Dependence of castration-resistant prostate cancer (CRPC) stem cells on CRPC-associated fibroblasts.

We previously established a role for cancer-associated fibroblasts (CAF) in enhancing the self-renewal and differentiation potentials of putative prostate cancer stem cells (CSC). Our published work focused on androgen-dependent prostate cancer (ADPC) using the conditional Pten deletion mouse model. Employing the same model, we now describe the interaction of CAF and CSC in castration-resistant prostate cancer (CRPC). CAF isolated from ADPC (ADPCAF) and from CRPC (CRPCAF) were compared in terms of their ability to support organoid formation and tumor initiation by CSC from CRPC (CRPCSC) in vitro and in vivo. CRPCSC formed spheroids in vitro and well-differentiated glandular structures under the renal capsules of recipient mice in vivo more effectively in the presence of CRPCAF compared to ADPCAF. Furthermore, whereas CSC with CAF from ADPC formed mostly well-differentiated tumors in our previous study, we now show that CRPCSC, when combined with CRPCAF (but not ADPCAF), can form aggressive, poorly-differentiated tumors. The potential of CRPCAF to support organoid/tumor formation by CRPCSC remained greater even when compared to 10-fold more ADPCAF, suggesting that paracrine factors produced specifically by CRPCAF preferentially potentiate the stemness and tumorigenic properties of the corresponding CSC. This apparently unique property of CRPCAF was notable when the CAF and CSC were grafted in either intact or castrated recipient mice. In both environments, CRPCAF induced in the epithelial compartment higher proliferative activity compared to ADPCAF, indicated by a higher Ki67 index. Factors released by CRPCAF to regulate CRPCSC may be targeted to develop novel therapeutic approaches to manage advanced prostate cancer.

A joint effect of new Western diet and retinoid X receptor α prostate-specific knockout with development of high-grade prostatic intraepithelial neoplasia in mice--a preliminary study.

BACKGROUND: The "New Western-style Diet" (NWD) characterized by high in fat and low in fiber, vitamin D, calcium, and methyl donors--are considered as a risk factor for prostate cancer. Previous studies have shown that premalignant lesions of human prostate have decreased expression of the Retinoid X Receptor alpha (RXRα). This study was to determine the effect of diet in RXRα knockout mice in developing high-grade prostate intraepithelial neoplasia (mPIN). METHODS: Male mice (n = 54) with or without the RXRα prostate null mutation were fed either NWD or AIN-76A control diet for 10 months; prostates were harvested at 11 months of age and examined for prostate mPIN. RESULTS: mPIN was seen in 79% of RXRα prostate null mice fed NWD (n = 19), 30.8% RXRα prostate null mice fed AIN-76A (n = 13), 42.9% RXRα wild-type mice fed NWD (n = 14), and 12.5% RXRα wild-type mice fed AIN-76A (n = 8). Unconditional Logistic analysis showed a significant joint effect of NWD and RXRα status in developing mPIN 26.3 (95% CI: 2.5-280), but interaction was not significant owing to the small sample size 1.6 (0.09-27.7, P = 0.7441). CONCLUSION: This study provides preliminary data to support a joint RXRα-diet effect in prostate carcinogenesis.

Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer.

The murine Pten prostate cancer model described in this study recapitulates the disease progression seen in humans: initiation of prostate cancer with prostatic intraepithelial neoplasia (PIN), followed by progression to invasive adenocarcinoma, and subsequent metastasis with defined kinetics. Furthermore, while Pten null prostate cancers regress after androgen ablation, they are capable of proliferating in the absence of androgen. Global assessment of molecular changes caused by homozygous Pten deletion identified key genes known to be relevant to human prostate cancer, including those "signature" genes associated with human cancer metastasis. This murine prostate cancer model provides a unique tool for both exploring the molecular mechanism underlying prostate cancer and for development of new targeted therapies.

Pten null prostate tumorigenesis and AKT activation are blocked by targeted knockout of ER chaperone GRP78/BiP in prostate epithelium.

