Because of the histological similarities with malignant melanoma, CCS is also known as malignant melanoma of soft parts [
31]. However, the genetic findings of the
EWS-ATF1 fusion gene support the supposition that CCS and malignant melanoma are 2 distinct entities [
13]. To the best of our knowledge, 12 CCS cell lines have been reported in the English literature [
17‐
27], and there are only 4 cell lines that have been shown to possess both tumorigenicity in immunodeficient mice and
EWS-ATF1 fusion transcripts (Table
1). Furthermore, there is only 1 cell line (UM-CCS-1) that has the type 2
EWS-ATF1 transcript (Table
1). However, UM-CCS-1 could be passaged only in nude mice. Therefore, Hewga-CCS is the first cell line that harbors the type 2 chimeric
EWS-ATF1 transcript and can be stably cultured
in vitro and xenografted in nude mice.
Table 1
Characterization of clear cell sarcoma cell lines
SU-CCS1 (1984) | 16/F | heel | 1 | yes |
HS-MM (1993) | 39/M | knee | - | yes |
NCS-1 (1994) | 38/M | foot | - | yes |
DTC1 (1995) | - | chest wall | 1 | - |
MST1 (1996) | 14/F | knee | - | yes |
Kao (1997) | 9/F | thigh | 1 | yes |
MP-CCS-SY (2002) | 17/F | ankle | 1 | - |
GG62 (2002) | 25/F | lower leg | 1 | - |
MST2 (2004) | 60/M | knee | 1 | - |
MST3 (2004) | 34/M | groin | 1 | - |
CCS292 (2006) | - | - | 1 | yes |
UM-CCS-1 (2002) | 60/F | thigh | 2 | yes (in vivo only) |
Hewga-CCS | 34/F | toe | 2 | yes |
It has been reported that
EWS-ATF1 directly activates the melanocyte transcription factor (
MITF)[
26], which in turn activates the c-MET gene [
32]. Furthermore, c-MET is widely activated in CCS in an autocrine fashion by its ligand HGF, and CCS strongly depends on HGF/c-MET signaling [
33,
34]. In agreement with previous reports, we identified a robust activation of c-MET in Hewga-CCS cells (Figure
4A). In addition, we found that Hewga-CCS cells secreted higher amounts of HGF and moderate amounts of VEGF into the culture media compared with the amount of SYO-1 (Additional file
8: Figure S6). These results indicated that Hewga-CCS produced autocrine ligand HGF to activate CCS driver kinase c-MET. Therefore, from the context of analyzing drug sensitivity, Hewga-CCS driven by c-MET signaling commonly observed in CCS can be useful for the accelerated development of targeted therapies for CCS.
Pazopanib is approved for the treatment of advanced renal cell carcinoma and advanced STS by the U.S. Food and Drug Administration [
16,
35]. However, there have only been a few reports that have demonstrated the molecular mechanism by which pazopanib inhibits the growth of a variety of tumors [
15,
36‐
40]. Kumar used a cell-free assay system to show kinase activity of pazopanib and found that pazopanib had an IC
50 value of 6 μmol/L for inhibiting c-MET activity [
15]. This value was much higher than the IC
50 values of <0.1 μmol/L for pazopanib target kinases, including the VEGFRs, PDGFRs, FGFRs, and c-Kit [
15]. Podar demonstrated that pazopanib inhibited multiple myeloma cell growth
in vitro by inhibiting VEGF signaling at IC
50 values of 10–30 μmol/L [
37]. Paesler demonstrated that pazopanib abrogated the survival of chronic lymphocytic leukemia cells at an IC
50 of 32.7 μmol/L through VEGF pathway suppression [
38]. These studies revealed significant differences in IC
50 values for pazopanib between cell growth assays and cell-free assays. A potential explanation for this discrepancy is the possibility that kinase activity may be different between living cell and cell-free conditions. In this study, the IC
50 value of pazopanib in terms of Hewga-CCS cell growth was approximately 8 μmol/L, and comparable concentrations were reportedly achieved after once-daily administration of ≥200 mg pazopanib [
41]. It was reported that the combination of pazopanib and lapatinib led to complete inhibition of c-MET by an unknown mechanism, although each of the inhibitors alone had marginal or partial effects [
36]. Further, Gotink suggested that low binding affinity of a tyrosine kinase inhibitor to a certain kinase may have a crucial impact on cell signaling, while the same inhibitor with a high binding affinity to another kinase may have no significant effect [
42]. We demonstrated the inhibition of c-MET in xenografts treated with pazopanib (Figure
6E). In addition, we showed no significant antitumor effects of bevacizumab
in vitro and
in vivo (Figure
5). These results indicated that pazopanib delayed xenograft development by direct antitumor activity through the inhibition of c-MET signaling, at least in part.