Background
Osteosarcoma is the most prevalent primary malignant bone tumor that affects mainly children and adolescents. Osteosarcoma treatment has undergone dramatic changed drastically over the past 20 years, whatever the survival rate shows limited. Thus far, the 5-year survival rate is approximately 20% with surgical treatment alone. Moreover, half of the patients often exhibit pulmonary metastasis, which results in high patient mortality [
1]. Thus, chemotherapy is typically employed in an adjuvant case for improving the prognosis and long-term survival. However, recurrence frequently manifests as pulmonary metastasis or, less frequently, metastasis to distant bones or as a local recurrence [
2‐
4]. Thus, a novel strategy that would effectively inhibit metastasis, particularly to the lungs, from the primary osteosarcoma site is highly desirable.
Cancer metastasis is a critical step in tumor progression and a major cause of mortality in cancer patients. Epithelial to mesenchymal transition (EMT) has received considerable attention as a conceptual paradigm for explaining invasive and metastatic behavior during cancer progression [
5]. EMT is a normal physiologic process in vertebrate development and tissue homeostasis, and is increasingly considered to be involved in disease states such as tissue fibrosis and cancer [
6]. EMT is a process through which epithelial cells lose their polarity and are converted into a mesenchymal phenotype [
5,
7]. A hallmark of EMT is the loss of epithelial characteristics, such as a decrease in the expression of the cell adhesion molecular E-cadherin and the acquisition of a mesenchymal phenotype accompanied by increased vimentin expression. EMT-related transcription factors such as TWIST-1, snail, slug, ZEB1, and ZEB2 orchestrate the EMT, and enable the early steps of metastasis, which consist primarily of local invasion and the subsequent dissemination of tumor cells to distant sites [
8]. These transcription factors repress E-cadherin expression by binding to the E-box in the E-cadherin gene promoter, and consequently promote EMT [
9‐
13]. Substantial evidence has indicated that osteosarcoma exhibits EMT-like states, characterized by changes in the expression of EMT-related transcription factors, such as TWIST-1, snail, and ZEBs, which are involved in the complex pathogenesis of osteosarcoma [
14]. Targeting these transcription factors may provide a novel opportunity in osteosarcoma treatment by controlling metastasis.
Cysteine-rich 61 (Cyr61) is the first discovered member of the CCN family [
15], which comprises Cyr61/CCN1, connective tissue growth factor (CTGF/CCN2), nephroblastoma overexpressed (Nov/CCN3), Wisp-1/elm1 (CCN4), Wisp-2/rCop1 (CCN5), and Wisp-3 (CCN6). All members of the CCN family share a common domain structure and exert variant cellular functions such as the regulation of cell division, chemotaxis, apoptosis, adhesion, motility, and ion transport [
16‐
18]. The Cyr61 protein has been reported to mediate cell adhesion, stimulate chemostasis, augment growth factor-induced DNA synthesis, foster cell survival, and enhance angiogenesis [
19,
20]. Previous studies have found that Cyr61 expression is associated with breast cancer, pancreatic cancer, and gliomas [
21‐
24]. Downregulated Cyr61 expression has been reported in prostate cancer, uterine leiomyoma, rhabdomyosarcoma, and non-small-cell lung carcinoma [
25‐
28]. The ambiguous expression of Cyr61 in different types of cancer suggests that Cyr61 may exert a sophisticated function depending on the cellular context.
Previous studies have shown that Cyr61 modulates cell migration and invasion in human cancer cells [
29‐
31]. Moreover, Cyr61 has been proposed to play a pivotal role in osteogenesis and an aberrant expression of Cyr61 is associated with osteosarcoma progression and lung metastasis [
32]. Nevertheless, whether Cyr61 promotes EMT in osteosarcoma remains unclear. This paper is the first to provide evidence that Cyr61 increases EMT in osteosarcoma and contributes to lung metastasis. In addition, Cyr61-promoted EMT is mediated by αvβ5 integrin, Raf-1, mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK), and Elk-1 signaling pathways, and may be involved in the regulation of EMT in osteosarcoma. Finally, we show that the knock-down of Cyr61 expression inhibits lung metastasis in osteosarcoma. In conclusion, our findings revealed that Cyr61 regulated EMT and thus promoted lung metastasis in osteosarcoma.
Discussion
Osteosarcoma is a high-grade malignant bone neoplasm that occurs mainly in children and adolescents. The chemotherapy regimens are not considerably effective, and result in the death of 20% of all patients because of lung metastasis. Therefore, developing an effective adjuvant therapy for preventing osteosarcoma metastasis is critical. EMT has received considerable attention as a conceptual paradigm for explaining invasive and metastatic behavior during cancer progression [
5]. In this study, we determined that Cyr61 induced EMT and promoted lung metastasis in osteosarcoma. Moreover, Cyr61-induced EMT was mediated by an integrin αvβ5 receptor and the Raf-1, ERK, and Elk-1 signaling pathways.
