Activated polyamine catabolism depletes acetyl-CoA pools and suppresses prostate tumor growth in TRAMP mice

The enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) regulates the catabolism and export of intracellular polyamines. We have previously shown that activation of polyamine catabolism by conditional overexpression of SSAT has antiproliferative consequences in LNCaP prostate carcinoma cells. Growth inhibition was causally linked to high metabolic flux arising from a compensatory increase in polyamine biosynthesis. Here we examined the in vivo consequences of SSAT overexpression in a mouse model genetically predisposed to develop prostate cancer. TRAMP (transgenic adenocarcinoma of mouse prostate) female C57BL/6 mice carrying the SV40 early genes (T/t antigens) under an androgen-driven probasin promoter were cross-bred with male C57BL/6 transgenic mice that systemically overexpress SSAT. At 30 weeks of age, the average genitourinary tract weights of TRAMP mice were approximately 4 times greater than those of TRAMP/SSAT bigenic mice, and by 36 weeks, they were approximately 12 times greater indicating sustained suppression of tumor outgrowth. Tumor progression was also affected as indicated by a reduction in the prostate histopathological scores. By immunohistochemistry, SV40 large T antigen expression in the prostate epithelium was the same in TRAMP and TRAMP/SSAT mice. Consistent with the 18-fold increase in SSAT activity in the TRAMP/SSAT bigenic mice, prostatic N(1)-acetylspermidine and putrescine pools were remarkably increased relative to TRAMP mice, while spermidine and spermine pools were minimally decreased due to a compensatory 5-7-fold increase in biosynthetic enzymes activities. The latter led to heightened metabolic flux through the polyamine pathway and an associated approximately 70% reduction in the SSAT cofactor acetyl-CoA and a approximately 40% reduction in the polyamine aminopropyl donor S-adenosylmethionine in TRAMP/SSAT compared with TRAMP prostatic tissue. In addition to elucidating the antiproliferative and metabolic consequences of SSAT overexpression in a prostate cancer model, these findings provide genetic support for the discovery and development of specific small molecule inducers of SSAT as a novel therapeutic strategy targeting prostate cancer.     

Clonal deletion of simian virus 40 large T antigen-specific T cells in the transgenic adenocarcinoma of mouse prostate mice: an important role for clonal deletion in shaping the repertoire of T cells specific for antigens overexpressed in solid tumors

In addition to their overexpression in cancer cells, most of the tumor-associated Ags are expressed at low but detectable levels in normal tissues. It is not clear whether the repertoire of T cells specific for unmutated tumor Ags is shaped by negative selection during T cell development. The transgenic adenocarcinoma of mouse prostate (TRAMP) model is transgenic for the SV40 large T Ag (Tag) under the control of the rat probasin regulatory elements. Although it has been established that T lymphocytes from TRAMP mice are tolerant to SV40 Tag, the mechanism of the tolerance is largely unknown. To examine whether the T cell clonal deletion is responsible for the tolerance, we crossed the TRAMP mice with mice transgenic for a rearranged TCR specific for SV40 Tag presented by the H-2K(k). Double transgenic TRAMP/TCR mice showed profound thymic deletion of SV40 Tag-reactive T cells, including a 6- to 10-fold reduction in the total thymocyte numbers and a >50-fold reduction in phenotypically mature T cells. Consistent with this finding, we observed that the SV40 Tag and endogenous mouse probasin genes are expressed at low levels in the thymus. These results demonstrate that clonal deletion is a major mechanism for tolerance to Ags previously regarded as prostate-specific, and provide direct evidence that the T cell repertoire specific for an unmutated tumor Ag can be shaped by clonal deletion in the thymus.     

