Malignant Prostate Cells Research Articles

Abstract 2176: Deletion of 3p14 is linked to ERG-fusion positive prostate cancers and harbors the FOXP1 tumor suppressor gene

Abstract Genetic rearrangements involving 3p14 are frequent alterations in prostate cancer, affecting approximately 15-20% of tumors. Both the clinical significance and the tumor suppressor at 3p14 locus are currently unknown. We performed SNP array analysis of 72 primary prostate cancers and show a commonly deleted region at 3p14 in 14 (19%) of the tumors. Our data indicate the existence of a small, recurrent deletion in a significant fraction of prostate cancers harboring 8 genes only. Ten of 14 tumors with 3p14 deletions detected in our study included the FOXP1 locus. To estimate the prevalence and clinical significance of FOXP1 deletions, we analyzed a tissue microarray containing 4,699 prostate cancers with histopathological and clinical follow-up data. Heterozygous FOXP1 deletions were found in 15% of tumors, and were strongly associated with ERG-fusion positive cancers (p<0.0001). No homozygous deletions were detected. FOXP1 deletions were unrelated to tumor stage or Gleason grade. FOXP1 deletions were linked to early PSA recurrence in ERG-positive cancers (p=0.0576) but not in ERG negative tumors (p=0.5425). To better understand the association between ERG fusion and FOXP1 expression, we performed FOXP1 immunohistochemistry on our TMA. FOXP1 expression was found in virtually all (95%) prostate cancers, and was particularly strong in ERG-fusion positive tumors. This finding argues for an important role of FOXP1 in prostate cancer in general and for ERG fusion positive tumors in particular. Since FOXP1 is a transcriptional repressor, we hypothesized that upregulation might be a consequence of ERG activation in order to control transcription of ERG target genes. However, neither expression of ERG in LNCaP cells, or depletion of ERG in VCaP cells had an effect on FOXP1 mRNA levels, strongly arguing against a direct link between ERG and FOXP1 at least in the tested cell lines. In addition neither the transcription nor the steady state levels of FOXP1 protein were modulated by activation of AR signaling. Finally, colony formation assays in both benign and malignant prostate cell lines revealed that FOXP1 may act as a tumor suppressor. In summary, our study demonstrates that FOXP1 functions as one of the tumor suppressor genes in prostate cancer that is targeted by genomic deletions at 3p14. 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 2176. doi:1538-7445.AM2012-2176

796 CORTISOL SERUM LEVELS ARE INCREASED AND INDUCE GENOMIC INSTABILITY IN ADVANCED PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Cortisol, also known as stress hormone, is involved with chronic stress; it appears to be associated with higher levels of prostate-specific antigen (PSA) and benign prostate enlargement (BPE). To assess the relationship between cortisol and prostate cancer (PCa), serum cortisol levels were measured in normal men and those with primary or metastatic PCa. In addition, the effect of cortisol on genomic instability was investigated by evaluating DNA mismatch repair (MMR) pathway in androgen-deprived benign or malignant prostate cells. METHODS: Serum cortisol and creatinine levels were determined using ELISA assay. Immunohistochemical staining for MMR proteins was performed on tissue microarray slides. Genomic instability was evaluated by analyzing the microsatellite instability (MSI) using PCR and the National Cancer Institute (NCI)-recommended panel of five MS markers including two mononucleotide repeat (BAT25 and BAT26) and three dinucleotide repeats (D2S123, D5S346, and D17S250). Genomic DNAs for PCR were extracted from AR-positive (LNCaP, E006AA, CWR-22RV1, C4-2B, MDA-PCa2b) and androgeninsensitive (PC-3 and DU145) PCa cell lines and normal prostate epithelial cells that were treated with cortisol and androgen-deprived for 2 to 20 weeks. Purified PCR fragments were separated on ABI 3730XL DNA Analyzer and genotyped using the ABI Gene Mapper ID, V3.2 software. MMR genes and protein expression levels in the cells were examined with qRT-PCR and western blotting, respectively. RESULTS: Serum cortisol levels were in rank order metastatic castrate-resistant prostate cancer (mCRPCa) primary PCa BPE. Serum cortisol levels were higher in African Americans (AAs) than Caucasian Americans (CAs) in BPE, primary PCa, and mCRPCa. Chronic treatment of PCa cells with cortisol increased growth rate and PSA expression. Cortisol induced high-frequency MSI as evidenced by the insertion or deletion of 1-5 bps in at least 2 of 5 MS markers in both androgen-responsive and androgen-insensitive PCa cell lines. Analysis of the DNA mismatch repair pathway in PCa cells and tissues originated from AAs and CAs showed alterations in the expression levels of PMS1, PMS2, MLH1, MSH2, MSH3, MSH6, that belong to the MMR system. CONCLUSIONS: These data support a non-immunological role for cortisol in promoting genomic instability in androgen-deprived PCa cells that might lead to hyper-mutator phenotype or accumulation of adoptive-genetic changes in response to the stress that results from androgen-deprivation therapy for advanced PCa.

