ABSTRACT
The original version of this article contained an error in Fig. 5a where the colours of the labels representing the Hinge and LBD of the AR were incorrect and did not match the corresponding exons. The corrected version of this Figure now appears in the article. The conclusions of this paper were not affected. The authors apologise for this error and any confusion caused.
ABSTRACT
Stem cell characteristics have been associated with treatment resistance and poor prognosis across many cancer types. The ability to induce and regulate the pathways that sustain these characteristic hallmarks of lethal cancers in a novel in vitro model would greatly enhance our understanding of cancer progression and treatment resistance. In this work, we present such a model, based simply on applying standard pluripotency/embryonic stem cell media alone. Core pluripotency stem cell master regulators (OCT4, SOX2 and NANOG) along with epithelial-mesenchymal transition (EMT) markers (Snail, Slug, vimentin and N-cadherin) were induced in human prostate, breast, lung, bladder, colorectal, and renal cancer cells. RNA sequencing revealed pathways activated by pluripotency inducing culture that were shared across all cancers examined. These pathways highlight a potential core mechanism of treatment resistance. With a focus on prostate cancer, the culture-based induction of core pluripotent stem cell regulators was shown to promote survival in castrate conditions-mimicking first line treatment resistance with hormonal therapies. This acquired phenotype was shown to be mediated through the upregulation of iodothyronine deiodinase DIO2, a critical modulator of the thyroid hormone signalling pathway. Subsequent inhibition of DIO2 was shown to supress expression of prostate specific antigen, the cardinal clinical biomarker of prostate cancer progression and highlighted a novel target for clinical translation in this otherwise fatal disease. This study identifies a new and widely accessible simple preclinical model to recreate and explore underpinning pathways of lethal disease and treatment resistance.
ABSTRACT
BACKGROUND: The ability to provide accurate prognostic and predictive information to patients is becoming increasingly important as clinicians enter an era of personalized medicine. For a disease as heterogeneous as epithelial ovarian cancer, conventional algorithms become too complex for routine clinical use. This study therefore investigated the potential for an artificial intelligence model to provide this information and compared it with conventional statistical approaches. METHODS: The authors created a database comprising 668 cases of epithelial ovarian cancer during a 10-year period and collected data routinely available in a clinical environment. They also collected survival data for all the patients, then constructed an artificial intelligence model capable of comparing a variety of algorithms and classifiers alongside conventional statistical approaches such as logistic regression. RESULTS: The model was used to predict overall survival and demonstrated that an artificial neural network (ANN) algorithm was capable of predicting survival with high accuracy (93 %) and an area under the curve (AUC) of 0.74 and that this outperformed logistic regression. The model also was used to predict the outcome of surgery and again showed that ANN could predict outcome (complete/optimal cytoreduction vs. suboptimal cytoreduction) with 77 % accuracy and an AUC of 0.73. CONCLUSIONS: These data are encouraging and demonstrate that artificial intelligence systems may have a role in providing prognostic and predictive data for patients. The performance of these systems likely will improve with increasing data set size, and this needs further investigation.
Subject(s)
Algorithms , Neoplasms, Glandular and Epithelial/surgery , Neural Networks, Computer , Ovarian Neoplasms/surgery , Area Under Curve , Bayes Theorem , Carcinoma, Ovarian Epithelial , Cytoreduction Surgical Procedures , Decision Trees , Female , Humans , Predictive Value of Tests , Prognosis , Proportional Hazards Models , ROC Curve , Support Vector Machine , Survival RateABSTRACT
BACKGROUND: Evidence increasingly supports that prostate cancer is initiated by the malignant transformation of stem cells (SCs). Furthermore, many SC-signalling pathways are shown to be shared in prostate cancer. Therefore, we planned transcriptome characterisation of adult prostate SCs as a strategy to consider new targets for cancer treatment. METHODS: Intuitive pathway analysis was used for putative target discovery in 12 matched selections of human prostate SCs, transiently amplifying cells and terminally differentiated cells. These were pooled into three groups according to the stage of differentiation for mRNA microarray analysis. Targets identified were validated using uncultured primary tissue (n=12), functional models of prostate cancer and a tissue microarray consisting of benign (n=42) and malignant prostate (n=223). RESULTS: A deficiency in class 1 UDP glucuronosyltransferase (UGT) enzymes (UGT1A) was identified in prostate SCs, which are involved in androgen catabolism. Class 1 UGT enzyme expression was also downregulated in cancer SCs and during progression to metastatic castration-resistant prostate cancer (CRPC). Reduction of UGT1A expression in vitro was seen to improve cell survival and increase androgen receptor (AR) activity, as shown by upregulation of prostate-specific antigen expression. INTERPRETATION: Inactivation of intracellular androgen catabolism represents a novel mechanism to maintain AR activity during CRPC.
