Todd Romigh

Resveratrol regulates the PTEN/AKT pathway through androgen receptor-dependent and -independent mechanisms in prostate cancer cell lines

The tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome 10) and the androgen receptor (AR) play important roles in tumor development and progression in prostate carcinogenesis. Among many functions, PTEN negatively regulates the cytoplasmic phosphatidylinositol-3-kinase/AKT anti-apoptotic pathway; and nuclear PTEN affects the cell cycle by also negatively regulating the MAPK pathway via cyclin D. Decreased PTEN expression is correlated with prostate cancer progression. Over-expression of AR and upregulation of AR transcriptional activity are often observed in the later stages of prostate cancer. Recent studies indicate that PTEN regulates AR activity and stability. However, the mechanism of how AR regulates PTEN has never been studied. Furthermore, resveratrol, a phytoalexin enriched in red grapes, strawberries and peanuts, has been shown to inhibit AR transcriptional activity in prostate cancer cells. In this study, we use prostate cancer cell lines to test the hypothesis that resveratrol inhibits cellular proliferation in both AR-dependent and -independent mechanisms. We show that resveratrol inhibits AR transcriptional activity in both androgen-dependent and -independent prostate cancer cells. Additionally, resveratrol stimulates PTEN expression through AR inhibition. In contrast, resveratrol directly binds epidermal growth factor receptor (EGFR) rapidly inhibiting EGFR phosphorylation, resulting in decreased AKT phosphorylation, in an AR-independent manner. Thus, resveratrol may act as potential adjunctive treatment for late-stage hormone refractory prostate cancer. More importantly, for the first time, our study demonstrates the mechanism by which AR regulates PTEN expression at the transcription level, indicating the direct link between a nuclear receptor and the PI3K/AKT pathway.

Differential regulation of PTEN expression by androgen receptor in prostate and breast cancers

Prostate cancer and breast cancer are the most common malignancies in the western world. Androgen receptor (AR) and PTEN both have been well documented to have important roles in prostate carcinogenesis. In contrast, AR and PTEN in breast carcinogenesis have not been well studied. Furthermore, the crosstalk and connection between those two pathways remain unclear. Increased AR expression in prostate cancers, combined with decreased PTEN expression, portends a poor clinical outcome. Paradoxically, both high AR and high PTEN levels, detected by immunohistochemistry, in primary breast carcinomas have been associated with better disease-free survival. Here, we performed in silico analysis of publicly available microarray data sets from prostate or breast carcinomas. We found an inverse correlation between AR and PTEN transcript expression in prostate cancer tissues in contrast to the positive correlation in breast cancer. These data led us to hypothesize that AR may directly affect PTEN transcriptional regulation in prostate and breast cancer cells. Here, we show for the first time that AR inhibits PTEN transcription in prostate cancer cells, whereas AR upregulates PTEN transcription in breast cancer cells, which mechanistically explains both the immunohistochemical PTEN–AR expressional data noted in clinical trials and in our in silico analysis of the transcriptomes of breast and prostate cancers. In addition, we have fine-mapped the AR-binding motif within the PTEN promoter. Here we show that, in patients with Cowden syndrome, an inherited cancer syndrome caused by germline mutations scattered throughout PTEN, point variants affecting the 3′ end of the AR-binding motif result in abrogation of androgen-mediated transcriptional regulation of PTEN expression. We may speculate that the differential AR effect on PTEN may begin to explain organ-specific and perhaps sex-specific neoplasia predisposition in Cowden syndrome, as well as why only a fraction of women with germline PTEN mutations develop breast cancer, depending on the androgen steroid milieu and levels.

ATP modulates PTEN subcellular localization in multiple cancer cell lines

The tumour suppressor gene PTEN plays an important somatic role in both hereditary and sporadic breast carcinogenesis. While the role of PTENs lipid phosphatase activity, as a negative regulator of the cytoplasmic phosphatidylinositol-3-kinase/Akt pathway is well known, it is now well established that PTEN exists and functions in the nucleus. Multiple mechanisms of regulating PTENs subcellular localization have been reported. However none are ubiquitous across multiple cancer cell lines and tissue types. We show here that adenosine triphosphate (ATP) regulates PTEN subcellular localization in a variety of different cancer cell lines, including those derived from breast, colon and thyroid carcinomas. Cells deficient in ATP show an increased level of nuclear PTEN protein. This increase in PTEN is reversed when cells are supplemented with ATP, ADP or AMP. In contrast, the addition of the non-hydrolyzable analogue ATPγS, did not reverse nuclear PTEN protein levels in all the cell types tested. To our knowledge, this is the first report that describes a regulation of PTEN subcellular localization that is not specific to one cell line or tissue type, but appears to be common across a variety of cell lineages.

