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17070 Potential chemopreventive effects of diosgenin in the TRAMP mouse model of prostate cancer are mediated through the suppression of multiple pro-inflammatory

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Gautam Sethi1, Muthu K Shanmugam2, Feng Li2, Sakshi Sikka2, Tina Ong1 and Kam Hui1

1National Cancer Centre, Singapore

2The National University of Singapore

Constitutive activation of pro-inflammatory transcription factors nuclear factor-kB (NF-kB) and signal transducers and activators of transcription 3 (STAT3) is frequently observed in prostate cancer and has been linked with tumor cell proliferation, invasion, metastasis and angiogenesis. In this study, we investigated the effect of diosgenin on NF-kB and STAT3 signaling pathways in both androgen dependent and independent prostate cancer cell lines and also tested its potential chemopreventive effects in the transgenic adenocarcinoma of mouse prostate (TRAMP) mice. We found that diosgenin inhibited constitutive and inducible activation of NF-kB in DU145 and LNCaP cells in a time dependent manner. The suppression was mediated through the inhibition of constitutive and TNF-a induced phosphorylation of IkBa, IkBa kinase (IKK) activation, phosphorylation of p65 and NF-kB dependent reporter activity. Furthermore, diosgenin suppressed both constitutive and inducible STAT3 activation in prostate cancer cells concomitant with suppression of activation of upstream kinases such as (Src and JAK2) and STAT3-dependent reporter gene activity. Diosgenin also downregulated the expression of various NF-kB and STAT3 regulated gene products involved in survival, metastasis, and angiogenesis. In vivo, we found that TRAMP mice fed with diosgenin diet displayed delayed formation of prostate intraepithelial neoplasia (PIN), slow progression of PIN to adenocarcinoma and markedly reduced tumor growth. Overall our results indicate that diosgenin mediates its anti-tumor effects through the suppression of NF-kB and STAT3 pathways in prostate cancer.

22270 Acquisition of epithelial-mesenchymal transition/cancer stem cell is associated with activation of PI3K pathway in prostate cancer radioresistance

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Lei Chang

St George clinical school, The University of New South Wales, Australia

Purpose

Radioresistance is a major problem in prostate cancer (CaP) radiotherapy (RT). This study was to investigate the association of epithelial-mesenchymal transition (EMT)/cancer stem cells (CSCs) with PI3K/Akt/mTOR signalling pathway in CaP radioresistance and the effects of a combination of a dual PI3K/mTOR inhibitor (BEZ235) with RT on CaP radiation-resistant (RR) cells in vitro.

Experimental Design

Three CaP-RR cell lines (PC-3RR, DU145RR and LNCaPRR) were established by RT. The phenotypic change of EMT/CSC markers and the activation of PI3K/Akt/mTOR signaling proteins were detected by immunofluorescence and Western blotting. Adual PI3K/mTOR inhibitor BEZ235 was used to examine whether the inhibitor can improve radiosensitivity of CaP-RR cells and affect EMT/CSC expression in vitrousing colony assay and western blotting analysis.

Results

We have developed three CaP-RR cell lines and confirmed their radioresistance with a clonogenic survival assay. These CaP-RR (PC-3RR, DU145RR and LNCaPRR) cells had increased proliferation, and enhanced colony and sphere formation, and invasion ability compared to CaP-control cells (P < 0.05). In addition, the enhanced EMT/CSC phenotypes and the activation of PI3K/Akt/mTOR pathway proteins were also found in CaP-RR cells by immunofluorescence and Western blotting. Furthermore, the BEZ235 dual inhibitor effectively increased radiosensitivity and induced more apoptosis/autophagy (Becline-1 and LC3/B) in CaP-RR cells in vitro proteins, which is specifically correlated with the enhanced EMT/CSC expression

Conclusions

CaP radioresistance is associated with enhanced EMT and CSC expression via the activation of PI3K/Akt/mTOR signaling pathway. The combination of a PI3K/mTOR inhibitor (BEZ235) with RT is a promising modality for the treatment of CaP to overcome radioresistance. This combination approach is worthwhile for future animal study and clinical trials.

22562 The role of macrophages in Docetaxel (DTX) resistance in castrate resistant prostate cancer (CRPC)

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Kate Mahon1, Mark Chatfield2, Samuel Breit3, David Brown3, Mark Molloy4, Gavin Marx5, Nick Pavlakis5, Michael Boyer1, Martin Stockler1 and Lisa Horvath1

1Sydney Cancer Centre, Royal Prince Alfred Hospital, Australia

2National Health and Medical Research Council Clinical Trials Centre, Australia

3St Vincent's Centre for Applied Medical Research, Australia

4Australian Proteome Analysis Facility, Macquarie University, Australia

5Royal North Shore Hospital, Australia

Objective

DTX improves symptoms and survival in advanced CRPC, however, ∼50% of patients have chemoresistant disease. Given the toxicity of cytotoxic treatment in this population, there is an urgent need for early chemoresistance markers, a greater understanding of chemoresistance mechanisms and the development of new therapies. This study examined whether early changes in cytokine levels predict chemoresistance and explored the role of macrophages in cytokine generation and chemoresistance in vitro.

Methods

28 cytokines were measured pre/post cycle 1 of chemotherapy from 59 men with metastatic CRPC. Cytokine levels were correlated with PSA response and overall survival. DTX-sensitive PC3 and DTX-resistant PC3-Rx cells were cultured alone and in co-culture with U937 monocytes +/− DTX treatment. Conditioned media (CM) was analysed by a 36 cytokine array panel and by ELISA for MIC1. CM from U937 cells exposed to DTX was used to treat PC3 cells combined with escalating DTX doses.

Results

Change in the levels of 10 cytokines over 1 cycle of chemotherapy (MIC-1 p = 0.003; IL1ra p = 0.004; IL1b p = 0.01; IL4 p < 0.001; IL5 p = 0.041; IL6 p = 0.003; IL7 p = 0.009; IL8 p = 0.017; IL12 p = 0.004; IFNÎ3 p = 0.001) was associated with clinical benefit group (PR + SD vs PD). The combination of changes in MIC1, IL4 and IL6 most strongly predicted PSA response (ROC AUC 0.88, 95% CI 0.79–0.97). PC3 and PC3-Rx cells produced low cytokine levels alone. PC3Rx/U937 co-culture expressed markedly more cytokines, chiefly markers of alternative macrophage differentiation, compared to PC3/U937 co-culture (IL10 17-fold; IL27 15-fold). DTX treatment enhanced cytokine production by PC3Rx/U937 co-culture while reducing cytokine levels in PC3/U937 CM. The IC70 for DTX in PC3 cells incubated with CM from DTX exposed U937 cells was 10 fold greater than with U937 CM without DTX exposure.

Conclusions

Early changes in circulating cytokine levels, especially those involved in macrophage activity, were associated with chemoresistance in men with CRPC. In vitro studies revealed a cytokine-mediated interaction between the macrophages and the DTX-resistant cells when treated with DTX that was not apparent in the DTX-sensitive cell model. When considered together, these data suggest a significant role for host macrophages in the development of DTX resistance in CRPC.

23878 Novel thiosemicarbazone iron chelators reverse the metastatic properties of prostate cancer cells via the iron regulated metastasis suppressor NDRG1

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Zaklina Kovacevic1, Zhiqiang Chen2, Sun Jing2, Daohai Zhang1 and Des R. Richardson1

1The University of Sydney, Australia

2General Surgery Department of Ruijin Hospital, Shanghai Jiao Tong University, Canada

Introduction

Prostate cancer mortality is largely attributed to metastasis of the primary tumour, migration and invasion to other sites in the body. One novel strategy for the treatment of cancer is to target proteins that have the ability to inhibit the metastatic and invasive properties of cancer cells. A promising new target molecule is the iron regulated metastasis suppressor, N-myc down-stream regulated gene 1 (NDRG1; (Kovacevic Z., et al. Mol. Pharmacol. 2011;80:598–609). Indeed, NDRG1 is often down-regulated in advanced prostate cancers and metastases, with its expression being correlated with increased survival of prostate cancer patients.

Novel thiosemicarbazone iron chelators Dp44mT and DpC are currently being developed as anti-cancer agents and were found to be highly effective at inhibiting the progression of numerous cancers both in vitro and in vivo (Richardson D.R. et al. PNAS 2006;103:14901–6). Apart from their ability to generate cytotoxic reactive oxygen species, these agents are also able to markedly up-regulate NDRG1 expression in prostate cancer (Kovacevic Z., et al. JBC 2012; 287:17016–28; Mol. Pharmacol. 2013;83:454–69).

Methods

We examined the molecular mechanisms behind the anti-metastatic effects of NDRG1 using prostate cancer cells. NDRG1 was over-expressed or silenced in these cells to determine its down-stream effects. Novel iron chelators Dp44mT and DpC were also utilized to determine their effects on cancer cell migration and invasion.

Results and Discussion

We found that NDRG1 was able to inhibit the key step in metastasis, namely the epithelial to mesenchymal transition (EMT) leading to reduced cell migration and invasion. NDRG1 also promoted the membrane expression of the adherens junction proteins E-cadherin and Î2-catenin, while inhibiting the ROCK1/pMLC2 pathway that promotes stress fiber formation and cell motility.

Considering the ability of Dp44mT and DpC to up-regulate NDRG1 expression in cancer cells, we further examined the effect of these agents on the NDRG1 down-stream targets. Indeed, we found that both Dp44mT and DpC induced similar effects to NDRG1 over-expression, resulting in inhibition of EMT and prostate cancer cell motility, migration and invasion. Using siRNA to silence NDRG1 expression, we found that the effects of the iron chelators on EMT and cell motility were largely mediated by NDRG1.

Conclusions

We have identified a promising new strategy for the treatment of prostate cancer, one that involves novel thiosemicarbazone iron chelators that target the iron regulated metastasis suppressor, NDRG1.

24038 Anthocyanin extracted from black soybean induces apoptosis of DU-145cells in vitro and inhibits xenograft growth

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Sung-Hoo Hong, U-Syn Ha, Hyuk Jin Cho, Ji Youl Lee, Tae-Kon Hwang and Sae Woong Kim

The Catholic University of Korea, Republic of Korea

Objective

This study investigated the effects ofanthocyanins extracted from black soybean, which have antioxidant activity,on apoptosis in vitro(in hormone refractory prostate cancer cells)and ontumor growth in in vivo (in athymic nude mouse xenograft model).

Methods

The growth and viability of DU-145 cells treated with anthocyanins were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and apoptosis was evaluated by DNA laddering. Immunoblotting was performed to evaluate changes in expression of p53, Bax, Bcl, androgen receptor (AR), and prostate specific antigen (PSA). To study the inhibitory effects of anthocyanins on tumor growth in vivo, DU-145 tumor xenografts were established in athymic nude mice.DU-145 cells were injected subcutaneously into the flank of 6-week-old male BALB/c nude mice after treatment with anthocyanin (8mg/kg) for 2 weeks. Tumor dimensions were measured every day using calipers and volumes were calculated according to the formula (length × width2)/2.

Results

Anthocyanin treatment of DU-145 cells (a) increased apoptosis in a dose-dependent manner, (b) caused G2-M phase cell cycle arrest, (c) significantly decreased p53 and Bcl-2expression (with increased Bax expression), and (d) significantly decreased PSA and AR expression. In the xenograft model, anthocyanin-treated mice had significantly less tumor growth.

Conclusions

This study suggeststhat anthocyanins from black soybean inhibit the progression of prostate cancer in vitro and in a xenograft model.

24214 The anti-tumor agent, Dp44mT, can ‘Hijack’ lysosomal P-glycoprotein to overcome multi-drug resistance

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Patric J. Jansson, Tetsuo Yamagishi, Danae Sharp and Des Richardson

The University of Sydney, Australia

Objective

Discovering new therapeutic targets for multidrug resistant (MDR) cancers such as prostate cancer (PCa) is crucial, as current therapies have very limited activity. The poor response to chemotherapy in PCa patients is linked to P-glycoprotein (Pgp) expression. P-gp is a drug transporter known to be localized on plasma membranes and is thought to be responsible for the efflux of internalized drugs out of cells. However, the distribution and the role of intracellular Pgp remain unclear and are important to elucidate. Interestingly, a breakthrough in our field has been the development of the di-2-pyridylketone thiosemicarbazone (DpT) class of anti-tumour agents, particularly di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), which overcomes resistance in MDR cells. However, the mechanism of how these agents overcome MDR is not known. The aim of the current study was to (1) characterize intracellular Pgp, (2) to investigate the role of intracellular Pgp in MDR and (3) if Pgp is linked to how Dp44mT can overcome MDR.

Methods

Intracellular localization of Pgp was assessed using co-localization with well characterized organelle-specific antibodies or dyes, namely lysosomal-associated membrane protein 2 (Lamp2) that is a common marker used for lysosomes, 4′,6-diamidino-2-phenylindole (DAPI) for the nucleus and Mitotracker® Deep Red (Mito DRed) for mitochondria. P-gp inhibitors Valspodar and Elacridar, as well as P-gp knockdown using siRNA, was used to study the function of P-gp by tracking the movement of the fluorescent PCa cytotoxic Doxorubicin (DOX) by using confocal microscopy and cellular organelle markers Lamp2 and DAPI. To elucidate if lysosomal Pgp was important for Dp44mT induced lysosomal damage, lysosomal integrity was assessed using two classical lysosomal markers, namely Acridine Orange and Lysotracker Red in the presence and absence of P-gp inhibitors. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] assays and Pgp inhibitors were used to study importance of Pgp for DOX and Dp44mT cytotoxicity.

Results

Using confocal microscopy, Pgp was shown to be expressed on the lysosomal membrane. The lysosomal P-gp was demonstrated to be functional since the sequestration of DOX (fluorescent), a Pgp substrate, was observed to co-localize with the lysosomal marker, Lamp2. The lysosomal sequestration of DOX was found to be directly Pgp-dependent, as inhibition of P-gp by using specific Pgp inhibitors, as well as Pgp knockdown using siRNA, prevented DOX sequestration into lysosomes and led to redistribution to the nucleus. Furthermore, this DOX sequestration into lysosomes was demonstrated to confer drug resistance, as redirection of DOX into the nucleus using the lysosomotropic agent, ammonium chloride, sensitised the cells to DOX (20-fold increase). In contrast, the lysosomal membrane Pgp drug pump was ‘hijacked’ by Dp44mT to enhance transport of the drug into this target organelle. This resulted in an increase in lysosomal damage and cytotoxicity toward Pgp-expressing tumour cells.

Conclusions

The significance of this contribution is that it demonstrates the lysosomal compartment can be manipulated to overcome multi-drug resistance. While the mitochondrion has drawn great attention as a therapeutic target in recent times, little work has focused on the lysosome, an organelle that is central to necrosis and apoptosis. For the first time, this study demonstrates that lysosomal Pgp is functional and contributes to drug resistance through Pgp-mediated sequestration of chemotherapeutics used in prostate cancer. Most importantly, these studies identified that lysosomal Pgp mediated resistance can be ‘hijacked’ by novel agents, offering a novel strategy of overcoming multi-drug resistance in prostate cancer.

24390 Differences in self-reported outcomes of open prostatectomy patients and robotic prostatectomy patients in an international web-based survey

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Peter O'Shaughnessy1, Thomas Laws1, Carole Pinnock2, Judd Moul3 and Adrian Esterman1

1The University of South Australia, Australia

2Repatriation General Hospital, Australia

3Duke University, United States

Objectives

To compare patient reported outcomes between robotic assisted surgery and non-robotic assisted surgery.

Methods

Based on this qualitative research and literature review, an internet based questionnaire was developed with approximately 70 items. The questionnaire included both closed and open-ended questions.

Results

Responses were received from 193 men of whom 86 had received either open (OP) or robotic (RALP) surgery. A statistically significant (p = 0.027), ranked analysis of covariance was found demonstrating higher recent distress in the robotic (RALP) surgery group. Although not statistically significant, there was a pattern of men having robotic (RALP) surgery reporting fewer urinary and bowel problems, but having a greater rate of sexual dysfunction.

Conclusions

Men who opt for robotic surgery have higher expectations for robotic (RALP) surgery, when these expectations are not fully met they may be less likely to accept the consequences of this major cancer surgery. Information regarding surgical choice needs to be tailored to ensure that men diagnosed with prostate cancer are fully informed of not only short term surgical and physical outcomes such as erectile dysfunction and incontinence, but also of potential issues with regards to masculinity, lifestyle and sexual health.

