Uterine serous carcinoma is a very aggressive cancer for which there are no effective therapeutic protocols. There is, therefore, an urgent need to identify new therapeutic regimens that will prolong survival of USC patients and that will transform response to treatment into cure. In this study we demonstrate that vorinostat, a histone deacetylase inhibitor, markedly induces the accumulation of acetylated histone H3, leading to transcriptional modulation of specific gene programs, including genes involved in IGF-IR and downstream mediators regulation. Our results demonstrate that vorinostat induced apoptosis, growth arrest, and inhibition of migration and colony formation in Type I and II endometrial cancer cell lines. In addition, we demonstrate that inhibition of HDAC modulates IGF-IR expression in Ishikawa and USPC-2 cells lines. The Ishikawa cell line was derived from a well differentiated adenocarcinoma and is a validated model for Type I endometrial cancer. Type I cancers express the estrogen and progesterone receptors 
, exhibit microsatellite instability, and include mutations in the ras
proto-oncogene, p53 
and pTEN tumor suppressor genes, resulting in deregulated PI3K signaling and activation of AKT. In addition, they secrete IL-6, TGF-alpha and IGF-II, but not IGF-I 
. By contrast, Type II tumors show mutations in p53 
, but almost never have microsatellite instability or ras
or pTEN mutations. Usually they overexpress the HER-2/neu oncogene, and secrete IGF's, IGFBP and IL-6.
PTEN was shown to downregulate IGF-II and IGF-IR expression in hepatoma and prostate cancer cells, respectively, suggesting that the anti-proliferative effects of pTEN are, at least in part, mediated through the regulation of expression of components of the IGF system 
. Vorinostat was shown to down-regulate autocrine IGF-I production and expression, and the IGF-IR in multiple myeloma 
. A number of studies have shown a correlation between components of the IGF system and endometrial cancer risk. The IGF system has autocrine and paracrine functions in the regulation of endometrial proliferation and differentiation 
. Results of migration assays suggest that both cell lines secrete growth factors and/or cytokines responsible for their proliferative effects. In particular, a highly mitogenic effect was seen in USPC-2 cells. Vorinostat, both in the presence or absence of IGF-I, inhibited migration of both cell lines. Western blot analyses showed that vorinostat robustly increased the IGF-I-stimulated IGF-IR phosphorylation in Ishikawa cells. Furthermore, alone or in combination with IGF-I, vorinostat up-regulated total IGF-IR levels. A potential explanation for the increase in IGF-IR phosphorylation in Ishikawa cells is the reported vorinostat-induced down-regulation of endogenous IGF-II. This reduction may shift the competition between secreted IGF-II and exogenous IGF-I, leading to an increase in IGF-I binding with ensuing enhanced activation. In addition, the expression of a constitutively-active AKT in Ishikawa cells (as a result of loss of pTEN) significantly increased the total IGF-IR expression. This effect might be mediated at the level of transcription, as shown by the large increase in IGF-IR promoter activity.
A number of epigenetic drugs have already been approved for the treatment of hematological malignancies 
. These drugs include demethylating agents and HDAC inhibitors such as trichostatin A. Previous studies showed that treatment of mice fibroblasts with trichostatin A induced partial relaxation of genomic imprinting as well as decreased DNA methylation of both IGF-IIR sense and antisense promoters. Hence, increases in histone acetylation can lead to decreased DNA methylation, thereby modulating the regulation of the imprinted expression of IGF-IIR sense and antisense transcripts. Interestingly, Risinger et al reported that promoter hypermethylation is common in Type I, but not Type II, endometrial tumors 
. HDAC inhibitors can also induce TNF-related apoptosis-inducing ligand (TRAIL) 
, suggesting the activation of the death receptor pathway without the requirement of exogenous TRAIL, leading to activation of caspase 8 
. Our data suggest that vorinostat may modulate apoptotic cell death by both the extrinsic and intrinsic pathways in USPC-2 cells because it changed the activation of caspase 8, the expression of Bcl2, and further contributed to caspase 3 activation, following activation of caspase 9. Vorinostat-induced apoptosis in Ishikawa cells might be related to caspases 3 and 9 activation. The activation of caspase 3 was accompanied by PARP cleavage into an 85-kDa fragment in both cells lines. Moreover, our data showed that the pro-apoptotic action of vorinostat is not correlated with IGF-IR levels.
Lu et al reported that a high level of mTor activity is a consistent feature of endometrial carcinomas. Activation of mTor occurred in tumors that had lost pTEN, resulting in deregulated PI3K signaling and activation of AKT. While pTEN alterations are well known in endometrial cancer, signaling defects involving the downstream effector mTor, or other tumor suppressors that regulate mTor signaling, including TSC2 and LKB1, are less documented 
. Constitutive mTor expression was observed in Ishikawa cells including an inactive pTEN. In addition, vorinostat-induced activation of mTor in USPC-2 cells might be mediated via the PI3K/AKT and p53/Ampk signaling pathways. Alternatively, activation of mTor expression might be induced by p53-independent mechanisms. No data, however, is available regarding the p53 status in USPC-2 cells. Previous investigations have provided evidence to support the view that the presence of an intact p53 is essential for an efficient HDAC inhibitor-induced apoptotic response 
. This dependence appears to vary with the agent used and may be due to differences in potency. Furthermore, acetylation of p53 occurs following HDAC inhibitor treatment and may increase its activity and reduce targeting of p53 for degradation 
. However, others have shown that HDAC inhibitors induce apoptosis via p53-independent mechanisms 
. More experiments are required to define the role of p53 in the vorinostat-mediated apoptosis of USPC-2 cells.
Studies have shown that HDAC inhibitors are relatively non-toxic to normal cells or tissues, exhibiting selective cytotoxicity against a wide range of cancer cells. A number of HDAC inhibitors are currently being investigated in clinical trials against hematological and solid tumors, both as single agents and in combination with other cytotoxic therapies 
. MTT analysis revealed a significant anti-proliferative activity of vorinostat in both cell lines assayed and a synergistic effect of combined treatment of vorinostat with cisplatin in USPC-2 cells. In addition, cell cycle analyses have shown that vorinostat is able to arrest cell cycle progression in endometrial cancer cells. Recent studies have demonstrated that the HDAC inhibitor-mediated induction of p21 was the result of increased H3 and H4 acetylation associated with the p21 gene promoter 
. These results are in line with preclinical and clinical data showing that H3 and H4 acetylation are important biomarkers for vorinostat action 
. P21 is member of the family of endogenous cyclin-dependent kinase inhibitors that negatively regulate cell cycle progression and cyclin D1, a positive regulator of cell cycle progression. Our data show that vorinostat increased p21 expression and inhibited cyclin D1 expression in both cell types, with ensuing growth arrest. In summary, our studies demonstrate that vorinostat exhibits a potent apoptotic and anti-proliferative effect in both Type I and II endometrial cancer cell lines, strongly suggesting that endometrial cancer may be therapeutically targeted by vorinostat.