Transgenic Adenocarcinoma Of The Mouse Prostate Model Research Articles

Ultrasound-Mediated Delivery of Chemotherapy into the Transgenic Adenocarcinoma of the Mouse Prostate Model

Ultrasound (US) in combination with microbubbles (MB) has had promising results in improving delivery of chemotherapeutic agents. However, most studies are done in immunodeficient mice with xenografted tumors. We used two phenotypes of the spontaneous transgenic adenocarcinoma of the mouse prostate (TRAMP) model to evaluate if US + MB could enhance the therapeutic efficacy of cabazitaxel (Cab). Cab was either injected intravenously as free drug or encapsulated into nanoparticles. In both cases, Cab transiently reduced tumor and prostate volume in the TRAMP model. No additional therapeutic efficacy was observed combining Cab with US + MB, except for one tumor. Additionally, histology grading and immunostaining of Ki67 did not reveal differences between treatment groups. Mass spectrometry revealed that nanoparticle encapsulation of Cab increased the circulation time and enhanced the accumulation in liver and spleen compared with free Cab. The therapeutic results in this spontaneous, clinically relevant tumor model differ from the improved therapeutic response observed in xenografts combining US + MB and chemotherapy.

Dietary Tomato or Lycopene Do Not Reduce Castration-Resistant Prostate Cancer Progression in a Murine Model

Dietary tomato products or lycopene protect against prostate carcinogenesis, but their impact on the emergence of castration-resistant prostate cancer (CRPC) is unknown. We hypothesized that tomato or lycopene products would reduce the emergence of CRPC. Transgenic adenocarcinoma of the mouse prostate (TRAMP) mice were castrated at 12-13 wk and the emergence of CRPC was monitored by ultrasound in each study. In Study 1, TRAMP mice (n=80) were weaned onto an AIN-93G-based control diet (Con-L, n=28), a 10% tomato powder diet (TP-L, 10% lyophilized w/w, n=26), or a control diet followed by a tomato powder diet after castration (TP-Int1, n=26). In Study 2, TRAMP mice (n=85) were randomized onto a control diet with placebo beadlets (Con-Int, n=29), a tomato diet with placebo beadlets (TP-Int2, n=29), or a control diet with lycopene beadlets (Lyc-Int, n=27) following castration (aged 12 wk). Tumor incidence and growth were monitored by ultrasound beginning at an age of 10 wk. Mice were euthanized 4 wk after tumor detection or aged 30 wk if no tumor was detected. Tissue weights were compared by ANOVA followed by Dunnett's test. Tumor volumes were compared using generalized linear mixed model regression. Ultrasound estimates for the in vivo tumor volume were strongly correlated with tumor weight at necropsy (R2=0.75 and 0.94, P <0.001 for both Studies 1 and 2, respectively). Dietary treatments after castration did not significantly impact cancer incidence, time to tumor detection, or final tumor weight. In contrast to studies of de novo carcinogenesis in multiple preclinical models, tomato components had no significant impact on the emergence of CRPC in the TRAMP model. It is possible that specific mutant subclones of prostate cancer may continue to show some antiproliferative response to tomato components, but further studies are needed to confirm this.

Dietary Tomato Varieties Similarly Inhibit Prostate Carcinogenesis in the TRAMP Model in Association with Distinct Transcriptomic and Metabolomic Profiles

Abstract Objectives Lycopene intake is associated with reduced risk of prostate cancer, especially lethal disease. Tangerine tomatoes contain highly bioavailable lycopene isomers, compared to red tomatoes, but impact of isomers on cancer preventive bioactivity are unknown. Our goals are: (1) to determine if feeding tomato containing diets with differing lycopene isomers inhibit transgenic adenocarcinoma of the mouse prostate (TRAMP) carcinogenesis, and (2) to determine key early metabolic and transcriptional pathways through which tomatoes act to prevent carcinogenesis. Methods We examine prostate carcinogenesis in the TRAMP model, comparing two tomatoes with distinct lycopene isomer profiles: all-trans-lycopene-rich red tomatoes and cis-lycopene-isomer-rich tangerine tomatoes. Weanling TRAMP and wild type (WT) mice were fed AIN-93 G control, 10% tangerine tomato + AIN-93 G (w/w), or 10% red tomato + AIN-93 G (w/w) diet until 10 or 18 wks. At 10 wks., prostate RNA-seq and plasma metabolomics were performed on TRAMP and WT samples. At 18 wks., plasma carotenoid analysis and histopathology was performed on TRAMP mice. Results At 18 wks., plasma lycopene concentrations were 2.84-fold greater in tangerine tomato-fed mice than red tomato-fed mice (p &amp;lt; 0.0001), yet both red and tangerine tomato diets similarly prevent carcinoma incidence compared to the control diet by 43% and 36% respectively (p &amp;lt; 0.001, both vs. control). Our investigation of early carcinogenesis (10 wks.) shows that both tomato diets similarly impact the plasma metabolome, and we define prostate transcriptomic profiles mediated by TRAMP genotype, such as inflammation- and immune-regulated pathways, that are inhibited by dietary tomato. We identify an inflammatory gene panel (Olr1, Il1a, Cscl9, and Ccl22) which is inhibited by tomato diets. In a human prostate cancer database, higher expression of this gene panel is associated with a more aggressive phenotype. Conclusions Together, our results implicate red and tangerine tomatoes as potentially beneficial for the inhibition of early prostate carcinogenesis through the modulation of transcriptional programs centered on inflammation and the immune response. Our findings support efforts to test novel tomato products for the inhibition of human prostate carcinogenesis. Funding Sources AICR, NIH P30, USDA NNF.

