[Show abstract][Hide abstract] ABSTRACT: 1alpha,25-dihydroxyvitamin D3, 1,25(OH)(2)D(3), regulates gene expression through the vitamin D receptor. The present studies identify the epidermal growth factor receptor, EGFR, as a target gene suppressed by 1,25(OH)(2)D(3) in human ovarian cancer cells. The suppression was detected at both mRNA and protein levels in vitamin D-sensitive human ovarian cancer cells. A novel vitamin D response element was identified in intron 1 of the EGFR genome, a known hotspot for its transcriptional regulation. Chromatin immunoprecipitations and reporter gene analyses showed that the intronic DNA element bound to vitamin D receptor and a co-repressor and was functional in mediating transcriptional suppression of EGFR promoter by 1,25(OH)(2)D(3) under stable transfection conditions. Consistent with the EGFR down regulation, 1,25(OH)(2)D(3) suppressed activation of the external signal regulated kinase by epidermal growth factors. Over expression of an active EGFR in vitamin D sensitive ovarian cancer cells caused resistance to 1,25(OH)(2)D(3)-induced growth suppression and diminished the hormonal regulation of cyclin D1, cyclin E, Skp2 and p27, a group of cell cycle regulators that mediate 1,25(OH)(2)D(3)-induced cell cycle arrest at G1-S checkpoint. Taken together, our studies demonstrate that 1,25(OH)(2)D(3) suppresses the response of human ovarian cancer cells to mitogenic growth factors and couple the suppression to the cell cycle arrest at G1-S checkpoint by the hormone.
Molecular and Cellular Endocrinology 03/2011; 338(1-2):58-67. · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Members of the FOXO (forkhead O) class of transcription factors are tumor suppressors that also control aging and organismal life span. Mammalian FOXO degradation is proteasome-mediated, although the ubiquitin E3 ligase for FOXO factors remains to be defined. We show that MDM2 binds to FOXO1 and FOXO3A and promotes their ubiquitination and degradation, a process apparently dependent on FOXO phosphorylation at AKT sites and the E3 ligase activity of MDM2. Binding of MDM2 to FOXO occurs through the region of MDM2 that directs its cellular trafficking and the forkhead box of FOXO1. MDM2 promotes the ubiquitination of FOXO1 in a cell-free system, and its knockdown by small interfering RNA causes accumulation of endogenous FOXO3A protein in cells and enhances the expression of FOXO target genes. In cells stably expressing a temperature-sensitive p53 mutant, activation of p53 by shifting to permissive temperatures leads to MDM2 induction and degradation of endogenous FOXO3A. These data suggest that MDM2 acts as an ubiquitin E3 ligase, downstream of p53, to regulate the degradation of mammalian FOXO factors.
Journal of Biological Chemistry 04/2009; 284(21):13987-4000. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: FoxO (mammalian forkhead subclass O) proteins are transcription factors acting downstream of the PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor. Their activity is negatively regulated by AKT-mediated phosphorylation. Our previous studies showed that the transcriptional activity of the androgen receptor (AR) was inhibited by PTEN in an AKT-sensitive manner. Here, we report the repression of the activity of the full-length AR and its N-terminal domain by FoxO1 and the participation of FoxO1 in AR inhibition by PTEN. Ectopic expression of active FoxO1 decreased the transcriptional activity of AR as well as androgen-induced cell proliferation and production of prostate-specific antigen. FoxO1 knock down by RNA interference increased the transcriptional activity of the AR in PTEN-intact cells and relieved its inhibition by ectopic PTEN in PTEN-null cells. Mutational analysis revealed that FoxO1 fragment 150-655, which contains the forkhead box and C-terminal activation domain, was required for AR inhibition. Mammalian two-hybrid and glutathione-S-transferase pull-down assays demonstrated that the inhibition of AR activity by PTEN through FoxO1 involved the interference of androgen-induced interaction of the N- and C-termini of the AR and the recruitment of the p160 coactivators to its N terminus and to the androgen response elements of natural AR target genes. These studies reveal new mechanisms for the inhibition of AR activity by PTEN-FoxO axis and establish FoxO proteins as important nuclear factors that mediate the mutual antagonism between AR and PTEN tumor suppressor in prostate cancer cells.
