Prostate Cancer Progression Model Research Articles

Economic Evaluation of Urine-Based or Magnetic Resonance Imaging Reflex Tests in Men With Intermediate Prostate-Specific Antigen Levels in the United States

ObjectivesFor men with intermediate prostate-specific antigen (PSA) levels (4-10 ng/mL), urine-based biomarkers and multiparametric magnetic resonance imaging (MRI) are increasingly used as reflex tests before prostate biopsy. We assessed the cost effectiveness of these reflex tests in the United States. MethodsWe used an existing microsimulation model of prostate cancer (PCa) progression and survival to predict lifetime outcomes for a hypothetical cohort of 55-year-old men with intermediate PSA levels. Urine-based biomarkers—PCa antigen (PCA3), TMPRSS2:ERG gene fusion (T2:ERG), and the MyProstateScore (MPS) for any PCa and for high-grade (Gleason score ≥7) PCa (MPShg)—were generated using biomarker data from 1112 men presenting for biopsy at 10 United States institutions. MRI results were based on published sensitivity and specificity for high-grade PCa. Costs and utilities were sourced from literature and Medicare reimbursement schedules. Outcome measures included life years, quality-adjusted life years (QALYs), and lifetime medical costs per patient. Incremental cost-effectiveness ratios were empirically calculated on the basis of simulated life histories under different reflex testing strategies. ResultsBiopsying all men provided the most life years and QALYs, followed by reflex testing using MPShg, MPS, MRI, T2:ERG, PCA3, and biopsying no men (QALY range across strategies 15.98-16.09). Accounting for costs, MRI and MPShg were dominated by other strategies. PCA3, T2:ERG, and MPS were likely to be the most cost-effective strategy at willingness-to-pay thresholds of $100 000/QALY, $125 000/QALY, and $150 000/QALY, respectively. ConclusionsUsing PCA3, T2:ERG, or MPS as reflex tests has greater economic value than MRI, biopsying all men, or biopsying no men with intermediate PSA levels.

Expression, Localization, and Function of the Nucleolar Protein BOP1 in Prostate Cancer Progression

Differentiating between indolent and aggressive prostate cancers (CaP) is important to decrease overtreatment and increase survival for men with the aggressive disease. Nucleolar prominence is a histologic hallmark of CaP; however, the expression, localization, and functional significance of specific nucleolar proteins have not been investigated thoroughly. The nucleolar protein block of proliferation 1 (BOP1) is associated with multiple cancers but has not been implicated in CaP thus far. Meta-analysis of publicly available data showed increased BOP1 expression in metastatic CaP and recurrent CaP, and was inversely associated with overall survival. Multiplexed immunohistochemistry was used to analyze expression and localization of BOP1 and nucleolar protein 56 in human tissue samples from various stages of CaP progression. Here, increased BOP1 expression was observed at later stages of CaP progression, coinciding with a localization change from nuclear to cytoplasmic. In patient samples, cytoplasmic BOP1 was also inversely associated with overall survival. In models of prostate cancer progression, BOP1 expression showed expression and localization similar to that in human patient samples. The functional significance of BOP1 in metastatic CaP was assessed by genetic knockdown, where BOP1 knockdown resulted in decreased proliferation and motility compared with control. Taken together, these data suggest prognostic significance of BOP1 expression and localization in CaP progression and provide a foundation for further investigation into the functional role of nucleolar proteins in advanced CaP.

Genome-wide gene expression analysis of a murine model of prostate cancer progression: Deciphering the roles of IL-6 and p38 MAPK as potential therapeutic targets.

BackgroundProstate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer-related deaths among adult males globally. The poor prognosis of PCa is largely due to late diagnosis of the disease when it has already progressed to an advanced stage marked by androgen-independence, thus necessitating new strategies for early detection and treatment. We construe that these direly needed advances are limited by our poor understanding of early events in the progression of PCa and that would thus represent ideal targets for early intervention. To begin to fill this void, we interrogated molecular “oncophenotypes” that embody the transition of PCa from an androgen-dependent (AD) to–independent (AI) state.MethodsTo accomplish this aim, we used our previously established AD and AI murine PCa cell lines, PLum-AD and PLum-AI, respectively, which recapitulate primary and progressive PCa morphologically and molecularly. We statistically surveyed global gene expressions in these cell lines by microarray analysis. Differential profiles were functionally interrogated by pathways, gene set enrichment and topological gene network analyses.ResultsGene expression analysis of PLum-AD and PLum-AI transcriptomes (n = 3 each), revealed 723 differentially expressed genes (392 upregulated and 331 downregulated) in PLum-AI compared to PLum-AD cells. Gene set analysis demonstrated enrichment of biological functions and pathways in PLum-AI cells that are central to tumor aggressiveness including cell migration and invasion facilitated by epithelial-to-mesenchymal transition (EMT). Further analysis demonstrated that the p38 mitogen-activated protein kinase (MAPK) was predicted to be significantly activated in the PLum-AI cells, whereas gene sets previously associated with favorable response to the p38 inhibitor SB203580 were attenuated (i.e., inversely enriched) in the PLum-AI cells, suggesting that these aggressive cells may be therapeutically vulnerable to p38 inhibition. Gene set and gene-network analysis also alluded to activation of other signaling networks particularly those associated with enhanced EMT, inflammation and immune function/response including, but not limited to Tnf, IL-6, Mmp 2, Ctgf, and Ptges. Accordingly, we chose SB203580 and IL-6 to validate their effect on PLum-AD and PLum-AI. Some of the common genes identified in the gene-network analysis were validated at the molecular and functional level. Additionally, the vulnerability to SB203580 and the effect of IL-6 were also validated on the stem/progenitor cell population using the sphere formation assay.ConclusionsIn summary, our study highlights pathways associated with an augmented malignant phenotype in AI cells and presents new high-potential targets to constrain the aggressive malignancy seen in the castration-resistant PCa.

