Transgenic Adenocarcinoma Of Mouse Prostate Research Articles

Timing of intervention with selenium and soy on cancer chemoprevention in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice

The purpose of this project was to determine at what age dietary intervention with supplemental selenium (Se) or soy must be started to maximize chemopreventive efficacy against prostate cancer in TRAMP mice. [C57BL/6 X FVB] F1 TRAMP male mice were fed stock diets high or low in soy, with or without a supplement of Se (4.0 mg Se/kg BW as Se‐methylselenocysteine) by gavage 5 d/wk in a 2 × 2 factorial design. Mice were exposed to their respective diets beginning at conception, or at 6 wk of age, and were killed at 18 wk. Three‐way ANOVA showed that supplemental Se increased serum and liver Se, with significant interactions with both time and soy intake. Selenium dosing decreased BW independent of soy intake and time of dietary intervention. Both Se and soy decreased epididymal fat pad weights, with Se's effects being more pronounced in mice exposed to diets from conception than from 6 wk. Urogenital tract weights, a measure of prostate proliferation and tumor volume, were significantly reduced by Se supplementation (P<0.001) and soy (p=0.044), independent of time of dietary intervention. These data suggest that, in this model, chemopreventive efficacy of Se and soy does not differ between prenatal and early post‐natal introduction. A comparison of these start points with 12 wks for animals killed at 18 wks, and for animals whose dosing began at all time points, with sacrifice at 24 weeks is in progress. Supported by NIH CA141385 to MJC.

β1 integrins mediate resistance to ionizing radiation in vivo by inhibiting c‐Jun amino terminal kinase 1

This study was carried out to dissect the mechanism by which β1 integrins promote resistance to radiation. For this purpose, we conditionally ablated β1 integrins in the prostatic epithelium of transgenic adenocarcinoma of mouse prostate (TRAMP) mice. The ability of β1 to promote resistance to radiation was also analyzed by using an inhibitory antibody to β1 , AIIB2, in a xenograft model. The role of β1 integrins and of a β1 downstream target, c-Jun amino-terminal kinase 1 (JNK1), in regulating radiation-induced apoptosis in vivo and in vitro was studied. We show that β1 integrins promote prostate cancer (PrCa) progression and resistance to radiation in vivo. Mechanistically, β1 integrins are shown here to suppress activation of JNK1 and, consequently apoptosis, in response to irradiation. Downregulation of JNK1 is necessary to preserve the effect of β1 on resistance to radiation in vitro and in vivo. Finally, given the established crosstalk between β1 integrins and type1 insulin-like growth factor receptor (IGF-IR), we analyzed the ability of IGF-IR to modulate β1 integrin levels. We report that IGF-IR regulates the expression of β1 integrins, which in turn confer resistance to radiation in PrCa cells. In conclusion, this study demonstrates that β1 integrins mediate resistance to ionizing radiation through inhibition of JNK1 activation.

Combination effects of dietary soy and methylselenocysteine in a mouse model of prostate cancer

High dietary intake of soy or selenium (Se) is associated with decreased risk of prostate cancer. Soy constituents and various chemical forms of Se have each been shown to downregulate expression of the androgen receptor (AR) and AR-regulated genes in the prostate. We hypothesized that downregulation of AR and AR-regulated genes by the combination of these dietary components would inhibit tumorigenesis in the TRansgenic Adenocarcinoma of Mouse Prostate (TRAMP) mouse. Male mice were exposed from conception to stock diets high or low in soy, with or without a supplement of Se-methylseleno-L-cysteine (MSC) in a 2 × 2 factorial design. Mice were sacrificed at 18 weeks. Prostate histopathology, urogenital tract (UGT) weight, hepatic activity of androgen-metabolizing enzymes, and expression of AR, AR-regulated, and AR-associated FOX family genes, in the dorsolateral prostate were examined. High soy intake decreased activity of hepatic aromatase and 5α-reductase, expression of AR, AR-regulated genes, FOXA1, UGT weight, and tumor progression, and upregulated protective FOXO3. Supplemental MSC upregulated AKR1C14, which reduces 5α-dihydrotestosterone. Soy is an effective pleiotropic dietary agent for prevention of prostate cancer. The finding of effects of soy on FOX family gene expression in animals is novel. Combination effects of supplemental MSC may depend upon the soy content of the basal diet to which it is added.