[Show abstract][Hide abstract] ABSTRACT: Recent studies suggest that growth inhibition by 1,25-dihydroxyvitamin D3 represents an innovative approach to ovarian cancer therapy. To understand the molecular mechanism of 1,25-dihydroxyvitamin D3 action, we profiled the hormone-induced changes in the transcriptome of ovarian cancer cells using microarray technology. More than 200 genes were identified to be regulated by 1,25-dihydroxyvitamin D3. Reverse transcription-PCR analyses confirmed the regulation of a group of apoptosis-related genes, including the up-regulation of the decoy receptor that inhibits tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) action, TRAIL receptor 4, and the down-regulation of Fas, the receptor that mediates the action of Fas ligand. The regulation was further confirmed at the protein level. Consistent with the regulation of the death receptors, pretreatment with 1,25-dihydroxyvitamin D3 decreased apoptosis induced by TRAIL and Fas ligand. Because persistent 1,25-dihydroxyvitamin D3 treatment has been shown to induce apoptosis in ovarian cancer, the hormone appears to exert a dual effect on the death of ovarian cancer cells. Knockdown of TRAIL receptor 4 by RNA interference or ectopic expression of Fas relieved the suppressive effect of 1,25-dihydroxyvitamin D3, showing that molecular manipulation of death receptors is a viable approach to overcome the protective effect of 1,25-dihydroxyvitamin D3 on the apoptosis of ovarian cancer. These strategies may allow ovarian cancer patients to benefit from therapy with both 1,25-dihydroxyvitamin D3 and ligands for death receptors, such as TRAIL, shown to selectively induce apoptosis in cancer but not normal cells.
Journal of Biological Chemistry 11/2005; 280(42):35458-68. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The poor response of advanced epithelial ovarian cancer to current treatments necessitates the development of alternative therapeutic strategies. Inhibition of cancer growth by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] compounds represents an innovative approach for cancer therapy. The current study evaluated the therapeutic potential of a synthetic 1,25(OH)2D3 analogue EB1089 in the treatment of ovarian cancer.
The response of human ovarian cancer cells to 1,25(OH)2D3 and EB1089 were first compared in cell growth, gene transcription, and apoptotic assays. Then, nude mice bearing OVCAR3 tumor xenografts were treated with EB1089 at different dosages, and tumor volumes were monitored. The effect of EB1089 and 1,25(OH)2D3 on the level of serum calcium was also examined. After the treatment, tumors were excised and processed for histologic examination, Ki-67 staining, and tissue terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays to evaluate the morphologic, proliferative, and apoptotic changes induced by EB1089, respectively.
The study shows that EB1089 suppresses the in vitro growth of ovarian cancer cells and transcriptionally activates the GADD45 reporter gene more effectively than 1,25(OH)2D3. Clinically more importantly, EB1089 suppresses the growth of OVCAR3 tumor xenografts in nude mice without inducing hypercalcemia. Ki-67 staining and tissue TUNEL assays showed that both inhibition of cell proliferation and induction of apoptosis contribute to the EB1089-induced tumor suppression in vivo.
This study is the first demonstration that ovarian cancer responds positively in vivo to treatment with a 1,25(OH)2D3 compound and thus supports continued development of 1,25(OH)2D3 analogues for possible use as an alternative or complementary therapy for human ovarian cancer.
Clinical Cancer Research 02/2005; 11(1):323-8. · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The maintenance of telomere length is required for continued cell proliferation, and approximately 85-90% of human cancers, including ovarian epithelial cancers (OCa), show high activity of telomerase. In the present study we report that 1,25-dihydroxyvitamin D(3) (1,25(OH)(2) VD)(3)induces OCa cell apoptosis by down-regulating telomerase. Quantitative reverse transcription-PCR analysis shows that 1,25(OH)(2)VD(3) decreases the level of human telomerase reverse transcriptase (hTERT) mRNA, the catalytic subunit of telomerase. The decrease is not due to transcriptional repression through the putative vitamin D response element present in the 5' regulatory region of hTERT gene. Instead, 1,25(OH)(2) VD(3) decreases the stability of the hTERT mRNA. Stable expression of hTERT in OCa cells decreases their response to 1,25(OH)(2)VD(3)-induced growth suppression. Although the cell cycle progression of these clones stably expressing hTERT is inhibited by 1,25(OH)(2)VD(3) to a similar degree as that of the parental cells, these clones are more resistant to apoptosis induced by 1,25(OH)(2)VD(3) .In contrast to parental cells, which lose proliferation potential after the 1,25(OH)(2)VD(3) treatment, hTERT-expressing clones resume rapid growth after withdrawal of 1,25(OH)(2)VD(3). Overall, the study suggests that the down-regulation of telomerase activity by 1,25(OH)(2)VD(3) and the resulting cell death are important components of the response of OCa cells to 1,25(OH)(2)VD(3)-induced growth suppression.
