Histone deacetylase inhibitor vorinostat suppresses the growth of uterine sarcomas in vitro and in vivo.

BACKGROUND: Uterine sarcomas are very rare malignancies with no approved chemotherapy protocols. Histone deacetylase (HDAC) inhibitors belong to the most promising groups of compounds for molecular targeting therapy. Here, we described the antitumor effects of suberoylanilide hydroxamic acid (SAHA; vorinostat) on MES-SA uterine sarcoma cells in vitro and in vivo. We investigated effects of vorinostat on growth and colony forming ability by using uterine sarcoma MES-SA cells. We analyzed the influence of vorinostat on expression of different HDACs, p21(WAF1) and activation of apoptosis. Finally, we examined the antitumor effects of vorinostat on uterine sarcoma in vivo. RESULTS: Vorinostat efficiently suppressed MES-SA cell growth at a low dosage (3 microM) already after 24 hours treatment. Decrease of cell survival was even more pronounced after prolonged treatment and reached 9% and 2% after 48 and 72 hours of treatment, respectively. Colony forming capability of MES-SA cells treated with 3 microM vorinostat for 24 and 48 hours was significantly diminished and blocked after 72 hours. HDACs class I (HDAC2 and 3) as well as class II (HDAC7) were preferentially affected by this treatment. Vorinostat significantly increased p21(WAF1) expression and apoptosis. Nude mice injected with 5 x 106 MES-SA cells were treated for 21 days with vorinostat (50 mg/kg/day) and, in comparison to placebo group, a tumor growth reduction of more than 50% was observed. Results obtained by light- and electron-microscopy suggested pronounced activation of apoptosis in tumors isolated from vorinostat-treated mice. CONCLUSIONS: Our data strongly indicate the high therapeutic potential of vorinostat in uterine sarcomas.

Valproate inhibition of histone deacetylase 2 affects differentiation and decreases proliferation of endometrial stromal sarcoma cells.

Covalent modifications of histone proteins, in particular deacetylation of lysine residues, are important for the regulation of gene transcription both in normal and malignant cells. These processes are controlled by histone acetyltransferases and histone deacetylases (HDAC) and have up to now not been described in solid mesenchymal tumors. The present study shows differences in the HDAC1 and HDAC2 expression in endometrial stromal sarcomas (ESS) and a cognate cell line (ESS-1) compared with nonneoplastic endometrial stroma. We show for the first time that HDAC2 expression is consistently increased in ESS. In contrast, HDAC1 expression is generally lower than HDAC2 both in nonneoplastic stroma and in ESS, suggesting that these two proteins, although closely related, are regulated in different ways. In vitro experiments with an ESS cell line showed that valproate, an inhibitor of the class I HDACs, led to significant HDAC2 decrease and to cell differentiation. HDAC2 inhibition in ESS-1 cells caused significant changes in the cell cycle by inhibiting G1-S transition and influencing expression of p21WAF1 and cyclin D1. Moreover, in ESS-1 cells, increased expression of the p21WAF1 was associated with reduction of HDAC2 expression after transfection with small interfering RNA directed against HDAC2. Our results suggest that HDAC2 might be considered as potential drug target in the therapy of ESS and that HDAC inhibitors should be further evaluated in clinical trials in ESS.

JAZF1/JJAZ1 gene fusion in endometrial stromal sarcomas: molecular analysis by reverse transcriptase-polymerase chain reaction optimized for paraffin-embedded tissue.

Endometrial stromal tumors are rare uterine neoplasms including benign stromal nodules, low-grade endometrial stromal sarcomas (ESS), and undifferentiated endometrial sarcomas (UES), the latter representing the most aggressive form. Morphological characteristics and cytogenetic abnormalities are heterogeneous, making diagnosis difficult. Recently, a gene fusion on chromosome 7 that includes two zinc-finger genes (JAZF1 and JJAZ1) has been discovered in these tumors. Hitherto only 31 cases, described by three different research groups, have shown JAZF1/JJAZ1 fusion in approximately 50% of all analyzed low-grade ESSs whereas it is less frequent in UESs. In this study we analyzed 20 ESS and 2 UES cases using two-step reverse transcriptase-polymerase chain reaction optimized for formalin-fixed, paraffin-embedded tissue. In our subset of samples, the JAZF1/JJAZ1 fusion transcript occurred in 80% of analyzed ESS cases and in none of two UES cases. In comparison to published data, our results identified the JAZF1/JJAZ1 gene fusion more frequently in endometrial stromal tumors than hitherto presumed. This cytogenetic abnormality was not present in normal endometria, leiomyomas, or leiomyosarcomas or in lung, gastric, or hepatic carcinomas, indicating its specificity for endometrial stromal tumors. In combination with other established methods, accurate reverse transcriptase-polymerase chain reaction analysis of JAZF1/JJAZ1 gene fusion may be useful in diagnosing difficult or unusual ESS/UES cases.

Inverse correlation of secreted frizzled-related protein 4 and beta-catenin expression in endometrial stromal sarcomas.

Endometrial stromal sarcomas are rare uterine tumours. Whereas the histology and immunohistochemistry of these tumours are well documented, almost nothing is known about the molecular mechanisms involved in their pathogenesis. To characterize the genes altered in these malignancies, a genome-wide cDNA library was generated by suppression subtractive hybridization and a set of differentially expressed clones was isolated. These were then used to produce custom-spotted cDNA arrays. Genes deregulated in endometrial stromal sarcomas were identified by cDNA array hybridization and were confirmed by quantitative real-time PCR analyses and in situ hybridization. Following cDNA array analysis, more than 300 genes deregulated in endometrial stromal sarcoma were selected and sequenced. Among the most significantly deregulated genes were those of secreted frizzled-related proteins (SFRPs), in particular secreted frizzled-related protein 4 (SFRP4). SFRPs are putative modulators of the Wnt-signalling pathway and play a role in different cellular events including cell proliferation. Compared with normal endometrium, the expression of SFRP4 was decreased in both low-grade endometrial stromal sarcoma (ESS; n = 10) and undifferentiated endometrial sarcoma (UES; n = 4), being lower in the latter more aggressive form. These results were verified on paraffin wax-embedded tissue by quantitative real-time PCR analysis and in situ hybridization. Furthermore, the expression of beta-catenin, an important component of the Wnt-signalling pathway, was regulated in an opposite manner to SFRP4, being particularly increased in undifferentiated sarcomas. The activation of the Wnt-signalling pathway was additionally supported by the immunohistochemical demonstration that beta-catenin was translocated to the nucleus in UES. SFRP4 may therefore be a putative tumour suppressor involved in deregulation of the Wnt pathway and in the pathogenesis of ESS and UES.