Abstract A69: Elucidating the cell type-specific mechanisms of the Ron receptor in the prostate tumor microenvironment

Abstract The incidence of advanced prostate cancer (PCa) in American men has grown by 72% within the last decade, yet current treatments fail to effectively treat advanced disease. We study the Ron receptor tyrosine kinase as a novel target for PCa treatment. Ron is primarily expressed in macrophages and epithelial cells and is found overexpressed in PCa. Ron expression increases with disease severity in humans and mouse models, suggesting Ron drives progression to advanced PCa. Our laboratory has shown that loss of Ron signaling in prostate epithelial cells or in myeloid cells markedly reduces prostate tumor growth and modulates the tumor microenvironment, particularly the functions of macrophages and CD8+ T cells. Thus, we hypothesize that the Ron receptor utilizes cell type-specific mechanisms to suppress the antitumor immune response. However, our previous studies were limited because they were performed separately in different orthotopic models. Therefore, we employed the well-established Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) PCa mouse model to study loss of Ron signaling in either cell type in a single autochthonous model. We used Cre-Lox technology to generate TRAMP mice containing conditional deletion of Ron signaling in prostate epithelial cells or in myeloid cells. At 30 weeks of age, prostate tumors are harvested and analyzed by weight, immunohistochemical staining, quantitative PCR, Western blot, and flow cytometry. To assess the effects of Ron modulation in each cell type and interrogate the interaction between tumor cells and macrophages, we perform in vitro co-culture assays utilizing an established prostate cancer epithelial cell line derived from the TRAMP model and bone marrow-derived macrophages. We observe that Ron signaling in either cell type is critical for prostate tumor growth in TRAMP mice. Furthermore, loss of Ron in either cell type leads to increased macrophage and CD8+ T-cell tumor infiltration, and our in vitro data show that loss of Ron in either cell type suppresses antitumor M1 macrophage activation and promotes tumor-supporting M2 macrophage activation. Additionally, preliminary data suggest loss of Ron in either cell type leads to increased activation of CD8+ tumor-infiltrating lymphocytes. Overall, our data suggest the Ron receptor coordinates immunosuppressive mechanisms in both the malignant tumor cells and the host macrophages to dampen antitumor immunity and promote prostate cancer. These studies implicate Ron targeting as a powerful therapeutic tool with broad utility for understanding and refining therapeutic strategies to treat advanced PCa. Citation Format: Camille Sullivan, Nicholas E. Brown, Susan E. Waltz. Elucidating the cell type-specific mechanisms of the Ron receptor in the prostate tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A69.

Extract From Yellow Passion‐Fruit (passilora edulis) Inhibits Prostate Cancer Cell Proliferation By TLR4/NFkB Pathway Regulation

The yellow passion‐fruit (passiflora‐edulis) is rich in piceatannol (PIC), a polyphenolic and resveratrol analog known by its beneficial effects on health such as anti‐inflammatory anti‐cancer properties. The present study aimed to investigate the passion‐fruit extract (PFE) mechanistic of action in prostate cancer (PCa) cell lines with different genetic backgrounds and in the transgenic adenocarcinoma of the mouse prostate (TRAMP). For in vitro experiments, LNCaP (androgen‐responsive) and PC‐3 (androgen‐independent) cells were treated with different doses of PFE (125–500 μg/mL) and used for cell viability assay and western blotting analysis. For in vivo experiments, TRAMP mice were gavaged with 20mg/Kg of PIC contained in the PFE during 4 or 10 weeks of treatment. The ventral prostate was collected for light microscopy, immunohistochemistry and western blotting analysis. Because inflammation is intrinsically evolved with prostate alterations, like hormonal imbalance and cell survival, we focused on if PFE could affect these pathways. PFE treatment caused a significant reduction of cell viability in both cell lines tested, as well as decreased nuclear factor kappa B (NFκB) and toll‐like receptor 4 protein level. Interestingly, LNCaP cells were more sensitive to the PFE action in all experiments and we verified that the androgen receptor was markedly reduced by the treatment in these cells. PFE delayed CaP progression in the TRAMP model in both periods of treatment, significantly decreasing high‐grade prostatic intraepithelial neoplasia and well‐differentiated adenocarcinoma. These results were confirmed by the decrease of proliferating cell nuclear antigen immunostaining. Also, treatment with PFE reduced inflammation markers such as tumor necrosis factor‐alpha (TNF‐α), NFκB, and modulated negatively cell cycle regulatory protein levels and signal transducer and activator of transcription 3. Therefore, the results herewith showed that PFE was able to modulate disease progression through the inhibition of the inflammatory pathways, delay of the cell cycle, especially in the hormone‐dependent profile, demonstrating its chemopreventive and therapeutic potential in PCa.Support or Funding InformationSao Paulo Research Foundation ( Number: 2017/01573‐5)

Ultrasound and magnetic resonance imaging for group stratification and treatment monitoring in the transgenic adenocarcinoma of the mouse prostate model.

