S-phase kinase-associated protein 2 promotes cell growth and motility in osteosarcoma cells

ABSTRACT

Skp2 (S-phase kinase-associated protein 2) plays an oncogenic role in a variety of human cancers. However, the function of Skp2 in osteosarcoma (OS) is elusive. Therefore, in the current study, we explore whether Skp2 exerts its oncogenic function in OS. The cell growth, apoptosis, invasion and cell cycle were measured in OS cells after Skp2 overexpression. We found that overexpression of Skp2 enhanced cell growth, and inhibited cell apoptosis in OS cells. Moreover, we observed that upregulation of Skp2 accelerated cell cycle progression in OS cells. Furthermore, the ability of migration and invasion was enhanced in Skp2 overexpressing OS cells. Mechanically, our Western blotting data suggested that Skp2 decreased the expression of E-cadherin, Foxo1, p21, and p57, but increased MMP-9 in OS cells. In conclusion, our study demonstrated that Skp2 exhibited an oncogenic function in OS cells, suggesting that inhibition of Skp2 may be a novel approach for the treatment of OS.

KEYWORDS:

• Apoptosis
• cell growth
• cell proliferation
• invasion
• osteosarcoma
• migration
• Skp2

Introduction

Osteosarcoma (OS) has become a common primary malignant bone tumor in adolescent and young adults.^1,2 Overall incidence accounts for one to 3 per million annually worldwide.³ Despite advance in both multi-agent chemotherapy and surgery of tumor resection, the survival rate of the OS patients has been remained to 60–80%.^4-6 Moreover, the 5-year survival rate of the OS patients with metastasis is approximate 30%.⁷ The effect of therapy also depends on the response rate, because the OS size may not become smaller even though it was treated with chemotherapeutic agents.^8,9 Thus, it is highly urgent to discover the molecular mechanism of OS development and to improve the poor survival rate of OS patients.

Skp2 (S-phase kinase-associated protein 2), an oncoprotein, consists of 4 distinct domains, known as destruction domain (D-box), nuclear localization signal (NLS), F-box domain, and C-terminal LRR domain.¹⁰ As a critical component of SCF (Skp, cullin, F-box containing complex), Skp2 could recognize and ubiquitinate various substrates to play its biologic role in cell cycle progression and other cellular processes.^11,12 The defined specific substrates of Skp2 consist of p21,¹³ p27,¹⁴ p57,¹⁵ p130,¹⁶ Tob1 (transducer of ERBB2),¹⁷ FOXO1 (Forkhead box O1),¹⁸ and Myc.^19-21 There is no doubt that Skp2 involves in many cellular processes including cell cycle regulation, cell proliferation, apoptosis, differentiation, and survival. All the processes have an intimate relationship with tumorigenesis and these need SCF^Skp2 E3 complex mediated ubiquitination and degradation.¹² Due to that most substrates are tumor suppressors, Skp2 is considered as an oncoprotein. In line with this notion, overexpression of Skp2 is found in multiple cancer cells like lymphomas,²² prostate cancer,²³ melanoma,²⁴ nasopharyngeal carcinoma,²⁵ pancreatic cancer,²⁶ breast carcinomas,^27,28 cervical cancer,²⁹ and human gastric cancer.³⁰ One report indicates that androgen signaling pathway promotes cell growth by upregulating Skp2 and targeting p27.^31,32 More importantly, the tumorigenesis caused by the loss of p19 Arf or the PTEN (phosphatase and tensin homolog) protein can be reversed by deleting the Skp2 in mice.³³ Moreover, another study shows that the acetylation of Skp2 and its cytoplasmic retention enhances cell migration by degrading the E-cadherin.^33,34 Chan et al. determines that Skp2-SCF E3 ligase regulates Akt ubiquitination, herceptin sensitivity and tumorigenesis.³⁵ The group also reports that Skp2-SCF complex restricts cancer stem cell traits and cancer progression.³⁶ SIRT2 (Sirtuin 2) inhibited cell growth via impairing Skp2-mediated p27 degradation in non-small cell lung cancer.³⁷ Moreover, phosphorylation by mTORC1 stabilized Skp2 and regulated its oncogenic function in gastric cancer.³⁸ Additionally, β-TRCP (β-transducin repeat-containing homolog protein)-FBXW2 (F-box and WD repeat domain containing 2)-Skp2 axis controlled cancer cell growth via promoting Skp2 degradation.³⁹ Another study reported that Skp2 loss destabilized EZH2 (enhancer of zeste homolog 2) through promoting TRAF6 (TNF receptor associated factor 6)-mediated ubiquitination to suppress prostate cancer.⁴⁰ All these studies indicate that knockdown of Skp2 may be a new way for clinical therapy.⁴¹ However, the exact mechanisms of Skp2 in modulation of tumorigenesis of OS are enigmatic.

