ESCC cells migration and invasion, respectively. Additionally, in vivo study showed knockdown of ETV5 inhibited tumor metastasis. Further experiments unveiled ETV5 could transcriptionally upregulate the expression of SKA1 and TRPV2 and further activate MMPs in ESCC progression. In conclusion, ETV5 was associated with ESCC tumor staging and ESCC prognosis clinically. ETV5 promoted metastasis of ESCC by activating MMPs through augmenting the transcription of SKA1 and TRPV2. ETV5 was likely to be a novel oncogene and therapeutic target in ESCC.


Results
ETV5 was observed upregulated in ESCC both from online database and our ESCC tissues and ETV5 was associated with tumor staging. Knockdown of ETV5 or its downstream genes SKA1 and TRPV2 signi cantly suppress ESCC cells migration and invasion, respectively. Additionally, in vivo study showed knockdown of ETV5 inhibited tumor metastasis. Further experiments unveiled ETV5 could transcriptionally upregulate the expression of SKA1 and TRPV2 and further activate MMPs in ESCC progression.

Conclusion
ETV5 promoted metastasis of ESCC by activating MMPs through augmenting the transcription of SKA1 and TRPV2. ETV5 was likely to be a novel diagnostic marker and therapeutic target in ESCC treatment.

Background
Esophageal cancer is a global health problem with an overall 5-year less than 20% [1]. So far, China occupied the most signi cant proportion in esophageal cancer cases and deaths worldwide and esophageal squamous cell carcinoma (ESCC) was the main histological type [2,3]. ESCC is notorious for its rapid progression, especially early tumor metastasis and lacking effective treatment. Therefore, much attention should be paid to explore the biological behavior, diagnosis markers, and approach targets of ESCC.
As one of the largest families of signal-dependent transcription factors, the E26 transformation-speci c (ETS) family comprises 28 homologs. The PEA3 subset of the ETS family, contains three factors: ETV1/ER81, ETV4/PEA3/E1AF, and ETV5/ERM [4]. Recently, ETV5 attracts much attention for its important role as an oncogenic transcription factor in multiple cancer types and its involvement in multiple biological processes. For example, ETV5 is related to EMT in papillary thyroid cancer [5]. ETV5 accelerates tumor growth by promoting cell cycle G1/S transition in colorectal cancer [6]. Additionally, ETV5 is linked to the maintenance of cancer stem cell (CSC) phenotype in breast cancer [7]. Yet to date, the role of ETV5 played in ESCC is still unknown. Through TCGA and GEO databases, we found ETV5 was signi cantly elevated in ESCC, but the true function of ETV5 in ESCC has never been studied. Thus, to evaluate and identify the role of ETV5 in ESCC development and progression, further study is needed.
In this study, we investigated the expression and function of ETV5 in ESCC and further identi ed its downstream targets by employing both in vitro and in vivo assays for the rst time. The results revealed the oncogenic role of ETV5 in ESCC progression.

Data collection
The Cancer Genome Atlas (TCGA) is a large-scale cancer genomics program, and it has molecularly characterized 33 primary cancer types comprising esophageal squamous cell carcinoma. GEO is a public genomics platform composited of array-and sequence-based data. Using UALCAN and Oncomine, the expression of ETV5 in ESCC was investigated from TCGA and GEO database.

Cell culture
We purchased human ESCC cell lines, ECA109, KYSE150 and TE1 from the Institute of Biochemistry and Cell Biology of the CAS (Shanghai, China). All cell lines were cultured in DMEM medium (GIBCO) supplemented with 10% FBS (GIBCO) in an incubator containing 5% CO2 at 37℃.

Cell transfection
For transient transfection, cells were seed in 6-well plates, 100pmol siRNA-ETV5 or siRNA-SKA1 or siRNA-TRPV2 (GenePharma, Shanghai, China) was transduced into each well. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting were applied to detect transfection e ciency at 48h after transfection. For stable transfection, lentivirus vectors that encode a shRNA targeting ETV5 or shRNA non-targeting control were used to transfect ECA109 cells following the manufacturer's instruction (Genechem, Shanghai, China). In brief, cells were seed in 6-well plates, medium containing viral uid but without serum was added when the cell density reached 30% and replaced with complete medium 24h later. The transfection e ciency of each vector was detected via western blotting.

