Association of FBXW11 levels with tumor development and prognosis in chondrosarcoma

Abstract

INTRODUCTION:

The E3 ubiquitin ligase FBXW11 exerts an oncogenic or tumor suppressive function in a cellular context-dependent manner. However, the clinical significance and biological role of FBXW11 in chondrosarcoma have not been clearly characterized. This study focuses on the expression profile, prognostic value and biological function of FBXW11 in chondrosarcoma.

METHODS:

FBXW11 expression was analyzed by qRT-PCR and Western blot in six cases of chondrosarcoma specimens and the matched adjacent non-tumor tissues. The expression profile and prognostic value of FBXW11 were investigated in sixty-three cases of chondrosarcoma patients. Cell viability, colony formation, migration, invasion and apoptosis assays were further detected in SW1353 chondrosarcoma cells with restored FBXW11 expression.

RESULTS:

Downregulation of FBXW11 was remarkably detected in human chondrosarcoma specimens compared with the corresponding non-tumor tissues and benign cartilage tumors. Downregulated FBXW11 expression significantly correlated with high-grade chondrosarcoma and poor prognosis. Furthermore, FBXW11 was identified as an independent prognostic factor for the overall survival of chondrosarcoma patients. Restored expression of FBXW11 significantly suppressed chondrosarcoma cell growth and induced apoptosis.

CONCLUSIONS:

These findings establish that FBXW11 was markedly downregulated and recognized as an independent prognostic factor for patients with chondrosarcoma, and restored FBXW11 expression can suppress chondrosarcoma growth and induce apoptosis, highlighting a novel biological marker and potential therapeutic target against chondrosarcoma.

1. Introduction

Chondrosarcoma is a common primary malignant cartilage-forming tumor that usually occurs in the long bones of the extremities such as proximal femur, characterized by a relatively high recurrence and mortality due to resistance to classic radiotherapy and chemotherapy [1]. In recent years, the molecular mechanisms of the pathogenesis of chondrosarcoma and targeted therapy have become research hotspots, providing pivotal theoretical and experimental basis for in-depth understanding of its biological behaviors [2, 3, 4]. Therefore, further exploration of the relationship between novel regulatory genes and chondrosarcoma development has important theoretical significance for unravelling its molecular mechanisms, designing reasonable molecular targeted drugs, and further improving chondrosarcoma treatment [5, 6].

The ubiquitin-proteasome system is a master regulator of targeting cellular proteins for ubiquitination-dependent protein turnover, participating in both physiological and pathological processes [7]. Cullin-RING E3 ubiquitin ligase complex such as SKP1-Cullin 1-F-box protein (SCF) play a central role in multiple cellular processes [8]. F-box proteins further divided into FBXW, FBXL and FBXO subfamilies based upon protein structure [9], are the substrate-recognition subunits of SCF E3 ligase complexes and involved in ubiquitylation-dependent protein degradation and also tumorigenesis [10, 11]. FBXW11 (F-box and WD-40 domain protein 11), also known as $\beta$ -TrCP2 or HOS, belongs to the FBXW family [9], which is involved in multiple developmental pathways such as the Wnt/ $\beta$ -catenin, IhH, and RAS signaling pathways [12], and also tumorigenesis in human cancers [10]. Recently, several authors have reported that FBXW11 is aberrantly increased and exerts an oncogenic activity in melanoma, cervical cancer, lymphocytic leukemia, and skin cancer [13, 14, 15, 16, 17]. In contrast, FBXW11 protein levels are markedly decreased in NSCLC, colorectal cancer, pancreatic caner, and thereby decreased FBXW11 expression dramatically promotes cell proliferation and invasion, supporting its tumor-suppressive functions [18, 19, 20, 21]. These findings have revealed FBXW11 exerts an oncogenic or tumor-suppressive function according to the specific cancer type or cellular context [11], suggesting that deregulation of FBXW11 may be a frequent event in tumorigenesis.

Dysfunction of FBXW11 could involve in tumorigenesis, however, its biological function in chondrosarcoma remains elusive. We hypothesized that FBXW11 may be implicated in the pathogenesis of chondrosarcoma. Thus, we determined whether and how FBXW11 implicates in chondrosarcoma development and exerts pro- or anti-neoplastic activity against chondrosarcoma.