Macrophage Inhibitory Cytokine-1 (MIC-1/GDF15) Slows Cancer Development but Increases Metastases in TRAMP Prostate Cancer Prone Mice

Macrophage inhibitory cytokine-1 (MIC-1/GDF15), a divergent member of the TGF-β superfamily, is over-expressed by many common cancers including those of the prostate (PCa) and its expression is linked to cancer outcome. We have evaluated the effect of MIC-1/GDF15 overexpression on PCa development and spread in the TRAMP transgenic model of spontaneous prostate cancer. TRAMP mice were crossed with MIC-1/GDF15 overexpressing mice (MIC-1(fms)) to produce syngeneic TRAMP(fmsmic-1) mice. Survival rate, prostate tumor size, histopathological grades and extent of distant organ metastases were compared. Metastasis of TC1-T5, an androgen independent TRAMP cell line that lacks MIC-1/GDF15 expression, was compared by injecting intravenously into MIC-1(fms) and syngeneic C57BL/6 mice. Whilst TRAMP(fmsmic-1) survived on average 7.4 weeks longer, had significantly smaller genitourinary (GU) tumors and lower PCa histopathological grades than TRAMP mice, more of these mice developed distant organ metastases. Additionally, a higher number of TC1-T5 lung tumor colonies were observed in MIC-1(fms) mice than syngeneic WT C57BL/6 mice. Our studies strongly suggest that MIC-1/GDF15 has complex actions on tumor behavior: it limits local tumor growth but may with advancing disease, promote metastases. As MIC-1/GDF15 is induced by all cancer treatments and metastasis is the major cause of cancer treatment failure and cancer deaths, these results, if applicable to humans, may have a direct impact on patient care.     

Anaplastic Renal Carcinoma Expressing SV40 T Antigen in a Female TRAMP Mouse

An 8-mo-old female transgenic adenocarcinoma of the mouse prostate (C57BL/6-Tg(TRAMP)8247Ng/J) mouse presented with abdominal distention, lethargy, and serosanguineous vaginal discharge. A large primary renal tumor with metastases to lung and liver was present at necropsy. The tumor was composed of poorly differentiated and crowded epithelial cells forming ducts, acini, and cribriform patterns, with comedonecrosis and frequent bizarre mitoses. Immunohistochemistry revealed that neoplastic cells expressed nuclear SV40 T antigen, confirming aberrant expression of the transgene. In addition, cells were positive for pancytokeratin and negative for synaptophysin and estrogen and progesterone receptors. This report details the first transgene-induced tumor in a female TRAMP mouse.Abbreviations: Tag, large T antigen; TRAMP, transgenic adenocarcinoma mouse prostateThe transgenic adenocarcinoma of the mouse prostate (TRAMP) strain is a genetically engineered mouse strain developed on a C57BL/6 background as a model of human prostate cancer. Human prostatic cancer is characterized by a loss of wildtype tumor protein 53 (TP53) and retinoblastoma (RB1) genes.^9,10,13 The TRAMP model was developed to mimic that mechanism by using the SV40 polyomavirus large T antigen (Tag) to act as an oncogene.¹⁰ Tag protein binds to and inactivates the tumor suppressor proteins TRP53 and RB1. In TRAMP mice, Tag expression is high and limited to the dorsolateral and ventral prostatic lobes, as well as the stromal cells of the seminal vesicles, through the use of the rat probasin promoter (Tg[rPb-SV40Tag]), and affects the secretory epithelial cells.^6,8,9,20 This gene–promoter combination results in epithelial transformation in vivo under the regulation of androgens and zinc.^9,20 Whereas TRAMP mice on an FVB background readily develop neuroendocrine tumors of the prostate, TRAMP mice with a B6 background are less likely to form neuroendocrine-type prostatic tumors and instead were shown to produce prostatic epithelial origin tumors that can be described as glandular, papillary, or cribriform.²⁰ Seminal vesicle tumors in TRAMP mice do not express Tag within the epithelial component but do express it in the mesenchymal component.²⁰Male TRAMP mice of B6 background begin expressing abnormal differentiation of prostatic epithelial cells at sexual maturity and by 10 to 12 wk of age have mild to severe prostatic hyperplasia with the formation of cribriform structures. By 24 to 30 wk of age, these mice typically develop primary prostatic adenocarcinomas.^9,10 Other abnormalities noted in male TRAMP mice include epithelial–stromal (phyllodes) tumors in the seminal vesicles involving mainly the stroma with low epithelial involvement.^20,22 Female mice are reported as being fertile, with no expression of the transgene or associated neoplasia.¹⁰ This report presents the first documented occurrence of a transgene-expressing neoplasm in a female TRAMP mouse.     

