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Immunotherapy approaches focusing on T cells have provided breakthroughs in treating solid tumors. However, there remains an opportunity to drive anticancer immune responses via other cell types, particularly myeloid cells. ATRC-101 was identified via a target-agnostic process evaluating antibodies produced by the plasmablast population of B cells in a patient with non-small cell lung cancer experiencing an antitumor immune response during treatment with checkpoint inhibitor therapy. Here, we describe the target, antitumor activity in preclinical models, and data supporting a mechanism of action of ATRC-101. Immunohistochemistry studies demonstrated tumor-selective binding of ATRC-101 to multiple nonautologous tumor tissues. In biochemical analyses, ATRC-101 appears to target an extracellular, tumor-specific ribonucleoprotein (RNP) complex. In syngeneic murine models, ATRC-101 demonstrated robust antitumor activity and evidence of immune memory following rechallenge of cured mice with fresh tumor cells. ATRC-101 increased the relative abundance of conventional dendritic cell (cDC) type 1 cells in the blood within 24 h of dosing, increased CD8+ T cells and natural killer cells in blood and tumor over time, decreased cDC type 2 cells in the blood, and decreased monocytic myeloid-derived suppressor cells in the tumor. Cellular stress, including that induced by chemotherapy, increased the amount of ATRC-101 target in tumor cells, and ATRC-101 combined with doxorubicin enhanced efficacy compared with either agent alone. Taken together, these data demonstrate that ATRC-101 drives tumor destruction in preclinical models by targeting a tumor-specific RNP complex leading to activation of innate and adaptive immune responses.

There is significant debate regarding whether B cells and their antibodies contribute to effective anti-cancer immune responses. Here we show that patients with metastatic but non-progressing melanoma, lung adenocarcinoma, or renal cell carcinoma exhibited increased levels of blood plasmablasts. We used a cell-barcoding technology to sequence their plasmablast antibody repertoires, revealing clonal families of affinity matured B cells that exhibit progressive class switching and persistence over time. Anti-CTLA4 and other treatments were associated with further increases in somatic hypermutation and clonal family size. Recombinant antibodies from clonal families bound non-autologous tumor tissue and cell lines, and families possessing immunoglobulin paratope sequence motifs shared across patients exhibited increased rates of binding. We identified antibodies that caused regression of, and durable immunity toward, heterologous syngeneic tumors in mice. Our findings demonstrate convergent functional anti-tumor antibody responses targeting public tumor antigens, and provide an approach to identify antibodies with diagnostic or therapeutic utility., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Isolation of prostate stem cells (PSCs) is crucial for understanding their biology during normal development and tumorigenesis. In this aim, we used a transgenic mouse model expressing GFP from the stem cell-specific s-SHIP promoter to mark putative stem cells during postnatal prostate development. Here we show that cells identified by GFP expression are present transiently during early prostate development and localize to the basal cell layer of the epithelium. These prostate GFP+ cells are a subpopulation of the Lin- CD24+ Sca-1+ CD49f+ cells and are capable of self-renewal together with enhanced growth potential in sphere-forming assay in vitro, a phenotype consistent with that of a PSC population. Transplantation assays of prostate GFP+ cells demonstrate reconstitution of prostate ducts containing both basal and luminal cells in renal grafts. Altogether, these results demonstrate that s-SHIP promoter expression is a new marker for neonatal basal prostate cells exhibiting stem cell properties that enables PSCs in situ identification and isolation via a single consistent parameter. Transcriptional profiling of these GFP+ neonatal stem cells showed an increased expression of several components of the Wnt signaling pathway. It also identified stem cell regulators with potential applications for further analyses of normal and cancer stem cells., Competing Interests: The authors declare no competing or financial interests that could be perceived as prejudicing the impartiality of the research reported.

The activating receptor NK cell group 2 member D (NKG2D) mediates antitumor immunity in experimental animal models. However, whether NKG2D ligands contribute to tumor suppression or progression clinically remains controversial. Here, we have described 2 novel lines of "humanized" bi-transgenic (bi-Tg) mice in which native human NKG2D ligand MHC class I polypeptide-related sequence B (MICB) or the engineered membrane-restricted MICB (MICB.A2) was expressed in the prostate of the transgenic adenocarcinoma of the mouse prostate (TRAMP) model of spontaneous carcinogenesis. Bi-Tg TRAMP/MICB mice exhibited a markedly increased incidence of progressed carcinomas and metastasis, whereas TRAMP/MICB.A2 mice enjoyed long-term tumor-free survival conferred by sustained NKG2D-mediated antitumor immunity. Mechanistically, we found that cancer progression in TRAMP/MICB mice was associated with loss of the peripheral NK cell pool owing to high serum levels of tumor-derived soluble MICB (sMICB). Prostate cancer patients also displayed reduction of peripheral NK cells and high sMIC levels. Our study has not only provided direct evidence in "humanized" mouse models that soluble and membrane-restricted NKG2D ligands pose opposite impacts on cancer progression, but also uncovered a mechanism of sMIC-induced impairment of NK cell antitumor immunity. Our findings suggest that the impact of soluble NKG2D ligands should be considered in NK cell-based cancer immunotherapy and that our unique mouse models should be valuable for therapy optimization.

The contextual signals that regulate the expansion of prostate tumor progenitor cells are poorly defined. We found that a significant fraction of advanced human prostate cancers and castration-resistant metastases express high levels of the β4 integrin, which binds to laminin-5. Targeted deletion of the signaling domain of β4 inhibited prostate tumor growth and progression in response to loss of p53 and Rb function in a mouse model of prostate cancer (PB-TAg mice). Additionally, it suppressed Pten loss-driven prostate tumorigenesis in tissue recombination experiments. We traced this defect back to an inability of signaling-defective β4 to sustain self-renewal of putative cancer stem cells in vitro and proliferation of transit-amplifying cells in vivo. Mechanistic studies indicated that mutant β4 fails to promote transactivation of ErbB2 and c-Met in prostate tumor progenitor cells and human cancer cell lines. Pharmacological inhibition of ErbB2 and c-Met reduced the ability of prostate tumor progenitor cells to undergo self-renewal in vitro. Finally, we found that β4 is often coexpressed with c-Met and ErbB2 in human prostate cancers and that combined pharmacological inhibition of these receptor tyrosine kinases exerts antitumor activity in a mouse xenograft model. These findings indicate that the β4 integrin promotes prostate tumorigenesis by amplifying ErbB2 and c-Met signaling in tumor progenitor cells.

Adoptive T cell therapy (ACT) for the treatment of established cancers is actively being pursued in clinical trials. However, poor in vivo persistence and maintenance of antitumor activity of transferred T cells remain major problems. TGF-β is a potent immunosuppressive cytokine that is often expressed at high levels within the tumor microenvironment, potentially limiting T cell-mediated antitumor activity. In this study, we used a model of autochthonous murine prostate cancer to evaluate the effect of cell-intrinsic abrogation of TGF-β signaling in self/tumor-specific CD8 T cells used in ACT to target the tumor in situ. We found that persistence and antitumor activity of adoptively transferred effector T cells deficient in TGF-β signaling were significantly improved in the cancerous prostate. However, over time, despite persistence in peripheral lymphoid organs, the numbers of transferred cells in the prostate decreased and the residual prostate-infiltrating T cells were no longer functional. These findings reveal that TGF-β negatively regulates the accumulation and effector function of transferred self/tumor-specific CD8 T cells and highlight that, when targeting a tumor Ag that is also expressed as a self-protein, additional substantive obstacles are operative within the tumor microenvironment, potentially hampering the success of ACT for solid tumors.