Abstract B62: Protein inhibitor of activated STAT1 (PIAS1) is expressed in prostate cancer and stimulates cellular proliferation through inhibition of p21

Abstract Introduction: PIAS (Protein Inhibitors of Activated STAT) proteins have originally been identified as inhibitors of the JAK/STAT pathway but they have a wide role in the regulation of transcription. Besides their DNA binding ability they also act as SUMO-E3 ligases, thus interacting with and modulating the activity of various proteins. Whilst PIAS proteins have previously been reported to regulate androgen receptor activity in prostate cancer, studies that systematically address PIAS protein expression patterns and their role in the regulation of cell growth and cell death in cancer have not been performed yet. The aim of this study is to investigate both in vivo and in vitro expression patterns of PIAS1 in prostate specimens and cell lines and to elucidate the functional role of PIAS1 in the regulation of proliferation, cell cycle progression, and apoptosis. Experimental procedures: In vivo expression of PIAS1 was determined by immunohistochemical analysis of a tissue microarray of benign and malignant human prostate cancer samples (n=90). For in vitro studies, the expression levels of mRNA and protein were determined by Western Blot and Real Time PCR. Localization studies were performed by immunofluorescence and cell fractionation. For down-regulation of PIAS1, cells were transfected with 25nM of siRNA. For overexpression of PIAS1, cells were transfected with 3μg of pEGFP-PIAS1. Proliferation was measured using [3H] thymidine incorporation assay. Apoptosis and cell cycle analysis were determined by FACS after propidium iodide staining. Clonogenicity was determined by a 2-D colony formation assay. Results: Tissue microarray data reveal that PIAS1 expression is significantly higher in malignant areas in comparison to benign tissue in clinical prostate cancer specimens. Furthermore, the staining pattern of PIAS1 correlates with the staining patterns of the proliferation markers PCNA and Ki67, thus suggesting a pro-proliferative role for PIAS1. Most benign and malignant prostate cell lines express PIAS1 mRNA and protein. PIAS1 predominantly shows nuclear localisation. Downregulation of PIAS1 leads to a ∼50% decrease in cell proliferation and clonogenicity after 96 h. At the same time, PIAS1 depletion does not significantly influence apoptosis. Decreased proliferation correlates with an increased nuclear expression of the cell-cycle regulator p21 at mRNA and protein level (up to 6-fold). Furthermore, PIAS1 down-regulation leads to a significant increase of the percentage of cells in G1-phase and a decrease of cells in S-phase of the cell cycle. Overexpression of PIAS1 decreases expression of p21, leading to a significant increase of cells in S-phase, and subsequently to an accelerated proliferation rate. Conclusion: Our data indicate that PIAS1 has pro-proliferative effects in prostate cancer cell lines and tissue. We demonstrate that PIAS1 influences cell cycle progression via the regulation of p21 expression and thereby affects cell proliferation. Our results clearly show that PIAS1 may be a new target for improved prostate cancer therapy. Citation Format: Julia Hoefer, Martin Puhr, Georg Schaefer, Helmut Klocker, Holger Erb, Ian G. Mills, Ludger Hengst, Zoran Culig. Protein inhibitor of activated STAT1 (PIAS1) is expressed in prostate cancer and stimulates cellular proliferation through inhibition of p21 [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B62.

Prostasomes are mediators of intercellular communication: from basic research to clinical implications

Prostasomes are nanosized microvesicles secreted by acinar epithelial cells of the prostate gland. Furthermore, they are intracellular microvesicles inside another larger vesicle, a so-called storage vesicle, equivalent to multivesicular bodies of late endosomal origin. Prostasomes are thought to play an important role in intercellular communication by direct interaction primarily between the immobile acinar cells of the prostate gland and the mobile spermatozoa. Prostasomes transfer not only membrane components but also genetic material to spermatozoa. They are rich in various transferable bioactive molecules (e.g., receptors and enzymes) that promote the fertilizing ability of spermatozoa. In this review, the pleiotropic biological effects of prostasomes that are relevant for successful fertilization will be discussed. The ability to synthesize and export prostasomes to the extracellular space is observed not only in normal prostate epithelial cells but also in malignant prostate cells. Release of prostasomes by prostate cancer cells suggests a role in malignant cell growth and proliferation. These findings may provide new therapeutic and diagnostic strategies.

Deacetylation of FOXO3 by SIRT1 or SIRT2 leads to Skp2-mediated FOXO3 ubiquitination and degradation