Increased CK5/CK8-positive intermediate cells with stromal smooth muscle cell atrophy in the mice lacking prostate epithelial androgen receptor

Results from tissue recombination experiments documented well that stromal androgen receptor (AR) plays essential roles in prostate development, but epithelial AR has little roles in prostate development. Using cell specific knockout AR strategy, we generated pes-ARKO mouse with knock out of AR only in the prostate epithelial cells and demonstrated that epithelial AR might also play important roles in the development of prostate gland. We found mice lacking the prostate epithelial AR have increased apoptosis in epithelial CK8-positive luminal cells and increased proliferation in epithelial CK5-positive basal cells. The consequences of these two contrasting results could then lead to the expansion of CK5/CK8-positive intermediate cells, accompanied by stromal atrophy and impaired ductal morphogenesis. Molecular mechanism dissection found AR target gene, TGF-β(1), might play important roles in this epithelial AR-to-stromal morphogenesis modulation. Collectively, these results provided novel information relevant to epithelial AR functions in epithelial-stromal interactions during the development of normal prostate, and suggested AR could also function as suppressor in selective cells within prostate.     

False-positive TUNEL staining observed in SV40 based transgenic murine prostate cancer models

The TRAMP (Transgenic Adenocarcinoma of the Mouse Prostate) and LADY (Probasin-large T antigen transgenic mouse) mice are widely used autochthonous models of prostate cancer. Both models utilise probasin promoters to direct androgen-regulated expression of oncogenic SV40 specifically to epithelial cells of the mouse prostate. The oncogenic processes and phenotypes which result mimic many features of human prostate cancer, making these transgenic mouse models useful experimental systems. The terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP in situ nick end labelling (TUNEL) assay is a commonly used method for the detection of cells undergoing apoptosis. In this study, we demonstrate false-positive TUNEL staining in frozen prostate tissue from TRAMP and LADY mice, which was not observed in non-transgenic control animals and is not due to non-specific binding of labelled-dUTP substrate. The false-positive signal co-localised with large SV40 T-antigen expression. False-positive signal was apparent using multiple commercial TUNEL kits with different detection systems. These results caution against the use of the TUNEL assay for detection of apoptosis in frozen prostate tissue of large T-antigen based autochthonous transgenic models of prostate cancer.     

Modulation of androgen receptor transactivation by the SWI3-related gene product (SRG3) in multiple ways

The SWI3-related gene product (SRG3), a component of the mouse SWI/SNF complex, has been suggested to have an alternative function. Here, we demonstrate that in the prostate transactivation of the androgen receptor (AR) is modulated by SRG3 in multiple ways. The expression of SRG3, which is developmentally regulated in the prostate, is induced by androgen through AR. SRG3 in turn enhances the transactivation of AR, providing a positive feedback regulatory loop. The SRG3 coactivation of AR transactivation is achieved through the recruitment of coactivator SRC-1, the protein level of which is upregulated by SRG3, providing another pathway of positive regulation. Interestingly, SRG3 coactivation of AR transactivation is fully functional in BRG1/BRM-deficient C33A cells and the AR/SRG3/SRC-1 complex formed in vivo contains neither BRG1 nor BRM protein, suggesting the possibility of an SRG3 function independent of the SWI/SNF complex. Importantly, the AR/SRG3/SRC-1 complex occupies androgen response elements on the endogenous SRG3 and PSA promoter in an androgen-dependent manner in mouse prostate and LNCaP cells, respectively, inducing gene expression. These results suggest that the multiple positive regulatory mechanisms of AR transactivation by SRG3 may be important for the rapid proliferation of prostate cells during prostate development and regeneration.     