24466 Investigating the epithelial to mesenchymal transition in prostate cancer invasion and metastasis

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Nataly Stylianou, Abhishek Kashyap, Lidija Jovanovic, Melanie Lehman, Chenwei Wang, Jennifer Gunter, Nelson Colleen and Brett Hollier

Queensland University of Technology, Australian Prostate Cancer Research Centre, Queensland, Australia

Introduction

The majority of prostate cancer (PCa) deaths occur due to the metastatic spread of tumour cells to distant organs and the establishment of secondary tumours. The epithelial to mesenchymal transition (EMT) is an important developmental pathway that becomes re-activated in many cancers and has been shown to play pivotal roles in cancer progression and metastasis. However the role of EMT in PCa has not been fully characterised. We previously engineered an inducible cell model with the inducible expression of known EMT inducer Snail, whereby EMT induction could be controlled and temporally studied. Here we have engineered a second model with the EMT inducer Slug, which we have used in combination with Snail to derive a PCa specific EMT signature.

Methods

We developed doxycycline inducible cell models whereby the induction and reversal of EMT can be tightly regulated with the inducible expression of Snail or Slug. Protein, RNA and morphological changes were recorded, in two dimensional tissue culture experiments and three dimensional (3D) Matrigel assays following EMT induction. We have a custom designed Agilent 180K probe microarray to identify a temporal transcriptional signature of EMT induction in LNCaP PCa cells. A number of candidate genes identified in our microarray studies have been validated using quantitative real time PCR (qRT-PCR) and western blotting.

Results

qRT-PCR and western blot analysis demonstrated robust expression of Snail and Slug when doxycycline was added to the culture media, resulting in a decrease in E-cadherin expression (a hallmark event of EMT). In 3D Matrigel cultures, LNCaP cells acquired a highly invasive phenotype upon induction of Slug or Snail expression. Biostatistical analysis of the microarray data revealed a common EMT-Invasion signature induced by both Snail and Slug models during 3D Matrigel invasion. Interrogation of multiple clinical PCa datasets has identified a number of these EMT-Invasion candidate genes to be dysregulated in metastatic and therapy resistant tumour tissue.

Conclusion

This study reports that for the first time a PCa specific EMT signature using novel inducible PCa models. Future studies will focus on determining the functional relevance of identified candidate genes on EMT and PCa metastasis using both in vitro and in vivo models.

24474 miRDeep*: an integrated application tool for miRNA identification from RNA sequencing data

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Jiyuan An, John Lai, Melanie Lehman and Colleen Nelson

Queensland University of Technology, Australia

miRDeep and its varieties are widely used to quantify known and novel micro RNA (miRNA) from small RNA sequencing (RNAseq). This work describes miRDeep*, our integrated miRNA identification tool, which is modeled off miRDeep, but the precision of detecting novel miRNAs is improved by introducing new strategies to identify precursor miRNAs. miRDeep* has a user-friendly graphic interface and accepts raw data in FastQ and Sequence Alignment Map (SAM) or the binary equivalent (BAM) format. Known and novel miRNA expression levels, as measured by the number of reads, are displayed in an interface, which shows each RNAseq read relative to the pre-miRNA hairpin. The secondary pre-miRNA structure and read locations for each predicted miRNA are shown and kept in a separate figure file. Moreover, the target genes of known and novel miRNAs are predicted using the TargetScan algorithm, and the targets are ranked according to the confidence score. miRDeep* is an integrated standalone application where sequence alignment, pre-miRNA secondary structure calculation and graphical display are purely Java coded. This application tool can be executed using a normal personal computer with 1.5 GB of memory. Further, we show that miRDeep* outperformed existing miRNA prediction tools using our LNCaP and other small RNAseq datasets. miRDeep* is freely available online at http://www.australianprostatecentre.org/%20research/software/mirdeep-star.

24554 mCOPA: analysis of heterogeneous features in cancer expression data

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Chenwei Wang1, Alperen Taciroglu2, Stefan Maetschke2, Colleen Nelson1, Mark Ragan2 and Melissa Davis2

1Australian Prostate Cancer Research Centre – Queensland, Australia

2Institute for Molecular Bioscience, The University of Queensland, Australia

Background

Cancer outlier profile analysis (COPA) has proven to be an effective approach to analyzing cancer expression data, leading to the discovery of the TMPRSS2 and ETS family gene fusion events in prostate cancer. However, the original COPA algorithm did not identify down-regulated outliers, and the currently available R package implementing the method is similarly restricted to the analysis of over-expressed outliers. Here we present a modified outlier detection method, mCOPA, which contains refinements to the outlier-detection algorithm, identifies both over- and under-expressed outliers, is freely available, and can be applied to any expression dataset.

Results

We compare our method to other feature-selection approaches, and demonstrate that mCOPA frequently selects more-informative features than do differential expression or variance-based feature selection approaches, and is able to recover observed clinical subtypes more consistently. We demonstrate the application of mCOPA to prostate cancer expression data, and explore the use of outliers in clustering, pathway analysis, and the identification of tumour suppressors. We analyse the under-expressed outliers to identify known and novel prostate cancer tumour suppressor genes, validating these against data in Oncomine and the Cancer Gene Index. We also demonstrate how a combination of outlier analysis and pathway analysis can identify molecular mechanisms disrupted in individual tumours.

Conclusions

We demonstrate that mCOPA offers advantages, compared to differential expression or variance, in selecting outlier features, and that the features so selected are better able to assign samples to clinically annotated subtypes. Further, we show that the biology explored by outlier analysis differs from that uncovered in differential expression or variance analysis. mCOPA is an important new tool for the exploration of cancer datasets and the discovery of new cancer subtypes, and can be combined with pathway and functional analysis approaches to discover mechanisms underpinning heterogeneity in cancers.

24566 Engineering a high-throughput prostate cancer stem cell niche mimic

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Michael Doran, Katarzyna Futrega, Karen Chambers, Judith Clements and Pamela Russell

Queensland University of Technology, Australia

Introduction

Metastatic prostate cancer cells home to the haematopoietic stem cell (HSC) bone marrow niche. The unique signal milieu within the niche enables prostate cancer “stem cells” to evade the immune system and chemotherapy. For this reason it is anticipated that effective in vitro bone marrow niche mimics will play a significant role in the development of future prostate cancer therapies. However, effectively mimicking this complex milieu remains untenable as evidenced by the failure of existing mimics to support HSC self-renewal in vitro.

Objective

Develop an effective high-throughput in vitro bone marrow stem cell niche mimic (micro-niche), and use the micro-niche as a tool to study prostate cancer-niche interactions and as a tool to screen therapeutics.

Methods

Using a novel high-throughput microwell platform we assemble hundreds of 3D micro-niches each composed of 95 bone marrow niche stromal cells and 5 stem cells. Unlike previously reported 3D microwell systems, our system is compatible with robotic fluidic platforms and this enables truly high-throughput drug screening. In preliminary work we validate the quality of the micro-niche by characterizing its HSC supportive capacity, and then demonstrate its utility in high-throughput drug screening.

Results

Micro-niche mimics self-assemble in microwells over the first 3-hours of culture. The micro-niche enables superior maintenance of HSC in vitro relative to gold standard cultures. The novel microwell design enables efficient high throughput screening of up to 36,000 niche mimics in a single plate.

Conclusions

The 3D micro-niche is a superior niche mimic and a promising tool for investigating prostate cancer bone marrow metastasis and drug screening.

24586 Genetic and epidemiological factors impacting significant increases in prostate cancer risk and aggressive disease associated with an African ancestry

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Vanessa Hayes1, Elizabeth Tindall2, Philip Venter3 and Riana Bornman4

1The Garvan Institute of Medical Research, Australia

2J. Craig Venter Institute, United States

3The University of Limpopo, South Africa

4The University of Pretoria, South Africa

Objective

Although African-ancestry is arguably one of the most significant risk factors for prostate cancer, including increased mortality, few studies have addressed the contributing factors, particularly within Africa. We recently established the Southern African Prostate Cancer Study (SAPCS) a unique resource to investigate both genetic and epidemiological factors associated with disease risk and outcome in African men.

Methods

Totaling more than 900 participants either with our without prostate cancer, we investigate the power for the most significant pre-defined prostate cancer risk alleles (n = 46) and epidemiological measures including demographic, lifestyle and environmental factors to predict prostate cancer and/or disease outcome in the SAPCS.

Results

We show evidence that no previously defined prostate cancer risk allele, largely identified through genome-wide association studies (GWAS), significantly predict prostate cancer risk within our study, while genetic risk profiles did not enhance the predictive power of prostate specific antigen testing. Several lifestyle/environmental factors associated with prostate cancer risk included a family history of cancer, diabetes, current sexual activity and erectile dysfunction, balding pattern, frequent aspirin usage and high PSA levels.

Conclusions

This is the first experimental data addressing the application of current risk alleles to predict prostate cancer within a pure African study. While we observe evidence of association with several epidemiological risk factors, no known genetic risk factor showed an association. The latter finding can be explained by the current bias in GWAS data towards European studies and calls for much needed focus on African resources as well as regionally relevant patterns of genomic diversity.

24674 Application of PSMA-targeting magnetic nanoparticles into MRI of prostate cancer

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Brian Tse1, Gary Cowin2, Carolina Soekmadji1, Lidija Jovanovic1, Benjamin Thierry3 and Pamela Russell1

1Australian Prostate Cancer Research Centre, Queensland, Australia

2National Imaging Facility, Centre for Advanced Imaging, Australia

3Ian Wark Institute, Australia

Objective

Novel imaging techniques for prostate cancer (PCa) are required to improve staging and real-time assessment of therapeutic response. We performed preclinical evaluation of newly-developed, biocompatible magnetic nanoparticles (MNPs) conjugated with J591, an antibody specific for prostate specific membrane antigen (PSMA), to enhance magnetic resonance imaging (MRI) of PCa. PSMA is expressed on ∼90% of PCa, including those that are castrate-resistant, rendering it as a rational target for PCa imaging.

Methods

The specificity of J591 for PSMA was confirmed by flow cytometric analysis of several PCa cell lines of known PSMA status. MNPs were prepared, engineered to the appropriate size, labeled with DiR fluorophore, and their toxicity to a panel of PC cells was assessed by in vitro Alamar Blue assay. Immunohistochemistry, fluorescence microscopy and Prussian Blue staining (iron uptake) were used to evaluate PSMA specificity of J591-MNP conjugates. In vivo MRI studies (16.4T MRI system) were performed using live immunodeficient mice bearing orthotopic LNCaP xenografts and injected intravenously with J591-MNPs or MNPs alone.

Results

MNPs were non-toxic to PCa cells. J591-MNP conjugates showed no compromise in specificity of binding to PSMA+ cells and showed enhanced iron uptake compared with MNPs alone. In vivo, tumour targeting (significant MR image contrast) was evident in mice injected with J591-MNPs, but not MNPs alone. Resected tumours from targeted mice had an accumulation of MNPs, not seen in normal control prostate.

Conclusion

Application of PSMA-targeting MNPs into conventional MRI has potential to enhance PCa detection and localization in real-time, improving patient management.

24682 The prevalence of osteoporosis in men with prostate cancer on androgen deprivation therapy: a systematic review and meta-analysis

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Annie-Claude Lassemillante1, John Hooper2, John Prins2, Suhail Doi3 and Olivia Wright1

1The University of Queensland, Australia

2Mater Medical Research Institute, Australia

3The Unversity of Queensland, Australia

Background

Androgen deprivation therapy (ADT), commonly used in the treatment of prostate cancer, is associated with increased morbidity such as metabolic syndrome, erectile dysfunction and bone loss. The decrease in bone mineral density has been quantified as 1.5–4% within 12 months of treatment initiation and 2% every year thereafter. Consequently, osteopenia and osteoporosis are highly prevalent in this patient group.

Objectives

The aim of this meta-analysis is to determine the prevalence of osteoporosis in men with prostate cancer on ADT. The secondary aim is to explain why the prevalence of osteoporosis varies in this patient group.

Methods

The Pubmed, EmBase and Scopus databases were searched for published literature on PCa and bone health, with a focus on osteoporosis. The studies were quality assessed and meta-analysed using the quality effects model.

Results

Twelve studies were reviewed and it was found that the prevalence of osteoporosis in men with prostate cancer on ADT varied between 9% and 53%. A higher prevalence of osteoporosis was identified in studies reporting on longer duration of ADT and anti-androgen mono-therapy. Ethnicity, DXA machine and skeletal region used to measure bone mass also explained the disparity in osteoporosis prevalence.

Conclusions

Variation in the prevalence of osteoporosis in men with prostate cancer on ADT, highlights the importance of bone monitoring early in this disease rather than when treatment is initiated.

24686 High-throughput 3D tissue culture for prostate cancer drug screening

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Futrega Katarzyna, Karen Chambers, William Lott, Judith Clements, Pamela Russell and Michael Doran

Queensland University of Technology, Australia

Introduction

3D tissue mimics formed through the aggregation of cells provide a superior in vitro model system, relative to classical 2D monolayer cultures. Historically, such studies required hanging drop cultures, which were laborious, and this prevented their widespread adoption. Recently, a number of high-throughput commercial products have enabled more efficient 3D culture, but efficiency still either remains modest and such products are expensive. Our group has developed an inexpensive high-throughput 3D tissue mimic platform that is compatible with liquid handling robots. This platform is suitable for prostate cancer drug screening.

Objective

Develop and test a 3D tissue culture platform that is compatible with high-throughput liquid handling robotics.

Methods

Discs with 150 microwells/cm2 were fabricated from polydimethylsiloxane (PDMS). A 40 μm nylon mesh was fixed to the PDMS, and microwell-mesh constructs were inserted into a 24 well plate. LnCAP prostate cancer cells were distributed into the wells via forced aggregation (5 min × 500 G) to form microaggregates of 300 cells each, and then an Eppendorf liquid handling robot was used to execute programed drug titration assays.

Results

Incorporation of a nylon mesh onto the microwell platform enabled single cells to be distributed into the microwells via forced aggregation, however it did not allow larger assembled 3D tissues to escape from the microwells. This feature makes this microwell platform uniquely robust even when using in combination with the liquid handling robot.

Conclusions

The microwell-mesh platform enabled high-throughput drug screening of 3D tissue mimics using liquid handling robotics.

24698 New tests improving the diagnostic accuracy of prostate biopsies

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John Mills1, Gianluca Severi2, Luis Martin3, David C. Muller2, Francois Guimont3, Brian Reguly3, Graham G. Giles2, Jennifer Creed4, Ryan Parr3 and John Pedersen1

1TissuPath Specialist Pathology, Australia

2Cancer Council, Victoria, Australia

3Mitomics Inc, Canada

4Mitomics Inc., United States

Objective

To develop and validate tests on diagnostic prostate tissue (biopsies or TURP “chips”) that can (1) identify cancers adjacent to the diagnostic tissue, but not present in them (that is, detecting a cancer-related “field effect”), and (2) predict the outcome of the cancer better than “conventional” variables such as Gleason patterns & scores using protein biomarkers assessed by immunohistochemistry – a technique easily translated into clinical practice.

Methods

(1) Using tissue blocks from radical prostatectomy specimens assessed at TissuPath, we took 1mm punch biopsies at varying intervals from the known cancer site which were then assessed quantitatively for the presence of a 3.4kb deletion in mitochondrial DNA (the MitomicsTM PCMT assay). (2) using a CCV cohort of patients that have died from prostate cancer and matched patients that have not died from prostate cancer, we evaluated the ability of a 5-protein biomarker panel (MUC1, ZAG, p53, PTEN, NKX3.1), assessed by immunohistochemistry, to predict survival beyond that predicted by Gleason score.

Results

(1) 3.4kb deletions in mitochondrial DNA were markedly more common in prostates with cancers than in those without. The “field effect” identified by this deletion was found throughout the gland, not just in proximity to the cancer, Prostates with larger tumors had more deletions in surrounding tissue than prostates with small tumors, but the differences were small. (2) ZAG expression in cancerous diagnostic tissue predicted good outcomes, while expression of either MUC1 or p53, or both, predicted worse outcomes. For example, Gleason score 8–10 tumours, which normally have a very bad prognosis, have a relatively high 5-year survival probability (90%) if they express only ZAG.

Conclusions

Quantifying the 3.4kb mitochondrial deletion in diagnostic prostate tissue appears to increase detection of cancers that would not have been detected by histopathology. For diagnostic tissue showing cancer, the ability to predict outcomes is improved by the addition of immunohistochemistry for ZAG, MUC1 and p53.