Genetic Ablation of the Tetraspanin CD151 Reduces Spontaneous Metastatic Spread of Prostate Cancer in the TRAMP Model

Tetraspanins are integral membrane proteins that associate with motility-related molecules such as integrins. Experimental studies have indicated that they may be important regulators of tumor invasion and metastasis, and high expression of the tetraspanin CD151 has been linked to poor prognosis in a number of cancers. Here, we show for the first time that genetic ablation of CD151 inhibits spontaneous metastasis in a transgenic mouse model of de novo tumorigenesis. To evaluate the effects of CD151 on de novo prostate cancer initiation and metastasis, a Cd151(-/-) (KO) murine model was crossed with the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model. Mice were analyzed for initiation of prostate tumor by palpation and primary tumors were analyzed by immunohistochemistry. Liver and lungs were examined for incidence and size of spontaneous metastatic lesions by histopathology. Knocking-out Cd151 had no significant effect on prostate cancer initiation or on expression of markers of proliferation, apoptosis, or angiogenesis in primary tumors. However, it did significantly decrease metastasis in a site-specific fashion, notably to the lungs but not the liver. Thus, CD151 acts principally as promoter of metastasis in this model. Prostate cancer is the second highest cause of cancer-related deaths in men in most Western countries, with the majority of deaths attributed to late-stage metastatic disease. CD151 may prove to be a valuable prognostic marker for treatment stratification and is a possible antimetastatic target.

Abstract B86: Immunomodulatory effects of LL-37/CRAMP in prostate cancer progression.

Abstract Prostate cancer (PCa) is the most common type of cancer among males in the United States. However, even with advanced diagnostic capacity and conventional therapies such as surgery and chemotherapy, the 5-year survival rate for disseminated disease is approximately 36 months suggesting the need for finding alterate targets and therapies to increase patient survival. We have recently demonstrated that LL-37, an antimicrobial peptide that plays an important role in innate immune response against microbial pathogens, and its mouse orthologue CRAMP (Cathelicidin-related antimicrobial peptide) plays an important role in the progression of PCa both in humans and in a spontaneously developing mouse PCa model, respectively. Over-expression of CRAMP has also been recently reported in a few human epithelial cancers including carcinomas of the breast and lung. Results of our recent studies also demonstrated that targeted knock-down of CRAMP expression in a mouse PCa cell line, derived from the transgenic adenocarcinoma mouse prostate (TRAMP) model, TRAMP C1, resulted in decreased level of myeloid-derived suppressor cells (MDSC) in the spleen upon syngeneic transplantation. Based on these findings, the present study determined the role of LL-37/CRAMP in immune modulation during PCa progression. C57BL/6 mice were subcutaneously implanted with 3 different PCa cell lines; TRAMP C1 (CRAMPhigh), TRAMP C1scrambled (CRAMPhigh) and TRAMP C1CRAMPshRNA (CRAMPlow). Mice in TRAMP C1 and TRAMP C1scrambled groups developed measurable tumors starting from 45 days post-implantation, whereas mice implanted with TRAMP C1CRAMPshRNA showed measurable tumors from 120 days post-implantation suggesting that high levels of CRAMP expression in TRAMP C1 and TRAMP C1scrambled groups promote prostate tumor growth in vivo. Immune cell phenotyping was performed on cells collected from spleen and tumor at day 50 and 55 post-implantation to identify modulation in infiltrating immune cells as a surrogate for decrease in tumor growth due to abrogation of CRAMP expression. The result indicated a higher number of neutrophils infiltrating to the tumor site from the spleen during the initial stages of tumor growth in TRAMP C1 and TRAMP C1scrambled groups, while high numbers of neutrophils remained in the spleen of TRAMP C1CRAMPshRNA group during that time point. Elevated numbers of MDSC were observed both in spleen and in tumor microenvironment in TRAMP C1 and TRAMP C1scrambled groups suggesting that MDSC may further promote PCa growth whereas mice in the TRAMP C1CRAMPshRNA group showed significantly low level of MDSC in spleen, even when compared to control mice, at earlier time point. The number of plasmacytoid dendritic cells (pDC) both in spleen and tumor in TRAMP C1 and TRAMP C1scrambled groups was low while significantly high numbers of pDC were observed in spleen in TRAMP C1CRAMPshRNA group suggesting that high numbers of pDC may suppress the tumor growth by exerting pro-inflammatory response through cytokines such as IL-12. Altogether, the data suggest that CRAMP promotes PCa growth and plays an important role in immunomodulation during PCa progression. The CRAMP induces infiltration of neutrophils to tumor site and increased number of MDSC in both spleen and tumor, which may induce immunosuppression. Since CRAMP triggers protumorigenic stimuli including angiogenesis, invasion and key immune modulation to promote the tumor growth, LL-37/CRAMP may be used as a possible therapeutic target for PCa treatment. Ongoing tumor challenge studies in CRAMP knockout mouse model will shed more light to confirm these observations and to possibly extend the findings in human patients as a potential therapeutic target. Citation Format: Ha-Ram Cha, Anandi Sawant, Carnella Lee, Jonathan Hensel, George Tsuladze, Selvarangan Ponnazhagan. Immunomodulatory effects of LL-37/CRAMP in prostate cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B86.