CCK8 assay
For evaluating the proliferation capacity of ESCC cells, the Cell Counting Kit 8 (CCK8) assay was used.
We planted 1x10 3 cells/well in the 96-well plates, and 10µl CCK8 reagent was used to mix cells in every well respectively after cells being incubated for 0, 24, 48, 72 and 96h. After 4h of incubation, we measured the absorbance of cells at 450nm by using a microplate reader.

Migration and invasion assay
To explore the migration ability, we resuspended ESCC cells in 200µl DMEM without serum and added them in upper chamber of the transwell device, with 5x10 4 cells/well. We then added 600µl complete medium into the lower chamber as the chemical attractants. After incubation for 48h at 37℃, cells on the lower surface of the non-coated membrane were xed by 4% paraformaldehyde and then stained by Giemsa. Images from ve representative elds of each membrane were taken by using a light microscope (100×). The number of migratory cells was counted and the relative migration rate can be calculated.
Invasion assay was similar to migration assay, but with the difference that 100µl of 200µg/ml diluted Matrigel matrix (Corning, 356234) was carefully added to the center of each transwell insert and incubated at 37℃ for 1 hour to form a gel before cells were plated.

Wound healing assay
The ECA109 and KYSE150 cells underwent a culturing process in six-well plates and when the cells reached 80% con uence, the monolayers were scraped by the tip of a 200µl pipette, and cells continued to be cultured in DMEM free of serum. At 0, 24 and 48h after scratch, cell migration was photographed by a light microscope (100×). Image J was used to calculate the closure. The formulas are: average scratch width = scratch gap area/length; the relative cell migration rate = (the scratch width at 0h-scratch width after culture)/the scratch width at 0h × 100%.
Real-time PCR assay and RNA sequencing Total RNAs were extracted from cells by using Trizol solution, according to the instruction of the manufacturer. We used Nanodrop2000 spectrophotometer to measure the RNA concentrations and synthesized complementary DNA (cDNA) from RNA by using a PrimeScript RT reagent kit (Takara, Japan). TaqMan real-time PCR assays for ETV5, SKA1 and TRPV2 were applied following the instruction of Takara Bio. The relevant primers were summarized in supplementary table 1. All reactions, including the no-template controls were run in triplicate. After the reactions, the CT values were determined using xed threshold settings. Library preparation for RNA sequencing was conducted. Generally, 1 µg highquality RNA was used, and sequencing was carried out by HiSeq2500 (Illumina Inc., San Diego, CA) at Genechem Biotechnology (Shanghai) Co., Ltd.

Chromatin immunoprecipitation (CHIP) assay
A CHIP assay was performed using an Upstate Biotechnology kit. Brie y, we successively subjected cells to the procedures containing DNA-protein cross-linking, disruption of membrane and cytosol. Samples were digested by MNase and sonicated and then precipitated with antibody against transcriptional factor ETV5. Quantitative real-time PCR was used to measure the amount of bound DNA. According to the relative amount of input and the IgG ratio, the enrichment value was calculated. The primers covering ETV5 binding site of SKA1 and TRPV2 gene promoter region were summarized in supplementary table 1.
Dual luciferase reporter assay ESCC cells were seeded into 12-well plates at a density of 2.5×10 4 cells/well. The reporter plasmid containing wild SKA1 or TRPV2 promoter and mutant SKA1 or TRPV2 promoter at the binding site, was transfected into cells, respectively. The cells were also transfected with Renilla luciferase reporter plasmids for signal normalization. After 24h, the luciferase activity was measured by the Dual Luciferase Assay System Kit (Promega, Madison, WI, USA).