Protein Expression Analysis of an In Vitro Murine Model of Prostate Cancer Progression: Towards Identification of High-Potential Therapeutic Targets.

Background: Prostate cancer (PC) is the most frequently diagnosed cancer among men worldwide. The poor prognosis of PC is largely due to late diagnosis of the disease when it has progressed to advanced stages marked by androgen-independence. We interrogated proteomic signatures that embody the transition of PC from an androgen-dependent (AD) to an androgen-independent (AI) state. Methods: We have previously established AD and AI murine PC cell lines, PLum-AD and PLum-AI, respectively, which recapitulate primary and progressive PC at phenotypic and subcellular levels. We statistically surveyed global protein expression profiles in these cell lines. Differential profiles were functionally interrogated by pathways and protein–protein interaction network analyses. Results: Protein expression pattern analysis revealed a total of 683 proteins, among which 99 were significantly differentially altered in PLum-AI cells as compared to PLum-AD cells (45 increased and 54 decreased). Principal component analysis (PCA) revealed that the two different cell lines clearly separated apart, indicating a significant proteome expression difference between them. Four of the proteins (vimentin, catalase, EpCAM, and caspase 3) that were differentially expressed in PLum-AI cells compared to PLum-AD cells were subjected to biochemical validation by Western blotting. Biological process gene ontology (GO) analysis of the differentially expressed proteins demonstrated enrichment of biological functions and pathways in PLum-AI cells that are central to PI3 kinase and androgen receptor pathways. Besides, other relevant biological processes that are enriched in PLum-AI cells included cell adhesion and cell migration processes, cell and DNA damage, apoptosis, and cell cycle regulation. Conclusions: Our protein expression analysis of a murine in vitro model of PC progression identified differential protein spots that denote this progression and that comprise high-potential targets for early treatment of PC with a personalized patient-specific approach. Efforts are underway to functionally assess the potential roles of these proteins as therapeutic targets for PC progression.

Lifetime Benefits and Harms of Prostate-Specific Antigen–Based Risk-Stratified Screening for Prostate Cancer

BackgroundStudies conducted in Swedish populations have shown that men with lowest prostate-specific antigen (PSA) levels at ages 44–50 years and 60 years have very low risk of future distant metastasis or death from prostate cancer. This study investigates benefits and harms of screening strategies stratified by PSA levels.MethodsPSA levels and diagnosis patterns from two microsimulation models of prostate cancer progression, detection, and mortality were compared against results of the Malmö Preventive Project, which stored serum and tracked subsequent prostate cancer diagnoses for 25 years. The models predicted the harms (tests and overdiagnoses) and benefits (lives saved and life-years gained) of PSA-stratified screening strategies compared with biennial screening from age 45 years to age 69 years.ResultsCompared with biennial screening for ages 45–69 years, lengthening screening intervals for men with PSA less than 1.0 ng/mL at age 45 years led to 46.8–47.0% fewer tests (range between models), 0.9–2.1% fewer overdiagnoses, and 3.1–3.8% fewer lives saved. Stopping screening when PSA was less than 1.0 ng/mL at age 60 years and older led to 12.8–16.0% fewer tests, 5.0–24.0% fewer overdiagnoses, and 5.0–13.1% fewer lives saved. Differences in model results can be partially explained by differences in assumptions about the link between PSA growth and the risk of disease progression.ConclusionRelative to a biennial screening strategy, PSA-stratified screening strategies investigated in this study substantially reduced the testing burden and modestly reduced overdiagnosis while preserving most lives saved. Further research is needed to clarify the link between PSA growth and disease progression.