Protective immunosurveillance and therapeutic antitumor activity of gammadelta T cells demonstrated in a mouse model of prostate cancer

In contrast to Ag-specific alphabeta T cells, gammadelta T cells can kill malignantly transformed cells in a manner that does not require the recognition of tumor-specific Ags. Although such observations have contributed to the emerging view that gammadelta T cells provide protective innate immunosurveillance against certain malignancies, particularly those of epithelial origin, they also provide a rationale for developing novel clinical approaches to exploit the innate antitumor properties of gammadelta T cells for the treatment of cancer. Using TRAMP, a transgenic mouse model of prostate cancer, proof-of-concept studies were performed to first establish that gammadelta T cells can indeed provide protective immunosurveillance against spontaneously arising mouse prostate cancer. TRAMP mice, which predictably develop prostate adenocarcinoma, were backcrossed with gammadelta T cell-deficient mice (TCRdelta(-/-) mice) yielding TRAMP x TCRdelta(-/-) mice, a proportion of which developed more extensive disease compared with control TRAMP mice. By extension, these findings were then used as a rationale for developing an adoptive immunotherapy model for treating prostate cancer. Using TRAMP-C2 cells derived from TRAMP mice (C57BL/6 genetic background), disease was first established in otherwise healthy wild-type C57BL/6 mice. In models of localized and disseminated disease, tumor-bearing mice treated i.v. with supraphysiological numbers of syngeneic gammadelta T cells (C57BL/6-derived) developed measurably less disease compared with untreated mice. Disease-bearing mice treated i.v. with gammadelta T cells also displayed superior survival compared with untreated mice. These findings provide a biological rationale for clinical trials designed to adoptively transfer ex vivo expanded autologous gammadelta T cells for the treatment of prostate cancer.     

Flt3 ligand expands dendritic cell numbers in normal and malignant murine prostate

We have developed a murine model that facilitates the structural and functional analysis in vivo of dendritic cell (DC)-mediated phagocytosis of prostate epithelial cells. Recombinant human Flt3 ligand (rhFL) expands the number of dendritic cells in lymphoid and non-lymphoid tissues of mice. We show that rhFL also induced the ingress of dendritic cells into murine prostate, which involutes via epithelial apoptosis after surgical castration. Intact or castrated C57BL/6 and syngeneic transgenic adenocarcinoma of mouse prostate (TRAMP) mice were treated with rhFL or PBS control. Prostate and spleen were then studied by flow cytometry and immunohistochemistry.The number of prostatic CD11c+ and CD11b+ dendritic cells increased significantly in rhFL-treated mice compared with PBS-treated control mice and this effect was greatly augmented by castration of the mice. The immunophenotype of rhFL-mobilized prostatic cells was consistent with that of Langerhans cells (MHC class II+, CD11c+,CD11b+, DEC-205+, CD8 alpha-).MHC class II+ and CD11c+ dendritic cells that were present in the prostate glands of rhFL-treated and castrated C57BL/6 mice were intimately associated with TUNEL+ inclusions, which suggests that Langerhans-type dendritic cells in prostate participated in the clearance of apoptotic cells. Expression of MHC class II, CD54, CD80 and CD86 by prostatic dendritic cells was not up-regulated after castration and freshly isolated rhFL-induced prostate cells were unable to prime allogeneicT cells unless they were activated by culture either on plastic or with recombinant soluble CD40 ligand. Our data suggest that rhFL-mobilized prostatic dendritic cells resemble the functionally immature dendritic cells, which reside in peripheral tissues and contribute to the maintenance of peripheral tolerance.     