Effects of dietary high fat on prostate intraepithelial neoplasia in TRAMP mice

Increased fat intake is known to be a major cause of prostate cancer. In this study, we investigated the effect of dietary high fat on prostate intraepithelial neoplasia using transgenic adenocarcinoma mouse prostate (TRAMP) mice. Six-week-old male TRAMP mice were fed AIN93G (control group, 4.0 kcal/kg, n=6) and AIN93G-HFD (experimental group, 4.8 kcal/kg, n=7) for 10 weeks. Prostate histopathology, urogenital tract (UGT) weight, epididymal white adipose tissue weight, argyrophilic nucleolar organizer regions (AgNORs) counts, and serum leptin levels were examined. AIN93G-HFD fed group showed progressed neoplastic lesions in the prostate (P<0.05) compared to AIN93G fed group. AIN93G-HFD intake resulted in a increase in the weight of UGT (P<0.05) and epididymal white adipose tissue. The number of Ag-NOR positive dots significantly increased in each prostate lobe and final serum leptin levels in AIN93G-HFD fed group were about twice those of AIN93G fed group (P<0.05). Dietary high fat was related to the prostate cancer progression in the early stage of TRAMP mice and increased serum leptin levels, suggesting that the regulation of dietary components could delay the progression of prostate cancer.

Role of OGR1 in myeloid-derived cells in prostate cancer

Ovarian cancer G-protein-coupled-receptor-1 (OGR1) is a tumor metastasis suppressor in prostate cancer (PCa). OGR1 knockout mice (ogr1(-/-)) are grossly normal under physiological conditions, however, reduced melanoma tumorigenesis has been observed, with the mechanisms of this reduction completely unknown. In this work, we demonstrated that OGR1 deficiency in host cells significantly reduced tumorigenesis of PCa in mice. Adoptive transfer of WT CD11b(+) Gr1(+) double positive (DP) cells, but not T cells, was sufficient to allow tumor development in ogr1(-/-) mice. The expression of an M1 macrophage marker, inducible nitric oxide synthase (iNOS) was higher and expression of an M2 macrophage marker, arginase-1 (Arg 1) was lower in tumors from ogr1(-/-) mice compared with WT mice. Furthermore, coinjection of transgenic adenocarcinoma mouse prostate (TRAMP)-C2 cells with WT, but not ogr1(-/-) macrophages, increased tumor incidence in ogr1(-/-) mice. T-cell depletion experiments suggested that T cells were required for tumor rejection in ogr1(-/-)mice, although OGR1 expression in T cells may not be necessary. In summary, the expression of OGR1 in myeloid-derived cells, especially in DP cells, was required for PCa tumor cell-induced immunosuppression.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Dendritic Cell-Directed Vaccination with a Lentivector Encoding PSCA for Prostate Cancer in Mice

Many studies have demonstrated that prostate stem cell antigen (PSCA) is an attractive target for immunotherapy based on its overexpression in prostate tumor tissue, especially in some metastatic tissues. In this study, we evaluated dendritic cell (DC)-directed lentiviral vector (DCLV) encoding murine PSCA (DCLV-PSCA) as a novel tumor vaccine for prostate cancer in mouse models. We showed that DCLV-PSCA could preferentially deliver the PSCA antigen gene to DC-SIGN-expressing 293T cells and bone marrow-derived DCs (BMDCs). Direct immunization with the DCLV-PSCA in male C57BL/6 mice elicited robust PSCA-responsive CD8+ and CD4+ T cells in vivo. In a transgenic adenocarcinoma mouse prostate cell line (TRAMP-C1) synergetic tumor model, we further demonstrated that DCLV-PSCA-vaccinated mice could be protected from lethal tumor challenge in a prophylactic model, whereas slower tumor growth was observed in a therapeutic model. This DCLV-PSCA vaccine also showed efficacy in inhibiting tumor metastases using a PSCA-expressing B16-F10 model. Taken together, these data suggest that DCLV is a potent vaccine carrier for PSCA in delivering anti-prostate cancer immunity.