Sirtuin deacetylases and FOXO (Forkhead box, class O) transcription factors have important roles in many biological pathways, including cancer development. SIRT1 and SIRT2 deacetylate FOXO factors to regulate FOXO function. Because acetylation and ubiquitination both modify the ɛ-amino group of lysine residues, we investigated whether FOXO3 deacetylation by SIRT1 or SIRT2 facilitates FOXO3 ubiquitination and subsequent proteasomal degradation. We found that SIRT1 and SIRT2 promote FOXO3 poly-ubiquitination and degradation. Proteasome-inhibitor treatment prevented sirtuin-induced FOXO3 degradation, indicating that this process is proteasome dependent. In addition, we demonstrated that E3 ubiquitin ligase subunit Skp2 binds preferentially to deacetylated FOXO3. Overexpression of Skp2 caused poly-ubiquitination of FOXO3 and degradation, whereas knockdown of Skp2 increased the amount of FOXO3 protein. We also present evidence that SCF-Skp2 ubiquitinates FOXO3 directly in vitro. Furthermore, mutating four known acetylated lysine residues (K242, K259, K290 and K569) of FOXO3 into arginines to mimic deacetylated FOXO3 resulted in enhanced Skp2 binding but with inhibition of FOXO3 ubiquitination; this suggests that some or all of these four lysine residues are likely the sites for ubiquitination. In the livers of mice deficient in SIRT1, we detected increased expression of FOXO3, indicating SIRT1 regulates FOXO3 protein levels in vivo. Furthermore, we found that the elevation of SIRT1 and Skp2 expression in malignant PC3 and DU145 prostate cells is responsible for the downregulation of FOXO3 protein levels in these cells. Taken together, our data support the notion that deacetylation of FOXO3 by SIRT1 or SIRT2 facilitates Skp2-mediated FOXO3 poly-ubiquitination and proteasomal degradation.

Chromosome-wide mapping of DNA methylation patterns in normal and malignant prostate cells reveals pervasive methylation of gene-associated and conserved intergenic sequences

BackgroundDNA methylation has been linked to genome regulation and dysregulation in health and disease respectively, and methods for characterizing genomic DNA methylation patterns are rapidly emerging. We have developed/refined methods for enrichment of methylated genomic fragments using the methyl-binding domain of the human MBD2 protein (MBD2-MBD) followed by analysis with high-density tiling microarrays. This MBD-chip approach was used to characterize DNA methylation patterns across all non-repetitive sequences of human chromosomes 21 and 22 at high-resolution in normal and malignant prostate cells.ResultsExamining this data using computational methods that were designed specifically for DNA methylation tiling array data revealed widespread methylation of both gene promoter and non-promoter regions in cancer and normal cells. In addition to identifying several novel cancer hypermethylated 5' gene upstream regions that mediated epigenetic gene silencing, we also found several hypermethylated 3' gene downstream, intragenic and intergenic regions. The hypermethylated intragenic regions were highly enriched for overlap with intron-exon boundaries, suggesting a possible role in regulation of alternative transcriptional start sites, exon usage and/or splicing. The hypermethylated intergenic regions showed significant enrichment for conservation across vertebrate species. A sampling of these newly identified promoter (ADAMTS1 and SCARF2 genes) and non-promoter (downstream or within DSCR9, C21orf57 and HLCS genes) hypermethylated regions were effective in distinguishing malignant from normal prostate tissues and/or cell lines.ConclusionsComparison of chromosome-wide DNA methylation patterns in normal and malignant prostate cells revealed significant methylation of gene-proximal and conserved intergenic sequences. Such analyses can be easily extended for genome-wide methylation analysis in health and disease.

Differing levels of testosterone and the prostate: a physiological interplay

The controversies surrounding testosterone replacement therapy (TRT) have been addressed in the past few years. Although the androgenic effects of TRT on normal and malignant prostate cells have been studied for over 70 years, little clinical prospective research exists on the physiological responses of prostate tissues to a wide range of serum testosterone levels. The prostate is both an androgen-dependent and an androgen-sensitive organ; active processes are triggered at a 'threshold' or 'saturation' level of testosterone. Despite decades of research, no compelling evidence exists that increasing testosterone beyond this threshold level has a causative role in prostate cancer, or indeed changes the biology of the prostate. Testosterone deficiency has marked physiological and clinical effects on men in middle age and beyond. With subnormal testosterone levels, the potential positive benefits of TRT on factors such as muscle mass, libido or erectile function are likely a dose-response phenomenon, and should be considered differently than the threshold influence on the prostate. This Review will re-examine classic androgen research and reflect on whether testosterone actually stimulates prostatic cellular growth and progression in a 'threshold' or a 'dose-response' (or both) manner, as well as discuss the influence of testosterone on prostate cells in the hypogonadal and eugonadal states.

Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer

Prostasomes are microvesicles (mean diameter, 150 nm) that are produced and secreted by normal and malignant prostate acinar cells. It has been hypothesized that invasive growth of malignant prostate cells may cause these microvesicles, normally released into seminal fluid, to appear in interstitial space and therewith into peripheral circulation. The suitability of prostasomes as blood biomarkers in patients with prostate cancer was tested by using an expanded variant of the proximity ligation assay (PLA). We developed an extremely sensitive and specific assay (4PLA) for detection of complex target structures such as microvesicles in which the target is first captured via an immobilized antibody and subsequently detected by using four other antibodies with attached DNA strands. The requirement for coincident binding by five antibodies to generate an amplifiable reporter results in both increased specificity and sensitivity. The assay successfully detected significantly elevated levels of prostasomes in blood samples from patients with prostate cancer before radical prostatectomy, compared with controls and men with benign biopsy results. The medians for prostasome levels in blood plasma of patients with prostate cancer were 2.5 to sevenfold higher compared with control samples in two independent studies, and the assay also distinguished patients with high and medium prostatectomy Gleason scores (8/9 and 7, respectively) from those with low score (≤ 6), thus reflecting disease aggressiveness. This approach that enables detection of prostasomes in peripheral blood may be useful for early diagnosis and assessment of prognosis in organ-confined prostate cancer.