2. Materials and methods

2.1 Clinical specimens and cell line

Clinical specimens such as six cases of fresh chondrosarcoma tissues and the matched non-tumor tissues next to chondrosarcoma, sixty-three cases of conventional chondrosarcomas and seventeen cases of benign cartilage tumors including osteochondroma and enchondroma were enrolled as described previously [4, 5, 22]. All chondrosarcoma cases in this study were pathologically diagnosed as conventional chondrosarcomas, the rare subtypes of chondrosarcomas including dedifferentiated, mesenchymal, juxtacortical, and clear-cell chondrosarcomas were excluded according to their distinctly different clinicopathological features. Clinical clinicopathological information such as histological grading and surgical staging system of the Musculoskeletal Tumor Society (MSTS), and follow-up data were reported previously [23, 24]. Signed informed consent for sample collection and analysis were obtained from all patients and was in full compliance with national legislation and the ethical standards as described previously (IRB00001052-08044) [23, 24]. Human chondrosarcoma SW1353 cell line was purchased from the American Type Culture Collection (Bethesda, MD, USA) and maintained in a humidified cell incubator with 5% CO ${}_2$ at 37 ${}^{\circ }$ C.

2.2 RNA extraction and real-time PCR

Total RNA extraction, cDNA synthesis, and qRT-PCR were performed as described previously [25]. Briefly, total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, USA). Total RNA (2 $\mu$ g) was used for the synthesis of first-strand cDNA using M-MLV reverse transcriptase (Invitrogen, Carlsbad, USA). Quantitative real-time PCR (qRT-PCR) was performed using the SYBR green mix and a 7500 Fast Real-Time PCR System (Applied Biosystems). The primers for FBXW11 were followed as Forward: 5’-GGAACATCATCTGTGATCGTCTC-3’; Reverse: 5’-TGGTAAAGCGGTAATAAAGTCCC-3’ as described previously [21]. The data were shown as 2 ${}^{-\mathrm{\Delta }\mathrm{\Delta }Ct}$ values and were representative of at least three independent experiments.

2.3 Protein extraction and Western blot analysis

Protein extraction and Western blot were performed as previously described [25]. Briefly, total protein extracts were separated using 12.5% SDS-polyacrylamide gel electrophoresis, transblotted onto nitrocellulose membranes, and blocked before incubating the membranes with primary antibodies overnight at 4 ${}^{\circ }$ C. After washing, bound primary antibodies were detected using a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG or anti-mouse IgG (Sigma, CA, USA). Primary antibodies against primary FBXW11 (Proteintech, 13149-1-AP) and $\beta$ -actin (Sigma-Aldrich, A5316) were used at 1:1000 dilution. Bound antibody was detected using an appropriate IRDye 700 or 800-conjugated secondary antibody and visualized using infrared fluorescence.

2.4 Immunohistochemistry and evaluation of staining

Detailed experimental protocols have been described previously [22, 23]. Briefly, all specimen slides (5 $\mu$ m) were deparaffinized and rehydrated, and then endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 9 min. For antigen retrieval, slides were incubated with 50 mM Tris-EDTA (pH 9.0) in a pressure cooker for 2 min. After antigen retrieval, FBXW11 (Proteintech, 13149-1-AP; 1:100) immunoreactivities were detected using the EnVision+, Peroxidase system (DAKO Diagnostics, Denmark). The slides were incubated with DAB substrate (DAKO Diagnostics, Denmark) and counterstained with hematoxylin. Cytoplamsic immunoreactivity of FBXW11 was assessed as follows: (1) a low expression level, recognized as scores of 0 for no staining and 1 $+$ for weak staining; and (2) a high expression level, recognized as a score of 2 $+$ for moderate staining and 3 $+$ for strong staining.

2.5 Generation of stable cell clones

Stable SW1353 cell clones with ectopic expression of FBXW11 were generated as described previously [23]. Specifically, human FBXW11 full-length cDNA was obtained by RT-PCR, and was cloned into a pLPCX (Clontech, CA) according to the manufacturer’s instructions. The Lenti-X HTX packaging system (Clontech, CA) produced lentiviral supernatants. Puromycin (2.5 $\mu$ g/mL) was selected for stable positive cell clones according to the manufacturer’s instructions.

2.6 Cell viability and clonogenic assays

Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) for cell viability, and clonogenic formation assays were conducted as described previously [23, 25]. Briefly, cell clones were incubated for the indicated time, and then CCK-8 was added to calculate cell viability. In addition, transfected cells were allowed to grow for an additional 10–14 days for clonogenic assay. Colonies were counted after methanol/acetone (1:1) fixation and Gentian Violet staining according to the previous experimental protocols [20].