18F-Choline PET/mpMRI for Detection of Clinically Significant Prostate Cancer: Part 2. Cost-Effectiveness Analysis

The objective of this study was to evaluate the cost-effectiveness of 18F-choline PET/multiparametric MRI (mpMRI) versus mpMRI alone for the detection of primary prostate cancer with a Gleason score of greater than or equal to 3 + 4 in men with elevated prostate-specific antigen levels. Methods: A Markov model of prostate cancer onset and progression was used to estimate the health and economic consequences of 18F-choline PET/mpMRI for the detection of primary prostate cancer with a Gleason score of greater than or equal to 3 + 4 in men with elevated prostate-specific antigen levels. Multiple simultaneous hybrid 18F-choline PET/mpMRI strategies were evaluated using Likert or Prostate Imaging Reporting and Data System version 2 (PI-RADSv2) scoring; the first was biopsy for Likert 5 mpMRI lesions or Likert 3-4 lesions with 18F-choline target-to-background ratios of greater than or equal to 1.58, and the second was biopsy for PI-RADSv2 5 mpMRI lesions or PI-RADSv2 3-4 mpMRI lesions with 18F-choline target-to-background ratios of greater than or equal to 1.58. These strategies were compared with universal standard biopsy, mpMRI alone with biopsy only for PI-RADSv2 3-5 lesions, and mpMRI alone with biopsy only for Likert 4-5 lesions. For each mpMRI strategy, either no biopsy or standard biopsy could be performed after negative mpMRI results were obtained. Deaths averted, quality-adjusted life years (QALYs), cost, and incremental cost-effectiveness ratios were estimated for each strategy. Results: When the results of 18F-choline PET/mpMRI were negative, performing a standard biopsy was more expensive and had lower QALYs than performing no biopsy. The best screening strategy among those considered in this study performed hybrid 18F-choline PET/mpMRI with Likert scoring on men with elevated PSA, performed combined biopsy (targeted biopsy and standard 12-core biopsy) for men with positive imaging results, and no biopsy for men with negative imaging results ($22,706/QALY gained relative to mpMRI alone); this strategy reduced the number of biopsies by 35% in comparison to mpMRI alone. When the same policies were compared using PI-RADSv2 instead of Likert scoring, hybrid 18F-choline PET/mpMRI cost $46,867/QALY gained relative to mpMRI alone. In a threshold analysis, the best strategy among those considered remained cost-effective when the sensitivity and specificity of PET/mpMRI and combined biopsy (targeted biopsy and standard 12-core biopsy) were simultaneously reduced by 20 percentage points. Conclusion: 18F-choline PET/mpMRI for the detection of primary prostate cancer with a Gleason score of greater than or equal to 3 + 4 is cost-effective and can reduce the number of unneeded biopsies in comparison to mpMRI alone.

Abstract 5217: RB loss reprograms AR and E2F1 signaling in models of prostate cancer progression

Abstract Prostatic adenocarcinoma (PCa) is the most frequently diagnosed non-cutaneous malignancy, and the third leading cause of cancer death in males in the United States. A crucial component to the development and progression of PCa is the activity of the androgen receptor (AR). As such, targeting the AR-signaling axis through androgen deprivation therapy (ADT) is the first line of therapy for PCa. However, cells invariably become resistant to this therapy and men relapse with the incurable form of the disease termed, castration-resistant prostate cancer (CRPC). In addition to AR, another principal component aiding in the progression of disease is the retinoblastoma tumor suppressor protein (RB). RB functions to repress tumor development by negatively regulating the activity of the E2F family of transcription factors, preventing cell cycle progression. RB is lost in roughly 30% of CRPC tumors and is sufficient to induce a CRPC phenotype in hormone-sensitive cells under ADT conditions. Analyses into the molecular significance of RB loss on disease progression identified a potential cooperation between AR and the RB-E2F1 signaling axis. Biological assessment performed in isogenic RB knockdown (i.e. hormone-sensitive and CRPC models) identified distinct functional consequences of RB loss depending on AR status and disease state. Transcriptome analysis identified divergently regulated gene signatures between disease stages in the presence of AR activation, which were not seen under ADT conditions, implicating a unique role of AR in transcriptional regulation with the loss of RB. Data to be discussed will also include further comparison of the E2F1 and AR cistromes in the absence of RB to identify the mechanism by which the AR-RB-E2F1 signaling axis function in promoting the progression of disease. Citation Format: Amy C. Mandigo, Chris McNair, Matthew J. Schiewer, Karen E. Knudsen. RB loss reprograms AR and E2F1 signaling in models of prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5217.

Abstract B003: NRH:quinone oxidoreductase 2 (NQO2) and glutaminase (GLS) both play a role in large extracellular vesicles (LEV) formation in preclinical LNCaP-C4-2B prostate cancer model of progressive metastasis

