cAMP-responsive element-binding protein regulates vascular endothelial growth factor expression: implication in human prostate cancer bone metastasis.

Aberrant expression of vascular endothelial growth factor (VEGF) is associated with human prostate cancer (PCa) metastasis and poor clinical outcome. We found that both phosphorylation of cyclic AMP-responsive element-binding protein (CREB) and VEGF levels were significantly elevated in patient bone metastatic PCa specimens. A PCa ARCaP progression model demonstrating epithelial-to-mesenchymal transition exhibited increased CREB phosphorylation and VEGF expression as ARCaP cells became progressively more mesenchymal and bone-metastatic. Activation of CREB induced, whereas inhibition of CREB blocked, VEGF expression in ARCaP cells. CREB may regulate VEGF transcription via a hypoxia-inducible factor-dependent mechanism in normoxic conditions. Activation of CREB signaling is involved in the coordinated regulation of VEGF and may pre-dispose to PCa bone metastasis.

Quantum dots-based multiplexed immunohistochemistry of protein expression in human prostate cancer cells.

Semiconductor quantum dots (QDs) are bright fluorescent nanoparticles that have been successfully used for the detection of biomarker expression in cells. The objective of the present study is to use this technology in a multiplexing manner to determine at a single cell level the expression of a cell-specific bio-marker, prostate-specific antigen (PSA) expressed by human prostate cancer LNCaP and ARCaP cell lines. Here we compared the sensitivity of immunohistochemistry (IHC) and QD-based detection of AR and PSA expression in these cell lines. Further, we conducted multiplexing QD-based detection of PSA and androgen receptor (AR) expression in LNCaP cells subjecting to androgen (R1881) stimulation. The involvement of AR in PSA regulation in LNCaP cells, at a single cell level, was confirmed by the co-incubation of LNCaP cells in the presence of both R1881 and its receptor antagonist, bicalutamide (Casodex). We showed here the superior quality of QDs, in comparison to IHC, for the detection of AR and PSA in cultured LNCaP and ARCaP cells. Multiplexing QDs technique can be used to detect simultaneously AR and PSA expression induced by R1881 which promoted AR translocation from its cytosolic to the nuclear compartment. We observed AR antagonist, bicalutamide, inhibited AR nuclear translocation and PSA, but not AR expression in LNCaP cells.

Androgen receptor mediates the reduced tumor growth, enhanced androgen responsiveness, and selected target gene transactivation in a human prostate cancer cell line.

The growth and development of the prostate gland are regulated by the androgen and the androgen receptor (AR). Despite our molecular understanding of the roles of the AR regulating; a downstream target gene transcription, the direct or indirect (stromally mediated) actions of the androgen in controlling prostate cell growth and differentiation are still unclear. In this report, an invasive; and metastatic human prostate tumor cell line, androgen-repressed human prostate cancer cell line (ARCaP), either transduced with wild-type human AR (hAR) or a control neomycin-resistant plasmid DNA, was used to evaluate the direct role of AR in regulating prostate tumor cell growth and gene transcription. Results showed that: (a) introduction of wild-type hAR to ARCaP cells restored positive androgen regulation of prostate tumor cell growth in vitro through an enhanced cell-cycle progression from G(0)/G(1) to S and G(2)-M phases; (b) hAR was shown to transactivate glucocorticoid-responsive element but not prostate-specific antigen promoter-directed reporter gene expression; and (c) hAR-transduced ARCaP cells exhibited reduced growth, invasion, and migratory behavior in vitro and tumor growth in vivo. These results suggest that the introduction of hAR into the invasive human prostate cancer ARCaP cell line restored its androgen-regulated cell growth, decreased the rate of tumor growth, and selectively activated AR target gene expression. These cellular functions in response to androgen are commonly associated with increased differentiation of prostate epithelial cells.

Epidermal growth factor receptor-mediated autocrine and paracrine stimulation of human transitional cell carcinoma.