Mutations in the androgen receptor (AR) have been detected in experimental and clinical prostate tumors. Mice with enforced prostate-specific expression of one such receptor variant, AR-E231G, invariably develop prostatic intraepithelial neoplasia by 12 weeks and metastatic prostate cancer by 52 weeks. The aim of this study was to identify genes with altered expression in the prostates of AR-E231G mice at an early stage of disease that may act as drivers of AR-mediated tumorigenesis. The gene expression profile of AR-E231G prostate tissue from 12-week-old mice was compared to an equivalent profile from mice expressing the AR-T857A receptor variant (analogous to the AR-T877A variant in LNCaP cells), which do not develop prostate tumors. One hundred and thirty-two genes were differentially expressed in AR-E231G prostates. Classification of these genes revealed enrichment for cellular pathways known to be involved in prostate cancer, including cell cycle and lipid metabolism. Suppression of two genes upregulated in the AR-E231G model, ADM and CITED1, increased cell death and reduced proliferation of human prostate cancer cells. Many genes differentially expressed in AR-E231G prostates are also deregulated in human tumors. Three of these genes, ID4, NR2F1 and PTGDS, which were expressed at consistently lower levels in clinical prostate cancer compared to nonmalignant tissues, formed a signature that predicted biochemical relapse (hazard ratio 2.2, p = 0.038). We believe that our findings support the value of this novel mouse model of prostate cancer to identify candidate therapeutic targets and/or biomarkers of human disease., (Copyright © 2011 UICC.)

A novel TRIM family member, TRIM59 gene was characterized to be upregulated in SV40 Tag oncogene-directed transgenic and knockout mouse prostate cancer models as a signaling pathway effector. We identified two phosphorylated forms of TRIM59 (p53 and p55) and characterized them using purified TRIM59 proteins from mouse prostate cancer models at different stages with wild-type mice and NIH3T3 cells as controls. p53/p55-TRIM59 proteins possibly represent Ser/Thr and Tyr phosphorylation modifications, respectively. Quantitative measurements by ELISA showed that the p-Ser/Thr TRIM59 correlated with tumorigenesis, whereas the p-Tyr-TRIM59 protein correlated with advanced cancer of the prostate (CaP). The function of TRIM59 was elucidated using short hairpin RNA (shRNA)-mediated knockdown of the gene in human CaP cells, which caused S-phase cell-cycle arrest and cell growth retardation. A hit-and-run effect of TRIM59 shRNA knockdown was observed 24 hours posttransfection. Differential cDNA microarrray analysis was conducted, which showed that the initial and rapid knockdown occurred early in the Ras signaling pathway. To confirm the proto-oncogenic function of TRIM59 in the Ras signaling pathway, we generated a transgenic mouse model using a prostate tissue-specific gene (PSP94) to direct the upregulation of the TRIM59 gene. Restricted TRIM59 gene upregulation in the prostate revealed the full potential for inducing tumorigenesis, similar to the expression of SV40 Tag, and coincided with the upregulation of genes specific to the Ras signaling pathway and bridging genes for SV40 Tag-mediated oncogenesis. The finding of a possible novel oncogene in animal models will implicate a novel strategy for diagnosis, prognosis, and therapy for cancer., (© 2011 American Association for Cancer Research.)

Purpose: Cell adhesion molecules (CADM) comprise a newly identified protein family whose functions include cell polarity maintenance and tumor suppression. CADM-1, CADM-3, and CADM-4 have been shown to act as tumor suppressor genes in multiple cancers including prostate cancer. However, CADM-2 expression has not been determined in prostate cancer., Experimental Design: The CADM-2 gene was cloned and characterized and its expression in human prostatic cell lines and cancer specimens was analyzed by reverse transcription-PCR and an immunohistochemical tissue array, respectively. The effects of adenovirus-mediated CADM-2 expression on prostate cancer cells were also investigated. CADM-2 promoter methylation was evaluated by bisulfite sequencing and methylation-specific PCR., Results: We report the initial characterization of CADM-2 isoforms: CADM-2a and CADM-2b, each with separate promoters, in human chromosome 3p12.1. Prostate cancer cell lines, LNCaP and DU145, expressed negligible CADM-2a relative to primary prostate tissue and cell lines, RWPE-1 and PPC-1, whereas expression of CADM-2b was maintained. Using immunohistochemistry, tissue array results from clinical specimens showed statistically significant decreased expression in prostate carcinoma compared with normal donor prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia, and normal tissue adjacent to tumor (P < 0.001). Adenovirus-mediated CADM-2a expression suppressed DU145 cell proliferation in vitro and colony formation in soft agar. The decrease in CADM-2a mRNA in cancer cell lines correlated with promoter region hypermethylation as determined by bisulfite sequencing and methylation-specific PCR. Accordingly, treatment of cells with the demethylating agent 5-aza-2'-deoxycytidine alone or in combination with the histone deacetylase inhibitor trichostatin A resulted in the reactivation of CADM-2a expression., Conclusions: CADM-2a protein expression is significantly reduced in prostate cancer. Its expression is regulated in part by promoter methylation and implicates CADM-2 as a previously unrecognized tumor suppressor gene in a proportion of human prostate cancers., (©2010 AACR.)

The progression of prostate cancer from an organ-confined, androgen-sensitive disease to a metastatic one is associated with dysregulation of androgen receptor (AR)-regulated target genes and with a decrease in insulin-like growth factor-I receptor (IGF-IR) expression. To investigate the differential effects of wild type (wt) and mutant AR on IGF-IR levels we employed a series of isogenic prostate-derived cell lines and human xenografts. We show that basal and phosphorylated IGF-IR levels progressively decreased as prostate cancer cells became more tumorigenic and metastatic. In addition, we show that wt, but not mutant, AR along with dihydrotestosterone treatment increased IGF-IR promoter activity and endogenous IGF-IR levels. ChIP analysis show enhanced AR binding to the IGF-IR promoter in AR-overexpressing cells. Finally, wt AR-overexpressing cells display an enhanced proliferation rate. In summary, we provide evidence that activated wt AR enhances IGF-IR transcription in prostate cancer cells via a mechanism that involves AR binding to the IGF-IR promoter. AR mutations alter the ability of the mutated protein to regulate IGF-IR expression. Our results suggest that prostate cancer progression is associated with a decrease in IGF-IR expression that could be the result of impaired ability of AR to stimulate IGF-IR gene expression., (2010 Elsevier Ireland Ltd. All rights reserved.)

We previously reported that naive, tumor-specific CD8(+) (TcR-I) T cells transferred into prostate tumor-bearing mice traffic to the prostate where they become tolerized. We now report that TcR-I cells suppress the proliferation of naive T cells. This suppression is mediated at least in part by secreted factors, and the suppressive activity can be blocked by Abs directed against TGF-beta. We further report that TcR-I cells must infiltrate the prostate to acquire suppressive activity. Delivery of tumor-specific CD4(+) T cells prevents the conversion of TcR-I cells into suppressor cells. Taken together, our findings may have critical implications for sustaining T cell responsiveness during immunotherapy, as the development of suppressor cells in the tumor microenvironment may eliminate the potency of T cells primed in the periphery or delivered during adoptive immunotherapy.

Scutellaria barbata (SB) has been used in Chinese medicine to treat various cancers. This study investigated the effects of SB on prostate cancer prevention. Male TRansgenic Adenocarcinoma Mouse Prostate (TRAMP) mice at 9 weeks were randomly divided into four groups and given daily oral feedings of 8, 16, or 32 mg SB or sterilized water. In the control group, palpable tumors initially appeared at 19 weeks of age and were present in all mice by 32 weeks. In the respective treatment groups, palpable tumor development was delayed by 2, 4, and 7 weeks and 22, 30, and 38% of the mice were free of palpable tumors. Palpable tumor development in 50% of the mice occurred at 25 weeks in the placebo group, 29 weeks in the low-dose and mid-dose treatment groups, and 33 weeks in the high-dose group (log rank, P = 0.0211). Histological assessment further showed that the SB treatment (32 mg) delayed prostate tumor progression in the TRAMP mice. Caspase 3 activation was observed in SB-treated prostate tissue. Positive TUNEL assay results were detected in TRAMP-C1 and LNCaP cells treated with SB (1 mg/ml), which indicated significant apoptosis induction. Western blotting of SB-treated LNCaP cells also showed elevated expression of Bax, p53, Akt, and JNK. In-vivo data showed that the SB delayed tumor development in TRAMP mice. Complementary in-vitro data indicated that SB might exert this function by upregulating the apoptotic pathway and downregulating the survival pathway in prostate cancer cells, thus suggesting that SB possesses chemopreventive properties and has potential for cancer treatment.

