Prostate stem cell antigen vaccination induces a long-term protective immune response against prostate cancer in the absence of autoimmunity

Prostate stem cell antigen (PSCA) is an attractive antigen to target using therapeutic vaccines because of its overexpression in prostate cancer, especially in metastatic tissues, and its limited expression in other organs. Our studies offer the first evidence that a PSCA-based vaccine can induce long-term protection against prostate cancer development in prostate cancer-prone transgenic adenocarcinoma mouse prostate (TRAMP) mice. Eight-week-old TRAMP mice displaying prostate intraepithelial neoplasia were vaccinated with a heterologous prime/boost strategy consisting of gene gun-delivered PSCA-cDNA followed by Venezuelan equine encephalitis virus replicons encoding PSCA. Our results show the induction of an immune response against a newly defined PSCA epitope that is mediated primarily by CD8 T cells. The prostates of PSCA-vaccinated mice were infiltrated by CD4-positive, CD8-positive, CD11b-positive, and CD11c-positive cells. Vaccination induced MHC class I expression and cytokine production [IFN-gamma, tumor necrosis factor-alpha, interleukin 2 (IL-2), IL-4, and IL-5] within prostate tumors. This tumor microenvironment correlated with low Gleason scores and weak PSCA staining on tumor cells present in hyperplastic zones and in areas that contained focal and well-differentiated adenocarcinomas. PSCA-vaccinated TRAMP mice had a 90% survival rate at 12 months of age. In contrast, all control mice had succumbed to prostate cancer or had heavy tumor loads. Crucially, this long-term protective immune response was not associated with any measurable induction of autoimmunity. The possibility of inducing long-term protection against prostate cancer by vaccination at the earliest signs of its development has the potential to cause a dramatic paradigm shift in the treatment of this disease.     

Inhibition of prostate carcinogenesis by combined active immunoprophylaxis

The aim of this study is to investigate whether an active immunoprophylactic approach combining specific antigens and adjuvant stimuli would be able to inhibit prostate carcinogenesis in transgenic TRAMP mice. A vaccine consisting of allogeneic large T antigen (TAg)-positive SV40-transformed cells combined with systemic recombinant IL-12 was administered to TRAMP mice, starting from when they were still tumor-free at 5-6 weeks of age. The combined vaccine significantly inhibited prostate carcinogenesis, giving a more than doubled median latency time of prostatic tumors (53 weeks in comparison to 26 weeks in control mice). Vaccination with cells alone or IL-12 treatment alone was poorly effective (median latency of 30 and 39 weeks, respectively). The combined vaccine induced a very high CD4 response biased toward the Th1 pathway, with the induction of a humoral response that included TAg-specific antibodies. Therefore, such active immunoprophylactic approach based on the combination of allogeneic SV40 TAg-positive cells and systemic administration of recombinant IL-12 significantly delayed autochthonous urogenital carcinogenesis driven by SV40 TAg in TRAMP mice.     

Potent induction of tumor immunity by combining tumor cryoablation with anti-CTLA-4 therapy

Thermal ablation to destroy tumor tissue may help activate tumor-specific T cells by elevating the presentation of tumor antigens to the immune system. However, the antitumor activity of these T cells may be restrained by their expression of the inhibitory T-cell coreceptor CTLA-4, the target of the recently U.S. Food and Drug Administration-approved antibody drug ipilumimab. By relieving this restraint, CTLA-4-blocking antibodies such as ipilumimab can promote tumor rejection, but the full scope of their most suitable applications has yet to be fully determined. In this study, we offer a preclinical proof-of-concept in the TRAMP C2 mouse model of prostate cancer that CTLA-4 blockade cooperates with cryoablation of a primary tumor to prevent the outgrowth of secondary tumors seeded by challenge at a distant site. Although growth of secondary tumors was unaffected by cryoablation alone, the combination treatment was sufficient to slow growth or trigger rejection. In addition, secondary tumors were highly infiltrated by CD4(+) T cells and CD8(+) T cells, and there was a significant increase in the ratio of intratumoral T effector cells to CD4(+)FoxP3(+) T regulatory cells, compared with monotherapy. These findings documented for the first time an effect of this immunotherapeutic intervention on the intratumoral accumulation and systemic expansion of CD8(+) T cells specific for the TRAMP C2-specific antigen SPAS-1. Although cryoablation is currently used to treat a targeted tumor nodule, our results suggest that combination therapy with CTLA-4 blockade will augment antitumor immunity and rejection of tumor metastases in this setting.     