Stromal-epithelial interactions may play a key role in tumor growth and metastasis. We have established a model to study the cellular and molecular basis of this paracrine interaction both in vivo and in vitro using a human transitional cell carcinoma cell line (WH). s.c. coinoculation of 1 x 10(6) WH cells with 1 x 10(6) nontumorigenic fetal rat urogenital sinus mesenchymal (rUGM) cells in athymic mice accelerated carcinoma growth 20 times faster than isolated WH cell inoculations and 4 times faster than coinoculations of the same number of NIH-3T3 or human bladder fibroblasts. Characterization of these chimeric tumors with immunohistochemical and DNA dot-blot analyses documented their predominantly human component. To evaluate the underlying mechanisms involved in this paracrine-mediated in vivo tumor growth acceleration, Northern analyses for growth factors (GFs) and extracellular matrix (ECM) expression in the different cell lines, as well as in vitro mitogenic assays, were performed. Northern analysis revealed basic fibroblast growth factor, transforming growth factor alpha, and epidermal growth factor receptor expression by WH cells but not rUGM cells; ECM components (fibronectin and collagens I and IV) were expressed only in the fibroblast cell lines. Cell type-specific paracrine growth factors are produced by cultured stromal and epithelial cells with a 2-3-fold bidirectional increase in WH and rUGM cell growth when cultured with reciprocal cell-type conditioned medium. An autocrine growth loop was observed for WH but not rUGM cells. WH cell growth is stimulated in vitro by low concentrations of transforming growth factor alpha and epidermal growth factor, while rUGM cell growth is stimulated 3-fold by basic fibroblast growth factor. Antiepidermal growth factor receptor antibodies completely inhibited autocrine and paracrine pathways stimulating WH cell growth, while anti-basic fibroblast growth factor antibodies had no inhibitory effect. These observations suggest that autocrine and paracrine growth factor stimulation of WH bladder carcinoma cell growth is most likely mediated by an epidermal growth factor receptor-related pathway. The predominant expression of ECM by fibroblasts in this model suggests that stromal cell ECM components may modulate tumor cell growth and angiogenesis possibly through mechanisms involving cellular adhesion, chemotaxis, or growth factor action.

Androgen-independent cancer progression and bone metastasis in the LNCaP model of human prostate cancer.

Our laboratory has previously reported on the derivation of LNCaP cell sublines from LNCaP tumors maintained in castrated and intact athymic male mice. These LNCaP sublines differ from the parental line in tumorigenicity and androgen dependence. This paper demonstrates that one of these sublines acquired metastatic potential. When inoculated either s.c. or orthotopically, the C4-2 subline metastasized to the lymph node and bone with an incidence of 11-50%. Interestingly, the incidence of osseous metastasis was higher in castrated than in intact male hosts. We evaluated the chromosomal, immunohistochemical, and biochemical characteristics of the LNCaP sublines derived from C4-2 tumors that metastasized to the lymph node and bone. Cytogenetic analysis showed that all sublines were human and shared common marker chromosomes with the parental LNCaP cells. This experimental human prostate cancer model may permit, for the first time, the study of the molecular mechanisms underlying human prostate cancer metastasis.

A conditional replication-competent adenoviral vector, Ad-OC-E1a, to cotarget prostate cancer and bone stroma in an experimental model of androgen-independent prostate cancer bone metastasis.

Prostate cancer has a high propensity to metastasize to bone, which often resists hormone, radiation, and chemotherapies. Because of the reciprocal nature of the prostate cancer and bone stroma interaction, we designed a cotargeting strategy using a conditional replication-competent adenovirus to target the growth of tumor cells and their associated osteoblasts. The recombinant Ad-OC-E1a was constructed using a noncollagenous bone matrix protein osteocalcin (OC) promoter to drive the viral early E1a gene with restricted replication in cells that express OC transcriptional activity. Unlike Ad-PSE-E1a, Ad-OC-E1a was highly efficient in inhibiting the growth of PSA-producing (LNCaP, C4-2, and ARCaP) and nonproducing (PC-3 and DU145) human prostate cancer cell lines. This virus was also found to effectively inhibit the growth of human osteoblasts and human prostate stromal cells in vitro. Athymic mice bearing s.c. androgen receptor-negative and PSA-negative PC-3 xenografts responded to a single intratumoral administration of 2 x 10(9) plaque-forming unit(s) of Ad-OC-E1a. In SCID/bg mice, intraosseous growth of androgen receptor-positive and PSA-producing C4-2 xenografts responded markedly to i.v. administrations of a single dose of Ad-OC-E1a. One hundred percent of the treated mice responded to this systemic Ad-OC-E1a therapy with a decline of serum PSA to an undetectable level, and 80% of the mice with PSA rebound responded to the second dose of systemic Ad-OC-E1a. Forty percent of the mice were found to be cured by systemic Ad-OC-E1a without subsequent PSA rebound or tumor cells found in the skeleton. This cotargeting strategy shows a broader spectrum and appears to be more effective than systemic Ad-PSE-E1a in preclinical models of human prostate cancer skeletal metastasis.