Abstract Advanced-stage prostate cancer is characterized by osseous metastases whose establishment involves the dynamic interplay of factors and exchange of cellular contents by constituents of the tumor bony microenvironment. Among the factors that transport cellular cargoes and facilitate the transmission of signaling complexes for establishment of the bone metastatic lesions are large extracellular vesicles (LEVs), also known as large oncosomes. Information on the contents of LEVs and the mechanisms by which LEVs are formed and regulated is incomplete. In the course of studies aimed at elucidating the mechanisms of bony metastases using the LNCaP-C4-2B prostate cancer progression model, we show that the expression and cellular distribution of nicotinamide riboside: quinone oxidoreductase 2 (NQO2) and glutaminase (GLS, kidney type) both participate in the induction of membrane-localized oxidative stress and the formation of LEVs. The salient features of our findings are the following: The LNCaP model system exhibits gradually increasing oxidative stress levels as it progresses to the bone metastatic derivative C4-2B, while at the same time showing a progressive decrease in the expression level of NQO2. Reverse transcription PCR studies showed that NQO2 mRNA levels were unchanged between LNCaP and its metastatic derivatives C4, C4-2 and C4-2B. As a control, the expression level of the analogous enzyme NADP(H):quinone oxidoreductase 1 (NQO1) was also unchanged. These results suggested the possibility that the NQO2 enzyme in the bone metastatic C4-2B derivative could be localized elsewhere, possibly at an extracellular location. Further studies showed that a fraction of the NQO2 enzyme was associated with the detergent resistant membrane fraction of the C4-2B prostate cancer cells. Molecular modeling studies showed that the NQO2 enzyme had two caveolin-1 and NQO1 enzyme had one caveolin-1 binding sites. While the caveolin-1 binding site of NQO1 is buried inside the molecule and hence inaccessible to caveolin-1, one of the two binding sites available in NQO2 was external and accessible to caveolin-1. We have validated the specific interaction between NQO2 and caveolin-1 using deletion constructs of caveolin-1 fused with glutathione S-transferase (GST). Mitochondrial preparations of LNCaP, C4, C4-2, and C4-2B cells showed increasing level of expression of the GLS enzyme, suggesting a correlation between increased oxidative stress, glutaminase activity, and metastatic potential. Moreover, we found that the metastatic C4-2B derivative extruded LEVs in which the enzyme NQO2 and caveolae specific caveolin-1 could be visualized as cargo using double immunofluorescence studies. The extrusion of these LEVs could be inhibited by the GLS specific inhibitor BP-TES. Characteristically, curcumin was also found to inhibit the extrusion of these LEVs. This naturally occurring plant-based compound was shown earlier to inhibit prostate cancer bone metastasis in vivo using the C4-2B model. These results indicate a role for the NQO2 enzyme at the membrane level, possibly increasing the oxidative stress in a localized manner by lipid peroxidation. These results also suggest that a glutamine/GLS-mediated metabolic reprogramming in the LNCaP model system as it progresses toward the bone metastatic C4-2B is an integral component of the force driving the metastatic process. Based on the results of our studies, a combinatorial strategy using antimetastatic therapies such as nitrogen-containing bisphosphonates (NBPs) and anti-GLS therapy or, alternatively, a naturally occurring plant compound-based anti-NQO2 and anti-GLS therapy, could be considered for treating advanced prostate cancer patients with bone metastatic complications. Citation Format: Thambi Dorai, Ankeeta B. Shah, Faith Summers, Rajamma Mathew, Jing Huang, Tze-chen Hsieh, Joseph M. Wu. NRH:quinone oxidoreductase 2 (NQO2) and glutaminase (GLS) both play a role in large extracellular vesicles (LEV) formation in preclinical LNCaP-C4-2B prostate cancer model of progressive metastasis [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B003.

NRH:quinone oxidoreductase 2 (NQO2) and glutaminase (GLS) both play a role in large extracellular vesicles (LEV) formation in preclinical LNCaP-C4-2B prostate cancer model of progressive metastasis.

In the course of studies aimed at the role of oxidative stress in the development of metastatic potential in the LNCaP-C4-2B prostate cancer progression model system, we found a relative decrease in the level of expression of the cytoplasmic nicotinamide riboside: quinone oxidoreductase (NQO2) and an increase in the oxidative stress in C4-2B cells compared to that in LNCaP or its derivatives C4 and C4-2. It was also found that C4-2B cells specifically shed large extracellular vesicles (LEVs) suggesting that these LEVs and their cargo could participate in the establishment of the osseous metastases. The level of expression of caveolin-1 increased as the system progresses from LNCaP to C4-2B. Since NQO2 RNA levels were not changed in LNCaP, C4, C4-2, and C4-2B, we tested an altered cellular distribution hypothesis of NQO2 being compartmentalized in the membrane fractions of C4-2B cells which are rich in lipid rafts and caveolae. This was confirmed when the detergent resistant membrane fractions were probed on immunoblots. Moreover, when the LEVs were analyzed for membrane associated caveolin-1 as possible cargo, we noticed that the enzyme NQO2 was also a component of the cargo along with caveolin-1 as seen in double immunofluorescence studies. Molecular modeling studies showed that a caveolin-1 accessible site is present in NQO2. Specific interaction between NQO2 and caveolin-1 was confirmed using deletion constructs of caveolin-1 fused with glutathione S-transferase (GST). Interestingly, whole cell lysate and mitochondrial preparations of LNCaP, C4, C4-2, and C4-2B showed an increasing expression of glutaminase (GLS, kidney type). The extrusion of LEVs appears to be a specific property of the bone metastatic C4-2B cells and this process could be inhibited by a GLS specific inhibitor BPTES, suggesting the critical role of a functioning glutamine metabolism. Our results indicate that a high level of expression of caveolin-1 in C4-2B cells contributes to an interaction between caveolin-1 and NQO2 and to their packaging as cargo in the shed LEVs. These results suggest an important role of membrane associated oxidoreductases in the establishment of osseous metastases in prostate cancer.