In the past decade, Cyr61 has been implicated in osteo/chondrogenesis. It promotes chondrogenic differentiation through the expression of type II collagen [
41]. In addition, Cyr61 expression promotes osteogenesis by increasing osteoblast differentiation and inhibiting osteoclast formation [
42]. Because Cyr61 is tightly regulated in osteogenesis, aberrant levels or altered forms of CCN proteins are associated with osteosarcoma progression. The Cyr61 expression level is correlated with a poor prognosis in osteosarcoma specimens, irrespective of whether the disease is metastatic [
33]. Moreover, Cyr61 expression was higher in patients with primary osteosarcoma than in those with normal bones, and was highly expressed in metastatic specimens. The knockdown of Cyr61 inhibited
in vitro osteosarcoma cell invasion and migration as well as
in vivo lung metastasis in mice [
43]. These results showed a great opportunity for Cyr61 to be used as a novel prognosis marker and therapeutic target in osteosarcoma. Consistent with previous studies, the current study showed that Cyr61 promoted cell migration and lung metastasis through EMT in osteosarcoma (Figure
2 and
6). The Cyr61 protein promoted mesenchymal transformation by upregulating mesenchymal markers (TWIST-1 and N-cadherin) and repressing the epithelial marker (E-cadherin). This study revealed a new molecular mechanism that elucidates the role of Cyr61 in osteosarcoma progression.
Integrin is the most cruciall cell surface regulator that links the extracellular matrix to intracellular signaling molecules, and could regulate numerous cellular biological functions such as cell adhesion, signaling, motility, survival, gene expression, growth, and differentiation. Most studies have shown that Cyr61 exerts its function by directly binding to integrins [
18,
44]. In this study, Cyr61-induced mesenchymal transformation was mediated by integrin αvβ5, but not by αvβ3. Moreover, treating osteosarcoma cells with the anti-αvβ5 integrin antibody inhibited Cyr61-induced mesenchymal transformation (Figure
2). The integrin αvβ3 and αvβ6 antibodies have been implicated in lung and oral cancers [
45,
46]. However, this paper is the first to present a discussion on the role of integrin αvβ5 in EMT.
Several intracellular signaling proteins such as the receptor tyrosine kinase family [
47], small GTPase family, and MAPK family [
48] are implicated in the process of EMT. In the MAPK family, ERK, JNK, and p38 promote EMT by repressing the expression of E-cadherin through distinct mechanisms [
49‐
51]. In this study, we determined that Cyr61 promoted mesenchymal transformation in osteosarcoma through the Raf-1/MEK/ERK signaling cascade (Figure
4). Both pathway inhibitors and dominant mutants could reduce the Cyr61-promoted mesenchymal transformation in osteosarcoma cells. Transforming growth factor-β (TGF-β), a multifunctional cytokine regulating various cellular processes, is a well-discussed EMT inducer. The TGF-β activates various signaling proteins such as Smads, phosphatidylinositol 3-kinase, and MAPK to regulate EMT [
52]. Our investigation showed that the MEK/ERK signaling pathway mediated Cyr61-induced EMT, suggesting that the other MAPK members may participate in this transition. Additional efforts will be expended to determine the detailed mechanism involved in Cyr61-induced EMT in osteosarcoma.
The Elk-1 belongs to the ETS-domain transcription factor family, which regulates cell growth, differentiation, and survival. However, recent studies have revealed that Elk-1 modulates the genes involved in transcript turnover and cell migration [
53]. By regulating the genes, such as plasminogen activator-1 and metalloproteinases-2 and -9, Elk-1 plays a crucial role in cancer progression. Numerous studies have indicated that Elk-1 is involved in the progression of several cancers. The Elk-1 is required for androgen-receptor-dependent growth and the survival of prostate cancer cells [
54]. The Elk-1 controls cell migration by targeting various genes [
55] or by regulating cell survival [
56] in breast cancer. Altough the correlation between Elk-1 and cancer progression has been strengthened, we examined the role of Elk-1 in EMT for the first time in this study. The Raf-1/MEK/ERK signaling cascades activated downstream transcription factor Elk-1, and thus, participated in Cyr61-induced EMT (Figure
5). The molecular mechanism involved in Elk-1-regulated EMT will be investigated in the future.