The transgenic adenocarcinoma of the mouse prostate (TRAMP) is a widely used genetically engineered spontaneous prostate cancer model. However, both the degree of malignancy and time of cancer onset vary. While most mice display slowly progressing cancer, a subgroup develops fast-growing poorly differentiated (PD) tumors, making the model challenging to use. We investigated the feasibility of using ultrasound (US) imaging to screen for PD tumors and compared the performances of US and magnetic resonance imaging (MRI) in providing reliable measurements of disease burden. TRAMP mice (n = 74) were screened for PD tumors with US imaging and findings verified with MRI, or in two cases with gross pathology. PD tumor volume was estimated with US and MR imaging and the methods compared (n = 11). For non-PD mice, prostate volume was used as a marker for disease burden and estimated with US imaging, MRI, and histology (n = 11). The agreement between the measurements obtained by the various methods and the intraobserver variability (IOV) was assessed using Bland-Altman analysis. US screening showed 81% sensitivity, 91% specificity, 72% positive predictive value, and 91% negative predictive value. The smallest tumor detected by US screening was 14 mm3 and had a maximum diameter of 2.6 mm. MRI had the lowest IOV for both PD tumor and prostate volume estimation. US IOV was almost as low as MRI for PD tumor volumes but was considerably higher for prostate volumes. US imaging was found to be a good screening method for detecting PD tumors and estimating tumor volume in the TRAMP model. MRI had better repeatability than US, especially when estimating prostate volumes.

The Impact of External Beam Radiation Therapy on Oxidative Damage in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) Prostate Cancer Model (P05-022-19)

ObjectivesExternal beam radiation therapy (EBRT) is one of the most common treatments for primary prostate cancer. The primary limitation for EBRT is the potential for damage to surrounding tissues. Epidemiological evidence indicates that tomato products are associated with reduced PCa progression. Tomatoes contain carotenoids and other potent antioxidants that may protect the surrounding tissue from the detrimental effects of EBRT. The objective of this pilot study is to determine if tomato feeding can alter early inflammatory and oxidative events following a single dose of radiation that may predict a more successful therapeutic outcome. MethodsTo test this hypothesis, male TRAMP mice (n = 60) were provided a powdered AIN-93 G diet (Control) or an AIN-93 G diet modified to contain 10% lyophilized tomato paste (TP; n = 30) beginning at 4 weeks of age. Mice were monitored by ultrasound biweekly for in vivo tumor detection and 3-D volumetric growth measurement. Tumors were irradiated with 7.5 gy (n = 15 per diet) or 0 gy (sham, n = 15 per diet) of radiation by a Cobolt-60 source once the animal had a 1000 mm3 tumor. Animals were euthanized after 24 hours and carotenoids were measured by high performance liquid chromatography (HPLC) in the tumor and liver. Sections of tumor, liver, kidneys, bladder, lymph, bladder and intestines were stained by hematoxylin and eosin-stained (H&E) and were assessed for tumor grade and inflammatory markers. Pro-inflammatory cytokines (IL-6 and TNFα) will be measured from the serum and tumor tissues by ELISA. Oxidative damage will be analyzed through the γH2AX assay and through measurement of reduced glutathione. ResultsThis study will be the first to explore the effects of tomato powder on the PCa tumor microenvironment following irradiation. We hypothesize that lifelong tomato consumption will delay tumor growth; will protect surrounding tissues from inflammatory and oxidative damage (reducing circulating pro-inflammatory cytokines); and will reduce histopathological markers of inflammation in surrounding tissues following a single exposure to 60-CO radiation in the TRAMP model. ConclusionsThis study will provide important preclinical data to inform future clinical trials that may positively affect dietary recommendations for EBRT to improve therapeutic outcomes. Funding SourcesUniversity of Illinois at Urbana-Champaign.

