Factors In Prostate Cancer Cells Research Articles

A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer

BackgroundmiR-346 was identified as an activator of Androgen Receptor (AR) signalling that associates with DNA damage response (DDR)-linked transcripts in prostate cancer (PC). We sought to delineate the impact of miR-346 on DNA damage, and its potential as a therapeutic agent.MethodsRNA-IP, RNA-seq, RNA-ISH, DNA fibre assays, in vivo xenograft studies and bioinformatics approaches were used alongside a novel method for amplification-free, single nucleotide-resolution genome-wide mapping of DNA breaks (INDUCE-seq).ResultsmiR-346 induces rapid and extensive DNA damage in PC cells - the first report of microRNA-induced DNA damage. Mechanistically, this is achieved through transcriptional hyperactivation, R-loop formation and replication stress, leading to checkpoint activation and cell cycle arrest. miR-346 also interacts with genome-protective lncRNA NORAD to disrupt its interaction with PUM2, leading to PUM2 stabilisation and its increased turnover of DNA damage response (DDR) transcripts. Confirming clinical relevance, NORAD expression and activity strongly correlate with poor PC clinical outcomes and increased DDR in biopsy RNA-seq studies. In contrast, miR-346 is associated with improved PC survival.INDUCE-seq reveals that miR-346-induced DSBs occur preferentially at binding sites of the most highly-transcriptionally active transcription factors in PC cells, including c-Myc, FOXA1, HOXB13, NKX3.1, and importantly, AR, resulting in target transcript downregulation. Further, RNA-seq reveals widespread miR-346 and shNORAD dysregulation of DNA damage, replication and cell cycle processes.NORAD drives target-directed miR decay (TDMD) of miR-346 as a novel genome protection mechanism: NORAD silencing increases mature miR-346 levels by several thousand-fold, and WT but not TDMD-mutant NORAD rescues miR-346-induced DNA damage. Importantly, miR-346 sensitises PC cells to DNA-damaging drugs including PARP inhibitor and chemotherapy, and induces tumour regression as a monotherapy in vivo, indicating that targeting miR-346:NORAD balance is a valid therapeutic strategy.ConclusionsA balancing act between miR-346 and NORAD regulates DNA damage and repair in PC. miR-346 may be particularly effective as a therapeutic in the context of decreased NORAD observed in advanced PC, and in transcriptionally-hyperactive cancer cells.

RANKL immunisation inhibits prostate cancer metastasis by modulating EMT through a RANKL-dependent pathway

Prostate cancer (PCa) morbidity in the majority of patients is due to metastatic events, which are a clinical obstacle. Therefore, a better understanding of the mechanism underlying metastasis is imperative if we are to develop novel therapeutic strategies. Receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL) regulates bone remodelling. Thus, agents that suppress RANKL signalling may be useful pharmacological treatments. Here, we used preclinical experimental models to investigate whether an inactive form of RANKL affects bone metastasis in RANKL-induced PCa. RANKL was associated with epithelial–mesenchymal transition (EMT) and expression of metastasis-related genes in PC3 cells. Therefore, we proposed a strategy to induce anti-cytokine antibodies using mutant RANKL as an immunogen. RANKL promoted migration and invasion of PC3 cells through EMT, and induced a significant increase in binding of β-catenin to TCF-4, an EMT-induced transcription factor in PCa cells, via mitogen-activated protein kinase and β-catenin/TCF-4 signalling. Thus, RANKL increased EMT and the metastatic properties of PC3 cells, suggesting a role as a therapeutic target to prevent PCa metastasis. Treatment with mutant RANKL reduced EMT and metastasis of PC3 PCa cells in an experimental metastasis model. Thus, mutant RANKL could serve as a potential vaccine to prevent and treat metastatic PCa.

