Human Molecular Genetics Advance Access published online on April 13, 2005
Human Molecular Genetics, doi:10.1093/hmg/ddi155
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1 Clinical Cancer Genetics Program, Human Cancer Genetics Program, Comprehensive Cancer Center, Division of Human Genetics, Department of Internal Medicine and Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, Ohio 43210, USA;
* To whom correspondence should be addressed. Epidemiological data suggest that consumption of phytoestrogens can be protective against the development of breast cancer. It may be logical to postulate that phytoestrogens may regulate proteins that control cellular division, such as the tumor suppressor PTEN. Germline, and more significantly, somatic PTEN mutations have been observed in a broad range of human cancers, especially those of the breast. Active PTEN results in decreased phosphorylation of Akt and MAPK, the up-regulation of p27 and down-regulation of cyclin D1 protein levels resulting in decreased proliferation and an increase in apoptosis. We hypothesized that phytoestrogen exposure regulates PTEN protein expression in the breast cancer cell line, MCF-7. When MCF-7 cells were stimulated with resveratrol, quercetin, or genistein, there was an increase in PTEN protein levels. Concomitantly, phytoestrogen stimulation resulted in decreased Akt phosphorylation and an increase in p27 protein levels, indicating active PTEN lipid phosphatase activity. In contrast, we found that MAPK phosphorylation and cyclin D1 levels, which are regulated by PTEN's protein phosphatase activity, were not altered. Using semi-quantitative RT-PCR, we found that mRNA levels were slightly increased in cells stimulated by phytoestrogens, suggesting that the mechanism for increased PTEN protein expression is dependent upon transcription. Concurrently, our data provide evidence that a mechanism for phytoestrogens' protective nature is partially through increased PTEN expression. More importantly, it provides a novel target for the regulation of PTEN expression and suggests that dietary changes may be adjunctive to traditional preventive and therapeutic strategies against breast cancer. The tumor suppressor gene PTEN, localized to 10q23.3, has been implicated in the pathogenesis of a variety of human cancers, including the inherited hamartoma-neoplasia syndromes, Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome, Proteus and Proteus-like syndromes (1). Cowden syndrome is an under-diagnosed syndrome with high risk of breast (28-50% in a lifetime) and thyroid (10%) cancers (2, 3). We have found that germline mutations of PTEN cause 85% of Cowden syndrome cases and there is a genotype-phenotype correlation with breast cancer (4, 5). More importantly, PTEN has been shown to play a role in sporadic cancers, including those of the breast. Since its discovery in 1997, abundant data show that PTEN, the protein product of PTEN, is a tumor suppressor in vitro and in vivo. In vitro, recombinant PTEN has been shown to dephosphorylate protein substrates on serine, threonine and tyrosine residues (6). In vivo, PTEN has been demonstrated to lie upstream of cellular signaling cascades [(1) and references within]. For example, it has been shown to dephosphorylate focal adhesion kinase (FAK), which inhibits cell spreading and migration. PTEN has also been shown to dephosphorylate lipid substrates, specifically phosphatidylinositol(3,4,5)triphosphate (PI(3,4,5)P3) yielding phosphatidylinositol(4,5)biphosphate (PI(4,5)P2). Consequently, PTEN is an antagonist of PI3K, the enzyme that phosphorylates PI(4,5)P2. Given that PI(3,4,5)P3 is required for Akt recruitment to the plasma membrane and its subsequent activation, PTEN inhibits the pro-proliferative Akt-dependent pathways (1). Accordingly, proper PTEN function leads to decreased phosphorylated-Akt (P-Akt) levels and apoptosis occurs. In contrast, absent or dysfunctional PTEN leads to high levels of P-Akt, which is pro-proliferative. PTEN also regulates cell survival by coordinating the cell cycle. In addition, PTEN coordinates G1 arrest through up-regulation of p27 and concomitant down-regulation of cyclin D1 (7) as well as regulating the activation of the MAPK pathway. PTEN appears to be constitutively active in vivo, suggesting that modulation of its activity is through regulation of both transcription and protein degradation. Activated PPAR Epidemiological data suggest that the consumption of moderate levels of soy, red wine and a diet rich in vegetables and fruits can be protective against breast cancer (13, 14). Previous research has shown that an important component of these foods are the phytoestrogens. However, how they elicit this effect is not completely known. Recently, there has been an emphasis on the elucidation of the interaction between phytoestrogens and regulatory proteins such as those encoded by tumor suppressor genes, as well as the cellular signaling cascades that regulate gene expression/activation. To date, there has not been direct evidence for phytochemicals interacting with PTEN. However, phytoestrogen exposure leads to cellular effects identical to those mediated by proper PTEN function and promotes apoptosis via a decrease in P-Akt levels. Also similar to PTEN, phytoestrogens play a role in regulating cell cycle and apoptosis such as MAPK (15-17) and cell cycle arrest (18). These similar cellular effects mediated by PTEN and phytoestrogens could be explained by independent mechanisms but it is much more plausible that one or more are the result of an interaction between PTEN and the phytoestrogens. We therefore investigated the effect of phytoestrogens on PTEN protein levels and activity.
Received February 24, 2005
Revised April 4, 2005
Accepted April 6, 2005
Article
Phytoestrogen exposure elevates PTEN levels
2 Clinical Cancer Genetics Program, Human Cancer Genetics Program, Comprehensive Cancer Center, Division of Human Genetics, Department of Internal Medicine and Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, Ohio 43210, USA;; Cancer Research UK Human Cancer Genetics Research Group, University of Cambridge, Cambridge CB2 1XZ, UK
Charis Eng, E-mail: eng-1{at}medctr.osu.edu
Abstract 
(8) p53 (9) and EGR-1(10) have been shown to up-regulate PTEN transcription. Factors that may down-regulate transcription have yet to be identified. Protein stability has been shown to be regulated in both a phosphorylation-dependent and -independent manner (11, 12) and modes of PTEN protein regulation are still to be elucidated.
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