Subject(s)
Adult Stem Cells/enzymology , Gene Expression Regulation, Neoplastic , Glucuronosyltransferase/genetics , Neoplastic Stem Cells/enzymology , Prostate/enzymology , Prostatic Neoplasms/enzymology , RNA, Messenger/analysis , Androgens/metabolism , Cell Line , Cell Line, Tumor , Cell Survival , Down-Regulation , Gene Expression Profiling , Glucuronosyltransferase/metabolism , Humans , Kallikreins/metabolism , Male , Prostate/cytology , Prostate-Specific Antigen/metabolism , Prostatic Neoplasms/genetics , Real-Time Polymerase Chain Reaction , Receptors, Androgen/metabolism , Transcriptome , Up-RegulationABSTRACT
BACKGROUND: Prostate cancer (PC) represents a global health issue. Treatment for locally advanced and metastatic PC remains unsatisfactory. The androgen receptor (AR) has been validated in having a key role in both naïve and castrate-resistant PC (CRPC). However, the significance of other signalling pathways in CRPC is less well validated. METHODS: To gain a better insight into the molecular signalling cascades involved in clinical CRPC, we performed gene expression profiling using the Illumina DASL assay and studied matched hormone-naive (HN) and CR prostate tumours (n=10 pairs). Ingenuity Pathways Analysis (IPA) was used to identify potential networks involved, and further validation was performed in in vitro cell models and clinical tumours. RESULTS: Expression of 50 genes was significantly different between HN and CRPC. IPA revealed two networks of particular interest, including AR and FGFR1, respectively. FGFR1 expression was confirmed to be significantly upregulated in CRPC (P ≤ 0.005), and abnormal FGFR1 expression was associated with shorter time to biochemical relapse in HNPC (P=0.006) and less favourable disease-specific survival in CRPC (P=0.018). CONCLUSION: For the first time, our gene expression profiling experiment on archival tumour materials has identified upregulated FGFR1 expression to be associated with PC progression to the CR state.
Subject(s)
Androgen Antagonists/pharmacology , Castration , Gonadotropin-Releasing Hormone/pharmacology , Prostatic Neoplasms/genetics , Receptor, Fibroblast Growth Factor, Type 1/genetics , Aged , Aged, 80 and over , Cell Line, Tumor , Gene Expression Profiling , Humans , Male , Middle Aged , Neoplasms, Hormone-Dependent/genetics , Orchiectomy , Receptors, Androgen/genetics , Recurrence , Signal Transduction , Up-RegulationABSTRACT
PURPOSE: Primary androgen deprivation therapy (PADT) is an important treatment modality for men with localized or locally advanced prostate cancer and without bone metastasis. There is, however, a lack of data on the biochemical relapse (BR) outcomes in these patients. Here, we studied the outcome of a contemporary series of men treated by PADT and investigated predictive risk factors for BR. METHODS: One hundred and fifty-five patients treated by PADT formed the initial study cohort, and BR outcomes in this group were reviewed. The outcomes of men with bone scan negative disease were specifically analysed. The predictive value of a panel of clinical risk factors for BR was evaluated using univariate and multivariate analysis. The results were further validated in a separate cohort of patients without bone metastasis from a second institution (n = 84). RESULTS: Median follow-up was 70 months. In the first study cohort, 109/155 men (70%) had bone scan negative disease. In these patients, only 45% developed BR during the follow-up period with only 28% relapsing within 5 years of initiating PADT. Key-independent factors predicting BR were a high PSA nadir (p = 0.001) and a shorter time to nadir (p < 0.001). A nadir of ≤0.1 ng/ml and time to nadir of >24 months specifically identified men with a very good outcome from PADT. In a second-independent cohort, very similar overall and 5-year BR rates were observed in men without bone metastasis (39 and 35%, respectively). PSA nadir thresholds identified in the first cohort were again able to define a good prognostic group in this re-test cohort (p = 0.005 and p = 0.01, respectively). CONCLUSION: Men treated by PADT and without bone metastasis can have very durable responses to PADT with the majority remaining BR free at 5 years. PSA nadir and time to nadir are key predictors of a good outcome in this group.