Androgen receptor: acting in the three-dimensional chromatin landscape of prostate cancer cells.

Androgen receptor (AR) acts as a hormone-controlled transcription factor that conveys the messages of both natural and synthetic androgens to the level of genes and gene programs. Defective AR signaling leads to a wide array of androgen insensitivity disorders, and deregulated AR function, in particular overexpression of AR, is involved in the growth and progression of prostate cancer. Classic models of AR action view AR-binding sites as upstream regulatory elements in gene promoters or their proximity. However, recent wider genomic screens indicate that AR target genes are commonly activated through very distal chromatin-binding sites. This highlights the importance of long-range chromatin regulation of transcription by the AR, shifting the focus from the linear gene models to three-dimensional models of AR target genes and gene programs. The capability of AR to regulate promoters from long distances in the chromatin is particularly important when evaluating the role of AR in the regulation of genes in malignant prostate cells that frequently show striking genomic aberrations, especially gene fusions. Therefore, in addition to the mechanisms of DNA loop formation between the enhancer bound ARs and the transcription apparatus at the target core promoter, the mechanisms insulating distally bound ARs from promiscuously making contacts and activating other than their normal target gene promoters are critical for proper physiological regulation and thus currently under intense investigation. This review discusses the current knowledge about the AR action in the context of gene aberrations and the three-dimensional chromatin landscape of prostate cancer cells.

Highlights of This Issue

Pioneer work showed Lin28a/28b targeting of pre-let-7g for degradation plays an important role in dysregulation of miRNA expression in cancer. Wang and colleagues discovered that the ribosomal protein RPS2 is aberrantly overexpressed in prostate cancer cells and specifically binds the stem loop domain (i.e., UAGGGUCAC) of pre-let-7a-1, blocking pre-let-7a-1 processing to let-7a/let-7f. Consequently, RPS2 promoted ras/myc expression to increase the tumorigenesis of primary and malignant prostate cells in vitro and in vivo. The overexpression of let-7a/7f in pBABE.pre-let-7a transfected PC-3ML variants blocked tumorigenesis, suggesting that RPS2 functions in a negative-feedback loop, which downregulates let-7a/7f to promote oncogene-dependent tumorigenesis.Activation of epithelial-to-mesenchymal transition (EMT) in cancer stem cells (CSCs) was implicated in the metastasis of various human tumors. Gavert and colleagues investigated the necessity for changes in EMT and CSC markers during L1-mediated metastasis of colorectal cancer (CRC) cells. The elevation or suppression of L1 levels had no effect on EMT and CSC markers in CRC cells and tissue. When EMT was induced in CRC cells by Slug or Twist overexpression, this was insufficient (unlike L1 overexpression) to cause metastasis. NF-κB signaling was essential for L1-mediated metastasis of CRC cells implying its critical role in human CRC progression.MYC oncoproteins are a family of transcription factors involved in the control of pivotal processes such as cell cycle progression, differentiation, carcinogenesis, metabolism, and angiogenesis. Although deregulation of their expression was shown to be critical in several human neoplasia, MYC overexpression might also lead to apoptosis. By gain and loss of function experiments, Petroni and coworkers show that sensitization to apoptosis by MYCN requires p53, its phosphorylation at serine 46, and the induction of the p53 proapoptotic kinase HIPK2. MYCN leads to a nontranscriptional HIPK2 accumulation by inhibiting its degradation via an ATM-dependent DNA damage response. Importantly, the HIPK2-p53 pro-apoptotic pathway is conserved in a large number of MYCN-amplified human neuroblastomas, where it can be targeted by the p53 reactivating compound Nutlin-3. Indeed, Nutlin-3 further induces HIPK2 accumulation, p53S46 phosphorylation, and a dramatic level of apoptosis, in MYCN-amplified neuroblastoma cells, suggesting its possible use in the treatment of an otherwise largely untreatable neoplastic disease.The transmembrane adaptor Cbp/PAG1 is a suppressor for c-Src-induced cell transformation. Kanou and colleagues examined its relevance to human lung cancers. Cbp was widely downregulated in non-small cell lung cancer (NSCLC) cells. Re-expression of Cbp suppressed growth, invasion, and metastasis of NSCLC cells, particularly in which c-Src is upregulated. Furthermore, there was an inverse correlation between Cbp levels and the ability of lymph node metastasis in lung cancers. These results indicate that Cbp acts as a suppressor for tumor progression in a subset of NSCLC. Thus, mechanistic analysis of Cbp downregulation would offer new opportunities for therapeutic intervention in NSCLC.

