Endogenous androgen receptor proteomic profiling reveals genomic subcomplex involved in prostate tumorigenesis.

Androgen receptor (AR) is a key player in prostate cancer development and progression. Here we applied immunoprecipitation mass spectrometry of endogenous AR in LNCaP cells to identify components of the AR transcriptional complex. In total, 66 known and novel AR interactors were identified in the presence of synthetic androgen, most of which were critical for AR-driven prostate cancer cell proliferation. A subset of AR interactors required for LNCaP proliferation were profiled using chromatin immunoprecipitation assays followed by sequencing, identifying distinct genomic subcomplexes of AR interaction partners. Interestingly, three major subgroups of genomic subcomplexes were identified, where selective gain of function for AR genomic action in tumorigenesis was found, dictated by FOXA1 and HOXB13. In summary, by combining proteomic and genomic approaches we reveal subclasses of AR transcriptional complexes, differentiating normal AR behavior from the oncogenic state. In this process, the expression of AR interactors has key roles by reprogramming the AR cistrome and interactome in a genomic location-specific manner.

Imaging heterogeneity of peptide delivery and binding in solid tumors using SPECT imaging and MRI.

BACKGROUND: As model system, a solid-tumor patient-derived xenograft (PDX) model characterized by high peptide receptor expression and histological tissue homogeneity was used to study radiopeptide targeting. In this solid-tumor model, high tumor uptake of targeting peptides was expected. However, in vivo SPECT images showed substantial heterogeneous radioactivity accumulation despite homogenous receptor distribution in the tumor xenografts as assessed by in vitro autoradiography. We hypothesized that delivery of peptide to the tumor cells is dictated by adequate local tumor perfusion. To study this relationship, sequential SPECT/CT and MRI were performed to assess the role of vascular functionality in radiopeptide accumulation. METHODS: High-resolution SPECT and dynamic contrast-enhanced (DCE)-MRI were acquired in six mice bearing PC295 PDX tumors expressing the gastrin-releasing peptide (GRP) receptor. Two hours prior to SPECT imaging, animals received 25 MBq (111)In(DOTA-(ßAla)2-JMV594) (25 pmol). Images were acquired using multipinhole SPECT/CT. Directly after SPECT imaging, MR images were acquired on a 7.0-T dedicated animal scanner. DCE-MR images were quantified using semi-quantitative and quantitative models. The DCE-MR and SPECT images were spatially aligned to compute the correlations between radioactivity and DCE-MRI-derived parameters over the tumor. RESULTS: Whereas histology, in vitro autoradiography, and multiple-weighted MRI scans all showed homogenous tissue characteristics, both SPECT and DCE-MRI showed heterogeneous distribution patterns throughout the tumor. The average Spearman's correlation coefficient between SPECT and DCE-MRI ranged from 0.57 to 0.63 for the "exchange-related" DCE-MRI perfusion parameters. CONCLUSIONS: A positive correlation was shown between exchange-related DCE-MRI perfusion parameters and the amount of radioactivity accumulated as measured by SPECT, demonstrating that vascular function was an important aspect of radiopeptide distribution in solid tumors. The combined use of SPECT and MRI added crucial information on the perfusion efficiency versus radiopeptide uptake in solid tumors and showed that functional tumor characteristics varied locally even when the tissue appeared homogenous on current standard assessment techniques.

Cross-resistance between taxanes and new hormonal agents abiraterone and enzalutamide may affect drug sequence choices in metastatic castration-resistant prostate cancer.