Abstract A72: Efficacy and safety of multiple doses of polyphenon E in prostate cancer using TRAMP mice

Abstract Purpose: The purpose of this study was to assess the efficacy and safety of polyphenon E as a chemopreventive agent against prostate cancer using the transgenic adenocarcinoma of mouse prostate (TRAMP) mice model. Experimental Design: Seven week old 128 male control C57BL/6 mice and 128 male TRAMP mice were treated with 3 different doses of polyphenon E orally at a human achievable dose. Animals were sacrificed at 12, 22, and 32 weeks of age. For the safety study, complete blood counts with leukocyte differentials and blood chemistry levels for liver and kidney functions were determined. For the efficacy study, weight of the prostate, tumor progression and metastasis, and histopathological evaluation of all organs were assessed. Prostate histology was assessed separately for each lobe of the prostate (i.e., the anterior, dorsal, lateral, and ventral lobes) since lesions progress differently in each lobe of the mouse. Results: Polyphenon E treatment significantly inhibited prostate cancer progression and metastasis in TRAMP mice model. As expected, metastases were observed in all untreated TRAMP mice (100%, 8 of 8) at 32 weeks of age. However, significant reductions were found in all three polyphenon E treated groups with a dose response manner. 50% (6 of 12), 27% (3 of 11), and 12.5% (2 of 12) of low, intermediate, and high dose treated groups exhibited metastasis of prostate cancer to distant organ sites, respectively. Furthermore, treated TRAMP mice resulted in significant delay in tumor progression from 12 to 32 weeks of age with a dose response manner. No adverse effect in polyphenon E treatment group was found in control C57BL/6 mice up to 32 weeks of age regardless amount of polyphenone E treatment. Conclusion: Polyphenon E was an effective chemopreventive agent in the progression of prostate cancer in the animal model. Polyphenon E also showed minimal toxicity in the mice model. Citation Format: Jick Hwan Ha, Seung Joon Kim, Hea Yon Lee, Chang Dong Yeo, Chan Kwon Park, Sang Haak Lee, Jong Y. Park. Efficacy and safety of multiple doses of polyphenon E in prostate cancer using TRAMP mice. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A72.

Combination of Vessel-targeting Agents With Fractionated Radiation Therapy: The Role of SDF-1/CXCR4 Pathway

To investigate the changes of structures and functions in tumor microvasculature during fractionated radiation therapy (F-RT), and examine the possibility of enhancing RT effects by targeting pericytes and bone marrow derived cells (BMDCs). Tumors in C57BL mice were generated by i.m. inoculation of TRAMP C-1 (Transgenic Adenocarcinoma of Mouse Prostate) cell line. Tumors at size of 4 mm were treated by a F-RT protocol with 4 Gy/per fraction (fx), 60 Gy in 15 fx, and 5 fx/week. Tumors were monitored in size and collected at indicated time points for studying the structural and functional changes of tumor microvasculature. Combination of Gefitinib (an epidermal growth factor receptor inhibitor) or AMD3100 (an antagonist of CXCR4) with F-RT was implanted to study the effects of targeting pericytes or BMDCs, respectively. The transplantation of green fluorescent protein-tagged bone marrow (GFP-BM) into lethally irradiated recipient mice was used to study the roles of BMDCs on vessel formation. In F-RT-treated tumors, the microvascular density (MVD) was reduced, but the remaining vessels were dilated, incorporated with GFP-positive cells, tightly adhered with pericytes, and well perfused with Hoechst 33342 dyes. These changes suggested remaining vessels are in a more mature structure and vasculogenesis is involved in their survival during F-RT. Although the combination of the EGFR inhibitor gefitinib with F-RT affected vascular structure by dissociating pericytes from vascular wall, it had no enhancement effect on tumor growth delay. This combination in fact protected tumor vessels from irradiation damage, in accord with the elevation of MVD and good vascular perfusion leading to less tumor necrosis and hypoxia. In contrast, combination of CXCR4 inhibitor AMD3100 with F-RT had a significant enhancement effect on tumor growth delay. As compared with those treated by F-RT alone, tumors treated by this combination had less infiltration of GFP-positive BM cells, lower MVD, poorer perfusion function in tumor vessels and more hypoxia; these tumor vessels were not covered by pericytes. Our results suggest that vasculogenesis is involved in surviving tumor vessels during fractionated radiation therapy. There are complex interactions between combination of vessel-targeting therapy with RT and enhancement effect do not always exist. However, blockage of the influx of BMDCs by CXCR4 inhibitor intervenes the vascular maturation via vasculogenesis and has an enhancement effect on RT.