Abstract 4436: Eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) is upregulated modulates prostate cancer growth and proliferation

Abstract Introduction: cap-dependent translation is necessary due to high protein requirement in cancer cells. An interaction between EIF4E and EIF4G is crucial for the formation of the EIF4F complex and initiation of cap-dependent translation. In the current study, we analyzed Human prostate cancer tissue microarray (TMA) and mRNA expression data in clinical datasets, and prostate tumor tissue from TRAMP (Transgenic Adenocarcinoma of the Mouse Prostate) model. We also assessed the functional role of EIF4G1 in commonly used PCa cell lines. Methods: Human prostate cancer tissue microarray was used to analyze the EIF4G1 level in patient samples. mRNA expression data for EIF4G1 was analyzed from TCGA and Trento/Cornell/Broad clinical data sets. PCa cells viz. LNCaP, C4-2b, 22Rv1, DU145, PC3 and normal human prostate cell line RWPE-1 were used. For an in-vivo model of PCa, we used TRAMP and wild-type mouse. Loss of function studies was performed by using siRNA/shRNA. Real-time PCR and Western Blot analysis were used to quantitate the relative mRNA and protein levels respectively. Analysis of polysome was performed by sucrose density gradient fractionation. Polysome-to-Monosome (P/M) ratios were determined for global translation activity. Cell cycle, cell proliferation, cell migration and Clonogenic activity were measured by standard methods. Results: Results from TMA analysis showed that protein levels of EIF4G1 are high in PCa as compared to normal prostate tissue, and there is a graded increase in EIF4G1 as the disease progresses. Results of our analysis of EIF4G1 expression in TCGA database revealed that increased expression of EIF4G1 positively correlated with higher tumor grade and stages (Gleason Score). Available data from Trento/Cornell/Broad clinical dataset revealed that 43% of castration-resistant prostate cancer (CRPC) patients have EIF4G1 mRNA up-regulation. PCa cells express a significantly higher level of EIF4G1 as compared to normal prostate cells. Similarly, prostate tumor tissue from TRAMP tissue showed higher EIF4G1 expression as compared to normal wild-type prostate tissue. Silencing of EIF4G1 causes G0/G1 cell cycle delay and decreases Cyclin D1 and p-Rb levels. There is a shift in polysome (P) to monosome (M) ratio with the siEIF4G1 knockdown in LNCaP and C4-2b. Loss of function studied by knockdown of EIF4G1 showed impaired Clonogenic activity as well as cell proliferation. Real-time PCR data suggests that EIF4G1 knockdown in LNCaP &amp; C4-2b decreases the level of EMT markers such as N-Cadherin, Vimentin &amp; Zeb1 and limits the cell migration in C4-2b cells. Conclusions: Taken all together, our data indicate that EIF4G1 may function as an oncoprotein and is a novel target for intervention in PCa and CRPC. Citation Format: Praveen K. Jaiswal, Sweaty Koul, Prakash Srinivasan Timiri Shanmugam, Hari K. Koul. Eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) is upregulated modulates prostate cancer growth and proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4436.

Chronic low dose ethanol induces an aggressive metastatic phenotype in TRAMP mice, which is counteracted by parthenolide.

Despite advances in prostate cancer therapy, dissemination and growth of metastases results in shortened survival. Here we examined the potential anti-cancer effect of the NF-κB inhibitor parthenolide (PTL) and its water soluble analogue dimethylaminoparthenolide (DMAPT) on tumour progression and metastasis in the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model of prostate cancer. Six-week-old male TRAMP mice received PTL (40mg/kg in 10% ethanol/saline), DMAPT (100mg/kg in sterile water), or vehicle controls by oral gavage thrice weekly until palpable tumour formation. DMAPT treatment slowed normal tumour development in TRAMP mice, extending the time-to-palpable prostate tumour by 20%. PTL did not slow overall tumour development, while the ethanol/saline vehicle used to administer PTL unexpectedly induced an aggressive metastatic tumour phenotype. Chronic ethanol/saline vehicle upregulated expression of NF-κB, MMP2, integrin β1, collagen IV, and laminin, and induced vascular basement membrane degradation in primary prostate tumours, as well as increased metastatic spread to the lung and liver. All of these changes were largely prevented by co-administration with PTL. DMAPT (in water) reduced metastasis to below that of water-control. These data suggest that DMAPT has the potential to be used as a cancer preventive and anti-metastatic therapy for prostate cancer. Although low levels of ethanol consumption have not been shown to strongly correlate with prostate cancer epidemiology, these results would support a potential effect of chronic low dose ethanol on metastasis and the TRAMP model provides a useful system in which to further explore the mechanisms involved.

Soy-enriched diet limits tumor progression and reduces myeloid derived suppressor cells in the murine TRAMP model of prostate cancer.