Murine six-transmembrane epithelial antigen of the prostate, prostate stem cell antigen, and prostate-specific membrane antigen: prostate-specific cell-surface antigens highly expressed in prostate cancer of transgenic adenocarcinoma mouse prostate mice

To identify genes that are differentially up-regulated in prostate cancer of transgenic adenocarcinoma mouse prostate (TRAMP) mice, we subtracted cDNA isolated from mouse kidney and spleen from cDNA isolated from TRAMP-C1 cells, a prostate tumor cell line derived from a TRAMP mouse. Using this strategy, cDNA clones that were homologous to human six-transmembrane epithelial antigen of the prostate (STEAP) and prostate stem cell antigen (PSCA) were isolated. Mouse STEAP (mSteap) is 80% homologous to human STEAP at both the nucleotide and amino acid levels and contains six potential membrane-spanning regions similar to human STEAP. Mouse PSCA (mPsca) shares 65% homology with human PSCA at the nucleotide and amino acid levels. mRNA expression of mSteap and mPsca is largely prostate-specific and highly detected in primary prostate tumors and metastases of TRAMP mice. Both mSteap and mPsca map to chromosome 5. Another known gene coding for mouse prostate-specific membrane antigen (mPsma) is also highly expressed in both primary and metastatic lesions of TRAMP mice. These results indicate that the TRAMP mouse model can be used to effectively identify genes homologous to human prostate-specific genes, thereby allowing for the investigation of their functional roles in prostate cancer. mSteap, mPsca, and mPsma constitute new tools for preventative and/or therapeutic vaccine construction and immune monitoring in the TRAMP mouse model that may provide insights into the treatment of human prostate cancer.     

Versatile prostate cancer treatment with inducible caspase and interleukin-12

To establish optimized conditions for immunity against prostate cancer, we compared the efficacy of multiple approaches in autochthonous and s.c. transgenic adenocarcinoma of the mouse prostate (TRAMP)-based models. Mice immunized with interleukin (IL)-12-containing apoptotic, but not necrotic TRAMP-C2 cell-based, vaccines were resistant to TRAMP-C2 tumor challenge and re-challenge, independently of the route of vaccination (s.c. or i.p.). Administration of gamma-irradiated TRAMP-C2 cells preinfected with adenovirus containing both B7-1 and IL-12 genes, unlike adenovirus containing B7-1 alone, considerably protected C57BL/6 mice from TRAMP-C2 tumor growth and extended the life span of TRAMP mice. Vaccines that included dendritic cells, instead of IL-12, were equally efficient. Whereas injections of ligand-inducible caspase-1- and IL-12-containing adenoviruses cured small s.c. TRAMP-C2 tumors, nanopump-regulated delivery of viruses led to elimination of much larger tumors. The antitumor immune responses involved CD4+-, CD8+-, and natural killer cells and were strengthened by increasing the number of vaccinations. Intraprostatic administration of inducible caspase-1- and IL-12-containing adenoviruses resulted in local cell death and improved survival of adenocarcinoma-bearing TRAMP mice. Thus, tumor cell apoptosis induced by caspase in situ and accompanied by IL-12 is efficient against prostate cancer in a preclinical model.     

