USP17 is upregulated in osteosarcoma and promotes cell proliferation,metastasis,and epithelial–mesenchymal transition through stabilizing SMAD4

Abstract

USP17 is upregulated in several cancers, indicating that USP17 might play essential functions in tumor development. However, the function of USP17 in osteosarcoma is still unknown. Our work aimed to investigate the function of USP17 in osteosarcoma. We found that the expression of USP17 was upregulated in osteosarcoma tissues and cell lines, including MG-63 and U2OS. Several functional experiments, such as colony formation analysis, Cell Counting Kit-8 assay, wound healing analysis, and transwell assay, showed that USP17 promoted cell proliferation, migration, and invasion. Moreover, we found that USP17 facilitated migration and invasion through promoting epithelial–mesenchymal transition. SMAD4 has been found to regulate epithelial–mesenchymal transition, co-immunopurification, and glutathione S-transferase pull-down analysis demonstrated that USP17 interacted with SMAD4. Furthermore, USP17 stabilized SMAD4 through its deubiquitinase activity. In conclusion, this study shows that USP17 enhances osteosarcoma cell proliferation and invasion through stabilizing SMAD4.

Keywords

USP17 proliferation epithelial–mesenchymal transition metastasis SMAD4

Introduction

Osteosarcoma (OS) has become one of the most aggressive malignant tumors of the bone among children and adolescents.¹ Although therapeutic strategies have been developed for OS, the 5-year survival rate is still very low.¹ The major problems of OS are metastasis and recurrence. Almost 80%–90% of patients died due to pulmonary metastasis.^2–4 Therefore, it is urgent to understand the molecular mechanism of metastasis and find an effective therapeutic target to inhibit cancer cells’ metastasis.

Cancer metastasis is a multistep and complex process, which involves local invasion, lymphatic, and vascular metastasis. Epithelial–mesenchymal transition (EMT) has been found to improve cancer cells’ metastasis.⁵ EMT is regarded as a crucial step of metastasis in several cancers including OS.^6,7

Ubiquitin modification is dynamically regulated by ubiquitin ligases and deubiquitinases (DUBs). Several reports demonstrate that DUBs play multiple functions in cancer development, including signal transduction, transcriptional regulation, and cell cycle and DNA repair.^8–11 Recently, increasing evidence show that DUBs serve as tumor activator or tumor suppressor.^12,13 The ubiquitin-specific protease (USP) family is the largest family of DUB. There are approximately over 60 members.⁵

USP17 was originally regarded as DUB3, a member of the murine hematopoietic system.⁵ USP17 is a 58-kDa protein; it has a catalytic domain near the N-terminus and two hyaluronan-binding motifs in the C-terminal region.^14,15 USP17 has been reported to play key functions in tumor development, such as cell proliferation and migration.^16,17 USP17 deubiquitinates and stabilizes CDC25A during the G1/S and G2/M checkpoints, thereby promoting cell cycle progression.⁵ USP17 has been found to regulate metastasis in non–small-cell lung cancer (NSCLC).⁵ But, the function of USP17 in OS is still unknown.

Here, our work shows that USP17 is upregulated in OS tissues and cell lines. USP17 serves as an oncogene and promotes cancer cell proliferation, migration, and invasion. Furthermore, USP17 also promotes EMT, thereby enhancing invasion ability of OS cells. USP17 also stabilizes SMAD4 through its DUB activity. Together, our work provides USP17 as a novel therapy target in OS.

Materials and methods

OS tissue samples

Our tissue sample experiments were approved by the Ethics Committee of the Fujian Medical University Union Hospital, and the patients had known and approved the experiments.

Cell lines and cell culture

The human OS cell lines U2OS and MG-63 and human immortalized osteoblast cell line hFOB1.19 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The hFOB1.19 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s F-12 supplemented with Geneticin (400 mg/mL) and 10% fetal bovine serum (FBS) at 34°C with 5% CO₂. The U2OS and MG-63 cells were cultured in RPMI 1640 containing 1% streptomycin and 10% FBS at 37°C with 5% CO₂.

Cell transfection

Cells were cultured to approximately 60%–70% confluence and transfected with vector, FLAG-USP17 or scramble RNA (SCR), USP17 small interfering RNA (siRNA) using the Lipofectamine 2000 Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.