24714 Curcumin targets unique transcriptional programs in prostate epithelial and fibroblast cell lines

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Lauren Giorgio, Andrew Trotta, Eleanor Need and Grant Buchanan

The University of Adelaide, Australia

Curcumin, a component of the spice turmeric, demonstrates anti-cancer efficacy in a range of solid malignancies. While curcumin action has been extensively characterised in prostate epithelial cells, its effect on the prostate cancer microenvironment, particularly fibroblasts, is not well understood. We therefore define the intracellular mechanisms of curcumin action in prostate fibroblasts.

Microarray analysis in fibroblasts treated with curcumin for 4, 8, 12 and 16 h revealed genes that were chronically upregulated by curcumin (increased and maintained over time) were enriched in stress response pathways (NRF2 and MAPK signalling) while chronically downregulated genes (decreased and maintained over time) were enriched for growth arrest pathways (DNA damage, double strand breaks and GADD45 signalling). Comparison of our data to publically available curcumin treated prostate cancer microarray data (LNCaP and C4-2B) demosntrated little crossover (52 genes) in curcumin targets between the three cell lines. Further, curcumin treated fibroblasts reveal enrichment of HGF signalling (p < 0.05); a pathway not targeted by curcumin in LNCaP or C4-2B cells. Given this pathway has recently been implicated in microenvironment mediated drug resistance, we developed curcumin resistant fibroblasts and confirmed enrichment of HGF signalling between curcumin-treated resistant and sensitive cells (p < 0.05).

Together, these data suggest disparate genetic modes of curcumin action between cancer cells and fibroblasts. Further, the development of curcumin resistance in fibroblasts may involve HGF signalling with implications to future treatment and disease progression remaining unknown. Therefore understanding curcumin action in both cellular compartments of the prostate is critical if curcumin is to be used as a preventative supplement in men with intact prostates.

24718 Expression of the TGF-b superfamily cytokine MIC-1/GDF15slows tumor growth but increases metastases in prostate cancer prone TRAMP mice

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Yasmin Husaini1, Min Ru Qiu2, Geln Lockwood1, Lele Jiang1, Vicky WW Tsai1, Pamela J Russell3, David A Brown1 and Samuel N Breit1

1St Vincents Centre for Applied Medical Research, St Vincents Hospital, Australia

2St Vincents Hospital, Australia

3Queensland University of Technology, Australia

Objective

Macrophage inhibitory cytokine-1 (MIC-1/GDF15), a divergent member of the TGF-Î2 superfamily, is over-expressed by many common cancers including those of the prostate (PCa). Its expression and consequently its serum levels, increase in many human cancers and in advanced cancers these serum levels can increase by 10–50x leading to cancer anorexia/cachexia. Our objective was to explore the effect of MIC-1/GDF15 overexpression on PCa development and spread in the TRAMP transgenic model of spontaneous prostate cancer.

Methods

We crossed the TRAMP mice with MIC-1/GDF15 overexpressing mice (MIC-1fms) to produce syngeneic TRAMPfmsmic-1 mice, which also over-expressed MIC-1/GDF15 under the control of the myeloid specific CSF-1 receptor promoter. We compared the survival rate, prostate tumor size, histopathological grades and extent of distant organ metastases of PCa in TRAMP mice and TRAMPfmsmic-1 mice. We also compared metastasis of TC1-T5, an androgen independent TRAMP cell line that lacks MIC-1/GDF15 expression, by injecting intravenously into MIC-1fms and syngeneic WT C57BL/6 mice.

Results

Whilst TRAMPfmsmic-1 survived about 7.4 weeks longer, had significantly smaller Genitourinary (GU) tumors and lower PCa histopathological grades than TRAMP mice, a much larger proportion of these mice developed distant organ metastases. Additionally, a higher number of TC1-T5 lung tumor colonies were observed in MIC-1fms mice than syngeneic WT C57BL/6 mice.

Conclusion

Our studies strongly suggest that MIC-1/GDF15 has complex actions on tumor behavior: It limits local tumor growth but with advancing disease, may promote metastases. As MIC-1/GDF15 is induced by all cancer treatments and metastasis is the major cause of cancer treatment failure, these results, if applicable to humans, may have a direct impact on patient care.

24730 Androgen influence on prostate cancer exosomes production and content

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Carolina Soekmadji1, Pamela J Russell1, Michelle Hill2, Jonathan C Dunne3, T William Jordan3 and Colleen C Nelson1

1Australian Prostate Cancer Research Centre, Queensland, Queensland University of Technology, Australia

2Diamantina Institute, The University of Queensland, Australia

3Victoria University of Wellington, New Zealand

There is a large body of evidence showing that prostate cancer and its progression to castrate resistant disease is driven by the action of androgens and the androgen receptor. Exosomes, nanosize vesicles secreted by cells, are shown to be involved in cell to cell communication, and evidence from studies in other types of cancer, suggests that the exosomes and microvesicles are regulators which mediate adaptive cancer processes including metastatic progression. We aim to investigate the involvement of androgens in the exosome secretion process in prostate cancer. Our data support the hypothesis that androgens through the androgen receptor play a regulatory role in the secretion process, both qualitatively and quantitatively determining exosome size, content, and number in the context of the androgen sensitive prostate cancer cell, LNCaP. We isolated the exosomes from LNCaP cells conditioned medium by serial ultracentrifugation and confirmed the presence of exosomes by electron microscopy. Treatment with dihydrotestosterone (DHT) changes the composition of protein content, as characterised by mass spectrometry. The presence of androgen receptor antagonist drug, MDV3100, alters the population of exosomes secreted by LNCaP cells. These data indicate that androgens not only mediate prostate cancer progression through regulating gene transcription, but also alter cell to cell communications between prostate cancer cells via exosomes. The data supports that exosome analysis has the potential to detect and differentiate prostate cancer response to androgen targeted therapies. Exosome isolation and characterisation from advanced prostate cancer patients undergoing systemic therapy could provide an indicator of treatment response and treatment resistance.

24738 Epithelial estrogen receptor α (ERα) regulates the proliferation of prostate cancer cells

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Itsuhiro Takizawa1, Mitchell Lawrence1, Helen Pearson2, Normand Pouliot2, John Pedersen3, Patrick Humbert2, Luc Furic1 and Gail Risbridger1

1Monash University, Australia

2Peter MacCallum Cancer Centre, Australia

3TissuPath Pathology, Australian Prostate Cancer BioResource, Monash University, Australia

Androgens and estrogens are both required for the development and progression of prostate cancer. Estrogens have dual roles in prostate cancer, either pro- or anti-tumourigenic depending on whether signaling occurs through the ERα or ERβ subtype, respectively. There is evidence that the pro-tumourigenic effects of ERα can be mediated by tumour stroma, but relatively little is known about the autocrine functions of ERα in prostate cancer cells. Nevertheless, epithelial ERα activity has been linked with PTEN loss in breast and endometrial cancer and, notably, the incidence and severity of tumours in prostate-specific PTEN null mice, correlates with the estrogen sensitivity of each prostatic lobe. Therefore, we hypothesized that this model could be used to study the role of ERα in prostate cancer progression. Immunohistochemistry revealed a consistent pattern of ERα expression in PTEN null mice: low in benign glands, moderate in tumours within the dorsal, lateral and ventral lobes, and high in tumours within the anterior prostate. This pattern significantly correlated with the levels of the proliferative marker Ki67. There was also a significant correlation in the distribution of ERα and Ki67 within individual malignant foci in the anterior prostate. Furthermore, the in vitro proliferation and colony formation of cells derived from PTEN null tumours was attenuated by ERα knockdown. These observations show that autocrine ERα signaling drives aberrant proliferation of aggressive tumour cells.

24742 Genome-wide analyses reveal specific functional repetitive DNA that are proximal to prostate cancer risk alleles

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John Lai1, Jiyuan An2, Colleen Nelson2, Zsofia Kote-Jarai3, Douglous Easton3, Ali Amin3, Rosalind Eeles3, Judith Clements2 and Jyotsna Batra2

1Australian Prostate Cancer Research Centre, Queensland. IHBI, Queensland University of Technology, Australia

2Queensland University of Technology, Australia

3The Royal Marsden NHS Foundation Trust, United Kingdom

Objective

Genome wide association studies (GWAS) account for ∼30% of heritable prostate cancers. This suggests that the majority of heritable prostate cancer risk lie in other single nucleotide polymorphisms (SNPs), or other genetic variations such as short tandem repeats (STRs). These lesser studied repetitive regions of the genome are increasingly recognised as important in (patho)-physiology. We aim to study the potential of STRs to account for some of the missing prostate cancer heritability.

Method

We analysed data generated from ChIPseq and RNAseq to identify potentially functional STRs.

Results

We show that the androgen receptor and RNA Pol II are preferentially recruited to different types of STR sequences, but importantly, we show that some of these STR are located within 200 bp of SNPs that we have recently found to confer prostate cancer risk using the custom Illumina iSelect genotyping array (iCOGs) platform. We also reveal that some STRs that are located near prostate cancer risk SNPs are highly expressed and are differentially responsive to androgens and therapeutic anti-androgens. Importantly, these differentially expressed STRs were found near SNPs that also discriminate between gleason score 6 and gleason score 8 prostate tumours.

Conclusion

We propose that these highly polymorphic STRs that are near prostate cancer risk SNPs should be prioritised for future case-control association studies. Further, we propose that STRs are critical functional elements of the prostate cancer phenotype, and that these repetitive regions of the genome may account for some of the missing prostate cancer heritability.

24754 PSA testing of asymptomatic men in general practice – a review

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Kristen Pickles, Stacy Carter and Lucie Rychetnik

The University of Sydney, Australia

Objective

Our objective was to review the literature on the use of PSA testing in general practice. The use of PSA as a screening tool for detecting prostate cancer remains controversial. However, PSA testing is common in general practice and it is important to understand how and why this occurs.

Methods

Web of Knowledge and Medline databases were searched from inception to February 2013. All empirical studies focused on PSA testing in general practice were included. Editorials, opinion pieces, reviews, and guidelines were excluded. Country of origin, year, sampling techniques, and methodologies were recorded.

Results

588 papers were identified, 65 met the inclusion criteria. Most studies used population-based surveys and routinely collected data; two used qualitative methods; most were from the US and UK. A majority of studies described the prevalence of PSA testing and some identified correlated variables. PSA testing has been associated with patient and consultation characteristics, GP demographics and GP attitudes, experience, and knowledge. Few studies examined how and why PSA testing occurs in general practice.

Conclusions

Existing research has highlighted potential explanatory variables associated with PSA testing. Future research should build on and augment this evidence to better understand and explain how and why decisions are made about PSA testing in general practice.

24778 Identification of novel androgen and antiandrogen regulated long noncoding RNAs and protein-coding RNAs in prostate cancer

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Melanie Lehman1, Chenwei Wang1, Gregor Tevz1, Jiyuan An1, John Lai1, Stephen Hendy2, Stephen McPherson1, Martin Sadowski1 and Colleen Nelson1

1Queensland University of Technology, Australia

2The University of British Columbia, Canada

Most prostate cancer depends on androgens for survival. Therefore, the removal of androgens is currently the most effective treatment for non-localized prostate cancer. Androgen deprivation therapy offers a temporary remission; however, cancer cells adapt to survive in castrate levels of circulating androgens leading to castrate resistant prostate cancer (CRPC). Mounting evidence suggests that intratumoral androgens acting through the androgen receptor (AR) continue to play a critical role in CRPC and underpin the mechanism of action for new clinically approved steroid synthesis inhibitors (e.g. Abiraterone) and improved AR antagonists (e.g. Enzalutamide). We have used strand-specific Illumina RNA sequencing (RNA-seq) followed by de novo transcriptome assembly to investigate transcripts regulated by androgens and AR antagonists in prostate cancer cell lines (LNCaP, LAPC4, 22RV1). We have observed distinct transcriptional signatures between androgens (R1881 and DHT) as well as differences between AR antagonists (Bicalutamide and Enzalutamide) with each having distinct signatures beyond the expected reversal an androgen signature. In addition to alternative variants of protein-coding RNAs, we have identified long noncoding RNAs (lncRNAs) expressed from intergenic regions as well as lncRNAs that are overlapping – and often mistaken for – protein-coding RNAs. RNA-seq is rapidly shifting our understanding of the complexity to the human transcriptome and proteome by identifying alternative variants of protein-coding RNAs and regulatory lncRNAs that were undetected by previous profiling technologies. The identification and functional characterization of these novel androgen and antiandrogen regulated RNAs is critical to develop therapies for men with late stage prostate cancer.

24790 Daxx regulates mitotic progression and prostate cancer predisposition

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Patrick Ling1 and Pak Shing Kwan2

1Queensland University of Technology, Australia

2The University of Hong Kong, Hong Kong

Mitotic progression of mammalian cells is tightly regulated by the E3 ubiquitin ligase APC/C. Deregulation of APC/C is frequently observed in cancer cells and is suggested to contribute to chromosome instability and cancer predisposition. Here, we identified Daxx as a novel APC/C inhibitor frequently overexpressed in prostate cancer. Daxx interacts with the APC/C coactivator Cdc20 and Cdh1 in vivo, with the binding of Cdc20 dependent on the consensus destruction boxes near the N-terminal of the Daxx protein. Ectopic expression of Daxx, but not the D-box deleted mutant (Daxxï„D-box), inhibited the degradation of APC/Cdc20 and APC/Cdh1 substrates, leading to a transient delay in mitotic progression. Daxx is frequently upregulated in prostate cancer tissues; the expression level positively correlated with the Gleason score and disease metastasis (p = 0.027 and 0.032, respectively). Furthermore, ectopic expression of Daxx in a non-malignant prostate epithelial cell line induced polyploidy under mitotic stress. Our data suggest that Daxx may function as a novel APC/C inhibitor, which promotes chromosome instability during prostate cancer development.

24814 Focal adhesion kinase: a mediator of docetaxel-resistance in castrate resistant prostate cancer

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Brian Yong Lee1, Falko Hochgräfe2, Hui-Ming Lin1, Lesley Castillo1, Jianmin Wu1, Mark Raftery3, S. Martin Shreeve4, Lisa Horvath5 and Roger Daly6

1The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Australia

2The University of Greifswald, Germany

3Bioanalytical Mass Spectrometry Facility, The University of New South Wales, Australia

4Pfizer Oncology, United States

5Sydney Cancer Centre, Royal Prince Alfred Hospital, Australia

6The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Australia

Background

Docetaxel remains the standard-of-care for men diagnosed with metastatic castrate resistant prostate cancer (CRPC). Inevitably, all patients develop resistance to chemotherapy. This study aimed to characterize global perturbations in tyrosine kinase signaling associated with Docetaxel-resistance and thereby develop a potential therapeutic strategy to overcome chemoresistance.

Methods

We compared tyrosine phosphorylation events in Docetaxel-sensitive and -resistant DU145 and PC3 prostate cancer cell lines using quantitative mass-spectrometry-based phosphoproteomics. Effects of the focal adhesion kinase (FAK) inhibitors, PF-00562271 and PF-04554878 on chemosensitivity were characterized. The antitumor efficacy of Docetaxel +/− PF-04554878 was determined in tumour-bearing Balb/c nude mice.

Results

Docetaxel-resistant cell lines exhibited increased FAK phosphorylation on Y397 and Y576, compared to parental controls. While treatment with Docetaxel or either of the FAK tyrosine kinase inhibitors (TKIs) reduced FAK phosphorylation in the resistant cells, co-treatment with Docetaxel and a FAK TKI was required to reduce FAK phosphorylation to that of drug-sensitive cells. Treatment with a FAK TKI alone did not affect cell viability of the resistant cells, but co-treatment with Docetaxel and a FAK TKI overcame chemoresistance. The enhanced efficacy of co-treatment was not due to increased apoptosis, but rather enhanced autophagic cell death. In tumor-bearing mice, co-administration of Docetaxel and PF-04554878 resulted in a greater reduction of tumor growth than either monotherapy, and was also associated with increased autophagy.

Conclusions

Enhanced activation of FAK mediates Docetaxel resistance in CRPC. Our study has identified a clinical niche for FAK TKIs, where co-administration with Docetaxel may be used in CRPC to overcome chemoresistance.

24822 Lack of a low-dose radiation adaptive response on prostate cancer progression in the TRAMP model

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Pamela Sykes1, Rebecca Ormsby1, Benjamin Blyth1, Eva Bezak2, Michelle Newman1, Wayne Tilley3 and Mark Lawrence1

1Flinders University, Australia

2Royal Adelaide Hospital, Australia

3The University of Adelaide, Australia

Current understanding of the biological mechanisms underlying the responses of prostate tissue to ionising radiation exposure, including cancer induction is surprisingly limited for both high and low dose exposures. Low dose radiation-induced adaptive responses for increased cancer latency and decreased cancer frequency have been demonstrated in mouse animal models, largely for haematological cancers. This study is the first examination of the effects of high- and low-dose whole-body radiation exposure on prostate cancer development using an autochthonous mouse model (TRAMP, TRansgenic Adenocarcinoma of the Mouse Prostate) of prostate cancer.