INTRODUCTION: Treatment options for patients with metastatic castration-resistant prostate cancer (mCRPC) have expanded in recent years with the introduction of cabazitaxel, abiraterone and enzalutamide. With new systemic therapies available, the optimal treatment sequence of these drugs in mCRPC becomes increasingly important. As shown recently, patients who had previously been treated with abiraterone showed impaired responses to docetaxel, suggesting clinical cross-resistance [1]. In the present study, we aimed to identify cross-resistance between taxanes (docetaxel and cabazitaxel) and the new hormonal agents abiraterone and enzalutamide. As a potential mechanism for cross-resistance, we investigated the effects on androgen receptor (AR) nuclear translocation of these compounds. METHODS: To identify cross-resistance, we determined the effects of docetaxel, cabazitaxel, abiraterone and enzalutamide on cell viability in prostate cancer cell lines with acquired resistance to abiraterone and enzalutamide. Time-lapse confocal microscopy was used to study the dynamics of AR nuclear translocation. RESULTS: We observed impaired efficacy of docetaxel, cabazitaxel and enzalutamide in the abiraterone-resistant cell line, compared to the non-resistant cell line, providing evidence for in vitro cross-resistance. Impaired efficacy of docetaxel, cabazitaxel and abiraterone was observed in the enzalutamide-resistant cell line. Furthermore, docetaxel and cabazitaxel inhibited AR nuclear translocation, which was also observed for abiraterone and enzalutamide. CONCLUSIONS: In conclusion we found substantial preclinical evidence for cross-resistance between the taxanes docetaxel and cabazitaxel, and AR targeting agents abiraterone and enzalutamide. Since these compounds all interfere with AR-signalling, this strongly suggests a common mechanism of action, and thus a potential mechanism for cross-resistance in mCRPC.

Improving radiopeptide pharmacokinetics by adjusting experimental conditions for bombesin receptor-targeted imaging of prostate cancer.

AIM: Prostate cancer (PC) is a major health problem. The Gastrin-Releasing Peptide Receptor (GRPR) offers a promising target for staging and monitoring of PC since it is overexpressed in PC and not in normal prostatic tissue. To improve receptor-mediated imaging we investigated the impact of various experimental conditions on pharmacokinetics using the Indium-111 labelled bombesin (BN) analogue AMBA. Besides frequently used androgen-resistant PC-3 also the clinically more relevant androgen sensitive VCaP celline was used as human PC xenograft in nude mice. METHODS: Non-purified [111In]AMBA was compared with HPLC-purified [111In]AMBA. Effect of specific activity was studied administering 0.1MBq [111In]AMBA supplemented with different amounts of AMBA (1-3000pmol). GRPR was saturated with Tyr4-BN 1 and 4h prior to injection of [111In]AMBA. RESULTS: GRPR-positive tissue showed a significant 2 to 3-fold increase in absolute uptake after HPLC-purification while keeping a stable tumor-to-pancreas ratio. Lowering specific activity resulted in decline in uptake to 43% in tumor, 49% in kidney and 92% in pancreas between 10 and 3000 pmol. Tumor-to-pancreas ratio improved six-fold from 0.1±0 after 10 pmol up to 0.6±0.2 after 3000 pmol (P<0.01). When saturating GRPR 4h prior to [111In]AMBA injection tumor-to-pancreas ratio improved from 0.10±0.3 to 0.22±0.2 (P<0.01) and tumor-to-kidney ratio increased from 0.92±0.16 to 3.45±0.5 (P<0.01). CONCLUSION: Besides specific peptide characteristics also the experimental conditions, such as HPLC-purification, variations in specific activity and saturation of the GRPR prior to [111In]AMBA administration essentially affect radiopeptide pharmacokinetics. Experimental conditions therefore need to be carefully selected in order to compose ideal standardised protocols for optimal targeting.

A transductionally retargeted adenoviral vector for virotherapy of Her2/neu-expressing prostate cancer.

The efficacy of adenovirus (Ad)-based gene therapy of solid tumors, such as prostate cancer, is limited. One of the many problems is that the virus infects many different cell types in the body, resulting in high toxicity, whereas the target cancer cells are often less prone to wild-type Ad infection. Our aim was to develop genetically de- and retargeted Ad vectors to reduce off-target effects and increase target infection for prostate cancer. We have previously reported an Ad5 vector specific for the cancer-associated receptor Her2/neu, created by inserting Her2/neu-reactive Affibody(®) molecules (ZH) into the HI loop of a coxsackievirus and adenovirus receptor binding-ablated fiber (Ad[ZH/1]). In addition to virus retargeting to Her2/neu, this virus was further modified from wild-type Ad by changing the RGD motif in the penton base to EGD and by substitution of the KKTK motif in the third shaft repeat to RKSK, resulting in the vector Ad[ZH/3]. The ZH-containing vectors could be produced to high titers and were specific for their target, resulting in efficient infection and killing of Her2/neu-positive androgen-dependent PC346C prostate cancer cells in vitro. Here we show that the oncolytic Ad[ZH/3] vector significantly prolonged survival time and reduced serum prostate-specific antigen levels in an orthotopic prostate tumor model in nude mice to the same extent as wild-type Ad5. Our results show that Her2/neu targeting using Ad-based vectors for prostate cancer is feasible and may serve as a basis for the development of gene therapy of human prostate cancer as well as other Her2/neu-expressing cancers.