SNAIL Transctiption factor in prostate cancer cells promotes neurite outgrowth

Neurite outgrowth involves reciprocal signaling interactions between tumor cells and nerves where invading tumor cells have acquired the ability to respond to pro-invasive signals within the nerve environment. Neurite outgrowth could serve as a mechanism leading to invasion of cancer cells into the nerve sheath and subsequent metastasis. Snail transcription factor can promote migration and invasion of prostate cancer cells. We hypothesized that prostate cancer cell interaction with nerve cells will be mediated by Snail expression within prostate cancer cells. For this study we utilized various prostate cancer cell lines: C4-2 non-silencing (NS, control); C4-2 Snail shRNA, (stable Snail knockdown); LNCaP Neo (empty vector control) and LNCaP Snail (stably over-expressing Snail). Cancer cell adhesion and migration towards nerve cells (snF96.2 or NS20Y) was examined by co-culture assays. Conditioned media (CM) collected from C4-2 cells was cultured with nerve cells (PC-12 or NS20Y) for 48 h followed by qualitative or quantitative neurite outgrowth assay. Our results showed that cancer cells expressing high levels of Snail (LNCaP Snail/C4-2 NS) displayed significantly higher migration adherence to nerve cells, compared to cells with lower levels of Snail (LNCaP Neo/C4-2 Snail shRNA). Additionally, LNCaP Snail or C4-2 NS (Snail-high) CM led to a higher neurite outgrowth compared to the LNCaP Neo or C4-2 Snail shRNA (Snail-low). In conclusion, Snail promotes migration and adhesion to nerve cells, as well as neurite outgrowth via secretion of soluble factors. Therefore, targeting cancer cell interaction with nerves may contribute to halting prostate cancer progression/metastasis.

The STAT3 inhibitor galiellalactone inhibits the generation of MDSC-like monocytes by prostate cancer cells and decreases immunosuppressive and tumorigenic factors.

BackgroundThe transcription factor signal transducer and activator of transcription 3 (STAT3) is implicated in cancer drug resistance, metastasis, and immunosuppression and has been identified as a promising therapeutic target for new anticancer drugs. Myeloid‐derived suppressor cells (MDSCs) play a major role in the suppression of antitumor immunity and STAT3 is involved in the accumulation, generation, and function of MDSCs. Thus, targeting STAT3 holds the potential of reversing immunosuppression in cancer. This study aims to investigate the effect of the small molecule STAT3 inhibitor galiellalactone on prostate cancer cell– induced generation of MDSCs from monocytes and the effect on immunosuppressive factors and inflammatory cytokines.MethodsPrimary human monocytes were cocultured with prostate cancer cells (DU145, PC3, and LNCaP‐IL6) or with conditioned medium (CM) from prostate cancer cells in the presence or absence of the STAT3 inhibitor galiellalactone. Monocytes were analyzed by flow cytometry for an MDSC‐like phenotype (CD14+ HLA‐DR−/lo). The secretion and gene expression of immunosuppressive factors and inflammatory cytokines from prostate cancer cells and monocytes were investigated.ResultsGaliellalactone blocked the prostate cancer cell–induced generation of MDSC‐like monocytes with an immunosuppressive phenotype ex vivo. Monocytes cultured with CM from prostate cancer cells showed increased expression of phosphorylated STAT3. Prostate cancer cells increased the expression of interleukin1β (IL1β), IL10, and IL6 in monocytes which was inhibited by galiellalactone. In addition, galiellalactone decreased indoleamine 2,3‐dioxygenase gene expression in monocytes. Galiellalactone reduced the levels of IL8 and granulocyte macrophage‐colony stimulating factor in prostate cancer cells per se.ConclusionThe STAT3 inhibitor galiellalactone may prevent the prostate cancer cell–induced generation of MDSCs and reverse the immunosuppressive mechanisms caused by the interplay between prostate cancer cells and MDSCs. This is a potential new immunotherapeutic approach for the treatment of prostate cancer.

IFNγ-Induced IFIT5 Promotes Epithelial-to-Mesenchymal Transition in Prostate Cancer via miRNA Processing.

IFNγ, a potent cytokine known to modulate tumor immunity and tumoricidal effects, is highly elevated in patients with prostate cancer after radiation. In this study, we demonstrate that IFNγ can induce epithelial-to-mesenchymal transition (EMT) in prostate cancer cells via the JAK-STAT signaling pathway, leading to the transcription of IFN-stimulated genes (ISG) such as IFN-induced tetratricopeptide repeat 5 (IFIT5). We unveil a new function of IFIT5 complex in degrading precursor miRNAs (pre-miRNA) that includes pre-miR-363 from the miR-106a-363 cluster as well as pre-miR-101 and pre-miR-128, who share a similar 5'-end structure with pre-miR-363. These suppressive miRNAs exerted a similar function by targeting EMT transcription factors in prostate cancer cells. Depletion of IFIT5 decreased IFNγ-induced cell invasiveness in vitro and lung metastasis in vivo. IFIT5 was highly elevated in high-grade prostate cancer and its expression inversely correlated with these suppressive miRNAs. Altogether, this study unveils a prometastatic role of the IFNγ pathway via a new mechanism of action, which raises concerns about its clinical application.Significance: A unique IFIT5-XRN1 complex involved in the turnover of specific tumor suppressive microRNAs is the underlying mechanism of IFNγ-induced epithelial-to-mesenchymal transition in prostate cancer.See related commentary by Liu and Gao, p. 1032.

