Prostate-derived Ets Transcription Factor Research Articles

Tumor metastasis suppressor functions of Ets transcription factor through integrin β3-mediated signaling pathway.

Prostate-derived Ets transcription factor (PDEF) is a member of the Ets transcription factor family originally identified in prostate tissue. PDEF functions like a tumor metastasis suppressor. Although the underlying mechanism is not very clear. In this study, a significantly higher expression level of integrin β3 was observed in aggressive prostate cancer (PCa) cells; PC3, that lack the expression of PDEF, was confirmed by quantitative real-time polymerase chain reaction and the immunoblot analysis. Dual immunofluorescence studies have confirmed the lower expression of PDEF and higher expression level of integrin β3 in PCa cells compared with non-tumorigenic prostate epithelial RWPE-1 cells. Then, it was attempted to identify integrin β3-mediated downstream signaling pathways that modulate actin cytoskeleton remodeling in PCa cells. Inhibition of PDEF by RNA interference (PDEFKD ) in DU145 cells confirmed by transcript and western blot analysis, exhibited significantly higher expression level of integrin β3 and its downstream signaling molecules talin and paxillin, associated with promoting migration and invasion of cells. Given the involvement of integrin-mediated invasion of cells, PDEFKD DU145 cells were treated with Echistatin, a potent integrin β3-specific antagonist and found that the cells significantly reduced the transcript and protein expression levels of talin and paxillin; and reduced the invasion of cells. Overall, the cytoskeleton remodeling by integrin β3, modulated by PDEF, may represent a novel molecular link with cell adhesion and migration leading to the suppression of metastasis in PCa cells.

Signatures of prostate-derived Ets factor (PDEF) in cancer.

The Ets proteins are a family of transcription factors characterized by an evolutionarily conserved DNA-binding domain and have diverse biological functions including tumor suppressor as well as tumor promoter functions. They are regulated via a complex and diverse number of mechanisms and control key cellular processes. Prostate-derived Ets transcription factor (PDEF), a unique member of the ETS family, is present in tissues with high epithelial content are hormone-regulated, such as prostate, breast, salivary glands, ovaries, colon, airways, and stomach tissues. PDEF (prostate-derived Ets factor) is also referred to as SPDEF (SAM pointed domain containing Ets transcription factor), PSE (mouse homolog), or hPSE (human PSE) in the literature and is the sole member of the PDEF ETS sub-family. The role of PDEF in cancer development is still not fully elucidated though. The present article focuses on the key findings about the PDEF's biological functions, interacting proteins, and its target genes. There is a strong urge to focus on the clinical studies in larger cohort, which elucidate the regulation of PDEF and its target genes, to determine the potential of PDEF as biomarker. Based on the studies discussed in the present article, one can anticipate that PDEF offers a great potential for developing therapeutics against cancer.

Epigenetic modifications of prostate-derived Ets transcription factor in breast cancer cells

The importance of epigenetic alterations such as DNA methylation, histone modification and nucleosome remodeling in breast cancer is well established. Epigenetic alterations are reversible, and much research has been focused on understanding these alterations with the aim of developing effective therapies. Prostate-derived Ets factor (PDEF) is a member of the Ets family of transcription factors and has long been under investigation for its key role in tumor development and progression. To date, no studies have been conducted to elucidate the epigenetic modifications of PDEF in cancer progression. Using breast and prostate cancer cells, we investigated the effect of the methylation inhibitor 5' azacytidine (AZA) on the expression of PDEF in these cells. The inhibition of methylation observed was specific to breast cancer cells as experiments with prostate cells did not exhibit any significant change. Notably, the expression of p21, a cyclin-dependent kinase (CDK) inhibitor 1 and also a target gene of PDEF, was found to be positively correlated with PDEF expression following 5'AZA treatment. Inhibition of methylation led to a decrease in the proliferation rate of MDA-MB-468 cells as revealed by MTT proliferation assay. Other epigenetic alterations such as histone modifications were not observed in these breast cancer cells following treatment with specific HDAC inhibitors. Our data suggest the possibility of epigenetic modification of PDEF due to DNA methylation and involvement of the cell cycle inhibitor p21. Future studies on the epigenetic alterations of PDEF in correlation with p21 or other targets may facilitate the development of effective therapies for the treatment of breast cancer.