Subject(s)
Androgen Antagonists/therapeutic use , Antineoplastic Agents, Hormonal/therapeutic use , Biomarkers, Tumor/blood , Orchiectomy , Prostate-Specific Antigen/blood , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/therapy , Aged , Aged, 80 and over , Analysis of Variance , Bone Neoplasms/diagnosis , Bone Neoplasms/secondary , Cohort Studies , Disease-Free Survival , Gonadotropin-Releasing Hormone/agonists , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Neoplasm Staging , Predictive Value of Tests , Prognosis , Prostatic Neoplasms/immunology , Prostatic Neoplasms/pathology , Reproducibility of Results , Risk Assessment , Risk FactorsABSTRACT
BACKGROUND: Cellular proliferation, driven by cyclin-dependent kinases (CDKs) and their cyclin partners, is deregulated in cancer. Anti-estrogens, such as tamoxifen, antagonise estrogen-induced ERalpha transactivation of cyclin D1, resulting in reduced CDK4/6 activity, p27(Kip1)-mediated inhibition of CDK2 and growth arrest. We hypothesised that direct inhibition of CDK2 and CDK1 may overcome the major clinical problem of anti-estrogen resistance. METHODS: The cellular effects of CDK2/1 siRNA knockdown and purine-based CDK2/1 inhibitors, NU2058 and NU6102, were measured in anti-estrogen-sensitive and resistant breast cancer cell lines. RESULTS: CDK2 knockdown caused G1 accumulation, whereas CDK1 depletion caused G2/M slowing, and dual CDK1/2 depletion resulted in further G2/M accumulation and cell death in both anti-estrogen-sensitive and resistant cells, confirming CDK2 and CDK1 as targets for breast cancer therapy. In contrast to tamoxifen, which only affected hormone-sensitive cells, NU2058 and NU6102 reduced CDK2-mediated phosphorylation of pRb, E2F transcriptional activity and proliferation, ultimately resulting in cell death, in both anti-estrogen-sensitive and resistant cells. Both drugs caused G2/M arrest, reflective of combined CDK2/1 knockdown, with a variable degree of G1 accumulation. CONCLUSION: These studies confirm the therapeutic potential of CDK2 and CDK1 inhibitors for cancer therapy, and support their use as an alternative treatment for endocrine-resistant breast cancer.