Cancer metastasis, the major cause of mortality in cancer patients, comprises several steps through which cells detach from the primary tumor and form a secondary tumor at a distant site [
57]. Abundant evidence for EMT associated with metastasis has been provided in recent studies [
7]. To determine the effect of Cyr61 on osteosarcoma progression directly, we knocked down Cyr61 expression in MG63 cells by using shRNA (Figure
6). The Cyr61 knockdown significantly reduced mesenchymal markers, induced epithelial marker expression, and inhibited migration in MG63 cells. Finally, the transition of the cell phenotype inhibited lung metastasis. These data indicated that Cyr61 plays a crucial role in osteosarcoma metastasis to the lung
in vivo.
Because lung metastasis is the major cause in mortality of patients with late-stage osteosarcoma, identifying the tumor-related factors involved in cancer metastasis is critical. In this study, we gained new insights into the Cyr61 function and its role in osteosarcoma progression. The Cyr61 expression was correlated with cell migratory potential in osteosarcoma cells, and its upregulation promotes EMT and tumor metastasis in vivo. Moreover, the Cyr61-promoted mesenchymal transition was mediated by the integrin αvβ5, Raf-1, MEK, ERK, and Elk-1 signaling pathways. Our observation provides a novel opportunity for treating osteosarcoma by targeting the Cyr61 gene.
Materials and methods
Material
Protein A/G beads, antimouse and antirabbit IgG-conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for Cyr61, TWIST-1, N-cadherin, E-cadherin, p-Raf-1, Raf-1, p-ERK, ERK, p-MEK, MEK, p-Elk, Elk, PCNA and β-Actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibodies specific for αvβ3 and αvβ5 integrin were purchased from Chemicon (Temecula, CA, USA). The recombinant human Cyr61, osteopontin and MFGE-8 were purchased from PeproTech (Rocky Hill, NJ, USA). All of the shRNAs plasmids used for gene knock down were purchased from the National RNAi Core Facility Platform (Taipei, Taiwan). All of the other chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA).
Cell culture
The human osteosarcoma cell line MG63 was purchased from the American Type Cell Culture Collection (Manassas, VA, USA). The cells were maintained in Dulbecco’s Modified Eagle’s Medium, which was supplemented with 20 mM HEPES, 10% heat-inactivated fetal bovine serum, 2 mM-glutamine, penicillin (100 U/mL), and streptomycin (100 μg/mL), at 37°C with 5% CO2.
To establish the Cyr61 stable knockdown MG63 cell line, Cyr61 shRNA plasmids were purchased from the National RNAi Core Facility Platform (Taipei, Taiwan). The Cyr61 shRNA plasmids were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and Cyr61 shRNA-expressing cells were puromycin selected (10 μg/mL). The surviving cells were selected and expanded to produce clonal cell populations. For monolayer growth curves, 104 cells were plated in 6-well plates and grown for 1–3 days. The cells were trypsinized, and the cell numbers were counted daily.
Establishment of migration-prone sublines
The subpopulations from MG63 cells were selected according to their differential migration ability [
58]; the cell culture insert system was used as described. After 24 h of migration, cells that penetrated pores and migrated to the underside of the filters were trypsinized and harvested for a second round of selection. The original cells that did not penetrate membrane pores were designated as M0. After 10 rounds of selection, the migration-prone subline was designated as M10.
Migration assay
The migration assay was performed using the Transwell assay (Costar, NY, USA; pore size: 8 μm) in 24-well dishes. Before the migration assay, the cells were pretreated with different concentrations of inhibitors, including Gw5047, PD98059, U0126, and the vehicle control (0.1% DMSO), or neutralized antibodies such as Cyr61, αvβ3, and αvβ5 integrin, and the vehicle control (IgG) for 30 min, or they were transfected with the indicated shRNA plasmids, including the TWIST-1, integrin αv, integrin β5, Raf-1, and Elk-1 for 24 h. After pretreatment, approximately 1 × 104 cells in 200 μL of a serum-free medium were placed in the upper chamber, and 300 μL of the same medium containing Cyr61, osteopontin or MFGE-8 was placed in the lower chamber. The plates were incubated for 24 h at 37°C in 5% CO2, and then the cells were fixed in methanol for 15 min and stained with 0.05% crystal violet in PBS for 15 min. The cells on the upper side of the filters were removed with cotton-tipped swabs, and the filters were washed with PBS. The cells on the underside of the filters were examined and counted using a microscope. Each experiment was repeated at least 3 times. The number of invading cells in each experiment was adjusted using the cell viability assay to correct for the proliferation effects of Cyr61 treatment (corrected invading cell number = counted invading cell number/percentage of viable cells).