Disconnecting the yin and yang relation of epidermal growth factor receptor (EGFR)-mediated delivery: a fully synthetic, EGFR-targeted gene transfer system avoiding receptor activation.

The epidermal growth factor receptor (EGFR) is upregulated within a high percentage of solid tumors and hence is an attractive target for tumor-targeted therapies including gene therapy. The natural EGFR ligand epidermal growth factor (EGF) has been used for this purpose, despite the risk of mitogenic effects due to EGFR activation. We have developed a fully synthetic, EGFR-targeted gene delivery system based on PEGylated linear polyethylenimine (LPEI), allowing evaluation of different EGFR-binding peptides in terms of transfection efficiency and EGFR activation. Peptide sequences directly derived from the human EGF molecule enhanced transfection efficiency with concomitant EGFR activation. Only the EGFR-binding peptide GE11, which has been identified by phage display technique, showed specific enhancement of transfection on EGFR-overexpressing tumor cells including glioblastoma and hepatoma, but without EGFR activation. EGFR targeting led to high levels of cell association of fluorescently labeled polyplexes after only 30 min of incubation. EGF pretreatment of cells induced enhanced cellular internalization of all polyplex types tested, pointing at generally enhanced macropinocytosis. EGF polyplexes diminished cell surface expression of EGFR for up to 4 hr, whereas GE11 polyplexes did not. In a clinically relevant orthotopic prostate cancer model, intratumorally injected GE11 polyplexes were superior in inducing transgene expression when compared with untargeted polyplexes.

Gastrin-releasing peptide receptor-based targeting using bombesin analogues is superior to metabolism-based targeting using choline for in vivo imaging of human prostate cancer xenografts.

PURPOSE: Prostate cancer (PC) is a major health problem. Overexpression of the gastrin-releasing peptide receptor (GRPR) in PC, but not in the hyperplastic prostate, provides a promising target for staging and monitoring of PC. Based on the assumption that cancer cells have increased metabolic activity, metabolism-based tracers are also being used for PC imaging. We compared GRPR-based targeting using the (68)Ga-labelled bombesin analogue AMBA with metabolism-based targeting using (18)F-methylcholine ((18)F-FCH) in nude mice bearing human prostate VCaP xenografts. METHODS: PET and biodistribution studies were performed with both (68)Ga-AMBA and (18)F-FCH in all VCaP tumour-bearing mice, with PC-3 tumour-bearing mice as reference. Scanning started immediately after injection. Dynamic PET scans were reconstructed and analysed quantitatively. Biodistribution of tracers and tissue uptake was expressed as percent of injected dose per gram tissue (%ID/g). RESULTS: All tumours were clearly visualized using (68)Ga-AMBA. (18)F-FCH showed significantly less contrast due to poor tumour-to-background ratios. Quantitative PET analyses showed fast tumour uptake and high retention for both tracers. VCaP tumour uptake values determined from PET at steady-state were 6.7 ± 1.4%ID/g (20-30 min after injection, N = 8) for (68)Ga-AMBA and 1.6 ± 0.5%ID/g (10-20 min after injection, N = 8) for (18)F-FCH, which were significantly different (p <0.001). The results in PC-3 tumour-bearing mice were comparable. Biodistribution data were in accordance with the PET results showing VCaP tumour uptake values of 9.5 ± 4.8%ID/g (N = 8) for (68)Ga-AMBA and 2.1 ± 0.4%ID/g (N = 8) for (18)F-FCH. Apart from the GRPR-expressing organs, uptake in all organs was lower for (68)Ga-AMBA than for (18)F-FCH. CONCLUSION: Tumour uptake of (68)Ga-AMBA was higher while overall background activity was lower than observed for (18)F-FCH in the same PC-bearing mice. These results suggest that peptide receptor-based targeting using the bombesin analogue AMBA is superior to metabolism-based targeting using choline for scintigraphy of PC.

Peptide receptor imaging of prostate cancer with radiolabelled bombesin analogues.