TRAMP mice were exposed to acute high (2 Gy), single low (50 mGy) and repeated (5 × 50 mGy) low doses of X-rays to evaluate both the potential prostate cancer inducing/promoting effects of high-dose radiation and low-dose adaptive response phenomena. Prostate weights, histopathology, and time-to-palpable tumour were examined after high dose X-rays. Proliferation (Ki-67), apoptosis and DNA damage (gamma-H2AX) and transgene expression (large T-antigen) were examined within TRAMP prostate sections after high and low doses.

Neither high- nor low-dose radiation-induced effects on prostate cancer progression were observed for any of the endpoints studied, suggesting that modulation of tumourigenesis in the TRAMP model is largely resistant to such exposures. However, further study is required to better assess the effects of radiation exposure using alternative prostate cancer models that incorporate normal prostate.

This work was funded by the Prostate Cancer Foundation of Australia (ID: GC-0810) and the Flinders Medical Centre Foundation.

24826 Culturing prostate cells as 3D micro-aggregates for drug testing

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Karen Chambers, Michael Doran, Pamela Russell and Judith Clements

Australian Prostate Cancer Research Centre, Queensland/ Queensland University of Technology, Australia

Introduction

Monolayer cultures, consisting of only tumour cells, do not accurately mimic the in vivo environment and as a result often function as poor predictors of whether a drug will ultimately yield clinical benefit. By contrast, when cancer cells are cultured as 3-dimensional (3D) aggregates their response to chemotherapy agents more frequently parallels what is observed in vivo.

Objective

Recently, we have developed a 3D prostate cancer cell micro-aggregate culture system for drug testing in which the aggregate size can be controlled.

Methods

Approximately 600 small or 150 large aggregates of uniform diameter are formed per cm2 of tissue culture surface. Prior to cell seeding, the PDMS micro-well surface is coated with poly-lysine and multi-layers of hyaluronan and chitosan, to prevent cell attachment. The prostate cancer cell lines, LNCaP and RWPE-2 were used in the model system and compared to the normal prostate cell lines, RWPE-1 and BPH-1.

Results and Conclusions

We have developed a novel culture system for cancer growth whereby the micro-aggregate size can be strictly regulated. Using this system the cancer cells survive over 14 days, however metabolism is reduced suggesting that the cells are becoming quiescent, a phenomenon associated with stem cells. Conversely, the non-cancerous cell types, BPH-1 and RWPE-1 undergo cell death after 14 days in culture. In summary, this system may be used to test the drug response of differently sized cancer cell aggregates in vitro.

24846 Association and functional analysis of Kallikrein genetic variants with prostate cancer

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Jyotsna Batra1, Srilakshmi Srinivasan1, Felicity Lose2, Kote-Jarai Zsofia3, Douglos Eston3, Rosalind Eeles4, Amanda Spurdle5 and Judith Clements1

1Australian Prostate Cancer Research Centre, Queensland/Queensland University of Technology, Australia

2Queensland Institute of Medical Research, Australia

3The Institute of Cancer Research & Cancer Genetics Unit, United Kingdom

4The Institute of Cancer Research & Cancer Genetics Unit, United Kingdom, The Australian Prostate Cancer Bioresource, Queensland University of Technology, Australia

5Queensland Institute of Medical Research, Australia

Objective

Kallikreins (KLKs), encoded by a gene cluster on chromosome 19q13.4, are secreted serine proteases with diverse expression patterns and physiological roles. In addition to applicability as cancer biomarkers(e.g. PSA/KLK3), KLKs are implicated in invasion, proliferation, and metastasis. SNP rs2735839 located close to KLK3 was found to be significantly associated with prostate cancer (PrCa) risk in a genome-wide association study. We have undertaken a KLK fine mapping studies and now intend to undertake functional validation.

Methods

Using a custom Illumina iSelect genotyping array (iCOGS) designed for the Collaborative Oncology Gene-Environment Study, we genotyped 438 SNPs spanning ∼400 kb around the KLK region in lymphocyte extracted DNA from 22,301 PrCa cases and 22,320 matched controls. Functional assays were undertaken as appropriate.

Results

Multiple stepwise logistic regression analysis and imputation identified four signals in the Kallikrein region that are independently associated with PrCa risk, two of which represents non-synonymous SNPs within the KLK3 gene. The rs17632542 SNP (Ile178Thr) is the most significantly associated likely causal variant (OR = 0.74, 95% CI = 0.70–0.78) at this locus as we showed KLK3 ‘Thr’ variant to be less stable compared to the ‘Ile’ isoform using thermodynamic stability assays, which suggests functional differences between the variant and wild-type alleles. Further, by analysing 17,680 PrCa patients with available data on PrCa SEER-staging, (883 with metastatic disease), we identified rs17632542 SNPto be significantly associated with metastasis (OR = 1.5, 95% CI = 1.3–1.8) of PrCa. These results are interesting as they indicate that while the KLK3 minor allele (‘Thr’ isoform) has a protective role in PrCa predisposition; it is more prevalent in metastatic disease.

Conclusion

SNPs in the KLK3 gene may be important in regulating the expression and/or activity of KLK3/PSA, an important mediator of prostate carcinogenesis and metastasis.

24862 Metformin modulates key insulin-induced survival pathways in prostate cancer cells

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Jenni Gunter1, Amy Lubik2, Raja Vasireddy1, Nataly Stylianou1, Stephen Hendy2, Brett Hollier1, Martin Gleave2, Michael Pollak3, Adrian Herington1 and Colleen Nelson1

1Queensland University of Technology and Translational Research Institute, Australia

2Vancouver Prostate Centre at Vancouver General Hospital, Canada

3McGill University, Canada

Androgen deprivation therapy (ADT) for advanced prostate cancer (PCa) results in tumour regression; however, ADT induces features of the metabolic syndrome, including persistent high levels of circulating insulin (hyperinsulinaemia), which is specifically associated with poor outcomes, including rapid progression and increased cancer mortality. We have recently demonstrated that insulin reactivates androgen receptor activity via increased de novo steroidogenesis, fuelling cancer growth, along with several other pathways that promote cell proliferation and survival. Metformin is widely used to treat diabetes and insulin resistance by normalising systemic insulin sensitivity. Recently, metformin has been considered as a potential cancer therapeutic, which may act directly on tumour cells to counter balance the effects of elevated insulin, disrupting crucial cancer survival pathways. We investigated the effect of metformin on insulin-induced steroidogenesis and other key, related prostate cancer survival pathways, including cholesterol synthesis and lipogenesis. We observed metformin reduced insulin-induced increases in androgen-regulated genes including PSA and TMPRSS2 and decreased steroid production (measured by radiometric HPLC) with accompanying reduction in steroidogenic enzyme expression, determined by QRT-PCR and western blot analysis, in LNCaP and 22RV1 PCa cells. Insulin increased expression of cholesterol synthesis and fatty acid mobilisation genes (HMGCR, FASN, ACC, HSL,) which was reduced by metformin as was expression of the transcriptional regulator of cholesterol, fatty acid and steroid synthesis, sterol regulatory element binding proteins (SREBP)s. Insulin activation of both steroidogenesis and fatty acid synthesis pathways may have important implications in PCa progression, and our data supports metformin as a potential adjuvant therapy in ADT-induced hyperinsulinaemia.

24866 CD151, a novel candidate for regulation of prostate cancer cell motility, invasion and angiogenesis

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Sujitra Detchokul1, Bradley Newell1, Elizabeth D. Williams2 and Albert G. Frauman1

1The University of Melbourne, Australia

2Monash Institute of Medical Research, Australia

Tumour metastasis is a multi-factorial process and tumour cells are sensitive to local microenvironmental cues that regulate normal physiological functions such as wound healing and epithelial morphogenetic changes leading to malignant behaviour1. CD151, a tetraspanin superfamily member, has been identified as a promoter of prostate cancer (PCa) migration and invasion and involves association with integrins on both cell-cell and cell-stroma levels2, 3. Furthermore, CD151 plays a role in endothelial cell motility 4–6. We investigated potential roles of CD151 in PCa metastasis.

CD151 is heterogeneously expressed across different PCa cell lines and the level of CD151 expression is significantly higher in the highly tumorigenic, androgen-insensitive cell lines PC-3 and DU-145 compared to the androgen-sensitive LNCaP (P < 0.001). This supports the previous findings that CD151 has value as a prognostic indicator for human PCa7. CD151 also has a role in the promotion of motility and invasion of PCa cell lines2. Orthotopic implantation of human PC-3 cells into SCID mice prostates was investigated and the majority developed lymph node metastases. Primary xenografts from mice that formed secondary metastases had higher CD151 expression and microvessel density (CD31 staining) compared to primary xenograft from those unable to form metastasis (P = 0.01 and P = 0.03, respectively). MVD were not correlated with tumour size. These findings underscore the potential role of CD151 and angiogenesis in the metastatic potential of PCa, as suggested by a previous report in which CD151 knock-out animals had impaired pathologic angiogenesis8.

CD151 is likely to be an important regulator of PCa cell communication with the surrounding microenvironment.

Quaranta V. Motility cues in the tumor microenvironment. Differentiation 2002; 70: 590598.

Ang J, Fang B-L, Ashman LK, Frauman AG. The migration and invasion of human prostate cancer cell lines involves CD151 expression. Oncol Rep 2010; 24(6): 15931597.

Nishiuchi R, Sanzen N, Nada S, et al. Potentiation of the ligand-binding activity of integrin alpha 3 beta 1 via association with tetraspanin CD151. Proc Natl Acad Sci U S A 2005; 102(6): 19391944.

Yáñez-Mó M, Alfranca A, Cabanas C, et al. Regulation of endothelial cell motility by complexes of tetraspan molecules CD81/TAPA-1 and CD151/PETA-3 with alpha3 beta1 integrin localized at endothelial lateral junctions. J Cell Biol 1998; 141(3): 791804.

Longo N, Yanez-Mo M, Mittelbrunn M, et al. Regulatory role of tetraspanin CD9 in tumor-endothelial cell interaction during transendothelial invasion of melanoma cells. Blood 2001; 98(13): 37173726.

Sadej R, Romanska H, Baldwin G, et al. CD151 regulates tumorigenesis by modulating the communication between tumor cells and endothelium. Mol Cancer Res 2009; 7(6): 787798.

Ang J, Lijovic M, Ashman LK, Kan K, Frauman AG. CD151 protein expression predicts the clinical outcome of low-grade primary prostate cancer better than histologic grading: a new prognostic indicator? Cancer Epidemiol Biomarkers Prevent 2004; 13(11): 17171721.

Takeda Y, Li QL, Kazarov AR, et al. Diminished metastasis in tetraspanin CD151-knockout mice. Blood 2011; 118(2): 464472.

24874 Insulin increases migration and invasion in androgen deprived prostate cancer cells

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Phoebe Sarkar1, Jennifer Gunter1, Amy Lubik2, Nataly Stylianou1, Brett Hollier1, Michael Pollak3 and Colleen Nelson1

1Queensland University of Technology and Translational Research Institute, Australia

2Vancouver Prostate Centre, Vancouver General Hospital, Canada

3McGill University and Jewish General Hospital, Canada

Androgen deprivation therapy (ADT) is commonly used to treat advanced prostate cancer (PCa). However, a major side effect of reduced testosterone is the onset of symptoms associated with metabolic syndrome including elevated insulin levels (hyperinsulinaemia) associated with more aggressive disease and more rapid PCa progression. These observations point towards a role of insulin signalling in promoting PCa progression. We have recently shown that insulin can promote de novo steroidogenesis to promote cancer progression; however insulin may be expected to upregulate a number of cancer progression pathways. Thus, we undertook in vitro studies to determine the direct actions of insulin on migration and invasion of PCa cells.

Insulin induced migration was investigated using migration assays, invasion assays into Matrigel, qRT-PCR, Western blot and microarray analysis. We have observed that in the absence of androgens, insulin promotes up to 70% increased migration and invasion of 22RV1 and LNCaP cell lines. Importantly, insulin induced migration and invasion was blocked significantly by an inhibitor of insulin receptor, BMS 745807-04 but not by IGF-1 receptor inhibitor CP 751871, indicating these responses are insulin specific. Additionally, we observed insulin-induced molecular changes within the cells reminiscent of an epithelial-to-mesenchymal transition (EMT); insulin increased expression of vimentin and the SNAIL and ZEB1 transcription factors. These novel results show insulin promotes PCa cell migration and invasion, possibly by inducing EMT-like changes within PCa cells and make a strong case for investigating the clinical efficacy of using insulin-sensitizing drugs as an adjuvant therapy for PCa treatment.

24882 Simvastatin suppresses insulin induced steroidogenesis and fatty acid synthesis in prostate cancer

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Amy Lubik1, Raja Vasireddy2, Jennifer Gunter2, Susan Ettinger1, Stephen Hendy1, Michelle Hill3, Martin Gleave1, Michael Pollak4, Adrian Herington2 and Colleen Nelson2

1Vancouver Prostate Centre at Vancouver General Hospital, Canada

2Queensland University of Technology and Translational Research Institute, Australia

3UQ and Translational Research Institute, Australia

4McGill University, Canada

As a result of androgen deprivation therapy, approximately 55% of prostate cancer (PCa) patients develop metabolic syndrome and hyperinsulinemia which shortens the time to incurable disease. We have recently shown that insulin can upregulate steroidogenesis in PCa cells, and insulin is proposed to stimulate other pathways, such as lipogenesis. In epidemiological studies, the use of statins correlates to decreased risk and severity of PCa, potentially through inhibition of these pathways. In the work presented we have demonstrated that simvastatin diminished insulin mediated increases in cholesterol, steroid, and fatty acid synthesis.

In LNCaP and 22RV1 cells, steroidogenesis was analyzed by radiometric HPLC and DHT ELISA. Intracellular cholesterol was examined by Amplex Red assay, and neutral lipid levels were analyzed by Oil red-O staining. Enzyme levels were investigated by western blot and QRT-PCR.

Simvastatin inhibited 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), decreasing cholesterol in PCa cells in the presence and absence of insulin; both insulin and simvastatin increased HMGCR mRNA message in LNCaP and 22RV1 cells. Protein levels of HMGCR increased in all treatments in 22RV1 cells, but the insulin induction of protein was quelled in LNCaPs. Furthermore, simvastatin treatment resulted in decreased protein and mRNA expression of key steroidogenesis enzymes, such as cytochrome p450 reductase 17A1 (CYP17A1). Additionally, insulin-induced steroid levels were suppressed by simvastatin as was lipogenesis in PCa cells.

We propose that insulin induction of steroidogenesis, cholesterol synthesis, and lipogenesis may contribute to PCa progression. Therefore, the inhibition of these pathways by simvastatin may prove a useful therapeutic tool.

24890 Insulin stimulates fatty acid synthesis and metabolism in androgen deprived human prostate cancer cell lines

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Colleen Nelson1, Amy Lubik2, Jennifer Gunter1, Melanie Lehman1, Carolina Soekmadji1, Chenwei Wang1, Martin Gleave2, Stephen Finn3, Michael Pollak4 and Adrian Herington1

1Queensland University of Technology and Translational Research Institute, Australia

2Vancouver Prostate Centre at Vancouver General Hospital, Canada

3Trinity College Dublin, Ireland

4McGill University, Canada

Fatty acid synthase (FASN) and lipid metabolism provides preferential fuel for prostate cancer (PCa) growth and increased FASN expression correlates with aggressive and high grade PCa. Androgens drive tumour growth and survival (including regulation of FASN) thus, androgen deprivation therapy is used in advanced PCa patients. A major side effect of reduced testosterone levels in men is the development of insulin resistance resulting in hyperinsulinaemia, which has also recently been associated with high grade PCa and more rapid progression to castrate resistant prostate cancer (CRPC). We hypothesised that, in androgen-deprived conditions, insulin may also drive FASN expression and activity, as in liver and adipose tissue. We identified by microarray analysis, several integrated lipogenic and lipid metabolic pathways (including lipid-activated steroidogenesis, ï ¢-oxidation and prostaglandin and leukotrine synthesis) significantly upregulated by insulin. We also identified expression of genes within these pathways are upregulated in clinical prostate tumour samples. We used QRT-PCR, western blot and immunofluorescent microscopy in LNCaP and 22RV1 PCa cell lines to demonstrate insulin upregulated genes and proteins which resulted in increased lipid synthesis and mobilisation from intracellular stores. Cellular fatty acid levels were measured by GC–MS and LC–MS and show insulin induces de novo fatty acid synthesis and promotes lipid accumulation, assessed by Oil-Red O assay. We propose that this insulin induction of fatty acid synthesis and lipid-mediated pathways may contribute to PCa progression and suggest that the fatty acid related pathways are attractive targets for PCa therapy, especially in patients with ADT-induced metabolic syndrome/ hyperinsulinaemia.