Immunohistochemistry (IHC)
This study was approved by the institutional review board of East Hospital, Tongji University. ESCC and corresponding healthy esophageal mucosa (CHEM) tissues of 98 patients were collected. After being xed by Formalin and embedded by para n, tissue sections were cut to 4µm thickness and then placed in xylene and graded alcohols for depara nization and hydration. We performed heat-induced antigen retrieval in EDTA (PH 8.0) buffer for 15 minutes by using a microwave oven. To reduce nonspeci c staining, we performed blocking with 10% goat serum. After the speci c primary anti-ETV5 (ab102010, Abcam) was dropped onto the sections and incubated overnight at 4℃, the slides were counterstained with light hematoxylin, dehydrated, and cover-slipped.
Animal studies This in vivo study was approved by the animal care and use committee of Tongji University. 20 female BALB/c nude mice (6 weeks old) were used for animal studies. The animals were randomly divided into 2 groups (control and treated groups, 10 mice per group). ECA109 cells were treated with stable transfection. After cell harvest, cells were resuspended and injected into the tail vein of each mouse (2×10 6 viable ECA109 cells/mouse). At 6 weeks, the lung metastasis was monitored. According to the AVMA Guidelines for the Euthanasia of Animals, we performed intraperitoneal injection of a three-fold dose of barbiturates to euthanize all the mice. After that, lungs were removed, and the lung colonization number was counted. Serial sections of lung tissue were stained with hematoxylin and eosin.

Statistical analysis
The statistical analysis was performed by SPSS 19.0 (IBM, Armonk, NY, USA). All the experiments were carried out repeatedly three times. Differences between groups were calculated using Student's t-test, Chisquare test, or Fisher's exact test. The p value < 0.05 was considered with statistical signi cance.

Results
ETV5 expression is upregulated in esophageal squamous cell carcinoma.
To investigate the expression of ETV5 in ESCC, we initially analyzed the online data from TCGA and GEO by using UALCAN and Oncomine, respectively. ETV5 is signi cantly upregulated in ESCC, not only supported by the RNA-sequencing data from TCGA, but also validated by data from Oncomine. The expression of ETV5 is higher in ESCC cells than that in normal esophageal epithelial cells. Besides, IHC analysis showed that upregulation of ETV5 was also found in our ESCC tissues. Moreover, by analyzing the correlation of clinicopathologic parameters with ETV5 level in ESCC tissues, we found that ETV5 protein level was higher in Stage III-IV cancers than in Stage 0-II cancers.
ETV5 silencing suppresses migration and invasion but not the proliferation of ESCC cells.
To further study how ETV5 affects the functions of ESCC cells, CCK8, migration, invasion and wound healing assays were performed. The expression of ETV5 was knocked down by transfecting speci c siRNA targeting ETV5, indicated by real-time PCR and western blot results. The results of CCK8 assays indicated that knockdown of ETV5 did not decrease the cell proliferation in ECA109 and KYSE150 cell lines. However, the migration and wound healing assays results indicated that cell migration in these two cell lines was obviously suppressed when ETV5 was knocked down. Additionally, the results of invasion assay also showed that knockdown of ETV5 evidently decreased cell invasion in these two cell lines.

ETV5 transcriptionally regulates SKA1 and TRPV2
In order to identify the molecular mechanism underlying the ETV5-mediated increase in cancer progression, RNA-seq analysis was performed to compare protein-coding transcripts levels in ECA109, KYSE150 and TE1 cells treated with or without siETV5. With P < 0.05 and FC > 2 as cutoff values, 100 downregulated genes in siETV5 transfected cells were found. Among the 100 genes, SKA1 and TRPV2 attracted our attention because though the mechanism was unclear, its' over-expression in ESCC had been reported [8,9]. We performed real-time PCR and western blot validation in ESCC cells after knockdown ETV5. In ECA109 and KYSE150 cells transfected with siETV5, SKA1 and TRPV2 mRNA were signi cantly downregulated. Considering ETV5 is a transcript factor, we proposed that ETV5 may exert its functions by regulating the transcription of SKA1 and TRPV2. CHIP assay was done to verify the binding site of ETV5 in the SKA1 and TRPV2. Besides, luciferase assays demonstrated that ETV5 could increase SKA1 and TRPV2 transcriptional activation. Moreover, MMP2 and MMP9, which were reported to be downstream of SKA1 or TRPV2 in many other cancers, were found to be downregulated in ESCC cells when SKA1 or TRPV2 was knocked down by siRNAs.