Abstract Epidemiological studies suggest diets enriched with soy confer a lower risk of prostate cancer. Data from our group and others indicate that soy has immunomodulatory properties which may contribute to their health promoting effects. Prior results from our group in a randomized phase II clinical trial revealed that men with asymptomatic prostate cancer on a soy-enriched diet had significantly reduced systemic inflammatory cytokines and myeloid-derived suppressor cells (MDSCs). We hypothesized that a soy-enriched diet limits systemic immunosuppression by reducing MDSC expansion. To understand how dietary soy modulates immune biomarkers in prostate cancer, we administered TRAMP (transgenic adenocarcinoma of the mouse prostate) mice a soy-enriched (n=28) or control diet (n=32) for 16 weeks. TRAMP mice administered a soy-enriched diet had a reduced tumor burden compared to control fed mice (p=0.0419). Splenic CD11b+Ly6GloLy6C− granulocytic (p=0.0195), CD11b+Ly6C+Ly6G− monocytic (p=0.0005), and total MDSCs (p=0.0009) were significantly reduced in TRAMP mice on a soy-enriched diet compared to control diet. However, we did not observe differences in circulating CD4+CD25+Foxp3+ T-regulatory cells or other circulating immune subsets such as CD4+ T cells, phenotypically defined T helper-1 (CD4+CXCR3+CCR4−CCR6−)/T helper-2 (CD4+CCR4+CXCR3+CCR6−) T cells, CD8+ T cells, and NK cells. Ongoing studies are examining plasma cytokine levels and immunosuppressive cell populations in the tumor microenvironment of mice from this study. Together, these data indicate that soy as a dietary natural product may have a modulatory effect during chemopreventive or immunotherapy in the setting of prostate cancer.

Epigenetic alterations in TRAMP mice: epigenome DNA methylation profiling using MeDIP-seq

PurposeWe investigated the genomic DNA methylation profile of prostate cancer in transgenic adenocarcinoma of the mouse prostate (TRAMP) cancer model and to analyze the crosstalk among targeted genes and the related functional pathways.MethodsProstate DNA samples from 24-week-old TRAMP and C57BL/6 male mice were isolated. The DNA methylation profiles were analyzed by methylated DNA immunoprecipitation (MeDIP) followed by next-generation sequencing (MeDIP-seq). Canonical pathways, diseases and function and network analyses of the different samples were then performed using the Ingenuity® Pathway Analysis (IPA) software. Some target genes with significant difference in methylation were selected for validation using methylation specific primers (MSP) and qPCR.ResultsTRAMP mice undergo extensive aberrant CpG hyper- and hypo-methylation. There were 2147 genes with a significant (log2-change ≥ 2) change in CpG methylation between the two groups, as mapped by the IPA software. Among these genes, the methylation of 1105 and 1042 genes was significantly decreased and increased, respectively, in TRAMP prostate tumors. The top associated disease identified by IPA was adenocarcinoma; however, the cAMP response element-binding protein (CREB)-, histone deacetylase 2 (HDAC2)-, glutathione S-transferase pi (GSTP1)- and polyubiquitin-C (UBC)-related pathways showed significantly altered methylation profiles based on the canonical pathway and network analyses. MSP and qPCR results of genes of interests corroborated with MeDIP-seq findings.ConclusionsThis is the first MeDIP-seq with IPA analysis of the TRAMP model to provide novel insight into the genome-wide methylation profile of prostate cancer. Studies on epigenetics, such as DNA methylation, will potentially provide novel avenues and strategies for further development of biomarkers targeted for treatment and prevention approaches for prostate cancer.

Abstract 5061: TET inhibits prostate cancer tumor growth, progression and metastasis in TRAMP mice

Abstract Introduction: Prostate cancer (PCa) is the 2nd most common malignancy in USA. Novel agents for treatment of advanced PCa are warranted. Transgenic adenocarcinoma of the mouse prostate (TRAMP) is an autochthonous mouse model exhibits both histological and morphological features that mimic human prostate carcinogenesis. Herein, for the first time, we evaluated the in vivo effects of TET, a derivative of Tetrandrine in TRAMP model. Methods: Beginning 12 weeks of age, male TRAMP mice were administrated with TET (30 mgm/kg body weight, orally, alternative days) in PBS or PBS alone (Control) till 30 weeks of age. Body weight (B) of animals was recorded weekly. At various time points animals were euthanized, genitourinary tract (G) were weighed. Prostate tissue was subjected to immunohistochemical analyses for SV40-TAg, epithelial-mesenchymal transition (EMT), proliferation, neuroendocrine differentiation (NED) and apoptosis. Multiple organs were examined for drug toxicity and lungs were analyzed for metastasis. Results: TRAMP mice exhibit to high-grade prostatic intraepithelial neoplasia (PIN) by 12 weeks and progresses to poorly differentiated adeno-carcinoma by 30 weeks with distant metastasis to lungs. TET feeding did not show any considerable difference body weight loss profiles during the entire treatment regimen. At the time of necropsy, there was no evidence of edema, abnormal organ size or appearance in non-target organs. TET gavage group showed (p&amp;lt;0.005) lower G/B ratio compared to the PBS treated group. These findings clearly indicate that TET dosing is non-toxic and restricts the abnormal growth of the prostate in TRAMP mice. TET repress the EMT as well as NED transition and inhibits cell proliferation (p&amp;lt;0.005) by Ki-67 staining. Oral administration of TET inhibits PCa growth and progression by increases (p&amp;lt;0.005) apoptosis in tumor tissues. Further, TET inhibited metastasis as there was significant (p&amp;lt;0.005) decrease in metastasis to lungs in TET treated animals. Conclusion: Human achievable dose of TET treatment to TRAMP mice bearing prostate tumor, exhibited no-observed-adverse-effect-level in toxicology evaluations and also significantly inhibited tumor growth, progression, local invasion and distant metastasis involving suppression of tumor, and thus could have potential against human PCa. Citation Format: Hari K. Koul, Prakash Srinivasan Timiri Shanmugam, Praveen K. Jaiswal, Sweaty Koul. TET inhibits prostate cancer tumor growth, progression and metastasis in TRAMP mice [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 5061. doi:10.1158/1538-7445.AM2017-5061