Prostate derived Ets transcription factor and Carcinoembryonic antigen related cell adhesion molecule 6 constitute a highly active oncogenic axis in breast cancer

We previously reported overexpression of Prostate derived Ets transcription factor (PDEF) in breast cancer and its role in breast cancer progression, supporting PDEF as an attractive target in this cancer. The goal of this research was to identify specific PDEF induced molecules that, like PDEF, show overexpression in breast tumors and a role in breast tumor progression. PDEF expression was down regulated by shRNA in MCF-7 human breast tumor cell line, and probes from PDEF down-regulated and control MCF-7 cells were used to screen the HG-U133A human gene chips. These analyses identified 1318 genes that were induced two-fold or higher by PDEF in MCF-7 cells. Further analysis of three of these genes, namely CEACAM6, S100A7 and B7-H4, in relation to PDEF in primary breast tumors showed that in 82% of ER+, 67% of Her2 overexpressing and 24% of triple-negative breast tumors both PDEF and CEACAM6 expression was elevated 10-fold or higher in comparison to normal breast tissue. Overall, 72% (94 of 131) of the primary breast tumors showed 10-fold or higher expression of both PDEF and CEACAM6. In contrast, S100A7 and B7-H4 failed to show concordant elevated expression with PDEF in primary tumors. To determine the significance of elevated PDEF and CEACAM6 expression to tumor phenotype, their expression was down regulated by specific siRNAs in human breast tumor cell lines. This resulted in the loss of viability of tumor cells in vitro, supporting an oncogenic role for both PDEF and CEACAM6 in breast cancer. Together, these findings show that PDEF-CEACAM6 is a highly active oncogenic axis in breast cancer and suggest that targeting of these molecules should provide novel treatments for most breast cancer patients.

The Transcription Factor SPDEF Suppresses Prostate Tumor Metastasis

Emerging evidence suggests that the SAM pointed domain containing ETS transcription factor (SPDEF) plays a significant role in tumorigenesis in prostate, breast, colon, and ovarian cancer. However, there are no in vivo studies with respect to the role of SPDEF in tumor metastasis. The present study examined the effects of SPDEF on tumor cell metastasis using prostate tumor cells as a model. Utilizing two experimental metastasis models, we demonstrate that SPDEF inhibits cell migration and invasion in vitro and acts a tumor metastasis suppressor in vivo. Using stable expression of SPDEF in PC3-Luc cells and shRNA-mediated knockdown of SPDEF in LNCaP-Luc cells, we demonstrate for the first time that SPDEF diminished the ability of disseminated tumors cells to survive at secondary sites and establish micrometastases. These effects on tumor metastasis were not a result of the effect of SPDEF on cell growth as SPDEF expression had no effect on cell growth in vitro or subcutaneous tumor xenograft-growth in vivo. Transcriptional analysis of several genes associated with tumor metastasis, invasion, and the epithelial-mesenchymal transition demonstrated that SPDEF expression selectively down-regulated MMP9 and MMP13 in prostate cancer cells. Further analysis indicated that forced MMP9 or MMP13 expression rescued the invasive phenotype in SPDEF expressing PC3 cells in vitro, suggesting that the effects of SPDEF on tumor invasion are mediated, in part, through the suppression of MMP9 and MMP13 expression. These results demonstrate for the first time, in any system, that SPDEF functions as a tumor metastasis suppressor in vivo.