Subject(s)
Antineoplastic Agents/pharmacology , CDC2 Protein Kinase/metabolism , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 2/metabolism , Protein Kinase Inhibitors/pharmacology , Breast Neoplasms , Cell Cycle/drug effects , Cell Line, Tumor , Estrogen Receptor Modulators/pharmacology , Female , HumansABSTRACT
BACKGROUND: The fibroblast growth factor (FGF) axis is an important mitogenic stimulus in prostate carcinogenesis. We have previously reported that transcript level of human similar expression to FGF (hSef), a key regulator of this pathway, is downregulated in clinical prostate cancer. In this study we further analysed the role of hSef in prostate cancer. METHODS: hSef function was studied in in vitro and in vivo prostate cancer models using stable over-expression clones. Protein expression of hSef was studied in a comprehensive tissue microarray. RESULTS: Stable over-expression of hSef resulted in reduced in vitro cancer cell proliferation, migration and invasive potential. In an in vivo xenograft model, the expression of hSef significantly retarded prostate tumour growth as compared with empty vector (P=0.03) and non-transfected (P=0.0001) controls. Histological examination further showed a less invasive tumour phenotype and reduced numbers of proliferating cells (P=0.0002). In signalling studies, hSef inhibited FGF-induced ERK phosphorylation, migration to the nucleus and activation of a reporter gene. Constitutively active Ras, however, was able to reverse these effects, suggesting that hSef exerts an effect either above or at the level of Ras in prostate cancer cells. In a large tissue microarray, we observed a significant loss of hSef protein in high-grade (P<0.0001) and metastatic (P<0.0001) prostate cancer. CONCLUSIONS: Considered together, the role of hSef in attenuating FGF signalling and evidence of downregulation in advanced tumours argue strongly for a tumour suppressor function in human prostate cancer.
Subject(s)
Fibroblast Growth Factors/antagonists & inhibitors , Prostatic Neoplasms/metabolism , Receptors, Interleukin/biosynthesis , Animals , Cell Growth Processes/physiology , Cell Line, Tumor , Down-Regulation , Fibroblast Growth Factors/metabolism , Gene Expression Regulation, Neoplastic , Humans , Male , Mice , Mice, Nude , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Interleukin/genetics , Receptors, Interleukin/metabolism , Signal Transduction , TransfectionABSTRACT
BACKGROUND: Epidemiological and in vitro data implicate androgens in the aetiology of ovarian cancer, but the mechanisms by which this is mediated are unclear. In this study, we wished to examine the effects of androgens on gene expression in ovarian cancer. METHODS: The expression of androgen receptor (AR) in OVCAR3 and OSEC2 cells was confirmed using immunoblotting and response to androgens was measured using flow cytometric assessment of S-phase fraction. The differential gene expression between androgen stimulated and unstimulated OVCAR3 ovarian cancer cells was examined with a cDNA microarray. The upregulation of a subset of these genes was then confirmed with reverse transcriptase PCR in both OVCAR3 and OSEC2, an ovarian epithelial cell line. Finally, the clinical significance of this upregulation was investigated by examining the expression of Rab25 and Rab35, two G-protein-related molecules in an ovarian cancer tissue microarray (TMA). RESULTS: OVCAR3 and OSEC2 cells were shown to express the AR and showed an increase in S-phase fraction in response to androgen treatment. Treatment of OVCAR3 cells with androgen resulted in a significant upregulation of 121 genes. These findings were confirmed for a subset of seven monomeric G-protein-related genes in both OVCAR3 and OSEC2 cells. After staining for Rab25 and Rab35, the majority of TMA sections examined showed expression for Rab25 (92%) and Rab35 (95%). The expression of Rab25 correlated with histological grade, and expression was higher in endometrioid (median histoscore 10.5) than serous (7.5) or mucinous (5.3) tumours. The expression of Rab25 correlated positively with AR expression supporting its role as an androgen responsive gene in ovarian cancer. CONCLUSIONS: These results suggest that androgens can effect expression of the oncogenic GTPases in ovarian cancer. We propose that the androgen responsive Rab35 may have clinical importance as a biomarker of AR function.
