Anait S. Levenson

Resveratrol enhances p53 acetylation and apoptosis in prostate cancer by inhibiting MTA1/NuRD complex

Dietary compounds and epigenetic influences are well recognized factors in cancer progression. Resveratrol (Res), a dietary compound from grapes, has anticancer properties; however, its epigenetic effects are understudied. Metastasis‐associated protein 1 (MTA1) is a part of the nucleosome remodeling deacetylation (NuRD) corepressor complex that mediates posttranslational modifications of histones and nonhistone proteins resulting in transcriptional repression. MTA1 overexpression in prostate cancer (PCa) correlates with tumor aggressiveness and metastasis. In this study, we have identified a novel MTA1‐mediated mechanism, by which Res restores p53‐signaling pathways in PCa cells. We show, for the first time, that Res causes down‐regulation of MTA1 protein, leading to destabilization of MTA1/NuRD thus allowing acetylation/activation of p53. We demonstrated that MTA1 decrease by Res was concomitant with accumulation of Ac‐p53. MTA1 knockdown further sensitized PCa cells to Res‐dependent p53 acetylation and recruitment to the p21 and Bax promoters. Furthermore, MTA1 silencing maximized the levels of Res‐induced apoptosis and pro‐apoptotic Bax accumulation. HDAC inhibitor SAHA, like MTA1 silencing, increased Res‐dependent p53 acetylation and showed cooperative effect on apoptosis. Our results indicate a novel epigenetic mechanism that contributes to Res anticancer activities: the inhibition of MTA1/NuRD complexes due to MTA1 decrease, which suppresses its deacetylation function and allows p53 acetylation and subsequent activation of pro‐apoptotic genes. Our study identifies MTA1 as a new molecular target of Res that may have important clinical applications for PCa chemoprevention and therapy, and points to the combination of Res with HDAC inhibitors as an innovative therapeutic strategy for the treatment of PCa.

Estrogenic activity is increased for an antiestrogen by a natural mutation of the estrogen receptor

The estrogen receptor (ER) functions as a ligand-activated transcription factor which mediates the actions of estrogens and antiestrogens in target tissues. Other investigators have shown that artificial point mutations in the transcriptional activation domain AF-2 of the ligand binding domain (LBD) of the ER can increase the estrogenic properties of antiestrogens, determined by transcriptional activation of estrogen-responsive reporter constructs cotransfected into cells. Although these data provide valuable information about ER function there is no evidence that these mutations occur naturally. We have taken a different approach and examined the naturally occurring codon 351 asp --> tyr mutation in the LBD of ER to stimulate the expression of an endogenous target gene. This approach avoids dependence on artificial reporter constructs and their idealized estrogen response elements (EREs). In this report we describe the regulation of transforming growth factor alpha (TGF alpha) mRNA by estradiol and the antiestrogens keoxifene and ICI 182,780 in our stable transfectants of ER-negative MDA-MB-231 breast cancer cells, which express either the wild-type (S30 cells) or codon 351 asp --> tyr mutant ER (BC-2 cells). The mutant receptor was identified in a tamoxifen-stimulated human breast tumor. Our results demonstrate, for the first time, that a naturally occurring mutation in the ER changes the pharmacology of the antiestrogen keoxifene by increasing estrogenic activity, and that keoxifene exhibits a gene-specific estrogen-like effect with mutant ER but not with wild-type ER. The pure antiestrogen ICI 182,780 maintained complete antagonistic activities in both ER transfectants, demonstrating that its action is unaffected by the mutation.

Post-transcriptional gene regulation by HuR promotes a more tumorigenic phenotype

In a breast tumor xenograft model, the MCT-1 oncogene increases the in vivo tumorgenicity of MCF7 cells by promoting angiogenesis and inhibiting apoptosis. Increases in the tumor microvascular density are accompanied by a strong reduction in the levels of the angiogenesis inhibitor thrombospondin-1 (TSP1), but the mechanisms underlying this process are unknown. We show that TSP1 expression is controlled, at least in part, by post-transcriptional events. Using RNA interference to knock down the expression of the RNA-binding protein HuR in MCF7 cells as well as HuR overexpression, we demonstrate that HuR plays an important role in translation of the TSP1 mRNA. Furthermore, employing the RIP-Chip assay yielded 595 transcripts with significantly altered binding to HuR in the more tumorigenic breast cancer clones compared with the weakly tumorigenic clones. These mRNAs clustered in several pathways implicated in the transformed phenotype, such as the RAS pathway (involved in mitogenesis), the PI3K pathway (evasion of apoptosis) and pathways mediating angiogenesis and the cellular response to hypoxia. These findings demonstrate for the first time that global changes in HuR-bound mRNAs are implicated in the evolution to a more tumorigenic phenotype in an in vivo tumor model and underscore the role of global mRNA-protein interactions toward tumor progression.