Prostate Cancer (PC) is a type of cancer that is often diagnosed at very early stages due to improved detection among man in the Western world. Current imaging techniques are not optimal to determine extent of minimal early stage PC even though this is of great clinical importance. Human PC and high-grade PIN have shown high Gastrin-Releasing Peptide Receptor (GRPR) expression, while normal prostate tissue and BPH revealed to be predominantly GRPR-negative. Radiolabelled Gastrin-Releasing Peptide (GRP) or bombesin (BN) analogues targeting the GRPR can be used as non-invasive tools to diagnose, monitor and potentially treat PC. These BN analogues have already proven to be able to image PC in both tumour-bearing mice and clinical patients showing no important side effects. It's desirable that new peptides require fast-track standardised comparative testing in relevant PC models to select the best performing BN analogues for further evaluation in patients. Although knowledge about GRPR expression and development of new BN analogues can be extended, it is time to study performance of BN analogues for peptide receptor based imaging in patients validating results of PC imaging using histopathology as a golden standard.

Human xenograft models as useful tools to assess the potential of novel therapeutics in prostate cancer.

With docetaxel as effective chemotherapy for hormone refractory prostate cancer (HRPC), the number of new treatment combinations for HRPC is expanding demanding a fast-track screening system. This review elaborates on the use of xenograft models to select the most promising combination therapies for entering into phase II clinical trials.

Overexpression and gene amplification of BAG-1L in hormone-refractory prostate cancer.

BAG-1L (Bcl-2-associated anthanogene 1) has been found to interact with androgen receptor (AR), and has been suggested to be involved in the development of prostate cancer. In order to determine the presence of genetic and/or expression alterations of BAG-1L in prostate cancer, we analysed human prostate cancer cell lines and xenografts as well as patient samples of untreated, hormone-naïve, and hormone-refractory prostate carcinomas for sequence variations using denaturing high-performance liquid chromatography (DHPLC), for gene copy number using fluorescence in situ hybridization (FISH), and for expression using both quantitative RT-PCR and immunostaining. Only one sequence variation was found in all 37 cell lines and xenografts analysed. BAG-1 gene amplification was detected in two xenografts. In addition, gene amplification was found in 6 of 81 (7.4%) hormone-refractory clinical tumours, whereas no amplification was found in any of the 130 untreated tumours analysed. Additionally, gain of the BAG-1 gene was observed in 27.2% of the hormone-refractory tumours and in 18.5% of the untreated carcinomas. In a set of 263 patient samples, BAG-1L protein expression was significantly higher in hormone-refractory tumours than in primary tumours (p = 0.002). Altogether, these data suggest that amplification and overexpression of BAG-1L may be involved in the progression of prostate cancer.

Validation of transrectal ultrasonographic volumetry for orthotopic prostate tumours in mice.

Orthotopic human prostate tumour models in athymic nude mice are regarded as being most suitable for fundamental and pre-clinical research on prostate cancer. The anatomic localization of the tumour in the pelvis, however, provides little possibility for monitoring tumour growth or regression. To assess time-related changes in orthotopic tumour volume, we applied transrectal ultrasonography (TRUS) to the murine prostate. This technique has the advantages of allowing accurate monitoring of tumours during therapeutic manipulations and a reduction of animal use due to a reduction of sacrificing endpoints. To validate the TRUS method, the mouse prostate reconstitution model, RM-9, and the prostate-specific antigen (PSA) producing human prostate cancer xenograft PC-346 were used. Volumetric calliper measurements were performed with a 30 MHz ultrasound probe designed for intra-arterial use in humans. Tumour weight, determined at various time-points, was found to be closely related to actual tumour weight (R = 0.99) and, in the PC-346 model, to the level of PSA in the plasma. Furthermore, the interobserver variation for TRUS was low for tumours above 50 mg. Thus, TRUS for murine prostate tumours proves to be an accurate, reproducible and sensitive method.

A novel gene on human chromosome 2p24 is differentially expressed between androgen-dependent and androgen-independent prostate cancer cells.

Identification of genes involved in the transition from androgen-dependent to androgen-independent prostate cancer is important to extend our current knowledge of the disease. Using differential display RT-PCR analysis between androgen-dependent and androgen-independent prostate cancer cells, we have identified a novel gene, designated GC109. GC109 harbours a putative Cys-His cluster, a nuclear localisation signal, a leucine zipper and a ret finger protein (rfp)-like domain. GC109 mRNA expression in normal human tissues was found not to be restricted to the prostate. However, using a variety of 15 human cancer cell lines, GC109 mRNA was preferentially expressed in androgen-dependent LNCaP-FGC, compared with androgen-independent LNCaP-LNO, DU145 and PC3 human prostate cancer cells. Finally, the GC109 gene was mapped on human chromosome 2p24. Based on its protein domain structure and chromosomal localisation, we hypothesise that GC109 may be involved in chromosomal rearrangements in prostate cancer.