Subject(s)
Dihydrotestosterone/pharmacology , Ovarian Neoplasms/metabolism , rab GTP-Binding Proteins/genetics , Cell Line, Tumor , Female , Humans , Ovarian Neoplasms/pathology , RNA, Messenger/analysis , Receptors, Androgen/genetics , Tissue Array AnalysisABSTRACT
The majority of epithelial ovarian cancers originate in the ovarian surface epithelium. The ovarian surface epithelium is a hormonally responsive tissue, and hormones are thought to play a key role in the development of this type of cancer. Gonadotrophin releasing hormone II is one of 2 isoforms which are thought to act through gonadotrophin releasing hormone receptor I, and gonadotrophin releasing hormone II has been shown to cause growth inhibition of cultured ovarian surface epithelium. The aim of this study was to investigate the expression levels and prognostic significance of gonadotrophin releasing hormone II and the gonadotrophin releasing hormone receptor I in epithelial ovarian cancer. Gonadotrophin releasing hormone II and gonadotrophin releasing hormone receptor I messenger RNA expression was examined in 23 cancers and 7 normal ovarian surface epithelium samples by quantitative real time polymerase chain reaction. An ovarian cancer tissue microarray containing 139 cases was constructed and immunohistochemical analysis of gonadotrophin releasing hormone II and gonadotrophin releasing hormone receptor I protein expression was performed and correlated with clinical outcome data. Gonadotrophin releasing hormone II messenger RNA expression was lower in cancer samples compared to normal ovarian surface epithelium samples (P < .05). Gonadotrophin releasing hormone II protein expression correlated with histologic subtype (25% serous versus 45% nonserous, P < .05) but not with overall survival. Gonadotrophin releasing hormone receptor I messenger RNA expression was highest in serous tumors when compared to non serous (P < .05) and normal tissue (P < .001). Expression of the gonadotrophin releasing hormone receptor I protein was also found to correlate with patient survival (P < .05). We have demonstrated gonadotrophin releasing hormone II and its receptor, gonadotrophin releasing hormone receptor I, are present in clinical ovarian samples, and that gonadotrophin releasing hormone receptor I protein expression is a favorable prognostic factor, suggesting these proteins play an important role in the development of epithelial ovarian cancer.
Subject(s)
Carcinoma/chemistry , Ovarian Neoplasms/chemistry , Receptors, LHRH/analysis , Adult , Aged , Aged, 80 and over , Female , Humans , Immunohistochemistry , Middle Aged , Ovary/chemistry , Prognosis , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, CulturedABSTRACT
The RNA-binding protein Sam68 has been reported to be up-regulated in clinical cases of prostate cancer (PCa), where it is thought to contribute to cell proliferation and survival. Consistent with this, we observed over-expression of Sam68 in a panel of clinical prostate tumours as compared with benign controls. Since Sam68 is implicated in a number of signalling pathways, we reasoned that its role in PCa may involve modulation of the androgen receptor (AR) signalling cascade, which drives the onset and progression of PCa. We found that Sam68 interacts with the AR in vivo in LNCaP cells, and is dynamically recruited to androgen response elements within the promoter region of the prostate-specific antigen (PSA) gene. Based on its known functions and nuclear location, Sam68 might either: (a) co-regulate AR-dependent transcription positively or negatively; or (b) modulate AR-dependent alternative splicing by enhancing incorporation of a Sam68-responsive exon transcribed under the control of an androgen-responsive promoter. We tested these possibilities using functional assays. Both wild-type Sam68 protein and the Sam68(V229F) mutant, which is impaired in RNA binding, functioned as a ligand-dependent AR co-activator on an androgen-regulated reporter gene. In contrast, splicing of a Sam68-responsive variable exon, transcribed under control of an androgen-responsive promoter, was strongly repressed in the presence of AR and androgens. This splicing inhibition was reversed by ectopic expression of Sam68 but enhanced by Sam68(V229F). These results demonstrate that Sam68 has separable effects on AR-regulated transcriptional activity and alternative splicing, both of which may affect PCa phenotypes.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Alternative Splicing , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic , Prostatic Neoplasms/metabolism , RNA-Binding Proteins/metabolism , Receptors, Androgen/metabolism , Transcription, Genetic/physiology , Androgens/metabolism , Case-Control Studies , Gene Expression , Gene Expression Profiling , Humans , Hyaluronan Receptors/genetics , Immunohistochemistry , Male , Mutation , Oligonucleotide Array Sequence Analysis , Prostate-Specific Antigen/metabolismABSTRACT