Abstract 2526: Modulation of metastatic factors in prostate cancer cells by DAX-1 (NR0B1), an androgen-induced orphan nuclear receptor

Abstract DAX-1, a member of the nuclear hormone receptor superfamily, has a pivotal role in adrenal and gonadal development. However, recent studies have shown that DAX-1 acts as a transcriptional repressor influencing the progression of many types of cancers. We hypothesize that DAX-1 has key roles in regulating metastatic genes during prostate cancer progression. We found that introducing DAX-1 into prostate cancer cells lacking endogenous expression of DAX-1 leads to a decrease in metastatic rates. Consequently, we analyzed DAX-1 transcriptional regulation over different markers associated with metastasis. Since the androgen receptor is a key regulator of prostate cancer progression, we used CRISPR to knockout DAX-1 expression in hormone-dependent prostate cancer cells, allowing us to understand the role DAX-1 has as a negative regulator of metastatic genes after androgen receptor activation. We have shown that treatment of hormone-dependent prostate cancer cells with a non-aromatizable androgen leads to upregulation of DAX-1 expression. Here we use the same mechanism to induce DAX-1 expression and analyze the effects on metastatic markers. Results have shown that DAX-1 is sufficient to decrease the expression of metastatic markers and inhibit metastatic rates in prostate cancer cells. These results further explain some of the intrinsic mechanisms involved in prostate cancer metastasis and could reveal new therapeutic routes in the treatment of metastatic prostate cancer. Citation Format: Roxxana Valeria Beltran Valencia, Christina Tzagarakis-Foster. Modulation of metastatic factors in prostate cancer cells by DAX-1 (NR0B1), an androgen-induced orphan nuclear receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2526.

MacroH2A1.2 inhibits prostate cancer-induced osteoclastogenesis through cooperation with HP1α and H1.2.

Osteoclasts are multinuclear bone-resorbing cells that differentiate from hematopoietic precursor cells. Prostate cancer cells frequently spread to bone and secrete soluble signaling factors to accelerate osteoclast differentiation and bone resorption. However, processes and mechanisms that govern the expression of osteoclastogenic soluble factors secreted by prostate cancer cells are largely unknown. MacroH2A (mH2A) is a histone variant that replaces canonical H2A at designated genomic loci and establishes functionally distinct chromatin regions. Here we report that mH2A1.2, one of the mH2A isoforms, attenuates prostate cancer-induced osteoclastogenesis by maintaining the inactive state of genes encoding soluble factors in prostate cancer cells. Our functional analyses of soluble factors identify lymphotoxin beta (LTβ) as a major stimulator of osteoclastogenesis and an essential mH2A1.2 target for its anti-osteoclastogenic activity. Mechanistically, mH2A1.2 directly interacts with HP1α and H1.2 and requires them to inactivate LTβ gene in prostate cancer cells. Consistently, HP1α and H1.2 have an intrinsic ability to inhibit osteoclast differentiation in a mH2A1.2-dependent manner. Together, our data uncover a new and specific role for mH2A1.2 in modulating osteoclastogenic potential of prostate cancer cells and demonstrate how this signaling pathway can be exploited to treat osteolytic bone metastases at the molecular level.