The Ron receptor promotes prostate tumor growth in the TRAMP mouse model

The Ron receptor tyrosine kinase (TK) is overexpressed in many cancers, including prostate cancer. To examine the significance of Ron in prostate cancer in vivo, we utilized a genetically engineered mouse model, referred to as TRAMP mice, that is predisposed to develop prostate tumors. In this model, we show that prostate tumors from 30-week-old TRAMP mice have increased Ron expression compared with age-matched wild-type prostates. Based on the upregulation of Ron in human prostate cancers and in this murine model of prostate tumorigenesis, we hypothesized that this receptor has a functional role in the development of prostate tumors. To test this hypothesis, we crossed TRAMP mice with mice that are deficient in Ron signaling (TK-/-). Interestingly, TK-/- TRAMP+ mice show a significant decrease in prostate tumor mass relative to TRAMP mice containing functional Ron. Moreover, TK-/- TRAMP+ prostate tumors exhibited decreased tumor vascularization relative to TK+/+ TRAMP+ prostate tumors, which correlated with reduced levels of the angiogenic molecules vascular endothelial growth factor and CXCL2. Although Ron loss did not alter tumor cell proliferation, a significant decrease in cell survival was observed. Similarly, murine prostate cancer cell lines containing a Ron deficiency exhibited decreased levels of active nuclear factor-κB, suggesting that Ron may be important in regulating prostate cell survival at least partly through this pathway. In total, our data show for the first time that Ron promotes prostate tumor growth, prostate tumor angiogenesis and prostate cancer cell survival in vivo.     

Gene therapy of prostate cancer with the soluble vascular endothelial growth factor receptor Flk1

A variety of novel therapeutic approaches have emerged recently for the treatment of human cancers. We have coupled two of these therapeutic approaches, gene therapy and antiangiogenic therapy and tested them in two murine prostate cancer models Recombinant adenovirus encoding the ligand-binding ectodomain of the VEGF receptor 2 (Flk1) fused to an Fc domain was administered to SCID mice carrying orthotopic human LNCaP tumors as well as to transgenic (TRAMP) mice with spontaneous prostate tumors. Ad Flk1-Fc injection reduced tumor growth by 66% for orthotopic LNCaP tumors and by 42% for spontaneous tumors in TRAMP mice. Microvessel density in the primary tumors was reduced by 68% and 40% in the two models respectively. A decrease in microvessel density was also observed in lymphatic metastases in Ad Flk1-Fc-treated TRAMP mice and was correlated with a decrease in the frequency of regional metastases in the treated animals. Survival time was also extended in the Ad Flk1-Fc-treated TRAMP mice relative to the control-treated animals. Our results suggest that adenoviral delivery of soluble Flk1 receptor can reduce vascular density and prostate tumor growth and prolong survival time in orthotopically implanted tumors as well as in spontaneous prostate tumors in transgenic animals.     

CD4+CD25+ regulatory T-cell-inactivation in combination with adenovirus vaccines enhances T-cell responses and protects mice from tumor challenge

Cancer vaccines are a promising approach to treating tumors or preventing tumor relapse through induction of an immune response against tumor-associated antigens (TAA). One major obstacle to successful therapy is the immunological tolerance against self-antigens which limits an effective antitumor immune response. As a transient reduction of immunological tolerance may enable more effective vaccination against self-tumor antigens, we explored this hypothesis in a CEA tolerant animal model with an adenovirus expressing CEA vaccine in conjunction with inactivation of CD4(+)CD25(+) regulatory T cells. This vaccination modality resulted in increased CEA-specific CD8(+), CD4(+) T cells and antibody response. The appearance of a CD4(+) T-cell response correlated with a stronger memory response. The combined CD25(+) inactivation and genetic vaccination resulted in significant tumor protection in a metastatic tumor model. Non-invasive tumor visualization showed that not only primary tumors were reduced, but also hepatic metastases. Our results support the viability of this cancer vaccine strategy as an adjuvant treatment to prevent tumor relapse in cancer patients.     

Peripheral T-cell tolerance associated with prostate cancer is independent from CD4+CD25+ regulatory T cells

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) are thought to suppress the natural and vaccine-induced immune response against tumor-associated antigens (TAA). Here, we show that Treg accumulate in tumors and tumor-draining lymph nodes of aging transgenic adenocarcinoma of the mouse prostate (TRAMP) male mice, which spontaneously develop prostate cancer. TAA overexpression and disease progression associate also with induction of TAA-specific tolerance. TAA-specific T cells were found in the lymphoid organs of tumor-bearing mice. However, they had lost the ability to release IFN-gamma and kill relevant targets. Neither in vivo depletion of Treg by PC61 monoclonal antibody followed by repeated vaccinations with antigen-pulsed dendritic cells nor the combined treatment with 1-methyl-L-tryptophan inhibitor of the enzyme indoleamine 2,3-dyoxigenase, PC61 antibody, and dendritic cell vaccination restored the TAA-specific immune response. Treg did not seem to control the early phases of tolerance induction, as well. Indeed, depletion of Treg, starting at week 6, the age at which TRAMP mice are not yet tolerant, and prolonged up to week 12, did not avoid tolerance induction. A similar accumulation of Treg was found in the lymph nodes draining the site of dendritic cell vaccination both in TRAMP and wild-type animals. Hence, we conclude that Treg accrual is a phenomenon common to the sites of an ongoing immune response, and in TRAMP mice in particular, Treg are dispensable for induction of tumor-specific tolerance.     