Abstract 3637: Loss of prostate derived Ets (E-twenty six) transcription factor (PDEF) predicts patients at the highest risk of metastatic prostate cancer

Abstract PSA has ability to detect prostate cancer (CaP) at an early stage, but many CaP tumors are slow to progress, and many patients are overtreated for a disease that will not become life-threatening. Since there remains no reliable way to distinguish indolent from aggressive disease, doctors who advise and patients on active surveillance worry about progression to incurable disease. Therefore, new prognostic tools must be developed to identify patients with potentially metastatic, life threatening CaP. Prostate derived Ets transcription factor (PDEF) has shown to be a master negative regulator of transcriptional networks controlling migration and invasion in many cancer tissues, including CaP. Therefore we studied the expression of PDEF in radical prostatectomy specimens to determine retrospectively whether PDEF expression is predictive of clinical outcome. Patients’ tumor PDEF expression was determined by immunohistochemical staining of a 724 patient tissue microarray (TMA). CaP patients were stratified into groups based on PDEF expression (high, mid, low, null) and a Cox proportional hazards model was used to compare clinical outcomes. Univariate analysis showed that loss of PDEF expression significantly increased risk of metastatic disease (HR = 5.38, PDEF mid; to HR = 43.48, PDEF null; p=0.0059), although it did not correlate with incidence of biochemical failure detected using the American Urological Association (AUA) or National Comprehensive Cancer Network (NCCN) definitions. This result was confirmed in the Kaplan-Meier survival analysis that showed that patients whose CaP were PDEF low/null had significantly shorter metastases-free survival (p=0.0151). Multivariate analysis also demonstrated CaP with low/null PDEF expression were associated with increased risk of developing metastatic disease (HR = 6.21/10.53), although it was not statistically significant, in part due to the low incidence of patients with metastatic disease in the TMA. Therefore, we studied PDEF and vimentin (stem-like marker, repressed by PDEF) tumor expression in a cohort of patients matched for Gleason score, stage, age, and follow-up who developed metastatic disease versus those who did not. Patients that developed metastatic disease had a significantly lower PDEF (3.9 vs 1.7, p<0.0001) and higher vimentin (0.43 vs. 4.1, p<0.0001) expression compared to matched controls. We propose that intact PDEF expression in CaP can be used to distinguish patients who are suitable for active surveillance from those who require aggressive treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3637. doi:1538-7445.AM2012-3637

Abstract 2871: Inhibition of androgen receptor-mediated expression of prostate derived ets transcription factor (PDEF) induces a loss of prostate cancer cell differentiation

Abstract While inhibition of androgen signaling has shown to extend the lives of many patients, there is a growing concern in the field that androgen ablation may select for a more aggressive and lethal phenotype of prostate cancer. It has long been known that androgen receptor (AR) mediates growth and cellular differentiation. Therefore, inhibition of AR may stem cancer growth, but concurrently cause cells to de-differentiate and gain migratory and invasive properties. Prostate derived ets transcription factor (PDEF) is an AR target gene highly expressed in normal prostate tissue and has shown to be sufficient to mediate terminal epithelial cellular differentiation in the lung and colon. Previous studies in our lab have demonstrated, through retrospective IHC staining of FFPE radical prostatectomy specimens, loss of PDEF expression significantly associated with increased incidence and patients’ shorter metastatic free survival time with early stage prostate cancer. Furthermore, previous studies in the literature have detected the PDEF promoter sequence through AR ChIP-Sequencing and ChIP-on-ChIP experiments. We therefore studied the regulation of PDEF expression by AR as a potential mediator of prostate epithelial cellular differentiation. Our data indicated that anti-androgens, androgen starvation or AR shRNA was sufficient to abrogate PDEF protein and mRNA expression. Stable expression of AR shRNA or PDEF shRNA by lentiviral infection displayed a coordinate decrease in e-cadherin expression and an increase in vimentin expression, indicating a potential shift to a mesenchymal-like phenotype. This epithelial-to-mesenchymal transition was also observed in cells acutely treated with anti-androgens bicalutamide or finasteride. Together, these results demonstrate that androgen ablation therapy may directly drive cells to de-differentiate and gain migratory/invasive capabilities, and therefore should only be indicated for those patients with a very short life expectancy or co-existing morbidities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2871. doi:1538-7445.AM2012-2871