Expression and shedding of endothelial protein C receptor in prostate cancer cells

BackgroundIncreasing evidences show that beyond its role in coagulation, endothelial protein C receptor (EPCR) interferes with carcinogenesis. Pro-carcinogenic effects of EPCR were linked with a raised generation of activated protein C (aPC) and anti-apoptotic signalling. This study was carried out to analyze the expression, cell surface exposition, and shedding of EPCR in normal and malignant prostate cell lines.ResultsEPCR expression is up-regulated both at the mRNA and protein levels in invasive prostate DU-145 and PC-3 cells in comparison to normal prostate epithelial cells (PrEC) and less-invasive LNCaP cells. Release of soluble EPCR (sEPCR) is induced by 12-myristate 13-acetate, ionomycin, H2O2, and disruptor of lipid rafts in PrEC, DU-145, and PC-3 cells. Furthermore, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), but not interleukin-6 or interferon-γ increase sEPCR release. In LNCaP cells, neither pharmacological agents nor IL-1β or TNF-α result in a significant increase of sEPCR release. The effects of IL-1β and TNF-α on EPCR shedding in DU-145 cells are mediated by MEK/ERK 1/2, JNK, and p38 MAPK signalling cascades. In PC-3 cells, however, the MEK/ERK 1/2 pathway is down-regulated and incubation with cytokines did not elevate the phosphorylated ERK-1/2 fraction as in the case of DU-145 cells. Treatment with 4-aminophenylmercuric acetate (APMA), an activator of metalloproteases, causes a disproportionately large increase of sEPCR release in DU-145 and PC-3 cells, compared to PrEC and LNCaP cells. Finally, an increased release of sEPCR mediated by APMA treatment is shown to be connected with reduced generation of activated protein C indicating the functionality of EPCR in these cells.ConclusionsThe study demonstrates a number of substantial differences in expression and shedding of EPCR in prostate cancer cell lines in comparison with normal cells that may be relevant for understanding the role of this receptor in carcinogenesis.

HOXC8 Inhibits Androgen Receptor Signaling in Human Prostate Cancer Cells by Inhibiting SRC-3 Recruitment to Direct Androgen Target Genes

HOX (homeobox) genes encode homeodomain-containing transcription factors critical to development, differentiation, and homeostasis. Their dysregulation has been implicated in a variety of cancers. Previously, we showed that a subset of genes of the HOXC cluster is upregulated in primary prostate tumors, lymph node metastases, and malignant prostate cell lines. In the present study, we show that HOXC8 inhibits androgen receptor (AR)-mediated gene induction in LNCaP prostate cancer cells and HPr-1 AR, a nontumorigenic prostate epithelial cell line. Mechanistically, HOXC8 blocks the AR-dependent recruitment of the steroid receptor coactivators steroid receptor coactivator-3 (SRC-3), and CREB binding protein to the androgen-regulated prostate-specific antigen gene enhancer and inhibits histone acetylation of androgen-regulated genes. Inhibition of androgen induction by HOXC8 is reversed upon expression of SRC-3, a member of the SRC/p160 steroid receptor cofactor family. Coimmunoprecipitation studies show that HOXC8 expression inhibits the hormone-dependent interaction of AR and SRC-3. Finally, HOXC8 expression increases invasion in HPr-1 AR nontumorigenic cells. These data suggest a complex role for HOXC8 in prostate cancer, promoting invasiveness while inhibiting AR-mediated gene induction at androgen response element-regulated genes associated with differentiated function of the prostate. A greater understanding of HOXC8 actions in the prostate and its interactions with androgen signaling pathways may elucidate mechanisms driving the onset and progression of prostate cancer.

Overexpression of Transforming Growth Factor β1 in Malignant Prostate Cells is Partly Caused by a Runaway of TGF-β1 Auto-induction Mediated Through a Defective Recruitment of Protein Phosphatase 2A by TGF-β Type I Receptor

To elucidate the mechanism of transforming growth factor (TGF)-β1 overexpression in prostate cancer cells. Malignant (PC3, DU145) and benign (RWPE1, BPH1) prostate epithelial cells were used. Phosphatase activity was measured using a commercial kit. Recruitment of the regulatory subunit, Bα, of protein phosphatase 2A (PP2A-Bα) by TGF-β type I receptor (TβRI) was monitored by coimmunoprecipitation. Blockade of TGF-β1 signaling in cells was accomplished either by using TGF-β-neutralizing monoclonal antibody or by transduction of a dominant negative TGF-β type II receptor retroviral vector. Basal levels of TGF-β1 in malignant cells were significantly higher than those in benign cells. Blockade of TGF-β signaling resulted in a significant decrease in TGF-β1 expression in malignant cells, but not in benign cells. Upon TGF-β1 treatment (10 ng/mL), TGF-β1 expression was increased in malignant cells, but not in benign cells. This differential TGF-β1 auto-induction between benign and malignant cells correlated with differential activation of extracellular signal-regulated kinase (ERK). Following TGF-β1 treatment, the activity of serine/threonine phosphatase and recruitment of PP2A-Bα by TβRI increased in benign cells, but not in malignant cells. Inhibition of PP2A in benign cells resulted in an increase in ERK activation and in TGF-β1 auto-induction after TGF-β1 (10 ng/mL) treatment. These results suggest that TGF-β1 overexpression in malignant cells is caused, at least in part, by a runaway of TGF-β1 auto-induction through ERK activation because of a defective recruitment of PP2A-Bα by TβRI.