Abnormal intracellular signaling contributes to carcinogenesis and may represent novel therapeutic targets. mitogen/extracellular signal-regulated kinase kinase-5 (MEK5) overexpression is associated with aggressive prostate cancer. In this study, we examined the role of extracellular signal-regulated kinase (ERK5, an MAPK and specific substrate for MEK5) in prostate cancer. ERK5 immunoreactivity was significantly upregulated in high-grade prostate cancer when compared to benign prostatic hyperplasia (P<0.0001). Increased ERK5 cytoplasmic signals correlated closely with Gleason sum score (P<0.0001), bony metastases (P=0.0044) and locally advanced disease at diagnosis (P=0.0023), with a weak association with shorter disease-specific survival (P=0.036). A subgroup of patients showed strong nuclear ERK5 localization, which correlated with poor disease-specific survival and, on multivariant analysis, was an independent prognostic factor (P<0.0001). Analysis of ERK5 expression in matched tumor pairs (before and after hormone relapse, n=26) revealed ERK5 nuclear expression was significantly associated with hormone-insensitive disease (P=0.0078). Similarly, ERK5 protein expression was increased in an androgen-independent LNCaP subline. We obtained the following in vitro and in vivo evidence to support the above expression data: (1) cotransfection of ERK5wt and MEK5D constructs in PC3 cells results in predominant ERK5 nuclear localization, similar to that observed in aggressive clinical disease; (2) ERK5-overexpressing PC3 cells have enhanced proliferative, migrative and invasive capabilities in vitro (P<0.0001), and were dramatically more efficient in forming tumors, with a shorter mean time for tumors to reach a critical volume of 1000 mm(3), in vivo (P<0.0001); (3) the MEK1 inhibitor, PD184352, blocking ERK1/2 activation at low dose, did not suppress proliferation but did significantly decrease proliferation at a higher dose required to inhibit ERK5 activation. Taken together, our results establish the potential importance of ERK5 in aggressive prostate cancer.
Subject(s)
MAP Kinase Kinase 5/genetics , Prostatic Neoplasms/enzymology , Aged , Aged, 80 and over , Cell Proliferation , Disease-Free Survival , Humans , Immunohistochemistry , Male , Middle Aged , Prognosis , Prostatic Neoplasms/pathologyABSTRACT
Bone morphogenetic protein-6 (BMP-6) has been strongly implicated in prostate cancer development and bone metastasis. Our previous data showed that BMP-6 mRNA was absent in patients with benign prostatic hyperplasia, but evident in primary tumours with established secondary skeletal metastases. To examine the role of BMP-6 in prostate cancer progression, we have developed a BMP-6-regulatable, doxycycline-inducible gene expression system. BMP-6 induction by doxycycline addition led to increased levels of BMP-6 RNA and protein, associated with nuclear translocation of SMADs and activation of the downstream target gene Id-1. BMP-6 protein did not enhance the proliferation rate of PC3M cells but did significantly increase the rate of migration and invasion in both PC3M and DU145 cells. Increased metalloproteinase (MMP-1 and MMP-9) mRNA levels were also observed following BMP-6 induction. Luciferase reporter assays confirmed BMP-6-mediated activation of MMP-1 and MMP-9 promoters, indicating direct transcriptional activation of MMPs by BMP-6. BMP-6 stimulation also led to an increase in phosphorylation levels of MAPK proteins. We next examined the effects of BMP-6 on the downstream gene Id-1 in a cohort of prostate cancer patients. A tissue microarray (TMA) was constructed and samples stained for BMP-6 and Id-1 expression. We observed a significant increase in the intensity of staining of epithelial BMP-6 in the cancer cases compared to the benign cases (Mann-Whitney U test, p < 0.0005) and in the intensity of staining of epithelial Id-1 in the cancer cases compared to the benign cases (Mann-Whitney U test, p = 0.015). We further observed a significant positive correlation between epithelial staining for Id-1 and BMP-6 (p = 0.001) across all samples for both benign and cancer cases. These data demonstrate that BMP-6 promotes migration and invasion of prostate cancer cells, potentially through activation of Id-1 and MMP activation.