PDEF downregulates stathmin expression in prostate cancer

The Ets proteins are a family of transcription factors characterized by an evolutionarily conserved DNA binding domain that controls key cellular processes. Prostate-derived Ets transcription factor (PDEF), a member of the Ets family, is reported to be present in tissues with high epithelial content, notably breast and prostate. However, the role of PDEF in cancer development is not fully understood. To gain insight into the molecular mechanisms associated with prostate cancer progression, we employed iTRAQ labeling followed by mass spectrometric (MS) analysis to identify candidate proteins that are differentially expressed in prostate cancer cells with or without PDEF. To this end, we overexpressed PDEF in PC3 human prostate cells using a tetracycline inducible system (Tet-On). Many differentially expressed proteins which play important roles in various cellular and biological processes were identified. Among them, stathmin (STMN), which is a microtubule (MT)-destabilizing protein, was found to be downregulated in multiple analyses. We demonstrated that re-expression of STMN reversed the antitumor properties of PDEF in PDEF-overexpressing PC3 cells. Using in vitro functional assays, we showed that STMN overexpression counteracted PDEF's effects against cell proliferation, colony formation and tumor migration. Similar results were further confirmed with the prostate cancer cell line CWR22rv1. In conclusion, many differentially expressed proteins were identified and STMN was found to be downregulated by PDEF. These results suggest that PDEF may inhibit prostate cancer progression by transcriptional downregulation of oncogenic STMN expression. Analyzing the association among differentially expressed proteins may provide a basis to better understand the molecular mechanisms underlying the process of cancer progression and development and further aid in designing therapeutics in the future.

Abstract 4173: Prostate derived Ets (E-twenty six) transcription factor (PDEF) as a prognostic biomarker for metastatic prostate cancer

Abstract While early stage prostate cancer (CaP) is unique in that many patients will take decades to progress to symptomatic disease; there exists a subset of patients whose disease may quickly become life-threatening. Unfortunately, due to the high incidence of CaP and clinicians’ inability to differentiate the majority of patients with disease that will never become symptomatic from the minority with aggressive life-threatening tumors, patients with CaP are often over-treated. Prognostic biomarkers, such as prostate specific antigen (PSA), have shown great efficacy in improving patients’ chances of surviving cancer by allowing physicians to identify the disease at an earlier stage, though there does not exist a biomarker for CaP that adequately differentiates disease that will quickly become metastatic. In order to facilitate prognosis, the expression of prostate derived Ets transcription factor (PDEF), a protein of significance to the progression of CaP, was studied. PDEF is expressed in normal epithelial cells and negatively regulates transcriptional networks controlling migration and invasion. Previous findings in breast cancer have also demonstrated that loss of PDEF expression results in upregulation of the antiapoptotic protein survivin, indicating a possible connection to growth and survival. Loss of PDEF expression has also been shown to increase the metastatic potential of several cancers. Patients PDEF expression was determined via immunohistochemistry staining of tissue microarray in set of over 700 archival CaP tissues taken at radical prostatectomy; and patients were subsequently stratified based on their tumor PDEF expression. The stratified CaP patients were then analyzed for the association of PDEF expression status with clinical outcomes. Univariate analysis showed that low PDEF expression is correlated with incidence of biochemical failure as defined by American Urological Association (AUA; OR=1.30, p=0.0493) or National Comprehensive Cancer Network (NCCN; OR=1.36, p=0.0164) definitions; and with metastatic disease incidence (OR=3.61, p=0.0047). Multivariate analysis also showed that low PDEF expression was associated with metastatic disease incidence (OR = 4.02, p=0.0146). Furthermore, Kaplan-Meier survival analysis showed that stratification of patients into subgroups based on PDEF expression (high, middle, low) was able to significantly predict relative time to biochemical failure (NCCN, p=0.0241) or metastatic disease (p=0.0084). This study demonstrates the potential usefulness of PDEF as a prognostic biomarker to differentiate those patients at the highest risk of developing life-threatening disease. We propose that the status of PDEF expression in CaP tissues could be used in conjunction with PSA to distinguish patients who are suitable for watchful waiting and patients who require aggressive treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4173. doi:10.1158/1538-7445.AM2011-4173

Prostate‐derived Ets transcription factor (PDEF) is a potential prognostic marker in patients with prostate cancer

