The EZC-prostate model: noninvasive prostate imaging in living mice

Recently, progress in the development of prostate-specific promoters and high resolution imaging techniques has made real-time monitoring of transgenic expression possible, opening a vista of potentially important in vivo models of prostate disease. Herein, we describe a novel prostate reporter model, called the EZC-prostate model that permits both ex vivo and in vivo imaging of the prostate using a sensitive charge-coupled device. Firefly luciferase and enhanced green fluorescent protein were targeted to the prostate epithelium using the composite human kallikrein 2 (hK2)-based promoter, hK2-E3/P. In EZC-prostate mice, the ventral and dorsal/lateral prostate lobes were brilliant green under fluorescence microscopy, with expression localized to the secretory epithelium. In contrast, enhanced green fluorescent protein was undetectable in the anterior lobes of prostate, seminal vesicles, testes, liver, lung, and brain. The kinetics of luciferase activity in intact and castrated living mice monitored with the IVIS charge-coupled device-based imaging system confirmed that firefly luciferase expression was largely prostate restricted, increased with age up to 24 wk, and was androgen dependent. Decreases in reporter expression after 24 wk may reflect well known, age-related decreases in androgen signaling with age in humans. Ex vivo imaging of microdissected animals further confirmed that the luminescence detected in living mice emanated predominately from the prostate, with minor signals originating from the testes and cecum. These data demonstrate that the hK2-E3/P promoter directs strong prostate-specific expression in a transgenic mouse model. Multigenic models, generated by crosses with various hyperplastic and neoplastic prostate disease models, could potentially provide powerful new tools in longitudinal monitoring of changes in prostate size, androgen signaling, metastases, or response to novel therapies without sacrificing large cohorts of animals.     

The discovery of a potent orally efficacious indole androgen receptor antagonist through in vivo screening

A series of novel 2-(1H-indol-2-yl)-propan-2-ols have been designed, synthesized, and screened for their ability to inhibit testosterone-induced prostate weight increases in immature rats. Through the use of this paradigm, we were able to identify compounds that exhibited in vivo potency equal to that of the marketed antiandrogen Casodex when orally administered.     

LncRNA PCBP1-AS1-mediated AR/AR-V7 deubiquitination enhances prostate cancer enzalutamide resistance

The refractory of castration-resistant prostate cancer (CRPC) is mainly reflected in drug resistance. The current research on the resistance mechanism of CRPC is still in its infancy. In this study, we revealed for the first time the key role of LncRNA PCBP1-AS1 in CRPC drug resistance. Through detailed in vivo and in vitro studies, we found that PCBP1-AS1 may enhance the deubiquitination of AR/AR-V7 by stabilizing the USP22-AR/AR-V7 complex, thereby preventing AR/AR-V7 from being degraded through the ubiquitin–proteasome pathway. Targeting PCBP1-AS1 can significantly restore the drug sensitivity of enzalutamide-resistant tumors in vivo and in vitro. Our research further expands the function of LncRNA in castration-resistant prostate cancer, which may provide new potential for clinical diagnosis and targeted therapy.Subject terms: Prostate cancer, Ubiquitylation, Long non-coding RNAs     

Caveolin-1 promotes tumor progression in an autochthonous mouse model of prostate cancer: genetic ablation of Cav-1 delays advanced prostate tumor development in tramp mice

Caveolin-1 (Cav-1) is the primary structural component of caveolae and is implicated in the processes of vesicular transport, cholesterol balance, transformation, and tumorigenesis. Despite an abundance of data suggesting that Cav-1 has transformation suppressor properties both in vitro and in vivo, Cav-1 is expressed at increased levels in human prostate cancer. To investigate the role of Cav-1 in prostate cancer onset and progression, we interbred Cav-1(-/-) null mice with a TRAMP (transgenic adenocarcinoma of mouse prostate) model that spontaneously develops advanced prostate cancer and metastatic disease. We found that, although the loss of Cav-1 did not affect the appearance of minimally invasive prostate cancer, its absence significantly impeded progression to highly invasive and metastatic disease. Inactivation of one (+/-) or both (-/-) alleles of Cav-1 resulted in significant reductions in prostate tumor burden, as well as decreases in regional lymph node metastases. Moreover, further examination revealed decreased metastasis to distant organs, such as the lungs, in TRAMP/Cav-1(-/-) mice. Utilizing prostate carcinoma cell lines (C1, C2, and C3) derived from TRAMP tumors, we also showed a positive correlation between Cav-1 expression and the ability of these cells to form tumors in vivo. Furthermore, down-regulation of Cav-1 expression in these cells, using a small interfering RNA approach, significantly reduced their tumorigenic and metastatic potential. Mechanistically, we showed that loss or down-regulation of Cav-1 expression results in increased apoptosis, with increased prostate apoptosis response factor-4 and PTEN levels in Cav-1(-/-) null prostate tumors. Our current findings provide the first in vivo molecular genetic evidence that Cav-1 does indeed function as a tumor promoter during prostate carcinogenesis, rather than as a tumor suppressor.     