Immunization against luteinizing hormone-releasing hormone fusion proteins does not decrease prostate cancer in the transgenic adenocarcinoma mouse prostate model

This study was undertaken to test the effect of immunization against luteinizing hormone-releasing hormone (LHRH) fusion proteins on the development and progression of prostate cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) model. Two LHRH fusion proteins, ovalbumin with seven LHRH peptides (OV-LHRH-7), and thioredoxin with seven LHRH peptides (TH-LHRH-7) were used in a cocktail vaccine. Two groups of male TRAMP mice were immunized with the cocktail. Primary immunizations were at either 4 or 8 weeks of age. LHRH immunized mice (n=19) were compared with castrated (n=19) and intact mice (n=18) for testosterone concentration, tumor weight, and lifespan. Immunization against LHRH in the TRAMP mice resulted in significant production of antibodies to LHRH compared with surgically castrated and intact control mice. Testicular weight was significantly reduced in the LHRH immunized groups compared with intact control mice. Serum testosterone was reduced (P<0.05) in the immunized mice compared with intact control mice and was not different from that of castrated mice (P>0.05). Tumor weight was variable and inconsistent throughout all treatment groups. Lifespan was not increased by immunization against LHRH or castration. Intact control mice (lived the longest (227+/-11 days), whereas immunized mice lived 206+/-11 days and castrated mice lived 213+/-13 days. Tumors from immunized TRAMP mice appeared more aggressive than tumors of castrated and intact mice, as demonstrated by 35% expression of gross lung tumors in the immunized mice whereas none were observed in the castrated or intact TRAMP mice. Prostate cancer is initially dependent upon androgens for growth and development, but cells have the ability to escape androgen dependence and progress to an androgen independent state, which was evident in this study. The TRAMP mouse model immunized against LHRH may have utility in future studies and treatments of the androgen independent prostate cancer.     

Overexpression of FGF9 in Prostate Epithelial Cells Augments Reactive Stroma Formation and Promotes Prostate Cancer Progression

Bone metastasis is the major cause of morbidity and mortality of prostate cancer (PCa). Fibroblast growth factor 9 (FGF9) has been reported to promote PCa bone metastasis. However, the mechanism by which overexpression of FGF9 promotes PCa progression and metastasis is still unknown. Herein, we report that transgenic mice forced to express FGF9 in prostate epithelial cells (F9TG) developed high grade prostatic intraepithelial neoplasia (PIN) in an expression level- and time-dependent manner. Moreover, FGF9/TRAMP bigenic mice (F9TRAMP) grew advanced PCa earlier and had higher frequencies of metastasis than TRAMP littermates. We observed tumor microenvironmental changes including hypercellularity and hyperproliferation in the stromal compartment of F9TG and F9TRAMP mice. Expression of TGFβ1, a key signaling molecule overexpressed in reactive stroma, was increased in F9TG and F9TRAMP prostates. Both in vivo and in vitro data indicated that FGF9 promoted TGFβ1 expression via increasing cJun-mediated signaling. Moreover, in silico analyses showed that the expression level of FGF9 was positively associated with expression of TGFβ1 and its downstream signaling molecules in human prostate cancers. Collectively, our data demonstrated that overexpressing FGF9 in PCa cells augmented the formation of reactive stroma and promoted PCa initiation and progression.     

CD8+ T cells specific for the androgen receptor are common in patients with prostate cancer and are able to lyse prostate tumor cells

The androgen receptor (AR) is a hormone receptor that plays a critical role in prostate cancer, and depletion of its ligand has long been the cornerstone of treatment for metastatic disease. Here, we evaluate the AR ligand-binding domain (LBD) as an immunological target, seeking to identify HLA-A2-restricted epitopes recognized by T cells in prostate cancer patients. Ten AR LBD-derived, HLA-A2-binding peptides were identified and ranked with respect to HLA-A2 affinity and were used to culture peptide-specific T cells from HLA-A2+ prostate cancer patients. These T-cell cultures identified peptide-specific T cells specific for all ten peptides in at least one patient, and T cells specific for peptides AR805 and AR811 were detected in over half of patients. Peptide-specific CD8+ T-cell clones were then isolated and characterized for prostate cancer cytotoxicity and cytokine expression, identifying that AR805 and AR811 CD8+ T-cell clones could lyse prostate cancer cells in an HLA-A2-restricted fashion, but only AR811 CTL had polyfunctional cytokine expression. Epitopes were confirmed using immunization studies in HLA-A2 transgenic mice, in which the AR LBD is an autologous antigen with an identical protein sequence, which showed that mice immunized with AR811 developed peptide-specific CTL that lyse HLA-A2+ prostate cancer cells. These data show that AR805 and AR811 are HLA-A2-restricted epitopes for which CTL can be commonly detected in prostate cancer patients. Moreover, CTL responses specific for AR811 can be elicited by direct immunization of A2/DR1 mice. These findings suggest that it may be possible to elicit an anti-prostate tumor immune response by augmenting CTL populations using AR LBD-based vaccines.     