Pharmacoeconomic study of the degarelix drug use for treatment of hormone-dependent prostate cancer compared to gonadotropin-releasing hormone analogs

Objective: to determine clinical and economic consequences of using the degarelix drug instead of gonadotropin-releasing hormone (GnRH) analogs for treatment of advanced hormone-sensitive prostate c ancer. Materials and methods. The main criteria of therapy effectiveness were overall survival and progression-free survival. Cost of the drugs being compared, as well as expenses associated with therapy after prostate cancer progression (per the dynamics of plasma prostate-specific antigen levels), were considered. A Markov model of prostate cancer progression was used including states “without progression”, “after progression”, and “death”. The modeling time horizon was 3 years. The main source of information on drug costs was the registry of maximal retail pricese for vital and essential drugs. The target population only included patients with high (>20 ng/ml) baseline level of prostate-specific antigen. Information on effectiveness was obtained based on analysis of the results of randomized comparative clinical trials of degarelix and leuprorelin. Results. Compared to leuprorelin, degarelix use was characterized by more favorable effectiveness and safety profiles (based on all criteria considered in the analysis). In the basic modeling script, progression-free survival was 27.9 and 20.1 months for degarelix and GnRH agonists, respectively. Despite higher cost of therapy prior to progression (359,000 and 268,000 rubles, respectively), degarelix use instead of GnRH agonists leads to 31 % decrease in direct budget medical expenses on drug provision under the state guarantee program (1,322,000 vs. 1,907,000 rubles), as well as to a decrease in the cost-effectiveness ratio (47,408 rubles/month without prostate cancer progression versus 94,651 rubles/ month without prostate cancer progression) Conclusion. Compared to treatment with GnRH agonists, use of degarelix as the 1st line therapy for treatment of prostate cancer leads to significant savings of healthcare budget expenses.

Abstract 2414: Fibromodulin expression in human prostate cancer tissue: Implications for prostate cancer progression

Abstract Background: Fibromodulin is a small leucine-rich proteoglycan important for extracellular matrix organization and essential for tissue repair in multiple organs. Recently, differential expression of fibromodulin has been shown in human prostate cancer cell lines with different cancer phenotypes and in human prostate cancer tissue compared to normal/benign tissue. Methods: The protein expression level of fibromodulin was analyzed by immunohistochemistry using a prostate tissue microarray (TMA), constructed from archival radical prostatectomy and trans-urethral prostatectomy tissue specimens, from patients with localized prostate cancer (PCa). Stained sections from the donor blocks were evaluated, and a representative area from PCa, high grade prostatic intraepithelial neoplasia (HGPIN), proliferative inflammatory atrophy (PIA), and benign prostate tissue (BPT) was included into the TMA. Staining for each lesion was scored, using H-score, by microscopic assessment of the percentage of epithelial cells with positive staining. H-score values were compared between groups by ANOVA test, and p < 0.05 was considered statistically significant. The Ethical Review Board of the academic institution approved this study. Results: Fibromodulin expression at protein level was evaluated in 378 cores representing PCa (143 cores), HGPIN (58), PIA (102), and BPT (78). Mean fibromodulin expression in cores with PCa, HGPIN, and PIA was significantly higher than mean of fibromodulin expression in normal glands (p<0.0001). There was a higher mean staining score in PCa (156, 95% CI:144–167), compared to HGPIN (131.4, 95% CI:109-153), PIA (128, 95% CI:117-138), and BPT (33.3, 95% CI:27-38). Thus, fibromodulin expression increased across the PCa progression model. Conclusions: The expression pattern of fibromodulin showed a significant increase from benign prostate tissue through pre-cancerous lesions to PCa. These findings suggest that this proteoglycan may be involved in the process of prostate cancer progression, with low levels in pre-cancerous lesions and higher levels in cancer tissue. Additional studies are required to determine the biological role and potential clinical implications of fibromodulin in prostate cancer. Citation Format: Estebana Ines Benedetti Padron, Angelo De Marzo, Javier Baena Del Valle, Jan Geliebter, Niradiz Reyes. Fibromodulin expression in human prostate cancer tissue: Implications for prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2414. doi:10.1158/1538-7445.AM2017-2414

Inhibition of cyclooxygenase-2-mediated matriptase activation contributes to the suppression of prostate cancer cell motility and metastasis.