Journal of Biological Chemistry 01/2005; 279(51):53213-21. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: p27(Kip1) (p27) is a tumor suppressor whose stability is controlled by proteasome-mediated degradation, a process directed in part by cyclin-dependent kinase 2 (CDK2)-mediated phosphorylation of p27 at Thr(187) and its subsequent interaction with the Skp1-Cullin-F-box protein/Skp2 (Skp2) ubiquitin ligase. The present study shows that 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) arrests ovarian cancer cells in G(1) by stabilizing the p27 protein. 1,25(OH)(2)D(3) initiates a chain of events by decreasing the amounts of cyclin E and cyclin E-associated CDK2 activity. As a result, p27 phosphorylation at Thr(187) and consequently the interaction with Skp2 are decreased. 1,25(OH)(2)D(3) also increases p27 stability by decreasing the abundance of Skp2. It is the combined effect of 1,25(OH)(2)D(3) on both the CDK2-dependent phosphorylation of p27, and thus its affinity for Skp2, and Skp2 expression that dramatically increases the stability of the p27 protein. Similar to its effects in ovarian cancer cells, 1,25(OH)(2)D(3) induces p27 accumulation in wild type mouse embryo fibroblasts and arrests wild type but not p27-null mouse embryo fibroblasts in G(1). Stable expression of Skp2 in OVCAR3 cells diminishes the G(1) arrest and decreases the growth response to 1,25(OH)(2)D(3). Taken together, the results of this study identify p27 as the key mediator of 1,25(OH)(2)D(3)-induced growth suppression in G(1) and show that the hormone achieves this by decreasing the activity of CDK2 and reducing the abundance of Skp2, which act together to degrade p27.
Journal of Biological Chemistry 07/2004; 279(24):25260-7. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 1,25-Dihydroxyvitamin D3 suppresses the growth of multiple human cancer cell lines by inhibiting cell cycle progression and inducing cell death. The present study showed that 1,25-dihydroxyvitamin D3 causes cell cycle arrest at the G2/M transition through p53-independent induction of GADD45 in ovarian cancer cells. Detailed analyses have established GADD45 as a primary target gene for 1,25-dihydroxyvitamin D3. A DR3-type vitamin D response element was identified in the fourth exon of GADD45 that forms a complex with the vitamin D receptor.retinoid X receptor heterodimer in electrophoresis mobility shift assays and mediates the dose-dependent induction of luciferase activity by 1,25-dihydroxyvitamin D3 in reporter assays. Chromatin immunoprecipitation assays have shown that the vitamin D receptor is recruited in a ligand-dependent manner to the exonic enhancer but not to the GADD45 promoter regions. In ovarian cancer cells expressing GADD45 antisense cDNA or GADD45-null mouse embryo fibroblasts, 1,25-dihydroxyvitamin D3 failed to induce G2/M arrest. Taken together, these results identify GADD45 as an important mediator for the tumor-suppressing activity of 1,25-dihydroxyvitamin D3 in human ovarian cancer cells.
Journal of Biological Chemistry 12/2003; 278(48):48030-40. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent studies suggested that the protection of cell apoptosis by AKT involves phosphorylation and inhibition of FKHR and related FOXO forkhead transcription factors and that androgens provide an AKT-independent cell survival signal in prostate cancer cells. Here, we report receptor-dependent repression of FKHR function by androgens in prostate cancer cells. Transcriptional analysis demonstrated that activation of the androgen receptor caused an inhibition of both wild-type FKHR and a mutant in which all three known AKT sites were mutated to alanines, showing that the repression is AKT independent. In vivo and in vitro coprecipitation studies demonstrated that the repression is mediated through protein-protein interaction between FKHR and the androgen receptor. Mapping analysis localized the interacting domains to the carboxyl terminus between amino acids 350 and 655 of FKHR and to the amino-terminal A/B region and the ligand binding domain of the receptor. Further analysis demonstrated that the activated androgen receptor blocked FKHR's DNA binding activity and impaired its ability to induce Fas ligand expression and prostate cancer cell apoptosis and cell cycle arrest. These studies identify a new mechanism for androgen-mediated prostate cancer cell survival that appears to be independent of the activity of the receptor on androgen response element-mediated transcription and establish FKHR and related FOXO forkhead proteins as important nuclear targets for both AKT-dependent and -independent survival signals in prostate cancer cells.
Molecular and Cellular Biology 02/2003; 23(1):104-18. · 5.04 Impact Factor