Reduced expression of prostate-derived Ets transcription factor (PDEF) leads to morphologic change as well as increased migration and invasiveness of prostate cancer cells. However, the clinical relevance of PDEF expression and its relationship to anti-apoptotic protein survivin is yet to be determined. Tissue microarrays of 73 prostate carcinomas and their adjacent benign prostate tissue, as well as 50 benign prostates were evaluated for PDEF expression by immunohistochemistry. Results were confirmed in available tumor tissues using Western blot and RT-PCR. Expression of survivin in prostate carcinoma and benign tissues were determined using Western blot. Results and correlation with clinical data were statistically analyzed. Patients' specimens with low Gleason scores (GS < 5) expressed higher levels of PDEF protein and lower levels of survivin protein when compared with moderate-to-high GS tumors (GS > 6). Patients with PDEF-positive tumor survived significantly longer (P < 0.0001) than patients with PDEF-negative tumor, and the 8-year survival rate was 94% and 40%, respectively. PDEF expression was detected at the highest levels in benign tissues and was down-regulated or lost in 30 recently diagnosed prostate carcinomas. Re-expression of PDEF in prostate cancer cells inhibited survivin expression. Treatment of prostate cancer cells with methylseleninic acid resulted in restoration of PDEF expression, down-regulation of survivin, and inhibition of tumor cell growth when compared with untreated controls (P < 0.05). These studies demonstrated an inverse correlation between PDEF and survivin expression, and that up-regulation of PDEF was associated with a favorable prognosis in patients with clinically localized prostate cancer.

Proteomic Analysis of a PDEF Ets Transcription Factor-Interacting Protein Complex

Ets proteins are a family of transcription factors that share an 85 amino acid conserved DNA binding domain, the ETS domain. The 27 known human Ets transcription factors control multiple biological processes, including cellular proliferation, differentiation, apoptosis, angiogenesis, transformation, and invasion. Overexpression of some Ets genes has been linked to numerous malignancies, including breast cancers. The prostate derived Ets transcription factor (PDEF) is reported to be a breast and prostate tumor-associated Ets factor. To understand the roles of PDEF in breast cancers, we transiently overexpressed PDEF in MDA-MB-231 human breast cancer cells by adenoviral-mediated gene delivery. PDEF binding protein complexes were isolated by immunoprecipitation and PDEF-interacting proteins were analyzed by LC-MS/MS. After subtracting the proteins binding nonspecifically to antibody-bead complexes, we identified 286 proteins in the PDEF-associated protein complex. By comparison to published protein-protein interactions, we selected 121 proteins for further analysis. PDEF interactors distribute not only in the nucleus, but also in the cytoplasm, as well as other subcellular compartments. Our data reveals that PDEF interacts with a variety of proteins involved in cell cycle, DNA repair, cytoskeleton organization, mRNA processing, tRNA biosynthesis, protein folding, and cell signaling. Furthermore, the EGFR1- (Erbb1) and Erbb2- (HER2) related proteins erbin, an ERBB2 interacting protein, catenin delta-1 (which interacts with Erbin), and EGFR (a HER2-homology receptor) were associated with PDEF. These findings indicate that PDEF may be regulated by Erbb2 or EGFR-activated signaling pathways in breast cancer cells. Further analysis of these proteins will identify the roles of PDEF-interacting proteins in breast tumorigenesis.

Prostate‐derived Ets transcription factor as a favorable prognostic marker in ovarian cancer patients