Abstract 4327: Dietary tomato reduces castration-resistant prostate cancer progression in TRAMP mice

Abstract Epidemiological data support the hypothesis that consumption of diets rich in tomato products are associated with a reduced risk of prostate cancer. Castration-resistant prostate cancer (CRPC) represents the late and lethal phase of human prostate carcinogenesis, and is defined as cancer progression after surgical castration or pharmacologic reduction of serum testosterone through androgen deprivation therapy. Mechanisms involved in the transition to CRPC include cancer cells’ acquired capacity for androgen synthesis or activation of ligand-independent signaling. We have previously shown that dietary tomato can inhibit prostatic androgen signaling and expression of androgen biosynthetic genes, as well as reduce primary cancer incidence in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Therefore, we examined whether dietary tomato might be an effective inhibitor of CRPC progression in mice. We hypothesized that lifelong dietary intake of tomato, as well as dietary tomato intervention following castration, would reduce tumor burden and growth rate in a mouse model of CRPC. TRAMP mice were fed an AIN-93G diet (CON) for one week and then randomized to the CON diet (n = 28) or a similar diet with 10% w/w lyophilized tomato paste (TP; n = 27) from 4 wks of age until euthanization. A third group, modeling dietary intervention, consumed CON from 4 to 12 wks of age, and the 10% w/w lyophilized tomato paste diet from wk 12 until euthanization (TP-I; n = 25). All animals were castrated at 12 wks of age. Beginning at 10 wks of age, mice were monitored longitudinally with ultrasound for tumor detection, tumor volume estimation, and calculation of tumor growth rate. Serial 2D image slices were used to generate a 3D volume estimate. Mice were euthanized after 5 consecutive tumor scans, or if no tumor had been detected by 30 weeks of age. Tumor growth area under the curve (AUC) was reduced approximately 46% by tomato intervention (TP-I) and 27% by lifelong tomato consumption (TP). At euthanization, TP-I reduced tumor weight by approximately 26%. Additionally, incidence of gross metastases to distant organs (lung, liver, kidney) was strongly inhibited by TP-I, compared to CON (0% and 26%, respectively). TP diet reduced incidence of distant organ gross metastasis to &amp;lt;10%. The consumption of tomato products following castration inhibits CRPC progression in the TRAMP model. Our findings support the development of human studies of dietary tomato interventions in combination with anti-androgen therapy for men with advanced prostate cancer. Citation Format: Joshua W. Smith, Joe L. Rowles, Rita J. Miller, Steven K. Clinton, William D. O’Brien, John W. Erdman. Dietary tomato reduces castration-resistant prostate cancer progression in TRAMP mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4327.

Diffusion‐weighted MRI for early detection and characterization of prostate cancer in the transgenic adenocarcinoma of the mouse prostate model

To improve early diagnosis of prostate cancer to aid clinical decision-making. Diffusion-weighted magnetic resonance imaging (DW-MRI) is sensitive to water diffusion throughout tissues, which correlates with Gleason score, a histological measure of prostate cancer aggressiveness. In this study the ability of DW-MRI to detect prostate cancer onset and development was evaluated in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. T2 -weighted and DW-MRI were acquired using a 7T MR scanner, 200 mm bore diameter; 10 TRAMP and 6 C57BL/6 control mice were scanned every 4 weeks from 8 weeks of age until sacrifice at 28-30 weeks. After sacrifice, the genitourinary tract was excised and sectioned for histological analysis. Histology slides registered with DW-MR images allowed for validation of DW-MR images and the apparent diffusion coefficient (ADC) as tools for cancer detection and disease stratification. An automated early assessment tool based on ADC threshold values was developed to aid cancer detection and progression monitoring. The ADC differentiated between control prostate ((1.86 ± 0.20) × 10(-3) mm(2) /s) and normal TRAMP prostate ((1.38 ± 0.10) × 10(-3) mm(2) /s) (P = 0.0001), between TRAMP prostate and well-differentiated cancer ((0.93 ± 0.18) × 10(-3) mm(2) /s) (P = 0.0006), and between well-differentiated cancer and poorly differentiated cancer ((0.63 ± 0.06) × 10(-3) mm(2) /s) (P = 0.02). DW-MRI is a tool for early detection of cancer, and discrimination between cancer stages in the TRAMP model. The incorporation of DW-MRI-based prostate cancer stratification and monitoring could increase the accuracy of preclinical trials using TRAMP mice.