2.7 Cell migration and invasion asssays

Cell migration and invasion assays were performed as described previously [23]. Cell migration was detected using a modified two chamber transwell migration assay. The number of invasive cells was determined by counting the leucocrystal violet-stained cells. For the cell invasion assay, cells that had migrated across the membrane were counted in five random visual fields using a light microscope.

Table 1

Downregulated protein expression of FBXW11 in human chondrosarcomas

Groups	Total No. of cases	FBXW11 expression		$P$ -value
		Low	High
Benign cartilage tumors	17	5	12	0.015 ${}^{*}$
Chondrosarcomas	63	40	23

$P$ values recorded are the results from Fisher’s Exact Tests. ${}^{*}P<$ 0.05.

2.8 Detection of cell apoptosis

Detailed experimental protocols have been previously described [23, 25]. Briefly, after treatment, cells were resuspended in 100 $\mu$ l of binding buffer containing 5 $\mu$ L of FITC-conjugated Annexin V and 5 $\mu$ L of PI (propidine iodide) according to the manufacturer’s instructions. Samples were immediately analyzed on a FACS Calibur flow cytometer (Becton Dickinson) and analyzed with Cell Quest Software (BD Bioscience) to calculate apoptotic cells. The results shown are representative of at least three separate experiments.

2.9 Statistical analysis

Data are presented as the Mean $\pm$ SD. The Student’s $t$ -test or the Pearson’s Chi-square test/Fisher’s exact test was utilized to analyze difference between groups with continuous variables or categorical variables, respectively. The Kaplan-Meier method and the log-rank test were applied for overall survival with chondrosarcoma patients. Univariate and multivariate analyses of clinicopathological factors, FBXW11 level and overall survival were performed using Cox regression model. Statistical analyses were performed using SPSS (SPSS, Chicago, IL, USA.) and $P<$ 0.05 was considered statistically significant. All reported $P$ values were two-sided.

3. Results

3.1 Downregulated expression of FBXW11 in chondrosarcoma specimens

To explore the potential role of FBXW11 in chondrosarcoma development, we performed RT-PCR and western blot in six cases of the fresh chondrosarcomas and the corresponding non-tumor tissues. Our results shown that the mRNA levels of FBXW11 in chondrosarcoma were abrrently decreased as compared to the matched adjacent non-tumor tissues (Fig. 1A, ${}^{**}P<$ 0.01). We also found that the FBXW11 protein expression displayed a remarkable decrease in fresh chondrosarcomas compared with the adjacent non-tumor tissues (Fig. 1B and 1C, ${}^{**}P<$ 0.01).

Next, we performed immunochemistry analysis and found that the positive signal of FBXW11 was preferentially recognized at the cytoplasm in human cartilage tumors such as osteochondroma and chondrosarcoma (Fig. 2A and 2B), and low protein expression of FBXW11 was identified as 63.5% (40/63) in chondrosarcomas samples as compared to 29.4% (5/17) in benign cartilage tumors ( $P=$ 0.015, Table 1).

Figure 1.

FBXW11 expression was significantly downregulated in chondrosarcomas. (A) The FBXW11 mRNA expression was analyzed by Real time PCR in six chondrosarcoma tissues and the paired corresponding adjacent non-tumor tissues. N, the corresponding non-tumor tissues; CS, chondrosarcoma tissues. ${}^{**}P<$ 0.01. (B) The diagram represented the statistical analysis of FBXW11 protein expression detected by Western blot. N, the corresponding non-tumor tissues; CS, chondrosarcoma tissues. ${}^{**}P<$ 0.01. (C) The FBXW11 protein levels were detected by Western blot in three representative pairs of tissues presented. To confirm the equal protein levels, the same blot was stripped and developed with anti $\beta$ -actin antibody.

3.2 Downregulation of FBXW11 correlated with high-grade chondrosarcoma and poor prognosis

To explore whether downregulated FBXW11 expression significantly correlated with clinicopathological factors in sixty-three chondrosarcoma patients, we analyzed their relationships, and displayed that low protein expression of FBXW11 significantly correlated with the increment of histological grade ( $P=$ 0.002) and MSTS stage ( $P=$ 0.004, Table 2). To evaluate the potential prognostic value of FBXW11 in patients with chondrosarcoma, we have performed the Kaplan-Meier analysis and the log-rank test, and found that the overall survival rate of patients with low FBXW11 protein expression was significantly lower than that of patients with high FBXW11 protein expression ( $P=$ 0.001, Fig. 2C).