Resveratrol acts as an estrogen receptor (ER) agonist in breast cancer cells stably transfected with ERα

Resveratrol (Res) is a phytoestrogen found in grapes and present in red wine. Res has been shown to function as an estrogen receptor (ER) agonist, but it remains unclear whether it may also exert antagonist activity. Our aim was to study the effects of Res at both the molecular (TGFα gene activation) and the cellular (cell growth) levels in breast cancer cells stably transfected with wild‐type (wt) ER(D351) and mutant (mut) ER (D351Y). TGFα mRNA induction was used as a specific marker of estradiol (E2) responsiveness. Res caused a concentration‐dependent (10−8–10−4 M) stimulation of TGFα mRNA, indicating that it acts as an estrogen agonist in these cell lines. The pure antiestrogen ICI 182,780 (ICI) blocked Res‐induced activation of TGFα, consistent with action through an ER‐mediated pathway. Further studies that combined treatments with E2 and Res showed that Res does not act as an antagonist in the presence of various (10−11−10−8 M) concentrations of E2. To determine whether Res can be classified as a type I or type II estrogen (Jordan et al., Cancer Res 2001;61:6619–23,), we examined Res with the D351G ER in the TGFα assay and found that Res belongs to the type I estrogens. Both Res and E2 had concentration‐dependent growth inhibitory effects in cells expressing wtER and D351Y ER. Although the pure antiestrogen ICI blocked the growth inhibitory effects of E2, it did not block the inhibitory effects of Res, suggesting that the antiproliferative effects of Res also involve ER‐independent pathways. Interestingly, Res differentially affected the levels of ER protein in these 2 cell lines: Res down‐regulated wtER levels while significantly up‐regulating the amount of mutD351Y ER. Co‐treatment with ICI resulted in strongly reduced ER levels in both cell lines. Gene array studies revealed Res‐induced up‐regulation of more than 80 genes, among them a profound activation of p21CIP1/WAF1, a gene associated with growth arrest. The p21CIP1/WAF1 protein levels measured by Western blotting confirmed Res‐induced significant up‐regulation of this protein in both cell lines. In summary, Res acts as an ER agonist at low doses but also activates ER‐independent pathways, some of which inhibit cell growth.

Resveratrol and prostate cancer: promising role for microRNAs.

SCOPE Resveratrol (Res) has anticancer activity in prostate cancer (PCa), which can be attributed to modulation of microRNAs (miRNAs/miRs). miRNAs/miRs are small non-coding RNAs that negatively regulate gene expression. We have analyzed differential miRNA expression in PCa cells treated with Res. METHODS AND RESULTS Using miRNA microarrays we found that 23 miRNAs were significantly down-regulated and 28 miRNAs were significantly up-regulated after Res treatment. The down-regulated miRs included miR-17-92 and miR-106ab clusters with well recognized oncogenic properties while the up-regulated miRs included several tumor suppressors. Selected miRs were verified by qRT-PCR, including miR-17, miR-20a, miR-20b, miR-106a and miR106b. Since these miRNAs target PTEN (phosphatase and tensin homolog deleted on chromosome 10), we performed Western blot to confirm up-regulation of PTEN in PCa cells. In addition, using TargetScan database, we have identified putative mRNA targets for Res-induced down- and up-regulated miRs. Using a bioinformatics approach, we generated gene networks specifically altered by Res-regulated miRNAs. CONCLUSION Our results indicate that the dietary compound Res may play an important role in prostate carcinogenesis through modulation of miRNA expression: Res down-regulated oncogenic miRs and up-regulated tumor suppressor miRs in PCa cells. Further in-depth studies are necessary in order to fully recognize the beneficial miRNA-mediated effects of Res in PCa.

MSRE-PCR for analysis of gene-specific DNA methylation.

Abnormal DNA methylation is observed in certain promoters of neoplastic cells, although the likelihood of methylation for each individual promoter varies. Simultaneous analysis of many promoters in the same sample can allow use of statistical methods for identification of neoplasia. Here we describe an assay for such analysis, based on digestion of genomic DNA with methylation-sensitive restriction enzyme and multiplexed PCR with gene-specific primers (MSRE-PCR). MSRE-PCR includes extensive digestion of genomic DNA (uncut fragments cannot be identified by PCR), can be applied to dilute samples (<1 pg/μl), requires limited amount of starting material (42 pg or genomic equivalent of seven cells) and can identify methylation in a heterogeneous mix containing <2% of cells with methylated fragments. When applied to 53 promoters of breast cancer cell lines MCF-7, MDA-MB-231 and T47D, MSRE-PCR correctly identified the methylation status of genes analyzed by other techniques. For selected genes results of MSRE-PCR were confirmed by methylation-specific PCR and bisulfite sequencing. The assay can be configured for any number of desired targets in any user-defined set of genes.