Chronic inflammation plays an important role in cancer development and progression. Cyclooxygenases-2 (COX-2) is a key enzyme in generating prostaglandins causing inflammation, is often found to be overexpressed in prostate cancer (PCa) and is correlated with PCa cell invasion and metastasis. We aim to investigate the molecular mechanism of how COX-2 promotes PCa cell invasion and metastasis and to evaluate the effect of COX-2 inhibitors in a selected model of PCa progression. Our results showed that the expression of COX-2 and Interleukin 1β (IL-1β) was upregulated in highly invasive PCa cells and was correlated with the activated levels of membrane-anchored serine protease matriptase. The expression levels of COX-2 were increased and were correlated with matriptase levels in PCa specimens. Moreover, results showed that COX-2 overexpression or a COX-2 product Prostaglandin E2 (PGE2) caused an increase in matriptase activation and PCa cell invasion, whereas COX-2 silencing antagonized matriptase activation and cell invasion. In addition, the inhibition of COX-2-mediated matriptase activation by Celebrex and sulindac sulfide suppressed the androgen-independent and COX2-overexpressing PCa PC-3 cell invasion, tumor growth and lung metastasis in an orthotopic xenograft model. Our results indicate that COX-2/matriptase signaling contributes to the invasion, tumor growth and metastasis of COX-2-overexpressing and androgen-independent PCa cells.

Probing the prostate tumour microenvironment I: impact of glucose deprivation on a cell model of prostate cancer progression.

In the developed world, prostate cancer is the most common cancer diagnosis in men. Although prostate cancer initially presents as a non life-threatening disease, 90% of patients will develop castration resistant prostate cancer (CRPC), which preludes distant metastasis and is largely accountable for prostate cancer associated deaths. This is because as yet, there are no viable molecular therapeutic targets for effective treatment of CRPC. It is now widely accepted that cancer cells can alter their metabolic profile during the course of tumourgenesis and metastasis such that they are able to survive in oxygen and nutrient-poor environments. This work was aimed towards gaining greater mechanistic understanding of how such stresses in the tumour microenvironment impact on both androgen sensitive (LNCaP) and androgen independent (LNCaP-abl and LNCaP-abl-Hof) prostate cancer cell lines. Here we have applied technically robust and reproducible label-free liquid chromatography mass spectrometry analysis for comprehensive proteomic profiling of prostate cancer cell lines under nutrient deficient (low glucose) conditions. This led to the identification of approximately 4,000 proteins - one of the largest protein datasets for prostate cancer cell lines established to date. The biological and clinical significance of proteins showing a significant change in expression as result of low glucose conditions was established. Novel, intuitive workflows were subsequently implemented to ensure the verification of selected proteins of interest in a robust, reproducible and high throughput manner. Overall, these data suggest that this strategy supports identification of protein biomarkers of prostate cancer progression and potential therapeutic targets for CRPC.

Probing the prostate tumour microenvironment II: Impact of hypoxia on a cell model of prostate cancer progression

Approximately one in six men are diagnosed with Prostate Cancer every year in the Western world. Although it can be well managed and non-life threatening in the early stages, over time many patients cease to respond to treatment and develop castrate resistant prostate cancer (CRPC). CRPC represents a clinically challenging and lethal form of prostate cancer. Progression of CRPC is, in part, driven by the ability of cancer cells to alter their metabolic profile during the course of tumourgenesis and metastasis so that they can survive in oxygen and nutrient-poor environments and even withstand treatment. This work was carried out as a continuation of a study aimed towards gaining greater mechanistic understanding of how conditions within the tumour microenvironment impact on both androgen sensitive (LNCaP) and androgen independent (LNCaP-abl and LNCaP-abl-Hof) prostate cancer cell lines. Here we have applied technically robust and reproducible label-free liquid chromatography mass spectrometry analysis for comprehensive proteomic profiling of prostate cancer cell lines under hypoxic conditions. This led to the identification of over 4,000 proteins – one of the largest protein datasets for prostate cancer cell lines established to date. The biological and clinical significance of proteins showing a significant change in expression as result of hypoxic conditions was established. Novel, intuitive workflows were subsequently implemented to enable robust, reproducible and high throughput verification of selected proteins of interest. Overall, these data suggest that this strategy supports identification of protein biomarkers of prostate cancer progression and potential therapeutic targets for CRPC.

XRP44X, an Inhibitor of Ras/Erk Activation of the Transcription Factor Elk3, Inhibits Tumour Growth and Metastasis in Mice

Transcription factors have an important role in cancer but are difficult targets for the development of tumour therapies. These factors include the Ets family, and in this study Elk3 that is activated by Ras oncogene /Erk signalling, and is involved in angiogenesis, malignant progression and epithelial-mesenchymal type processes. We previously described the identification and in-vitro characterisation of an inhibitor of Ras / Erk activation of Elk3 that also affects microtubules, XRP44X. We now report an initial characterisation of the effects of XRP44X in-vivo on tumour growth and metastasis in three preclinical models mouse models, subcutaneous xenografts, intra-cardiac injection-bone metastasis and the TRAMP transgenic mouse model of prostate cancer progression. XRP44X inhibits tumour growth and metastasis, with limited toxicity. Tumours from XRP44X-treated animals have decreased expression of genes containing Elk3-like binding motifs in their promoters, Elk3 protein and phosphorylated Elk3, suggesting that perhaps XRP44X acts in part by inhibiting the activity of Elk3. Further studies are now warranted to develop XRP44X for tumour therapy.