GRP78/BiP has recently emerged as a novel biomarker for aggressive prostate cancer. Here, we report that homozygous deletion of Grp78 specifically in mouse prostate epithelium suppresses prostate tumorigenesis without affecting postnatal prostate development and growth. Mouse prostates with double conditional knockout of Grp78 and Pten exhibit normal histology and cytology, in contrast to the invasive adenocarcinoma in mouse prostates with Pten inactivation. AKT activation in Pten null prostate epithelium is inhibited by Grp78 homozygous deletion, corresponding with suppression of AKT phosphorylation by GRP78 knockdown in prostate cancer cell line. Thus, inactivation of GRP78 may represent a previously undescribed approach to stop prostate cancer and potentially other cancers resulting from the loss of PTEN tumor suppression and/or activation of the oncogenic AKT.

Runx2 regulates survivin expression in prostate cancer cells.

Previously we described that bone morphogenetic protein-7 (BMP7) could protect prostate cancer C4-2B cells from serum starvation-induced apoptosis via survivin induction. Here, for the first time, we identify Runx2 as a key regulator of survivin transcription. In C4-2B cells grown normally, suppression of Runx2 reduced survivin expression. Using ChIP assays, two regions of the survivin promoter, -1953 to -1812 (I) and -1485 to -1119 (II) encompassing consensus Runx-binding sites were examined. Runx2 was found to be associated with both regions, with a stronger affinity to region-I. In serum-starved cells neither region was occupied, but BMP7 restored association to region-II and not region-I. In reporter assays, transcription activity by BMP7 was significantly reduced when sequences including binding sites of region-II were deleted. Additionally, Runx2 expression was enhanced by BMP7 in these cells. Along with a strong survivin expression, a trend in increased Runx2 expression in human prostate cancer cells and tissues was noted. In the conditional Pten-knockout mouse, Runx2 level increased with growth of prostate tumor. The data define a novel role of Runx2 in regulating survivin expression in malignant epithelial cells and identify it as a critical factor in BMP signaling that protects cancer cells against apoptosis.

Activation of beta-Catenin in mouse prostate causes HGPIN and continuous prostate growth after castration.

BACKGROUND: The role of Wnt/beta-Catenin signaling in embryogenesis and carcinogenesis has been extensively studied in organs such as colon, lung and pancreas, but little is known about Wnt/beta-Catenin signaling in the prostate. Although stabilizing mutations in APC and beta-Catenin are rare in primary prostate tumors, recent studies suggest that cytoplasmic/nuclear beta-Catenin is associated with advanced, metastatic, hormone-refractory prostate carcinoma. METHODS: To better understand the role of beta-Catenin in prostatic development and carcinogenesis, we studied Wnt expression during prostate development and activated Wnt/beta-Catenin signaling in the developing and adult prostate. RESULTS: Our results demonstrated that during prostate development Wnt ligands display a dynamic expression pattern. Activation of beta-Catenin during prostate development caused epithelial hyperplasia followed by prostatic intraepithelial neoplasia (PIN) in prostate. In the adult prostate, activation of beta-Catenin resulted in high grade PIN (HGPIN) and continuous prostatic growth after castration. As a result of activation of beta-Catenin, AR was first up-regulated with the emergence of epithelial hyperplasia, but was later down-regulated when HGPIN developed. Furthermore, activation of beta-Catenin induced Foxa2 re-expression in adult prostate which normally is only expressed in the embryonic budding stage during prostate development. CONCLUSIONS: The results from this study strongly suggest that Wnt/beta-Catenin signaling is involved in the regulation of prostate development and confirm that constitutive activation of this pathway enables the mouse prostate to grow after castration.

PTEN knockout prostate cancer as a model for experimental immunotherapy.