Plumbagin inhibits prostate cancer development in TRAMP mice via targeting PKC , Stat3 and neuroendocrine markers

Plumbagin (PL), 5-hydroxy-2-methyl-1,4-naphthoquinone, is a quinoid constituent isolated from the roots of the medicinal plant Plumbago zeylanica L. (also known as chitrak). PL has also been found in Juglans regia (English Walnut), Juglans cinerea (whitenut) and Juglans nigra (blacknut). The roots of P. zeylanica have been used in Indian and Chinese systems of medicine for more than 2500 years for the treatment of various types of ailments. We were the first to report that PL inhibits the growth and invasion of hormone refractory prostate cancer (PCa) cells [Aziz,M.H. et al. (2008) Plumbagin, a medicinal plant-derived naphthoquinone, is a novel inhibitor of the growth and invasion of hormone-refractory prostate cancer. Cancer Res., 68, 9024-9032.]. Now, we present that PL inhibits in vivo PCa development in the transgenic adenocarcinoma of mouse prostate (TRAMP). PL treatment (2 mg/kg body weight i.p. in 0.2 ml phosphate-buffered saline, 5 days a week) to FVB-TRAMP resulted in a significant (P < 0.01) decrease in prostate tumor size and urogenital apparatus weights at 13 and 20 weeks. Histopathological analysis revealed that PL treatment inhibited progression of prostatic intraepithelial neoplasia (PIN) to poorly differentiated carcinoma (PDC). No animal exhibited diffuse tumor formation in PL-treated group at 13 weeks, whereas 75% of the vehicle-treated mice elicited diffuse PIN and large PDC at this stage. At 20 weeks, 25% of the PL-treated animals demonstrated diffuse PIN and 75% developed small PDC, whereas 100% of the vehicle-treated mice showed large PDC. PL treatment inhibited expression of protein kinase C epsilon (PKCε), signal transducers and activators of transcription 3 phosphorylation, proliferating cell nuclear antigen and neuroendocrine markers (synaptophysin and chromogranin-A) in excised prostate tumor tissues. Taken together, these results further suggest PL could be a novel chemopreventive agent against PCa.

Characterization of the prostate cancer susceptibility gene KLF6 in human and mouse prostate cancers

Krüppel-like factor (KLF) 6 is a candidate tumor suppressor gene in prostate cancer, but the mechanisms contributing to its loss of expression are poorly understood. We characterized KLF6 expression and DNA methylation status during prostate tumorigenesis in humans and mice. KLF6 expression was assessed in matched human non-malignant (NM) and tumor prostate tissues (n = 22) by quantitative real-time PCR (qPCR) and in three independent human prostate cancer cohorts bioinformatically. QPCR for KLF6 expression and methylation-sensitive PCR (MSP) were performed in human prostate LNCaP cancer cells after 5-aza-2'-deoxycytidine treatment. Klf6 protein levels and DNA promoter methylation were assessed in TRansgenic Adenocarcinoma of Mouse Prostate (TRAMP) tumors by immunohistochemistry and MSP, respectively. KLF6 splice variants expression was increased (P = 0.0015) in human prostate tumors compared to NM tissues. Overall, KLF6 was decreased in metastatic compared to primary prostate cancers and reduced expression in primary tumors was associated with a shorter time to relapse (P = 0.0028). Treatment with the demethylating agent 5-aza-2'-deoxycytidine resulted in up-regulation of KLF6 expression (two-fold; P = 0.002) and a decrease in DNA methylation of the KLF6 promoter in LNCaP cells. Klf6 protein levels significantly decreased with progression in the TRAMP model of prostate cancer (P < 0.05), but there was no difference in Klf6 promoter methylation. KLF6 expression was decreased in both clinical prostate cancer and the TRAMP model with disease progression, but this could not be explained by DNA methylation of the KLF6 promoter.