We have previously shown that ovarian tumors express prostate-derived Ets transcription factor (PDEF). However, the precise role of PDEF in the prognosis of ovarian cancer is unknown. In our study, we report for the first time that expression of PDEF in tumor lesions of patients with ovarian cancer is associated with favorable prognosis. Evaluation of samples from 40 patients with ovarian cancer showed that early stage (IA) and borderline (IIB, III) ovarian tumors expressed higher levels of PDEF mRNA and protein and lower levels of survivin compared to late stage ovarian tumors (IIIC and IV, p < 0.05). Normal ovarian tissues expressed the highest levels of PDEF mRNA and protein when compared to tumor tissues (p < 0.05). A Log-Rank test showed that overall survival of patients with PDEF-positive and survivin-negative ovarian tumors was significantly longer than those with PDEF-negative and survivin-positive tumors (p < 0.01). Forced expression of PDEF in PDEF-negative ovarian tumor cells inhibited tumor cell growth, induced apoptosis, downregulated survivin expression and its promoter activity. Furthermore, treatment of ovarian cancer cells with vitamin D or a selenium compound resulted in re-expression of PDEF, downregulation of survivin, induction of apoptosis and inhibition of tumor cell growth when compared to untreated controls (p < 0.05). Together, these observations showed an inverse correlation between PDEF and survivin expression and suggested that increased PDEF expression along with reduced survivin was associated with prolonged survival of patients with ovarian cancer.

Prostate-Derived Ets Transcription Factor Overexpression is Associated with Nodal Metastasis, Hormone Receptor Positivity in Invasive Breast Cancer

Prostate-derived Ets transcription factor (PDEF) has recently been associated with invasive breast cancer, but no expression profile has been defined in clinical specimens. We undertook a comprehensive PDEF transcriptional expression study of 86 breast cancer clinical specimens, several cell lines, normal tissues. PDEF expression profile was analyzed according to standard clinicopathologic parameters, compared with hormonal receptor, HER-2/neu status, to the expression of the new tumor biomarker Dikkopf-1 (DKK1). Wide ranging PDEF overexpression was observed in 74% of tested tumors, at higher levels than the average expression found in normal breasts. High PDEF expression was associated with hormone receptor positivity (P < .001), moderate to good differentiation (less than grade III, P = .01), dissemination to axillary lymph nodes (P = .002). PDEF was an independent risk factor for nodal involvement (multivariate analysis, odds ratio 1.250, P = .002). It was expressed in a different subgroup compared to DKK1-expressing tumors (P < .001). Our data imply that PDEF mRNA expression could be useful in breast cancer molecular staging. Further insights into PDEF functions at the protein level, possible links with hormone receptors biology, bear great potential for new therapeutic avenues.

Phytoestrogens from Belamcanda chinensis regulate the expression of steroid receptors and related cofactors in LNCaP prostate cancer cells

To investigate the changes in expression underlying the marked reduction of tumour growth in vivo, by analysing the effect of Belamcanda chinensis extract (BCE) on LNCaP cells in vitro, as phytoestrogens are chemopreventive in prostate cancer, and in previous studies we examined the effects of the isoflavone tectorigenin isolated from B. chinensis on LNCaP prostate cancer cells, and a BCE consisting of 13 phytoestrogenic compounds on tumour-bearing nude mice. LNCaP cells were treated with 100, 400 or 1400 microg/mL BCE; proliferation was assessed with an Alamar Blue assay. We used real-time reverse transcription-polymerase chain reaction to quantify mRNA expression of the androgen receptor (AR), the AR coactivator prostate derived Ets transcription factor (PDEF), NKX3.1, prostate specific antigen (PSA) and oestrogen receptor-beta (ER-beta) compared with the expression of the housekeeping gene porphobilinogen deaminase (PBGD). PSA secretion from LNCaP cells was measured and protein expression of the AR investigated by Western blot analysis. Concomitant with a marked decrease of tumour cell proliferation BCE down-regulated the expression of the AR, PDEF, NKX3.1 and PSA. In the same experiments, the expression of PBGD was unaltered, whereas ER-beta expression increased. Furthermore, AR protein and PSA secretion were markedly diminished after treatments with the BCE. BCE, comprising 13 different phytoestrogens, decreases the expression of the AR and its co-activator PDEF concomitant with diminished cell proliferation and PSA secretion. NKX3.1 expression was also reduced by BCE. We hypothesise that the positive effects of BCE are initiated by up-regulation of the ER-beta, a putative tumour-suppressor gene.