The hydroxyl functional group of N-(4-hydroxyphenyl)retinamide mediates cellular uptake and cytotoxicity in premalignant and malignant human epithelial cells

In a previous study, we demonstrated that the anticancer synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) redox cycles at the mitochondrial enzyme dihydroorotate dehydrogenase to trigger anomalous reactive oxygen species (ROS) production and attendant apoptosis in transformed human epithelial cells. Furthermore, we speculated that the hydroxyl functional group of 4HPR was required for this pro-oxidant property. In this study, we investigated the role of the hydroxyl functional group in the in vitro cytotoxicity of 4HPR. Using 4HPR, its primary in vivo metabolite N-(4-methoxyphenyl)retinamide (4MPR), and the synthetic derivative N-(4-trifluoromethylphenyl)retinamide (4TPR), we examined the pro-oxidant and apoptotic effects, as well as the cellular uptake, of these three N-(4-substituted-phenyl)retinamides in premalignant and malignant human skin, prostate, and breast epithelial cells. Compared to 4HPR, both 4MPR and 4TPR were ineffective in promoting conspicuous cellular ROS production, mitochondrial disruption, or DNA fragmentation in these transformed cells. Interestingly, both 4MPR and 4TPR were not particularly cell permeative relative to 4HPR in skin or breast epithelial cells, which implied an additional role for the hydroxyl functional group in the cellular uptake of 4HPR. Moreover, the short-term uptake of 4HPR was directly proportional to cell size, but this characteristic, in obvious contrast to cellular bioenergetic status and/or dihydroorotate dehydrogenase expression, was not fundamentally influential in the overall sensitivity to the promotion of cellular ROS production and apoptosis induction by this agent. Together, these results strongly implicate the hydroxyl functional group in the cytotoxic effects of 4HPR.

Vitamin D pathway gene variants and prostate cancer prognosis

Observational studies linking vitamin D deficiency with increased prostate cancer (PCa) mortality and the pleiotropic anticancer effects of vitamin D in malignant prostate cell lines have initiated trials examining potential therapeutic benefits of vitamin D metabolites. There have been some successes but efforts have been hindered by risk of inducing hypercalcemia. A limited number of studies have investigated associations between variants in vitamin D pathway genes with aggressive forms of PCa. Increased understanding of relevant germline genetic variation with disease outcome could aid in the development of vitamin-D-based therapies. We undertook a comprehensive analysis of 48 tagging single-nucleotide polymorphisms (tagSNPs) in genes encoding for vitamin D receptor (VDR), vitamin D activating enzyme 1-alpha-hydroxylase (CYP27B1), and deactivating enzyme 24-hydroxylase (CYP24A1) in a cohort of 1,294 Caucasian cases with an average of 8 years of follow-up. Disease recurrence/progression and PCa-specific mortality risks were estimated using adjusted Cox proportional hazards regression. There were 139 cases with recurrence/progression events and 57 cases who died of PCa. Significantly altered risks of recurrence/progression were observed in relation to genotype for two VDR tagSNPs (rs6823 and rs2071358) and two CYP24A1 tagSNPs (rs927650 and rs2762939). Three VDR tagSNPs (rs3782905, rs7299460, and rs11168314), one CYP27B1 tagSNP (rs3782130), and five CYP24A1 tagSNPs (rs3787557, rs4809960, rs2296241, rs2585428, and rs6022999) significantly altered risks of PCa death. Genetic variations in vitamin D pathway genes were found to alter both risk of recurrence/progression and PCa-specific mortality.

HIF1α isoforms in benign and malignant prostate tissue and their correlation to neuroendocrine differentiation

BackgroundNeuroendocrine (NE) differentiation in prostate cancer has been correlated with a poor prognosis and hormone refractory disease. In a previous report, we demonstrated the presence of immunoreactive cytoplasmic hypoxia inducible factor 1α (HIF1α), in both benign and malignant NE prostate cells. HIF1α and HIF1β are two subunits of HIF1, a transcription factor important for angiogenesis. The aim of this study was to elucidate whether the cytoplasmic stabilization of HIF1α in androgen independent NE differentiated prostate cancer is due to the presence of certain HIF1α isoforms.MethodsWe studied the HIF1α isoforms present in 8 cases of benign prostate hyperplasia (BPH) and 43 cases of prostate cancer with and without NE differentiation using RT-PCR, sequencing analysis, immunohistochemistry and in situ hybridization.ResultsWe identified multiple isoforms in both benign and malignant prostate tissues. One of these isoforms, HIF1α1.2, which was previously reported to be testis specific, was found in 86% of NE-differentiated prostate tumors, 92% of HIF1α immunoreactive prostate tumors and 100% of cases of benign prostate hyperplasia. Immunohistochemistry and in situ hybridization results showed that this isoform corresponds to the cytoplasmic HIF1α present in androgen-independent NE cells of benign and malignant prostate tissue and co-localizes with immunoreactive cytoplasmic HIF1β.ConclusionOur results indicate that the cytoplasmic stabilization of HIF1α in NE-differentiated cells in benign and malignant prostate tissue is due to presence of an HIF1α isoform, HIF1α1.2. Co-localization of this isoform with HIF1β indicates that the HIF1α1.2 isoform might sequester HIF1β in the cytoplasm.