PURPOSE: Testing immunotherapeutic strategies for prostate cancer has been impeded by the lack of relevant tumor models in immunocompetent animals. This opportunity is now provided by the recent development of prostate specific PTEN knockout mice, which show spontaneous development of true adenocarcinoma arising from prostate epithelium and more faithfully recapitulate the human disease than any previous model. We investigated the feasibility of using tumor cells derived from this model to test tumor vaccination and adoptive immunotherapeutic strategies for prostate cancer. MATERIALS AND METHODS: PTEN-CaP8 adenocarcinoma cells derived from the biallelic PTEN knockout prostate cancer model were used to vaccinate nontumor bearing litter mates. Tumor specific effector cells were generated from splenocytes of vaccinated mice by mixed lymphocyte-tumor reactions, and antiproliferative effects and cytokine generation were examined in vitro. The effect of vaccination or adoptive immunotherapy on luciferase marked PTEN-CaP8 subcutaneous tumors was monitored by tumor volumetric measurements and noninvasive bioluminescence imaging. RESULTS: Vaccination of litter mate mice with irradiated PTEN-CaP8 cells showed a significant prophylactic effect against the subsequent tumor challenge. Effector cells harvested from vaccinated litter mates showed significant interferon-gamma secretion upon co-incubation with PTEN-CaP8 target cells and they were capable of efficient target cell growth inhibition in vitro. Intratumor adoptive transfer of effector cells resulted in significant growth inhibition of preestablished prostate tumors in vivo. CONCLUSIONS: The PTEN knockout model serves as a highly useful model in which to investigate tumor cell vaccination and adoptive immunotherapeutic strategies in the context of true adenocarcinoma of the prostate. This model should accelerate efforts to develop effective immunotherapies for human prostate cancer.

Mouse models of prostate adenocarcinoma with the capacity to monitor spontaneous carcinogenesis by bioluminescence or fluorescence.

The application of Cre/loxP technology has resulted in a new generation of conditional mouse models of prostate cancer. Here, we describe the improvement of the conditional Pten deletion model of prostate adenocarcinoma by combining it with either a conditional luciferase or enhanced green fluorescent protein reporter line. In these models, the recombination mechanism that inactivates the Pten alleles also activates the reporter gene. In the luciferase reporter model, the growth of the primary cancer can be followed noninvasively by bioluminescence imaging (BLI). Surgical castration of tumor-bearing animals leads to a reduced bioluminescence signal corresponding to tumor regression that is verified at necropsy. When castrated animals are maintained, the emergence of androgen depletion-independent cancer is detected using BLI at times varying from 7 to 28 weeks postcastration. The ability to monitor growth, regression, or relapse of the tumor with the use of BLI lead to the collection of tumors at different stages of development. By comparing the distribution of phenotypically distinct populations of epithelial cells in cancer tissues, we noted that the degree of hyperplasia of cells with neuroendocrine differentiation significantly increases in the recurrent cancer relative to the primary cancer, a characteristic which may parallel the appearance of a neuroendocrine phenotype in human androgen depletion-independent cancer. The enhanced green fluorescent protein model, at necropsy, can provide an opportunity to locate or assess tumor volume or to isolate enriched populations of cancer cells from tumor tissues via fluorescence-based technologies. These refined models should be useful in the elucidation of mechanisms of prostate cancer progression, and for the development of approaches to preclinical intervention.

Inactivation of Apc in the mouse prostate causes prostate carcinoma.

Alterations of the Wnt/beta-catenin signaling pathway are positively associated with the development and progression of human cancer, including carcinoma of the prostate. To determine the role of activated Wnt/beta-catenin signaling in mouse prostate carcinogenesis, we created a mouse prostate tumor model using probasin-Cre-mediated deletion of Apc. Prostate tumors induced by the deletion of Apc have elevated levels of beta-catenin protein and are highly proliferative. Tumor formation is fully penetrant and follows a consistent pattern of progression. Hyperplasia is observed as early as 4.5 weeks of age, and adenocarcinoma is observed by 7 months. Continued tumor growth usually necessitated sacrifice between 12 and 15 months of age. Despite the high proliferation rate, we have not observed metastasis of these tumors to the lymph nodes or other organs. Surgical castration of 6-week-old mice inhibited tumor formation, and castration of mice with more advanced tumors resulted in the partial regression of specific prostate glands. However, significant areas of carcinoma remained 2 months postcastration, suggesting that tumors induced by Apc loss of function are capable of growth under conditions of androgen depletion. We conclude that the prostate-specific deletion of Apc and the increased expression of beta-catenin associated with prostate carcinoma suggests a role for beta-catenin in prostate cancer and offers an appropriate animal model to investigate the interaction of Wnt signaling with other genetic and epigenetic signals in prostate carcinogenesis.

