Trabectedin is a promising antitumor agent potentially inducing melanocytic differentiation for clear cell sarcoma

Abstract

Clear cell sarcoma is an aggressive soft tissue sarcoma and highly resistant to conventional chemotherapy and radiation therapy. This devastating disease is defined by EWSR1-ATF1 fusion gene resulting from chromosomal translocation t(12;22)(q13;q12) and characterized by melanocytic differentiation. A marine-derived antineoplastic agent, trabectedin, inhibits the growth of myxoid liposarcoma and Ewing sarcoma by causing adipogenic differentiation and neural differentiation, respectively. In this study, we examined the antitumor effects and mechanism of action of trabectedin on human clear cell sarcoma cell lines. We showed that trabectedin decreased the cell proliferation of five clear cell sarcoma cell lines in a dose-dependent manner in vitro and reduced tumor growth of two mouse xenograft models. Flow cytometry and immunoblot analyses in vitro and immunohistochemical analysis in vivo revealed that trabectedin-induced G2/M cell cycle arrest and apoptosis. Furthermore, trabectedin increased the expression of melanocytic differentiation markers along with downregulation of ERK activity in vitro and the rate of melanin-positive cells in vivo. These results suggest that trabectedin has potent antitumor activity against clear cell sarcoma cells by inducing cell cycle arrest, apoptosis, and, in part, by promoting melanocytic differentiation through inactivation of ERK signaling. Our present study indicates that trabectedin is a promising differentiation-inducing agent for clear cell sarcoma.

Keywords: Clear cell sarcoma; differentiation therapy; melanocytic differentiation; trabectedin.

Figure 1

Trabectedin inhibits CCS cell growth in vitro. The CCS cells were incubated with various concentrations of trabectedin for 72 h. Cell proliferation was determined by WST‐1 assay. The IC ₅₀ values were calculated and shown in the table. Bars: SD.

Figure 2

Trabectedin induces G2/M cell‐cycle arrest and apoptosis in CCS cells. (A) Hewga‐CCS and KAS cells were exposed to 0.1–10 nmol/L trabectedin or vehicle for 48 h. After exposure, cells were stained with PI and analyzed by flow cytometry. (B) After treatment of trabectedin or vehicle in CCS cells, the protein expressions were observed by Immunoblot analyses.

Figure 3

Melanocytic differentiation markers in Hewga‐CCS and KAS were upregulated by the treatment of trabectedin and MTX, but not DOX. Hewga‐CCS and KAS were treated with (A) 5 nmol/L trabectedin for 0–24 h, (B) 1 uM MTX for 0–72 h and (C) 1 uM DOX for 0–24 h. The protein expressions were evaluated by Immunoblot analyses.

Figure 4

Trabectedin did not enhance the mRNA expression of MITF. Moreover, the drug did not affect the expression of EWSR1‐ATF1 fusion protein. Both trabectedin and a selective ERK inhibitor, SCH772984, decreased the ERK signaling and increased the protein level of MITF. Hewga‐CCS and KAS were treated with 5 nmol/L trabectedin or 100 nmol/L SCH772984 for 0–24 h. (A) Total RNA was extracted, and MITF transcription was quantified by qRT‐PCR. Values mean ± SD. (B, C) The protein expressions were assessed by Immunoblot analyses.

Figure 5

Trabectedin abrogated the growth of Hewga‐CCS and KAS xenograft tumors. (A) Hewga‐CCS (n = 8/group each) and KAS (n = 7/group each) xenograft tumors were treated with 0.15 mg/kg trabectedin intravenously injected. (B) The rate of PCNA‐positive tumor cells, cleaved caspase‐3‐positive cells, and melanin‐positive cells were counted in Hewga‐CCS xenograft tumors. *P < 0.01.