Downregulation of miR-17-92a cluster promotes autophagy induction in response to celastrol treatment in prostate cancer cells

Celastrol has potential application for the treatment of prostate cancer. However it causes autophagy as a protective response in prostate and other types of cancers, thus unveiling the underlying mechanisms may benefit its future application. In the present study, we demonstrate that the miR-17-92a cluster plays a negative role in celastrol induced-autophagy. Dissection of miR-17-92a cluster revealed the role of miR-17 seed family (miR-20a and miR-17) in autophagy inhibition in the context of prostate cancer cells. Autophagy-related gene ATG7 was validated as a target of miR-17 seed family by dual-luciferase assay and qPCR. Celastrol induced autophagy was inhibited by miR-20a or miR-17, while the inhibitory effects were rescued in the presence of pcDNA-ATG7 lacking 3′ UTR, demonstrating that these two members target ATG7 to inhibit celastrol-induced autophagy. As celastrol degrades androgen receptor (AR), a key transcription factor in prostate cancer cells, we further investigated whether AR affected miR-17-92a expression in prostate cancer cells. AR binding sites were found in the promoter and two introns of miR-17-92a. In addition, higher expression levels of miR-17-92a were observed in AR positive cells compared with AR negative cells. Ectopic expression of AR could enhance the expression of miR-17-92a cluster in AR-negative prostate cancer cells while knockdown of AR decreased miR-17-92a expression in AR-positive cells, demonstrating the regulation of AR on miR-17-92a transcription. In summary, our results demonstrate that celastrol downregulates AR and its target miR-17-92a, leading to autophagy induction in prostate cancer cells.

Important role of SUMOylation of Spliceosome factors in prostate cancer cells.

Sentrin/SUMO (small ubiquitin-like modifier)-specific proteases (SENPs) have been implicated in the development of prostate cancer. However, due to the low abundance of SUMO-modified proteins and high activity of SENPs, the SUMO substrates affected by SENPs in prostate cancer cells are largely unknown. Here, we identified SI2, a novel cell-permeable SENP-specific inhibitor, by high-throughput screening. Using SI2 as a way of inhibiting the activity of SENPs and the SUMO stably transfected PC3 cells as a prostate cancer model, in combination with the stable isotope labeling with amino acids (SILAC) quantitative proteomic technique, we identified more than 900 putative target proteins of SUMO, in which 231 proteins were further subjected to bioinformatic analysis. In the highly enriched spliceosome pathway, we validated that USP39, HSPA1A, and HSPA2 were novel target proteins of SUMO. Furthermore, we demonstrated that K6, K16, K29, K51, and K73 were the SUMOylation sites of USP39. Mutation of these SUMO modification sites of USP39 further promoted the proliferation-enhancing effect of USP39 on prostate cancer cells. This study provides the SUMOproteome of PC3 cells and reveals that SUMOylation of spliceosome factors may be implicated in the pathogenesis of prostate cancer. Optimization of SI2 for isotype-specific SENP inhibitors warrants further investigation.

FOXM1 and androgen receptor co-regulate CDC6 gene transcription and DNA replication in prostate cancer cells

CDC6 is a key component of the DNA replication initiation machinery, and its transcription is regulated by E2F or androgen receptor (AR) alone or in combination in prostate cancer (PCa) cells. Through both overexpression and knockdown approaches, we found that in addition to its effects on the E2F pathway, the cell proliferation specific transcription factor FOXM1 stimulated CDC6 transcription in cooperation with AR. We have identified a forkhead box motif in the CDC6 proximal promoter that is occupied by FOXM1 and is sufficient to drive FOXM1-regulated transcription. Indirectly, FOXM1 elevated AR protein levels and AR dependent transcription. Furthermore, FOXM1 and AR proteins physically interact. Using synchronized cultures, we observed that CDC6 expression is elevated near S phase of the cell cycle, at a time coinciding with elevated FOXM1 and AR expression and CDC6 promoter occupancy by both AR and FOXM1 proteins. Androgen increased the binding of AR protein to CDC6 promoter, and AR and FOXM1 knockdown decreased AR binding. These results provided new evidence for the regulatory mechanism of aberrant CDC6 oncogene transcription by FOXM1 and AR, two highly expressed transcription factors in PCa cells. Functionally, the cooperation of FOXM1 and AR accelerated DNA synthesis and cell proliferation by affecting CDC6 gene expression. Furthermore, siomycin A, a proteasome inhibitor known to inhibit FOXM1 expression and activity, inhibited PCa cell proliferation and its effect was additive to that of bicalutamide, an antiandrogen commonly used to treat PCa patients.

A milk protein, casein, as a proliferation promoting factor in prostate cancer cells.