24894 Evaluation of the fatty acid synthase inhibitor triclosan in prostate cancer cells

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Martin Sadowski1, Rebecca Pouwer2, Amy Lubik3, Stephen McPherson1 and Colleen Nelson1

1Australian Prostate Cancer Research Centre, Queensland, Australia

2Eskitis Institute, Australia

3Vancouver Prostate Centre, Canada

Inhibition of the metabolic oncogene fatty acid synthase (FASN) has emerged as a promising target for the prevention and treatment of cancer, and numerous natural and synthetic FASN inhibitors have been investigated. However, chemical instability, low solubility and oral bioavailability, appetite suppression, weight loss, or general toxicity challenge their suitability for clinical testing. The synthetic FASN inhibitor triclosan (TCS), which was initially developed as an antibacterial agent, is not troubled by these pharmacological limitations. Yet, little is known about its mechanism and efficacy in inhibiting growth of cancer cells. In this study, we tested TCS for the first time in a panel of five prostate cancer cell lines and compared its efficacy to the well known synthetic FASN inhibitor C75. TCS was cytotoxic and induced apoptosis with a several-fold lower half-maximal inhibitory concentration (IC50) than C75. Rescue experiments confirmed that end product starvation was a major cause of cytotoxicity. Consistent with this, reduction of FASN expression and fatty acid synthesis exacerbated the cytotoxic effect of TCS. Interestingly, TCS induced different changes to cell morphology, cell cycle and lipid content in LNCaP cells when compared to C75, suggesting that inhibition of different partial catalytic activities of FASN can generate distinct phenotypes. Analysis of a panel of TCS derivatives with substitutions to the hydroxyl group of the phenol ring highlighted the importance of this group to reduce cellular lipid levels and to induce cytotoxicity. Together, these results establish that TCS is a potent FASN inhibitor and cytotoxin in prostate cancer cells.

24902 Tetraspanin protein turnover rates vary in prostate cancer cell lines

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Judith Weidenhofer1, Danielle Bond2, Ben Copeland2 and Leonie Ashman2

1The University of Newcastle, Hunter Medical Research Institute, Australia

2The University of Newcastle, Australia

Objective

Prostate cancer is notable for its heterogeneity in the molecular characteristics of tumours as well as its disease progression and therefore patient outcomes. The tetraspanins are a family of proteins of which many are implicated in the progression of a number of cancers generally through having altered expression compared to controls. In particular CD151 (metastasis enhancer) and CD9 (metastasis suppressor) have shown prognostic potential in patient samples as well as a direct involvement in prostate cancer metastasis in mouse models. However it is currently unknown what drives the alterations in expression of CD151 and CD9 seen in the different stages of prostate (and other) cancer. For CD151 and CD9 to be translated to clinical prognostic use and potentially as novel therapeutic targets it is critical to determine what mechanisms control their deregulated expression.

Methods

Normal prostate and prostate cancer cell lines were examined for the rate at which CD151 and CD9 protein expression was turned over via the lysosomal or proteasomal pathways using inhibitors of these processes and western blotting for CD151 and CD9. The role of ubiquitin in these processes is being examined with immunoprecipitation and modulation of the expression of specific ubiquitin E3 ligases in prostate cancer cell lines.

Results

Generally CD151 and CD9 were processed via the lysosome to the greatest extent. However differing rates were observed within the 16h of proteasome or lysosome inhibition in different cell lines. The WPE1-NB26 cell line showed no alteration in CD151 levels suggesting CD151 is very stable in this highly tumourigenic line. Whereas CD151 was predominantly turned over via the lysosome in the normal RWPE1 line. Conversely CD9 showed similar turnover rates in RWPE1 and WPE1-NB26 cells.

Conclusions

Different prostate cancer cells control the levels of tetraspanins CD151 and CD9 via different mechanisms. This suggests that CD151 and CD9 could be used as prognostic biomarkers with greater specificity if a co-marker such as the level of their ubiquitin E3 ligase was also determined allowing patients with similar levels of CD151 for instance to be further stratified based on the mechanism leading to the altered expression.

24910 Androgen responsive non-coding RNA regulates the expression of prostate cancer associated CTBP1in LNCaP

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Gregor Tevz1, Melanie L. Lehman2, John Lai1, Jiyuan An1, Chenwei Wang1, Stephen C. Hendy2 and Colleen C. Nelson1

1Queensland University of Technology, Institute of Health and Biomedical Innovation, Australia

2Vancouver Prostate Centre, Canada

The growth of prostate cancer (PCa) initially depends on androgens, however, androgen deprivation therapy invariably selects for prostate cancers that can survive in castrate levels of androgens. The mechanisms that orchestrate this transition are still unknown. The most studied mechanism of androgen receptor action is through binding to hormone response elements upstream of responsive genes. However, genome-wide analyses have revealed that there are many androgen-regulated genes that do not recruit AR to their promoters or enhancer regions, suggesting that AR-mediated transcriptional regulation might be more complex than classical dogma. Strand specific RNA sequencing (ssRNA-seq) has unveiled ubiquitous overlapping RNA transcripts in humans, which can interfere with each other via transcriptional interference, post-transcriptional silencing or antisense-induced methylation. The regulatory mechanisms within such multitasking genomic loci (MGL) could extend the reach of androgen regulation to overlapping transcripts shifting the paradigm of gene regulation by androgens. We used ssRNA-seq to identify MGLs in LNCaP cells and demonstrated the principle of action on CTBP1, a co-repressor that is overexpressed in aggressive and metastatic PCa, and a repressor of many tumour suppressors. We reveal that androgen-regulated antisense non-coding RNA (CTBP1as) is transcribed from the opposite strand, which could interfere with the expression of CTBP1. We validated the expression of the overlapping transcripts by qPCR and western blotting, and investigated the linked expression by silencing CTBP1 or CTBP1as. While androgen treatment simultaneously increased CTBP1as and decreased CTBP1 levels, the knock-down of CTBP1as increased mRNA and protein levels of CTBP1. The current study challenges the classical dogma of androgen regulated transcription in PCa and sheds new light on the previously overlooked mechanism of androgen regulation within multitasking genomic loci.

24922 Novel LAT3 inhibitor suppresses prostate cancer cell proliferation

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Qian (Kevin) Wang1, Josep Font1, Tanja Grkovic2, Rebecca Pouwer2, Mika Jormakka1, Renae Ryan3, John Rasko1, Ronald Quinn2 and Jeff Holst1

1Centenary Institute, Australia

2Griffith University, Australia

3The University of Sydney, Australia

Objective

L-type amino acid transporter 3 (LAT3) is a leucine transporter which is highly expressed in primary prostate cancer. We have shown that androgen receptor signaling up-regulates the expression of LAT3 and thereby intracellular leucine levels, cooperating with PTEN mutations to activate mTORC1 signaling, regulating protein translation and cell growth in vitro and in vivo. In this study, we screened and identified novel compounds that inhibit LAT3.

Methods

we used a high throughput leucine uptake assay, followed by liquid chromatography-mass spectrometry and 1H NMR to screen and identify the compounds blocking LAT3 from Nature Bank, a natural compound library containing biota samples from Australia, China and Papua New Guinea. We performed leucine uptake assay in Xenopus oocytes and LNCaP cells to characterise the compounds. We also examined the cell proliferation using MTT assay and BrdU incorporation assay.

Results

we screened more than 4,500 fractions from Natural Bank and identified two compounds (2-4-2 and 2-4-6) that inhibited leucine uptake. Using oocytes overexpressing LAT1, LAT2, LAT3 and LAT4, we identified that compound 2-4-6 specifically inhibits LAT3 transport activity. Using the prostate cancer cell line LNCaP, which expresses high levels of LAT3, the IC50 of compound 2-4-6 was determined to be 8.12 ± 1.42 μM. We showed that cell viability was decreased ∼20% in the presence of 50 μM 2-4-6 in LNCaP cells, due to suppression of cells from entering S phase.

Conclusion

Our study suggests that compound 2-4-6 is a specific LAT3 inhibitor that can suppress prostate cancer growth.

24926 Targeting copper transport for improving chemotherapy of prostate and breast cancer

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Bobby Boumelhem, Natalie Wee, Janine Street, Stuart Fraser and Stephen Assinder

The University of Sydney, Australia

Objective

Whilst platinum-based drugs (eg. cisplatin) are an effective treatment for a number of cancers, prostate and breast cancers are unresponsive. The copper transporters CTR1 and CTR2 mediate cellular influx of cisplatin. However, the understanding of copper transporter regulation in cancer is poor. Cisplatin resistance is possibly due to altered expression of CTRs. The aims of this study were to compare CTR1 and 2 levels with cisplatin sensitivity and determine if copper chelation can increase sensitivity.

Methods

Proliferation and cell cycle of DU145, PC3 and LNCaP cells treated with either 10 μM cisplatin, 10 μM cisplatin/30 μM tetrathiomolybdate (TTM; copper chelator) or vehicle-treated control were assayed by crystal violet and flow cytometry respectively. Sensitivity to cisplatin was compared with levels of CTR1 and CTR2 as determined by western blot analysis and confocal microscopy.

Results and Conclusions

Both PC3 and DU145 cell lines were sensitive to cisplatin, with cell numbers significantly reduced with increased sub-G1 phase cells. LNCaPs were unresponsive. Both PC3 and DU145 had low levels of CTR1 and mid/high levels of CTR2. CTR2 levels were lower in LNCaPs and CTR1 was undetectable. Whilst TTM did not increase sensitivity, cisplatin treatment altered cellular localisation of CTR2. In conclusion, the presence of CTR1 and CTR2 correlated with cisplatin sensitivity. In contrast to observations in ovarian cancer chemosensitivity was not increased by copper chelation in the cell lines examined. Our future work is focused on understanding regulation of CTR1 and 2 expression in prostate and in breast cancer, and in cancer stem cells.

24970 A novel and inhibitable pro-metastatic mechanism in prostate cancer

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John Hooper1, Yaowu He1, Andreas Wortmann1, Berta Casar2, Mark Adams1 and James Quigley2

1Mater Research Institute, Australia

2The Scripps Research Institute, United States

Objective

Uncontrolled proteolytic activation of the cell surface glycoprotein CUB domain containing protein 1 (CDCP1) occurs in a number of malignancies including prostate cancer. Our objective was to examine whether this dysregulated activation is accompanied by a functional role for CDCP1 in cancer progression.

Methods

A role for CDCP1 in metastasis was examined using two animal models of vascular dissemination of tumor cells – chicken embryos and mice. These assays used a highly metastatic variant of prostate cancer PC3 cells (PC3-hi/diss) that expresses high levels of CDCP1, HeLa cervical cancer cells overexpressing CDCP1 (HeLa-CDCP1) and HEK293 kidney cells expressing mutated CDCP1 that can not be proteolytically activated. Molecular and cell biology approaches were employed to identify the mechanisms leading to proteolytic activation of CDCP1 and downstream signalling pathways that promote cancer cell survival. Further in vivo experiments explored the importance of CDCP1's proteolytic activation in malignant transformation. Blockade of CDCP1 function was achieved using molecular approaches as well as monoclonal antibodies that are exquisitely specific for this cell surface receptor.

Results

Silencing of CDCP1 substantially reduced the ability of PC3-hi/diss cells to metastasize via the chicken embryo vasculature. Consistently, CDCP1 expression markedly increased the ability of HeLa cells to metastasise via the vasculature of mice following injection into the tail vein. Also supporting a functional role for CDCP1, vascular metastasis of PC3-hi/diss and HeLa-CDCP1 cells in chicken embryos and mice was effectively blocked using anti-CDCP1 antibody 41–2. Our data indicate that CDCP1 facilitates survival soon after tumor cell arrest within capillaries and that blockade of CDCP1 with antibody 41–2 induces tumor cell apoptosis. Using proteomics approaches we identified R368 and K369 as the sites at which CDCP1 is activated by serine proteases in prostate and other cancer cell lines. Comparison of the metastatic ability of HEK293 cells stably expressing wildtype CDCP1 or activation deficient CDCP1 (CDCP1-R368A/K369A), demonstrated that proteolytic activation of this receptor is essential for its role in cancer progression. By using biochemical approaches we demonstrated that CDCP1's role in metastasis requires initiation of pro-survival signalling within cancer cells that involves the kinases Src, PKCδ and Akt.

Conclusions

These data indicate that the cell surface receptor CDCP1 promotes the dissemination of cancer cells via the blood stream. They also indicate that blockade of CDCP1 activation is an effective way to reduce metastasis. In conclusion, targeting the CDCP1 pro-metastatic pathway may be a realistic approach to reduce distant metastasis in patients who have undergone apparently curative surgical or radiation therapy but are at risk of having subclinical residual disease.

24982 Prostate cancer protein markers to determine optimum treatment strategy at diagnosis

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Jonathan Dunne1, Judy Murray2, Brett Delahunt2, Bill Jordan1 and David Lamb2

1Victoria University of Wellington, New Zealand

2Wellington School of Medicine and Health Sciences, New Zealand

Objective

Serum PSA dynamics after radiotherapy of locally advanced CaP stratifies patients into two groups that differ markedly in disease behaviour, implying fundamental differences between cancers exhibiting indolent or aggressive clinical course. We aim to define a set of proteins whose abundance will accurately predict the risks associated with an individual's cancer. The research is distinctive because the samples are from formalin-fixed paraffin-embedded (FFPE) biopsies from patients with long-term outcome data.

Methods

1D-GeLC, 2DE, and LC-MALDI-MS/MS were used to establish a FFPE prostate protein database. Candidate markers were selected according to their potential role in tumour progression. Western blot (WB) analysis was used to establish the relative abundance of each marker in matched tumour/control tissue from patients with favourable/poor long-term survival. WB results are validated by iTRAQTM analysis of the same tissue.

Results

Using LC-MALDI, 1D-GeLC, and 2DE >250 proteins were detected in FFPE CaP tissue, including proteins believed to mediate CaP metastasis. The relative abundance of several candidates varies according to tissue/tumour type. We are validating these results using iTRAQTM analysis of the same tissue set. Several PSA isoforms are also detected by 2D-WB. We are examining the correlation between these isoforms and tissue/tumour type, and if they may improve risk prediction.

Conclusion

This research is clinically significant as it may allow tailoring of CaP treatment in ways that are not currently possible. Patients with an aggressive protein profile can be treated immediately using a systemic/local treatment strategy, whereas those with an indolent profile can be confidently recommended watchful-waiting.

24990 Kallikrein-related peptidase 4initiates tumour-stroma cross-talk in prostate cancer

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Ruth Fuhrman-Luck1, Marcus Hastie2, Scott Stansfield1, Thomas Stoll2, Anja Rockstroh3, Melanie Lehman3, Colleen Nelson3, Daniela Loessner1, Jeffrey Gorman2 and Judith Clements1

1Institute of Health and Biomedical Innovation, Queensland University of Technology, Australia

2Queensland Institute for Medical Research, Australia

3Australian Prostate Cancer Research Centre – Queensland, Queensland University of Technology, Australia

Background

The prostate stromal niche is increasingly been recognised as necessary for prostate cancer progression. Cancer-associated fibroblasts (CAFs) lining the tumour boundary can transform benign epithelium, in vivo, and factors secreted by epithelial cancer cells can ‘activate’ normal fibroblasts to become CAFs. Understanding this cross-talk is essential in developing novel therapies.

Kallikrein-related peptidase 4 (KLK4) is a serine protease which is over-expressed in prostate cancer and in pre-cancerous lesions, from which CAFs are believed to originate. KLK4 is expressed by both luminal and basal prostate epithelial cells, thus likely mediating interactions with surrounding stroma.

Objective

We aimed to elucidate the role of KLK4 in tumour-stroma cross-talk, by identifying its fibroblast-secreted substrates and resulting fibroblast gene expression changes.

