BMP and TGFbeta pathways in human central chondrosarcoma: enhanced endoglin and Smad 1 signaling in high grade tumors

The expression of genes for BMP and TGFβ ligands and receptors was measured in central chondrosarcoma and normal cartilage samples by quantitative RT-PCR (Figure 1). All of the genes analyzed were found to be expressed in chondrosarcoma samples. While among the ligands analyzed the BMP2, BMP4, BMP6, BMP7, TGFB1 and TGFB2 genes did not show significant differences between chondrosarcomas of different histological grades, TGFB3 was significantly higher expressed in grade III compared to grade I chondrosarcoma (2-fold, p=0.006). From the receptors analyzed, only the type I receptor ALK2 showed differential expression and was significantly higher in grade III than in grade I chondrosarcoma (2.5-fold, p=0.012).

Compared to normal cartilage, chondrosarcoma showed altered expression levels for BMP2 and BMP7. BMP2 was significantly higher expressed in normal cartilage samples than in chondrosarcoma (37.8-fold, p<0.001), while BMP7 was not detected or found at very low expression levels in normal cartilage samples and was significantly higher expressed in chondrosarcoma (29.4-fold, p=0.005). The expression of BMP6 (data not shown) was similar in all sample groups.

In order to establish whether the BMP and TGFβ signaling pathways are active in central chondrosarcoma, the presence of nuclear phosphorylated Smad1/5/8 and Smad2 was evaluated by immunohistochemical analysis. Phosphorylated Smad1/5/8 and Smad2 was detected in all chondrosarcoma samples analyzed (Figure 2A, B). Highly phosphorylated Smad1/5/8, corresponding to a sum score higher than 3, was significantly more frequent in high-grade tumors compared to low grade while for highly phosphorylated Smad2 there was only a trend which did not reach significance (Table 1). There was a trend close to significance for a longer metastasis-free survival in patients with low phosphorylated Smad2, corresponding to a sum score lower or equal to 3 (p=0.055) (Figure 2D). This correlation was not independent from the histopathological grade of the tumors.

Endoglin / CD105 is a TGFβ co-receptor with the ability to modulate TGFβ signaling through Smad1/5/8 or Smad2/3 in various cell types including chondrocytes [7, 8, 15]. In order to establish whether endoglin could influence TGFβ signaling in chondrosarcoma, we have assessed its expression in chondrosarcoma by immunohistochemical analysis. Endoglin is an established marker of tumor vasculature [16]. Endoglin was detected in the cytoplasm and on the membrane of tumor and vascular cells. Only expression in tumor cells and not in the vasculature was scored in this study (Figure 2C). Only one grade I chondrosarcoma showed a sum score for endoglin higher than 3 and high endoglin expression was significantly more frequent in high-grade tumors (Table 1). From the 10 chondrosarcoma samples with high endoglin expression, 9 showed endoglin expression in more than 50% of tumor cells. There was a trend close to significance for a shorter metastasis-free survival in patients with high endoglin expression in more than 50% of the tumor cells (p=0.052) (Figure 2E). This correlation was not independent from the histopathological grade of the tumors. Notably, among the samples with low endoglin expression only 33% showed highly phosphorylated Smad1/5/8 while from the samples with high endoglin expression more than 80% also showed highly phosphorylated Smad1/5/8 (Figure 2F). High endoglin expression correlated with highly phosphorylated Smad1/5/8 (p=0.036, Pearson’s chi-square test) but not with highly phosphorylated Smad2.

Functional activity of the TGFβ- and BMP pathways was tested in the chondrosarcoma cell lines SW1353 and JJ012 using luciferase reporter assays with two reporter plasmids carrying pSmad2 (CAGA-luc) and pSmad1 (BRE-luc) responsive promoter elements (Figure 3). Pathway activity was shown by activation of the luciferase reporter genes, as shown by bioluminescence. Bioluminescence intensity could be inhibited by specific inhibitors, SB-431542 for TGFβ (Figure 3A) or LDN-193189 for BMP (Figure 3C). Stimulation of the pathways could also be achieved by TGFβ1 (Figure 3A) or BMP4 (Figure 3C). There was more variation in SW1353 than JJ012 in stimulation of both pathways when comparing three separate assays. Despite responsiveness of chondrosarcoma cells to specific manipulation of TGFβ and BMP activity there was no effect on proliferation of the cells upon inhibition or stimulation of the pathways (Figure 3B, D).