Fibroblast growth factor 2 promotes tumor progression in an autochthonous mouse model of prostate cancer

Fibroblast growth factor (FGF) 2 (or basic FGF) is expressed at increased levels in human prostate cancer. FGF2 can promote cell motility and proliferation, increase tumor angiogenesis, and inhibit apoptosis, all of which play an important role in tumor progression. To determine whether FGF2 plays a critical role in prostate cancer progression, we have used the transgenic adenocarcinoma of the mouse prostate (TRAMP) model system. A high percentage of TRAMP mice develop metastatic prostate cancer, and thus the TRAMP model is useful for evaluating cancer progression. TRAMP mice were crossed with FGF2 knockout (FGF2(-/-)) mice, and tumor progression in TRAMP mice that were either hemi- or homozygous for inactivation of the FGF2 allele was compared with progression in wild-type TRAMP mice. Inactivation of even one FGF2 allele resulted in increased survival, a decrease in metastasis, and inhibition of progression to the poorly differentiated phenotype in primary prostatic tumors. When compared with wild-type mice, poorly differentiated tumors arising in FGF(+/-) and FGF(-/-) mice expressed higher levels of vascular endothelial growth factor and, in some cases, increased levels of acidic FGF intracellular binding protein, a nuclear FGF1-binding protein. These findings suggest that both FGF2-mediated angiogenesis and intranuclear FGF2 activities may promote tumor progression and support the hypothesis that FGF2 plays a significant role in prostate cancer progression in vivo.     

Systemic therapy of spontaneous prostate cancer in transgenic mice with oncolytic herpes simplex viruses

Oncolytic viruses are an innovative therapeutic strategy for cancer, wherein viral replication and cytotoxicity are selective for tumor cells. Here we show the efficacy of systemically administered oncolytic viruses for the treatment of spontaneously arising tumors, specifically the use of oncolytic herpes simplex viruses (HSV) administered i.v. to treat spontaneously developing primary and metastatic prostate cancer in the transgenic TRAMP mouse, which recapitulates human prostate cancer progression. Four administrations of systemically delivered NV1023 virus, an HSV-1/HSV-2 oncolytic recombinant, to TRAMP mice at 12 or 18 weeks of age (presence of prostate adenocarcinoma or metastatic disease, respectively) inhibited primary tumor growth and metastases to lymph nodes. Expression of interleukin 12 (IL-12) from NV1042 virus, a derivative of NV1023, was additionally effective, significantly reducing the frequency of development of prostate cancer and lung metastases, even when the mice were treated after the onset of metastasis at 18 weeks of age. NV1042-infected cells, as detected by 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside staining for Lac Z expressed by the virus, were present in prostate tumors 1 week after the final virus injection and viral DNA was detected at 2 weeks after final virus injection by real-time PCR in primary and metastatic tumors but not in liver or blood. No toxicity was observed in any of the treated mice. The efficacy of the IL-12-expressing NV1042 virus in this aggressive prostate cancer model using a clinically relevant treatment paradigm merits its consideration for clinical studies.     

Effects of sustained antiangiogenic therapy in multistage prostate cancer in TRAMP model