Characterization of a zinc-finger protein and its association with apoptosis in prostate cancer cells.

BACKGROUND: The transition from androgen-dependent to androgen-independent prostate cancer is not fully understood but appears to involve multiple genetic changes. We have identified a gene, GC79, that is more highly expressed in androgen-dependent LNCaP-FGC human prostate cancer cells than in androgen-independent LNCaP-LNO human prostate cancer cells. Physiologic levels (0.1 nM:) of androgens repress expression of GC79 messenger RNA (mRNA) in LNCaP-FGC cells. To determine the role of GC79, we cloned its complementary DNA (cDNA) and functionally characterized its product. METHODS: The differentially expressed GC79 gene was cloned from human prostate cDNA libraries, sequenced, and transfected into mammalian cells to study its function. Expression of GC79 was analyzed in various adult and fetal human tissues and in prostate glands of castrated rats. The association of GC79 expression and apoptosis was investigated in COS-1 and LNCaP cells transfected with GC79 cDNA. All statistical tests are two-sided. RESULTS: Sequence analysis indicates that GC79 encodes a large, complex, multitype zinc-finger protein, containing nine C(2)H(2)-type zinc-finger domains, a cysteine-rich region, and a GATA C(4)-type zinc-finger domain. Castration-induced androgen withdrawal increased the expression of GC79 mRNA in the regressing rat ventral prostate, suggesting that the expression of GC79 mRNA is associated with the process of apoptotic cell death in the rat ventral prostate. Transfection and induction of GC79 cDNA in both COS-1 and LNCaP prostate cancer cells led to an apoptotic index that was eightfold higher (P:<.001, two-sided Student's t test) than that observed in uninduced transfected cells. CONCLUSIONS: We have cloned an androgen-repressible gene, GC79, that is potentially involved in apoptosis. This finding may have implications for the development of androgen-independent prostate cancer and, ultimately, for the treatment of prostate cancer.

Silencing of CD44 expression in prostate cancer by hypermethylation of the CD44 promoter region.

Loss of the CD44 transmembrane glycoprotein in primary prostate cancer has been shown to be associated with unfavorable clinical behavior. Moreover, the majority of prostate cancer metastases lack expression of this molecule. The mechanism of CD44 silencing in prostate cancer was investigated using both patient material and in vivo-propagated human prostate cancer xenografts. In 9 of 11 lymph node metastases of prostate cancer, we demonstrated by methylation-sensitive restriction enzyme digestion that the promoter region of the CD44 gene is methylated, indicating that this represents a major mechanism of CD44 silencing. Similarly, in 6 out of 12 in vivo-growing human prostate carcinoma xenograft models, hypermethylation of the CD44 gene was found. The extent of CpG island methylation was investigated by nucleotide sequencing after bisulphite modification of the CD44 promoter region. In the xenografts displaying hypermethylation, the examined 14 CpG sites in the CD44 transcription regulatory domain, including a Sp1 binding site, were consistently methylated. This correlated with reduced CD44 expression or lack of CD44 expression at mRNA and protein levels. In the xenografts lacking hypermethylation of the CD44 gene, high levels of CD44 mRNA and protein were expressed in some models, whereas in others CD44 mRNA expression was only detectable by RT-PCR and the CD44 protein could hardly be detected or was not detected at all. The results indicate that, in most prostate cancers, loss of CD44 expression is associated with extensive hypermethylation of the CpG island of the CD44 promoter region, but other, posttranscriptional mechanisms may also lead to CD44 loss.

Use of nude mouse xenograft models in prostate cancer research.

