1751 Histone deacetylase inhibitors induce growth arrest, apoptosis, and differentiation in clear cell sarcoma models Shuzhen Liu, Hongwei Cheng, Wanda Kwan, inhibitors increased expression of SOX9, MYOD1, and Joanna M. Lubieniecka, and Torsten O. Nielsen PPARG and decreased EWS-ATF1 expression in clear cell sarcoma cells. Histone deacetylase inhibitors show prom- Genetic Pathology Evaluation Centre, University of British ising preclinical activity in multiple clear cell sarcoma Columbia, Vancouver, British Columbia, Canada models. [Mol Cancer Ther 2008;7(6):1751–61] Abstract Introduction Clear cell sarcoma is an aggressive malignancy occurring Clear cell sarcoma typically presents in the distal extrem- most commonly in the distal extremities of young adults, ities of young adult patients and is characterized by characterized by t(12;22)(q13;q12) creating the chimeric t(12;22)(q13;q12) (ref. 1). This translocation fuses the 5¶ fusion oncoprotein EWS-ATF1. We assessed growth portion of the Ewing sarcoma (EWSR1) oncogene on inhibition and differentiation effects of histone deacety- chromosome 22q with the 3¶ portion of the activating lase inhibitors MS-275 and romidepsin (depsipeptide, transcription factor 1 (ATF1) oncogene on chromosome FK228) on clear cell sarcoma cells and evaluated drug 12q, resulting in the EWS-ATF1 chimeric fusion oncopro- sensitivity among related translocation-associated sarco- tein. Treatment for clear cell sarcoma is wide excision of mas and other cell models. Three clear cell sarcoma cell the tumor as soon as the diagnosis is established, with lines, seven other sarcomas, six nonsarcoma malignant neoadjuvant or adjuvant radiation therapy to improve the cell lines, and two nonneoplastic mesenchymal cell rate of local control. Nevertheless, more than half of models were treated with MS-275 or romidepsin. Growth patients develop metastases and 5-year disease-specific inhibition was assayed by monolayer 3-(4,5-dimethylth- survival is <50%. No targeted chemotherapies are available iazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Induc- and conventional agents offer little benefit (2). tion of cell cycle arrest and apoptosis were assessed by Clear cell sarcoma, formerly termed ‘‘malignant melano- propidium iodide/Annexin V flow cytometry in monolayer ma of soft parts,’’ is now recognized to fall into a category and spheroid cultures and by immunoblotting analysis. also including Ewing sarcoma, synovial sarcoma, desmo- Expression levels of key genes involved in mesenchymal plastic round cell tumor, myxoid liposarcoma, and several differentiation and of EWS-ATF1 were measured by other malignancies, which can be grouped together as the quantitative real-time PCR in clear cell sarcoma cells ‘‘fusion oncogene sarcomas of young adults.’’ The defining treated with histone deacetylase inhibitors. MS-275 and translocations in many of these sarcomas have a common romidepsin inhibited growth in clear cell sarcoma cells by structure: the non–DNA-binding EWSR1 protein or a FUS, TAF2N inducing cell cycle arrest and apoptosis in a time- and homologue ( ) forms the NH2 terminus, and a dose-dependent manner. Sarcomas showed greater sen- DNA binding transcription factor forms the COOH sitivity than other tumor types, with clear cell sarcomas terminus. In doing so, EWSR1 loses its RNA binding most sensitive of all, whereas nonmalignant mesenchymal domain, and the transcription factor its regulatory domain, cells were highly resistant. MS-275 at 1 Mmol/L and resulting in the EWSR1 trans-regulatory domain being romidepsin at 1 nmol/L induced histone H3 acetylation, aberrantly fused to the DNA binding domain of an cell cycle arrest, apoptosis, and differentiation in clear cell otherwise unrelated transcription factor (3). The retained sarcoma cells within 24 hours. Histone deacetylase NH2-terminal SYQG domain of EWSR1 is thought to function as a transcriptional activator (4), the functions of which could involve changes in histone acetylation status. EWS-FLI1 expression in myoblasts blocks mesenchymal Received 8/14/07; revised 1/18/08; accepted 4/4/08. differentiation and induces neural crest genes and Wnt Grant support: The Terry Fox Foundation, Canadian Cancer Society, and signaling pathway members (5). In myxoid liposarcoma Canadian Institutes ofHealth Research. Torsten O. Nielsen is a scholar of FUS the Michael Smith Foundation for Health Research. and rare cases of Ewing sarcoma, substitutes for its EWSR1 The costs ofpublication ofthis article were defrayed in part by the homologue (6), and the SYT-SSX oncoprotein in payment ofpage charges. This article must thereforebe hereby marked synovial sarcoma may also invoke similar effects by advertisement in accordance with 18 U.S.C. Section 1734 solely