Anterior prostate epithelial AR inactivation modifies estrogen receptor expression and increases estrogen sensitivity

Androgens influence prostate growth and development, so androgen withdrawal can control progression of prostate diseases. Although estrogen treatment was originally used to induce androgen withdrawal, more recently direct estrogen effects on the prostate have been recognized, but the nature of androgen-estrogen interactions within the prostate remain poorly understood. To characterize androgen effects on estrogen sensitivity in the mouse prostate, we contrasted models of castration-induced androgen withdrawal in the prostate stromal and epithelial compartments with a prostate epithelial androgen receptor (AR) knockout (PEARKO) mouse model of selective epithelial AR inactivation. Castration markedly increased prostate epithelial estrogen receptor (ER)α immunoreactivity compared with very low ERα expression in intact males. Similarly, strong basal and luminal ERα expression was detected in PEARKO prostate of intact males, suggesting that epithelial AR activity regulated epithelial ERα expression. ERβ was strongly expressed in intact, castrated, and PEARKO prostate. However, strong clusters of epithelial ERβ positivity coincided with epithelial stratification in PEARKO prostate. In vivo estrogen sensitivity was increased in PEARKO males, with greater estradiol-induced prostate growth and epithelial proliferation leading to squamous metaplasia, featuring markedly increased epithelial proliferation, thickening, and keratinization compared with littermate controls. Our results suggest that ERα expression in the prostate epithelial cells is regulated by local, epithelia-specific, androgen-dependent mechanisms, and this imbalance in the AR- and ER-mediated signaling sensitizes the mature prostate to exogenous estrogens.     

Fas-mediated T cell deletion potentiates tumor antigen-specific tolerance in a mouse model of prostate cancer

A pivotal obstacle to cancer immunotherapy is peripheral T cell tolerance to tumor-associated antigens (TAAs). Tolerance induction among mature T cells in the periphery operates through a variety of mechanisms, including anergy and apoptosis. Although Fas-FasL-mediated apoptosis is a well-defined tolerance inducing mechanism, direct evidence of its interference with TAA-specific immunity in vivo is still lacking. In this report, we used the TRAMP mouse, which expresses SV40 large T antigen (Tag) preferentially in the prostate and develops prostate tumors, as a model system to address the role of Fas-mediated apoptosis in regulating peripheral T cell tolerance. Using RT-PCR and tetramer staining to quantify TAA-specific TCR-expressing cytolytic T lymphocytes (CTLs), we have shown the presence of TAA-specific CTLs at higher levels in TRAMP mice than in syngeneic C57Bl/6 mice. Tag-specific immunization led to the expansion of Tag-specific CTLs in C57Bl/6 mice, and to their elimination in TRAMP mice. Interestingly, in TRAMP mice with deficient Fas (Hybrid TRAMP-lpr/lpr), Tag-specific CTL elimination in response to Tag immunization did not take place. The results of cytolytic-function assays were consistent with induction and elimination patterns of TAA-specific CTLs and those of RT-PCR and tetramer staining. In conclusion, our data show that Fas-mediated TAA-specific CTL apoptosis contributes to T cell tolerance and suggest that such tolerance could be potentiated following TAA-specific immunization.