Germline and somatic cancer-associated mutations in the ATP-binding motifs of PTEN influence its subcellular localization and tumor suppressive function

Germline and somatic PTEN mutations are found in Cowden syndrome (CS) and multiple sporadic malignancies, respectively. PTEN function appears to be modulated by subcellular compartmentalization, and mislocalization may affect function. We have shown that cellular ATP levels affect nuclear PTEN levels. Here, we examined the ATP-binding capabilities of PTEN and functional consequences, relevant to cancer-associated mutations. PTEN mutation analysis of CS patients and sporadic colorectal carcinomas and comparative aminoacid analysis were utilized to identify mutations in ATP-binding motifs. The ability of wild-type (WT) or mutant PTEN to bind ATP was assessed by ATP–agarose-binding assays. Subcellular fractionation, western blotting, confocal microscopy and growth assays were used to determine relative nuclear-cytoplasmic localization and function. Somatic colorectal carcinoma-derived PTEN missense mutations were associated with nuclear mislocalization. These mutations altered cellular proliferation, apoptosis and anchorage-dependent growth. Examination of PTENs amino acid sequence revealed these mutations resided in previously undescribed ATP-binding motifs (c.60–73; c.122–136). In contrast to WT PTEN, both cancer-associated somatic and germline-derived PTEN missense mutations, which lie within the ATP-binding motifs, result in mutant PTEN that does not bind ATP efficiently. We also show that CS patients with germline ATP-binding motif-mutations had nuclear PTEN mislocalization. Of four unrelated patients with functional germline ATP-binding domain mutations, all three female patients had breast cancers. Germline and somatic mutations within PTENs ATP-binding domain play important pathogenic roles in both heritable and sporadic carcinogenesis by PTEN nuclear mislocalization resulting in altered signaling and growth. Manipulation of ATP may represent novel therapies in tumors with such PTEN alterations.

Disruption of Transforming Growth Factor-β Signaling by Five Frequently Methylated Genes Leads to Head and Neck Squamous Cell Carcinoma Pathogenesis

Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with low survival rates in advanced stages. To facilitate timely diagnosis and improve outcome, early detection markers (e.g., DNA methylation) are crucial for timely cancer diagnosis. In a recent publication, an epigenome-wide screen revealed a set of genes that are commonly methylated and downregulated in head and neck cancers (SEPT9, SLC5A8, FUSSEL18, EBF3, and IRX1). Interestingly, these candidates are potentially involved in the transforming growth factor-beta (TGF-beta) signaling pathway, which is often disrupted in HNSCC. Therefore, we sought to determine coordinated epigenetic silencing of these candidate genes in HNSCC as potential key disruptors of TGF-beta signaling, which could ultimately result in HNSCC progression. Through immunoprecipitation studies, all five of the investigated candidate genes were found to interact with components of the TGF-beta pathway. Overexpression of SLC5A8, EBF3, and IRX1 resulted in decreased mitotic activity and increased apoptosis. In addition, EBF3 was found to increase p21 promoter activity, and SMAD2 significantly increased IRX1 promoter activity. These findings are significant because they reveal a set of genes that interact with components of the TGF-beta pathway, and their silencing via methylation in HNSCC results in coordinated decrease in apoptosis, increased proliferation, and decreased differentiation.

Naturally occurring germline and tumor-associated mutations within the ATP-binding motifs of PTEN lead to oxidative damage of DNA associated with decreased nuclear p53

Somatic and germline mutations in PTEN (phosphatase and tensin homolog deleted on chromosome 10) are found in sporadic cancers and Cowden syndrome patients, respectively. Recent identification of naturally occurring cancer and germline mutations within the ATP-binding motifs of PTEN (heretofore referred to as PTEN ATP-binding mutations) has revealed that these mutations disrupted the subcellular localization and tumor-suppressor activity of PTEN. However, very little is known about the underlying mechanisms of PTEN ATP-binding mutations in tumorigenesis. Here we show that these mutations impair PTENs function both qualitatively and quantitatively. On the one hand, PTEN ATP-binding mutants lose their phosphatase activity and the effect of downregulation of cyclin D1. On the other, the mislocalized mutant PTEN results in a significantly decreased nuclear p53 protein level and transcriptional activity, enhanced production of reactive oxygen species, induction of Cu/Zn superoxide dismutase as well as dramatically increased DNA double-strand breaks (DSBs). When compared with wild-type PTEN, the ATP-binding mutant PTEN has reduced half-life in vitro and decreased protein expression levels in vivo. Our data, thus, reveal a novel mechanism of tumorigenesis in patients with germline or somatic mutations affecting PTEN ATP-binding motifs, i.e. qualitative and quantitative impairment of PTEN due to the loss of its phosphatase activity, and nuclear mislocalization, resulting in rapid PTEN protein degradation, suppression of p53-mediated transcriptional activity, loss of protection against oxidative stress as well as accumulation of spontaneous DNA DSBs.