BACKGROUND: Our understanding of the mechanisms of (progressive) growth of prostatic cancer has been largely obtained through the study of experimental animal models. To be able to validate new concepts, representative model systems of human origin that mimic the clinical process of the disease in patients are essential. Unfortunately, the limited number of human prostate tumor models has considerably hampered research. METHODS: Various research groups have put much effort in the development of human prostate tumor xenograft models, and large numbers of clinical prostate tumors were heterotransplanted in immune-deficient host animals. This huge effort has resulted in a number of tumor lines which are reviewed here. RESULTS: Up to now, approximately 25 xenograft models of human prostate cancer have been established and reported in the literature. The available xenografts seem to represent the various stages of clinical prostate cancer, such as early progression and transition from androgen-dependent to androgen-independent growth. In addition, recent efforts are concentrating on the establishment of in vitro cell lines from these xenografts as well as on the development of (bone) metastatic variants. CONCLUSIONS: Xenograft models are important for elucidating regulatory pathways of tumor growth and progression and are indispensible for testing of new treatment modalities.

Androgen deprivation of the PC-310 [correction of prohormone convertase-310] human prostate cancer model system induces neuroendocrine differentiation.

Neuroendocrine (NE) cells are androgen-independent cells and secrete growth-modulating neuropeptides via a regulated secretory pathway (RSP). We studied NE differentiation after androgen withdrawal in the androgen-dependent prostate cancer xenograft PC-310. Expression patterns of chromogranin A, secretogranin III, and prohormone convertase-1 were analyzed at both protein and mRNA level to mark the kinetics of NE differentiation both in vivo and in vitro. PC-310 tumor-bearing nude mice were killed at 0, 2, 5, 7, 14, and 21 days postcastration. PC-310C cultures initiated from collagenase-treated tumor tissue could be maintained up to four passages, and androgen-deprivation experiments were performed similarly. PC-310 tumor volumes decreased by 50% in 10 days postcastration. Proliferative activity and prostate-specific antigen (PSA) serum levels decreased to zero postcastration, whereas PSA levels in PC-310C culture media first decreased and subsequently increased after 5 days. In vivo, androgen receptor (AR) expression decreased initially but returned to control level from 5 days postcastration on. CgA, secretogranin III, and secretogranin V expression increased in vivo from 5 days postcastration on. Subsequently, prohormone convertase-1 and peptidyl alpha-amidating monooxygenase as well as the vascular endothelial growth factor were expressed from 7 days postcastration on, and, finally, growth factors such as gastrin-releasing peptide and serotonin were expressed in a small part of the NE cells 21 days postcastration. The PC-310 tumors did not show colocalization of the AR on the NE cells in the tumor residues after 21 days. As in the PC-310 xenograft, NE differentiation was induced and AR expression relapsed after prolonged androgen suppression in PC-310C. For PC-310C cells, this relapse was associated with the secretion of PSA. PC-310C is the first culture of human prostatic cancer cells having the NE phenotype. The PC-310 model system is a potential androgen-dependent model for studying the role of NE cells in the progression of clinical prostate cancer. Androgen deprivation of NE-differentiated prostate cancer may induce the formation of both NE- and AR-positive dormant tumor residues, capable of actively producing NE growth factors via a RSP, possibly leading to hormone refractory disease.

Variations in activin receptor, inhibin/activin subunit and follistatin mRNAs in human prostate tumour tissues.

The possible role of activin in the regulation of malignant prostatic growth was studied using RNAase protection assays of activin receptors, inhibin/activin subunits and follistatin mRNAs in the human prostatic carcinoma cell lines LNCaP-FGC, -R and -LNO, in human prostatic carcinoma xenografts and in human prostatic tissue. Activin receptor types IA (ActRIA), IB (ActRIB), IIA (ActRIIA) and IIB (ActRIIB) mRNAs were generally expressed in prostate epithelial cells, with significantly lower levels of ActRIB mRNA in prostate tumour material when compared to non-malignant tissue (P < 0.05; Mann-Whitney U-test). Inhibin/activin betaA- and betaB-subunit mRNA expression was also found in prostate tissue. Androgen-independent xenografts expressed significantly lower amounts of betaB-subunit mRNA when compared to androgen-dependent xenografts (P< 0.05). While betaB-subunit mRNA was expressed by LNCaP-FGC and -LNO cells, virtually no expression was found in the androgen-independent LNCaP-R line. Inhibin alpha-subunit mRNA levels were low or undetectable in all samples investigated. Follistatin mRNA was undetectable in LNCaP-sublines, while low levels were found in prostatic tissues. In androgen-independent LNCaP-R cells, activin inhibited cell growth in a dose-dependent manner. These results suggest that prostate tumour progression is accompanied by a decrease of the inhibitory effect of locally produced activin by either a decrease in the expression of activin betaB-subunit mRNA or by a decrease of ActRIB mRNA levels.