Effects of hyperprolactinemia on rat prostate growth: evidence of androgeno-dependence

The effects of the polypeptide hormone prolactin (PRL) in the development and regulation of benign prostate hyperplasia (BPH) and also in prostate cancer are not very well characterized. This study examines the action of PRL, either alone or in association with androgens [testosterone (T) or dihydrotestosterone (DHT)], in the rat prostate gland. The effects of PRL and androgens were investigated after 30 and 60 days in control, castrated, castrated with a substitutive implant of T or DHT, and sham-operated Wistar rats. To enhance PRL release, we induced hyperprolactinemia by administering chronic injections of sulpiride (40 mg. kg(-1). day(-1)). Chronic hyperprolactinemia induces enlargement and inflammation of the lateral rat prostate without any histological changes on ventral and dorsal lobes. We also demonstrate that hyperprolactinemia induces Bcl-2 overexpression in the lateral rat prostate and that this could inhibit the level of apoptosis. The in vivo model established here is a useful in vivo approach for studying the hormonal regulation of normal and pathological prostate development.     

MYC cooperates with AKT in prostate tumorigenesis and alters sensitivity to mTOR inhibitors

MYC and phosphoinositide 3-kinase (PI3K)-pathway deregulation are common in human prostate cancer. Through examination of 194 human prostate tumors, we observed statistically significant co-occurrence of MYC amplification and PI3K-pathway alteration, raising the possibility that these two lesions cooperate in prostate cancer progression. To investigate this, we generated bigenic mice in which both activated human AKT1 and human MYC are expressed in the prostate (MPAKT/Hi-MYC model). In contrast to mice expressing AKT1 alone (MPAKT model) or MYC alone (Hi-MYC model), the bigenic phenotype demonstrates accelerated progression of mouse prostate intraepithelial neoplasia (mPIN) to microinvasive disease with disruption of basement membrane, significant stromal remodeling and infiltration of macrophages, B- and T-lymphocytes, similar to inflammation observed in human prostate tumors. In contrast to the reversibility of mPIN lesions in young MPAKT mice after treatment with mTOR inhibitors, Hi-MYC and bigenic MPAKT/Hi-MYC mice were resistant. Additionally, older MPAKT mice showed reduced sensitivity to mTOR inhibition, suggesting that additional genetic events may dampen mTOR dependence. Since increased MYC expression is an early feature of many human prostate cancers, these data have implications for treatment of human prostate cancers with PI3K-pathway alterations using mTOR inhibitors.     

2-Methoxyestradiol induces G2/M arrest and apoptosis in prostate cancer

Few therapeutic treatment options are available for patients suffering from metastatic androgen-independent prostate cancer. We investigated the ability of the estrogen metabolite 2-methoxyestradiol to inhibit the proliferation of a variety of human prostate cancer cell lines in vitro and to inhibit the growth of androgen-independent prostate cancer in a transgenic mouse model in vivo. Our results showed that 2-methoxyestradiol is a powerful growth inhibitor of LNCaP, DU 145, PC-3, and ALVA-31 prostate cancer cells. Cell flow cytometry of 2-methoxyestradiol-treated DU 145 cells showed a marked accumulation of cells in the G2/M phase of the cell cycle and an increase in the sub-G1 fraction (apoptotic). In addition, staining for annexin V, changes in nuclear morphology, and inhibition of caspase activity support a role for apoptosis. More importantly, we showed that 2-methoxyestradiol inhibits prostate tumor progression in the Ggamma/T-15 transgenic mouse model of androgen-independent prostate cancer without toxic side effects. These results in cell culture and an animal model support investigations into the clinical use of 2-methoxyestradiol in patients with androgen-independent prostate cancer.     

Design and synthesis of an androgen receptor pure antagonist (CH5137291) for the treatment of castration-resistant prostate cancer

A series of 5,5-dimethylthiohydantoin derivatives were synthesized and evaluated for androgen receptor pure antagonistic activities for the treatment of castration-resistant prostate cancer. Since CH4933468, which we reported previously, had a problem with agonist metabolites, novel thiohydantoin derivatives were identified by applying two strategies. One was the replacement of the alkylsulfonamide moiety by a phenylsulfonamide to avoid the production of agonist metabolites. The other was the replacement of the phenyl ring with a pyridine ring to improve in vivo potency and reduce hERG affinity. Pharmacological assays indicated that CH5137291 (17b) was a potent AR pure antagonist which did not produce the agonist metabolite. Moreover, CH5137291 completely inhibited in vivo tumor growth of LNCaP-BC2, a castration-resistant prostate cancer model.     

