EZH2/hSULF1 axis mediates receptor tyrosine kinase signaling to shape cartilage tumor progression

Reviewer #1 (Recommendations for the authors):

Below are the comments that can help further strengthen the study.

1. Is EZH2 enzymatic activity required for hSULF1 repression?

To address this point, we performed the new experiment (new Figure 4D in the revised version, attached below) and the results support “EZH2 enzymatic activity required for hSULF1 repression”. In brief, we explored hSULF1 expression in chondrosarcoma cell line treated with EPZ-6438, an EZH2 inhibitor, to block EZH2 enzymatic activity. The expression of hSULF1 was increased in a dose dependent manner in chondrosarcoma cell line in present of EPZ6438 (new Figure 4D). Also, the phosphorylation of MET was repressed while treated with EZH2 inhibitor of chondrosarcoma cell line. In addition, In the experiment shown in Figure 2E and F, we assessed the expression of hSULF1 in chondrosarcoma cell lines with EZH2 specific shRNA, and hSULF1 expression was augmented while EZH2 was knockdown. Together, the data showed that EZH2 enzyme activity is required for the expression of hSULF1 in chondrosarcoma cell lines.

2. Does the loss of EZH2, which induces hSULF1 expression, also impair c-Met phosphorylation as hSULF1 overexpression?

We thank the reviewer for the comments. Following the original data shown in Figure 2E and F, we repeated the experiment and included analysis of phosphorylation of cMET, the results demonstrated “the loss of EZH2, which induces hSULF1 expression, also impairs c-Met phosphorylation as hSULF1 overexpression (New in Figure 4E in revised version).

3. It will further strengthen the concept that SULF1 acts through its phosphatase activity to restrict tumorigenesis by including the SULF1 enzymatic dead mutant in the tumorigenesis assay presented in Figure 3F.

We appreciate the reviewer for the comments and performed the suggested experiments (New Figure 4I in revised version) which allowed us to identify an unexpected phosphorylation of Y1003 of cMET in tumors that may be worthy of further pursuing in the near future.

Chondrosarcoma stable cell lines with vector control, wild type SULF1 (WT), and enzymatic inactive mutant SULF1 (CA) were subcutaneously injected into the nude mice. Tumor volume was measured twice per week. The results indicated that WT SULF1, associated with full sulfatase activity inhibited tumor development, consistent to what was observed in vitro culture. But the CA mutant, which lost sulfatase activity, thought to recover tumorigenicity, still associated with partial tumor suppression activity as it could not completely recover tumor volume similar to that of vector control (New Figure 4I in revised version).

To further elucidate the possible underling mechanism for why the CA mutant which lost sulfatase activity still associated with partial tumor suppression activity. We examined the different phosphorylation site of cMET and found that CA mutant retained the similar phosphorylated profile of Y1234/1235 of cMET, a catalytic activation site, in cell lysates and tumors (New Figure 4 – supplement 1A in revised version). However unexpectedly, the gain of phosphorylation of Y1003 of cMET was markedly detected in tumors, but not in the in vitro cell culture (New New Figure 4 – supplement 1B in revised version). The phosphorylation of Y1003 of cMET has been implicated to associate tumor suppressive activity (Organ and Tsao, 2011). It is not yet clear how the tumor microenvironment might cause the unexpected results of CA mutant in the animal study (New Figure 4I) and it is certainly interesting to pursue further how tumor microenvironment might affect p-Y1003 of cMET in the future.

4. It will be beneficial for this study to compare the efficacy of EZH2 inhibitor and c-Met inhibitor in suppressing tumor growth in an in vivo tumor model in Figure 7C.

We thanks the reviewer comments. The treatment of EZH2 inhibitor in chondrosarcoma mice model was important. However, we had been tested several EZH2 inhibitors including GSK343 and EPZ-6438(Tazmetostat) in vitro (SFigure 3) and indicated that range of IC50 was 6 -14μM, much higher than cMET inhibitor (Crizotinib; IC50 = ~3μM) (SFigure 4). Since the IC50 of EZH2 inhibitors are much higher than that of c-Met inhibitors, we tested in vivo tumor assay using c-Met inhibitor ( Figure 7). In the future, if more advanced EZH2 inhibitors are developed further, it can be tested in the future.

Reference:

Organ, S. L., and Tsao, M. S. (2011). An overview of the c-MET signaling pathway. Ther Adv Med Oncol, 3(1 Suppl), S7-S19. doi:10.1177/1758834011422556