We have shown for the first time that the BMP signaling pathway is active in conventional central chondrosarcoma and that the activity correlates to the histopathological grade of the tumors as there were significantly more high-grade than low-grade chondrosarcomas with highly nuclear phosphorylated Smad1/5/8. Nuclear phosphorylated Smad2 was also detected but did not correlate to grade. Activity of both signaling pathways was furthermore confirmed through functional assays in 2 chondrosarcoma cell lines. Both pathways were found to be inducible upon stimulation with TGFβ1 or BMP4. Interestingly, changes in pathway activity did not affect cell proliferation.
Smad1/5/8 activation can on one hand be driven by BMPs through the ALK1/2/3/6 receptors. Our gene expression analysis of BMPs suggests that transcriptional regulation of BMPs is not relevant for the progression of chondrosarcoma. Higher expression of the type I receptor ALK2 in high-grade chondrosarcoma could however contribute to enhanced BMP signaling and phosphorylated Smad1/5/8 in these tumors compared to grade I. On the other hand, Smad1/5/8 can also be activated by TGFβ driven ALK1 activation as it has been shown in endothelial cells, neurons, hepatic stellate cells and chondrocytes [7
]. In that case, elevated TGFβ3 expression in grade III chondrosarcoma compared to grade I could contribute to Smad1/5/8 activation in these tumors. Our gene expression profiles suggest that the BMP and TGFβ signaling pathways are regulated very differently between normal cartilage and chondrosarcoma. As the crosstalk between TGFβ and BMP signaling pathways is known to be highly context-dependent [17
], it should be elucidated whether mechanisms described in chondrocytes could also be relevant in chondrosarcoma cells. This could be performed in the chondrosarcoma cell lines, for which we have shown activity of both signaling pathways.
In endothelial cells, it has been described that TGFβ/ALK5/Smad2/3 signaling antagonizes TGFβ/ALK1/Smad1, and that the balance of TGFβ/ALK1 versus TGFβ/ALK5 represents a determinant of the pro- and anti-angiogenic effects of TGFβ . It has also been proposed that the ratio of ALK1/ALK5 expression is a determinant of TGFβ signaling in chondrocytes and that high ratios result in a stronger activation of Smad1/5/8 [18
]. ALK5 was significantly lower expressed in chondrosarcoma in comparison to cartilage while expression levels of ALK1 were equal. The ALK1/ALK5 ratio in chondrosarcoma could thus favor Smad1 activation in comparison to normal cartilage. Smad1/5/8 signaling is strongly associated with chondrocyte terminal differentiation and hypertrophy [18
]. Transgenic mouse models have shown that a deletion of Smad1 and Smad 5 results in chondrodysplasia and inhibition of the differentiation of proliferating chondrocytes [19
]. However, in chondrosarcoma no hypertrophic differentiation occurs and we have observed that phosphorylated Smad1/5/8 was elevated in high-grade tumors with a less differentiated phenotype. Other mechanisms such as elevated PTHrP signaling in chondrosarcoma may be blocking hypertrophy in these tumors [21
The TGFβ co-receptor endoglin has been described as a central modulator of these signaling pathways in endothelial cells and chondrocytes [7
]. In human articular chondrocytes, endoglin interacts with ALK1 [22
] and was shown to enhance TGFβ1-induced Smad1/5 phosphorylation and to inhibit TGFβ1-induced Smad2 phosphorylation [8
]. In central chondrosarcoma, we found significantly higher expression of endoglin in high-grade tumors and a correlation of endoglin expression to Smad1/5/8 activity. This correlation suggests that endoglin expression in high-grade chondrosarcoma could represent a determinant of elevated Smad1/5/8 activation in these tumors. This could involve TGFβ as well as BMP signaling, as in Ewing sarcoma and melanoma cell lines endoglin was shown to lead also to higher BMP induced Smad1 phosphorylation [23
]. On the other hand, endoglin is not exclusively modulating the Smad1/5/8 activation. In bone marrow stromal cells, endoglin appears to be a positive regulator of both ALK1/Smad1/5/8 and ALK5/Smad2 pathways [24
]. The dissection of signaling pathways in chondrosarcoma cells would be necessary to determine whether the correlation of endoglin expression to Smad1/5/8 phosphorylation in these cells truly reflects an enhanced activation of this signaling axis in high grade chondrosarcoma.
Endoglin / CD105 is one of the classical markers expressed by mesenchymal stem cells and used for the definition of these cells [25
]. Endoglin expression is up-regulated during the dedifferentiation of chondrocytes [26
] and conversely down-regulated during the chondrogenic differentiation of mesenchymal stem cells [27
]. In bone marrow stromal cell lines, endoglin was shown to stimulate proliferation [24
]. In this context, thus, endoglin and Smad1 signaling correlate to undifferentiated states of proliferating chondrogenic precursors, which is in line with higher expression levels in high-grade chondrosarcoma. Our reporter assay indicates that the Smad1 and Smad2 signaling pathways may not be relevant for proliferation of chondrosarcoma cells. Thus, while endoglin / Smad1 signaling seem important for loss of differentiation, it is not crucial for proliferation.
Endoglin has furthermore been described to have a pivotal function in vascular development and disease [28
]. Endoglin expression is stimulated by hypoxia through the transcription factor HIF1α [29
]. It is a marker of activated endothelial cells and its expression has been established as a specific marker for tumor endothelium in several tumor types [16
]. Its expression was however not found exclusively in tumor endothelium but also in tumor cells in melanoma, ovary and prostate tumors [28
] and now in chondrosarcoma. We have previously described a constitutive activation of HIF1α in high-grade chondrosarcoma as well as elevated expression of HIF1α target genes in these tumors [30
]. The expression pattern of endoglin, as a further HIF1α target gene, is in line with these results. Therefore, the hypothesis can be made that endoglin could represent an important mediator of tumor angiogenesis in high-grade chondrosarcoma. It is known that high grade chondrosarcomas demonstrate increased microvessel density [30
] and this phenomenon is also clinically used in dynamic MRI and to diagnose chondrosarcoma. A correlation between microvessel density and endoglin is therefore likely, but would not prove a causal relation between these two phenomena. An association between angiogenesis and endoglin expression could only be approached in vitro in chondrosarcoma cells and animal models.
Since central chondrosarcoma is a rare tumor type and the isolation of good quality RNA is difficult due to low cellularity and extracellular matrix [32
], one limitation of this study is the restricted number of samples which allowed reaching only levels of significance close to the threshold. The analysis of larger patient groups would be necessary to establish the robustness of the correlations found in this study and would especially be interesting to assess whether high endoglin expression significantly correlates to a high tumor vascularization and to a low metastasis-free survival.