Cooperation between FGF8b overexpression and PTEN deficiency in prostate tumorigenesis.

Two commonly occurring genetic aberrations of human prostate cancer [i.e., overexpression of a mitogenic polypeptide (fibroblast growth factor 8, isoform b or FGF8b) and loss of function of PTEN tumor suppressor] were recapitulated into a new combinatorial mouse model. This model harboring the Fgf8b transgene and haploinsufficiency in Pten, both in a prostate epithelium-specific manner, yielded prostatic adenocarcinoma with readily detectable lymph node metastases, whereas single models with each of the defects were shown earlier to progress generally only up to prostatic intraepithelial neoplasia (PIN). In addition to late age-related development of typical adenocarcinoma, the model also displayed a low incidence of mucinous adenocarcinoma, a rare variant type of human prostatic adenocarcinoma. The cooperation between FGF8b activation and PTEN deficiency must be linked to acquisition of additional genetic alterations for the progression of the lesions to primary adenocarcinoma. Here, we identified loss of heterozygosity at the Pten gene leading to bialleic loss, as a necessary secondary event, indicating that a complete loss of PTEN function is required in the development of invasive cancer in the model. Analyses of expression of downstream mediators phospho-AKT (p-AKT) and p27(KIP1), in various types of lesions, however, revealed a complex picture. Although PIN lesions displayed relatively strong expression of p-AKT and p27(KIP1), there was a notable heterogeneity with variable decrease in their immunostaining in adenocarcinomas. Together, the results further underscore the notion that besides activation of AKT by loss of PTEN function, other PTEN-regulated pathways must be operative for progression of lesions from PIN to adenocarcinoma.

Bone morphogenetic protein 7 protects prostate cancer cells from stress-induced apoptosis via both Smad and c-Jun NH2-terminal kinase pathways.

We reported earlier that exposure to exogenous bone morphogenetic protein 7 (BMP7) could strongly inhibit serum starvation-induced apoptosis to C4-2B cell line, a variant of the LNCaP human prostate cancer cell line with propensity for bone metastasis. Whereas serum starvation suppressed the expression of survivin, a member of the inhibitor of apoptosis protein family, its expression was sustained in the presence of BMP7. In this study, we present evidence that BMP7 exposure up-regulated survivin promoter activity, an effect that was associated with activation of Smad, and could be repressed by dominant-negative Smad5. Additionally, serum starvation-induced suppression of c-jun NH2-terminal kinase (JNK) activity in C4-2B cells could be mostly restored by BMP7, and a JNK inhibitor could counteract the antiapoptotic effect of BMP7, without a significant effect on the level of survivin expression. Thus, we identified JNK pathway as another signaling mode for the antiapoptotic function of BMP7. To test the effect of endogenous up-regulation of BMP7, we genetically modulated the C4-2B cell line to overexpress BMP7 protein. Not only was this altered cell line resistant to serum starvation-induced apoptosis but it also exhibited patterns of Smad activation, survivin up-regulation, and JNK activation similar to those of the parental C4-2B cells exposed to exogenous BMP7. Consistent with these in vitro findings of BMP7 action, we acquired correlative results of Smad activation, survivin expression, and JNK activation in the progression of prostate cancer in the conditional Pten deletion mouse model, in which we first obtained the evidence of BMP7 overexpression.

Increased expression of osteopontin contributes to the progression of prostate cancer.