Table 2

Downregulated FBXW11 expression associated with clinicopathological factors in 63 patients with chondrosarcomas

Clinicopathological factors	Total No. of cases	FBXW11 expression		$P$ -value
		Low	High
Gender				0.793
Male	36	22	14
Female	27	18	9
Age (years)				0.223
$⩾$ 40	49	29	20
$<$ 40	14	11	3
Anatomical location				0.580
Limb bone	21	12	9
Axial bonne	42	28	14
Historical grade				0.002 ${}^{*}$
Well-differentiated (I)	32	15	17
Moderately (II)	19	15	4
Poorly (III)	12	10	2
MSTS stage				0.004 ${}^{*}$
I A $+$ I B	34	16	18
II A $+$ II B	29	24	5

$P$ values recorded are the results from Chi-square Tests or Fisher’s Exact Tests. ${}^{*}P<$ 0.05. MSTS stage represents Musculoskeletal Tumor Society stage.

Figure 2.

Downregulation of FBXW11 was associated with poor prognosis in patients with chondrosarcomas. (A–B) Representative images of FBXW11 staining were presented in human cartilage tumors. Osteochondroma with high cytoplasm immunostaining of FBXW1 (A). Negative cytoplasm immunostaining of FBXW11 (B) were detected in chondrosarcoma tissues. (C) Prognostic values of FBXW11 protein expression in sixty-three patients with chondrosarcomas by Kaplan-Meier survival curves and the log-rank test. Probability of overall survival in patients with chondrosarcoma with regard to FBXW11 expression demonstrated that low FBXW11 expression remarkably correlated with poor prognosis ( $P=$ 0.001).

3.3 FBXW11 idenfitied as an independent factor

Table 3

Results of Cox Regression analysis of the prognostic factors for overall survival in 63 patients with chondrosarcomas

Clinicopathological factors	Unfavorable vs. favorable	Univariate analysis		Multivariate analysis
		Hazard ratio	$P$ value	Hazard ratio	$P$ value
Gender	Male vs. female	1.281	0.582	1.498	0.470
Age	$⩾$ 40 vs. $<$ 40	1.700	0.396	1.315	0.760
Anatomical location	Axial vs. limb bone	1.298	0.588	3.112	0.075
Histological grade	Grade 3 vs. Grade 1 and 2	5.186	0.000 ${}^{*}$	6.236	0.000 ${}^{*}$
MSTS stage	II A $+$ II B vs. I A $+$ I B	3.818	0.003 ${}^{*}$	3.540	0.010 ${}^{*}$
FBXW11 expression	Low vs. high	4.719	0.014 ${}^{*}$	10.221	0.003 ${}^{*}$

$P$ values recorded are the results from Cox Regression Analysis. ${}^{*}P<$ 0.05.

To identify the potential independent prognostic factors for the overall survival of chondrosarcoma patients, we had conducted Cox regression model and shown that low FBXW11 level (HR, 10.221, $P=$ 0.003) was identified as an independent prognostic factor for poor survival of chondrosarcoma patients, in accordance with histological grade (HR, 6.236, $P=$ 0.000) and MSTS stage (HR, 3.540, $P=$ 0.010) as shown in Table 3.

3.4 Restored expression of FBXW11 suppressed chondrosarcoma development

Figure 3.

Ectopic expression of FBXW11 suppressed chondrosarcoma cell growth and induced apoptosis. (A) FBXW11 was markedly upregulated by pLPCX-FBXW11. The overexpressed expression of FBXW11 was detected by Western blot from stable SW1353 cell clones with pLPCX vector or pLPCX-FBXW11 (left panel). The histograms exhibited the gray intensity analysis to the bands of Western blot (right panel, ${}^{**}P<$ 0.01). (B–D) The inhibitory effects of ectopic FBXW11 expression on cell viability, colony formation, migration and invasion. Stable SW1353 cell clones with pLPCX vector or pLPCX-FBXW11 were incubated for the indicated time, and cell viability (B), colony formation (C), migration, and invasion (D) were detected by CCK-8, colony formation assay, and migration and invasion assay as indicated in Material and Methods. Data are plotted as the mean $\pm$ SD ( ${}^{*}P<$ 0.05, ${}^{**}P<$ 0.01). (E) Ectopic FBXW11 expression remarkably enhanced chondrosarcoma cell apoptosis. Stable SW1353 cell clones with pLPCX vector or pLPCX-FBXW11 were incubated for 72 h. Apoptotic cells were measured by FITC-Annexin V and PI staining followed by flow cytometry analysis. The representative data from triplicate independent experiments were shown. ${}^{**}P<$ 0.01.