Abstract A59: Dietary tomato and lycopene inhibition of prostate carcinogenesis in the TRAMP Model is β,β-carotene 9',10'-oxygenase (BCO2)-dependent

Abstract Background: Epidemiology and laboratory studies have implicated diets rich in tomatoes and the carotenoid lycopene in prostate cancer risk reduction. However, the specific roles of lycopene, lycopene metabolites, or other tomato phytochemicals are uncertain. The discovery of carotenoid metabolizing enzymes has opened the door to mechanistic studies. The enzyme β,β-carotene 9',10'-oxygenase (BCO2) has been shown to asymmetrically cleave lycopene and other carotenoids, yielding metabolites. We hypothesize that these cleavage products may be more biologically active and convey anti-cancer activity. Therefore, we defined the impact of BCO2 ablation on the efficacy of dietary tomato and lycopene to inhibit cancer progression in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse model of prostate carcinogenesis. Methods: BCO2-/- (B6; 129S6-Bcdo2tm1Dnp) mice were crossed with TRAMP+/- (C57BL/6-Tg(TRAMP)8247Ng/J) transgenic mice leading to the generation of TRAMP+/-:BCO2+/+ and TRAMP+/-:BCO2-/-. Male TRAMP:BCO2+/+ and TRAMP:BCO2-/- mice were randomized to three dietary treatments: AIN-93G control diet, 10% tomato powder diet, or 0.25% lycopene beadlet-containing diet from 3 until 18 weeks of age. Serum was collected for carotenoid analysis by HPLC, and prostate tissues were processed for histopathologic assessment of prostate carcinogenesis. Results: TRAMP:BCO2 +/+ mice fed tomato powder and lycopene attained similar serum lycopene concentrations (0.141μM +/- 0.007μM vs. 0.139μM +/- 0.007μM, respectively). The presence of the tomato carotenoids phytofluene (0.13μM +/- 0.002μM) and phytoene (0.025μM +/- 0.003μM) in serum was only observed with tomato feeding as anticipated. Loss of BCO2 enzyme resulted in significantly increased serum concentrations of total, total cis, 5-cis, and all-trans lycopene in both tomato and lycopene fed mice, and the accumulation of phytoene, phytofluene, and zeta-carotene in the tomato fed mice compared to the BCO2+/+ counterparts. For example, total serum lycopene levels were 0.229μM +/- 0.029μM and 0.223μM +/- 0.030μM in tomato- and lycopene-fed TRAMP:BCO2-/- mice, respectively. Dietary tomato powder and lycopene inhibited cancer progression. Incidence of cancer was 80% in control diet-fed TRAMP:BCO2+/+ mice, while tomato and lycopene feeding decreased incidence to 15% and 11%, respectively. Ablation of BCO2 (TRAMP:BCO2-/-) partially attenuated the anti-cancer efficacy of tomato and lycopene observed in TRAMP:BCO2+/+ mice as the incidence was 24% for tomato-fed and 46% for lycopene-fed mice. Conclusions: Overall, dietary tomato and lycopene inhibited the progression of prostate cancer in TRAMP in a BCO2-dependent manner. Future human studies to examine the role of lycopene metabolism and BCO2 genotype in cancer risk reduction are warranted. Citation Format: Hsueh-Li Tan, Jennifer M. Thomas-Ahner, Nancy E. Moran, Gregory S. Young, John W. Erdman, Jr., Steven K. Clinton. Dietary tomato and lycopene inhibition of prostate carcinogenesis in the TRAMP Model is β,β-carotene 9',10'-oxygenase (BCO2)-dependent. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr A59.

Immunosuppression via Tenascin-C.

Immunosuppression via Tenascin-C.

Low-lycopene containing tomato powder diet does not protect against prostate cancer in TRAMP mice

Previously, tomato powder (TP) diets initiated postweaning have been shown to be effective in reducing prostate cancer in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. The TRAMP model develops and progresses through all stages of carcinogenesis similarly to humans. We hypothesized that a 10% TP diet intervention after puberty would reduce carcinogenesis at 12, 16, and 20 weeks of age in TRAMP mice. Eight-week-old male C57BL/6 X FVB F1 TRAMP mice were randomized to consume either an AIN-93G + 10% TP diet (n = 90) or the AIN-93G control diet (n = 88) and randomized to 1 of 3 end point ages: 12 (n = 59), 16 (n = 60), or 20 (n = 59) weeks of age. There was no difference between diets in overall cancer incidence at any time point. However, at 16 weeks of age, TP significantly increased high-grade PIN (P = .014) and significantly decreased poorly differentiated (P = .024) lesions compared with the control diet suggesting a delay in the progression of prostate cancer. Two variables that may explain the modest effect of TP in this study are as follows: the low amount of lycopene in the TP diet (12.3 ppm) and the timing of the intervention (8 weeks of age). The TP diet contained 30-fold less lycopene than previous studies in our laboratory. In addition, the initiation of the diet intervention time of 8 weeks of age instead of 4 weeks of age may have been too late in cancer progression to substantially impact carcinogenesis. In conclusion, a low-lycopene TP intervention failed to reduce carcinogenesis in TRAMP mice.