From a collection of 30 conventional central chondrosarcoma cases, 26 fresh frozen tumor samples from the archives of the Department of Pathology of the Leiden University Medical Center and from the tumor bank of the Orthopaedic University Hospital Heidelberg, including 10 grade I, 10 grade II and 6 grade III tumors, were available for gene expression analysis. For immunohistochemical analysis, from the same collection of central tumors, formalin-fixed, paraffin-embedded material from 27 cases including 10 grade I, 11 grade II and 6 grade III tumors was retrieved from the files of the Leiden University Medical Center. In 23 of the cases, both gene expression and immunohistochemical analysis were performed. Histological grading was performed for all cases according to Evans by the same pathologist to avoid interobserver variability [35]. Except for one case of Ollier disease, all chondrosarcomas analyzed were solitary. Fresh frozen normal articular cartilage samples (n=6) obtained from patients undergoing amputation were used as normal controls for gene expression analysis. Specimens from Leiden were handled according to the ethical guidelines described in "Code for Proper Secondary Use of Human Tissue in The Netherlands" of the Dutch Federation of Medical Scientific Societies. For the cases from Heidelberg, the study was approved by the local ethics committee (medical faculty of Heidelberg) and informed consent was obtained from all individuals included in the study.

All tissue samples were processed centrally in one lab following the same protocol. Haematoxylin and eosin-stained frozen sections were used to ensure the presence of at least 70% of tumor cells in the material used for RNA isolation. Shock-frozen tumor and cartilage tissue was pulverized mechanically and consecutively dissolved in lysis/binding buffer for direct poly(A)⁺-mRNA isolation using oligo-d(T)-coupled beads (Dynabeads; Invitrogen). mRNA was subjected to first strand cDNA synthesis using reverse transcriptase (Sensiscript, Qiagen, Hilden, Germany) and oligo-d(T) primers. Expression levels of individual genes were analyzed by quantitative RT-PCR (Lightcycler, Roche). Aliquots of first-stranded cDNA were amplified using gene-specific primer sets (Table 2) obtained from Eurofins (Ebersberg, Germany) and real-time fluorimetric intensity of SYBR green I was monitored. The candidate normalization genes described for gene expression analysis of chondrosarcoma [21] SRPR, CPSF6, CAPNS1 and HNRPH1 were used as reference. For each gene, the number of cDNA copies was correlated with the apparent threshold cycle (Ct). Building the difference between Ct of the gene of interest and the mean Ct of the reference genes for each sample gave ∆Ct values that were expressed as a percentage of reference genes. Melting curves and agarose gel electrophoresis of the PCR products were used for quality control.

Immunohistochemistry was performed as described previously [36]. Details of primary antibodies are described in Table 3. As negative controls, slides were incubated with PBS/BSA 1% instead of primary specific antibodies. An IHC protocol optimized for cartilaginous tissue was applied to avoid detaching of sections. Antigen retrieval was performed using citrate buffer, pH6.0 at 98°C for 10 minutes in a microwave followed by cooling down for 2 h. The antibodies were incubated over night at room temperature. They were visualized using the DAB+ substrate-chromogen system (Dako, Heverlee, Belgium).

Semi quantitative scoring of immunohistochemical staining for phosphorylated Smad1/5/8 (pSmad1/5/8), phosphorylated Smad2 (pSmad2) and endoglin was performed as described previously [36]. Slides were evaluated blinded towards clinicopathological data. In short, staining intensities (0 = negative, 1 = weak, 2 = moderate, and 3 = strong intensity) and the percentage of positive cells (0 = 0%, 1 = 1–24%, 2 = 25–49%, 3 = 50–74%, and 4 = 75–100% positive) were assessed. For statistical analysis slides were scored as “high expression” when the sum score of the staining intensity and the percentage of positive cells were greater than 3.