Western blotting

The whole protein lysates were prepared with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Jiangsu, People’s Republic of China). Next, protein lysates were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes. Subsequently, the membranes were blocked with 5% skim milk at room temperature for 1 h and incubated with specific antibodies at 4°C overnight and then washed with phosphate-buffered saline with Tween 20 (PBST) for three times. Next, the membranes were incubated with horseradish peroxidase (HRP)–conjugated secondary antibodies at room temperature for 1 h and washed with PBST three times. Finally, enhanced chemiluminescence (ECL) reagent (Beyotime) was used for detection.

Real-time polymerase chain reaction

Total RNA was extracted from cells and tumor tissue samples using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s method. Next, complementary DNA (cDNA) was synthesized by reverse transcription and subjected to real-time polymerase chain reaction (PCR) with the specific primers using SYBR Green Mix (Thermo Fisher Scientific). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. Each experiment was performed three times. All data were calculated using the 2^−ΔΔCt method.

Colony formation analysis

Approximately, 3 × 10³ cells were placed in six-well plates. Cells were cultured with serum-free RPMI 1640 medium at 37°C. After 2 weeks, cells were fixed with ice-cold methanol and then stained with 0.5% crystal violet. The colonies containing over 60 cells were counted. Each experiment was performed three times.

Cell Counting Kit-8 assay

Cell Counting Kit-8 (CCK-8) assay was used to detect the effect of USP17 on cell growth. Briefly, 48 h after transfection, cells were placed in 96-well plates at the density of 2500 cells each well. About 20 µL of CCK-8 (Beyotime) was added into each well every 12 h. After 1 h of incubation, the absorbance was measured at 450 nm using a microplate spectrophotometer (Thermo 3001; Thermo Fisher Scientific). Each experiment was performed three times.

Transwell assay

Transwell assay was used to determine the invasion ability of cells. In brief, 24-well invasion chamber (Corning, New York, NY, USA) was coated with Matrigel (Corning) before being used. MG-63 cells were transfected with vector, FLAG-USP17 or SCR, USP17 siRNA, respectively. After 48 h of transfection, cells were counted and placed in the upper chamber at a density of 4 × 10⁴ cells with serum-free medium. The lower chamber was filled with 600 µL RPMI 1640 containing 10% FBS. Cells were incubated 24 h at 37°C with 5% CO₂. Migrated cells on the lower surface were fixed with methanol and stained with 0.5% crystal violet. The number of invaded cells was counted under microscope. Each experiment was performed three times.

In vivo deubiquitination analysis

FLAG-SMAD4, HA-ubiquitin, and Myc-USP17 were transfected in U2OS cells. After 48 h of transfection, cells were treated with MG132 (10 mM) for 5 h and then lysed. The ubiquitinated status of SMAD4 was determined by western blot with HA antibody.

In vitro deubiquitination analysis

The in vitro deubiquitination analysis was performed as previously described.¹⁸ In brief, FLAG-SMAD4 and HA-ubiquitin were co-expressed in U2OS cells. Next, cells were treated with proteasome inhibitor MG132 (10 mM) for 5 h, and FLAG antibody was used to isolate ubiquitinated SMAD4 through immunoprecipitation (IP) analysis. In a parallel experiment, vector or Myc-USP17 (wt or mutation) was expressed in U2OS cells and purified by IP with anti-Myc Affinity Matrix (Roche, Pleasanton, CA, USA). Myc-USP17 was eluded with Myc peptide and then dialyzed. The ubiquitinated SMAD4 was incubated with purified USP17 in a deubiquitination reaction buffer (5% glycerol, 50 mM HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) (pH 7.5), 5 mM MgCl₂, 1 mM dithiothreitol (DTT), 1 mM adenosine triphosphate (ATP), and 100 mM NaCl) at 30°C. The ubiquitinated status of SMAD4 was determined by western blot with HA antibody.

Wound healing analysis

USP17 was overexpressed or knocked down in MG-63 cells, 48 h after transcription; 5 × 10⁵ cells were placed in six-well plate; and cells were scratched by pipette tip when cell density reached 90%–100%. Subsequently, phosphate-buffered saline (PBS) solution was used to remove the detached cells. The migration distance was measured under a light microscope. Each experiment was performed three times.