Targeting prostate cancer angiogenesis through metastasis-associated protein 1 (MTA1)

Metastasis‐associated protein 1 (MTA1) is overexpressed in many forms of cancer types but its role in prostate cancer (PCa) progression and metastasis has not been explored. In this study, we addressed the functional and biological role of MTA1 in PCa.

Estrogenic effects of resveratrol in breast cancer cells expressing mutant and wild-type estrogen receptors: Role of AF-1 and AF-2

Resveratrol, a hydroxystilbene found in grapes and wine, has previously been shown to be a non-flavonoid phytoestrogen, and to act as an estrogen receptor (ER) superagonist in MCF-7 cells transiently transfected with estrogen-responsive reporter constructs. Several additional hydroxystilbenes, including diethylstilbestrol (DES) and piceatannol, were tested, and all showed ER agonism or partial agonism, but superagonism was specific to resveratrol. Moreover, superagonism was observed in cells carrying a stably integrated reporter gene, indicating that this phenomenon is not a result of transient transfection. To examine the role of the transcriptional activation function (AF) domains of ERalpha in resveratrol agonism, we compared the effects of resveratrol and estradiol (E2) on expression of exogenous reporter genes and an endogenous estrogen-regulated gene (TGFalpha) in MDA-MB-231 cells stably transfected with wild-type (wt) ERalpha or mutants with deleted or mutated AF domains. In reporter gene assays, cells expressing wtERalpha showed a superagonistic response to resveratrol. Deletion of AF-1 or mutation of AF-2 attenuated the effect of resveratrol disproportionately compared to that of E2, while deletion of AF-2 abrogated the response to both ligands. In TGFalpha expression assays, resveratrol acted as a full agonist in cells expressing wtERalpha. Deletion of AF-1 attenuated stimulation by E2 more severely than that by resveratrol, as did deletion of AF-2. In contrast, mutation of AF-2 left both ligands with a limited ability to induced TGFalpha expression. In summary, the effect of modifying or deleting AF domains depends strongly on the ligand and the target gene.

Pterostilbene Acts through Metastasis-Associated Protein 1 to Inhibit Tumor Growth, Progression and Metastasis in Prostate Cancer

The development of natural product agents with targeted strategies holds promise for enhanced anticancer therapy with reduced drug-associated side effects. Resveratrol found in red wine, has anticancer activity in various tumor types. We reported earlier on a new molecular target of resveratrol, the metastasis-associated protein 1 (MTA1), which is a part of nucleosome remodeling and deacetylation (NuRD) co-repressor complex that mediates gene silencing. We identified resveratrol as a regulator of MTA1/NuRD complex and re-activator of p53 acetylation in prostate cancer (PCa). In the current study, we addressed whether resveratrol analogues also possess the ability to inhibit MTA1 and to reverse p53 deacetylation. We demonstrated that pterostilbene (PTER), found in blueberries, had greater increase in MTA1-mediated p53 acetylation, confirming superior potency over resveratrol as dietary epigenetic agent. In orthotopic PCa xenografts, resveratrol and PTER significantly inhibited tumor growth, progression, local invasion and spontaneous metastasis. Furthermore, MTA1-knockdown sensitized cells to these agents resulting in additional reduction of tumor progression and metastasis. The reduction was dependent on MTA1 signaling showing increased p53 acetylation, higher apoptotic index and less angiogenesis in vivo in all xenografts treated with the compounds, and particularly with PTER. Altogether, our results indicate MTA1 as a major contributor in prostate tumor malignant progression, and support the use of strategies targeting MTA1. Our strong pre-clinical data indicate PTER as a potent, selective and pharmacologically safe natural product that may be tested in advanced PCa.

Trimethoxy-Resveratrol and Piceatannol Administered Orally Suppress and Inhibit Tumor Formation and Growth in Prostate Cancer Xenografts

Resveratrol (Res) is recognized as a promising cancer chemoprevention dietary polyphenol with antioxidative, anti‐inflammatory, and anticancer properties. However, the role of its analogues in prostate cancer (PCa) chemoprevention is unknown.