to binding an EWS homologue called SIP/CoAA (4). The indicate this fact. features of these translocation-associated sarcomas, includ- Requests for reprints: Torsten O. Nielsen, Anatomical Pathology, Room JP1401, 855 West 12th Avenue, Vancouver, British Columbia, Canada ing clear cell sarcoma, have many similarities to the V5Z 1M9. E-mail: [email protected] translocation-associated leukemias, for which related Copyright C 2008 American Association for Cancer Research. translocations have been implicated in transforming com- doi:10.1158/1535-7163.MCT-07-0560 mitted hematopoietic progenitors back to a stem cell state Mol Cancer Ther 2008;7(6).June 2008 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2008 American Association for Cancer Research. 1752 HDAC Inhibitors Induce Apoptosis in Clear Cell Sarcoma (7), and a model has been described linking fusion deacetylation, inhibits the level of EWS-FLI1 mRNA and oncoproteins to aberrant chromatin remodeling and im- protein in Ewing sarcoma cells (18), and induces differen- paired differentiation (8). tiation in chondrosarcoma cells (15). Phase I/II studies have Gene transcription is regulated by coactivator and shown that both MS-275 and romidepsin can be used in corepressor complexes. Whereas acetylaton of histones is humans (19, 20). associated with chromatin relaxation and transcriptional For most soft tissue sarcomas, no adjuvant chemotherapy activation, histone deacetylases repress transcription by offers proven benefit to overall or even progression-free condensing chromatin. Thus, histone deacetylases are survival, and metastatic disease is generally fatal (21). enzymatic effectors of transcriptional regulatory complexes Finding new approaches to targeted therapy is a priority performing epigenetic histone modifications, repressing for improving patient outcomes. Recent efforts focusing key genes, many of which control growth and induce on Ewing sarcoma (18), chondrosarcoma, and synovial differentiation (9). Histone deacetylases have been shown sarcoma (22) indicate that the histone deacetylase inhibitor to associate with promoters and repress transcription of romidepsin inhibits growth of those tumor cells in vitro and neuronal, intestinal, and muscle differentiation genes; in xenograft models. To date, there have been no studies such epigenetic alterations may represent very early events testing the effect of histone deacetylase inhibitors against in the development of cancer (10). Overexpression of clear cell sarcomas. We conducted this study to investigate histone deacetylases can induce cell proliferation (11), and the growth inhibition and differentiation activities of the deregulation of histone deacetylase recruitment seems to histone deacetylase inhibitors MS-275 and romidepsin on play an important role in tumorigenesis. clear cell sarcoma cell line models. We compare the For these reasons, inhibition of histone deacetylases is sensitivity translocation-associated sarcomas and other cell considered a promising antineoplastic targeted interven- line models to these histone deacetylase inhibitors, and tion in many cancer models and several such drugs are evaluate the effect of MS-275 and romidepsin on the in active development. Histone deactylase inhibitors are expression of genes associated with mesenchymal cell effective in leukemias, including those bearing fusion growth and differentiation. oncogenes, where they induce cell growth arrest and differentiation (12). Histone deacetylase inhibitors induce markers of differentiation in myeloid leukemia, bone, Materials and Methods and chondrosarcoma models (13–15). MS-275, a recently Drugs and Cell Lines developed class I histone deacetylase inhibitor, represses MS-275 and romidepsin (depsipeptide, FK228) were growth of human malignancies such as leukemias and generously provided by Berlex Pharmaceuticals (Montville, solid tumors in preclinical models (16, 17). Another histone NJ) and the Developmental Therapeutics Program of the deacetylase inhibitor, romidepsin (depsipeptide, FK228), National Cancer Institute (Bethesda, MD). Doxorubicin was inhibits proliferation, reverses EWS-FLI1 mediated histone purchased from Sigma and was used as an experimental Table 1. Cell lines and IC50 of MS-275 and romidepsin at 72 h of treatment Cell line Provider Tumor type Fusion gene IC50 of MS-275 IC50 of romidepsin (Amol/L) (nmol/L) DTC1 Dr. KL. Schaefer Clear cell sarcoma EWS-ATF1 0.28 0.51 KAO Dr. KL. Schaefer Clear cell sarcoma EWS-ATF1 0.38 0.91 SU-CCS-1 Dr. AL. Epstein Clear cell sarcoma EWS-ATF1 0.63 0.89 SYO-1 Dr. A. Kawai Synovial sarcoma SYT-SSX2 0.44 0.67 FUJI Dr. K. Nagashima Synovial sarcoma SYT-SSX2 2.51 1.31 SKNMC ATCC Ewing sarcoma EWS-FLI1 0.79 1.17 402-91 Dr. P. Aman Myxoid liposarcoma FUS-DDIT3