Subject(s)
Biomarkers, Tumor , Bone Morphogenetic Proteins/genetics , Gene Expression Regulation, Neoplastic , Inhibitor of Differentiation Protein 1/genetics , Prostatic Neoplasms/metabolism , Biomarkers, Tumor/analysis , Bone Morphogenetic Protein 6 , Bone Morphogenetic Proteins/analysis , Case-Control Studies , Cell Line, Tumor , Doxycycline/pharmacology , Gene Expression Profiling , Humans , Immunohistochemistry , Inhibitor of Differentiation Protein 1/analysis , Male , Matrix Metalloproteinase 1/genetics , Matrix Metalloproteinase 9/genetics , Neoplasm Invasiveness , Oligonucleotide Array Sequence Analysis , Phenotype , Prostate/metabolism , Prostatic Neoplasms/genetics , RNA, Messenger/analysis , Smad1 Protein/genetics , Statistics, Nonparametric , Transfection/methodsABSTRACT
GnRH II has important functional effects in steroid hormone-dependent tumours. Here we investigated the expression and regulation of GnRH II in prostate cancer. GnRH II protein was equally expressed in benign (73%) and malignant (78%) biopsies studied in a prostate tissue microarray (P = 0.779). There was no relationship between expression and clinical parameters in the cancer cohort. GnRH II was, however, significantly reduced in tumour biopsies following hormone ablation. This was further investigated in a prostate xenograft model where androgens increased GnRH II levels, while their withdrawal reduced it. In cell lines, we confirmed high levels of GnRH II in androgen receptor (AR)-positive LNCaP cells but low levels in AR-negative PC3 cells. In LNCaP cells, GnRH II induction by androgens was blocked by the AR inhibitor casodex, but not by cycloheximide treatment. Sequence analysis subsequently revealed a putative androgen response element in the upstream region of the GnRH II gene and direct interaction with the AR was confirmed in chromatin immunoprecipitation experiments. Finally, to test whether the effects of GnRH II were dependent on AR expression, LNCaP and PC3 cells were exposed to exogenous peptide. In both cell lines, GnRH II inhibited cell proliferation and migration, suggesting that its function is independent of AR status. These results provide evidence that GnRH II is widely expressed in prostate cancer and is an AR-regulated gene. Further studies are warranted to characterise the effects of GnRH II on prostate cancer cells and investigate its potential value as a novel therapy.
Subject(s)
Gene Expression Regulation, Neoplastic , Gonadotropin-Releasing Hormone/analogs & derivatives , Prostatic Neoplasms/genetics , Receptors, Androgen/physiology , Androgens/pharmacology , Animals , Base Sequence , Binding Sites , Cell Proliferation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Gonadotropin-Releasing Hormone/genetics , Gonadotropin-Releasing Hormone/metabolism , Humans , Male , Mice , Mice, Nude , Molecular Sequence Data , Neoplasm Invasiveness , Neoplasms, Hormone-Dependent/genetics , Neoplasms, Hormone-Dependent/metabolism , Neoplasms, Hormone-Dependent/pathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Receptors, Androgen/metabolism , Response Elements , Transplantation, Heterologous , Tumor Cells, CulturedABSTRACT
Fibroblast growth factor receptors (FGFRs) mediate the tumourigenic effects of FGFs in prostate cancer. These receptors are therefore potential therapeutic targets in the development of inhibitors to this pathway. To identify the most relevant targets, we simultaneously investigated FGFR1-4 expression using a prostate cancer tissue microarray (TMA) and in laser capture microdissected (LCM) prostate epithelial cells. In malignant prostates (n = 138) we observed significant FGFR1 and FGFR4 protein over-expression in comparison with benign prostates (n = 58; p < 0.0001). FGFR1 was expressed at high levels in the majority of tumours (69% of grade 3 or less, 74% of grade 4 and 70% of grade 5), while FGFR4 was strongly expressed in 83% of grade 5 cancers but in only 25% of grade 1-3 cancers (p < 0.0001). At the transcript level we observed a similar pattern, with FGFR1 and FGFR4 mRNA over-expressed in malignant epithelial cells compared to benign cells (p < 0.0005 and p < 0.05, respectively). While total FGFR2 was increased in some cancers, there was no association between expression and tumour grade or stage. Transcript analysis, however, revealed a switch in the predominant isoform expressed from FGFR2IIIb to FGFR2IIIc among malignant epithelial cells. In contrast, protein and transcript expression of FGFR3 was very similar between benign and cancer biopsies. The functional effect of targeting FGFR4 in prostate cancer cells has not previously been investigated. In in vitro experiments, suppression of FGFR4 by RNA interference effectively blocked prostate cancer cell proliferation (p < 0.0001) and invasion (p < 0.001) in response to exogenous stimulation. This effect was evident regardless of whether the cells expressed the FGFR4 Arg388 or Gly388 allele. In parallel experiments, FGFR3 suppression had no discernible effect on cancer cell behaviour. These results suggest evidence of selective over-expression of FGFR1 and FGFR4 in clinical prostate cancer and support the notion of targeted inhibition of these receptors to disrupt FGF signalling.