Capsaicin reduces the metastatic burden in the transgenic adenocarcinoma of the mouse prostate model

Capsaicin, the active compound in chili peppers, has demonstrated anti- carcinogenic properties in vitro in a number of malignancies, including the prostate. In the present study, we investigate the chemopreventive potential of capsaicin on prostate cancer using the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. The TRAMP is a murine model that resembles the progression of human disease. Thirty-five 6-week-old TRAMP x C57BL/6 mice were randomized between treatment with capsaicin (5 mg/kg body weight) or control (saline) three times a week by oral gavage until 30 weeks of age. Body weight of animals was recorded thrice weekly. At termination, all tumors were extracted, recorded, and analyzed for histopathological analysis. To understand the effect of capsaicin on migration and invasion, in vitro experiments were carried out using PC3 cells. Mice in the control group expressed an overall trend of higher-grade disease with 37.5% poorly differentiated (PD), 18.75% moderately differentiated (MD), and 44% of well-differentiated (WD) adenocarcinoma, compared to the capsaicin-treated group with only 27.7% PD, 61.0% of WD, and 11.1% of intraepithelial neoplasia (PIN). The treatment group demonstrated a higher incidence of noncancerous PIN lesions compared to the control group. The capsaicin group also demonstrated a significant reduction (P < 0.05) in the metastatic burden compared to the controls, which correlated to a reduction in p27(Kip) (1) expression and neuroendocrine differentiation in prostate tumors. Furthermore, there were no differences in body weight between groups overtime, and no pathological toxicities in the liver and gastrointestinal tract with capsaicin consumption. In vitro studies revealed a dose-dependent reduction in the invasion and migration capacity of PC3 cells. The following study provides evidence supporting the safety and chemopreventive effects of capsaicin in the TRAMP model.

Low‐Lycopene Tomato Powder Alters Prostate Biology in TRAMP Mice

The Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model develops and progresses through all stages of carcinogenesis. We, (Zuniga et al., Cancer Prev. Res. 2013) previously demonstrated a high lycopene tomato powder (TP) was effective in reducing carcinogenesis in the TRAMP model. The objective of the current study was to determine if a low-lycopene TP (20 fold less than previously tested) impacted carcinogenesis and androgen biology at 3 time points. 8-week-old male C57BL/6 X FVB TRAMP mice were randomized to consume either an AIN-93G + 10% TP diet (N=90) or the AIN-93G control diet (N=88) and assigned to one of three sacrifice ages: 12 (N=59), 16 (N=60), or 20 (N=59) weeks. There was no difference between diets in overall cancer incidence at each time point. TP significantly increased serum testosterone (p=0.01) and expression of prostatic androgen receptor in high-grade PIN lesions (p=0.01) at 20 weeks of age compared to the control, suggesting an interaction between tomato components and androgen status in pre-neoplastic prostate tissue. The results suggest that lycopene content of TP is a crucial modulator of PCa in TRAMP mice. We have shown that a low-lycopene TP is ineffective in reducing carcinogenesis in the TRAMP model. Support was provided by USDA Hatch grant #ILLU-971-348 and UIUC Margin of Excellence Research award.

Dietary methionine restriction inhibits prostatic intraepithelial neoplasia in TRAMP mice.

Prostate cancer (PCa) is a major aging-related disease for which little progress has been made in developing preventive strategies. Over the past several years, methionine restriction (MR), the feeding of a diet low in methionine (Met), has been identified as an intervention which significantly extends lifespan and reduces the onset of chronic diseases, including cancer, in laboratory animals. We, therefore, hypothesized that MR may be an effective strategy for inhibiting PCa. Control (0.86% Met) or MR (0.12% Met) diets were fed to 5-week old TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, a well-characterized model for PCa. The mice were sacrificed at 16 weeks of age and prostate and other tissues were harvested for histological and biochemical analyses. As previously reported, MR was associated with a decrease in body weight which was not associated with lowered food intake. MR led to significant reductions in the development of Prostatic Intraepithelial Neoplasia (PIN) lesions, specifically in the anterior and dorsal lobes of the prostate where the incidence of high-grade PIN was reduced by ∼50% (P < 0.02). The reduction in PIN severity was associated with 46-64% reductions in cell proliferation rates (P < 0.02) and plasma IGF-1 levels (P < 0.0001), which might, in part, explain the effects on carcinogenesis. Additionally, no adverse consequences of MR on immune function were observed in the TRAMP mice. Overall, these findings indicate that MR is associated with a reduction in prostate cancer development in the TRAMP model and supports the continued development of MR as a potential PCa prevention strategy.