In vitro deregulation of markers characteristic of human prostate epithelial cells

We have screened primary cultures of human prostate for the expression of markers reported to be characteristic of specific cell lineages in vivo, in order to ascertain whether human prostate cells in vitro maintain and reflect their in vivo differentiated phenotypes and to evaluate the homogeneity of the populations of cells that can be derived from this tissue. Using single and dual stain immunofluorescent microscopy to analyse very early organoid and subsequently derived monolayer stage cultures, we have observed that expression of markers characteristic of human prostate epithelial cells in vivo is deregulated within 48h, indicating that dissociation of human prostate tissue and cultivation of prostate epithelial cells in culture can result in promiscuous expression of cell type specific markers of prostate epithelial cells. These observations have important implications for studies of cell lineage and differentiation of prostate cells in vitro.     

紫杉醇抑制前列腺癌增殖的体内实验研究

目的探讨紫杉醇对人前列腺癌细胞PC-3增殖的体内抑制作用。方法建立体内绿色荧光蛋白（GFP）标记的人雄激素非依赖性前列腺癌细胞PC-3裸鼠原位移植瘤模型,观察紫杉醇对裸鼠前列腺癌原位移植瘤的体积、重量的影响。结果裸鼠模型体内实验显示,与对照组（100μL生理盐水）相比,紫杉醇处理组（0.5 mg/kg）在给药第18天后能显著抑制前列腺肿瘤的体积（P〈0.05）;紫杉醇处理组在抑制前列腺肿瘤重量方面与对照组相比亦有明显抑制作用（P〈0.05）。与对照组相比G31P处理组VEGF（P〈0.05）的表达差异具有统计学意义（免疫组化法）。结论紫杉醇在体内实验中能明显抑制人雄激素非依赖性前列腺癌细胞系PC-3的增殖。     

Identification of CD166 as a Surface Marker for Enriching Prostate Stem/Progenitor and Cancer Initiating Cells

New therapies for late stage and castration resistant prostate cancer (CRPC) depend on defining unique properties and pathways of cell sub-populations capable of sustaining the net growth of the cancer. One of the best enrichment schemes for isolating the putative stem/progenitor cell from the murine prostate gland is Lin(-);Sca1(+);CD49f(hi) (LSC(hi)), which results in a more than 10-fold enrichment for in vitro sphere-forming activity. We have shown previously that the LSC(hi) subpopulation is both necessary and sufficient for cancer initiation in the Pten-null prostate cancer model. To further improve this enrichment scheme, we searched for cell surface molecules upregulated upon castration of murine prostate and identified CD166 as a candidate gene. CD166 encodes a cell surface molecule that can further enrich sphere-forming activity of WT LSC(hi) and Pten null LSC(hi). Importantly, CD166 could enrich sphere-forming ability of benign primary human prostate cells in vitro and induce the formation of tubule-like structures in vivo. CD166 expression is upregulated in human prostate cancers, especially CRPC samples. Although genetic deletion of murine CD166 in the Pten null prostate cancer model does not interfere with sphere formation or block prostate cancer progression and CRPC development, the presence of CD166 on prostate stem/progenitors and castration resistant sub-populations suggest that it is a cell surface molecule with the potential for targeted delivery of human prostate cancer therapeutics.     

The use of genetically engineered mouse models of prostate cancer for nutrition and cancer chemoprevention research

The ability to modify the expression of specific genes in the mouse through genetic engineering technologies allows for the generation of previously unavailable models for prostate cancer prevention research. Although animal models have existed for some time for the study of prostate cancer prevention (primarily in the rat), it is uncertain if the mechanisms that drive prostate carcinogenesis in these models are relevant to those in human prostate cancer. Cell culture studies are of limited usefulness because the conditions are inherently artificial. Factors such as relevant physiologic concentrations and metabolism of putative chemoprevention compounds are difficult to model in an in vitro system. These studies also preclude the types of interactions known to occur between multiple cell types in vivo. In addition, all prostate cancer cell lines are already highly progressed and are not representative of the type of cells to which most preventive strategies would be targeted. Due to the advent of genetically engineered mouse (GEM) models, we now have models of prostate cancer that are dependent on molecular mechanisms already implicated in human prostate carcinogenesis. With these models we can perform a variety of experiments that could previously only be done in cell culture or in prostate cancer cell line xenografts. The currently available GEM models of prostate cancer have been extensively reviewed therefore, this review will focus on the types of models available and their usefulness for various types of preclinical studies relevant to prostate cancer prevention.     