We reported previously that tumor-specific CD8(+) T cells (TcR-I) become tolerant in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. In this study, we show that CD4(+) TcR transgenic (TcR-II) T cells transferred into TRAMP mice became activated in lymph nodes, trafficked to the prostate, and initially functioned as T(H)1 cells. Although a single cotransfer of TcR-II cells delayed TcR-I cell tolerization, repeated transfer of TcR-II cells was required to prevent TcR-I cell tolerization and significantly slowed progression of TRAMP prostate tumors. After transfer of TcR-II cells, dendritic cells within the tumor expressed higher levels of costimulatory molecules and displayed an enhanced ability to stimulate proliferation of naive T cells. Blockade of CD40-CD40L interactions during TcR-II transfer resulted in a profound reduction in dendritic cell stimulatory capacity and a partial loss of TcR-I effector functions and tumor immunity. These data show that sustained provision of activated tumor-specific CD4(+) T cells alters the immunosuppressive tumor microenvironment, ultimately leading to the control of tumor growth. These findings will assist in the design of more effective immunotherapeutic approaches for cancer.

Molecular imaging techniques used to detect the initiation of disease have the potential to provide the best opportunity for early treatment and cure. This report aimed at testing the possibility that Flk1+ (vascular endothelial growth factor receptor 2), a crucial angiogenesis factor of most tumor cells, could be a molecular targeted imaging marker for the diagnosis and prognosis of cancer. We performed Flk1-targeted microbubble-enhanced ultrasound (US) imaging of prostate cancer in a genetically engineered mouse model with normal-appearing intact US (negative) prostates and with three different tumor sizes (small, medium, and large). Higher levels of Flk1+ molecular signals were identified in the intact US (negative) prostate group by US-targeted imaging and immunohistochemical analysis. The increase in Flk1+ expression occurred prior to the angiogenesis switch-on phase and vascularity peak. After this peak accumulation stage of Flk1+ molecules, lower and stabilized levels of Flk1+ signals were maintained together with tumor growth from small, to medium, to large size. In a longitudinal observation in a subset (n = 5) of mice with established tumors, elevated Flk1+ signals were observed in tissues surrounding the prostate cancer, for example, the ipsilateral boundary zones between two developing tumor lobes, new tumor blood vessel recruits, the urethra border, and the pelvic node basin. The potential of Flk1-targeted US imaging as a predictive imaging tool was confirmed by correlation studies of three-dimensional US B-mode imaging, gross pathology, and histology analyses. The results of the application in a genetically engineered mouse model with prostate cancer of molecular Flk1-targeted US imaging support the contention that Flk1 can be used as a molecular imaging marker for small tumors undetectable by microimaging and as a molecular diagnostic and prognosis marker for tumor metastasis and progression.

Background: Poor penetration and uneven distribution of doxorubicin in tumors limits the efficacy of this drug in patients with prostate cancer (PC). Aim of the study was to investigate whether pre-treatment with NGR-TNF, a tumor necrosis factor-alpha derivative able to target tumor vessels and alter vessel permeability, increases the penetration and the efficacy of doxorubicin in pre-clinical models of PC., Methods: Wild type C57BL/6 mice bearing androgen-independent TRAMP-C1 PC and transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, which spontaneously develop PC and metastasis, were treated with repeated cycles of doxorubicin, administered either alone or following NGR-TNF. Tumor growth and drug uptake by cancer cells was evaluated., Results: Doxorubicin as a single agent blocked the growth of TRAMP-C1 cells in vitro but not in vivo. Pre-treatment of mice bearing subcutaneous TRAMP-C1 tumors with NGR-TNF favored doxorubicin penetration into the tumor mass, and in both TRAMP-C1 and TRAMP models significantly delayed tumor growth without increasing drug-related toxicity., Conclusions: Pre-treatment with NGR-TNF significantly expanded the therapeutic index of doxorubicin in mouse models of hormone-dependent and -independent PC., ((c) 2008 Wiley-Liss, Inc.)

Insulin-like growth factor-I (IGF-I) is a polypeptide hormone that can influence growth, differentiation, and survival of cells expressing the cognate type 1 receptor (IGF-IR). To better understand cell autonomous IGF-IR signaling in the epithelial compartment of the prostate gland, we generated a conditional (Cre/loxP) prostate-specific IGF-IR knockout mouse model. In contrast to epidemiologic studies that established a correlation between elevated serum IGF-I and the risk of developing prostate cancer, we show that abrogation of IGF-IR expression in the dorsal and lateral prostate could activate extracellular signal-regulated kinase 1/2 signaling and cause cell autonomous proliferation and hyperplasia. Moreover, persistent loss of IGF-IR expression in dorsal and ventral lobes induced p53-regulated apoptosis and cellular senescence rescue programs, predicting that titration of IGF-IR signaling might facilitate growth of tumors with compromised p53 activity. Therefore, we crossed the mice carrying the prostate-specific IGF-IR knockout alleles into the transgenic adenocarcinoma of the mouse prostate model that is driven, in part, by T antigen-mediated functional inactivation of p53. Consistent with our prediction, prostate epithelial-specific deletion of IGF-IR accelerated the emergence of aggressive prostate cancer when p53 activity was compromised. Collectively, these data support a critical role for IGF-IR signaling in prostate tumorigenesis and identify an important IGF-IR-dependent growth control mechanism.

The insulin-like growth factor-1 (IGF-1) signaling axis is important for cell growth, differentiation and survival and increased serum IGF is a risk factor for prostate and other cancers. To study IGF-1 action on the prostate, we created transgenic (PB-Des) mice that specifically express human IGF-1(des) in prostate epithelial cells. This encodes a mature isoform of IGF-1 with decreased affinity for IGF binding proteins (IGFBP) due to a 3-amino acid deletion in the N terminus. Expression of IGF-1(des) was sufficient to cause hyperplastic lesions in all mice, however the well-differentiated lesions did not progress to adenocarcinoma within a year. Remarkably, crossing the PB-Des mice to an established model of prostate cancer delayed progression of organ-confined tumors and emergence of metastatic lesions in young mice. While dissemination of metastatic lesions was widespread in old bigenic mice we did not detect IGF-1(des) in poorly differentiated primary tumors or metastatic lesions. Expression of endogenous IGF-1 and levels of P-Akt and P-Erk were reduced independent of age. These data suggest that increased physiologic levels of IGF-1 facilitate the emergence of hyperplastic lesions while imposing a strong IGF-1-dependent differentiation block. Selection against IGF-1 action appears requisite for progression of localized disease and metastogenesis.

Prostate cancer continues to be a major cause of morbidity and mortality in men around the world. The field of prostate cancer research continues to be hindered by the lack of relevant preclinical models to study tumorigenesis and to further development of effective prevention and therapeutic strategies. The Prostate Cancer Foundation held a Prostate Cancer Models Working Group (PCMWG) Summit on August 6th and 7th, 2007 to address these issues. The PCMWG reviewed the state of prostate cancer preclinical models and identified the current limitations of cell line, xenograft and genetically engineered mouse models that have hampered the transition of scientific findings from these models to human clinical trials. In addition the PCMWG identified administrative issues that inhibit the exchange of models and impede greater interactions between academic centers and these centers with industry. The PCMWG identified potential solutions for discovery bottlenecks that include: (1) insufficient number of models with insufficient molecular and biologic diversity to reflect human cancer, (2) a lack of understanding of the molecular events that define tumorigenesis, (3) a lack of tools for studying tumor-host interactions, (4) difficulty in accessing model systems across institutions, and (5) addressing why preclinical studies appear not to be predictive of human clinical trials. It should be possible to apply the knowledge gained molecular and epigenetic studies to develop new cell lines and models that mimic progressive and fatal prostate cancer and ultimately improve interventions.