Abstract A11: Downregulation of NRH:quinone oxidoreductase 2(NQO2) in the LNCaP prostate cancer progression model system: Implications to metastasis

Abstract Prostate cancer is characterized by an innate capacity to produce higher concentrations of reactive oxygen species (ROS). ROS is also a hallmark for highly aggressive disease. Our laboratory has focused on the molecular mechanisms of inhibition of prostate cancer bone metastasis by the naturally occurring plant based anti-oxidant compound curcumin, arising from the rhizomes of Curcuma Longa. These studies highlight the importance of studying the mechanisms of redox homeostasis in normal cells and how they are subverted in cancer cells. The LNCaP prostate cancer progression model system as exemplified by the development of C4, C4-2 and C4-2B ( bone metastatic) sublines derived from the parental LNCaP prostate cancer cells offer an unique avenue to study the role of redox systems in the progression to metastatic disease, culminating in the establishment of osseous metastases. The mechanisms of ROS production and destruction, the cellular machinery and the enzyme systems involved, the relationship of ROS to cellular oncogene activation and the antagonistic duality of oncogenes and tumor suppressor genes with respect to ROS homeostasis have been subjects of intense investigation. How the prostate cancer cells metabolize ROS to achieve their proliferative and metastatic potential is not completely understood. We chose to study one redox system orchestrated by the expression of NADPH:Quinone Oxidoreductase1 (NQO1) and the Nicotinamide Riboside (NRH): Quinone Oxidoreductase 2 (NQO2) in the LNCaP progression model system. Particularly, the enzyme NQO2 has been shown earlier to be the target of another polyphenolic chemopreventive antioxidant resveratrol. The role of NQO2 as a phase II detoxification enzyme acting on several xenobiotics is well characterized. However, the role of NQO2 in regulating ROS and hence cancer progression is only beginning to be understood. With this background, we investigated the potential role of NQO2/NQO1 expression in the transition from androgen dependence to androgen independence and to acquiring bone metastatic potential in C4-2B prostate cancer cells. Our studies indicate that 1) there is a down regulation of NQO2 as the androgen dependent LNCaP cells progress towards androgen independence in C4 and C4-2 cells. In C4-2B cells, there is a barely detectable level of NQO2. In contrast, there is very little change in the level of expression of NQO1; 2) Characteristically, the intracellular ROS production progressively increases from LNCaP to C4-2B; 3) In parallel, there is an up regulation of caveolin-1 as the LNCaP cells progress to bone metastatic C4-2B and 4) a small fraction of NQO2 could be detected in the detergent resistant membrane (DRM) fraction consisting of lipid rafts and caveolae. Our studies suggest that caveolin-1 may play a role in the membrane sequestration of the largely cytoplasmic NQO2 in LNCaP cells and that this interaction could be lost in bone metastatic C4-2B cells. The significance of these findings in relation to the metastatic potential of C4-2B cells will be discussed. Citation Format: Ankeeta Shah, Tze-Chen Hsieh, Rajamma Mathew, Joseph M. Wu, Thambi Dorai. Downregulation of NRH:quinone oxidoreductase 2(NQO2) in the LNCaP prostate cancer progression model system: Implications to metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A11.

Combined Akt and MEK pathway blockade in pre-clinical models of enzalutamide-resistant prostate cancer.

192 Background: Despite recent advances with newer androgen receptor(AR) pathway inhibitors, treatment resistance in castrate resistant prostate cancer continues to remain a clinical problem. Co-targeting approaches are of significant interest to slow the progression of disease and delay the onset of resistance. With both Akt and MEK pathways becoming activated as prostate cancer develops resistance to AR-targeted therapy, this study explores co-targeting these pathways in AR positive prostate cancer models. Methods: Using in vitro models of prostate cancer progression from androgen dependent to castrate resistant and ENZ-resistant disease, we evaluated the effect of Akt and/or MEK inhibition with AZD5363 and PD0325901, respectively, on cell proliferation, apoptosis and downstream signalling pathways. For in vivo models, we tested the combination of Akt and MEK inhibition castrated mice bearing MR49F and 22RV1 cells which are resistant to ENZ. Results: Inhibition of Akt reduced S6 phosphorylation, inhibited growth and induced apoptosis in LNCaP-derived cells; the addition of a MEK inhibitor increased apoptosis and cell cycle arrest, but did not further decrease cell proliferation. In contrast, PTEN wild-type 22RV1 cells demonstrated a greater sensitivity to MEK inhibition, but were generally insensitive to Akt inhibition. In vivo, combination of Akt and MEK inhibition resulted in more consistent tumour growth inhibition of MR49F xenografts and longer disease specific survival than Akt inhibitor monotherapy, with induction of pERK staining being evident in some AZD5363 monotherapy treated tumours. Conversely, 22RV1 xenografts had greater resistance to Akt inhibition and greater sensitivity to MEK inhibition. Conclusions: Our data suggest that combination of Akt and MEK inhibition may improve prostate cancer control in ENZ-resistant prostate cancer. Further, with the major effect in both MR49F and 22RV1 xenografts attributable to one inhibitor, our results also suggest the importance of characterizing the dominant oncogenic pathway in each patient’s tumour in order to select optimal therapy.

The 21st Annual Prostate Cancer Foundation Scientific Retreat report.

