Histone deacetylase inhibitors induce growth arrest, apoptosis, and differentiation in clear cell sarcoma models

Abstract

Clear cell sarcoma is an aggressive malignancy occurring most commonly in the distal extremities of young adults, characterized by t(12;22)(q13;q12) creating the chimeric fusion oncoprotein EWS-ATF1. We assessed growth inhibition and differentiation effects of histone deacetylase inhibitors MS-275 and romidepsin (depsipeptide, FK228) on clear cell sarcoma cells and evaluated drug sensitivity among related translocation-associated sarcomas and other cell models. Three clear cell sarcoma cell lines, seven other sarcomas, six nonsarcoma malignant cell lines, and two nonneoplastic mesenchymal cell models were treated with MS-275 or romidepsin. Growth inhibition was assayed by monolayer 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Induction of cell cycle arrest and apoptosis were assessed by propidium iodide/Annexin V flow cytometry in monolayer and spheroid cultures and by immunoblotting analysis. Expression levels of key genes involved in mesenchymal differentiation and of EWS-ATF1 were measured by quantitative real-time PCR in clear cell sarcoma cells treated with histone deacetylase inhibitors. MS-275 and romidepsin inhibited growth in clear cell sarcoma cells by inducing cell cycle arrest and apoptosis in a time-and dose-dependent manner. Sarcomas showed greater sensitivity than other tumor types, with clear cell sarcomas most sensitive of all, whereas nonmalignant mesenchymal cells were highly resistant. MS-275 at 1 Mmol/L and romidepsin at 1 nmol/L induced histone H3 acetylation, cell cycle arrest, apoptosis, and differentiation in clear cell sarcoma cells within 24 hours. Histone deacetylase inhibitors increased expression of SOX9, MYOD1, and PPARG and decreased EWS-ATF1 expression in clear cell sarcoma cells. Histone deacetylase inhibitors show promising preclinical activity in multiple clear cell sarcoma models.

Novel Therapeutic Targets in Soft Tissue Sarcomas

“…146 HDAC inhibitor will reverse the repression by the Polycomb protein, leading to de-repression of EGR1 and followed by apoptosis. Similar preclinical anti-tumour effect has been observed in Ewing sarcoma, [147][148][149] clear cell sarcoma, 150 endometrial stromal sarcoma, 151 mxyoid chondrosarcoma 152 and alveolar rhabdomyosarcoma. 149 Treatment of HDAC inhibitor leads to increase in expression of p21 and G2/S arrest and decrease in mTOR expression and activity, leading to apoptosis and autophagy.…”

Histone deacetylase 1 and 2 in mesenchymal tumors

“…35 By analogy, a dependence on HDAC2 activity for cell cycle progression in translocationassociated sarcomas could contribute to their particular sensitivity to HDAC inhibitors. 18 In summary, HDAC2, one of the HDAC isoforms and a therapeutic target for HDAC inhibitors, is present and intensely expressed in a very high proportion of mesenchymal tumors. Expression is highest among sarcomas associated with fusion transcription factors.…”

“…Total protein lysates from SYO-1 and DTC1 cell lines 18 were prepared using RIPA buffer containing sodium chloride 150 mM/l, 1% NP-40, 0.5% sodium deoxycolate, 0.1% sodium dodecyl sulfate, 50 mM/l Tris pH 8.0 and ready-to-use complete protease inhibitor cocktail (Roche, Mannheim, Germany). Protein extracts were run in SDS-PAGE and analyzed on western blots incubated with primary mouse monoclonal anti-HDAC1 1:2500 (Millipore, Billerica, MA, USA; 05-100), mouse monoclonal anti-HDAC2 1:4000 (Abcam, Cambridge, MA, USA; ab12169), mouse monoclonal anti-HDAC3 1:1000 (Becton Dickinson, Franklin Lakes, CA, USA; 611124), rabbit monoclonal anti-HDAC3 1:7500 (Abcam; ab32369) and rabbit polyclonal anti-HDAC6 1:200 (Abcam; ab1440).…”

“…13 Several HDAC inhibitors have been shown to be effective in Ewing preclinical models, [14][15][16] and comparable findings have now been reported in other translocation-associated sarcomas, including synovial sarcoma, 17 clear cell sarcoma, myxoid liposarcoma and desmoplastic small-blue round-cell tumor. 18 Despite this body of preclinical evidence supporting HDAC inhibitors as therapeutic agents in translocation-associated sarcomas, the well-documented involvement of class I HDACs in cancer biology, 19 and the recent opening of phase II clinical trials testing these agents in sarcomas, little information exists on the expression of the enzymes targeted by these drugs in mesenchymal neoplasms. In this study, we assay, by immunohistochemistry, the expression of HDAC 1 and 2 isoforms in a broad collection of mesenchymal neoplasms, to assess if there is high expression of specific HDAC isoforms in translocation-associated sarcomas that may help explain their sensitivity to this class of drugs and provide information to help determine which agents may be most effective.…”

SOX9 determines RUNX2 transactivity by directing intracellular degradation

“…(20,21) The functions of RUNX2 and SOX9 are controlled by transcriptional and posttranscriptional regulation, including ubiquitination, phosphorylation, and acetylation. (12,(22)(23)(24)(25)(26)(27)(28)(29)(30) The temporal and spatial overlap of RUNX2 and SOX9 expression during embryonic development indicates a close coordination between these two key transcription factors during bone formation. (1,10,31) Indeed, it has been found that SOX9 exerts a dominant function over RUNX2 in mesenchymal precursors.…”