Soft agar colony formation analysis

Approximately, 3 × 10⁴ cells were resuspended with 2.5 mL growth medium which contained 0.4% agarose and next layered onto 2.5 mL of 0.75% agarose/medium in the six-well plates. Cells were cultured with 2.5 mL fresh growth medium every 2 days for 2 weeks. Finally, the colonies were stained with 0.5% crystal violet and counted under a microscope. Each experiment was performed three times.

Co-immunopurification assay

Cell lysis was prepared by 0.3% IP buffer (0.3% NP40, 50 mM Tris-HCl (pH: 7.5), 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA)). About 2 µg anti-USP17 antibody was added and incubated at 4°C overnight. Next, approximately 70 µL 50% protein A beads was added and incubated at 4°C for 2 h with rotation. Beads were washed with 0.1% IP buffer (0.3% NP40, 50 mM Tris-HCl (pH: 7.5), 150 mM NaCl, 5 mM EDTA) three times. The USP17-interacted proteins were binding in the beads. Next, western blotting was performed to detect the interaction between USP17 and SMAD4.

Glutathione S-transferase pull-down analysis

Glutathione S-transferase (GST) pull-down analysis was performed as previously described.⁵ Briefly, 60 µL of 50% Glutathione Sepharose 4B beads (Amersham Biosciences, Piscataway, NJ, USA) was used to immobilize equal amounts of GST-fusion protein in 1.5 mL binding buffer (100 mM KCl (pH: 7.6), 10 mM HEPES, 0.5% CA630, 5 mM EDTA, 5% glycerol, 3 mM MgCl₂). Beads were incubated at 4°C for 90 min with rotation and then washed with binding buffer three times. Subsequently, 5 µL of in vitro–transcribed/translated SMAD4 was added and incubated at 4°C for 2 h with rotation. The beads were washed with ice-cold PBS three times and boiled in 30 µL 2× loading buffer.

Statistical analysis

All data were represented as mean ± standard deviation (SD). Comparisons between control group and experimental group were done using Student’s t test. p < 0.05 was considered as a statistically significant difference.

Results

USP17 is elevated in human OS tissues and cell lines

Initially, in order to detect the level of USP17 in OS, we performed quantitative reverse transcription polymerase chain reaction (qRT-PCR) in MG-63 and U2OS OS cell lines, and hFOB1.19 osteoblast cell line was used as a control. The results showed the expression of USP17 was obviously higher in OS cell lines than that in osteoblast cells (Figure 1(a)). Subsequently, we assessed the protein level of USP17 in OS cell lines and osteoblast cell line. As shown in Figure 1(b), the expression of USP17 was upregulated in OS cell lines. Moreover, we also collected 32 pairs of tumor adjacent normal tissues (ANT) and human OS tissues. The messenger RNA (mRNA) level of USP17 was assessed by qRT-PCR. It was found that the expression of USP17 was upregulated in OS tissues (Figure 1(c)). Subsequently, we analyzed the association between the expression of USP17 and clincopathological factors. High expression of USP17 was obviously correlated with tumor size and metastasis but not correlated with gender, age, differentiation, and tumor–node–metastasis (TNM) stage (Table 1). Furthermore, high expression of USP17 predicted poor prognosis of OS (Figure 1(d), p < 0.05). In conclusion, USP17 might play a key function in OS.

Figure 1.

USP17 is elevated in human osteosarcoma tissues and cell lines. (a) The mRNA level of USP17 was detected in the osteosarcoma cells and osteoblast cells by qRT-PCR. (b) The protein level of USP17 was detected in the osteosarcoma cells and osteoblast cells by western blotting. (c) The total RNA was extracted from 32 pairs of tumor adjacent normal tissues (ANT) and human osteosarcoma tissues, and the mRNA level of USP17 was determined by qRT-PCR. (d) The correlation between USP17 expression and overall survival of patients with osteosarcoma was detected by Kaplan–Meier analysis.

Table 1.

Clinicopathologic variables in 32 osteosarcoma patients.

Variables	No. (n = 32)	USP17 protein expression		p value
Variables	No. (n = 32)	Low (n = 10)	High (n = 22)	p value
Gender
Male	21	6	15	0.652
Female	11	4	7	0.652
Age
<18	19	5	14	0.467
≥18	13	5	8	0.467
Tumor size (cm)
<5	13	7	6	0.023
≥5	19	3	16	0.023
Differentiation
Well/moderate	14	4	10	0.773
Poor	18	6	12	0.773
TNM stage
I–II	14	5	9	0.631
III–IV	18	5	13	0.631
Metastasis
Yes	18	3	15	0.044
No	14	7	7	0.044

TNM: tumor–node–metastasis.