Wound-healing assay
For wound-healing migration assays, the cells were seeded on 12-well plates at a density of 2 × 105 cells/ per well in a culture medium. At 24 h after seeding, the cells were treated with the indicated inhibitors or a neutralized antibodies for 30 min or transfected with shRNA plasmids for 24 h. After pretreatment, the confluent monolayer of the culture was scratched using a fine pipette tip, and incubated with recombinant Cyr61 for 24 h and migration was observed using microscopy. The rate of wound closure was observed at the indicated times.
Quantitative real-time polymerase chain reaction
Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed using Taqman® one-step PCR Master Mix (Applied Biosystems, Foster City CA, USA). Furthermore, 100 ng of total cDNA was added to every 25-μL reaction with sequence-specific primers and Taqman® probes. The sequences for all target gene primers and probes were purchased commercially (β-actin was used as the internal control) (Applied Biosystems, Foster City CA, USA). The qRT-PCR assays were conducted in triplicate on a StepOnePlus sequence detection system. The cycling conditions were 10-min of polymerase activation at 95°C, followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. The threshold was set higher than the non-template control background and within the linear phase of target gene amplification for calculating the cycle number at which the transcript was detected (denoted as CT).
Western blot analysis
The celllysates were prepared, and proteins were then resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to Immobilon polyvinyldifluoride membranes. The blots were blocked with 4% BSA for 1 h at room temperature and then probed with rabbit antihuman antibodies against Cyr61, TWIST-1, N-cadherin, E-cadherin, p-Raf-1, Raf-1, p-ERK, ERK, p-MEK, MEK, p-Elk, and β-Actin (1:1000) for 1 h at room temperature. After 3 washes, the blots were subsequently incubated with a donkey antirabbit peroxidase-conjugated secondary antibody (1:1000) for 1 h at room temperature. The blots were observed using enhanced chemiluminescence by employing Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY, USA). Quantitative data were obtained using a computing densitometer and ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA).
Nuclear extracts were prepared as described previously [
58]. The cells were suspended in buffer A for 10 min on ice. The lysates were separated into cytosolic and nuclear fractions through centrifugation at 12000 g for 10 min. The supernatants containing cytosolic proteins were collected. A pellet containing nuclear fractions was resuspended in buffer C for 30 min on ice. The supernatants containing nuclear proteins were collected through centrifugation at 13 000 g for 20 min, and stored at -80°C.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation analysis was performed as described previously [
59]. DNA immunoprecipitated using the anti-Elk antibody was purified. The DNA was then extracted using phenol–chloroform. The purified DNA pellet was subjected to PCR, and the PCR products were then resolved by conducting 1.5% agarose gel electrophoresis, and they were observed using ultraviolet. The primers 5′-AGCCCCAGCAATCCAAATC-3′ and 5′-TCGGAGGAGACTGTCCTGG-3′ were used for amplification across the human TWIST-1 promoter region (1466 to 1613).
Immunofluorescence microscopy
The MG63 cells grown on glass coverslips were rinsed once with PBS, and fixed in 3.7% paraformaldehyde for 10 min in RT. The cells were then washed 3 times with PBS, and blocked with 4% BSA for 15 min. The cells were then incubated with rabbit antimouse Elk (1:100) for 1 h in RT, rewashed, and incubated with FITC-conjugated goat anti-rabbit IgG for 1 h. Finally, the cells were washed, mounted, and examined using a Leica TCS SP2 Spectral Confocal System.
In vivo tumor xenograft study
Four-week-old male SCID mice were purchased from Lasco (Taipei, Taiwan) and maintained under pathogen-free conditions. All of the animal experiments were performed according to a protocol approved by the Shin Kong Wu Ho-Su Memorial Hospital (Taipei, Taiwan) Institutional Animal Care and Use Committee. Male CB17/SCID mice (4 wk old) were used. Seven animals per group, were used, and the experiment was repeated twice. For experimental metastasis assays, 1 × 106 cells were resuspended in 0.1 mL of PBS, and injected into the lateral tail vein. After 4 wk, the mice were euthanized using an overdose of the anesthetic agent. The lungs were removed and fixed in 10% formalin. The number of lung tumor metastases was counted using a dissecting microscope.
Statistical analysis
Data are presented as the mean ± standard error of the mean (SEM). Statistical comparisons between two samples were performed using Student’s t test. Statistical comparisons of more than 2 groups were performed using one-way analysis of variance with Bonferroni’s post hoc test. A P-value less than 0.05 was considered statistically significant.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JFL and SMH conceived and designed the experiments. CHH, FLL, and JFL performed the experiments. CHH, SMH and JFL analyzed the data. CHH, FLL, SMH and JFL contributed reagents/materials/analysis tools. JFL and SMH wrote the paper. All authors read and approved the final manuscript.