To further explore the underlying role of FBXW11 in chondrosarcoma development, we had successfully generated stable chondrosarcoma SW1353 cell clones with ectopic expression of FBXW11, as confirmed by western blot (Fig. 3A). Next, we examined the effects of restored expression of FBXW11 on chondrosarcoma cell survival, and found that restored expression of FBXW11 significantly suppressed chondrosarcoma cell viability (Fig. 3B). Our results also shown that overexpression of FBXW11 markedly inhibited SW1353 cell colony formation as displayed in Fig. 3C. As shown in Fig. 3D, the supression of migration and invasion of SW1353 cells infected with pLPCX-FBXW11 was significantly greater than that with pLPCX vector. To clarify the potential reasons for restored FBXW11 expression mediated the growth arrest, we tested apoptosis in these cells, and found that overexpression of FBXW11 significantly induced cell apoptosis (Fig. 3E). Our data unravelled that restored expression of FBXW11 remarkably suppressed chondrosarcoma cell growth, at least in part, attributed to induction of apoptosis.

4. Discussion

Emerging data suggest dysfunction of many F-box proteins results in aberrant cellular ubiquitination and contributes to tumorigenesis and progression [11], whereas some inhibitors targeting F-box proteins have shown promising therapeutic potential [26]. Belonging to F-box protein family that recognizes and degrades substrates such as $\beta$ -catenin, NF- $\kappa$ B and YAP et al. [11, 27, 28], FBXW11 contributes to carcinogenesis and thus play a pro-neoplastic activity against tumors [13, 14, 15, 16, 17, 18, 19, 20, 21]. However, some reports have documented that FBXW11 is markedly decreased in NSCLC tissues and decreased protein level of FBXW11 dramatically facilitates tumor proliferation and invasion in NSCLC cells and pancreatic cancer cells [18, 19, 21]. In adddition, FBXW11 can facilitate ZNF281 ubiquitination and degradation, thereby inhibiting colorectal cancer progression [20]. These reports have presented the evidence that FBXW11 exerts anti-neoplastic activity. In regard to human chondrosarcoma, the potential role of FBXW11 in the pathogenesis of chondrosarcoma remains poorly unravelled. In this study, we first demonstrate that FBXW11 is significantly downregulated in chondrosarcoma tissues as compared to the corresponding adjacent non-tumor tissues and benign cartilage tumors such as osteochondroma and enchondroma. These findings suggest that FBXW11 can exert a tumor-suppressive role in the pathogenesis of chondrosarcoma in vivo. Previous mutation analysis of FBXW11 in gastric cancer is suggestive of its tumor-suppressive role [29, 30], suggesting that downregulated FBXW11 in chondrosarcoma might be due to gene mutation, but the molecular mechanism that FBXW11 is downregulated in chondroarcoma requires futher investigations. We previously reported that $\beta$ -catenin accumulation was increased in chondrosarcoma that had significant impacts on the prognosis [31], and aberrant activation of Wnt/ $\beta$ -catenin signaling drives proliferation of chondrosarcoma cells [32]. In addition, YAP nuclear accumulation was significantly elevated in chondrosarcoma, associated with high-grade chondrosarcoma and poor prognosis [23]. Given the ability of FBXW11 to degrade oncogenes such as $\beta$ -catenin and YAP [28, 33], downregulation of FBXW11 in chondrosarcoma in vivo could result in $\beta$ -catenin and YAP nuclear accumulation in chondrosarcoma cells, representing a potential mechanism for the pathogenesis of chondrosarcoma.