Osteopontin is a secreted glycosylated phosphoprotein known to be involved in numerous physiologic functions and associated with the late stages of various cancers. We used preneoplastic and neoplastic mouse models of prostate cancer to determine the onset of elevated expression of osteopontin in the development of this disease. Osteopontin alterations occurred early in the disease with dysregulated expression observed in lesions of low-grade prostatic intraepithelial neoplasia (PIN). Over time, osteopontin expressing dysplastic cells seemed to increase in number in high-grade PIN and increased further in adenocarcinoma, and in metastasis, almost all of the cancer cells immunohistochemically stained positive for osteopontin overexpression. We examined the biological properties of human prostate cancer cell lines LNCaP and PC-3, in which osteopontin overexpression was achieved via lentiviral gene transduction. Evidence was obtained that osteopontin could contribute to a proliferative advantage in both cell types, although more significantly in LNCaP than PC-3. Osteopontin also influenced their in vitro invasive ability, and again, most strikingly in the weakly oncogenic LNCaP. Furthermore, excess osteopontin induced the LNCaP cells to acquire a strong intravasation potential in vivo in the chicken embryo chorioallantoic membrane assay for blood vessel penetration. These results establish a correlation between an increased gradient of osteopontin expression throughout the stages of murine prostate cancer, beginning from the preneoplastic lesions to distant metastases that suggests a proliferative and invasive advantages to those prostate tumor cells overexpressing osteopontin. Together, these findings support a strategy designed to target osteopontin in the context of prostate cancer therapy.

Pten dose dictates cancer progression in the prostate.

Complete inactivation of the PTEN tumor suppressor gene is extremely common in advanced cancer, including prostate cancer (CaP). However, one PTEN allele is already lost in the vast majority of CaPs at presentation. To determine the consequence of PTEN dose variations on cancer progression, we have generated by homologous recombination a hypomorphic Pten mouse mutant series with decreasing Pten activity: Pten(hy/+) > Pten(+/-) > Pten(hy/-) (mutants in which we have rescued the embryonic lethality due to complete Pten inactivation) > Pten prostate conditional knockout (Pten(pc)) mutants. In addition, we have generated and comparatively analyzed two distinct Pten(pc) mutants in which Pten is inactivated focally or throughout the entire prostatic epithelium. We find that the extent of Pten inactivation dictate in an exquisite dose-dependent fashion CaP progression, its incidence, latency, and biology. The dose of Pten affects key downstream targets such as Akt, p27(Kip1), mTOR, and FOXO3. Our results provide conclusive genetic support for the notion that PTEN is haploinsufficient in tumor suppression and that its dose is a key determinant in cancer progression.

Diverse biological effect and Smad signaling of bone morphogenetic protein 7 in prostate tumor cells.

We found that bone morphogenetic protein (BMP) 7, a member of the BMP family, was strikingly up-regulated during the development of primary prostatic adenocarcinoma in the conditional Pten deletion mouse model. To determine the relevance of this finding to human prostate cancer, we examined the expression of BMPs and BMP receptors (BMPR) as well as the responsiveness to recombinant human BMP7 in a series of human prostate tumor cell lines. All prostatic cell lines tested expressed variable levels of BMP2, BMP4, and BMP7 and at least two of each type I and II BMPRs. In all cases, BMP7 induced Smad phosphorylation in a dose-dependent manner, with Smad5 activation clearly demonstrable. However, the biological responses to BMP7 were cell type specific. BPH-1, a cell line representing benign prostatic epithelial hyperplasia, was growth arrested at G1. In the bone metastasis-derived PC-3 prostate cancer cells, BMP7 induced epithelial-mesenchymal transdifferentiation with classic changes in morphology, motility, invasiveness, and molecular markers. Finally, BMP7 inhibited serum starvation-induced apoptosis in the LNCaP prostate cancer cell line and more remarkably in its bone metastatic variant C4-2B line. Each of the cell lines influenced by BMP7 was also responsive to BMP2 in a corresponding manner. The antiapoptotic activity of BMP7 in the LNCaP and C4-2B cell lines was not associated with a significant alteration in the levels of the proapoptotic protein Bax or the antiapoptotic proteins Bcl-2, Bcl-xl, and X-linked inhibitor of apoptosis. However, in C4-2B cells but not in LNCaP cells, a starvation-induced decrease in the level of survivin was counteracted by BMP7. Taken together, these findings suggest that BMPs are able to modulate the biological behavior of prostate tumor cells in diverse and cell type-specific manner and point to certain mechanisms by which these secreted signaling molecules may contribute to prostate cancer growth and metastasis.

EphB4 expression and biological significance in prostate cancer.