PurposeDespite most epidemiologic studies reporting that an increase in milk intake affects the growth of prostate cancer, the results of experimental studies are not consistent. In this study, we investigated the proliferation of prostate cancer cells treated with casein, the main protein in milk.Materials and MethodsProstate cancer cells (LNCaP and PC3), lung cancer cells (A459), stomach cancer cells (SNU484), breast cancer cells (MCF7), immortalized human embryonic kidney cells (HEK293), and immortalized normal prostate cells (RWPE1) were treated with either 0.1 or 1 mg/mL of α-casein and total casein extracted from bovine milk. Treatments were carried out in serum-free media for 72 hours. The proliferation of each cell line was evaluated by an 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.Resultsα-Casein and total casein did not affect the proliferations of RWPE1, HEK293, A459, SNU484, MCF7, HEK293, or RWPE1 cells. However, PC3 cells treated with 1 mg/mL of α-casein and casein showed increased proliferation (228% and 166%, respectively), and the proliferation of LNCaP cells was also enhanced by 134% and 142%, respectively. The proliferation mechanism of α-casein in PC3 and LNCaP cells did not appear to be related to the induction of Insulin-like growth factor-1 (IGF-1), since the level of IGF-1 did not change upon the supplementation of casein.ConclusionsThe milk protein, casein, promotes the proliferation of prostate cancer cells such as PC3 and LNCaP.

Abstract 5453: YK-4-279 inhibits ETS-positive prostate cancer cell metastasis in a mouse xenograft model.

Abstract Chromosomal translocations involving the ETS family of transcription factors are found in a majority of prostate cancers, including the most clinically aggressive forms. These translocations produce a chimeric gene, which fuses the promoter region of an androgen responsive gene to the coding region of ETS factors, most frequently ETV1 or ERG. Over-expression of ETS factors in prostate cancer cells results in a more invasive phenotype. The high prevalence of these rearrangements, and their biological significance represents a novel therapeutic target for the treatment of prostate cancer. We recently demonstrated that the small molecule YK-4-279 inhibits ERG and ETV1 biological activity in fusion-positive prostate cancer cells leading to decreased motility and invasion in-vitro. Here we present our findings in an in-vivo mouse xenograft model. SCID-beige mice were subcutaneously injected with LNCaP and PC-3 xenografts. Mice were treated with YK-4-279 and its effect on primary tumor size and lung metastasis was observed. YK-4-279 treatment resulted in reduced metastasis of the tumor from primary site to lungs. YK-4-279 also affected expression of ETV1 target genes such as MMP7 and MMP13. ETS fusion-negative PC-3 xenografts were unresponsive to YK-4-279. Our results demonstrate that YK-4-279 decreases tumor growth and inhibits metastasis in fusion-positive prostate cancer cell xenografts. Therefore, YK-4-279 may have an impact on preventing metastasis in prostate cancer patients, which is a leading cause of death. Citation Format: Said Rahim, Sarah Justvig, Sung-Hyeok Hong, Yali Kong, Milton L. Brown, Colm Morrissey, Jeffrey A. Toretsky, Aykut Uren. YK-4-279 inhibits ETS-positive prostate cancer cell metastasis in a mouse xenograft model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5453. doi:10.1158/1538-7445.AM2013-5453

Sequencing the transcriptional network of androgen receptor in prostate cancer

The progression of prostate cancer is largely dependent on the activity of the androgen receptor (AR), which in turn, correlates with the net output of the AR transcriptional regulatory network. A detailed and thorough understanding of the AR transcriptional regulatory network is therefore critical in the strategic manipulation of AR activity for the targeted eradication of prostate cancer cells. In this mini-review, we highlight some of the novel and unexpected mechanistic and functional insights of the AR transcriptional network derived from recent targeted sequencing (ChIP–Seq) studies of AR and its coregulatory factors in prostate cancer cells.