To characterize the clinical significance of FBXW11 in chondrosarcoma, we present the first evidence that low expression of FBXW11 was significantly correlated with histological grade and MSTS stage, rather than age, gender and anatomical location (Table 2). In addition, we also found that low expression of FBXW11 had significantly unfavourable impact on the overall survival of chondrosarcoma patients (Fig. 2C). Multivariate analysis by Cox regression model had demonstrated that FBXW11 is identified as an independent prognostic factor for the overall survival of chondrosarcoma patients (Table 3). It is rather definite that the histological grading and MSTS stage are the most important predictor for clinical behavior of chondrosarcoma [34]; and the prognosis of chondrosarcoma is associated with Enneking grades, histological grades and local recurrence rather than distant metastasis, surgical approaches or the size of the tumor [35]. Notably, the histological evaluation of tumor grade is somewhat subjective because chondrosarcoma represents a collective group of cartilage-originating tumor. Thus, alternative methods, including the evaluation of DNA synthesis and content, cytogenetics and molecular markers, have been sought to assess the prognosis for patients with chondrosarcomas [34]. For other factors which affect the prognosis of chondrosarcoma, controversies arise. Therefore, we conducted this study to screen prognosis-related factors. Our findings suggest that FBXW11 can be identified as an independent prognostic biomarker for chondrosarcoma patients. Previously, many researchers reported that several molecular markers have been recognized that manifest a significant correlation with histological grade and predictive for the prognosis of chondrosarcoma patients [34, 36]. Thus, our results might help the clinician to determine the prognosis of chondrosarcoma based upon the protein level of FBXW11, indicating the potential of FBXW11 as a novel biomarker in chondrosarcoma patients. However, further investigations for the clinical value of FBXW11 in larger cohorts of chondrosarcoma are indeed needed to conduct, which validates its clinical significance and prognosis in patients with chondrosarcomas.

Accumulated data suggest that FBXW11 can exert as a potent tumor-suppressive role in tumorigenesis [18, 19, 20, 21, 29, 30]. Consistent with these results, our findings also reveal that restored expression of FBXW11 can suppress chondrosarcoma cell viability, colony proliferation, migration and invasion, and induce apoptosis in vitro (Fig. 3), supporting the evidence for its tumor-suppressive role in the pathogenesis of chondrosarcoma, at least in part, attributed to induction of apoptosis. Previous studies have documented that FBXW11 can act as an oncogene promoting melanoma development [13]. Moreover, ectopic expression of FBXW11 in lymphocytic leukemia cells can remarkably stimulate tumor proliferation, mediated by the stimulation of cell cycle progression rather than the induction of apoptosis [15]. By constract, FBXW11 can induce proteasome-mediated degradation of Mcl-1, an antiapoptotic Bcl-2 family member and overexpressed in several human cancers, and thus facilitate GSK-3 $\beta$ -induced apoptosis [37]. Overall, these findings implicate FBXW11 to the proliferative and apoptosis signaling pathway. Our findings, combination with the previous studies [18, 19, 20, 21, 37], suggest that FBXW11 acts as a tumor suppressor, inhibiting chondrosarcoma cell growth via induction of apoptosis. In fact, defects in apoptotic pathways are though to contribute to the pathogenesis of human cancers, and may result in the survival of malignant tumors [38]. Therefore, induction of apoptosis mediated by restored expression of FBXW11 in chondrosarcoma cells represents a novel treatment strategy targeting chondrosarcoma. However, the precise molecular mechanisms by which FBXW11 suppressess chondrosarcoma cell growth resulting from induction of apoptosis certainly merit further investigations.

5. Conclusion

Our work identifies downregulation of FBXW11 significantly associated with high-grade chondrosarcoma and poor prognosis in patients with chondrosarcoma. We have unravelled that FBXW11 is recognized as an independent prognostic factor and restored FBXW11 expression can suppress chondrosarcoma growth and induce apoptosis, highlighting a novel biomarker and attractive target for against chondrosarcoma.

Data sharing statement

The datasets used and/or analyzed during this study are available from the corresponding author on reasonable request.

Author contributions

Conception: Changbao Chen, Xinlong Ma.

Interpretation or analysis of data: Hua Zhou, Xiaolin Zhang, Zhongjun Liu.

Preparation of the manuscript: Changbao Chen, Hua Zhou.

Revision for important intellectual content: Changbao Chen, Xinlong Ma.

Supervision: Xinlong Ma.

Ethics approval and consent to participate

Clinical samples such as benign cartilage tumors and chondrosarcomas were handled in accordance with the Declaration of Helsinki and the Human Tissue Act. The samples used in the present study were collected as discarded tissues after written informed consent was obtained from all patients, and the use of biological tissue material in this present project was covered by the ethical approvals from the Ethics Commission Peking University Third Hospital and Tianjin Hospital as described previously (IRB00001052-08044) [24, 25].

Consent for publication

Not applicable.

Competing interests

All authors declared that they have no competing interest in this work.