In the current study, we explore whether Skp2 exerts its oncogenic function in OS. We found that overexpression of Skp2 led to acceleration of cell growth and inhibition of cell apoptosis. Furthermore, Skp2 promoted cell cycle entry into S phase. Notably, we found that Skp2 inhibited the expression of E-cadherin, FOXO1, p21, and p57 and increased MMP-9 (Matrix metallopeptidase 9) in OS cells. Taken together, our work provides a conceptual framework that Skp2 may be a therapeutic target for OS treatment. Inhibition of Skp2 could be helpful for achieving better treatment outcomes in patients with OS.

Results

Overexpression of Skp2 enhanced cell proliferation

To investigate the role of Skp2 in OS cells, we transfected the Skp2 cDNA into OS cells and measured the Skp2 expression at mRNA and protein level in MG63 and U2OS cells. The real-time RT-PCR analysis results suggested that Skp2 mRNA level was increased in both MG63 and U2OS cells after Skp2 cDNA transfection (Fig. 1A). Furthermore, our Western blotting data demonstrated that Skp2 protein level was upregulated with the Skp2 cDNA transfection in OS cells (Fig. 1B and 1C). The CTG assays were performed to measure the cell viability in both MG63 and U2OS cells transfected with Skp2 cDNA for 48 hours and 72 hours, respectively. Our result implied that overexpression of Skp2 enhanced cell proliferation in both OS cells (Fig. 1D).

Figure 1. Overexpression of Skp2 enhanced cell proliferation. (A) The Skp2 mRNA level was detected by real-time PCR in both MG63 and U2OS cells with Skp2 cDNA transfection. (B) Western blot was performed to analyze the protein level of Skp2 in OS cells with Skp2 cDNA transfection. (C) Quantification of the Skp2 protein level was performed. *P < 0.05; ***P < 0.001. (D) Cell viability was measured by CTG solution at 48 hours and 72 hours in MG63 and U2OS cells after Skp2 overexpression.

Overexpression of Skp2 inhibited cell apoptosis

To further investigate the role of Skp2 in OS cells, the PI-FITC-annexin assays were conducted to explore whether Skp2 could inhibit cell apoptosis. As we expected, the cell apoptotic death of both MG63 and U2OS cells was inhibited by Skp2 overexpression. Specifically, the percentage of cell apoptosis decreased from 13.35% in control cDNA group to 5.54% in Skp2 cDNA transfection group in MG63 cells (Fig. 2A). Similarly, the cell apoptosis was inhibited from 13.05% to 5.94% after Skp2 overexpression in U2OS cells (Fig. 2B). Our work indicated that Skp2 overexpression could inhibit cell apoptosis, which could partly led to cell growth promotion.

Figure 2. Overexpression of Skp2 inhibited cell apoptosis. (A) Cell apoptosis was determined by Flow cytometry in MG63 cells transfected with Skp2 cDNA. (B) Cell apoptosis was measured by Flow cytometry in U2OS cells with Skp2 overexpression.