Upregulation of USP17 promotes cell proliferation

To investigate the function of USP17 on OS cells, we overexpressed or knocked down USP17 in MG-63 and U2OS cells, respectively. The expression of USP17 was detected by qRT-PCR and western blotting. As shown in Figure 2(a) and (b), USP17 was significantly upregulated when USP17 was overexpressed. And the expression of USP17 was dramatically decreased when USP17 was silenced by USP17 siRNA, and the USP17 siRNA#2 was more efficiency than USP17 siRNA#1, so USP17 siRNA#2 was used for the further experiments. In order to investigate the effect of USP17 on cell proliferation, we performed CCK-8 analysis and colony formation analysis. The data suggested that ectopic expression of USP17 promoted the growth of MG-63 and U2OS cells (Figure 2(c)). However, knockdown of USP17 suppressed the growth of MG-63 and U2OS cells (Figure 2(c)). Next, the result of colony formation analysis revealed that the number of colonies was significantly increased when USP17 was overexpressed; however, the number of colonies was decreased when USP17 was knocked down in MG-63 cells (Figure 2(d)). The similar results were observed in U2OS cells (Figure 2(d)). In brief, our work shows that USP17 facilitates cell proliferation in OS cells.

Figure 2.

Upregulation of USP17 promotes cell proliferation. (a) USP17 was overexpressed or knocked down in MG-63 and U2OS cells, respectively. The mRNA level of USP17 was assessed by qRT-PCR. (b) USP17 was overexpressed or knocked down in MG-63 and U2OS cells, respectively. The protein level of USP17 was assessed by western blotting. (c) MG-63 and U2OS cells were treated with vector, FLAG-USP17 or scramble siRNA (SCR), USP17 siRNA (siUSP17), respectively. The cell viability was determined by CCK-8 assay. (d) MG-63 and U2OS cells were treated with vector or FLAG-USP17; scramble siRNA (SCR) or USP17 siRNA (siUSP17), respectively. The cell proliferation was determined by colony formation assay.

USP17 facilitates migration and invasion in MG-63 cells

Tumor progression is regulated by cancer cell proliferation and invasion. For this reason, we next explored the function of USP17 on cell invasion. We first performed wound healing analysis to detect the effect of USP17 on cell migration. The result indicated that ectopic expression of USP17 strongly promoted cell migration in MG-63 cells; however, inhibition of USP17 significantly suppressed cell migration in MG-63 cells, compared with control group (Figure 3(a)). Subsequently, transwell analysis was performed. As shown in Figure 3(b), overexpression of USP17 dramatically increased the number of invaded cells. However, USP17 inhibition decreased the number of invaded cells (Figure 3(b)). Moreover, we detected whether USP17 regulated the anchorage-independent cell growth of MG-63 cells. As shown in Figure 3(c), the colony formation in soft agar was obviously increased when USP17 was overexpressed in MG-63 cells. On the contrary, the colony formation in soft agar was decreased when USP17 was knocked down in MG-63 cells. Together, USP17 facilitates migration, invasion, and anchorage-independent growth in MG-63 cells.

Figure 3.

USP17 facilitates migration and invasion in MG-63 cells. (a) USP17 was overexpressed or knocked down in MG-63 cells. Wound healing assay was used to detect the effect of USP17 on cell migration. (b) USP17 was overexpressed or knocked down in MG-63 cells. Transwell invasion assay was used to detect the effect of USP17 on cell invasion. (c) USP17 was overexpressed or knocked down in MG-63 cells. Soft agar assay was performed to determine the effect of USP17 on OS cells’ anchorage-independent growth.

USP17 promotes the EMT in OS

Increasing evidence indicate that EMT promotes cancer cells’ invasion.^19,20 The main characteristic of EMT is loss of E-cadherin and gain of N-cadherin.^6,21 In order to explore whether USP17 regulated EMT in MG-63 cells, we overexpressed or knocked down USP17 in MG-63 cells and detected the mRNA and protein levels of epithelial and mesenchymal markers. The results of qRT-PCR and western blotting demonstrated that the expression of epithelial markers, including E-cadherin and α-catenin, was significantly decreased when USP17 was overexpressed, but the expression of mesenchymal markers, including N-cadherin and fibronectin, was obviously increased when USP17 was overexpressed (Figure 4(a) and (b)). However, when USP17 was knocked down in MG-63 cells, the opposed results were observed. The expression of epithelial markers was increased, and the expression of mesenchymal markers was decreased (Figure 4(c) and (d)). In conclusion, USP17 promotes cell invasion through facilitating EMT in MG-63 cells.

Figure 4.

USP17 promotes the EMT in osteosarcoma. (a) MG-63 cells were transfected with vector or FLAG-USP17. After 48 h of transfection, qRT-PCR was performed to detect relative mRNA levels of EMT-associated gene. (b) MG-63 cells were transfected with vector or FLAG-USP17. After 48 h of transfection, western blotting was performed to detect relative protein levels of EMT-associated protein. (c) MG-63 cells were transfected with scramble siRNA (SCR) or USP17 siRNA (siUSP17). After 48 h of transfection, qRT-PCR was performed to detect relative mRNA levels of EMT-associated gene. (d) MG-63 cells were transfected with scramble siRNA (SCR) or USP17 siRNA (siUSP17). After 48 h of transfection, western blotting was performed to detect relative protein levels of EMT-associated protein. (e) SMAD4 was overexpressed in USP17-depleted MG-63 cells, 48 h after transfection, and qRT-PCR was performed to detect relative mRNA levels of EMT-associated gene. (f) SMAD4 was knocked down in USP17-overexpressed MG-63 cells, 48 h after transfection, and qRT-PCR was performed to detect relative mRNA levels of EMT-associated gene.

USP17 interacts with SMAD4 and stabilizes SMAD4 through its DUB activity

Several previous reports indicate that SMAD4 is highly expressed in multiple cancers, including skull base chordomas and OS.^22,23 Moreover, SMAD4 has been found to play a key role in EMT.^24–26 In order to determine the detailed mechanism of USP17 on EMT, we performed co-immunoprecipitation (co-IP) analysis to detect the interaction between USP17 and SMAD4. The results indicated that USP17 interacted with SMAD4, but there is no interaction between USP17 and SMAD3 (Figure 5(a)). Reciprocal immunoprecipitation with anti-SMAD4 and immunoblotting with anti-USP17 also demonstrated that USP17 interacted with SMAD4 (Figure 5(a)). To further explore whether USP17 directly interacted with SMAD4, GST pull-down assay was performed. The results showed that USP17 directly interacted with SMAD4 (Figure 5(b)). Collectively, our work reveals that USP17 physically associates with SMAD4.

Figure 5.

USP17 interacting with SMAD4 stabilizes SMAD4 through its deubiquitinase activity. (a) Co-IP analysis was used to determine the interaction between USP17 and SMAD4 in vitro. (b) GST pull-down analysis was used to determine the interaction between USP17 and SMAD4 in vivo. (c) USP17 was knocked down in U2OS cells, 48 h after transfection, and the protein and mRNA levels of SMAD4 were detected by western blotting and qRT-PCR, respectively. (d) Wild-type USP17 (wt-USP17) and catalytically inactive USP17 (USP17/C89S) were overexpressed in U2OS cells, respectively. After 48 h of transfection, the expression of USP17 and SMAD4 was detected by western blotting. (e) USP17 was knocked down in U2OS cells, and MG132 was incubated with cells. The protein level of SMAD4 was detected by western blotting. (f) HA-ubiquitin and FLAG-SMAD4 were co-expressed with SCR or USP17 siRNA in U2OS cells. Next, cells were incubated with MG132 for 5 h, SMAD4 was subjected to IP, and the poly-ubiquitination of SMAD4 assessed by western blot using HA antibody. (g) Ubiquitinated SMAD4 was purified from MG132-treated U2OS cells expressing FLAG-SMAD4 and then incubated with purified Myc-tagged wt-USP17 or USP17/C89S in a deubiquitination assay buffer. The poly-ubiquitinated state of SMAD4 was assessed by western blot using HA antibody.

USP17 has already been reported that it can deubiquitinate histone deacetylase (HDAC) and interleukin 33 (IL-33),^27,28 so we assume that USP17 might regulate the expression of SMAD4 through its DUB activity. In order to verify our hypothesis, we knocked down USP17 in U2OS cells. We found that the protein level of SMAD4 was dramatically decreased (Figure 5(c)), but the mRNA level had little change (Figure 5(c)). This indicated that USP17 might influence SMAD4 expression through post-transcriptional modification. To further decipher whether USP17 regulated SMAD4 through its DUB activity, next, we overexpressed wild-type USP17 and catalytically inactive USP17 (USP17/C89S) in U2OS cell. As shown in Figure 5(d), the expression of SMAD4 was increased when wild-type USP17 was overexpressed. However, ectopic expression of USP17/C89S had no effect on SMAD4 expression (Figure 5(d)). Moreover, we found MG132, a proteasome-specific inhibitor, rescued SMAD4 degradation in USP17-depleted cells (Figure 5(e)). Together, these results indicate that USP17 interacts with SMAD4 and stabilizes SMAD4 through its DUB activity.

In order to explore whether USP17 promoted EMT through stabilizing SMAD4, we overexpressed SMAD4 in USP17-depleted MG-63 cells and knocked down SMAD4 in USP17-overexpressed MG-63 cells. As shown in Figure 4(e) and (f), while SMAD4 was overexpressed in USP17-delpeted cells, the expression of E-cadherin and α-catenin was decreased, and the expression of N-cadherin and fibronectin was increased. On the contrary, while SMAD4 was knocked down in USP17-overexpressed cells, the expression of E-cadherin and α-catenin was increased, and the expression of N-cadherin and fibronectin was decreased. Together, USP17 promotes EMT through stabilizing SMAD4.

Discussion

Following the recent reports identifying the essential function of USP17 in the regulation of cell proliferation and migration,^10,17 the underlying mechanism of USP17 in OS is still unclear. Here, we found that USP17 was upregulated in OS tissues and cell lines. Moreover, high expression of USP17 was correlated with tumor size and metastasis.

Ubiquitination plays a crucial function in regulating cell cycle progression. Multiple components of the cell cycle progression are regulated by ubiquitination, such as various cyclin-dependent kinase inhibitors (CDKIs) and cyclins.⁵ Similar to other covalent modifications, including methylation and phosphorylation, ubiquitination is a reversible post-translational modification. Many DUBs have been found to regulate the cell cycle. For instance, USP44 facilitates the anaphase initiation and regulated mitosis through deubiquitinating CDC20.⁵ Moreover, USP9 regulates chromosomal alignment and segregation through deubiquitination of surviving cells.⁵ We found that USP17 facilitated cell proliferation in OS, but the detailed mechanism of USP17 on cell proliferation in OS was unknown.

Previous report has revealed that the expression of USP17 is regulated by chemokines and coordinates the chemotaxis, indicating that USP17 can potentially facilitate cancer metastasis.⁵ This work corroborates this hypothesis. Wound healing assay and transwell assay demonstrated that USP17 promoted OS cell migration and invasion. Recently, a report has showed that USP17 promotes tumorigenesis and invasion in NSCLC.⁵ Our work was consistent with previous reports. Several studies showed that SMAD4 plays important role in tumorigenesis and EMT.^29,30 Although many studies indicate that SMAD4 also serves as a tumor suppressor in multiple types of cancers,^31,32 multiple reports have demonstrated that SMAD4 acts as an oncogene in several cancers.⁵ SMAD4 has been found to promote colony formation and migration in hepatocellular carcinoma.⁵ Moreover, SMAD4 inhibition significantly increases E-cadherin expression and decreases N-cadherin expression in prostate cancer.³⁰ In this study, we found that USP17 interacted with SMAD4 and stabilized it through its DUB activity. Moreover, USP17 promoted EMT through regulation of SMAD4. Meanwhile, several reports have indicated that microRNA regulates SMAD4, such as miR-34a, miR-130a-3p, and miR-205.^25,26,29 Interestingly, although their mechanisms are different, whether SMAD4 is regulated by microRNA and USP17 or by different mechanisms in different cancers to regulate SMAD4 remains unknown. Moreover, USP17 has been found to regulate EMT through stabilizing Snail1 in breast cancer.³³ Snail1 is a key transcriptional factor which associated with EMT. USP17 might promote EMT in different cancers through different mechanisms.

In conclusion, our work first detects the function of USP17 in OS. USP17 serves as an oncogene in OS. And, we investigated the detailed mechanism of USP17 on promoting EMT. We provide USP17 as a novel biomarker in OS.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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