Inhibition of JNK-1 by small interfering RNA induces apoptotic signaling in PC-3 prostate cancer cells

Previous studies have shown that c-Jun-N-terminal kinase-1 (JNK-1) is involved in the transformation of primary fibroblasts and plays a role in tumor cell growth. A number of observations suggest that JNK-1 is a growth promoting factor in prostate cancer cells and blocking its function may induce apoptosis. To test this further, we used a small interfering RNA (siRNA) against JNK-1 mRNA that efficiently inhibits JNK-1 expression in the prostate cancer cell line, PC-3. The application of siRNA against JNK-1 decreased the expression of JNK-1 and affected the expression of p21, XIAP and Bcl-2, but had no effect on the expression of VEGF. In contrast, a control scramble siRNA did not affect the expression of the above indicated proteins. The downregulation of JNK-1 expression at both the mRNA and protein levels was detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. Cell proliferation inhibition rates were determined by the MTT assay. The effect of JNK-1-siRNA on cell cycle distribution and cell apoptosis was determined by flow cytometry, DNA fragmentation and caspase activity. Our data showed that siRNA against JNK-1 mRNA, could efficiently suppress the expression of JNK-1 in PC-3 cells. After 5 days of transfection, the cell death rate was 52%, the apoptotic rate 26% and the viability rate 22%. In conclusion, downregulation of JNK-1 expression by siRNA against JNK-1 mRNA induces apoptotic signaling in prostate cancer PC-3 cells. The use of siRNA against JNK-1 as a novel approach to cancer therapy deserves further investigation.

PDGF upregulates Mcl-1 through activation of β-catenin and HIF-1α-dependent signaling in human prostate cancer cells.

BackgroundAberrant platelet derived growth factor (PDGF) signaling has been associated with prostate cancer (PCa) progression. However, its role in the regulation of PCa cell growth and survival has not been well characterized.Methodology/Principal FindingsUsing experimental models that closely mimic clinical pathophysiology of PCa progression, we demonstrated that PDGF is a survival factor in PCa cells through upregulation of myeloid cell leukemia-1 (Mcl-1). PDGF treatment induced rapid nuclear translocation of β-catenin, presumably mediated by c-Abl and p68 signaling. Intriguingly, PDGF promoted formation of a nuclear transcriptional complex consisting of β-catenin and hypoxia-inducible factor (HIF)-1α, and its binding to Mcl-1 promoter. Deletion of a putative hypoxia response element (HRE) within the Mcl-1 promoter attenuated PDGF effects on Mcl-1 expression. Blockade of PDGF receptor (PDGFR) signaling with a pharmacological inhibitor AG-17 abrogated PDGF induction of Mcl-1, and induced apoptosis in metastatic PCa cells.Conclusions/SignificanceOur study elucidated a crucial survival mechanism in PCa cells, indicating that interruption of the PDGF-Mcl-1 survival signal may provide a novel strategy for treating PCa metastasis.

Abnormal Expression of the ERG Transcription Factor in Prostate Cancer Cells Activates Osteopontin

Osteopontin (OPN) is an extracellular matrix glycophosphoprotein that plays a key role in the metastasis of a wide variety of cancers. The high level of OPN expression in prostate cells is associated with malignancy and reduced survival of the patient. Recent studies on prostate cancer (PCa) tissue have revealed recurrent genomic rearrangements involving the fusion of the 5' untranslated region of a prostate-specific androgen-responsive gene with a gene coding for transcription factors from the ETS family. The most frequently identified fusion gene is TMPRSS2:ERG, which causes ERG protein overexpression in PCa cells. ERG is a transcription factor linked to skeletogenesis. This study was designed to test whether ERG and the product of the TMPRSS2:ERG fusion gene modulate OPN gene expression in PCa cells. To characterize ERG and TMPRSS2:ERG transcriptional activity of OPN, we focused on ETS binding sites (EBS) localized in conserved regions of the promoter. Using in vitro and in vivo molecular assays, we showed that ERG increases OPN expression and binds to an EBS (nt -115 to -118) in the OPN promoter. Moreover, stable transfection of prostate tumor cell lines by TMPRSS2:ERG upregulates endogenous OPN expression. Finally, in human prostate tumor samples, detection of the TMPRSS2:ERG fusion gene was significantly associated with OPN overexpression. Taken together, these data suggest that OPN is an ERG-target gene in PCa where the abnormal expression of the transcription factor ERG, due to the TMPRSS2:ERG fusion, disturbs the expression of genes that play an important role in PCa cells and associated metastases.