Identification of a novel FAS/ER-alpha fusion transcript expressed in human cancer cells.

A semi-nested reverse transcriptase-polymerase chain reaction (RT-PCR) was applied to evaluate the presence of estrogen receptor-alpha (ER-alpha) in human prostate cancer cells. Unexpectedly, a novel fatty acid synthase (FAS)/ER-alpha fusion transcript was identified, in which the N-terminus of FAS was fused in-frame with the C-terminus of ER-alpha. The existence of the FAS/ER-alpha transcript was further confirmed by RT-PCR analysis using various sets of amplification primers and different reverse-transcribed primers in the presence of dimethyl sulfoxide to eliminate the secondary structure of RNA. The predicted FAS/ER-alpha protein would contain largely domain I of FAS and the entire ligand binding domain of ER-alpha. The FAS/ER-alpha was expressed in a variety of human cancer cell lines including prostate, breast, cervical and bladder cancer cell lines. Our data suggest that the presence of FAS/ER-alpha may complicate the FAS and the ER-alpha signalling pathway.

Stromal fibroblast-derived miR-409 promotes epithelial-to-mesenchymal transition and prostate tumorigenesis.

Tumor-stromal interaction is a dynamic process that promotes tumor growth and metastasis via cell-cell interaction and extracellular vesicles. Recent studies demonstrate that stromal fibroblast-derived molecular signatures can be used to predict disease progression and drug resistance. To identify the epigenetic role of stromal noncoding RNAs in tumor-stromal interactions in the tumor microenvironment, we performed microRNA profiling of patient cancer-associated prostate stromal fibroblasts isolated by laser capture dissection microscopy and in bone-associated stromal models. We found specific upregulation of miR-409-3p and miR-409-5p located within the embryonically and developmentally regulated DLK1-DIO3 (delta-like 1 homolog-deiodinase, iodothyronine 3) cluster on human chromosome 14. The findings in cell lines were further validated in human prostate cancer tissues. Strikingly, ectopic expression of miR-409 in normal prostate fibroblasts conferred a cancer-associated stroma-like phenotype and led to the release of miR-409 via extracellular vesicles to promote tumor induction and epithelial-to-mesenchymal transition in vitro and in vivo. miR-409 promoted tumorigenesis through repression of tumor suppressor genes such as Ras suppressor 1 and stromal antigen 2. Thus, stromal fibroblasts derived miR-409-induced tumorigenesis, epithelial-to-mesenchymal transition and stemness of the epithelial cancer cells in vivo. Therefore, miR-409 appears to be an attractive therapeutic target to block the vicious cycle of tumor-stromal interactions that plagues prostate cancer patients.

Molecular therapy with recombinant p53 adenovirus in an androgen-independent, metastatic human prostate cancer model.