Ligands for the NKG2D stimulatory receptor are frequently upregulated on tumor lines, rendering them sensitive to natural killer (NK) cells, but the role of NKG2D in tumor surveillance has not been addressed in spontaneous cancer models. Here, we provided the first characterization of NKG2D-deficient mice, including evidence that NKG2D was not necessary for NK cell development but was critical for immunosurveillance of epithelial and lymphoid malignancies in two transgenic models of de novo tumorigenesis. In both models, we detected NKG2D ligands on the tumor cell surface ex vivo, providing needed evidence for ligand expression by primary tumors. In a prostate cancer model, aggressive tumors arising in NKG2D-deficient mice expressed higher amounts of NKG2D ligands than did similar tumors in wild-type mice, suggesting an NKG2D-dependent immunoediting of tumors in this model. These findings provide important genetic evidence for surveillance of primary tumors by an NK receptor.

Discovery of immunologically relevant antigens in prostate cancer forms the basis for developing more potent active immunotherapy. We report here a strategy using the transgenic adenocarcinoma of mouse prostate (TRAMP) model, which allows for the functional identification of immunogenic prostate tumor antigens with relevance for human immunotherapy. Using a combination of active tumor vaccination in the presence of CTL-associated antigen 4 (CTLA-4) in vivo blockade, we elicited tumor-specific T cells used to expression clone the first T cell-defined TRAMP tumor antigen, called Spas-1 (stimulator of prostatic adenocarcinoma specific T cells-1). Spas-1 expression was increased in advanced primary TRAMP tumors. We show that the immunodominant SPAS-1 epitope SNC9-H(8) arose from a point mutation in one allele of the gene in TRAMP tumor cells, and that immunization with dendritic cells pulsed with SNC9-H(8) peptide resulted in protection against TRAMP-C2 tumor challenge. In humans, the Spas-1 ortholog SH3GLB2 has been reported to be overexpressed in prostate cancer metastases. Additionally, we identified a nonmutated HLA-A2-binding epitope in the human ortholog SH3GLB2, which primed T cells from healthy HLA-A2(+) individuals in vitro. Importantly, in vitro-primed T cells also recognized naturally processed and presented SH3GLB2. Our findings demonstrate that our in vivo CTLA-4 blockade-based T cell expression cloning can identify immunogenic cancer antigens with potential relevance for human immunotherapy.

The fibroblast growth factor (FGF) regulates a broad spectrum of biological activities by activation of transmembrane FGF receptor (FGFR) tyrosine kinases and their coupled intracellular signaling pathways. FGF receptor substrate 2alpha (FRS2alpha) is an FGFR interactive adaptor protein that links multiple signaling pathways to the activated FGFR kinase. We previously showed that FGFR2 in the prostate epithelium is important for branching morphogenesis and for the acquisition of the androgen responsiveness. Here we show in mice that FRS2alpha is uniformly expressed in the epithelial cells of developing prostates, whereas it is expressed only in basal cells of the mature prostate epithelium. However, expression of FRS2alpha was apparent in luminal epithelial cells of regenerating prostates and prostate tumors. To investigate FRS2alpha function in the prostate, the Frs2alpha alleles were ablated specifically in the prostatic epithelial precursor cells during prostate development. Similar to the ablation of Fgfr2, ablation of Frs2alpha disrupted MAP kinase activation, impaired prostatic ductal branching morphogenesis and compromised cell proliferation. Unlike the Fgfr2 ablation, disrupting Frs2alpha had no effect on the response of the prostate to androgens. More importantly, ablation of Frs2alpha inhibited prostatic tumorigenesis induced by oncogenic viral proteins. The results suggest that FRS2alpha-mediated signals in prostate epithelial cells promote branching morphogenesis and proliferation, and that aberrant activation of FRS2-linked pathways might promote tumorigenesis. Thus, the prostate-specific Frs2alpha(cn) mice provide a useful animal model for scrutinizing the molecular mechanisms underlying prostatic development and tumorigenesis.

Herein, for the first time, we evaluated the chemopreventive efficacy of dietary silibinin against prostate cancer (PCa) growth and progression in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice from two different genetic backgrounds [C57BL/6 (TRAMP) x FVB; C57BL/6 (TRAMP) x C57BL/6]. At 4 weeks of age, mice were fed control or 0.1% to 1% silibinin-supplemented diets until 23 to 24 weeks of age. Silibinin-fed groups had a lower tumor grade and higher incidence of prostatic intraepithelial neoplasia (PIN) at the expense of a strong decrease in adenocarcinoma incidence. Prostate tissue showed a 47% (P < 0.001) decrease in proliferating cell nuclear antigen (PCNA)-positive cells and an approximately 7-fold (P < 0.001) increase in apoptotic cells at the highest silibinin dose. As potential mechanisms of silibinin efficacy, an approximately 50% (P < 0.05) decrease in insulin-like growth factor (IGF) receptor type I beta and an approximately 13-fold (P < 0.001) increase in IGF-binding protein 3 (IGFBP-3) protein levels were also observed. These changes were specific to tumors as they were not reflected in circulating IGF-IGFBP-3 system. Additionally, silibinin decreased protein expression of cyclin-dependent kinases (Cdk) by more than 90% (P < 0.001) with a concomitant increase in Cdk inhibitors, Cip1/p21 and Kip1/p27 (P < 0.05, for both). A dose-dependent decrease was also observed in cyclin B1, cyclin E, and cyclin A protein levels by silibinin. Together, these findings suggest that oral silibinin blocks PCa growth and progression at PIN stage in TRAMP mice via modulation of tumor IGF-IGFBP-3 axis and cell cycle regulation, and therefore it has practical and translational potential in suppressing growth and neoplastic conversion of PIN to PCa in humans.

Following castration, the transgenic adenocarcinoma of mouse prostate (TRAMP) model demonstrates rapid development of SV40-Tag-driven poorly differentiated tumors that express neuroendocrine cell markers. The cell population dynamics within the prostates of castrated TRAMP mice were characterized by analyzing the incorporation of 5-bromodeoxyuridine (BrdUrd) and the expression of SV40-Tag, synaptophysin, and androgen receptor (AR). Fourteen days postcastration, the remaining epithelial cells and adenocarcinoma cells were nonproliferative and lacked detectable SV40-Tag or synaptophysin expression. In contrast, morphologically distinct intraglandular foci were identified which expressed SV40-Tag, synaptophysin, and Ki67, but that lacked AR expression. These proliferative SV40-Tag and synaptophysin-expressing intraglandular foci were associated with the rare BrdUrd-retaining cells. These foci expanded rapidly in the postcastration prostate environment, in contrast to the AR- and SV40-Tag-expressing adenocarcinoma cells that lost SV40-Tag expression and underwent apoptosis after castration. Intraglandular foci of synaptophysin-expressing cells were also observed in the prostates of intact TRAMP mice at a comparable frequency; however, they did not progress to rapidly expanding tumors until much later in the life of the mice. This suggests that the foci of neuroendocrine-like cells that express SV40-Tag and synaptophysin, but lack AR, arise independent of androgen-deprivation and represent the source of the poorly differentiated tumors that are the lethal phenotype in the TRAMP model.