Germline disruption of Pten localization causes enhanced sex-dependent social motivation and increased glial production

PTEN Hamartoma Tumor Syndrome (PHTS) is an autosomal-dominant genetic condition underlying a subset of autism spectrum disorder (ASD) with macrocephaly. Caused by germline mutations in PTEN, PHTS also causes increased risks of multiple cancers via dysregulation of the PI3K and MAPK signaling pathways. Conditional knockout models have shown that neural Pten regulates social behavior, proliferation and cell size. Although much is known about how the intracellular localization of PTEN regulates signaling in cancer cell lines, we know little of how PTEN localization influences normal brain physiology and behavior. To address this, we generated a germline knock-in mouse model of cytoplasm-predominant Pten and characterized its behavioral and cellular phenotypes. The homozygous Pten(m3m4) mice have decreased total Pten levels including a specific drop in nuclear Pten and exhibit region-specific increases in brain weight. The Pten(m3m4) model displays sex-specific increases in social motivation, poor balance and normal recognition memory-a profile reminiscent of some individuals with high functioning ASD. The cytoplasm-predominant protein caused cellular hypertrophy limited to the soma and led to increased NG2 cell proliferation and accumulation of glia. The animals also exhibit significant astrogliosis and microglial activation, indicating a neuroinflammatory phenotype. At the signaling level, Pten(m3m4) mice show brain region-specific differences in Akt activation. These results demonstrate that differing alterations to the same autism-linked gene can cause distinct behavioral profiles. The Pten(m3m4) model is the first murine model of inappropriately elevated social motivation in the context of normal cognition and may expand the range of autism-related behaviors replicated in animal models.

Nuclear PTEN levels and G2 progression in melanoma cells

The phosphatase and tensin homolog (PTEN) exerts its function, in part, by negatively regulating the well-known phosphatidylinositol-3-kinase/AKT signaling pathway. Previous histological work has suggested that alterations in the nuclear/cytoplasmic compartmentalization of PTEN may play a role in the development and progression of melanoma. In this study, we examined the nuclear/cytoplasmic compartmentalization of PTEN in melanoma cell lines and its correlation with the cell cycle. Studies were performed in melanoma cells lines using classic cell biological techniques. In contrast to breast cancer cell lines, we found that increased levels of nuclear PTEN levels correlate with G2 rather than with G1 arrest. In WM164 and SKmel28 cells, overexpression of PTEN protein did not significantly increase the number of cells in the G2 phase. Differential CDC2 phosphorylation levels in cells that overexpressed PTEN compared with those where PTEN was downregulated suggest some involvement of PTEN in G2 checkpoint regulation. The data suggest that although nuclear PTEN levels correlate with the G2 phase, the role of PTEN in modulating G2/M arrest is not limiting. Further, the specific cell cycle phase regulated by nuclear PTEN is cell-type dependent. Taken together, our observations suggest that in melanoma, nuclear PTEN is involved in G2 progression possibly through the modulation of CDC2, opening up a new arena for investigation.

Cowden Syndrome-Related Mutations in PTEN Associate with Enhanced Proteasome Activity

Germline mutations in PTEN have been described in a spectrum of syndromes that are collectively known as PTEN hamartoma tumor syndrome (PHTS). In addition to being mutated in the germline in PHTS, somatic loss-of-function PTEN mutations are seen in a wide range of sporadic human tumors. Here, we show evidence of upregulated proteasome activity in PHTS-derived lymphoblasts, Pten knock-in mice and cell lines expressing missense and nonsense PTEN mutations. Notably, elevated nuclear proteasome activity occurred in cells expressing the nuclear mislocalized PTEN-K62R mutant, whereas elevated cytosolic proteasome activity was observed in cells expressing the cytosolic-predominant mutant PTEN (M3M4 and C136R). Treatment with proteasome inhibitor MG-132 was able to restore both nonsense and missense mutant PTEN protein levels in vitro. PHTS patients with destabilizing PTEN mutations and proteasome hyperactivity are more susceptible to develop neurologic symptoms such as mental retardation and autism than mutation-positive patients with normal proteasome activity. A detailed molecular and functional analysis shows that PTEN mutants most likely cause proteasome hyperactivity via 2 different mechanisms, namely, induction of proteotoxic stress and loss of protein phosphatase activity. These results provide novel insights into the cellular functions of PTEN and reveal molecular mechanisms whereby PTEN mutations increase proteasome activity and lead to neurologic phenotypes.

Activator protein 2 alpha (AP2α) suppresses 42 kDa C/CAAT enhancer binding protein α (p42 cEBPα) in head and neck squamous cell carcinoma

The tumor suppressor C/CAAT enhancer binding protein alpha (C/EBPα) is a transcription factor involved in cell cycle control and cellular differentiation. A recent study showed that C/EBPα is frequently downregulated in head and neck squamous cell carcinoma (HNSCC) by DNA methylation in an upstream regulatory region. Here, we investigated how DNA methylation in the upstream regulatory region disrupts the transcriptional regulation of C/EBPα in HNSCC. The results reveal that aberrant methylation correlates with methyl binding domain protein binding and repressive histone modifications. This methylated region contains previously uninvestigated AP2α binding sites. AP2α suppresses C/EBPα promoter activity and protein expression. Interestingly, silencing AP2α by shRNA increases the antiproliferative isoform of C/EBPα (p42C/EBPα). Furthermore, growth analysis revealed that these 2 isoforms yield very different proliferative properties in HNSCC.