Early and late passages of the cell lines SW1353 [37] and JJ012 [38] were tested for their STR loci using the Powerplex CellIDTM system (Promega) in order to obtain a genetic profile. For SW1353, the genetic profiles according to these loci were identical to the profile submitted to the DSMZ database (http://​www.​dsmz.​de). For JJ012 no genetic profile is submitted to the DSMZ database. Early and late passage had identical profiles and did not match with any other cell line in the DSZM database.

The BMP-responsive element (BRE)-luciferase construct that drives a luciferase gene was obtained from Prof. ten Dijke [39]. The TGFβ pathway responsive plasmid containing (CAGA)12-luciferase reporter, which is exclusively activated by TGF-β-induced complex, has been described previously [40]. pRL-CAGGS expresses Renilla luciferase under a constitutive CAGGS promoter and was obtained from Promega.

TGFβ activity is inhibited by SB-431542 (Tocris Bioscience) at different concentrations (0.1, 1 and 10 μM) and stimulated by TGFβ1 (Sigma) (0.5, 2.5 and 5 ng/ml). BMP activity is manipulated by LDN-193189 (Stemgent Inc.) (10, 100 and 200 nm) and BMP4 (R&D systems). Mouse osteoblastic cells C2C12 were used as positive control for TGFβ and BMP activity. Untreated and manipulated C2C12 cells showed luciferase reporter activity in the same range as chondrosarcoma cells.

The number of viable cells was determined by using a Cell Titer-96 Aqueous One Solution Cell Proliferation Assay (MTS) from Promega, Madison, USA. Cells were seeded at a density of 2000 cells per well in 96-well flat-bottom plates. The next day, medium was replaced by fresh medium containing drug as indicated or DMSO, each condition in triplicate. The MTS assay was performed according to the manufacturer’s instructions and absorbance was measured at 490 nm using a Victor3 Multilabel Counter 1420–042 (Perkin Elmer, MA, USA).

Cells were seeded at a density of 5000 cells per well in 96-well flat-bottom plates. Next day, 100μl transfection complex was prepared with 1.95 μg of each plasmid driving luciferase expression from the corresponding BMP or TGFβ responsive promoters and 0.05 μg of pRL-CAGGS, an internal control for transfection efficiency driving renilla expression from a constitutive promoter. 5μl of the mix was added per well using Fugene HD transfection reagent (Roche, Mannheim, Germany) according to the manufacturer’s protocol. After 24 hours the medium was replaced by medium supplemented with 300ng/ml BMP4 or 10, 100, 200nM LDN-193189. After 24 h incubation, cells were harvested and luciferase activity was measured with a Victor 3 Multilabel Counter 1420–042 using the Dual-luciferase Reporter Kit (Promega). The ratio of firefly to renilla fluorescence was calculated to normalize reporter activity to the transfection efficiency. Three independent transfections were performed, each in triplicate.

Data analysis was performed with SPSS for Windows (SPSS, Chicago, USA). Median values of gene expression levels as assessed by quantitative RT-PCR were calculated. The Mann–Whitney test was chosen to evaluate significant differences in gene expression levels between sample groups. For the comparison of gene expression levels between chondrosarcoma of different grades and between cartilage samples and chondrosarcoma in Figure 1, the bonferroni correction was used and p<0.0125 was considered significant. For the analysis of immunohistochemical data, the Pearson chi-square test/Fisher’s exact test, two-sided was used for comparison between low- and high-grade chondrosarcoma. Since the number of samples of grade III chondrosarcoma (n=6) alone was considered too low for this test the clinically more relevant comparison between low-grade (grade I) and high-grade (grade II + III) chondrosarcoma was considered. Total survival and metastasis-free survival curves based on Kaplan–Meier estimates were compared using log rank test. For all tests a p value <0.05 was considered significant.