The expression of fibroblast growth factor receptor (FGFR)-1 correlates with angiogenesis and is associated with prostate cancer (CaP) progression. To more precisely define the molecular mechanisms whereby FGFR1 causes angiogenesis in the prostate we exploited a transgenic mouse model, JOCK-1, in which activation of a conditional FGFR1 allele in the prostate epithelium caused rapid angiogenesis and progressive hyperplasia. By labeling the vasculature in vivo and applying a novel method to measure the vasculature in three dimensions, we were able to observe a significant increase in vascular volume 1 week after FGFR1 activation. Although vessel volume and branching both continued to increase throughout a 6-week period of FGFR1 activation, importantly, we discovered that continued activation of FGFR1 was not required to maintain the new vasculature. Exploring the molecular mediators of the angiogenic phenotype, we observed consistent upregulation of HIF-1alpha, vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang-2), whereas expression of Ang-1 was lost. Further analysis revealed that loss of Ang-1 expression occurred in the basal epithelium, whereas the increase in Ang-2 expression occurred in the luminal epithelium. Reporter assays confirmed that the Ang-2 promoter was regulated by FGFR1 signaling and a small molecule inhibitor of FGFR activity, PD173074, could abrogate this response. These findings establish a method to follow spontaneous angiogenesis in a conditional autochthonous system, implicate the angiopoietins as downstream effectors of FGFR1 activation in vivo, and suggest that therapies targeting FGFR1 could be used to inhibit neovascularization during initiation and progression of CaP.

Toward the goal of developing an optical imaging contrast agent that will enable surgeons to intraoperatively distinguish cancer foci from adjacent normal tissue, we developed a chlorotoxin:Cy5.5 (CTX:Cy5.5) bioconjugate that emits near-IR fluorescent signal. The probe delineates malignant glioma, medulloblastoma, prostate cancer, intestinal cancer, and sarcoma from adjacent non-neoplastic tissue in mouse models. Metastatic cancer foci as small as a few hundred cells were detected in lymph channels. Specific binding to cancer cells is facilitated by matrix metalloproteinase-2 (MMP-2) as evidenced by reduction of CTX:Cy5.5 binding in vitro and in vivo by a pharmacologic blocker of MMP-2 and induction of CTX:Cy5.5 binding in MCF-7 cells following transfection with a plasmid encoding MMP-2. Mouse studies revealed that CTX:Cy5.5 has favorable biodistribution and toxicity profiles. These studies show that CTX:Cy5.5 has the potential to fundamentally improve intraoperative detection and resection of malignancies.

Although the amplified-in-breast cancer 1 (AIB1; SRC-3, ACTR, or NCoA3) was defined as a coactivator for androgen receptor (AR) by in vitro studies, its role in AR-mediated prostate development and prostate cancer remained unexplored. We report here that AIB1 is expressed in the basal and stromal cells but not in the epithelial cells of the normal mouse prostates. AIB1 deficiency only slightly delayed prostate growth and had no effect on androgen-dependent prostate regeneration, suggesting an unessential role of AIB1 in AR function in the prostate. Surprisingly, when prostate tumorigenesis was induced by the SV40 transgene in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, AIB1 expression was observed in certain epithelial cells of the prostate intraepithelial neoplasia (PIN) and well-differentiated carcinoma and in almost all cells of the poorly differentiated carcinoma. After AIB1 was genetically inactivated in AIB1-/-/TRAMP mice, the progression of prostate tumorigenesis in most AIB1-/-/TRAMP mice was arrested at the well-differentiated carcinoma stage. Wild-type (WT)/TRAMP mice developed progressive, multifocal, and metastatic prostate tumors and died between 25 and 34 weeks. In contrast, AIB1-/-/TRAMP mice only exhibited PIN and early-stage well-differentiated carcinoma by 39 weeks. AIB1-/-/TRAMP prostates showed much lower cell proliferation than WT/TRAMP prostates. Most AIB1-/-/TRAMP mice could survive more than 35 weeks and died with other types of tumors or unknown reasons. Our results indicate that induction of AIB1 expression in partially transformed epithelial cells is essential for progression of prostate tumorigenesis into poorly differentiated carcinoma. Inhibition of AIB1 expression or function in the prostate epithelium may be a potential strategy to suppress prostate cancer initiation and progression.

Background: Several of the proposed mechanisms for the actions of the liposterolic extract of saw palmetto (SPE) are exerted on known risk factors for prostate cancer (CaP). This study investigated whether SPE could prevent the progression of CaP in a transgenic adenocarcinoma of the mouse prostate (TRAMP) model., Methods: Two different doses of SPE designed to deliver 50 mg/kg/day SPE and 300 mg/kg/day SPE were administered in a custom diet to TRAMP mice for 12 or 24 weeks. Body and organ weights were used to evaluate toxicity, and radioimmunoassay was used to measure plasma and tissue androgen levels to monitor effects of SPE on 5alpha reductase activity. Prostate tissues were evaluated histologically to determine the effect of treatment on tumor grade, cell proliferation, and apoptosis., Results: Treatment with 300 mg/kg/day SPE from 4 to 24 weeks of age significantly reduced the concentration of 5alpha-dihydrotestosterone (DHT) in the prostate and resulted in a significant increase in apoptosis and significant decrease in pathological tumor grade and frank tumor incidence., Conclusions: Dietary supplementation with SPE may be effective in controlling CaP tumorigenesis. SPE suppression of prostatic DHT levels lends support to the hypothesis that inhibition of the enzyme 5alpha-reductase is a mechanism of action of this substance., ((c) 2007 Wiley-Liss, Inc.)

We report the first application of high-frequency three-dimensional power Doppler ultrasound imaging in a genetically engineered mouse (GEM) prostate cancer model. We show that the technology sensitively and specifically depicts functional neoangiogenic blood flow because little or no flow is measurable in normal prostate tissue or tumors smaller than 2-3 mm diameter, the neoangiogenesis "switch-on" size. Vascular structures depicted by power Doppler were verified using Microfil-enhanced micro-computed tomography (micro-CT) and by correlation with microvessel distributions measured by immunohistochemistry and enhanced vascularity visualized by confocal microscopy in two GEM models [transgenic adenocarcinoma of the mouse prostate (TRAMP) and PSP94 gene-directed transgenic mouse adenocarcinoma of the prostate (PSP-TGMAP)]. Four distinct phases of neoangiogenesis in cancer development were observed, specifically, (a) an early latent phase; (b) establishment of a peripheral capsular vascular structure as a neoangiogenesis initiation site; (c) a peak in tumor vascularity that occurs before aggressive tumor growth; and (d) rapid tumor growth accompanied by decreasing vascularity. Microsurgical interventions mimicking local delivery of antiangiogenesis drugs were done by ligating arteries upstream from feeder vessels branching to the prostate. Microsurgery produced an immediate reduction of tumor blood flow, and flow remained low from 1 h to 2 weeks or longer after treatment. Power Doppler, in conjunction with micro-CT, showed that the tumors recruit secondary blood supplies from nearby vessels, which likely accounts for the continued growth of the tumors after surgery. The microsurgical model represents an advanced angiogenic prostate cancer stage in GEM mice corresponding to clinically defined hormone-refractory prostate cancer. Three-dimensional power Doppler imaging is completely noninvasive and will facilitate basic and preclinical research on neoangiogenesis in live animal models.

The androgen receptor signaling axis plays an essential role in the development, function and homeostasis of male urogenital structures including the prostate gland although the mechanism by which the AR axis contributes to the initiation, progression and metastatic spread of prostate cancer remains somewhat enigmatic. A number of molecular events have been proposed to act at the level of the AR and associated coregulators to influence the natural history of prostate cancer including deregulated expression, somatic mutation, and post-translational modification. The purpose of this article is to review the evidence for deregulated expression and function of the AR and associated coactivators and corepressors and how such events might contribute to the progression of prostate cancer by controlling the selection and expression of AR targets.