Abstract 64: Differences in lipogenesis between cancer cells in culture and solid tumors

Abstract Elevated lipogenesis is a characteristic feature of cancer. Fatty acid synthase (FASN) overexpression has been found in many human cancers. Tumor cells in culture and solid tumor models are essential tools to study cancer biology. A comparison of the models can reveal tumor microenvironmental influences on lipogenesis. Here we have characterized differences in lipid components such as total fatty acid (tFA) and phosphatidylcholine (PtCho) levels between tumor cells in culture and solid tumors, with 1H MR spectroscopy (MRS) of lipid-soluble cell or tumor extracts derived from breast and prostate cancer cell lines. MCF-12A, nonmalignant human mammalian epithelial cells, MDA-MB-231, an estrogen receptor (ER)/progesterone receptor (PR)-negative metastatic human breast cancer cell line and MCF-7, an ER/PR-positive poorly metastatic human breast cancer cell line, as well as PC-3 and DU-145, which are both androgen independent malignant human prostate cell lines were used in this study. Fully relaxed 1H MR spectra of lipid-soluble extracts from cells in culture and tumors were acquired on a Bruker Avance 500 MR spectrometer using tetramethylsilane (TMS) as an internal concentration standard. Total FA and PtCho levels were quantified as A.U./cell or A.U./g using integrals of the 1.3 ppm and 3.2 ppm signals in the 1H NMR spectra, respectively, normalized to the cell number or tumor weight (g). Patterns of PtCho and tFA levels were similar in cells, but different in tumors. Both PtCho and tFA levels were significantly higher in MCF-7 cells compared to MCF-12A and MDA-MB-231 cells. Interestingly, the levels of PtCho and tFA in nonmalignant MCF-12A cells and malignant MDA-MB-231 cells were comparable. However, the level of tFA in MDA-MB-231 tumors was significantly higher than in MCF-7 tumors. It has been known that hypoxia and low pH conditions activate FASN activity. FASN or/and other lipogenic enzymes in MDA-MB-231 tumors may be more sensitive to those microenvironmental factors. The tFA level of DU-145 cells was significantly higher compared with PC-3 cells. However, PtCho and tFA levels in PC-3 and DU-145 tumors were not significantly different. FASN protein levels in MCF-12A and MCF-7 cells were not significantly different. Since MCF-12A medium contains epidermal growth factor, which activates FASN, we need to further investigate the involvement of FASN without its activators and with only de novo fatty acid. Our results revealed that tFA levels of cells and tumors do not show similar patterns. The differences between cells and tumors can arise from environmental factors found in solid tumors such as depletion of nutrients and oxygen, changes in pH, as well as cancer cell and stromal/endothelial cell interactions. Additional studies are necessary to unravel the tumor microenvironmental factors responsible for the differences in lipid metabolism observed between cells grown in culture as compared to the same cells grown as solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 64.

Abstract 2591: Differential oncolysis by vesicular stomatitis virus in prostate cancers arising in transgenic mice

Abstract Prostate cancer (PCa) is the most prevalent malignancy and the second leading cause of cancer related mortality in North American men. Survival prospects for patients with locally advanced or metastatic prostate cancer are low and the side effects associated with current therapies seriously compromise quality of life in PCa patients. Oncolytic viral therapy provides an opportunity to efficiently target and kill primary and metastatic cancer cells while sparing normal cells. Vesicular Stomatitis Virus (VSV) is an oncolytic virus which can infect and kill cells that have defects in their cellular anti-viral immunity such as the interferon (IFN) response pathway. In this study VSV (AV3) strain with enhanced IFN sensitivity was injected intra-prostatically into two different transgenic mouse models (prostate specific PTEN null mice and transgenic adenocarcinoma mice [TRAMP]) and monitored for tissue bioluminescence over 96 h. While the virus readily dispersed throughout the body of each group after only 3 h, it persisted >72 h in prostates of tumor bearing mice compared to low levels ∼48 h in control mice. Plaque assay provided a similar pattern, with much higher concentrations of replicating virus in tumor bearing transgenic mice than in control. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis of paraffin embedded tissues demonstrated that VSV(AV3) is capable of selectively infecting and killing malignant prostate cells while sparing normal cells, specifically at the 48h time point. This cell death is due to disrupted IFN response in the PTEN null prostates. Moreover, > 60 % of TRAMP mice treated with VSV(AV3) did not present any metastasis while approximately 70% of TRAMP mice treated with inactivated virus presented metastatic lesions either in lymph nodes or lungs. In summary, our data demonstrates that VSV(AV3) may be used as a potential oncolytic viral therapy to target prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2591.