SKA1 or TRPV2 silencing suppresses migration and invasion of ESCC cells.
To explore the function of SKA1 and TRPV2 in ESCC cells, migration, invasion and wound healing assays were performed. The expressions of SKA1 and TRPV2 were knocked down by transfecting speci c siRNA, respectively, indicated by real-time PCR and western blot results. The migration and wound healing assays results indicated that cell migration in these two cell lines was suppressed when whatever SKA1 or TRPV2 was knocked down. It could also be easily detected that cell invasion in these two cell lines was decreased when whatever SKA1 or TRPV2 was knocked down from the invasion assays.

Effect of ETV5 on the Vivo metastasis of ECA109
We further studied the ETV5 effect on cancer metastasis by mice modes. In the tail vein injection nude mouse model, the average number of lung metastasis per mouse was signi cantly reduced in shETV5transfected ECA109 cells compared with the control cells. Histological analysis demonstrated that the metastatic nodules formed by shETV5-transfected cells were smaller than those formed by controlled cells.

Discussion
ESCC always has a poor prognosis because of its inconspicuous symptoms and metastasis in the early stage, which limits effective therapies to a great extent. Thus, understanding its distinct biological process and nd novel diagnostic markers and therapeutic targets is imperative. In this study, we found that the expression level of ETV5 was higher in ESCC than that in the normal esophagus, both from tissue and cell aspects, which was validated by analyzing the data from TCGA and GEO database. Furthermore, the expression level of ETV5 was correlated to tumor stage and prognosis from the IHC and the analysis of online data. These results demonstrated that ETV5 might act as an oncogene similar to ETS-1 [10] and ETV4 [11] in ESCC. We further applied in vitro and in vivo studies and found that knockdown of ETV5 signi cantly suppressed ESCC migration and invasion, which indicated that ETV5 could augment ESCC metastasis.
Considering ETV5 is a transcription factor, ETV5 should regulate some downstream targets during ESCC progression. We further exerted RNA-seq analysis and found two interesting genes SKA1 and TRPV2. Though they were ever reported to be overexpressed in ESCC, the concrete function and underlying mechanism are still obscure. Kinetochore-associated complex includes SKA1, SKA2 and SKA3. This complex plays a leading role in stabilizing the attachment of spindle microtubules to kinetochores and maintaining the metaphase plate during mitosis [12,13]. Acting as an oncogene in many cancer types, SKA1 contributes to multiple biological behaviors, including cell circle, EMT, and Wnt/β-catenin pathways [14,15]. Thus, its proper function in ESCC is worth exploring. Transient receptor potential Vanilloid family is a non-selective calcium-permeable channel and acts as a cellular sensor for heat, stretch and osmosis [16]. Recently, it has raised much attention that TRPV2 acts as a cancer biomarker and potential therapeutic target for many cancer types [17], but the speci c function in ESCC is not very clear. Herein, we speculated that SKA1 and TRPV2 could promote ESCC progression, transcriptionally regulated by ETV5. Firstly, the results of in vitro studies veri ed the oncogenic roles of SKA1 and TRPV2 in migration and invasion of ESCC cells, so they could also augment ESCC metastasis. Additionally, the results of Real-time PCR and western blotting analysis indicated ETV5 could regulate the expression of SKA1 and TRPV2. Moreover, the CHIP assays and luciferase assays results further told us that ETV5 could directly bind to the promotor of SKA1 and TRPV2 and activate their transcription.
As mentioned above, ETV5 could promote ESCC metastasis by directly regulating SKA1 and TRPV2, but the involved biological process was not clear.      Effects of ETV5 knockdown on colonization in the mice lungs after tail vein injection of ECA109-derived cells, including shETV5 or negative control group. Lung images were performed to detect metastasis foci (C). Metastasis quanti cation was evaluated (D). Representative hematoxylin and eosin (H&E) staining of lung sections (E). *P 0.05; **P 0.01; ***P 0.001.