Antiangiogenic therapy is a promising alternative for prostate cancer growth and metastasis and holds great promise as an adjuvant therapy. The present study evaluated the potential of stable expression of angiostatin and endostatin before the onset of neoplasia and during the early and late stages of prostate cancer progression in transgenic adenocarcinoma of mouse prostate (TRAMP) mice. Groups of 5-, 10-, and 18-week-old male TRAMP mice received recombinant adeno-associated virus-6 encoding mouse endostatin plus angiostatin (E+A) by i.m. injection. The effects of therapy were determined by sacrificing groups of treated mice at defined stages of tumor progression and following cohorts of similarly treated mice for long-term survival. Results indicated remarkable survival after recombinant adeno-associated virus-(E+A) therapy only when the treatment was given at an earlier time, before the onset of high-grade neoplasia, compared with treatment given for invasive cancer. Interestingly, early-stage antiangiogenic therapy arrested the progression of moderately differentiated carcinoma to poorly differentiated state and distant metastasis. Immunohistochemical analysis of the prostate from treated mice indicated significantly lower endothelial cell proliferation and increased tumor cell apoptosis. Vascular endothelial growth factor receptor (VEGFR)-2 expression was significantly down-regulated in tumor endothelium after treatment but not VEGFR-1. Analysis of the neuroendocrine marker synaptophysin expression indicated that antiangiogenic therapy given at an early-stage disease reduced neuroendocrine transition of the epithelial tumors. These studies indicate that stable endostatin and angiostatin gene therapy may be more effective for minimally invasive tumors rather than advanced-stage disease.     

Review: anti-CTLA-4 antibody ipilimumab: case studies of clinical response and immune-related adverse events

The immune system is a powerful natural agent against cancer. Cytotoxic T lymphocyte antigen 4 (CTLA-4), a key negative regulator of T-cell responses, can restrict the antitumor immune response. Ipilimumab (MDX-010) is a fully human, monoclonal antibody that overcomes CTLA-4-mediated T-cell suppression to enhance the immune response against tumors. Preclinical and early clinical studies of patients with advanced melanoma show that ipilimumab promotes antitumor activity as monotherapy and in combination with treatments such as chemotherapy, vaccines, or cytokines. Emerging data on the kinetics of response to ipilimumab and associated adverse events are increasing our understanding about how to manage patients treated with this therapy. For example, short-term tumor progression prior to delayed regression has been observed in ipilimumab-treated patients, and objective responses may be of prolonged duration. In some patients clinical improvement manifests as stable disease, which may also extend for months or years. Immune-related adverse events (IRAEs) have been observed in patients after CTLA-4 blockade and most likely reflect the drug mechanism of action and corresponding effects on the immune system. Early clinical data suggest a correlation between IRAEs and response to ipilimumab treatment. This paper briefly reviews the results from several ongoing and completed ipilimumab clinical trials, provides a synopsis of current trials, and presents several cases that demonstrate the kinetics of antitumor responses and the relationship to IRAEs in patients receiving ipilimumab.     

Targeting the inhibitory receptor CTLA-4 on T cells increased abscopal effects in murine mesothelioma model

We previously demonstrated that blockade of immune suppressive CTLA-4 resulted in tumor growth delay when combined with chemotherapy in murine mesothelioma. Tumor-infiltrating T cells (TIT) after local radiotherapy (LRT) play critical roles in abscopal effect against cancer. We attempt to improve the local and abscopal effect by modulating T cell immunity with systemic blockade of CTLA-4 signal. The growth of primary tumors was significantly inhibited by LRT while CTLA-4 antibody enhanced the antitumor effect. Growth delay of the second tumors was achieved when the primary tumor was radiated. LRT resulted in more T cell infiltration into both tumors, including Treg and cytotoxic T cells. Interestingly, the proportion of Treg over effector T cells in both tumors was reversed after CTLA-4 blockade, while CD8 T cells were further activated. The expression of the immune-related genes was upregulated and cytokine production was significantly increased. LRT resulted in an increase of TIT, while CTLA-4 blockade led to significant reduction of Tregs and increase of cytotoxic T cells in both tumors. The abscopal effect is enhanced by targeting the immune checkpoints through modulation of T cell immune response in murine mesothelioma.     

Utilization of bone marrow-derived endothelial cell precursors in spontaneous prostate tumors varies with tumor grade