Overexpression of Skp2 enhanced cell cycle progression

To further determine how Skp2 promoted cell growth in OS cells, the cell cycle analysis was performed by PtdIns staining and flow cytometry in MG63 and U2OS cells. Our data revealed that the percentage of S phase was increased from 30.78% to 39.82% in MG63 cells after Skp2 cDNA transfection (Fig. 3A). Meanwhile, the proportion of S phase was increased from 29.15% in control cDNA group to 39.31% in Skp2 overexpressing U2OS cells (Fig. 3B). These data showed that Skp2 distinctly accelerated cell cycle into S phase in OS cells.

Figure 3. Overexpression of Skp2 enhanced cell cycle progression. (A) The cell cycle analysis was performed in MG63 cells with Skp2 cDNA transfection. X axis: DNA content. (B) The cell cycle analysis was conducted in U2OS cells after Skp2 cDNA transfection. X axis: DNA content.

Overexpression of Skp2 promoted cell invasion and migration

The motility of OS cells with Skp2 overexpression was detected by invasion and migration assays. The Skp2 overexpressing cells increased penetration via matrigel-coated membrane compared with the control cells (Fig. 4A). The transwell experiment was conducted to dissect the migration ability of OS cells. The number of cells was increased after Skp2 transfection in both MG63 and U2OS cells (Fig. 4B). Taken together, Skp2 overexpression promoted the ability of cell motility in OS cell.

Figure 4. Overexpression of Skp2 promoted cell invasion and migration. (A) Left panel: The ability of cell invasion was measured via transwell assay in OS cells after Skp2 cDNA transfection. Right panel: Quantitative results are illustrated for left panel. (B) Left panel: The ability of cell migration was determined via transwell assay without Matrigel in OS cells after Skp2 cDNA transfection. Right panel: Quantitative results are illustrated for left panel.

Overexpression of Skp2 regulated its downstream genes in OS cells

To further investigate the molecular mechanisms of Skp2-induced cell growth and invasion in OS cells, we performed Western blotting to detect the expression of Skp2 downstream genes in MG63 and U2OS cells. It has been reported that E-cadherin, FOXO1, p21 and p57 are the substrates of Skp2.⁴¹ In line with this, we found that expression of E-cadherin and Foxo1 was significantly decreased in both MG63 and U2OS cells after Skp2 overexpression (Fig. 5 and 6). Importantly, the protein level of cell cycle related genes p21 and p57 was markedly reduced, indicating that Skp2 was involved in cell cycle in part through targeting p21 and p57 in OS cells (Fig. 5 and 6). Several studies have revealed that Skp2 enhanced MMP-9 expression in cancer cells.^42,43 Therefore, we also measured the expression of MMP-9 in OS cells after Skp2 overexpression. We observed that MMP9 protein was increased markedly after Skp2 cDNA transfection (Fig. 5 and 6). Altogether, these data implied that Skp2 exerted its oncogenic function partly through regulation of its downstream proteins in OS cells.

Figure 5. Overexpression of Skp2 regulated the related genes in OS cells. (A) The expression of downstream targets of Skp2 was detected by Western blotting in MG63 cells after Skp2 cDNA transfection. (B) Western blotting was performed to measure the expression of Skp2 downstream targets in U2OS cells with Skp2 overexpression.

Figure 6. Overexpression of Skp2 governed the downstream genes in OS cells. Quantitative results are illustrated for Fig. 5. ** P < 0.05 vs Control.

Discussion

A wealth of evidence has demonstrated that Skp2 was critically involved in tumorigenesis including OS. It has been reported that Skp2 was highly expressed in OS tissue samples. Moreover, Skp2 expression was correlated with the relapse, metastasis, and survival rate in OS.⁴⁴ This finding implied that Skp2 could be a key oncoprotein in the occurrence and development of OS, and might be a prognostic indicator in OS.⁴⁴ One study has shown that knockdown of GLI2, one key driver in Hedgehog pathway, enhanced cell cycle arrest via reduction of Skp2 in OS cells.⁴⁵ Overexpression of GLI2 promoted cell proliferation and accelerated cell cycle progression via overexpression of Skp2 in OS cells, indicating that Skp2 played a pivotal role in regulation of cell growth in OS cells.⁴⁵ Another study found that Forkhead box M1 controlled the transcriptional network of genes essential for mitotic progression and genes encoding the SCF (Skp2-Cks1) ubiquitin ligase.⁴⁶ Consistent with the oncogenic role of Skp2 in OS cells, we found that overexpression of Skp2 enhanced cell growth and invasion, inhibited apoptosis and accelerated cell cycle progression in OS cells. Our study provided the direct evidence for oncogenic function of Skp2 in OS cells.