Background: Prostate cancer is an important public health problem. It is an excellent candidate disease for chemoprevention because prostate cancer is typically slow growing and is usually diagnosed in elderly males. Pygeum africanum (Prunus africana or Rosaceae) is an African prune (plum) tree found in tropical Africa. An extract from the bark of Pygeum africanum has been used in Europe as a prevention and treatment of prostate disorders including benign prostatic hypertrophy (BPH). More recently in the USA, the phytotherapeutic preparations of Pygeum africanum and Saw palmetto have been marketed for prostate health including prostate cancer prevention and treatment., Methods: The anti-cancer potential of Pygeum africanum has been tested both in vitro (PC-3 and LNCaP cells) and in vivo (TRAMP mouse model)., Results: In tissue culture, ethanolic extracts (30%) of Pygeum africanum inhibited the growth of PC-3 and LNCaP cells; induced apoptosis and altered cell kinetics; down regulated ERalpha and PKC-alpha protein, and demonstrated good binding ability to both mouse uterine estrogen receptors and LNCaP human androgen receptors. TRAMP mice fed Pygeum africanum showed a significant reduction (P = 0.034) in prostate cancer incidence (35%) compared to casein fed mice (62.5%)., Conclusion: Pygeum africanum, which is widely used in Europe and USA for treatment of BPH, has a significant role in regulation of prostate cancer both in vitro and in vivo and therefore may be a useful supplement for people at high risk for developing prostate cancer.

In this report, we studied T cell responses to a prostate cancer Ag by adoptively transferring tumor Ag-specific T cells into prostate tumor-bearing mice. Our findings demonstrate that CD8(+) T cells initially encountered tumor Ag in the lymph node and underwent an abortive proliferative response. Upon isolation from the tumor, the residual tumor-specific T cells were functionally tolerant of tumor Ag as measured by their inability to degranulate and secrete IFN-gamma and granzyme B. We next sought to determine whether providing an ex vivo-matured, peptide-pulsed dendritic cell (DC) vaccine could overcome the tolerizing mechanisms of tumor-bearing transgenic adenocarcinoma of the mouse prostate model mice. We demonstrate that tumor Ag-specific T cells were protected from tolerance following provision of the DC vaccine. Concurrently, there was a reduction in prostate tumor size. However, even when activated DCs initially present tumor Ag, T cells persisting within the tolerogenic tumor environment gradually lost Ag reactivity. These results suggest that even though a productive antitumor response can be initiated by a DC vaccine, the tolerizing environment created by the tumor still exerts suppressive effects on the T cells. Furthermore, our results demonstrate that when trying to elicit an effective antitumor immune response, two obstacles must be considered: to maintain tumor Ag responsiveness, T cells must be efficiently primed to overcome tumor Ag presented in a tolerizing manner and protected from the suppressive mechanisms of the tumor microenvironment.

Background: A major focus of prostate cancer research has been to identify genes that are deregulated during tumor progression, potentially providing diagnostic markers and therapeutic targets., Methods: We have employed serial analysis of gene expression (SAGE) and microarray hybridization to identify alterations that occur during malignant transformation in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model. Many of these alterations were validated by real-time PCR (rtPCR)., Results: We identified several hundred mRNAs that were deregulated. Cluster analysis of microarray profiles with samples from various stages of the disease demonstrated that androgen-independent (AI) primary tumors are similar to metastases; 180 transcripts have expression patterns suggesting an involvement in the genesis of late-stage tumors, and our data support a role for phospholipase A2 group IIA in the acquisition of their highly aggressive characteristics., Conclusions: Our analyses identified well-characterized genes that were previously known to be involved in prostate cancer, validating our study, and also uncovered transcripts that had not previously been implicated in prostate cancer progression., ((c) 2006 Wiley-Liss, Inc.)

Rodents do not naturally develop prostate cancer. Currently, most widely used genetically engineered mouse prostate cancer models use SV40 T/tag oncogene. To understand the mechanism underlying prostate cancer development in transgenic and knock-in SV40 Tag mouse models, we did cDNA microarray analyses, comparing gene expression profiles of prostate cancer tissues from early-, late-, and advance-stage androgen-independent prostate cancers. Of the 67 genes that were up-regulated by > or = 10-fold, 40 are known to be required for chromosome stability. In particular, the spindle checkpoint component Bub1 was persistently up-regulated from early to advanced androgen-independent prostate cancer lesions. Significantly, Bub1, which is required for accurate chromosome segregation during mitosis, has recently been reported to bind SV40 Tag. Consistent with a spindle checkpoint defect, flow cytometry experiments indicate that advanced androgen-independent prostate cancer tumors exhibit aneuploidy, along with up-regulation of levels of both Bub1 mRNA and Bub1 protein or hyperphosphorylation. Importantly, up-regulation and hyperphosphorylation of Bub1 were also observed in established human prostate cancer cell lines and in clinical studies. Furthermore, analysis of human prostate cancer lines showed impaired spindle checkpoint function and endoreduplication following exposure to spindle toxins. Small interfering RNA-mediated repression of Bub1 in the human prostate cancer line PC-3 restrained cell proliferation, an effect mimicked by inhibition of mitogen-activated protein kinase, an upstream activator of Bub1. Thus, by perturbing Bub1 function, our observations suggest a new mechanism whereby the SV40 Tag oncoprotein promotes chromosomal instability and aneuploidy in transgenic mouse prostate cancer models. Whereas the exact details of this mechanism remain unclear, our novel findings raise the possibility of exploiting Bub1 as a new therapeutic target in the treatment of prostate cancer, the most common cancer in adult men in North America.

Background: There has been a determined search for therapies specifically aimed at eradicating tumor cells while leaving normal host cells unaffected. This goal can potentially be accomplished by engaging tumor antigen-specific T-cell repertoire to attack the tumor. A pre-requisite for a successful T-cell-mediated attack against tumors or pathogens is that the target tissues express major histocompatibility complex (MHC) molecules. Using newer anti-MHC class I and MHC class II antibody reagents, we re-examined the expression of MHC in both human and mouse prostate tumors and their microenvironments., Methods: Using immunocytochemistry, we examined the expression of MHC class I, class II, and CD3 molecules on cryopreserved human and mouse prostate tumor samples., Results: MHC class I molecules are expressed by the entire spectrum of different stages of both human and mouse prostate tumor cells. Additionally, cells of the hematopoietic lineage, dispersed in the tumor microenvironment, showed significant expression of MHC class II molecules. Human prostate tumors also show a significant infiltrate of CD3+ T cells., Conclusions: Expression of MHC class I and class II molecules within the prostate tumor microenvironment are consequential for T-cell-mediated immunotherapeutic approaches against prostate cancer., ((c) 2006 Wiley-Liss, Inc.)

Transforming growth factor-beta (TGF-beta) is a potent immunosuppressant. Overproduction of TGF-beta by tumor cells leads to evasion of host immune surveillance and tumor progression. Results of our early studies showed that adoptive transfer of tumor-reactive, TGF-beta-insensitive CD8(+) T cells into immunocompetent mice was able to eradicate lung metastasis of mouse prostate cancer. The present study was conducted with three objectives. (a) We tested if this technology could be applied to the treatment of solid xenograft tumors in allogeneic immunodeficient hosts. (b) We determined relevant variables in the tumor microenvironment with the treatment. (c) We tested if immune cells other than CD8(+) T cells were required for the antitumor effect. Mouse prostate cancer cells, TRAMP-C2 of the C57BL/6 strain, grown in immunodeficient allogeneic hosts of BALB/c strain, were used as a xenograft model. Tumor-reactive CD8(+) T cells from C57BL/6 mice were isolated, expanded ex vivo, and rendered insensitive to TGF-beta by introducing a dominant-negative TGF-beta type II receptor vector. Seven days following s.c. injection of TRAMP-C2 cells (5 x 10(5)) into the flank of male BALB/c-Rag1(-/-) mice, tumor-reactive, TGF-beta-insensitive CD8(+) T cells (1.5 x 10(7)) were transferred with and without the cotransfer of an equal number of CD8-depleted splenocytes from C57BL/6 donors. Naive CD8(+) T cells or green fluorescent protein-empty vector-transfected tumor-reactive CD8(+) T cells were transferred as controls. Forty days following the transfer, the average tumor weight in animals that received cotransfer of tumor-reactive, TGF-beta-insensitive CD8(+) T cells and CD8-depleted splenocytes was at least 50% less than that in animals of all other groups (P < 0.05). Tumors in animals of the former group showed a massive infiltration of CD8(+) T cells. This was associated with secretion of relevant cytokines, decreased tumor proliferation, reduced angiogenesis, and increased tumor apoptosis. Based on these results, we postulated a concept of antitumor immune response cycle in tumor immunology.