Methods

Substrate screening utilised the ‘Protein Topography and Migration Analysis Platform’ [1]. Briefly, fibroblast-secreted proteins were digested with active recombinant KLK4 (catalytically inactive KLK4 control) and separated via SDS-PAGE. The gel was sliced into equivalent horizontal sections and proteins extracted and sequenced (LC-MS/MS) to identify those KLK4 cleavage products with increased migration (treated lane) relative to the intact parent protein (control lane). Gene expression changes were analysed via microarray.

Results and Conclusions

The approach identified 72 putative novel KLK4 substrates in fibroblast secretions and 3 established targets, whose detection served to validate our method. KLK4-treated fibroblasts expressed elevated levels of a number of genes consistent with a CAF genotype. Cell-based validation of the effects of KLK4-stimulated fibroblast secretions on prostate cancer epithelial cells is currently being undertaken.

[1] Dix et al., Cell, 2008.

25038 YKL40as a therapeutic target for the treatment of metastatic Prostate Cancer

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Varinder Jeet and Colleen Coyne Nelson

Queensland University of Technology, Australia

YKL40 is a secreted glycoprotein shown to be highly expressed in patients with advanced cancers including breast cancer, colorectal cancer, glioblastoma and prostate cancer (PCa). The exact function of YKL40 is poorly understood but it is presumed to play an important role in promoting tumour angiogenesis and metastasis of cancer cells. However, the therapeutic value and biological functioning of YKL40 is unknown in PCa. In our study, we examined the expression and function of YKL40 for the first time in PCa. Methods: YKL40 mRNA and protein expression profiles were analysed in PCa cell lines and tissues representing progressive stages of human disease. Cells were treated with androgens and anti-androgens to determine the androgen specific regulation of YKL40. siRNA mediated YKL40 specific knockdown and lentivirus mediated overexpression was performed in LNCaP and C42-B cell lines and the effects on malignant transformation were analysed by real time PCR, immunoblotting, scratch wound healing, matrigel invasion, and anchorage independence assays. Results: The results show that 1.) YKL40 is responsive to androgens 2.) Gene and protein expression of YKL40 increases in cell lines and PCa tissues representative of advanced disease as compared to less aggressive cancer 3.) Knockdown of YKL40 significantly decreases invasion and migration of PCa cells whereas overexpression renders them more invasive. Conclusion: Our study characterises the role of YKL40 for the first time in PCa. This has the potential to lead to a new targeting approach for therapeutic intervention in advanced prostate cancers that have thus far proved difficult to treat.

25054 Parthenolide as a selective radiosensitiser in the treatment and prevention of prostate cancer

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Katherine Morel1, Rebecca Ormsby1, Eva Bezak2, Wayne Tilley3, Mark Lawrence1 and Pamela Sykes1

1Flinders University and Medical Centre, Australia

2Royal Adelaide Hospital, Australia

3The University of Adelaide and Hansen Institute, Australia

Current prostate cancer therapies are associated with undesirable side effects and treatment resistance is common. New approaches for prevention and treatment are required. Parthenolide, a component of the herbal medicine feverfew, displays anti-inflammatory and anti-tumour properties in several cancer types. In vitro prostate cancer studies have demonstrated that parthenolide also selectively exhibits a radiosensitisation effect on prostate cancer cells, but not in primary prostate epithelial cell lines. We are utilising the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model to observe in vivo changes in tumour onset and radiosensitivity induced by parthenolide treatment.

We hypothesise that parthenolide treatment will inhibit prostate tumourigenesis (initiation and/or progression) by increasing tumour latency, increasing the anticancer effects of radiotherapy (high dose radiation) while reducing DNA damage to normal surrounding tissues. Endpoints will include time to palpable tumour, tumour size, histopathology, proliferation, DNA damage/repair (gamma-H2AX) and apoptosis frequency.

Our pilot studies on C57BL/6J mice have identified basal levels of apoptosis and proliferation in the prostate and surrounding tissues following three 40 mg/kg parthenolide treatments. Current and future in vivo experiments will investigate the effect of parthenolide on prostate cancer progression in the TRAMP model, and its potential radiosensitisation when administered prior to high dose radiation.

A better understanding of the role of parthenolide on the tumourigenic process and its selective radiosensitisation response in prostate could be utilised for cancer prevention and to improve radiotherapy methods.

This work is funded by the Flinders Medical Centre Foundation and a Smiling for Smiddy PhD Scholarship to KLM.

25134 Targeting epithelial-mesenchymal plasticity for improved treatments of advanced prostate cancer

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Brett Hollier1, Nataly Stylianou1, Abhishek Kashyap2, Lidija Jovanovic1, Melanie Lehman1, Chenwei Wang1, Jennifer Gunter1 and Colleen Nelson1

1Australian Prostate Cancer Research Centre, Queensland, Queensland University of Technology, Australia

2Institute of health and BIomedical Innovation, Queensland University of Technology, Australia

Objective

Recent evidence suggests a role for the epithelial-mesenchymal plasticity (EMP) in facilitating prostate cancer (PCa) progression and metastasis. EMP encompasses both the epithelial-mesenchymal transition (EMT) and the reverse process of mesenchymal-epithelial transition (MET) and the interplay between these two phenotypic states endows tumour cells with full metastatic competency. The role of EMP in PCa is only just starting to be revealed and has been hampered by a lack of experimental models capable of replicating the full spectrum of EMP as it occurs in vivo. The objective of the study is to develop and characterise unique cellular models able to better recapitulate EMP in PCa cells both in vitro and in vivo.

Results

We have successfully developed EMT-inducible and reversible PCa cell models capable of reflecting the complex phenotypic alterations thought to occur during EMP. Using these models we have developed novel 3D in vitro models of EMT-mediated cell invasion and genome-wide profiles of the temporal transcriptional changes occurring during EMP in PCa. These studies have identified preliminary candidates for therapeutic intervention that are part of ongoing studies to test the inhibition of EMP-mediated cell invasion and metastasis.

Conclusions

Targeting EMP is a promising strategy for inhibiting PCa metastasis. This project has developed unique cell lines to better model the process of EMP in the context of PCa invasion and metastasis. It is hoped that this will identify novel therapeutic targets that may have clinical utility in halting the metastatic spread of PCa and improve patient outcomes.

25230 The expression and function of GHSROS, a long non-coding RNA encoded by the opposite strand of the ghrelin receptor gene, in prostate cancer

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Patrick Thomas1, Carina M. Walpole1, Inge Seim1, Penny L. Jeffery2, Lidija Jovanovic3, Adrian C. Herington1, Colleen C. Nelson3, Eliza J. Whiteside1 and Lisa K. Chopin1

1Queensland University of Technology, Australia

2Mater Medical Research Institute, Australia

3Australian Prostate Cancer Research Centre, Australia

Prostate cancer is the most common cancer in men in Australia and will affect one in eight men during their lifetime. Treatments for advanced prostate cancer have improved considerably in recent years, however, metastatic prostate cancer remains incurable. The roles of long non-coding RNA (lncRNA) in disease are becoming increasingly appreciated, particularly in cancer. Non-coding RNAs have emerged as important orchestrators of gene expression and have significant potential as biomarkers for cancer. We have discovered a natural antisense long non-coding RNA (lncRNA), encoded by the opposite strand of the growth hormone secretagogue receptor (GHSR) gene, which we have termed GHSROS (GHSR opposite strand).

Objective

The objective of this study was to investigate the expression and function of GHSROS in prostate cancer.

Methods

GHSROS expression was investigated using strand-specific, quantitative real-time RT-PCR (qRT-PCR) in prostate cancer cell lines and clinical prostate cancer specimens. Using the xCELLigence real-time cell analysis system the effects of forced overexpression of GHSROS on proliferation, cell migration and invasion were measured (and compared to vector-only controls) in the PC3 prostate cancer cell line.

Results

GHSROS is expressed in prostate cancer cell lines and in clinical prostate cancer specimens and forced overexpression of GHSROS significantly increased cell proliferation and migration in PC3 prostate cancer cells, compared to vector controls.

Conclusions

Our study indicates that GHSROS may have clinical significance in prostate cancer progression as it is expressed in clinical cases of prostate cancer and functional studies indicate that it plays a role in cell proliferation, migration and invasion. GHSROS may provide a useful target for the development of novel antisense therapies for prostate cancer treatment.

25286 Germline HOXB13G84E mutation and prostate cancer risk: a systematic review and meta-analysis

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Wee Loon Ong

The University of Cambridge/ Radiation Oncology, AlfredHealth, United Kingdom

Objective

A novel rare germline mutation HOXB13 G84E was recently reported to be associated with increased prostate cancer risk. Numerous replication studies have been conducted to verify this association, with a wide range of odds ratio (OR) being reported. The aim of this review is to critically appraise all literature on the G84E mutation and prostate cancer risk published to date, and to obtain a more precise effect estimate of the G84E mutation.

Methods

A systematic literature search on the PubMed database was performed (up to March 2013), searching for the term “prostate cancer” and “HOXB13”. Pooled OR and 95% confidence interval were estimated using a random-effects model. Analyses were limited to population of European descent to control for population stratification.

Results

Nine studies investigating G84E mutation and prostate cancer risk were included in this review, with a total of 24064 prostate cancer cases and 21414 controls. The G84E mutation carriers are more prevalent among prostate cancer cases compared to controls (2.2% vs. 0.5%). The OR for prostate cancer was 8.4 (95%CI = 3.5–6.5) among G84E mutation carriers. Strongest associations were observed among cases with positive family history (OR = 9.3; 95%CI = 5.4–16.1) and early-onset disease (OR = 12.7; 95%CI = 7.6–21.3).

Conclusions

The findings confirm that the G84E mutation is strongly associated with increased prostate cancer risk. With further characterization of penetrance and lifetime risk associated with the mutation, there is a potential role for HOXB13 G84E genetic testing in clinical settings for individuals with a family history or early-onset prostate cancer.

25290 Single v/s multiple fraction radiotherapy for palliation of painful vertebral bone metastases in prostate cancer: a prospective study

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Prashant kumbhaj and Dr Rameshwaram Sharma

S.M.S Medical College, India

Objectives

Metastatic bone disease is a commonly encountered problem in oncology practice. It is most commonly seen in harmone refractory advanced carcinoma of prostate. The most cost effective treatment is radiotherapy (RT). Different fractionation schedule of RT can be used to treat such condition.

Pain response assessment in patients with vertebral bone metastasis after treating them with various radiation fractionations and to compare the toxicity profile in the treatment arms.

Methods and Meterials

A prospective randomized study was done to include total 51 patients coming to department of radiotherapy S.M.S Hospital jaipur,from July 2010 to jan 2013. Patients with histopathologically proven Prostatic carcinoma having symptomatic secondary deposits to vertebra were selected for the study. Patients with age >75 years, Karnofsky Performance Status (KPS) <40, features of cord compression were excluded from study. Patients were randomized to two arms (A) receiving multiple fraction of RT with 30 Gy in 10 fractions and (B) 625 cGy in 1 fraction.

Initial pain response was assessed using Visual Analogue Scale (VAS) and compared using the same scale at weekly interval up to 1 month after treatment completion.

Results

Arm A comprised of 25 patients while 26 patients were enrolled in Arm B. Baseline patient characteristics were comparable. Initial pain scores were 7.22 ± 0.754 and 7.52 ± 0.55 in arm A and arm B, respectively. Pain scores reduced significantly in both the arms after 1 month (4.27 ± 1.82 in arm A; 5.16 ± 2.39 in arm B). Time of initiation of pain response was earlier in arm A, statistically significant. Mild G-I toxicity was noted in both the arms but differences in two arms were not statistically significant, no interruption of treatment was required because of side effects.

Conclusions

Different fractionation of radiation has same response and toxicity in treatment of vertebral bone metastasis. Single fraction RT may be safely used to treat these cases as this is more cost effective and less time consuming for overburdened radiotherapy department. Studies may be conducted to find out particular subgroup of patients to be benefitted more by either fractionation schedule; however, our study cannot comment on that issue.

25310 Australian prostate cancer BioResource, 2013

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Trina Yeadon

Australian Prostate Cancer BioResource, Australia

Objectives

The Australian Prostate Cancer BioResource (APCB) provides an unique quality assured facility for the collection, storage and access to tissue to support research into the treatment and improved clinical management of men with prostate cancer. The APCB manages four federated nodes located in Brisbane, Sydney, Melbourne and Adelaide.

The APCB is jointly funded by the National Health and Medical Research Council (NHMRC) and Prostate Cancer Foundation of Australia (PCFA).

Results

The APCB provides a significant number of unique services which include:

  • fresh tissue
  • paraffin embedded tissue sections,
  • buffy coat cells, serum, plasma
  • tissue microarrays

To date the APCB has collected 110,496 samples from 4,379 men and has distributed 3,505 samples to 41different researcher groups on 70 occasions.

The APCB is firmly embedded in translational prostate cancer research within Australia and provides resources to build and contribute Australasian cohorts in large scale genetic, genomic and proteomic biomarkers studies. This includes the following research programs; PRACTICAL Genome Wide Association Study, Irish Biomarker Consortium, ICGC, the Movember GAP initiative. The APCB also provides expertise and infrastructure to bank samples from the RAVES trial.

Conclusions

The APCB has a collection of high quality prospectively collected prostate cancer tissue with clinical and pathological data acquired at diagnosis and surgery. This collection has become an important resource for prostate cancer research both nationally and internationally. With maturity and recruitment of 5,500 men by 2014 with 8 year clinical follow up it will become an even more significant research resource.

25354 Inactivation of ATM/ATR DNA damage checkpoint promotes androgen induced chromosomal instability in prostate epithelial cells

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Kai Dun Tang1, Yung Tuen Chiu2, Pak Shing Kwan2 and Patrick Ling1

1Queensland University of Technology, Australia

2The University of Hong Kong, Hong Kong

The ATM/ATR DNA damage checkpoint functions in the maintenance of genetic stability and some missense variants of the ATM gene has been shown to confer a moderate increased risk of prostate cancer. However, whether inactivation of this checkpoint contributes directly to prostate specific cancer predisposition is still unknown. Here, we show that exposure of non-malignant prostate epithelial cells (HPr-1AR) to androgen led to activation of the ATM/ATR DNA damage response and induction of cellular senescence. Notably, knockdown of the ATM gene expression in HPr-1AR cells can promote androgen induced TMPRSS2: ERG rearrangement, a prostate-specific chromosome translocation frequently found in prostate cancer cells. Intriguingly, unlike the non-malignant prostate epithelial cells, the ATM/ATR DNA damage checkpoint appears to be defective in prostate cancer cells, since androgen treatment only induced a partial activation of the DNA damage response. This mechanism appears to preserve androgen induced auto phosphorylation of ATM and phosphorylation of H2AX, lesion processing and repair pathway yet restrain ATM/CHK1/CHK2 and p53 signaling pathway. Our findings demonstrate that ATM/ATR inactivation is a crucial step in promoting androgen-induced genomic instability and prostate carcinogenesis.

25362 Loss of sprouty genes induce prostatic intraepithelial neoplasia

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Stephen Assinder, Daniella Beniamen, Marissa Perera and Frank Lovicu

The University of Sydney, Australia

Objective

Sprouty proteins are key antagonists of receptor tyrosine kinase (RTK) signalling. Dysregulation of FGF is a common cause of prostate cancer, with subsequent RTK hyperactivation of multiple signal transduction pathways. To limit the strength of RTK signalling, induction of Sprouty family (Spry) antagonists occurs. Concomitant inactivation of Spry1 and Spry2 in the mouse prostate has recently been shown to cause prostatic intraepithelial neoplasia (PIN). We hypothesised that suppression of either Spry1 or Spry2 alone would induce PIN.

Methods

Prostates from 16- and 24-week-old Spry1- or Spry2-deficient mice, either hemizygous (+/−) or homozygous (−/−) for the null allele, were histologically scored for PIN and compared with wild type (wt) mice prostates. PCNA immunohistochemistry was used to determine differences in cell proliferation between groups.

Results and Conclusion

Both Spry1−/− and Spry1+/− mice displayed significantly greater incidence of PIN at 24 weeks of age compared to wt mice, consistent with increased PCNA-positive cells in these prostates. Similarly Spry2−/− and Spry2+/− had greater proportions of PIN compared with wt mice, as early as 16 weeks of age. It is concluded that loss of either Spry1 or Spry2 results in the development of prostate intraepithelial neoplasia, consistent with our preliminary finding that levels of both Spry1 and Spry2 are significantly less in metastatic human prostate cancers. Overall, our findings clearly demonstrate the importance of negative regulators of RTK signalling, such as Spry, in the clinical setting.