The lethal phenotypes of advanced prostate cancer are androgen independent (AI) and metastatic to the axial skeleton. Our laboratory has developed an AI mouse model of metastatic human prostate cancer. In this communication, we report the development of tumor suppressor gene therapy in this AI and metastatic (C4-2) cancer model. By using recombinant adenovirus as a delivery vehicle, we introduced a wild-type p53 tumor suppressor gene into prostate cancer cell lines. Despite a silent mutation at codon 152 of the p53 gene, C4-2 cells express functional, but low, levels of p53 protein. However, the other prostatic cell lines, PC-3 and DU145, have a deletion mutation and two point mutations of the p53 gene, respectively. In vitro studies showed that cell growth, as measured by the thymidine incorporation assay, was inhibited in the C4-2, PC-3, and DU145 cells infected with wild-type p53 adenovirus in comparison to control viruses. Recombinant wild-type p53 adenovirus inhibited prostate tumor growth and its production of prostate-specific antigen (PSA) when injected into C4-2 tumors in nude mice. All p53-treated mice were tumor free as long as 12 weeks after cessation of the 8-week treatment regimen. Two of 8 p53-treated mice developed small tumors growing at distant sites after a prolonged period of follow-up observation. Moreover, other AI prostate cancer cells, PC-3 and DU145, treated with Ad5-CMV-p53 failed to develop into tumors in vivo. This gene therapy strategy may be used against AI prostatic cancer regardless of p53 gene mutation status.

Identification and characterization of estrogen receptor variants in prostate cancer cell lines.

A sensitive semi-nested reverse transcriptase-polymerase chain reaction (RT-PCR) amplification was performed to evaluate estrogen receptor-alpha (ER-alpha) mRNA expression in prostate cancer cell lines. We demonstrated the presence of wild-type ER-alpha (wt ER-alpha) and five ER-alpha variants, designated ER-alphaA, B, C, D, and E. Unlike ER-alphaA and D, ER-alphaB, C, and E were not previously reported in normal or cancerous mammalian cells. DNA sequencing analysis of these ER-alpha variants revealed the genetic changes to be either in-frame or out-of-frame deletions. The expression of each ER-alpha variant differs significantly depending on the androgen responsiveness, tumorigenic and metastatic potentials of each prostate cancer cell line. The potential functional significance of ER-alpha variants was assessed in yeast two-hybrid and ERE promoter-reporter mammalian transcription assay systems. The results of these studies indicated that none of the ER-alpha variants can form homo- or heterodimers either with wt ER-alpha or among themselves in vivo, and that these ER-alpha variants have no demonstrable transcriptional or dominant-negative activity, as assessed in vitro.

Comparative studies of prostate cancers among United States, Chinese, and Japanese patients: characterization of histopathology, tumor angiogenesis, neuroendocrine factors, and p53 protein accumulations.

Although interracial differences of prostate cancer progression are well recognized, their underlying molecular and cellular mechanisms remain obscure. We compared the histopathologic, immunohistochemical, and molecular characteristics of unselected prostate cancer tissues obtained from U.S., Chinese, and Japanese men. Histopathologic analyses indicated that 74.4% of the prostate cancers in Chinese men were poorly differentiated, compared with 28.6% and 32.8% of the prostate cancers in U.S. and Japanese men, respectively. These differences cannot be attributed to patient age, clinical stage of disease, or methods of tissue sampling. The high proportion of poorly differentiated prostate cancer tissues in the Chinese group was not related to the patients' access to medical service or to geographic background within China. Significantly higher levels of tumor angiogenesis (2- to 4-fold), serotonin (2- to 20-fold), and bombesin (7- to 16-fold), but not chromogranin A, were found in the tissue specimens obtained from Chinese prostate cancer patients compared with those from U.S. and Japanese patients. We also observed marked interracial differences in p53 protein accumulation. The protein was present in 90.2% of Chinese specimens; 17.4% of specimens from U.S. whites; 7.1% of specimens from Japanese men; and 3.7% of specimens from U.S. blacks. Results from multivariate logistic regression analysis demonstrated that p53 protein accumulation, angiogenesis, and serotonin expression in the normal stroma area correlate independently with Chinese versus non-Chinese patient populations.

Establishment of a three-dimensional human prostate organoid coculture under microgravity-simulated conditions: evaluation of androgen-induced growth and PSA expression.