A Regulatory Feedback Loop Between Ca2+/Calmodulin-dependent Protein Kinase Kinase 2 (CaMKK2) and the Androgen Receptor in Prostate Cancer Progression

The androgen receptor (AR) plays a critical role in prostate cancer (PCa) progression, however, the molecular mechanisms by which the AR regulates cell proliferation in androgen-dependent and castration-resistant PCa are incompletely understood. We report that Ca(2+)/calmodulin-dependent kinase kinase 2 (CaMKK2) expression increases and becomes nuclear or perinuclear in advanced PCa. In the TRAMP (transgenic adenocarcinoma of mouse prostate) model of PCa, CaMKK2 expression increases with PCa progression with many cells exhibiting nuclear staining. CaMKK2 expression is higher in human castration-resistant tumor xenografts compared with androgen-responsive xenografts and is markedly higher in the AR-expressing, tumorigenic cell line LNCaP compared with cell lines that are AR-nonexpressing and/or nontumorigenic. In LNCaP cells, dihydrotestosterone induced CaMKK2 mRNA and protein expression and translocation of CaMKK2 to the nucleus. Conversely, androgen withdrawal suppressed CaMKK2 expression. Knockdown of CaMKK2 expression by RNAi reduced LNCaP cell proliferation and increased percentages of cells in G(1) phase, whereas correspondingly reducing percentages in S phase, of the cell cycle. CaMKK2 knockdown reduced expression of the AR target gene prostate-specific antigen at both mRNA and protein levels, AR transcriptional activity driven by androgen responsive elements from the prostate-specific probasin gene promoter and levels of the AR-regulated cell cycle proteins, cyclin D1 and hyperphosphorylated Rb. Our results suggest that in PCa progression, CaMKK2 and the AR are in a feedback loop in which CaMKK2 is induced by the AR to maintain AR activity, AR-dependent cell cycle control, and continued cell proliferation.

Aggressive Prostate Cancer Is Prevented in ERαKO Mice and Stimulated in ERβKO TRAMP Mice

Previous evidence suggests soy genistein may be protective against prostate cancer, but whether this protection involves an estrogen receptor (ER)-dependent mechanism is unknown. To test the hypothesis that phytoestrogens may act through ERα or ERβ to play a protective role against prostate cancer, we bred transgenic mice lacking functional ERα or ERβ with transgenic adenocarcinoma of mouse prostate (TRAMP) mice. Dietary genistein reduced the incidence of cancer in ER wild-type (WT)/transgenic adenocarcinoma of mouse prostate mice but not in ERα knockout (KO) or ERβKO mice. Cancer incidence was 70% in ERWT mice fed the control diet compared with 47% in ERWT mice fed low-dose genistein (300 mg/kg) and 32% on the high-dose genistein (750 mg/kg). Surprisingly, genistein only affected the well differentiated carcinoma (WDC) incidence but had no effect on poorly differentiated carcinoma (PDC). No dietary effects have been observed in either of the ERKO animals. We observed a very strong genotypic influence on PDC incidence, a protective effect in ERαKO (only 5% developed PDC), compared with 19% in the ERWT, and an increase in the incidence of PDC in ERβKO mice to 41%. Interestingly, immunohistochemical analysis showed ERα expression changing from nonnuclear in WDC to nuclear in PDC, with little change in ERβ location or expression. In conclusion, genistein is able to inhibit WDC in the presence of both ERs, but the effect of estrogen signaling on PDC is dominant over any dietary treatment, suggesting that improved differential targeting of ERα vs. ERβ would result in prevention of advanced prostate cancer.

Exercise modulation of the host-tumor interaction in an orthotopic model of murine prostate cancer

The purpose of this study is to investigate the effects of exercise on cancer progression, metastasis, and underlying mechanisms in an orthotopic model of murine prostate cancer. C57BL/6 male mice (6-8 wk of age) were orthotopically injected with transgenic adenocarcinoma of mouse prostate C-1 cells (5 × 10(5)) and randomly assigned to exercise (n = 28) or a non-intervention control (n = 31) groups. The exercise group was given voluntary access to a wheel 24 h/day for the duration of the study. Four mice per group were serially killed on days 14, 31, and 36; the remaining 38 mice (exercise, n = 18; control, n = 20) were killed on day 53. Before death, MRI was performed to assess tumor blood perfusion. Primary tumor growth rate was comparable between groups, but expression of prometastatic genes was significantly modulated in exercising animals with a shift toward reduced metastasis. Exercise was associated with increased activity of protein kinases within the MEK/MAPK and PI3K/mTOR signaling cascades with subsequent increased intratumoral protein levels of HIF-1α and VEGF. This was associated with improved tumor vascularization. Multiplex ELISAs revealed distinct reductions in plasma concentrations of several angiogenic cytokines in the exercise group, which was associated with increased expression of angiogenic and metabolic genes in the skeletal muscle. Exercise-induced stabilization of HIF-1α and subsequent upregulation of VEGF was associated with "productive" tumor vascularization with a shift toward suppressed metastasis in an orthotopic model of prostate cancer.