25414 The cross-talk between androgens and cell cycle regulators in prostate tumorigenesis

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Joanna Dulinska-Litewka1, James A. McCubrey2, Zygmunt Dobrowolski1, Wacław Lipczyński1, Robert Jach1, Krzyszof Okon1 and Piotr Laidler1

1Jagiellonian University Medical College, Poland

2Department of Microbiology and Immunology, United States

Objective

The progression of prostate cancer from an hormone sensitive to a hormone resistant is likely due to mechanisms involving alterations in AR expression, amplification, mutations, and/or AR activity. Here we discuss the roles of key signaling pathways in prostate cancer in concern to their possible interaction with AR signaling.

Methods

Studies were carried out on human prostate cell lines and prostate tissues. Secretion of metalloproteinases was analyzed by zymography, and the invasive potential using Boyden chambers. Expression of cell signaling proteins in wild and siRNA transfected cells was analyzed at mRNA and protein level.

Results

Silencing of AR+ prostate cells with siRNA significantly reduced proliferation, expression of nuclear β-catenin, metalloproteinases and increased expression of p21, p27 and E-cadherin. Expression of N-cadherin increased in the absence of androgens in primary tumors indicating a possible involvement of the AR in the regulation of N-cadherin. Its expression was associated with Gleason score and was increased in castration-resistant tumors in patients with established metastases. Silencing of N-cadherin in PC-3 cell line resulted in re-expression of E-cadherin.

Conclusions

In prostate cells either AR or Akt signaling prevails, depending on their initial androgen sensitivity as well as its availability. In androgen-independent prostate cancer cells, Akt takes over the role of AR and more effectively contributes through the same signaling molecule, β-catenin, to cancer progression. Akt can be a key target in prostate cancer. In addition we suggest that the expression of N-cadherin was influenced by androgen deprivation and was associated with metastasis in prostate cancer.

This work was supported by MNiSzW grants: K/ZDS/003700, K/PBW/000561 – Jagiellonian University Medical College, Krakow, Poland.

25434 Post-transcriptional regulation of CD151& CD9 by miRNAs in prostate cancer

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Danielle Bond, Murray Cairns, Leonie Ashman and Judith Weidenhofer

The University of Newcastle, Australia

Objective

Tetraspanins are involved in many prostate cancer processes involving cell adhesion and migration. The tetraspanin CD151 (pro-metastatic) is typically over-expressed in prostate cancers which correlates with poor patient outcomes, whereas CD9 (metastasis suppressor) is generally under-expressed and this is associated with poor patient prognosis. However, progress in developing CD151 and CD9 as prognostic biomarkers and therapeutic targets is hampered by a lack of understanding of how they are regulated in cancers. Bioinformatics analysis has identified around 250 miRNA which are predicted to bind CD151 or CD9. Thus we aim to investigate if miRNAs are able to regulate the expression of tetraspanins in prostate cancer in vitro.

Methods and Results

A dual luciferase reporter assay using the 3'UTR of CD151 and CD9 provided evidence that prostate cell lines endogenously express miRNA that regulate tetraspanins and that the level of protein expression regulation varies across the cell lines. Analysis of miRNA expression in normal and cancer cell lines using miRNA arrays and qPCR has confirmed that some miRNAs which are predicted to target CD9, are up-regulated in many prostate cancer cell lines compared to non-tumourigenic cells. Conversely, miRNA were identified with low expression that are predicted to target CD151. These miRNA have also been shown to directly regulate CD151 or CD9 using the dual luciferase 3'UTR reporter assay.

Conclusion

Altered miRNA expression may contribute to prostate cancer by manipulating tetraspanins to allow progression and metastasis. Therefore, miRNA may be effective prognostic and/or therapeutic targets for prostate cancer in the future.

25486 Development of a bioengineered model to quantitatively measure the tumorigenic properties of cancer-associated fibroblasts in human prostate cancer

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Ashlee Clark1, Anna Taubenberger2, Renea Taylor1, John Pederson3, Mark Frydenberg1, David Pook1, Mitchell Lawrence1, Stuart Ellem1, Dietmar Hutmacher2 and Gail Risbridger1

1Monash University, Australia

2Queensland University of Technology, Australia

3TissuPath, Australia

Stromal-epithelial cell interactions play an important role in cancer and the tumor stroma is regarded as a therapeutic target. In vivo xenografting is commonly used to study cellular interactions not mimicked or quantified in conventional 2D culture systems. To interrogate the effects of tumor stroma (cancer-associated fibroblasts or CAFs) on epithelia, we created a bioengineered microenvironment using human prostatic tissues. Patient-matched CAFs and non-malignant prostatic fibroblasts (NPFs) from men with moderate (Gleason 7) and aggressive (Gleason 8–9 or castrate-resistant) prostate cancer were cultured with non-tumorigenic BPH-1 epithelial cells. Changes in the morphology, motility and phenotype of BPH-1 cells in response to CAFs and NPFs were analysed using immunofluorescence and quantitative cell morphometric analyses. The matrix protein gene expression of CAFs, with proven tumorigenicity in vivo, had a significantly different gene expression profile of matrix proteins compared to patient matched NPFs. In co-culture with CAFs (but not NPFs), BPH-1 cells had a more invasive, elongated phenotype with increased motility and a more directed pattern of cell migration. CAFs from more aggressive tumors (Gleason 8–9 or CRPC) were not quantitatively different to moderate grade CAFs. Overall, our bioengineered microenvironment provides a novel in vitro platform to systematically investigate the effects of tumor stroma on prostate cancer progression.

25534 Protein expression alterations in a unique gleason prostate cancer progression cohort

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Lisa Philp, Tanya Day, Shalini Jindal, Marie Pickering, Lisa Butler and Wayne Tilley

The University of Adelaide, Australia

Objective

Recognizing key events involved in prostate cancer (PCa) initiation and progression is critical to improve diagnosis and prognosis. Few robust clinical cohorts are available to analyse multiple disease-stages. We assembled a patient cohort using Gleason grade as a marker of progression. Tissue microarrays (TMA) were constructed to study factors potentially altered during PCa progression.

Methods

TMAs featured duplicate cores of non-malignant prostate, prostate intraepithelial neoplasia (PIN), Gleason score (G) 6 PCa (grade (g) 3 + 3), G7 PCa (3 + 4, 4 + 3), G8 PCa (3 + 5, 4 + 4) and G9 PCa (4 + 5, 5 + 4). Protein expression was evaluated for proliferation marker, Ki-67; androgen receptor (AR); and AR co-chaperone, SGTA. Staining was scored manually and/or using Definiens Tissue Studio image analysis software.

Results

Ki-67 expression increased with PCa progression, in line with previous reports, validating our cohort. AR expression increased with increasing grade. SGTA, which influences AR's subcellular localisation, increased in PIN and PCa, irrespective of grade. AR and SGTA expression were influenced by both primary and secondary grade. Specifically, primary g4 samples exhibited greater staining when the secondary grade was 4/5, and less staining when secondary grade was 3. The interaction between primary and secondary Gleason grades has the potential to direct future clinical decisions and can only be detected using our TMA design. We showed concordance between manual scoring and Tissue Studio analyses.

Conclusions

Using a novel progression cohort, we demonstrate stage-specific expression of factors involved in prostate tumorigenesis and provide novel evidence of an interaction between primary and secondary Gleason grades in clinical samples.

25538 Use of the CellSearch® platform in enumeration and profiling of circulating tumour cells

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Lidija Jovanovic1, Mackenzie Mills1, Allison Allison2, Simon Wood3, Margot Lehman4, Elizabeth McCaffrey5, Irina Oleinikova3 and Colleen Nelson1

1Australian Prostate Cancer Research Centre, Queensland, Australia

2Australian Prostate Cancer BioResource, Australia

3Department of Urology, Princess Alexandra Hospital, Queensland Health, Australia

4Department of Radiation Oncology, Princess Alexandra Hospital, Queensland Health, Australia

5Department of Medical Oncology, Princess Alexandra Hospital, Queensland Health, Australia

Objective

Circulating tumour cells (CTCs) detach from primary tumours and circulate in blood of patients with metastatic carcinomas. The presence and number of CTCs, as detected on the CellSearch® system (Veridex, LLC), are associated with patient's prognosis, and predictive of progression-free and overall survival. Patients with high CTC counts at baseline have more aggressive disease and shorter overall survival than patients with lower baseline CTC count. However, decrease in CTCs during treatment is associated with better overall survival, indicating that CTC numbers can be used to monitor treatment response. We evaluated CTC enumeration and user-defined marker detection in different carcinoma types using the CellSearch® platform.

Methods

Blood samples from cancer patients were collected in CellSave tubes. CellSearch Epithelial kits were applied for enrichment and enumeration, and CXC kits were used for detection of user-defined markers on CTCs. Immunomagnetic capture and fluorescent staining of CTCs was performed on the CellTracks ®Autoprep® system, and the CellTracks Analyzer II® was used for enumeration and phenotyping of CTCs.

Results

CTCs have been successfully detected in patients with prostate, breast, colorectal, renal, non-small lung carcinomas and carcinoma of unknown primary. Using fluorescently-labelled monoclonal antibodies we developed tests for detection of putative biomarkers on CTCs which may have a role in cancer progression.

Conclusions

We successfully detected CTCs in a number of patients with different carcinoma types. Our results indicate that CellSearch® platform is reliable and highly reproducible tool for

CTC enumeration which we used in a in a number of clinical, basic research and commercial projects.

25566 Development of automated, multiplexed protein and gene profiling assay for circulating tumor cells (CTCs) of metastatic

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Dea Nagy, Karl Garsha, Steven Yun, Janice Riley, Elias Liabotis, Michael Otter, Eric Walk, Ryan Dittamore, Raymond Nagle and Thomas Grogan

Ventana Medical Systems, A Member of the Roche Group, United States

Background

Next Generation Sequencing (NGS) leads toward the identification of molecular-heterogeneity of mCRPC which can be correlated with clinical-heterogeneity.

CTCs offer plausible targets for continuous monitoring the mCRPC progress and therapeutic response due to their relatively easy accessibility.

Molecular characterization of CTCs with multiple biomarkers offers further tools in patient risk stratification and for companion diagnostics.

In these studies we sought to characterize CTCs for the simultaneous presence of selected biomarkers using immunofluorescence (IF) and fluorescence in situ hybridization (FISH).

Methods

CTCs from mCRPC patients were attached to glass slides utilizing different CTC-isolation technologies. The attached cells offer a critical interphase for IF/FISH assay on automated staining instrument (Ventana). Cytokeratin-positive/CD45-negative cells with an intact nucleus were identified as CTC and were further interrogated by either multiplexed IF with anti-AR, -ERG, and -PTEN antibodies, or multiplexed Quantum Dot FISH with 5′ERG, 3′ERG, PTEN, and Cen10 probes. The assays were validated in FFPET prostate cancer cell line xenografts, and biopsy samples. IF and FISH signals were visualized by spectral imaging (Ventana).

Results

The automated IF/FISH staining procedure facilitated multiplex characterization of individual CTCs for both protein (AR, ERG, and PTEN) and genomic biomarkers 5'ERG, 3'ERG, PTEN, and Cen10.

Conclusion

This method for automated, multiplex molecular characterization of CTCs in mCRPC patients might aid oncologists in identifying and stratifying patients likely to respond to targeted therapies. The method could provide high throughput option for companion diagnostics, and analysis of future biomarkers.

25582 Tetraspanins as potential biomarkers in prostate cancer

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Ben Copeland and Leonie Ashman

The University of Newcastle and Hunter Medical Research Institute, Australia

Objective

Prostate cancer (PCa) in Australia is the most commonly diagnosed cancer and second most common cause of mortality in males. However, PCa is a very heterogeneous disease and in many cases follows an indolent course. Current classification systems such as Gleason grade have limited prognostic values in some cases. A key issue is deciding on treatment strategies, which can be invasive and result in possible side effects. Hence new prognostic markers are urgently needed to guide PCa treatment.

Methods

Tissue microarrays (TMAs) containing tumours and matched normal tissue samples from 150 patients were obtained from the Australian Prostate Cancer Collaboration. We examined expression patterns of the tetraspanin surface protein molecules CD151 (x2), CD82, CD9 and Tspan 8 by immunohistochemistry and quantified the IHC intensity by manual pathological scoring. Analysis of the associated clinical follow-up data is has commenced.

Results

Expression of CD151 (both) and Tspan 8 was positively correlated with advanced PCa, while expression of CD82 and CD9 was negatively correlated with PCa progression. Wilcoxen test between matched tumour/normal samples for CD151 (RLM30) p = 0.0167, CD151 (11B1) p < 0.0001, Tspan 8 p < 0.0001, CD9 p < 0.0001, CD82 p < 0.0001 MS. CD151 expression was significantly associated with Gleason score (p = 0.043) and showed a trend towards relapse (p = 0.060). CD82 expression is significantly correlated with age at diagnosis (0.003) and relapse (p = 0.049).

Conclusions

Adjunct IHC for tetraspanins of biopsies may prove useful when incorporated into the PCa diagnostic process to provide additional information to help stratify patients and delineate appropriate treatment modalities.

25610 A Novel exosome based test for prostate cancer

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Vicki Velonas1, Cris dos Remedios1, Henry Woo2, Elena Kosobrodova3, YingYing Su4 and Stephen Assinder1

1Bosch Institute, The University of Sydney, Australia

2Sydney Adventist Hospital Clinical School, The University of Sydney, Australia

3Department of Applied Plasma and Physics, School of Physics, The University of Sydney, Australia

4Advanced Microscopy Facility, Bosch Institute, The University of Sydney, Australia

Objective

The prostate specific antigen (PSA) test used in the diagnosis of prostate cancer (PCa) lacks both sensitivity and specificity. Furthermore, it cannot predict outcome or treatment options. We are developing a new test based on changes in circulating exosomes that distinguishes patients who are healthy from those who have varying stages of malignant disease. Exosomes are small (30–100 nm diameter) vesicles secreted by most cell types but significantly increased in cancer. They contain intracellular and extracellular proteins that promise to be potential diagnostic and prognostic PCa cell markers. Our aim is to identify a “signature” of PCa using antibody microarray analysis of secreted exosomes derived from patient blood samples.

Methods

Blood samples (10 mL) were collected from 4 groups of patients with varying stages of malignancy enrolled at the Sydney Adventist Hospital. Exosomes were isolated from plasma (4 mL) by differential centrifugation. Nanosight LM10-HS was used to count and analyse the isolated particles using exosome specific fluorophores. Furthermore, exosomes were applied to plasma immersion ion implanted polycarbonate microarrays containing PCa related Cluster of Differentiation (CD) antibodies. SEM was carried out to visually examine the morphology of exosomes.

Results

On the basis of 20 test samples, statistical analysis identified proteins that are differentially expressed between healthy, clinically significant and clinically insignificant PCa subjects with an AUROC value >0.75. These proteins may promote tumour progression in PCa.

Conclusion

Nanosight and antibody microarray analysis of candidate exosome markers can be used for stratification of PCa patients from non- cancer individuals.

25626 Nuclear localization of the receptor tyrosine kinase EphB4in prostate cancer cells

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Sally-Anne Stephenson, Inga Mertens-Walker, Jessica Lisle, William Nyberg, Carson Stephens, Leslie Burke, Raphael Rutkowski and Adrian Herington

Queensland University of Technology, Australia

The EphB4 receptor tyrosine kinase is over-expressed in 66% of prostate cancers and contributes to viability, migration and invasion. Abundant evidence reported in the last decade shows that many receptor tyrosine kinases can be translocated to the nucleus where they have further biological functions including regulation of gene expression. We have identified EphB4 in the nucleus of prostate cancer cells.

Objective

A well described mechanism for nuclear translocation is the binding of importin proteins to an epitope within the cargo protein called a nuclear localization signal (NLS) with import via the nuclear pore complex. Two potential NLS sequences were identified in EphB4. The aim of this study was to determine whether both putative NLS sequences were functional and explore the function of EphB4 in the nucleus.

Methods

Fragments of the EphB4 sequence containing each NLS were cloned into pEGFP-N1 to create NLS-GFP fusion proteins. Immunofluorescence was used to examine cellular localisation after expression in prostate cancer cell lines. Chromatin immunoprecipitation was used to identify genomic DNA sequences to which nuclear EphB4 binds.

Results

Both NLS-GFP proteins moved into the nuclei of transfected cells demonstrating both NLS sequences are functional. A pull-down approach was used to demonstrate a direct interaction between EphB4 and α-importin. Several interesting EphB4-interacting genomic loci have been identified, some of which contain genes already linked to prostate cancer.