A novel in vitro human prostate cancer model was established by using a coculture technique in which isolated human prostate fibroblasts were observed to grow as a mixed culture with isolated human prostate cancer cells (LNCaP) on microcarrier beads under microgravity-simulated conditions. This model appears to be promising and deserves further exploration because: (a) cocultured human prostate fibroblasts and cancer epithelial cells appear to undergo patterns of histogenesis similar to those observed in human prostate tumors and (b) unlike the conventional cell culture on plastic dishes, cocultured human prostate fibroblasts and LNCaP cells in microgravity-simulated conditions responded to the inductive signals of growth and differentiation from dihydrotestosterone in a manner similar to that observed in the in vivo condition. These results offer an opportunity to examine molecular mechanisms of cellular signaling in response to androgen stimulation during normal and aberrant human prostate development. The microgravity-simulated three-dimensional prostate epithelial cell culture with prostate fibroblasts can be further explored as an ideal in vitro model for the study of normal and neoplastic prostate development. This model could also be adopted as a drug screening program for the discovery of novel therapeutic agents in the treatment of human prostate cancer and benign hyperplastic growth.

Permanent phenotypic and genotypic changes of prostate cancer cells cultured in a three-dimensional rotating-wall vessel.

A three-dimensional (3D) integrated rotating-wall vessel cell-culture system was used to evaluate the interaction between a human prostate cancer cell line, LNCaP, and microcarrier beads alone, or microcarrier beads previously seeded with either prostate or bone stromal cells. Upon coculture of LNCaP cells with microcarrier beads either in the presence or in the absence of prostate or bone stromal cells, 3D prostate organoids were formed with the expected hormonal responsiveness to androgen, increased cell growth, and prostate-specific antigen production. In this communication, we define permanent phenotypic and genotypic changes of LNCaP cells upon coculture with microcarrier beads alone, or with microcarrier beads previously seeded with either prostate or bone stromal cells. Most notably, we observed selective genetic changes, i.e., chromosomal losses or gains, as evaluated by both conventional cytogenetic and comparative genomic hybridization, in LNCaP sublines derived from the prostate organoids. Moreover, the derivative LNCaP cells appear to have altered growth profiles, and exhibit permanent and stable changes in response to androgen, estrogen, and growth factors. The derivative LNCaP sublines showed increased anchorage-independent growth rate, and enhanced tumorigenicity and metastatic potential when inoculated orthotopically in castrated athymic mice. Our results support the hypothesis that further nonrandom genetic and phenotypic changes in prostate cancer epithelial cells can occur through an event that resembles "adaptive mutation" such as has been described in bacteria subjected to nutritional starvation. The occurrence of such permanent changes may be highly contact dependent, and appears to be driven by specific microenvironmental factors surrounding the tumor cell epithelium grown as 3D prostate organoids.

Human prostate cancer progression models and therapeutic intervention.

Our laboratory has developed two cellular models of human prostate cancer progression. The LNCaP prostate cancer progression model is based upon the well-known cellular interaction between human prostate or bone stromal cells and LNCaP cells in vivo. The marginally tumorigenic LNCaP cells acquired tumorigenic and metastatic potential upon cellular interaction with either prostate or bone fibroblasts. A subline termed C4-2 was observed to grow readily in castrated animals and acquired metastatic potential spreading from the primary tumor site to the lymph node, the seminal vesicles, and the axial skeleton, resulting in an intense osteoblastic reaction. The second model is ARCaP, where prostate cancer cells derived from the ascites fluid of a man with metastatic disease exhibited an Androgen- and estrogen-Repressed Prostate Cancer cell growth and tumor formation in either a hormone-deficient or a castrated environment. However, the growth of either the tumor cells in vitro or the tumors in vivo was suppressed by both estrogen and androgen. While the tumor cells expressed low levels of androgen receptor and prostate-specific antigen (PSA), they were highly metastatic when inoculated orthotopically. Distant metastases to a number of organs were detected, including the liver, lung, kidney, and bone. We have employed a human prostate cancer progression model as a system to study the efficacy of gene therapy. Results of the study show that whereas universal promoters, such as Cytomegalovirus (CMV) and Rous Sarcoma Virus (RSV) promoter-driven tumor suppressors (e.g. p53, p21, and p16), were effective in inhibiting prostate tumor growth, the advantages of driving the expression of therapeutic toxic genes using a tissue-specific promoter prostate-specific antigen (PSA) and a tumor--but not tissue-specific promoter, osteocalcin (OC), are preferred. In the case of the PSA promoter, we can achieve cell-kill in PSA-producing human prostate cancer cells. To circumvent the supporting role of bone stroma for prostate cancer epithelial growth, we have recently developed a novel concept where the expression of therapeutic toxic genes is driven by a tumor--but not a tissue-specific OC promoter. Osteocalcin-thymidine kinase (OC-TK) was found to efficiently eradicate the growth of osteosarcoma, prostate, and brain tumors both in vitro and in vivo. We observed that androgen-independent human prostate cancer cells lines expressed OC-TK at higher levels than androgen-dependent human prostate cancer cell lines. We have obtained data to suggest that Ad-OC-TK plus a pro-drug acyclovir (ACV) may be used as an effective therapy to treat prostate cancer bone metastasis in models where the growth of androgen-independent PC-3 and C4-2 tumors in the bone has occurred.