The 21st Annual Prostate Cancer Foundation (PCF) Scientific Retreat was held from October 23-25, 2014, in Carlsbad, CA. This event is the world's foremost scientific meeting focusing on prostate cancer and brings together leading basic, translational and clinical researchers in prostate cancer and other diverse disciplines to discuss the newest findings most likely to advance the understanding of prostate cancer and the clinical care of prostate cancer patients. This year's meeting highlighted themes including: (i) research integrity and standards for scientific reproducibility; (ii) prostate cancer disparities; (iii) mechanisms and models of prostate cancer progression and dormancy; (iv) mechanisms of therapeutic resistance; and (v) advancements in precision medicine treatments, treatment models, and predictive and prognostic biomarkers.

The heat shock protein 90 inhibitor, (-)-epigallocatechin gallate, has anticancer activity in a novel human prostate cancer progression model.

(-)-Epigallocatechin gallate (EGCG), a major tea polyphenol, elicits anticancer effects. However, the mechanism of action is not fully understood. Our laboratory previously showed that EGCG inhibits heat shock protein 90 (HSP90). We used nontumorigenic (NT), tumorigenic, and metastatic cancer cells from a novel human prostate cancer progression model to test the hypotheses that certain stages are more or less sensitive to EGCG and that sensitivity is related to HSP90 inhibition. Treatment of cells with EGCG, novobiocin, or 17-AAG resulted in more potent cytotoxic effects on tumorigenic and metastatic cells than NT cells. When tumorigenic or metastatic cells were grown in vivo, mice supplemented with 0.06% EGCG in drinking water developed significantly smaller tumors than untreated mice. Furthermore, EGCG prevented malignant transformation in vivo using the full prostate cancer model. To elucidate the mechanism of EGCG action, we performed binding assays with EGCG-Sepharose, a C-terminal HSP90 antibody, and HSP90 mutants. These experiments revealed that EGCG-Sepharose bound more HSP90 from metastatic cells compared with NT cells and binding occurred through the HSP90 C-terminus. In addition, EGCG bound HSP90 mutants that mimic both complexed and uncomplexed HSP90. Consistent with HSP90 inhibitory activity, EGCG, novobiocin, and 17-AAG induced changes in HSP90-client proteins in NT cells and larger differences in metastatic cells. These data suggest that EGCG may be efficacious for the treatment of prostate cancer because it preferentially targets cancer cells and inhibits a molecular chaperone supportive of the malignant phenotype.

Abstract IA11: The role of bone microenvironment in the lethal progression of prostate cancer

Abstract Lethal prostate cancer (PCa) is clinically characterized by bone-homing and bone- forming metastases, and bone is frequently the initial organ involved in castrate-resistant progression. These clinical observations led investigators to develop bone-tropic radiopharmaceuticals to treat men with advanced PCa. The ability of the bone-homing alpha emitter (RAD-223) to prolong progression free and overall survival establishes the therapeutic relevance of targeting bone 1. The long accepted paradigm of PCa progression has been dominated by the model of evolution from androgen dependence to androgen independence under the selective pressure of castration. Thus, Src family kinases (SFKs) were investigated experimentally and clinically for the treatment of putative “androgen independent” PCa, as SFKs play a role in both tumor growth and bone turnover. The roles of SFKs in migration, invasion, survival of tumor cells, and osteoclast and osteoblast function in tumor microenvironment were supported by numerous clinical and laboratory observations 2, 3. Despite promising phase I /II reports of dasatinib, a multikinase and potent SFK inhibitor, in men with advanced PCa 4, 5, a phase III trial failed to yield the expected survival advantage 6. The result of this trial highlights the difficulties in translating data from cell line models to biologically heterogeneous clinical PCa and in transitioning from phase I/II to phase III clinical trials. The efficacy of dasatinib was not observed likely due to persistent AR signaling, which remains the primary driver of progression in bone by the transition from paracrine to intracrine androgen signaling 7. Based on these considerations, we are testing the efficacy of an androgen biosynthesis inhibitor in combination with dasatinib to better understand the relevance of mechanism(s). A co–clinical investigational strategy is being applied to better define biomarkers to predict clinical efficacy. These studies also highlight the importance of preclinical work that utilizes models that better reflect the heterogeneity of PCa. This problem is partly overcome by patient- derived xenografts (PDX) 8, 9 10. These models reflect many aspects of PCa in bone and can be used to identify therapy targets and better identify both mechanisms of drug efficacy and development of resistance. Studies in PDX have led to identification of FGF signaling as a therapy target 11. Additional studies in PDX implicated the tumor microenvironment in the striking bone responses observed with cabozantinib 13. These observations led us to propose a model of prostate cancer progression founded on the hypothesis that cancer progresses from being an endocrine-driven cancer (Dihydrotestosterone-dependent) to paracrine/intracrine driven (microenvironment-dependent), and then finally to a cell autochthonous phase 12. Continued studies should lead to decisions on therapy treatment that are based on an understanding of markers that predict disease progression. This new paradigm will also result in more effective therapy combinations.