Maspin is an angiogenesis inhibitor

Maspin, a unique member of the serpin family, is a secreted protein encoded by a class II tumor suppressor gene whose downregulation is associated with the development of breast and prostate cancers. Overexpression of maspin in breast tumor cells limits their growth and metastases in vivo. In this report we demonstrate that maspin is an effective inhibitor of angiogenesis. In vitro, it acted directly on cultured endothelial cells to stop their migration towards basic fibroblast growth factor and vascular endothelial growth factor and to limit mitogenesis and tube formation. In vivo, it blocked neovascularization in the rat cornea pocket model. Maspin derivatives mutated in the serpin reactive site lost their ability to inhibit the migration of fibroblasts, keratinocytes, and breast cancer cells but were still able to block angiogenesis in vitro and in vivo. When maspin was delivered locally to human prostate tumor cells in a xenograft mouse model, it blocked tumor growth and dramatically reduced the density of tumor-associated microvessels. These data suggest that the tumor suppressor activity of maspin may depend in large part on its ability to inhibit angiogenesis and raise the possibility that maspin and similar serpins may be excellent leads for the development of drugs that modulate angiogenesis.     

Ectopical expression of human MUC18 increases metastasis of human prostate cancer cells

MUC18, a cell adhesion molecule (CAM), has been reported to be a diagnostic marker for the early detection of the metastatic potential of prostate cancers as well as implicated to be an important determinant for mediating the tumorigenesis and metastasis of prostate cancer. To test the hypothesis, we further investigated the possible role of MUC18 in the malignant progression of human prostate cancer. The human MUC18-minus, non-metastatic human prostate cancer LNCaP cells were transfected with the human cytomegalovirus immediate-early gene (HCMV-IE) promoter-driven human MUC18 (huMUC18) cDNA. The G418-resistant (G418R)-LNCaP clones that expressed a high level of huMUC18 were selected and used for testing the effect of huMUC18 expression on the in vitro growth, motility, and invasiveness as well as on the in vivo metastasis (via orthotopical injection) in a xenograft nude mouse model. HuMUC18 expression increased by four- to fivefold of in vitro motility and invasiveness of LNCaP cells. Anti-huMUC18 antibody significantly inhibited the in vitro motility and invasiveness of huMUC18-expressing LNCaP clones, but not the control clones. We suggest that huMUC18 expression is responsible for increasing these behaviors of LNCaP cells. HuMUC18 expression also directly increased the in vivo metastatic abilities of the LNCaP cells from the prostate gland to multiple distant organs. Western blot and immunohistochemistry analyses showed that the prostatic tumors as well as metastatic lesions expressed high levels of MUC18, indicating that they originated from the injected huMUC18-expressing LNCaP cells. We therefore conclude that HuMUC18 is an important determinant in increasing metastasis of human prostate cancer LNCaP cells to distant organs in a nude mouse model.     

Mechanisms for 2-methoxyestradiol-induced apoptosis of prostate cancer cells

Prostate and breast carcinomas are sex hormone-related carcinomas, which are known to be associated with an over-expression of the proto-oncogene Bcl-2. Here, we report that 2-methoxyestradiol (2-ME), an endogenous metabolite of estrogen that does not bind to nuclear estrogen receptors, effectively induces apoptosis in Bcl-2-expressing human prostate and breast carcinoma cells in vitro and in a rat prostate tumor model in vivo. In several cell lines derived from prostate, breast, liver and colorectal carcinomas, 2-ME treatment led to an activation of c-Jun N-terminal kinase (JNK) and phosphorylation of Bcl-2, which preceded the induction of apoptosis. In summary, our data suggest that 2-ME induces apoptosis in epithelial carcinomas by causing phosphorylation of JNK, which appears to be correlated with phosphorylation of Bcl-2.     

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

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

Resveratrol–zinc combination for prostate cancer management

Zinc, an essential trace element, plays a critical role in cell signaling, and defect(s) in zinc homeostasis may contribute to adverse physiological and pathological conditions, including cancer. Zinc is present in healthy prostate at a very high concentration, where it is required for important prostatic functions. However, zinc levels are significantly diminished in cancerous tissue, and intracellular zinc level is inversely correlated with prostate cancer progression. During neoplastic transformation, zinc-accumulating, citrate-producing normal prostate cells are metabolically transformed to citrate oxidizing cells that lose the ability to accumulate zinc. Interestingly, zinc has been shown to function as chemopreventive agent against prostate cancer, albeit at high doses, which may lead to many adverse effects. Therefore, novel means to enhance bioaccumulation of sufficient zinc in prostate cells via increasing zinc transport could be useful against prostate cancer. On the basis of available evidence, we present a possibility that the grape antioxidant resveratrol, when given with zinc, may lead to retuning the zinc homeostasis in prostate, thereby abolishing or reversing malignancy. If experimentally verified in in vivo model(s) of prostate cancer, such as transgenic mouse models, this may lead to novel means toward management of prostate cancer and other conditions with compromised zinc homeostasis.