Role of resveratrol and nitric oxide in the modulation of murine prostate cancer cells (162.9)

Abstract Resveratrol (RES), a polyphenolic compound found in the skin of red fruits, exhibits anti-inflammatory, anti-oxidative, anti-cancer, and anti-proliferative characteristics. RES is known to affect nitric oxide (NO) production. NO is produced by nitric oxide synthase, a regulator of innate and adaptive immunity and displays cytostatic and cytotoxic activity against pathogens and cancer cells. Prior reports suggest that RES and NO mediate the killing of cancer cells in vivo as well as in vitro. In this study, we hypothesized that the RES and NO, either alone or synergistically, modulates the molecular mechanisms in murine prostate cancer (PCa) cells. This hypothesis has been tested on mouse PCa cell lines, derived from transgenic adenocarcinoma of mouse prostate (TRAMP), in C57/B6 mice. Interestingly, TRAMP-C1 and TRAMP-C2 are tumorigenic while TRAMP-C3 is non-tumorigenic. TRAMP cells were treated with different concentrations of RES/NO at different time points and analyzed for cell viability/proliferation, cytokines and growth factors production. The results show that the RES/NO-induced molecular mechanisms in TRAMP-C1 and C2 are significantly different while compared to TRAMP-C3 suggesting that TRAMP-C3 cells may use distinct pathways that regulate “eat me” signal in comparison to TRAMP-C1 and C2. This data also demonstrates that RES/NO mode of action is very distinct and exclusive for each TRAMP cell lines in the modulation of molecular pathways.

Abstract 609: Benzyl isothiocyanate inhibits prostate cancer development in the transgenic adenocarcinoma mouse prostate model

Abstract Benzyl isothiocyanate (BITC) is a hydrolysis product of glucotropaeolin, a compound found in cruciferous vegetables, and has also been shown to have anti-tumor properties. In the present study, we attempted to determine whether BITC inhibits the development of prostate cancer using the transgenic adenocarcinoma mouse prostate (TRAMP) model. Male TRAMP mice and their nontransgenic (normal) littermates at 5 wk of age were randomly divided into control and BITC-treatment groups and gavage-fed with 0 (vehicle), 5, or 10 mg/kg of BITC every other day. At the time of sacrifice (24 wk of age), BITC did not affect body weight of normal or transgenic animals. The genitourinary tract weight of TRAMP mice was increased markedly as compared to normal mice, and this increase was suppressed significantly via the oral administration of 5 or 10 mg/kg of BITC. H&amp;E staining of the dorsolateral lobes of the prostate demonstrated that well-differentiated carcinoma (WDC) was a predominant feature in the 24 week old vehicle-fed TRAMP mice, whereas number of lobes with WDC was reduced and the number of lobes with prostatic intraepithelial neoplasia was increased by feeding with 5 or 10 mg/kg of BITC. BITC feeding reduced the number of cells expressing the Ki67 (a proliferation marker) in the prostatic tissue. Additionally, BITC feeding reduced the expression of cyclin E, cyclin D1, cyclin A, and cyclin-dependent kinase (CDK)2. Our in vitro cell culture results revealed that BITC decreased DNA synthesis and CDK2 activity in TRAMP-C2 mouse prostate cancer cells. However, BITC did not affect apoptosis either in transgenic mice in vivo or in TRAMP-C2 cells in vitro. These results indicate that inhibition of cell cycle progression contributes to the inhibition of prostate cancer development in animals treated with BITC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 609. doi:1538-7445.AM2012-609

Abstract 2584: Notch1 and Notch2 activation by phenethyl isothiocyanate hinders its inhibitory effect on prostate cancer cell migration