Id1 and Id3 genes are required for vascularization, growth, and metastasis of xenograft tumors. In Id-deficient mice, tumor transplantation and proangiogenic factors fail to mobilize and recruit circulating endothelial precursor cells (CEPs) and hematopoietic cells, leading to defective tumor angiogenesis in various models. To investigate the requirement of Id genes and bone marrow incorporation in spontaneous prostate tumors, we crossbred Id mutant mice with the transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. Id1-/- Id3+/- TRAMP mice display delayed tumor growth at 24 weeks compared with wild-type TRAMP mice. Id1 and Id3 were strongly expressed in the endothelial cells of poorly differentiated prostate adenocarcinoma but not in the vasculature of well-differentiated tumors, a finding that is corroborated in human prostate tumor samples. In Id-deficient TRAMP mice, the poorly differentiated tumors show extensive hemorrhage, whereas well-differentiated tumors exhibit none. Transplantation with Id wild-type bone marrow significantly reduced the hemorrhage in poorly differentiated prostate adenocarcinomas with bone marrow-derived endothelial cells contributing to 14% of the tumor blood vessels. However, in well-differentiated prostate adenocarcinomas, there was little evidence of bone marrow-derived endothelial cell incorporation. These differences in the expression of Id genes, the effects of Id loss, and the recruitment of bone marrow-derived endothelial precursor cells in tumor vasculature between well-differentiated and poorly differentiated prostate adenocarcinoma suggest that tumor angiogenesis varies depending on the tumor grade.     

Hepatocyte growth factor-like protein is required for prostate tumor growth in the TRAMP mouse model

The Ron receptor is deregulated in a variety of cancers. Hepatocyte growth factor-like protein (HGFL) is the ligand for Ron and is constitutively secreted from hepatocytes into the circulation. While a few recent reports have emerged analyzing ectopic HGFL overexpression in cancer cells, no studies have examined the effect of host-produced HGFL in tumorigenesis. To examine HGFL function in prostate cancer, the TRAMP mouse model, which is predisposed to develop prostate tumors, was utilized. Prostate tumors from TRAMP mice exhibit elevated levels of HGFL, which correlated with upregulation in human prostate cancer. To directly implicate HGFL in prostate tumorigenesis, TRAMP mice deficient in HGFL (HGFL-/-TRAMP+) were generated. HGFL-/- TRAMP+ mice developed significantly smaller prostate tumors compared to controls. Analysis of HGFL-/- tumors revealed reduced tumor vascularization. No differences in cancer cell proliferation were detected between HGFL-/- TRAMP+ and HGFL+/+ TRAMP+ mice. However, a significant increase in cancer cell death was detected in HGFL-/- TRAMP+ prostates which correlated with decreased pro-survival targets. In vitro analysis demonstrated robust STAT3 activation resulting in Bcl2-dependent survival following treatment of prostate cancer cells with HGFL. These data document a novel function for endogenous HGFL in prostate cancer by imparting a critical survival signal to tumor cells.     

Caveolin-1 is required for the upregulation of fatty acid synthase (FASN), a tumor promoter, during prostate cancer progression

Prostate cancer is the second leading cause of cancer-related deaths in men. Fatty acid synthase (FASN) is normally upregulated during human prostate cancer onset and metastatic progression and its expression positively correlates with the development of advanced metastatic disease. However, it remains unknown what molecular factor(s) control FASN expression. It has been hypothesized that FASN functions as a tumor promoter during prostate cancer progression in humans. Consistently, an established mouse of model of prostate cancer, termed TRAMP mice, also shows the progressive upregulation of FASN levels during prostate cancer development. Here, we examine the role of caveolin-1 (Cav-1) in regulating FASN expression during prostate cancer progression. For this purpose, we crossed Cav-1-/- null mice with TRAMP mice to generate TRAMP/Cav-1+/+ and TRAMP/Cav-1-/- mice. Then, we assessed the expression of FASN in Cav-1+/+ and Cav-1-/- prostate tumors by immuno-histochemistry and Western blot analysis. Interestingly, our results indicate that FASN fails to be upregulated in Cav-1-/- tumors. Importantly, the tumors examined were the same morphological grade, but Cav-1-/- tumors were dramatically smaller and did not metastasize efficiently. We conclude that Cav-1 expression is normally required for the upregulation of FASN during prostate cancer progression. These results also mechanistically explain why TRAMP/Cav-1-/- mice are dramatically resistant to the development of prostate tumors and lung metastases, as they lack the expression of the FASN tumor promoter. Thus, TRAMP/Cav-1-/- mice will provide a novel model system to elucidate the role of FASN in prostate tumor progression. In addition, our results provide the first molecular genetic evidence that Cav-1 functions upstream of FASN during prostate cancer progression.     