Abstract 5758: Deletions of olfactomedin 4 gene is associated with progression of prostate cancer

Abstract The human olfactomedin 4 gene (OLFM4) encodes an olfactomedin-related glycoprotein, which our group first cloned and characterized in myeloid cells and mapped to chromosome 13q14.3. In a subsequent study, we have found that OLFM4 was expressed in normal prostate tissue, but its expression was lost in 10.9% (6/55) of well-differentiated prostate caner (Gleason score 4-6), 22.2% (8/36) of intermediately differentiated prostate cancer (Gleason score 7) and 58.8% (40/68) of advanced prostate cancer tissues (Gleason score 8-10). The loss of expression of OLFM4 was significantly associated with a higher Gleason score for prostate cancer (p<0.001). To study the mechanisms of loss of expression of OLMF4 gene in prostate cancer, we investigated alterations in the genetics and epigenetics of the OLFM4 gene in human prostate cancer patients. We looked for deletions and mutations of the OLFM4 gene and the status of methylation of the OLFM4 promoter region in benign and malignant prostate cell lines and in the paired normal and cancerous prostate epithelial cells that were obtained from whole mount prostate tissues by laser capture microdissection (LCM). The results showed that deletions and mutations of the OLFM4 gene is the primary cause of loss of expression of OLFM4 gene in human prostate cancer. We found that 28% (4/14) of primary prostate cancer cells had loss of heterozygosity (LOH) at rs 2298231 of the OLFM4 gene and 14% (2/14) of primary prostate cancer cells revealed deletion in exon 5 of the OLFM4 gene. We also found deletions in the promoter region in tumor cell lines, LNCaP and DU145, as well as exon 5 deletions in DU145 cells. There were no significant differences in the methylation status compared to adjacent normal (n=8), Prostatic Intraepithelial Neoplasia (PIN, n=3), low grade prostate cancer cells (Gleason score 4-7) and high grade prostate cancer cells (Gleason score 8-10, n=7) from the LCM samples studied. To further explore the function of the OLMF4 gene in prostate cancer, we have developed and studied the pathological changes in an OLFM4 knockout mouse model. Histopathological analysis of prostate tissue from OLMF4 wild type mice revealed the following: 5 out of 5 mice were normal at ages of 10 to 12 months, and 1 out of 5 showed prostate dysplastic changes at 18-20 month of age. The prostate from OLFM4 homozygous knock-out (KO) mice revealed that 7 out of 9 mice developed prostatic epithelial hyperplasia, and lower grade prostatic intraepithelial neoplasia (low-grade PIN) in mice at age of 10 to 12 months. By the age of 18 to 20 months, we found that 12 out of 18 KO mice developed high grade prostate intraepithelial neoplasia (high-grade PIN) in which 4 out of 18 developed into prostate cancer and 1 out of 18 showed evidence of lung metastasis. Our results suggest that OLFM4 gene is a candidate for the putative prostate cancer likely tumor suppressor gene located on chromosome 13q and is involved in the progression of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5758.

Abstract 2061: Vitamin D-regulates microRNAs in primary cultures of normal prostatic epithelial cells

Abstract Recent discoveries in cancer find that endogenous non-coding RNAs, microRNAs (miRNA), bind to the 3’ untranslated region of target mRNAs and function to translationally repress or decrease mRNA stability. Additionally, a number of studies show that aberrant miRNA expression is linked to various cancers, such as prostate cancer. Cancer-related changes in miRNA levels may result in either repression of tumor suppressor genes and/or activation of oncogenes. MiRNAs pose a potential role as targets in the treatment of cancers. In prostate cancer research, studies suggest a role of vitamin D as a chemopreventive agent. Vitamin D inhibits the growth and differentiation of malignant prostate cells and tumors. Studies show that a reduction in serum levels of vitamin D metabolites is linked to PC occurrence. Research from our laboratory shows that vitamin D treatment alters mRNA stability. This suggests a potential role for miRNAs in the vitamin D prevention of prostate cancer. We hypothesize that vitamin D's prevention of prostate cancer involves up-regulating tumor suppressive miRNAs and down-regulating oncogenic miRNAs. Using normal human prostatic epithelial cells as a model, we investigated the miRNA expression profile changes in cells that were treated with 1,25-dihydroxyvitamin D for time points ranging from 30 min to 24 hours. Our results demonstrated that approximately 50% of the miRNAs are expressed in the normal epithelial cells. A smaller percentage of miRNAs, approximately 10%, were changed by vitamin D treatment. We found that vitamin D down-regulated a small number of miRNAs (<0.5 fold) commonly up-regulated in cancer (miR-181d, miR-378) and up-regulated miRNAs that are down-regulated in cancer (miR-29a, miR-16). Of the miRNAs altered by vitamin D, we observed an interesting down-regulation of the let-7 family of tumor suppressor miRNAs. Six of the seven let-7 family members present on the array (let-7 a-g) were down-regulated across the time points with four of the let-7 miRNAs downregulated as early as 30 minutes. This is a somewhat discrepant finding since let-7 is known as a tumor suppressor miRNA that is decreased in many cancers and we know that vitamin D is a cancer preventive agent. Further studies will determine the role of vitamin D on let-7 in the prostatic epithelial cells and the implications in cancer. Ongoing research will also determine the mechanism of vitamin D regulation of miRNAs to help further understand the chemopreventive role of vitamin D in prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2061.