Prostate-derived Ets transcription factor (PDEF) downregulates survivin expression and inhibits breast cancer cell growth in vitro and xenograft tumor formation in vivo

Previous studies using immunohistochemistry suggest that loss of the expression of the prostate-derived Ets transcription factor (PDEF) is a strong indicator for cancer cell malignancy. However, the underlying mechanism for this has not been well elucidated. We determined the role of PDEF in breast cancer cell growth and tumor formation using a series of experiments including Western blotting, promoter-luciferase reporter assay, RNA interference technology and a mouse xenograft model. We also determined the relationship between PDEF expression in human breast tumor specimen and cancer patient survivability. These studies revealed that PDEF expression is inversely associated with survivin expression and breast cancer cell xenograft tumor formation. PDEF-specific shRNA-mediated silencing of PDEF expression resulted in the upregulation of survivin expression in MCF-7 cells, which was associated with increased cell growth and resistance to drug-induced DNA fragmentation (apoptosis). In contrast, survivin-specific siRNA-mediated silencing of survivin expression decreased MCF-7 cell growth. Ectopic expression of PDEF inhibited both survivin promoter activity and endogenous survivin expression. Importantly, shRNA-mediated silencing of PDEF expression in MCF-7 breast cancer cells enhanced survivin expression and xenograft tumor formation in vivo. Furthermore, loss of PDEF expression in breast cancer tissues tends to be associated with unfavorable prognosis. These studies provide new information for the role of PDEF and survivin in breast cancer cell growth and tumor formation.

Prostate derived Ets transcription factor shows better tumor-association than other cancer-associated molecules

We previously reported that prostate derived Ets transcription factor (PDEF) is a breast tumor-associated molecule. To obtain further insights into PDEF expression in other human tumor types, a cDNA library database from human adult normal and tumor tissues was compiled and searched for PDEF distribution. The results showed that PDEF is present at relative higher frequencies in the cDNA libraries from brain, breast, lung and ovarian tumors in comparison to those from the corresponding normal tissues. RT/PCR analysis of PDEF expression in ovarian tumors confirmed that PDEF is expressed in 36 out of 51 (71%) ovarian tumors. Further comparison of the distribution of PDEF with other widely recognized cancer-associated molecules showed that PDEF has more restricted distributions than Her-2/neu, Bcl-2, survivin or telomerase in cDNA libraries from normal human tissues and more increased distribution than Her-2/neu, CA-125, Bcl-2, survivin and telomerase in cDNA libraries from brain (except survivin), breast, lung and ovarian tumors. These data together show a better tumor-association for PDEF and suggest that PDEF is a more suitable target for developing specific cancer therapies.

Expressional changes after histone deacetylase inhibition by valproic acid in LNCaP human prostate cancer cells

Pathogenesis of prostate cancer is paralleled by aberrant transcriptional regulation which involves gene silencing by histone deacetylases. In cancer cells, inhibitors of histone deacetylases such as valproic acid can act as differentiation agents which relieve pro-apoptotic factors from transcriptional repression. We investigated the potential of the well-tolerated anticonvulsant valproic acid in prostate cancer cell line LNCaP and analyzed the activation of pro-apoptotic factors and resulting apoptosis. We used real time RT-PCR to quantify the mRNA expression of prostate-specific antigen, prostate-derived Ets transcription factor, tissue inhibitor of matrix metalloproteinase-3 and insulin-like growth factor binding protein-3. An automated sandwich-ELISA was used to measure secretion of prostate-specific antigen in conditioned cell culture media of LNCaP prostate cancer cells. Apoptotic cells were detected cytochemically and by applying immunocytochemistry. Activity of histone deacetylases in nuclear extracts was measured with a colorimetric assay kit. Valproic acid treatment caused a marked inhibition of histone deacetylases activity. Expression of prostate-derived Ets transcription factor and consequently prostate-specific antigen were down-regulated to basal levels in LNCaP cells. Pro-apoptotic factor caspase-3, tissue inhibitor of matrix metalloproteinase-3 and insulin-like growth factor binding protein-3 were up-regulated resulting in apoptosis of tumor cells. Valproic acid mediates marked effects on the expression of genes relevant in proliferation and apoptosis. Our study provides strong evidence that prostate cancer may benefit particularly from anti-proliferative stimuli from this well established drug.