Prostate cancer is the most common cancer in men. Advanced prostate cancer spreading beyond the gland is incurable. Identifying factors that regulate the spread of tumor into the regional nodes and distant sites would guide the development of novel diagnostic, prognostic, and therapeutic targets. The aim of our study was to examine the expression and biological role of EphB4 in prostate cancer. EphB4 mRNA is expressed in 64 of 72 (89%) prostate tumor tissues assessed. EphB4 protein expression is found in the majority (41 of 62, 66%) of tumors, and 3 of 20 (15%) normal prostate tissues. Little or no expression was observed in benign prostate epithelial cell line, but EphB4 was expressed in all prostate cancer cell lines to varying degrees. EphB4 protein levels are high in the PC3 prostate cancer cell line and several folds higher in a metastatic clone of PC3 (PC3M) where overexpression was accompanied by EphB4 gene amplification. EphB4 expression is induced by loss of PTEN, p53, and induced by epidermal growth factor/epidermal growth factor receptor and insulin-like growth factor-I/insulin-like growth factor-IR. Knockdown of the EphB4 protein using EphB4 short interfering RNA or antisense oligodeoxynucleotide significantly inhibits cell growth/viability, migration, and invasion, and induces apoptosis in prostate cancer cell lines. Antisense oligodeoxynucleotide targeting EphB4 in vivo showed antitumor activity in murine human tumor xenograft model. These data show a role for EphB4 in prostate cancer and provide a rationale to study EphB4 for diagnostic, prognostic, and therapeutic applications.

Fibroblast growth factor 8 isoform B overexpression in prostate epithelium: a new mouse model for prostatic intraepithelial neoplasia.

Fibroblast growth factor 8 isoform b (FGF8b), a mitogenic and transforming polypeptide, was demonstrated to be naturally up-regulated in prostatic premalignant and malignant lesions in men. We generated four independent lines of transgenic mice with targeted overexpression of FGF8b in the prostatic epithelium using an improved rat probasin promoter, ARR(2)PB. Transgene expression in the prostate tissue was readily demonstrated by reverse transcription-PCR and localized to the prostatic epithelium by in situ hybridization. The histopathology of the prostate tissues was followed in different age groups of the various lines but most extensively in one line (line 3), starting from 1 month of age up to 24 months. Prostatic hyperplasia appeared in the lateral and ventral prostates in some animals as early as 2-3 months and in other lobes between 6 and 16 months. Beginning at 5-7 months, dysplasia, akin to what may be considered low-grade prostatic intraepithelial neoplasia (LGPIN) in humans, was detected. During the first 14 months, 100% of animals exhibited multifocal epithelial hyperplasia; 35% also had areas of LGPIN. This profile changed in subsequent months (15-24 months) to a higher incidence of LGPIN (66%) along with high-grade PIN (HGPIN) lesions (51%). Similar to HGPIN, stromal proliferation and appearance of papillary hyperplasia with atypia displayed a delayed pattern. The affected stroma consisted primarily of the smooth muscle cell component. The incidence of chronic inflammation, mostly involving T cells, was higher in the prostate of the transgenic mice relative to controls; however, the presence of a direct correlation between inflammation and hyperplasia or preneoplastic lesions was not identified. These transgenic mice represent a "natural" animal model for investigating the mechanism of development and progression of prostatic diseases, such as prostatic hyperplasia and preneoplastic lesions.

Prostatic intraepithelial neoplasia in mice with conditional disruption of the retinoid X receptor alpha allele in the prostate epithelium.

Retinoids, which are important regulators of cell growth, differentiation, and apoptosis, have been used in treatment or chemoprevention of multiple cancers including prostate cancer. To elucidate the mechanism of action of retinoids in the context of the prostate, we used the Cre-loxP system to disrupt the retinoid X receptor alpha (RXRalpha) gene specifically in the prostatic epithelium of the mouse. Evidence for tissue-specific gene inactivation was obtained at DNA, RNA, and protein levels. Phenotypic changes in the prostate in the homozygous animals of different age groups ranging from 1 to 15 months were investigated. Developmentally, prostatic ductal branching appeared to be increased from the loss of RXRalpha function. There was also a significant change in the profile of secretory proteins in the RXRalpha mutant prostate relative to littermate controls with intact RXRalpha allele. Histopathologically, homozygous RXRalpha-deficient prostates showed multifocal hyperplasia as early as 4 months of age. Lesions, which could be described as low-grade prostatic intraepithelial neoplasias, were detected after 5 months. Subsequently, beginning at approximately 10 months, high-grade prostatic intraepithelial neoplasias developed in some animals. The incidences of low-grade prostatic intraepithelial neoplasias and high-grade prostatic intraepithelial neoplasias among the animals 10-15 months of age were 62 and 17%, respectively. The heterozygous mutant mice also developed similar prostatic phenotypes but in a delayed manner, implying a role of haploinsufficiency. Together, these results indicated for the first time that a major component of retinoid action in the prostate is mediated by a retinoid receptor, RXRalpha, the inactivation of which in the prostatic epithelium leads to the development of preneoplastic lesions.