[Cloning and characterization of a specific cytokeratin-8 cDNA from rat prostatic epithelium].

In this study, we report the cloning and sequencing of a full-length cytokeratin-8 (CK-8) cDNA from a rat prostatic epithelial cDNA library. The effects of androgen on CK-8 expression in rat prostate were also studied. The data indicated that the steady-state level of CK-8 mRNA was elevated by about 5- to 10-fold in both prostate and seminal vesicles in castrated rats, and the elevated levels persisted during a 23-day experimental period. Androgen but not estrogen administration repressed the expression of CK-8 mRNA. This effect could be antagonized by the simultaneous administration of an antiandrogen, flutamide, indicating that CK-8 is a new class of androgen-repressed genes whose regulation is presumably mediated by androgen receptor mechanisms.

EPLIN downregulation promotes epithelial-mesenchymal transition in prostate cancer cells and correlates with clinical lymph node metastasis.

Epithelial-mesenchymal transition (EMT) is a crucial mechanism for the acquisition of migratory and invasive capabilities by epithelial cancer cells. By conducting quantitative proteomics in experimental models of human prostate cancer (PCa) metastasis, we observed strikingly decreased expression of EPLIN (epithelial protein lost in neoplasm; or LIM domain and actin binding 1, LIMA-1) upon EMT. Biochemical and functional analyses demonstrated that EPLIN is a negative regulator of EMT and invasiveness in PCa cells. EPLIN depletion resulted in the disassembly of adherens junctions, structurally distinct actin remodeling and activation of ß-catenin signaling. Microarray expression analysis identified a subset of putative EPLIN target genes associated with EMT, invasion and metastasis. By immunohistochemistry, EPLIN downregulation was also demonstrated in lymph node metastases of human solid tumors including PCa, breast cancer, colorectal cancer and squamous cell carcinoma of the head and neck. This study reveals a novel molecular mechanism for converting cancer cells into a highly invasive and malignant form, and has important implications in prognosis and treating metastasis at early stages.

Establishment of human prostate carcinoma skeletal metastasis models.

BACKGROUND: Prostate carcinoma progression from an androgen dependent (AD) state to an androgen independent (AI) state occurs clinically in patients who undergo hormonal therapy. In their laboratory, the authors developed two human prostate carcinoma skeletal metastasis models, the LNCaP progression model and the ARCaP model, to investigate phenotypic and genotypic changes of prostate carcinoma cells during disease progression and to understand molecular pathways for potential therapeutic targeting. METHODS: LNCaP or ARCaP cells were inoculated in athymic mice and were exposed to selective hormonal conditions both in vivo and in vitro. The effects of various hormonal treatment regimens on tumor volumes and distant metastasis and the effects of bone stromal cells on prostate specific antigen (PSA) expression by prostate carcinoma cells were evaluated. RESULTS: The authors propose that prostate carcinoma progression from the AD state to the AI state assumes three AI phenotypes: AI that remains androgen responsive, AI that is unresponsive to androgen stimulation, and AI that is suppressed by or hypersensitive to androgen. AI prostate carcinoma cells interacted reciprocally with osteoblasts to produce enhanced tumor growth and osteoblastic reaction when they are deposited in bone. Bone stromal cell conditioned media stimulated prostate carcinoma cell growth and suppressed its PSA expression, as also evidenced by androgen receptor-mediated transactivation of PSA promoter reporter activity. Conditioned media obtained from prostate carcinoma cells also stimulated osteoblastic cell growth in vitro. A novel gene therapy strategy is being developed to target prostatic tumor epithelium and its supporting stroma using tissue specific and tumor-restricted, promoter-directed toxic gene expression in both cellular compartments. In addition, new strategies are being designed to target the tumor endothelial system in the stroma and tumor cell-extracellular matrix interaction mediated by isotype specific integrins. CONCLUSIONS: Prostate carcinoma skeletal metastasis models may prove useful in developing a new targeting strategy for the prevention and treatment of patients with prostate carcinoma.