Abstract Practical and safe modalities for chemoprevention of prostate cancer are clinically attractive because of high mortality associated with this malignancy in American men. Plant products, including constituents of fruits and vegetables, continue to attract attention for the discovery of novel cancer chemopreventive agents. Phenethyl isothiocyanate (PEITC) is one such promising cancer chemopreventive agent abundant in edible cruciferous vegetables such as watercress. Evidence for protective effect of cruciferous vegetables and their components, including PEITC, against prostate cancer derives from population-based observational studies as well as laboratory investigations. For example, we have shown previously that PEITC administration in the diet (3 µmol/g diet) significantly inhibits incidence as well as burden of poorly-differentiated prostate cancer in the dorsolateral prostate of Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) transgenic mice. Cancer chemopreventive response to PEITC is attributed to its ability to inhibit multiple oncogenic signaling pathways, including nuclear factor-βB, Akt, and androgen receptor. The present study demonstrates, for the first time, that PEITC treatment activates Notch1 and Notch2 in malignant as well as normal human prostate cells. The Notch1 and Notch2 belong to a family of transmembrane receptors implicated in prostate cancer development and metastasis. Exposure of human prostate cancer cells (LNCaP, PC-3, and DU145) and a normal human prostate epithelial cell line (PrEC) to PEITC resulted in cleavage (active form) of Notch1 and Notch2, which was associated with an increase in transcriptional activity of Notch. PEITC treatment caused induction of Notch ligands Jagged1 and Jagged2, overexpression of γ-secretase complex components Presenilin1 and Nicastrin, nuclear enrichment of cleaved Notch2, and/or up-regulation of Notch1, Notch2, Jagged1, and/or Jagged2 mRNA in cancer cells. PEITC-induced apoptosis in LNCaP and PC-3 cells was significantly attenuated by RNA interference of Notch2, but not by pharmacological inhibition of Notch1. Inhibition of PC-3 and LNCaP cell migration resulting from PEITC exposure was significantly augmented by RNA interference of Notch2 as well as pharmacological inhibition of Notch1 activation. Nuclear expression of cleaved Notch2 protein was significantly higher in PC-3 xenografts from PEITC-treated athymic mice and dorsolateral prostates from PEITC-fed TRAMP mice compared with the respective control. Because Notch signaling is implicated in epithelial-mesenchymal transition and metastasis, the present study suggests that anti-metastatic effect of PEITC may be augmented by a combination regimen involving PEITC and a Notch inhibitor. This investigation was supported by the US PHS grant RO1 CA101753-08. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2584. doi:1538-7445.AM2012-2584

Abstract 5173: The human homologue of frog (Xenopus laevis) anterior gradient protein promotes metastasis via regulation of cellular adhesion

Abstract Our recent studies indicated a significant up-regulation of anterior gradient protein-2 (AGR-2) in bone metastatic human prostate cancer cells, PC3, following growth in bone marrow conditioned medium. Evidence indicates that AGR2 belongs to the family of protein disulfide isomerases and has been linked to intestinal mucus production, tumor growth and metastasis of various adenocarcinomas and poor survival of patients with breast and prostate cancers. Few recent reports have also reported the regulatory elements of AGR2 function, but its specific role(s) in tumorigenesis and metastasis is still unclear. In the present study, we have analyzed the role of AGR2 in cancer progression and metastasis using prostate cancer as a model. Human prostate cancer tissue microarrays and autopsy tissues, obtained from University of Alabama at Birmingham consortiums were immunostained for AGR-2 expression. Results were also confirmed in a spontaneously developing transgenic adenocarcinoma of mouse prostate (TRAMP) model. Further characterization of AGR-2 function was determined in PC3 cells with targeted silencing of AGR-2 expressions for tumor growth and metastases in vitro and in vivo. Results of our study found AGR-2 expression mainly in the prostate luminar epithelial cells, but no difference was observed between normal prostate gland, BPH and early stages of prostate cancer in both human and TRAMP mouse. Significant reduction in AGR-2 expression was observed in grade IV &amp; V prostate cancers. Interestingly, although declining AGR-2 was evident in higher grade prostate tumors, significantly elevated AGR-2 expressions were observed in the metastatic sites of the same individuals. AGR-2-silenced PC3 cells formed tumors in SCID mice similar to control cells whereas their ability to remain attached to various ECM proteins were greatly compromised. Several key integrins, which mediates ECM binding, were also found to be down-regulated in the AGR-2 silenced PC3 cells, suggesting loss of AGR-2 may be associated with their detachment from the primary tumor and initiation of metastasis. AGR-2 is further up-regulated in metastatic sites indicating its necessity for restoration of integrins for attachment in a new microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5173. doi:1538-7445.AM2012-5173