Androgens and prostate cancer: are the descriptors valid?

The term androgen-independent cancer has now become a misnomer. Given that the androgen receptor can be activated by even low androgen concentrations or via protein modifications or other protein-protein interactions, a growing prostate cancer has the chance of assuming an androgen depletion-independent state, without necessarily bypassing the androgen signaling processes. It is thus suggested that "androgen-independent (AI)" cancer should be more accurately termed "androgen depletion-independent (ADI)" cancer.

In vivo expression of GFP transgene delivered via a replicating feline leukemia virus.

We previously described replication-competent feline leukemia virus (FeLV) vectors with high-level and stable expression of a green fluorescent protein (GFP) or a suicide transgene in cell cultures in vitro. Considering that FeLV might potentially be used to deliver therapeutic genes in vivo, we first evaluated the expression of the GFP gene introduced in cats by the FeLV, Rickard subgroup A (FRA) construct. Eight newborn kittens were either inoculated with pFRA-GFP plasmid DNA intradermally, or challenged intraperitoneally with FRA-GFP-infected feline fibroblasts. During a 12-week observation period, five cats were shown to be progressively viremic. Quantitative PCR and RT-PCR analyses of plasma and tissue samples from these cats showed that GFP was retained in FeLV DNA or RNA to a variable degree, ranging from 0.002 to 27.890%. Tissue DNA samples were analyzed by PCR for the status of GFP and the env-transgene complex. While the proviruses carrying the GFP transgene were shown to be minor species, all tissues, however, retained the full-length GFP transgene. Despite the occurrence of predominant species with various deletions in the viral genome, approximately 1-3% of the total cell population was GFP-positive in the lymphoid tissues as visualized by laser confocal microscopy. Co-localization of immunofluorescent cells indicated that CD3-positive T cells, dendritic cells and macrophages were the major targets for GFP expression. These findings on the detectable in vivo expression of GFP for as long as a period of 3 months could be viewed positively for contemplating a therapeutic strategy for control of FeLV infection in the cats.

Identification of Androgen Receptor Splice Variants in the Pten Deficient Murine Prostate Cancer Model.

Androgen receptor (AR) variants are associated with resistance to anti androgen therapy both in human prostate cancer cell lines and clinical samples. These observations support the hypothesis that AR isoform accumulation is a consequence of selective therapeutic pressure on the full length AR. The Pten deficient prostate cancer model proceeds with well-defined kinetics including progression to castration resistant prostate cancer (CRPC). While surgical castration and enzalutamide treatments yield an initial therapeutic response, Pten-/-epithelia continue to proliferate yielding locally invasive primary tumor pathology. That most epithelium remains AR positive, but ligand independent, suggests the presence of oncogenic AR variants. To address this hypothesis, we have used a panel of recently described Pten-/- tumor cell lines derived from both from hormone intact (E4, E8) and castrated Pten mutants (cE1, cE2) followed by RACE PCR to identify and characterize three novel truncated, amino terminus containing AR variants (mAR-Va, b, c). Variants appear not only conserved throughout progression but are correlated with nearly complete loss of full length AR (AR-FL) at castrate androgen levels. The overexpression of variants leads to enhanced transcriptional activity of AR while knock down studies show reduced transcriptional output. Collectively, the identification of truncated AR variants in the conditional PTEN deletion model supports a role for maintaining the CRPC phenotype and provides further therapeutic applications of this preclinical model.