Considering the oncogenic role of Skp2 in various cancer cells, it may be a good alternative to target Skp2 or to find Skp2 inhibitor for clinical cancer therapy. The MG132, a normal proteasome inhibitor, could inhibit cell proliferation and promote cell apoptosis though downregulating Skp2 in lymphoma cells.⁴⁷ However, the patients treated with proteasome inhibitor have many side effects.⁴⁸ It is urgent to develop small molecular inhibitors without side effects to suppress Skp2. Cyclopamine, a specific inhibitor of SMO, slowed the cell growth and promoted cell cycle arrest via reducing the expression of Skp2 and subsequent induction of p21 in OS cells.⁴⁹ Moreover, inhibition of Notch pathway by its gamma secretase inhibitor prevents OS cell growth by cell cycle regulation via reduction of Skp2 expression.⁵⁰ One study reports that CpdA (compound A) could block Skp2 binding to the SCF complex and suppress cell proliferation by inhibiting cell cycle and promoting apoptosis in myeloma cells.⁵¹ Moreover, CpdA is intensive to chemotherapeutic agents such as dexamethasone, doxorubicin, and melphalan, as well as proteasome inhibitor bortezomib in multiple myeloma.⁵¹ Interestingly, SMIP0004, one chemical compound, downregulates Skp2 in prostate cancer cells and accumulates protein p27.⁵² Recently, a new Skp2 inhibitor, compound 25, was found to restrict cancer stem cell traits and cancer progression.³⁶ There is limitation to use chemical compounds to suppress Skp2 due to the inappropriate in vivo for human cancer.

It is thought that “natural agents” may overcome these limitations and side effects. Recently, researchers found that curcumin, quercetin, lycopene, silibinin, epigallocatechin-3 gallate, could inhibit cell cycle progression and decrease the level of Skp2 in human cancers.^53-56 Saurolactam, a natural compound isolated from the aerial portions of Saururus chinensis, was reported to inhibit proliferation, migration, and invasion via reduction of Skp2 expression in human OS cells.⁵⁷ Additionally, 15,16-dihydrotanshinone I (DHTI), a lipophilic tanshinone extracted from Danshen root, was found to induce apoptosis and inhibit the cell proliferation, migration via suppression of Skp2 expression in OS cells.⁵⁸ Recently, rottlerin was found to exert its antitumor activity through inhibition of Skp2 in human cancer cells.^59,60 Matrine derivative YF-18 inhibited cell proliferation and migration via downregulation of Skp2 in lung cancer.⁶¹ It is required to discover new Skp2 inhibitors for the treatment of OS. In conclusion, our work validated the oncogenic role of Skp2 in OS, suggesting that targeting Skp2 could be an effective approach to treat OS.

Materials and methods

Cell culture and agents

The MG63 and U2OS cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin in 5% CO₂ and 37°C atmosphere. Primary antibodies for Skp2 and p57 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The E-cadherin was bought form Abcam. The antibodies for FOXO1 and p21 were obtained from Cell Signaling Technology (Danvers, MA, USA). The second antibodies used were purchased from Thermo Scientific. Lipofectamine 2000 was obtained from Invitrogen. Anti-tubulin and CTG were bought from Sigma-Aldrich (St. Louis, MO).

Transfection

Cells were seeded in 96-well or 6-well plates for overnight and transfected with control cDNA and Skp2 cDNA via lipofectamine 2000 following the instruction's protocol.⁶² After the transfection, cells were used to further analysis as described under the results sections.