Subject(s)
Gene Expression Regulation, Neoplastic , Prostatic Neoplasms/genetics , Receptors, Fibroblast Growth Factor/genetics , Case-Control Studies , Cell Line, Tumor , Cell Proliferation , Gene Expression Profiling , Humans , Immunohistochemistry , Male , Microdissection , Microscopy, Confocal , Oligonucleotide Array Sequence Analysis , Polymorphism, Single Nucleotide , Prostatic Neoplasms/metabolism , Protein Isoforms/genetics , RNA Interference , RNA, Small Interfering/pharmacology , Receptor, Fibroblast Growth Factor, Type 1/analysis , Receptor, Fibroblast Growth Factor, Type 1/genetics , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Receptor, Fibroblast Growth Factor, Type 2/analysis , Receptor, Fibroblast Growth Factor, Type 2/genetics , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Receptor, Fibroblast Growth Factor, Type 3/analysis , Receptor, Fibroblast Growth Factor, Type 3/genetics , Receptor, Fibroblast Growth Factor, Type 3/metabolism , Receptor, Fibroblast Growth Factor, Type 4/analysis , Receptor, Fibroblast Growth Factor, Type 4/genetics , Receptor, Fibroblast Growth Factor, Type 4/metabolism , Receptors, Fibroblast Growth Factor/analysis , Receptors, Fibroblast Growth Factor/metabolism , Transcription, GeneticABSTRACT
Antiandrogens are initially effective in controlling prostate cancer (CaP), the second most common cancer in men, but resistance, associated with the loss of androgen-regulated cell cycle control, is a major problem. At present there is no effective treatment for androgen-independent prostate cancer (AIPC). Cellular proliferation is driven by cyclin-dependent kinases (CDKs) with kinase inhibitors (for example, p27) applying the breaks. We present the first investigation of the therapeutic potential of CDK inhibitors, using the guanine-based CDK inhibitor NU2058 (CDK2 IC(50)=17 microM, CDK1 IC(50)=26 microM), in comparison with the antiandrogen bicalutamide (Casodex) in AIPC cells. A panel of AIPC cells was found to be resistant to Casodex-induced growth inhibition, but with the exception of PC3 (GI(50)=38 microM) and CWR22Rv1 (GI(50)=46 microM) showed similar sensitivity to NU2058 (GI(50)=10-17 microM) compared to androgen-sensitive LNCaP cells (GI(50)=15 microM). In LNCaP cells and their Casodex-resistant derivative, LNCaP-cdxR, growth inhibition by NU2058 was accompanied by a concentration-dependent increase in p27 levels, reduced CDK2 activity and pRb phosphorylation, a decrease in early gene expression and G1 cell cycle phase arrest in both cell lines. In response to Casodex, there were similar observations in LNCaP cells (GI(50)=6+/-3 microM Casodex) but not in LNCaP-cdxR cells (GI(50)=24+/-5 microM Casodex).
Subject(s)
Anilides/therapeutic use , Cyclin-Dependent Kinases/antagonists & inhibitors , Guanine/analogs & derivatives , Nitriles/therapeutic use , Prostatic Neoplasms/drug therapy , Protein Kinase Inhibitors/therapeutic use , