Adenocarcinoma Of Mouse Prostate Model Research Articles

Combined maternal and post-weaning high fat diet inhibits male offspring's prostate cancer tumorigenesis in transgenic adenocarcinoma of mouse prostate model.

High fat diet (HFD) and its responsive change of gut microbiota are associated with numerous diseases in human and animal studies. But how maternal and post-weaning HFD may influence the tumorigenesis of prostate cancer (PCa) in male offspring has not yet been reported. We studied both solitary and combined effects of maternal or/and post-weaning HFD on the tumorigenesis of prostate in offspring using a transgenic adenocarcinoma of mouse prostate (TRAMP) model. TRAMP male mice (n = 24) were born to and fostered onto control diet or HFD-fed mothers, the weaned pups were further fed control or fat diet. All mice were sacrificed at the 28th week. The prostate histopathology was performed. The gut microbiota was analyzed by fecal 16S rRNA pyrosequencing, and the serum metabolites were determined by LC-MS/MS. Our results indicated that post-weaning HFD could significantly promote the PCa tumorigenesis in offspring (P = 0.031), and similar results were found in maternal HFD group but with no significant differences (P = 0.056). However, combined maternal and post-weaning HFD could significantly reduce the PCa tumorigenesis in offspring (P = 0.019). Significant differences in specific microbial populations and serum metabolites were observed across groups. For example, mice with combined maternal and post-weaning HFD had higher abundance of gut Lachonospiraceae, Roseburia, and Amycolatopsis, increased serum L-methionine (P = 0.0052), and decreased serum α-linolenic acid (P < 0.001). This study highlights the inhibitory effects of combined maternal and post-weaning HFD on PCa tumorigenesis, and provides new insights into the interactions between diet, gut microbiota, serum metabolites, and PCa.

Nimbolide-Induced Oxidative Stress Abrogates STAT3 Signaling Cascade and Inhibits Tumor Growth in Transgenic Adenocarcinoma of Mouse Prostate Model.

Prostate cancer (PCa) is one of the most commonly diagnosed cancers worldwide. Currently available therapies for metastatic PCa are only marginally effective, hence novel treatment modalities are urgently required. Considerable evidence(s) suggest that deregulated activation of oncogenic transcription factor, signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in the development and progression of PCa. Thus, agents that can abrogate STAT3 activation could form the basis of novel therapy for PCa patients. In the present study, we analyzed whether the potential anticancer effects of nimbolide (NL), a limonoid triterpene derived from Azadirachta indica, against PCa cell lines and transgenic adenocarcinoma of mouse prostate (TRAMP) model are mediated through the negative regulation of STAT3 pathway. Data from the in vitro studies indicated that NL could significantly inhibit cell viability, induce apoptosis, and suppress cellular invasion and migration. Interestingly, NL also abrogated STAT3 activation and this effect was found to be mediated via an increased production of reactive oxygen species (ROS) due to GSH/GSSG imbalance. Oral administration of NL significantly suppressed the tumor growth and metastasis in TRAMP mouse model without exhibiting any significant adverse effects. The present study demonstrates the critical role of GSH/GSSG imbalance-mediated ROS production contributing to the STAT3 inhibitory and tumor-suppressive effect of NL in PCa. Overall, our findings indicate that NL exhibits significant anticancer effects in PCa that may be primarily mediated through the ROS-regulated inhibition of STAT3 signaling cascade.

Abstract 2133: Suppressing effect of a Kava fraction on two lineages of prostate carcinogenesis in the transgenic adenocarcinoma of mouse prostate model

Abstract Kava (Piper methysticum Forest) is a crop historically originating from west Pacific Islands. The epidemiologic studies for Pacific Island populations indicated that their cancer incidence rates were inversely related to the local consumptions of kava. Whereas chemopreventive efficacy of Kava ethanol extract has been shown in chemically-induced lung carcinogenesis models, its efficacy against prostate carcinogenesis is not known. In this study, we aim to evaluate the efficacy of Kava Fraction B, which is mostly kavalactones and free of flavokawains and most potent in lung carcinogenesis models, on prostatic carcinogenesis in the well-characterized C57BL/6 transgenic adenocarcinoma of mouse prostate (TRAMP) preclinical model. Previous studies by us (The Prostate, 2011) and others have suggested a paradigm of distinct lineages of carcinogenesis in this model: i.e., at least, androgen receptor (AR)-dependent glandular epithelia lesions (usually arise from the dorsal-lateral prostate [DLP] lobes) and AR-independent neuroendorine (NE)-like poorly differentiated (PD) carcinomas (usually derive from the ventral prostate [VP] lobe). Experimentally, male C57BL/6 TRAMP mice were fed AIN93M purified diet without or with 0.4% of Kava fraction B from 8 weeks of age (WOA). Mice were euthanized at either 16 WOA or 28 WOA unless large tumors necessitated earlier sacrifice. The growth of the DLP in Kava-treated TRAMP mice were inhibited by 70% (P&amp;lt;0.05) from control TRAMP mice by 16 WOA. By 28 WOA, the inhibition of TRAMP DLP growth were 76% (P&amp;lt;0.001). Furthermore, Kava-fed TRAMP mice cohort had lower incidence of palpable and dissectable tumors (2 out of 14 mice) than control mice (7 out of 14 mice). Therefore, Kava fraction B appeared to inhibit both epithelial lineage lesions and NE-carcinogenesis. Future work will focus on 1) profiling cellular processes and molecular targets of Kava fraction B and 2) identifying active compounds in Kava Fraction B that inhibit epithelial lineage lesions and NE-carcinogenesis. Supported in part by NCCAM AT007395 R01 and NCI CA142649 R01 grants. Citation Format: Su-Ni Tang, Palika Datta, Peixin Jiang, Pablo Leitzman, Cheng-guo Xing, Cheng Jiang, Junxuan Lu. Suppressing effect of a Kava fraction on two lineages of prostate carcinogenesis in the transgenic adenocarcinoma of mouse prostate model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2133. doi:10.1158/1538-7445.AM2014-2133

Knockout of the tetraspaninCd9in the TRAMP model ofde novoprostate cancer increases spontaneous metastases in an organ-specific manner

Prostate cancer is an extremely heterogeneous disease; patients that do progress to late-stage metastatic prostate cancer have limited treatment options, mostly palliative. Molecules involved in the metastatic cascade may prove beneficial in stratifying patients to assign appropriate treatment modalities and may also prove to be therapeutic antimetastatic targets. The tetraspanin group of molecules are integral membrane proteins that associate with motility-related proteins such as integrins. Clinical studies have mostly shown that reduced expression levels of the tetraspanin CD9 are correlated with tumour progression in a range of cancers. Furthermore, functional studies have shown CD9 to be involved in cell motility and adhesion and that it may influence metastasis. The effects of endogenous CD9 on prostate cancer initiation and progression were analysed by crossing a Cd9-/- (KO) murine model with a model of de novo developing and spontaneously metastasising prostate cancer, namely the transgenic adenocarcinoma of mouse prostate model. Our study demonstrates for the first time that ablation of Cd9 had no detectable effect on de novo primary tumour onset, but did significantly increase metastasis to the liver but not the lungs.

Abstract LB-184: A paradigm of carcinogenesis lineage specificities of cancer chemoprevention: Korean Angelica extract and its pyranocoumarins in the transgenic adenocarcinoma of mouse prostate model.

Abstract Oriental medicinal herbals hold great promise for low cost and non-toxic chemopreventive remedies for cancers of prostate and other organs. Rigorous efficacy assessment in preclinical models of prostate carcinogenesis is necessary and essential for evidence-based “translational” preventive oncology with such herbal natural products. Our international collaborative team had recently shown Korean Angelica gigas Nakai (AGN) ethanolic extract with anti-cancer efficacy in a number of xenograft or allograft cancer models. However the active chemical species and cellular and molecular targets remain to be identified. In the present study we aim to test the hypothesis that orally administered AGN extract or its major pyranocoumarin compounds decursin/decursinol angelate (D/DA) inhibit prostatic carcinogenesis in the well-characterized C57BL/6 transgenic adenocarcinoma of mouse prostate (TRAMP) mice as the preclinical model. Previous studies by us (The Prostate, 2011) and others have suggested a new paradigm of distinct lineages of carcinogenesis in this model: i.e., androgen receptor (AR)-dependent glandular epithelial lesions (usually arise from the dorsal-lateral prostate [DLP] lobes) and AR-independent neuroendocrine (NE)-like poorly differentiated (PD) carcinomas (usually from the ventral prostate [VP] lobes). Experimentally, 3 cohorts of male C57BL/6 TRAMP mice (35-36 mice per group) were fed AIN93M purified diet and gavaged with either 0.15 mL of vehicle (Ethanol: PEG400: Tween 80: 5% glucose = 3:6:1:20), AGN (5 mg per mouse) or D/DA (3 mg per mouse, equi-molar to that in AGN) 5 days per week from 8 weeks of age (WOA). Mice were euthanized at either 16 WOA (n=12 mice per group) or 28 WOA unless large tumors necessitated earlier sacrifice. The results show that the growth of the TRAMP DLP lobes in AGN- and D/DA-treated mice were inhibited by 71% and 61%, respectively (ANOVA, P&amp;lt;0.0001), from vehicle-control mice by 16 WOA. By 28 WOA, the inhibition of TRAMP DLP growth was 73% and 75%, respectively (ANOVA, P&amp;lt;0.001). Whereas AGN decreased the burden of palpable prostate tumors (i.e., NE-Ca), D/DA did not. In summary, AGN extract and equi-molar intake of D/DA inhibit epithelial lineage lesions, the extract is much superior to D/DA compounds at suppressing NE-carcinogenesis, lending credence to the lineage specific targeting hypothesis. Future work will focus on 1) identifying non-pyranocoumarin compounds in AGN extract that inhibit NE-carcinogenesis; 2) testing whether D/DA-hydrolysis product decursinol mediates the epithelial targeting inhibitory action. Supported by NCCAM AT 005383 R21 and AT007395 R01 grants. Citation Format: Suni Tang, Yong Zhang, Peixin Jiang, Jinhui Zhang, Chengguo Xing, Sung-Hoon Kim, Cheng Jiang, Junxuan Lu. A paradigm of carcinogenesis lineage specificities of cancer chemoprevention: Korean Angelica extract and its pyranocoumarins in the transgenic adenocarcinoma of mouse prostate model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-184. doi:10.1158/1538-7445.AM2013-LB-184

Targeting and Killing of Metastatic Cells in the Transgenic Adenocarcinoma of Mouse Prostate Model With Vesicular Stomatitis Virus

Vesicular stomatitis virus (VSV) is an oncolytic virus which selectively infects and kills cancer cells. The goal of the present study was to determine whether VSV is capable of targeting metastatic lesions that arise in situ in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. The interferon (IFN)-responsive luciferase containing VSV(AV3) strain was injected intraprostatically into both control and TRAMP mice. Distribution, infectivity, apoptosis, and status of the IFN response were evaluated at the site of viral injection (prostate), as well as in metastatic lesions (lymph nodes), through plaque, polymerase chain reaction (PCR), and immunohistochemical analysis. Bioluminescence analyses demonstrated that VSV(AV3) persisted at high levels in the prostate region of TRAMP mice for up to 96 hours, but at relatively low levels and for only 48 hours in control mice. Live virus was discovered in the lymph nodes of TRAMP mice, but not in control mice. TUNEL staining revealed increased cell death in VSV(AV3) infected metastatic cells present in the lymph nodes of TRAMP mice. There was an evidence of IFN activation in lymph nodes containing metastatic cells. Our results indicate that intraprostatic injections of VSV(AV3) can be used as a means to infect and kill metastatic lesions associated with advanced prostate cancer.

Systemic Buffers Inhibit Carcinogenesis in TRAMP Mice

Hypoxia and acidosis develop in in situ tumors as cellular expansion increases the diffusion distance of substrates and metabolites from blood vessels deep to the basement membrane. Prior studies of breast and cervical cancer revealed that cellular adaptation to microenvironmental hypoxia and acidosis is associated with the transition from in situ to invasive cancer. We hypothesized that decreased acidosis in intraductal tumors would alter environmental selection pressures for acid adapted phenotypes and delay or prevent evolution to invasive cancer. A total of 37 C57BL/6 TRAMP mice were randomized to a control group or to 1 of 4 treatment groups. In the latter groups 200 mM sodium bicarbonate were added to drinking water starting between ages 4 and 10 weeks. In all 18 controls prostate cancer developed that was visible on 3-dimensional ultrasound at a mean age of 13 weeks. They died within 52 weeks (median 37). When sodium bicarbonate therapy commenced before age 6 weeks in 10 mice, all reached senescence (age 76 weeks) without radiographic evidence of prostate cancer. Histological sections of the prostates in this cohort showed hyperplasia but no cancer in 70% of mice and minimal well differentiated cancer in the remainder. When therapy commenced after age 6 weeks in 9 mice, prostate cancer development was no different from that in controls. Immunohistochemical staining for carbonic anhydrase 9 in regions of ductal hyperplasia showed increased expression in controls vs the early treatment group. Regional pH perturbation in in situ tumors may be a simple, inexpensive and effective cancer prevention strategy.

Inhibition of CXCR4/CXCL12 signaling axis by ursolic acid leads to suppression of metastasis in transgenic adenocarcinoma of mouse prostate model

Increasing evidences indicate that CXCR4/CXCL12 signaling pathway plays a pivotal role in the process of distant site metastasis that accounts for more than 90% of prostate cancer related deaths in patients. Thus, novel drugs that can downregulate CXCR4/CXCL12 axis have a great potential in the treatment of metastatic prostate cancer. In this report, we tested an agent, ursolic acid (UA) for its ability to modulate CXCR4 expression in prostate cancer cell lines and inhibit metastasis in vivo in transgenic adenocarcinoma of mouse prostate (TRAMP) model. We observed that UA downregulated the expression of CXCR4 in prostate cancer cells irrespective of their HER2 status in a dose- and time-dependent manner. Neither proteasome inhibitor nor lysosomal stabilization had any effect on UA-induced decrease in CXCR4 expression. When investigated for the molecular mechanisms, it was observed that the downregulation of CXCR4 was due to transcriptional regulation as indicated by downregulation of mRNA expression, inhibition of NF-κB activation and modulation of chromatin immunoprecipitation activity. Suppression of CXCR4 expression by UA further correlated with the inhibition of CXCL12-induced migration and invasion in prostate cancer cells. Finally, we also found that UA treatment can inhibit metastasis of prostate cancer to distal organs, including lung and liver and suppress CXCR4 expression levels in the prostate tissues of TRAMP mice. Overall, our experimental findings suggest that UA exerts its antimetastatic effects through the suppression of CXCR4 expression in prostate cancer both in vitro and in vivo.

Abstract 1614: Genetic loss of PKC epsilon inhibits development and metastasis of prostate cancer in transgenic adenocarcinoma of mouse prostate model

Abstract Protein kinase C epsilon (PKC∈), a novel PKC isoform is over-expressed in prostate cancer (PCa) development in both human and mouse. PKC∈ expression correlates with the aggressiveness of human PCa. However, the role of PKC∈ plays in the course of development and metastasis of PCa in vivo is unknown. To obtain the first molecular genetic evidence that PKC∈ is linked to development and metastasis of PCa, we generated PKC∈-deleted mice by crossbreeding transgenic adenocarcinoma of mouse prostate (TRAMP) (TG//WT) mice with PKC∈ knock out (TG//KO) mice. Sixteen weeks old TG Wild-type (TG//WT), TG//PKC∈ Heterozygous (TG//Het) and TG//PKC∈ KO (TG//KO) were evaluated for PCa development and metastasis. A significant decrease in the PKC∈ protein level was observed in the prostate tissues of TG//Het and TG//KO mice compared to TG//WT mice. PKC∈ deletion in TRAMP mice did not affect on the expression of other PKC isoforms neither in prostate nor in brain tissues. We performed MicroPET/CT imaging, using a tumor selective radiopharmaceutical agent 124I-NM404, of 16 wks old mice from each of TG//WT, and TG//KO mice. Results illustrated a lack of focal uptake of 124I-NM404 in TG//KO mice compared to TG//WT mice. TG//WT mice showed metastasis in proximal lymph node as evident by uptake of 124I-NM404. Histopathology data revealed PCa metastasis into the lungs, livers and lymph nodes of TG//WT mice. However, no metastasis was observed in TG//Het and TG//KO mice suggesting the role of PKC∈ in development and metastasis. We have previously shown that PKC∈ associates with Stat3 and this association increases with the progression of the diseases in TRAMP mice and in human PCa. Deletion of PKC∈ in TRAMP mice inhibited 1) phosphorylated forms of both Stat3Ser727 and Stat3Tyr705, 2) DNA binding activity of Stat3, 3) expression levels of markers of proliferation (PI3K85 and PCNA), anti-apoptosis (BclxL), and metastasis (VEGF and COX-2) and 4) IL-6 receptor expression and serum IL-6 levels. To obtain clues about the genes regulated by PKC∈ and linked to the Stat3 signaling pathway, we performed focused PCR arrays of JAK/STAT signaling in excised PCa tissues from TG//WT and TG//KO mice. This array contained 84 genes related to JAK/STAT family members, the receptors that activate them, nuclear co-factor and co-activators associated with the Stat proteins, Stat inducible genes, and negative regulators. Data revealed a significant down-regulation in the m-RNA expression of CCAAT/enhancer binding protein (CEBP) β (2.5 fold), C-reactive protein (4.34 fold), epidermal growth factor receptor (EGFR) (4.54 fold), gp130 (2.5 fold), jun B (2.5 fold), and Stat3 (2.85 fold) in TG//KO mice compared to TG//WT mice. Taken together, these results provide first molecular genetic evidence of the role of PKC∈ in the development and metastasis of PCa in a transgenic mouse model. (Support: NIH Grant, CA35368). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1614. doi:10.1158/1538-7445.AM2011-1614

Abstract 851: Interactions among transcription factors as a potential mechanism for inhibiting castrate-resistant prostate cancer (CRPCa) in transgenic adenocarcinoma of mouse prostate (TRAMP) through regulation of FLIP

Abstract Androgens are critical regulators of prostate differentiation and function as well as prostate cancer growth and survival. Prostate tumors initially regress in response to androgen-ablation therapy. However, most cancers eventually relapse with an androgen-depletion-independent phenotype that is often more aggressive than the original androgen-dependent tumor. Studies from our laboratory demonstrated that intervention with 2-ME2[2-methoxyestradiol] prevents early stage prostate cancer development and causes regression of established prostate tumors in the transgenic adenocarcinoma of mouse prostate model. Although 2-ME2 was found to be safe, well tolerated, reduce or stabilize PSA levels when given to hormone refractory prostate cancer patients who had failed other treatments including hormone therapy, it was found to be less bioavailable. Accordingly second generation 2-ME2 was developed using nanocrystal colloidal dispersion technology and has increased its bioavailability by 5-10 fold (EntreMed, Inc, Rockville, MD). In the current study we tested the ability of this newly formulated 2-ME2 on the development and progression of castrate-resistant prostate cancer in TRAMP mice. 10-12 week old TRAMP mice were castrated and fed water containing different amounts of 2-ME2 for 6 weeks. Efficacy was evaluated by magnetic resonance imaging, determining the prostate seminal vesicle complex volume and histological analysis of prostate tumor or tissue. Our results also show that 2-ME2 inhibited development of CRPCa in these mice. Further molecular analysis using cell lines demonstrated 2-ME2 treatment significantly inhibited DHT-stimulated cell proliferation in androgen-responsive LNCaP cells (p&amp;lt;0.001). Further DHT-induced FLIP, PSA and AR transcriptional activity (as measured by promoter activities) were also reduced by 2-ME2 (p&amp;lt;0.001). Transient expression assays coupled with gel-shift and ChIP assays show binding of multiple transcription factors including Sp1, Sp3, NFκB and AR to the FLIP promoter. 2-ME2 treatment reduced such binding to the FLIP promoter as evidenced by ChIP. 2-ME2 treatment also modulated the interactions among these transcription factors as evidenced by Re-ChIP assays. Interestingly transcription factor CREB but not Sp1 seems to play a predominant role in androgen regulation of FLIP expression. Our findings suggest that 2-ME2 affects androgen induced survival of prostate cancer cells through regulation of FLIP involving potential interactions between multiple transcription factors. Supported by ACS RSG-04-169 and NIH CA 135451 (APK). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 851. doi:10.1158/1538-7445.AM2011-851

Abstract 1165: PKC epsilon modulated miRNAs profiling during development of prostate cancer in transgenic adenocarcinoma of mouse prostate model

Abstract MicroRNAs (miRNAs) are small nucleotides endogenously expressed non-coding RNAs that have been implicated in a wide range of cellular processes. Dysregulation of miRNA expression has been identified in a number of cancers including prostate. Evidence indicates that miRNAs are involved in prostate cancer (PCa) development and metastasis via activating several oncogenes and inhibiting tumor suppressor genes. However, the molecules’ targeting these microRNAs is poorly understood. Protein kinase C epsilon (PKC∈), a novel PKC isoforms, is over-expressed in both human and mouse PCa. We have previously shown that PKC∈ expression correlates with the aggressiveness of PCa. We generated PKC∈-deleted TRAMP mice by crossbreeding transgenic adenocarcinoma of mouse prostate (TRAMP) (TG//WT) mice with PKC∈ knock out (TG//KO) mice. Sixteen weeks old TG Wild-type (TG//WT), TG//PKC∈ Heterozygous (TG//Het) and TG//PKC∈ KO (TG//KO) mice were evaluated for PCa development and metastasis. We observed that deletion of PKC∈ in TRAMP mice inhibited PCa development and metastasis as observed by CT-PET scanning, tumor weight and histopathology of livers, lungs, kidneys and lymph nodes. To determine the clues about the miRNAs, which are modulated by PKC∈, we performed global miRNA profiling in prostate tissues of TG//WT and TG//KO mice by using miRNA Microarray chip containing transcripts of 750 miRNAs (Listed in Sanger miRBase Release 15.0). We observed a significant down-regulation of 7 miRNAs (miRNA762, miR125b-5p, miR-16, miR-467a-1, miR-574-3p, miRNA-451 and miR-669f) and up-regulation of 6 miRNAs (miR-1187, miR-1195, miR-2133, miR-574-5p, miR-690 and miR-709) in prostate tissues of TG//KO mice compared to TG//WT mice. It has been reported that miRNA-451 plays a role in the induction of mTOR signaling, which is well documented for its role in human PCa progression. Importantly, we observed 20-fold decreased expression of miRNA-451 in prostate tissues of TG//KO mice compared to TG//WT mice. It has been reported that up-regulation of miRNA-709 inhibits expression of BORIS, a tumor promoting transcription factor. We observed two fold induction of miRNA-709 in TG//KO mice. Taken together these finding suggest that PKC∈ plays an important role in PCa development and metastasis via up-regulating oncogenic miRNA and down regulating tumor suppressing miRNA. We suggest that these miRNAs modulated by PKC∈ have role in PCa development and metastasis. However further studies warranted to understand the functional implication of these miRNAs in human PCa. (Support: NIH Grant, CA35368). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1165. doi:10.1158/1538-7445.AM2011-1165

Proteomic Profiling of Potential Molecular Targets of Methyl-Selenium Compounds in the Transgenic Adenocarcinoma of Mouse Prostate Model

Because the Selenium (Se) and Vitamin E Cancer Prevention Trial (SELECT) failed to show the efficacy of selenomethionine for prostate cancer prevention, there is a critical need to identify safe and efficacious Se forms for future trials. We have recently shown significant preventive benefit of methylseleninic acid (MSeA) and Se-methylselenocysteine (MSeC) in the transgenic adenocarcinoma mouse prostate (TRAMP) model by oral administration. The present work applied iTRAQ proteomic approach to profile protein changes of the TRAMP prostate and to characterize their modulation by MSeA and MSeC to identify their potential molecular targets. Dorsolateral prostates from wild-type mice at 18 weeks of age and TRAMP mice treated with water (control), MSeA, or MSeC (3 mg Se/kg) from 8 to 18 weeks of age were pooled (9-10 mice per group) and subjected to protein extraction, followed by protein denaturation, reduction, and alkylation. After tryptic digestion, the peptides were labeled with iTRAQ reagents, mixed together, and analyzed by two-dimensional liquid chromatography/tandem mass spectrometry. Of 342 proteins identified with >95% confidence, the expression of 75 proteins was significantly different between TRAMP and wild-type mice. MSeA mainly affected proteins related to prostate functional differentiation, androgen receptor signaling, protein (mis)folding, and endoplasmic reticulum-stress responses, whereas MSeC affected proteins involved in phase II detoxification or cytoprotection, and in stromal cells. Although MSeA and MSeC are presumed precursors of methylselenol and were equally effective against the TRAMP model, their distinct affected protein profiles suggest biological differences in their molecular targets outweigh similarities.

A Novel Sulindac Derivative Lacking Cyclooxygenase-Inhibitory Activities Suppresses Carcinogenesis in the Transgenic Adenocarcinoma of Mouse Prostate Model

Nonsteroidal anti-inflammatory drugs including sulindac are well documented to be highly effective for cancer chemoprevention. However, their cyclooxygenase (COX)-inhibitory activities cause severe gastrointestinal, renal, and cardiovascular toxicities, limiting their chronic use. Recent studies suggest that COX-independent mechanisms may be responsible for the chemopreventive benefits of nonsteroidal anti-inflammatory drugs and support the potential for the development of a novel generation of sulindac derivatives lacking COX inhibition for cancer chemoprevention. A prototypic sulindac derivative with a N,N-dimethylammonium substitution called sulindac sulfide amide (SSA) was recently identified to be devoid of COX-inhibitory activity yet displays much more potent tumor cell growth-inhibitory activity in vitro compared with sulindac sulfide. In this study, we investigated the androgen receptor (AR) signaling pathway as a potential target for its COX-independent antineoplastic mechanism and evaluated its chemopreventive efficacy against prostate carcinogenesis using the transgenic adenocarcinoma of mouse prostate model. The results showed that SSA significantly suppressed the growth of human and mouse prostate cancer cells expressing AR in strong association with G(1) arrest, and decreased AR level and AR-dependent transactivation. Dietary SSA consumption dramatically attenuated prostatic growth and suppressed AR-dependent glandular epithelial lesion progression through repressing cell proliferation in the transgenic adenocarcinoma of mouse prostate mice, whereas it did not significantly affect neuroendocrine carcinoma growth. Overall, the results suggest that SSA may be a chemopreventive candidate against prostate glandular epithelial carcinogenesis.

Androgen receptor levels during progression of prostate cancer in the transgenic adenocarcinoma of mouse prostate model

Aim To construct tissue microarrays (TMAs) that consisted of prostate tumours from the transgenic adenocarcinoma of mouse prostate (TRAMP) mice and non-transgenic murine prostates and to assess androgen receptor (AR) levels during progression of prostate cancer in TRAMP mice by immunohistochemistry. Methods Haematoxylin and eosin (H&E) sections from the ventral and dorso-lateral prostate lobes of non-transgenic, intact TRAMP and castrated TRAMP were used to demarcate regions of interest for TMAs construction. The samples on TMAs were used to evaluate AR expression using video image analysis (VIA). Results AR was expressed during cancer progression, but AR levels were reduced or absent in late stage disease. Further¬more, when AR levels were compared in tumours from intact and castrate animals, a significant increase in AR levels was observed following androgen ablation. Conclusion Similar to clinical prostate cancer, in the TRAMP model, prostate tumours evolve mechanisms to maintain AR expression and AR responsive gene pathways following castration to facilitate continued tumour growth. (Med J lndones 2010; 19:5-13)

Targeting lymphotoxin-mediated negative selection to prevent prostate cancer in mice with genetic predisposition.

The identification of individuals genetically susceptible to cancer calls for preventive measures to minimize the cancer risk in these high-risk populations. Immune prevention is made necessary by the anticipated health threat, but lack of enough high-affinity T cells against tumor-associated antigens and the unpredictability of tumor antigens make antigen-based immune prevention untenable for cancer. To address this issue, we explored a non-antigen-based cancer immune prevention strategy using the transgenic adenocarcinoma of mouse prostate model that spontaneously develops prostate cancer with 100% penetrance. We show that targeted mutation of the lymphotoxin alpha (LTalpha) gene efficiently rescued tumor-reactive T cells, drastically reduced cancer incidence, and almost completely ablated metastasis. Remarkably, short-term treatments with the fusion protein consisting of constant region of IgG and extracellular domain of lymphotoxin beta receptor (LTbetaRIg) interrupted clonal deletion, reduced the size of the primary cancer, and completely prevented metastasis later in life. Our data demonstrated the value of non-antigen-based immune prevention for those with a genetic predisposition to cancer.

Differential requirement for focal adhesion kinase signaling in cancer progression in the transgenic adenocarcinoma of mouse prostate model

Increasing evidence indicates that adhesion signaling plays an important role in the tumor microenvironment, contributing to cancer progression, invasion, and metastasis. Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that regulates adhesion-dependent cell signaling and has been implicated in mediating steps in cancer progression and metastasis in many human cancers, including prostate. We have investigated the role of FAK in the appearance of adenocarcinoma (atypical epithelial hyperplasia of T antigen) and neuroendocrine carcinomas in the transgenic adenocarcinoma of mouse prostate (TRAMP) model using either Cre-mediated recombination to genetically ablate FAK expression or pharmacologic inhibition of FAK activity with the small-molecule inhibitor, PF-562,271. We provide evidence that loss of FAK or its inhibition with PF-562,271 does not alter the progression to adenocarcinoma. However, continued FAK expression (and activity) is essential for the androgen-independent formation of neuroendocrine carcinoma. These data indicate that integrin signaling through FAK is an important component of cancer progression in the TRAMP model and suggest that treatment modalities targeting FAK may be an appropriate strategy for patients with castrate-resistant cancer.

Methyl-Selenium Compounds Inhibit Prostate Carcinogenesis in the Transgenic Adenocarcinoma of Mouse Prostate Model with Survival Benefit

Chemoprevention of prostate cancer by second-generation selenium compounds in reference to selenomethionine holds strong promise to deal with the disease at the root. Here we used the transgenic adenocarcinoma mouse prostate (TRAMP) model to establish the efficacy of methylseleninic acid (MSeA) and methylselenocysteine (MSeC) against prostate carcinogenesis and to characterize potential mechanisms. Eight-week-old male TRAMP mice (C57B/6 background) were given a daily oral dose of water, MSeA, or MSeC at 3 mg Se/kg body weight and were euthanized at either 18 or 26 weeks of age. By 18 weeks of age, the genitourinary tract and dorsolateral prostate weights for the MSeA- and MSeC-treated groups were lower than for the control (P < 0.01). At 26 weeks, 4 of 10 control mice had genitourinary weight >2 g, and only 1 of 10 in each of the Se groups did. The efficacy was accompanied by delayed lesion progression, increased apoptosis, and decreased proliferation without appreciable changes of T-antigen expression in the dorsolateral prostate of Se-treated mice and decreased serum insulin-like growth factor I when compared with control mice. In another experiment, giving MSeA to TRAMP mice from 10 or 16 weeks of age increased their survival to 50 weeks of age, and delayed the death due to synaptophysin-positive neuroendocrine carcinomas and synaptophysin-negative prostate lesions and seminal vesicle hypertrophy. Wild-type mice receiving MSeA from 10 weeks did not exhibit decreased body weight or genitourinary weight or increased serum alanine aminotransferase compared with the control mice. Therefore, these selenium compounds may effectively inhibit this model of prostate cancer carcinogenesis.