Cell viability assay

The cells were seeded in 96 wells for overnight. Skp2 cDNA and empty cDNA plasmid were transfected into cells. After 48 hours and 72 hours, 20ul of CTG were added to the wells and incubated for 10 min at 37°C. Then the reaction mixture was detected by the microplateau at 490 nm.

Cell apoptosis assay

Cells were seeded in 6-well plates and transfected with empty cDNA and Skp2 cDNA for 48 hours. Then the cells were collected and washed with 1 x PBS. Subsequently, cells were suspended in 500μl binding buffer consisting of 5 μl Propidium iodide (PtdIns) and 5 μl FITC-conjugated anti-Annexin V antibody. Apoptosis was measured by a FACScalibur flow cytometer (BD, USA) as described before.⁶³

Cell cycle analysis

Exponentially growing cells were seeded in a 6-well plate for overnight and then transfected with Skp2 cDNA and control cDNA. After 48 hours, cells were harvested and washed with cold PBS. The cells were resuspended in 70% cold alcohol and kept at 4°C overnight. On the next day, cells were washed with cold PBS and suspended in 1 × 10⁶ cells/ml in PBS. Subsequently, cells were incubated with 0.1mg/ml RNase I and 50 mg/ml PtdIns at 37°C for 30 min. Cell cycle was further detected with a FACScalibur flow cytometer (BD, USA).

Cell migration assay

Cells were seeded and transfected with control cDNA and Skp2 cDNA. The transfected cells were seeded on the upper chamber without the matrigel-coated membrane in 200 μl serum-free medium. After 24h, the migrated cells were stained with 4 ug/mL calcein AM in Hanks’ buffered saline at 37°C for 1 h. Migration was assessed by calculating the migrating cells with a microscope.

Cell invasion assay

Trans-well chamber was conducted to measure tumor cell invasion. MG63 and U2OS were transfected with control cDNA and Skp2 cDNA. The transfected cells were seeded on the upper chamber with the matrigel-coated membrane in 200 μl serum-free medium. After 24h, the invaded cells were stained with 4 ug/mL calcein AM at 37°C for 1 h. Invasiveness was photographed by a microscope.

Quantitative real-time reverse transcription-PCR analysis

Total RNA was extracted from OS cells. Then the cDNA was generated by reverse transcription (RT) using RevertAid TM First Strand cDNA Synthesis Kits (Fermentas Life Sciences, Chicago, IL, USA) and oligo (dT) primers. PCR were performed using Power SYBR Green PCR Master Mix and the results were calculated by 2-ΔΔCt method as described previously.⁶² The primers used in the PCR reaction are: Skp2, forward primer (5′– GCT GCT AAAGGT CTC TGG TGT -3′) and reverse primer (5′- AGG CTT AGA TTC TGC AAC TTG -3′); GAPDH, forward primer (5′- ACC CAG AAG ACT GTG GAT GG -3′) and reverse primer (5′- CAG TGA GCT TCC CGT TCA G- 3′).

Western Blotting analysis

Proteins were extracted and the cell lysates were loaded into each lane in equal amounts and resolved by SDS-PAGE transferred to nitrocellulose membranes. Then the membranes were incubated with the primary antibodies (anti-Skp2: 1:2000; anti-E-cadherin: 1:1000; anti-FOXO1: 1:1000; anti-MMP-9: 1:500; anti-p21: 1:1000; anti-p57: 1:1000; anti-tubulin: 1:5000) at 4°C overnight. On the second day, the primaries antibodies were washed and then second antibodies were added to the membranes for 1 hour. Subsequently, the expression of protein was measured by electrochemiluminescence assay.

Statistic analysis

All statistical data were conducted by GraphPad Prism 5.0 (Graph Pad Software, La Jolla, CA). Student t test was used to evaluate significance. The results were presented as means ± SD. P < 0.05 was considered as statistically significant.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgment

We thank our colleagues for their critical comments and help.

Funding

This work is supported by the program for graduate research innovation of Xinjiang Medical University (CXCY2017033) and the research innovation program of Xinjiang Medical University (XYDCX201695).