In vivo imaging advances have greatly expanded the use of animal cancer models. Herein, we describe two new models that permit prostate imaging ex vivo, in vivo, and in utero. Further, we show the use of these models for detecting small metastasis and testing reagents that modulate the androgen receptor (AR) axis. A luciferase reporter gene was directed to the prostate epithelium using three composite promoters called human kallikrein 2 (hK2)-E3/P, PSA-E2/P, and ARR2PB, derived from hK2, PSA, and rat probasin regulatory elements, to generate the EZC1, EZC2, and EZC3-prostate mice, respectively. EZC2 and EZC3-prostate display robust expression in the prostate with only minimal detectable expression in other organs, including testes and epididymis. Luciferase expression was detected as early as embryonic day 13 (E13) in the urogenital track. To image prostate cancer progression, lines of EZC mice were bred with prostate cancer models TRAMP and JOCK1, and imaged longitudinally. When crossed with prostate cancer models, EZC3 facilitated detection of metastatic lesions although total prostate luciferase expression was static or reduced due to weakening of AR-regulated promoters. Castration reduced luciferase expression by 90% and 97% in EZC2 and EZC3 mice, respectively, and use of GnRH antagonist also led to extensive inhibition of reporter activity. The EZC-prostate model permits prostate imaging in vivo and should be useful for imaging prostate development, growth, metastasis, and response to treatment noninvasively and longitudinally. These models also provide powerful new reagents for developing improved drugs that inhibit the AR axis.

Inappropriate expression of the Aurora kinases can induce aberrant mitosis, centrosome irregularities, and chromosomal instability, which lead to anueploidy and cell transformation. Here, we report that Aurora-A and Aurora-B are highly expressed in primary human and mouse prostate cancers and prostate cancer cell lines. In clinical samples, levels of Aurora-A and Aurora-B were significantly elevated in prostatic intraepithelial neoplasia lesions and prostate tumors when compared with the non-neoplastic samples. Interestingly, expression of Aurora-A in non-neoplastic prostates correlated with seminal vesicle invasion (rho = 0.275, P = 0.0169) and in prostate tumor with positive surgical margins (rho = 0.265, P = 0.0161). In addition, nuclear expression of Aurora-B in prostatic intraepithelial neoplasia lesions correlated with clinical staging of the tumor (rho = -0.4, P = 0.0474) whereas cytoplasmic expression in tumors correlated with seminal vesicle invasion (rho = 0.282, P = 0.0098). Cell lines and primary tumors derived from the TRAMP model were also found to express high levels of Aurora-A and Aurora-B. When human PC3, LNCaP, and mouse C1A cells were treated with the potent Aurora kinase inhibitor VX680, which attenuates phosphorylation of histone H3, cancer cell survival was reduced. VX680 could further reduce cell viability >2-fold when used in combination with the chemotherapy drug doxorubicin. Our findings support a functional relationship between Aurora kinase expression and prostate cancer and the application of small-molecule inhibitors in therapeutic modalities.

Background: TGF-beta is a potent immunosuppressant. High levels of TGF-beta produced by cancer cells have a negative inhibition effect on surrounding host immune cells and leads to evasion of the host immune surveillance and tumor progression. In the present study, we report a distinct ability of tumor reactive, TGF-beta-insensitive CD8+ T cells to infiltrate into established tumors, secrete relevant cytokines, and induce apoptosis of tumor cells., Methods: CD8+ T cells were isolated from the spleens of C57BL/6 mice, which were primed with irradiated mouse prostate cancer cells, the TRAMP-C2 cells. After ex vivo expansion, these tumor reactive CD8+ cells were rendered TGF-beta-insensitive by infection with a retroviral (MSCV)-mediated dominant negative TGF-beta type II receptor (TbetaRIIDN). Control CD8+ cells consist of those transfected with the GFP-only empty vector and naïve CD8+ T cells. Recipient mice were challenged with a single injection of TRAMP-C2 cells 21 days before adoptive transfer of CD8+ T cells was performed. Forty days after the adoptive transfer, all animals were sacrificed. The presence of pulmonary metastases was evaluated pathologically. Serial slides of malignant tissues were used for immunofluorescent staining for different kinds of immune cell infiltration, cytokines, and apoptosis analysis., Results: Pulmonary metastases were either eliminated or significantly reduced in the group receiving adoptive transfer of tumor-reactive TGF-beta-insensitive CD8+ T cells (3 out of 12) when compared to GFP controls (9 out of 12), and naïve CD8+ T cells (12 out of 12). Results of immunofluorescent studies demonstrated that only tumor-reactive TGF-beta-insensitive CD8+ T cells were able to infiltrate into the tumor and mediate apoptosis when compared to CD4+ T cells, NK cells, and B cells. A large amount of cytokines such as perforin, nitric oxide, IFN-gamma, IL-2, TNF-alpha were secreted in tumor tissue treated with tumor-reactive TGF-beta-insensitive CD8+ T cells. No immune cells infiltration and cytokine secretion were detected in tumor tissues treated with naïve T cells and GFP controls., Conclusions: Our results demonstrate the mechanism of anti-tumor effect of tumor-reactive TGF-beta-insensitive CD8+ T cells that adoptive transfer of these CD8+ T cells resulted in infiltration of these immune cells into the tumor parenchyma, secretion of relevant cytokines, and induction of apoptosis in tumor cells. These results support the concept that tumor-reactive TGF-beta-insensitive CD8+ T cells may prove beneficial in the treatment of advanced cancer patients., (Copyright (c) 2005 Wiley-Liss, Inc.)

Current prostate cancer research in both basic and preclinical trial studies employ genetically engineered mouse models. However, unlike in human prostate cancer patients, rodents have no counterpart of prostatic-specific antigen (PSA) for monitoring prostate cancer initiation and progression. In this study, we established a mouse serum tumor marker from a mouse homologue of human prostate secretory protein of 94 amino acids (PSP94). Immunohistochemistry studies on different histologic grades from both transgenic and knock-in mouse prostate cancer models showed the down-regulation of tissue PSP94 expression (P < 0.001), the same as for PSA and PSP94 in humans. The presence of mouse serum PSP94 was shown by affinity column and immunoprecipitation purification using a polyclonal mouse PSP94 antibody. A competitive ELISA protocol was established to quantify serum PSP94 levels with a sensitivity of 1 ng/mL. Quantified serum levels of mouse PSP94 ranged from 49.84 ng/mL in wild-type mice to 113.86, 400.45, and 930.90 ng/mL in mouse prostatic intraepithelial neoplasia with microinvasion, well differentiated, moderately differentiated, and poorly differentiated prostate cancer genetically engineered prostate cancer mice, respectively (P < 0.01, n = 68). This increase in serum PSP94 is also well correlated with age and tumor weight. Through longitudinal monitoring of serum PSP94 levels of castrated mice (androgen ablation therapy), we found a correlation between responsiveness/refractory prostate tissues and serum PSP94 levels. The utility of mouse serum PSP94 as a marker in hormone therapy was further confirmed by three-dimensional ultrasound imaging. The establishment of the first rodent prostate cancer serum biomarker will greatly facilitate both basic and preclinical research on human prostate cancer.