Suppression of prostate carcinogenesis by dietary supplementation of celecoxib in transgenic adenocarcinoma of the mouse prostate model

Epidemiological studies and clinical observations suggest that nonsteroidal anti-inflammatory drugs and certain selective cyclooxygenase (COX)-2 inhibitors may reduce the relative risk of clinically evident prostate cancer. This prompted us to investigate the chemopreventive potential of celecoxib, a selective COX-2 inhibitor, against prostate carcinogenesis in a transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Similar to prostate cancer in humans, prostate malignancies in TRAMP mice progress from precursor intraepithelial lesions, to invasive carcinoma that metastasizes to lymph nodes, liver, lungs, and occasionally to bone. The basal enzyme activity and protein expression of COX-2 is significantly higher (>4-fold) in the dorsolateral prostate of TRAMP mice up to 24 weeks of age compared with their nontransgenic littermates. Eight-week-old TRAMP mice were randomly divided and fed either control diet (AIN 76A) or a custom prepared AIN 76A diet containing 1500-ppm celecoxib ad libitum for 24 weeks, a dosage that would compare with the normal recommended dose for the treatment of human disease. Studies from two independent experiments, each consisting of 10 mice on test, showed that the cumulative incidence of prostate cancer development at 32 weeks of age in animals fed with AIN 76A diet was 100% (20 of 20) as observed by tumor palpation, whereas 65% (13 of 20), 35% (7 of 20), and 20% (4 of 20) of the animals exhibited distant site metastases to lymph nodes, lungs, and liver. Celecoxib supplementation to TRAMP mice from 8-32 weeks of age exhibited significant reduction in tumor development (5 of 20) with no signs of metastasis. Celecoxib feeding resulted in a significant decrease in prostate (56%; P < 0.0003) and genitourinary weight (48%; P < 0.008). Sequential magnetic resonance imaging analysis of celecoxib-fed mice documented lower prostate volume compared with the AIN 76A-fed group. Histopathological examination of celecoxib-fed animals showed reduced proliferation, and down-modulation of COX-2 and prostaglandin E2 levels in the dorsolateral prostate and plasma, respectively. These results correlated with retention of antimetastasis markers, viz E-cadherin, and alpha- and beta-catenin, along with a significant decrease in vascular endothelial growth factor protein expression. Celecoxib supplementation also resulted in enhanced in vivo apoptosis in the prostate as monitored by several techniques including a recently perfected technique of 99mTc-labeled annexin V in live animals followed by phosphor imaging. One striking observation in an additional study was that celecoxib feeding to mice with established tumors (16 weeks of age) significantly improved their overall survival (P = 0.014), compared with AIN 76A-fed group. Our findings suggest that celecoxib may be useful in chemoprevention of prostate cancer.     

A null-mutation in the Znt7 gene accelerates prostate tumor formation in a transgenic adenocarcinoma mouse prostate model

Decrease of cellular zinc in the epithelium of the prostate has been implicated in the development of prostate cancer. To investigate whether ZnT7, a zinc transporter involved in intracellular zinc accumulation, played a role in prostate cancer development, we generated and characterized a transgenic adenocarcinoma mouse prostate (TRAMP) model with a Znt7-null genetic background. TRAMP mice (TRAMP/Znt7^−/− and TRAMP/Znt7^+/+) were euthanized at 6, 8, 16, and 28 weeks of age for histopathological analysis of the prostates and for the presence of prostate tumors and metastasis. At 6 and 8 weeks of age, TRAMP/Znt7^−/− mice displayed higher frequencies of low grade prostatic intraepithelial neoplasia (PIN) and high grade PIN, respectively, in the prostates than the age-matched TRAMP/Znt7^+/+ mice. At 16 weeks of age, 33% TRAMP/Znt7^−/− mice had prostate tumors and one half of the mice with prostate tumors had tumor metastasized to the draining lymph nodes while no prostate tumor was detected in the control TRAMP mice. By 28 weeks, 67% TRAMP/Znt7^−/− mice developed prostate tumors while only 22% control TRAMP mice had prostate tumors. Furthermore, apoptosis was reduced in the prostates of TRAMP/Znt7^−/− mice. In conclusion, a null-mutation of the Znt7 gene accelerates prostate tumor formation in TRAMP mice.