Identification of a gene on human chromosome 8q11 that is differentially expressed during prostate-cancer progression.

Using differential-display RT-PCR analysis between androgen-dependent LNCaP-FGC and androgen-independent LNCaP-LNO human prostate-cancer cells, we have identified a gene not previously described as being expressed in prostate. The gene is more highly expressed in androgen-independent than in androgen-dependent LNCaP prostate-cancer cells. Sequence analysis showed that the gene has already been cloned as a transcript present in embryonic brain, with unknown functions. Expression of the gene was found not to be restricted to the prostate, and not regulated by androgens in androgen-independent prostate-cancer cells. In concert with the cell-culture system, Northern-blot analysis of gene expression in vivo, using a panel of human prostate-cancer xenografts, demonstrated that the gene is more highly expressed in androgen-independent than in androgen-dependent prostate-cancer xenografts. The gene could be mapped on human chromosome 8q11. The 8q arm is known to be frequently amplified during prostate-cancer progression and harbors several proto-oncogenes potentially involved in cancer development. Since expression of the gene is positively correlated with prostate-cancer progression and its 8q11 chromosomal localization, we hypothesize that the gene may be involved in the development and progression of prostate cancer.

Regulation of expression of Na+,K+-ATPase in androgen-dependent and androgen-independent prostate cancer.

The beta 1-subunit of Na+,K+-ATPase was isolated and identified as an androgen down-regulated gene. Expression was observed at high levels in androgen-independent as compared to androgen-dependent (responsive) human prostate cancer cell lines and xenografts when grown in the presence of androgens. Down-regulation of the beta 1-subunit was initiated at concentrations between 0.01 nM and 0.03 nM of the synthetic androgen R1881 after relatively long incubation times (> 24 h). Using polyclonal antibodies, the concentration of beta 1-subunit protein, but not of the alpha 1-subunit protein, was markedly reduced in androgen-dependent human prostate cancer cells (LNCaP-FGC) cultured in the presence of androgens. In line with these observations it was found that the protein expression of total Na+,K+-ATPase in the membrane (measured by 3H-ouabain binding) was also markedly decreased. The main function of Na+,K+-ATPase is to maintain sodium and potassium homeostasis in animal cells. The resulting electrochemical gradient is facilitative for transport of several compounds over the cell membrane (for example cisplatin, a chemotherapeutic agent experimentally used in the treatment of hormone-refractory prostate cancer). Here we observed that a ouabain-induced decrease of Na+,K+-ATPase activity in LNCaP-FGC cells results in reduced sensitivity of these cells to cisplatin-treatment. Surprisingly, androgen-induced decrease of Na+,K+-ATPase expression, did not result in significant protection against the chemotherapeutic agent.

Kinetics of neuroendocrine differentiation in an androgen-dependent human prostate xenograft model.

It was previously shown in the PC-295 xenograft that the number of chromogranin A (CgA)-positive neuroendocrine (NE) cells increased after androgen withdrawal. NE cells did not proliferate and differentiated from G0-phase-arrested cells. Here we further characterized NE differentiation, androgen receptor status, and apoptosis-associated Bcl-2 expression in the PC-295 model after androgen withdrawal to assess the origin of NE cells. PC-295 tumor volumes decreased by 50% in 4 days. Intraperitoneal bromodeoxyuridine (BrdU) incorporation and MIB-1 labeling decreased to 0%, and the apoptosis was maximal at day 4. Androgen receptor expression and prostate-specific antigen (PSA) serum levels decreased rapidly within 2 days. The number of NE cells increased 6-fold at day 4 and 30-fold at day 7. Five and ten percent of the CgA-positive cells were BrdU positive after continuous BrdU labeling for 2 and 4 days, respectively. However, no MIB-1 expression was observed in CgA-positive cells. NE cells expressed the regulated secretory pathway marker secretogranin III but were negative for androgen receptor and Bcl-2. Bcl-2 expression did increase in the non-NE tumor cells. In conclusion, androgen withdrawal leads to a rapid PC-295 tumor regression and a proliferation-independent induction of NE differentiation. The strictly androgen-independent NE cells that were still present after 21 days differentiated mainly from G0-phase-arrested cells.