Abstract 971: Obesity initiates a self-perpetuating inflammatory response in LNCaP prostate cancer cells

Abstract Background: Obesity increases prostate cancer (PCa) mortality but the mechanism remains unclear. Obesity induced chronic inflammation may be an important factor in advanced PCa. Studies have shown a local inflammatory microenvironment can promote prostate tumorigenesis. Obese men with advanced PCa have high levels of cytokines in their prostate and plasma. Clinical studies promoting weight loss in obese PCa patients have not demonstrated significant benefit in reducing disease progression, suggesting that once initiated, progression is self-sustaining. The role of obesity in stimulating the release of inflammatory autocrine factors in PCa cells and its effect on metastasis remains unclear. Previously, we have shown PCa cells exposed to obese sera increased migration, invasion, proliferation, MMP -9 activity and changes in EMT markers. Our aim was to determine whether PCa cells sustain an inflammatory response initiated by obesity and autocrine factors secreted by PCa cells sustain the metastatic phenotype shown in our previous studies. We hypothesize obesity initiates a self-perpetuating inflammatory response in PCa cells increasing metastatic potential. Methods: We developed an in vitro model which mimics the systemic effect of obesity. 6 week old C57bl/6 male mice were fed a diet induced obesity chow (60% calories from fat) or control chow (10% calories from fat). After 12 weeks, mice were sacrificed, sera was collected and used for in vitro studies. To determine if the initial effects of obesity on PCa invasion are sustained by soluble factors secreted by PCa cells, we treated LNCaP cells with obese or control sera for 30 minutes. Treatments were replaced with serum free media for 18 hours. After 18 hours, conditioned media (CM) was harvested and used for in vitro studies. Cytokine protein array was used to detect a cytokines in conditioned media. Biological assays were used to investigate the effect of conditioned media on invasion, changes in EMT markers, proliferation and cell migration. Results: After stimulation with obese sera, PCa cells secreted high levels of cytokines that mediate inflammatory processes including CCL2, IL-6, IL-8, Rantes and SDF-1. PCa cells treated with obese-stimulated CM resulted in a 2-fold increase in invasive capacity. Obese-stimulated CM resulted in a dispersion of e-cadherin from the cell membrane and nuclear translocation of β-catenin. CM from cells treated with obese sera modulated migration and proliferation promoting an aggressive prostate cancer phenotype. Conclusion: PCa cells treated with obese sera sustain an inflammatory response in the absence of sera. Autocrine factors secreted by obesity treated cells sustain the metastatic phenotype similar to that of PCa cells treated directly with obese sera. The preliminary results from these in vitro studies warrant further in vivo research into the role of weight loss either through diet or exercise on PCa progression. 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 971. doi:10.1158/1538-7445.AM2011-971

Abstract 663: YK-4-279 inhibits ERG and ETV1 mediated prostate cancer cell invasion

Abstract Chromosomal translocations involving the ETS family of transcription factors are found in a majority of prostate cancers, including the most clinically aggressive forms. These translocations produce a chimeric gene, which fuses the promoter region of an androgen responsive gene, such as TMPRSS2, to the coding region of ETS factors, most frequently ETV1 or ERG. Over-expression of ETS factors in prostate cancer cells results in a more invasive phenotype. The high prevalence of these rearrangements, and their biological significance represents a novel therapeutic target for the treatment of prostate cancer. We recently reported the development of YK-4-279, a small molecule inhibitor of EWS-FLI1 oncoprotein in Ewing's Sarcoma. ERG and ETV1 belong to the same class of ETS factors as FLI1. Due to the close homology of FLI1 with ERG and ETV1, we tested the ability of YK-4-279 to inhibit ETS biological activity in prostate cell-lines such as VCaP and LNCaP that demonstrate androgen dependent ERG and ETV1 expression, respectively. YK-4-279 inhibited ERG and ETV1 mediated transcription of target genes such as PLAU, PLAT, ADAM19 and MMP-13, which are involved in breakdown of the extracellular matrix and metastasis. YK-4-279 reduced the motility and invasive phenotype of ETV1-fusion positive LNCaP cells and ERG fusion-positive VCaP cells in in vitro invasion assays. However, ETS fusion-negative PC-3 cells were unresponsive to YK-4-279. ERG knockdown in VCaP cells resulted in a loss of drug responsiveness. Transient ERG expression in PC-3 cells resulted in an increased invasive phenotype, which was reduced by YK-4-279. Our results demonstrate that YK-4-279 inhibits ERG and ETV1 biological activity in fusion-positive prostate cancer cells leading to decreased motility and invasion. Therefore, YK-4-279 may have an impact on metastasis in prostate cancer, which is a leading cause of death, and it may be further evaluated for its clinical applications in prostate cancer, in addition to Ewing's sarcoma. 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 663. doi:10.1158/1538-7445.AM2011-663