Involvement of FLIP in 2-Methoxyestradiol–Induced Tumor Regression in Transgenic Adenocarcinoma of Mouse Prostate Model

The purpose of this study is to investigate whether Fas-associated death domain interleukin-1 converting enzyme like inhibitory protein (FLIP) inhibition is a therapeutic target associated with 2-methoxyestradiol (2-ME2)-mediated tumor regression. Expression and levels of FLIP were analyzed using (a) real-time PCR and immunoblot analysis in androgen-independent PC-3 cells treated with the newly formulated 2-ME2 and (b) immunohistochemistry in different Gleason pattern human prostate tumors. Transient transfections and chromatin immunoprecipitation (ChIP) assays were used to identify the transcription factors that regulate FLIP. Involvement of FLIP in 2-ME2-induced tumor regression was evaluated in transgenic adenocarcinoma mouse prostate (TRAMP) mice. High Gleason pattern (5+5) human prostate tumors exhibit significant increase in FLIP compared with low Gleason pattern 3+3 (P=or<0.04). 2-ME2 reduced the levels and promoter activity of FLIP (P=0.001) in PC-3 cells. Transient expression assays show sequences between -503/+242 being sufficient for 2-ME2-induced inhibition of FLIP promoter activity. Cotransfection experiments show that overexpression of Sp1 activated, whereas Sp3 inhibited, Sp1 transactivation of FLIP promoter activity (P=0.0001). 2-ME2 treatment reduced binding of Sp1 to the FLIP promoter as evidenced by ChIP. Further, levels of FLIP associated with Fas or FADD decreased, whereas cleavage of caspase-8, levels of Bid, and apoptosis increased in response to 2-ME2 treatment in PC-3 cells. Administration of 2-ME2 regressed established prostate tumors in TRAMP mice that were associated with reduced expression of FLIP and Sp1. Targeting Sp1-mediated FLIP signaling pathway may provide a novel approach for prostate cancer management.

Stage-Specific Alterations of DNA Methyltransferase Expression, DNA Hypermethylation, and DNA Hypomethylation during Prostate Cancer Progression in the Transgenic Adenocarcinoma of Mouse Prostate Model

We analyzed DNA methyltransferase (Dnmt) protein expression and DNA methylation patterns during four progressive stages of prostate cancer in the transgenic adenocarcinoma of mouse prostate (TRAMP) model, including prostatic intraepithelial neoplasia, well-differentiated tumors, early poorly differentiated tumors, and late poorly differentiated tumors. Dnmt1, Dnmt3a, and Dnmt3b protein expression were increased in all stages; however, after normalization to cyclin A to account for cell cycle regulation, Dnmt proteins remained overexpressed in prostatic intraepithelial neoplasia and well-differentiated tumors, but not in poorly differentiated tumors. Restriction landmark genomic scanning analysis of locus-specific methylation revealed a high incidence of hypermethylation only in poorly differentiated (early and late) tumors. Several genes identified by restriction landmark genomic scanning showed hypermethylation of downstream regions correlating with mRNA overexpression, including p16INK4a, p19ARF, and Cacna1a. Parallel gene expression and DNA methylation analyses suggests that gene overexpression precedes downstream hypermethylation during prostate tumor progression. In contrast to gene hypermethylation, genomic DNA hypomethylation, including hypomethylation of repetitive elements and loss of genomic 5-methyldeoxycytidine, occurred in both early and late stages of prostate cancer. DNA hypermethylation and DNA hypomethylation did not correlate in TRAMP, and Dnmt protein expression did not correlate with either variable, with the exception of a borderline significant association between Dnmt1 expression and DNA hypermethylation. In summary, our data reveal the relative timing of and relationship between key alterations of the DNA methylation pathway occurring during prostate tumor progression in an in vivo model system.

Dissociation of Epithelial and Neuroendocrine Carcinoma Lineages in the Transgenic Adenocarcinoma of Mouse Prostate Model of Prostate Cancer

The transgenic adenocarcinoma of mouse prostate (TRAMP) model is widely used in prostate cancer research because of rapid tumor onset and progression. The transgenic mouse is on a C57BL/6 (B6) background and expresses SV40 T-antigen under the probasin promoter. The strong genetic component of susceptibility to prostate cancer in humans prompted us to investigate the effect of mouse strain background (FVB and B6) on incidence, progression, and pathology of prostate cancer in this model. Because TRAMP lesions are unique but differ from conventional prostatic intraepithelial neoplasia because the epithelium and stroma are affected diffusely, we designated them as "atypical hyperplasia of Tag." Although the incidence and severity of atypical hyperplasia of Tag is similar, FVB-TRAMP mice live significantly shorter lives than B6-TRAMP mice because of the rapid development and progression of neuroendocrine carcinomas. This is associated with an increased frequency of neuroendocrine precursor lesions in young TRAMP mice, detectable at 4 weeks after birth. These lesions show properties of bipotential stem cells and co-express markers of epithelial (E-cadherin) and neuroendocrine (synaptophysin) lineages, as well as the transcription factors Foxa1 and Foxa2. Transplantation studies using TRAMP prostatic ducts suggested that neuroendocrine carcinomas arise independently from atypical hyperplasias or other epithelial lesions. Adenocarcinomas were not seen in our cohort. Thus, neuroendocrine carcinomas are the principal malignancy in this model and may develop from bipotential progenitor cells at an early stage of prostate tumorigenesis.