A fetal rat urogenital sinus mesenchymal cell line (rUGM): accelerated growth and conferral of androgen-induced growth responsiveness upon a human bladder cancer epithelial cell line in vivo.

A cell-cell interaction model was developed to examine the intercellular communication between mesenchymal and epithelial cells in vivo, and to define the role of androgen and paracrine growth factors in promoting growth and differentiation of the target epithelial cells. Using this model system, we have demonstrated that, in the presence of androgenic steroids, a fetal urogenital sinus mesenchymal cell line exhibited androgen-induced growth responses which resulted in an induction of growth of a non-androgen target epithelial cell line derived from human urinary bladder. Our results show that: (1) a rat fetal urogenital sinus mesenchyme-derived cell line (rUGM) accelerated growth and conferred androgen-induced growth responsiveness upon a non-androgen target cell line, WH, derived from a human bladder transitional-cell carcinoma (TCC); this induction of epithelial tumor growth in vivo occurred in a fibroblast-specific manner; (2) live fetal rUGM cells are required to promote WH tumor growth in vivo, which suggests that continuous production of factors that may serve as mediators for paracrine/autocrine pathways are responsible for androgen stimulation of WH tumor growth in vivo; and (3) although WH tumor growth, mediated by the presence of rUGM cells, was markedly accelerated by the presence of androgen in vivo, androgen and rUGM cells failed to promote the expression of a human prostate-specific antigen (PSA) by WH tumors in vivo. Our results emphasize the importance of organ-specific fibroblasts that promote tumor growth and mediate androgen-induced growth responses; the accelerated growth of the bladder epithelium was not accompanied by the expression of PSA, a known differentiated gene product produced by human prostatic epithelial cells. This report also discusses the potential significance of mesenchymal-epithelial cellular interaction which mediates androgen action and may play an important role by influencing human prostate tumor growth, progression and differentiation.

Development of human prostate cancer models for chemoprevention and experimental therapeutics studies.

The progression of human prostate cancer from histomorphologic to clinical expression often requires several decades. This study emphasizes the importance of developing relevant human prostate cancer models to study the molecular events leading to prostate cancer progression. These models will provide a rational basis for chemopreventive and treatment strategies to retard the progression of human prostate cancer from its localized to its metastatic state. In our laboratory, we have established the LNCaP progression and ARCaP models and the in vitro three-dimensional growth models involving prostate cancer and bone stroma to study the progression of prostate cancer. We propose that prostate cancer may progress from an androgen-dependent to an androgen-independent state. While existing as androgen-independent tumors (defined as tumors capable of growing in castrated hosts and secreting PSA in serum), prostate cancer may assume three different phenotypes as it progresses: androgen-independent while remaining androgen-responsive; androgen-independent and unresponsive to androgen stimulation; and androgen-independent but suppressed by androgen. It is conceivable that any androgen-independent human prostate cancer may contain variable proportions of cells that exhibit these three phenotypes. This concept may have important implications in determining strategies for chemopreventive and therapeutic trials. We have established three-dimensional growth models of prostate cancer cells either in collagen gel or microgravity-simulated growth conditions to form viable and functional organoids which contain prostate cancer epithelial cells admixed with prostate or bone stromal cells. These in vitro models combined with the in vivo models described above will enhance our understanding of the regulatory mechanism of prostate cancer growth and progression, and hence could improve efficiency in screening chemopreventive and therapeutic agents which alter the biologic behaviors of human prostate cancer.