Serotonin synthesis and metabolism-related molecules in a human prostate cancer cell line

Prostate cancer is one of the most common tumors in males and its incidence is steadily increasing worldwide. Serotonin or 5-hydroxytryptamine (5-HT) is a well-known neurotransmitter that mediates a wide variety of physiological effects. An increase in the number of 5-HT-releasing neuroendocrine (NE) cells has been correlated with tumor progression. However, it is particularly unclear whether released 5-HT or the release of 5-HT has a role in tumor cell growth. We hypothesized that 5-HT synthesis and metabolism in NE cells regulate the growth of prostate cancer cells. In the present study, 5-HT was found to play a role as a cell growth factor in prostate cancer cells. Moreover, the pharmacological inhibition of 5-HT synthesis and metabolism interrupted the growth of prostate cancer cells. To confirm the existence of 5-HT in prostate cancer cells, we performed ELISA, HPLC, RT-PCR and immunohistochemical analyses. A high expression of tryptophan hydroxylase (TPH-1), dopa decarboxylase (DDC) and monoamine oxidase A (MAO-A) was noted in the prostate cancer cells when compared with normal prostate cells. Previous studies showed that 5-HT stimulated the proliferation of prostate cancer cells mediated by 5-HT receptors 5-HTR1A and R1B. However, cell proliferation was significantly inhibited when siRNA for both DDC and TPH-1 was transfected to the cells. Consequently, we propose that the secretion system of prostate NE cells capable of 5-HT synthesis and metabolism plays a significant role in prostate tumor generation and progression. These findings provide crucial clues for the development of potential pharmacotherapeutics to slow prostate tumor progression.

Resveratrol induces growth arrest and apoptosis through activation of FOXO transcription factors in prostate cancer cells.

BackgroundResveratrol, a naturally occurring phytopolyphenol compound, has attracted extensive interest in recent years because of its diverse pharmacological characteristics. Although resveratrol possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. The present study was carried out to examine whether PI3K/AKT/FOXO pathway mediates the biological effects of resveratrol.Methodology/Principal FindingsResveratrol inhibited the phosphorylation of PI3K, AKT and mTOR. Resveratrol, PI3K inhibitors (LY294002 and Wortmannin) and AKT inhibitor alone slightly induced apoptosis in LNCaP cells. These inhibitors further enhanced the apoptosis-inducing potential of resveratrol. Overexpression of wild-type PTEN slightly induced apoptosis. Wild type PTEN and PTEN-G129E enhanced resveratrol-induced apoptosis, whereas PTEN-G129R had no effect on proapoptotic effects of resveratrol. Furthermore, apoptosis-inducing potential of resveratrol was enhanced by dominant negative AKT, and inhibited by wild-type AKT and constitutively active AKT. Resveratrol has no effect on the expression of FKHR, FKHRL1 and AFX genes. The inhibition of FOXO phosphorylation by resveratrol resulted in its nuclear translocation, DNA binding and transcriptional activity. The inhibition of PI3K/AKT pathway induced FOXO transcriptional activity resulting in induction of Bim, TRAIL, p27/KIP1, DR4 and DR5, and inhibition of cyclin D1. Similarly, resveratrol-induced FOXO transcriptional activity was further enhanced when activation of PI3K/AKT pathway was blocked. Over-expression of phosphorylation deficient mutants of FOXO proteins (FOXO1-TM, FOXO3A-TM and FOXO4-TM) induced FOXO transcriptional activity, which was further enhanced by resveratrol. Inhibition of FOXO transcription factors by shRNA blocked resveratrol-induced upregulation of Bim, TRAIL, DR4, DR5, p27/KIP1 and apoptosis, and inhibition of cyclin D1 by resveratrol.Conclusion/SignificanceThese data suggest that FOXO transcription factors mediate anti-proliferative and pro-apoptotic effects of resveratrol, in part due to activation of extrinsic apoptosis pathway.