Conclusion

The discovery of EphB4 in the nuclei of cells may indicate a potential function in gene regulation adding further complexity to the biology of this important cancer-associated protein.

25686 Role of GADD45G in transcriptional regulation of androgen-responsive genes and prostate cancer cell survival

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Raja Vasireddy1, Dwan Cochrane2, Amy Lubik2, Ladan Fazil2, Brett Hollier1, Melanie Lehman1 and Colleen Nelson1

1Australian Prostate Cancer Research Centre, Queensland, Australia

2Vancouver Prostate Centre, Vancouver General Hospital, BC, Canada

GADD45G is a member of the Growth Arrest and DNA Damage inducible-45 family and GADD45 proteins were previously shown to play a role in DNA demethylation. GADD45 proteins were recently shown to be recruited to regulatory elements of androgen receptor (AR)-target genes such as the Prostate Specific Antigen (PSA) promoter.

Objective

To determine the role of GADD45G in regulation of transcriptional expression of AR-target genes and prostate cancer cell survival.

Methods

Westernblot was used to measure GADD45G protein levels. qPCR and northern blot analysis were performed to measure the levels of mRNA. GADD45G knock-down was performed using siRNA (Ambion inc.). GADD45G protein levels in tissues were measured using immuno-histochemistry (IHC) protocol. Cell survival was measured using WST1 assay.

Results

Our microarray data analysis showed upregulation of GADD45G in response to androgens in LNCaP cells. Knock-down of GADD45G in LNCaP cells led to reduced expression of typical androgen-responsive genes such as PSA, TMPRSS2 and FKBP5. Analysis of IHC data from patient samples undergoing radicals showed relapse of GADD45G protein after ADT, suggesting the role of GADD45G in conferring treatment resistance and tumour recurrence. Knock-down of GADD45G in presence of docetaxel decreased the survival of LNCaP cells by down-regulation of key cell survival pathways.

Conclusion

Based on our results, we suggest that stress-activated GADD45G plays a critical role in regulating the transcription of AR-target genes and also plays a role in cell survival pathways.

25690 Do proteases regulate EphB4In prostate cancer?

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Jessica Lisle, Inga Mertens-Walker Inga, Carson Stephens, Scott Stansfield, Adrian Herington, Judith Clements and Sally-Anne Stephenson

Queensland University of Technology, Australia

Objective

EphB4, a member of the largest family of receptor tyrosine kinases, is over-expressed in 66% of prostate cancers (PCa) where it promotes tumour angiogenesis, increases cancer cell survival and facilitates cell invasion and migration. We have identified 2 protease-mediated cleavage events that liberate fragments of both the extracellular and intracellular domains of EphB4 from PCa cells.

Methods and Results

Previous studies in our laboratory determined that the PCa-associated serine protease KLK4 was the mediator of the first cleavage event and this occurred in the extracellular domain of EphB4 at arginine 508. This was consistent with the identified fragments of 70 and 50 kDa however there was a second C-terminal fragment of 47 kDa which was also generated after KLK4 incubation of the PCa cell line 22Rv1-B4 which exogenously over-expresses EphB4. In this study treatment of these cells with the Î3-secretase inhibitor compound E resulted in a loss of the 47 kDa fragment and an accumulation of the 50 kDa fragment. Subcellular fractionation was also performed on the KLK4 positive PCa cell line LNCaP and the 47 kDa fragment was seen in the nuclear fraction.

Conclusion

This study provides the first evidence of proteolytic regulation of EphB4 in PCa. KLK4 cleaves the ectodomain of EphB4 which leads to a subsequent second cleavage event by the intracellular protease Î3-secretase. The liberated C-terminal fragment then translocates to the nucleus where it may have functions in PCa promotion. This study identifies a further mechanism by which EphB4 may contribute to PCa.

25694 Analysis of ERG and its metastatic potential in prostate cancer

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David Gillatt1, Rachel Hagen2, Patricia Adamo2, Jon Oxley3, Raj Persad3, Jeff Holly4, Michael Ladomery2 and Anthony Rhodes2

1Bristol Urological Institute, United Kingdom

2The University of West of England, United Kingdom

3North Bristol NHS Trust, United Kingdom

4The University of Bristol, United Kingdom

Background

The oncogene ERG is consistently overexpressed in prostate cancer. Alternatively spliced variants of ERG have variable biological activities and specific variants containing the 72 bp exon 11 are associated with aggressiveness and progression of prostate cancer. The 81 bp exon 10 has also been shown to be alternatively spliced. Our objective was to determine whether we could reproducibly obtain high quality RNA from archived formalin-fixed, paraffin-embedded (FFPE) prostate tissue sections to further characterise the clinical significance of ERG with respect to prostate cancer metastasis and the differential expression of ERG splice variants in prostate cancer.

Methods

We isolated RNA from a cohort of patients using FFPE archival tissue sections (Benign n = 12, Cancer n = 42). ERG mRNA and protein expression were assessed using Real Time PCR (RT-PCR) and immunohistochemistry (IHC) respectively. Metastatic gene expression (MMP7, Osteopontin, Septin9, E/N-Cadherin) was analysed using RT-PCR. We used the linear equation method in order to quantitatively determine relative proportions of ERG variants (ERG72/Δ72, ERG81/Δ81) for each sample.

Results

ERG mRNA and protein expression is increased between benign and localised prostate cancer and is further increased in advanced prostate cancer as assessed by qPCR and IHC. Genes involved in cell migration and invasiveness (MMP7, osteopontin, septin9 and N-Cadherin) were increased in cancers that overexpress ERG suggesting that overexpression of ERG may increase the invasive potential of the prostate cancer cell. In addition we demonstrate a shift in the relative proportions of ERG variants with a significant reduction in skipping and an upregulation of inclusion of exon 10 and 11 of ERG in prostate cancer compared with benign. In addition there appears to be a trend that as prostate cancer advances there is a further reduction in relative proportions of ERGΔ72 and ERGΔ81. Analysis of the relative proportions of ERG variants present rather than total ERG may be valuable in determining prognosis and development of prostate cancer.

25710 Hyperglycaemia induced-resistance of prostate cancer cells to docetaxel chemotherapy is mediated via IGFBP-2

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David Gillatt1, Kalina Biernacka2, Claire Perks2, Jeff Holly2, Uzoh Chris2, Raj Persad3 and Zeng Li2

1Bristol Urological Institute, Southmead Hospital, United Kingdom

2The University of Bristol, United Kingdom

3North Bristol NHS Trust, United Kingdom

Clinically relevant prostate cancer is more frequent in Westernised societies and increasingly men have co-morbidities associated with a Western lifestyle primarily diabetes, characterised by hyperinsulinemia and hyperglycaemia. Insulin-like growth factors and their binding proteins are important mediators of the effects of nutrition on growth and play a key role in the development of prostate cancer.

We used DU145, PC3 and LNCaP prostate cancer cell lines to examine how hyperglycaemia altered their response to docetaxel. Trypan blue dye-exclusion assay was used to determine the percentage cell death. Protein abundance was determined using Western immunoblotting. Levels of insulin-like growth factor binding protein-2(IGFBP-2) were measured using an Enzyme-linked Immunoassay. IGFBP-2 gene silencing was achieved using siRNA technology. DNA methylation was assessed using combined bisulfite restriction analysis (COBRA). Acetylation status of histones H3 and H4 associated with IGFBP-2 gene was assessed using Chromatin Immunoprecipitation assay.

Hyperglycemia reduced Docetaxel-induced apoptosis by 40% for DU145 cells and by 88% for LNCaP cells. This reduced cell death was mediated by a glucose-induced up-regulation of IGFBP-2, as silencing IGFBP-2 negated the survival effect of high glucose. Glucose increased IGFBP-2 via increasing the acetylation of histones associated with the IGFBP-2 gene promoter.

This finding could have important implications in relation to therapeutic strategies as epigenetic modulation could be reversible.

Acknowledgements

  1. Top of page

We thank the Bristol Urological Institute (BUI) and The European Foundation for the Study of Diabetes (EFSD) for funding this work.

25810 The origin of lymph node metastases in prostate cancer

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Matthew Hong1, Jessica Chung2, Geoff Macintyre2, Xiaowen Chin3, John Pedersen4, Anthony Costello3, Christopher Hovens3 and Niall Corcoran3

1The University of Melbourne, Australia

2NICTA Victoria Research Laboratory, The University of Melbourne, Australia

3Australian Prostate Cancer Research Centre Epworth, Australia

4Tissupath Specialist Pathology, Mount Waverley, Australia

Objective

To determine the origin of lymph node metastases from advanced prostate cancers.

Methods

Prostate cancer patients with lymph node metastases who underwent radical prostatectomy were selected. Inclusion criteria included pT3 cancer, at least one positive lymph node, and availability of archival tissue. Lymph node metastases and different parts of interest within the tumours were manually microdissected from slides and genomic DNA was extracted (QIAmp DNA FFPE, Qiagen). The DNA was restored using the Infinium HD FFPE Restore Protocol (Illumina) and hybridised to the HumanOmniExpress-FFPE-12v1.0 BeadChip (Illumina). Copy number was called using Allele-Specific Copy Number Analysis of Tumours. The chromosomes were divided into 500 kbp segments and ploidy values generated for each. Indices of relatedness were generated between samples using a combination of similarity and complexity calculations and Pearson correlation.

Results

Three patients with large locally advanced prostate cancer were included in the study. In two patients the lymph node metastases were related to areas of extraprostatic extension as well as seminal vesicle involvement. The lymph node metastasis in the third patient was related to the central part of the tumour with areas of extraprostatic extension being distinct. Different sides of the same tumour in each case were distinct, suggesting that smaller cancer foci coalesced to form larger unifocal cancers in our series.

Conclusions

Lymph node metastasis may occur at different times during the evolution of any given prostate cancer, but is more often related to clones giving rise to extraprostatic spread and seminal vesical invasion.

25874 β-catenin signalling in prostate cancer

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Joanna Dulińska-Litewka, Paulina Dudzik, Iwona Hryniewicz, Krzysztof Okoń and Piotr Laidler

Jagiellonian University Medical College, Poland

Objective

Targeted cancer chemotherapy has become one of the major successes in recent years. One of the main target are tyrosine kinases as they play an important role in the modulation of growth factor signaling. Inhibitor type IV, IX of Akt as well as FH535, as an inhibitor of the WNT signaling pathway were all shown to decrease growth of cancer cells suggesting that those pathways plays important role in tumor progression and metastasis.

Methods

Studies were carried out on normal and cancer human prostate cell lines. Expression of cell signaling proteins in wild and inhibitor treated cells was analyzed at mRNA and protein level.

Results

Akt IV inhibitor caused prostate cells to lose their ability to adhere to the plate. In addition it decreased N-cadherin expression with parallel increase of AR and PTEN expression in androgen-independent cells. Inhibition of β-catenin or Akt decreased up to 80% the proliferation of cells while promoting apoptosis. The FH535 was a potent growth inhibitor for androgen-dependent prostate cancer cell lines more than for androgen-independent cells and markedly inhibited the expression of PSA. It was also shown that GSK-3beta regulation by phosphorylation is important for cell morphology and growth.

Conclusions

The obtained results indicate that the inhibition of prostate cancer cell growth is mediated by β-catenin level which seems to play crucial role in progression of human prostate cancer but the final effect depend on their initial androgen sensitivity as well as its availability.

This work was supported by MNiSzW grants: K/ZDS/003700, K/PBW/ 000561 – Jagiellonian University Medical College, Krakow, Poland.

26546 Functional validation of a KLK3variant, rs17632542, with prostate cancer risk

  1. Top of page

Srilakshmi Srinivasan1, Carson Stephens2, Amanda Spurdle3, Judith Clements4 and Jyotsna Batra4

1Queensland University of Technology

2Queensland University of Technology, United States

3Queensland Institute of Medical Research, Australia

4Queensland University of Technology, Australia

Objective

Fine-mapping studies identified a missense SNP rs17632542 (Ile178Thr) in exon-4 of kallikrein 3 (KLK3) to be associated with prostate cancer (PCa) risk. We aim to delineate the molecular consequences of this missense rs17632542 SNP.

Methods

In-silico tools were employed to analyse the effect of the SNP on splicing, protein stability and glycosylation. Mini-gene assays verifying the differential splicing effect for the SNP is being performed in LNCaP and PC3 cell lines. Protein stability analysis by differential scanning fluorimetry (DSF) and substrate activity assays using a fluorescent peptide were performed.

Results and Conclusions

In-silico analysis for rs17632542 SNP suggests an alteration in a KLK3 splice site, KLK3 protein stability and glycosylation for the risk allele. Recombinant KLK3 protein harbouring the variant (Thr) and wild-type (Ile) alleles were made in insect cell lines and interestingly, DSF technique suggested lower stability for the ‘Thr’ variant compared to the wild-type. Fluorescent peptide activity assays have also shown the ‘Thr’ variant to have lower activity compared to ‘Ile’ isoform. These results indicate the missense rs17632542 SNP to have biological effect on the expression and/or function of the KLK3 protein which has role in PCa prognosis.

26610 Targeting Eph receptors and their ephrin ligands in prostate cancer

  1. Top of page

Carolin Offenhäuser, Jennifer K. Ellacott, Fiona M. Smith, Kirrilee Miller, Bryan W Day, Kathleen S Ensbey, Kate Bastick and Andrew W Boyd

Queensland Institute of Medical Research, Australia

Eph receptors and their ephrin ligands are aberrantly expressed in a range of cancers and have emerged as potential molecular targets for cancer therapies, with first clinical trials in leukaemia already being underway. In this study, we investigate the feasibility of targeting Ephs and ephrins in prostate cancer. Using mRNA expression screening, we find EphA2, EphA3, EphB4, ephrin-A1 and ephrin-A5 are expressed in a subset of PCa cell lines. Functional analysis of ephrin-A5 showed that reducing ephrin-A5 expression, using shRNA knockdown, inhibits cell motility and anchorage-independent cell growth in vitro and tumour formation and growth in vivo. EphA3 appears to be expressed in normal prostate and early stages of prostate cancer and we are currently investigating EphA3 as a potential tumour suppressor. In contrast, EphA2 expression, which has been linked to poorly differentiated prostate cancer and poor patient outcome, promotes cell motility and proliferation in the absence of activation; however, activation of the receptor has diametrically opposite effects. This has led us to develop agonistic monoclonal antibodies to EphA2, which successfully inhibit pro-oncogenic signalling and cell motility in vitro, and are now being tested in vivo using an orthotopic prostate cancer model. We conclude, Eph- and/or ephrin-specific therapies have the potential to target prostate cancer, including late stages of prostate cancer where the tumour has spread. We propose that using antibody-based therapies to target these molecules has the potential to inhibit tumour metastasis and progression and improve patient outcome.

31314 Isolation and characterization of prostate cancer initiating cells based on aldehyde dehydrogenase activity

  1. Top of page

Jin H (Angela) Xie1, Frédéric Hollande2, John M Haynes1, Colin W Pouton1, Erica K Sloan1 and Sab Ventura1

1Monash University, Australia

2The University of Melbourne, Australia

Background

Prostate cancer is the most common male malignancy in western society. It has been suggested that a small population of cells (tumour initiating cells) in prostate cancer harbour intrinsic characteristics that make them resistant to androgen deprivation treatment and chemotherapy. In this study, a small population of cells from the prostate cancer PC3 cell line were isolated as having high aldehyde dehydrogenase activity using ALDEFLUOR assay to assess whether they display the characteristics of tumour initiating cells.

Methods

Real-time time PCR was performed on ALDHhigh,medium,low and ungated PC3 cells to detect the gene expression level of 10 potential tumor initiating cell markers. Sorted cells were also treated with the chemotherapy drugs vincristine, docetaxel and doxorubicin for 24 hours to assess drug resistance. In vivo, tumorigenicity studies were performed by injecting NOD/SCID mice with 5 × 104 ALDHhigh,low or ungated PC3 cells in the left flank. Tumour incidence and growth rate were recorded. Tumour morphology and angiogenesis were investigated using H&E and CD31 staining.

Results

We demonstrated that the embryonic stem cell marker oct-4 and the regulator of tumour suppressor genes DCLK1 were overexpressed in ALDHhigh cells which were resistant to chemotherapy drugs. Our in vivo work showed that ALDHhigh cells had the ability to initiate tumours earlier with a shorter survival time. Tumours derived from ALDHhigh cells had increased angiogenesis and lower non-cellular tissue rates. Gene up-regulation of Trop2 and DCLK1 which are indicators of tumorigenicity was also observed in tumours arising from ALDHhigh cells.