Malignantly transformed stem cells represent a potential common nidus for the primary cancer and the recurrent cancer that arises after treatment failure. Putative prostate stem cells and prostate tumor stem cells in benign and malignant human prostate tissue, in primary human prostate xenografts, and in the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model of prostate cancer, are defined by expression of breast cancer resistance protein (BCRP), a marker of pluripotent hematopoietic, muscle, and neural stem cells, and by an absence of androgen receptor (AR) protein. Inhibition of BCRP-mediated efflux of dihydrotestosterone by novobiocin or fumitremorgin C in a rat prostate progenitor cell line that expresses BCRP and AR mRNAs, but minimal AR protein, results in stabilization and nuclear translocation of AR protein, providing a mechanism for lack of AR protein in BCRP-expressing stem cells. In both benign and malignant human prostate tissue, the rare epithelial cells that express BCRP and lack AR protein are localized in the basal cell compartment, survive androgen deprivation, and maintain proliferative potential in the hypoxic, androgen-deprived prostate. Putative prostate tumor stem cells that express BCRP but not AR protein in TRAMP are the source of a BCRP-negative and AR-negative, Foxa2- and SV40Tag-expressing, transit amplifying compartment that progresses to the poorly differentiated carcinomas that arise rapidly after castration. Therefore, BCRP expression isolates prostate stem/tumor stem cells from the prostate tissue microenvironment through constitutive efflux of androgen, protecting the putative tumor stem cells from androgen deprivation, hypoxia, or adjuvant chemotherapy, and providing the nidus for recurrent prostate cancer.

The cells' ability to proliferate in response to growth factor stimulation is significantly altered during cancer progression. To investigate the mechanisms underlying these alterations in prostate cancer, the role and expression of beta1A integrin and type 1 insulin-like growth factor receptor (IGF-IR), known to contribute to cell proliferation and transformation, were analyzed. Using small interfering RNA oligonucleotides to down-regulate beta1A, we show that beta1A expression is required for IGF-IR-mediated prostate cancer cell proliferation and anchorage-independent growth. In vivo, using age-matched transgenic adenocarcinoma of mouse prostate (TRAMP) mice at different stages of prostate cancer [prostatic intraepithelial neoplasia, PIN; well-differentiated adenocarcinoma, WD; and poorly differentiated adenocarcinoma, PD], the expression of beta1A and of IGF-IR was studied. beta1A and IGF-IR expression levels were concurrently up-regulated in high PIN and WD, whereas their expression did not correlate in late-stage PD. In contrast to the up-regulated expression of beta1A, the levels of beta1C, a beta1 cytoplasmic variant that inhibits cell proliferation, were down-regulated in all stages of prostate cancer. A similar expression pattern was observed for a beta1C downstream effector, Grb2-associated binder-1 (Gab1) which is known to inhibit IGF-IR phosphorylation. To analyze in vitro the mechanistic implications of beta1A, beta1C, and Gab1 deregulation in prostate cancer, we investigated whether expression of either beta1 variant in beta1-null cells affected IGF-IR localization. We found that IGF-IR and beta1A were colocalized in highly specialized integrin signaling compartments, designated focal contacts. However, in the presence of beta1C, IGF-IR remained diffuse on the cell surface and did not localize to focal contacts. The findings that beta1 integrins and IGF-IR are concurrently deregulated and that expression of beta1 integrins is necessary to achieve appropriate IGF-IR intracellular distribution point to the important role that the cross-talk between these receptors may have during prostate cancer progression and will be helpful in formulating new therapeutic strategies.

Prostate cancer is the most common cancer in adult men in North America. Preclinical studies of prostate cancer employ genetically engineered mouse models, because prostate cancer does not occur naturally in rodents. Widespread application of these models has been limited because autopsy was the only reliable method to evaluate treatment efficacy in longitudinal studies. This article reports the first use of three-dimensional ultrasound microimaging for measuring tumor progression in a genetically engineered mouse model, the 94-amino acid prostate secretory protein gene-directed transgenic prostate cancer model. Qualitative comparisons of three-dimensional ultrasound images with serial histology sections of prostate tumors show the ability of ultrasound to accurately depict the size and shape of malignant masses in live mice. Ultrasound imaging identified tumors ranging from 2.4 to 14 mm maximum diameter. The correlation coefficient of tumor diameter measurements done in vivo with three-dimensional ultrasound and at autopsy was 0.998. Prospective tumor detection sensitivity and specificity were both >90% when diagnoses were based on repeated ultrasound examinations done on separate days. Representative exponential growth curves constructed via longitudinal ultrasound imaging indicated volume doubling times of 5 and 13 days for two prostate tumors. Compared with other microimaging and molecular imaging modalities, the application of three-dimensional ultrasound imaging to prostate cancer in mice showed advantages, such as high spatial resolution and contrast in soft tissue, fast and uncomplicated protocols, and portable and economical equipment that will likely enable ultrasound to become a new microimaging modality for mouse preclinical trial studies.

Circulating insulin-like growth factor-I (IGF-I) levels have been shown to be related to risk of prostate cancer in epidemiologic studies. While specific genetic loci responsible for interindividual variation in circulating IGF-I levels in normal men have not been identified, candidate genes include those involved in the growth hormone (GH)-IGF-I axis such as the hypothalamic factors GH releasing hormone (GHRH) and somatostatin and their receptors. To investigate the role of the GH-IGF-I axis on in vivo prostate carcinogenesis and neoplastic progression, we generated mice genetically predisposed to prostate cancer (the TRAMP model) to be homozygous for lit, a mutation that inactivates the GHRH receptor (GHRH-R) and reduces circulating levels of GH and IGF-I. The lit mutation significantly reduced the percentage of the prostate gland showing neoplastic changes at 35 weeks of age (P=0.0005) and was also associated with improved survival (P<0.01). These data provide an example of a germ line mutation that reduces risk in an experimental prostate carcinogenesis model. The results suggest that prostate carcinogenesis and progression may be influenced by germ line variation of genes encoding signalling molecules in the GH-IGF-I axis.

Studies in vitro suggest that osteopontin (OPN), an extracellular matrix protein secreted by macrophages infiltrating prostate tumors, and by tumor cells, may have a role in the transition from clinically insignificant tumors to metastatic prostate cancer (PC). Latent PC occurs at equal rates in Western and Asian men, but the incidence of advanced PC is many-fold higher in Western men. Our earlier studies in TRAnsgenic Mouse Prostate adenocarcinoma (TRAMP) mice showed that genistein, an isoflavone found in soybeans, lowered the incidence of advanced PC. This suggested that lower intake of dietary soy may be one possible cause for higher incidence of advanced PC in Western men. The objective of the present study was to test the hypothesis that genistein may exert its preventive effect by inhibiting OPN expression. From 5 to 28 wk of age, 80, 68, and 30 TRAMP mice were fed AIN-76A diet containing 0, 250, or 500 mg genistein/kg body weight, respectively. Organ weights were measured. The steady-state level of OPN mRNA was evaluated by RT-PCR in a longitudinal study in 74 TRAMP and 32 nontransgenic litter mates (NTM). Administration of 250 and 500 mg genistein/kg AIN-76A improved survival (P = 0.008 and P = 0.005, respectively) and reduced mean weight of prostates with poorly differentiated cancer (PD) (P < 0.001), as well as the mean weight of periaortic lymph nodes (LN), although the latter was not significant. OPN was upregulated 10-fold in PD compared with prostates with a lower pathological score from TRAMP or NTM of any age (P = 0.003). OPN mRNA levels in the dorsolateral prostate and metastasis to LN were significantly correlated (r = 0.643; P = 0.00006). Genistein had a dose-dependent, significant inhibitory effect on OPN transcript levels in prostates displaying advanced prostate cancer (PD; score 6; P = 0.05). Studies are consistent with the possibility that dietary genistein may delay the progression from benign to malignant tumors by inhibiting OPN expression.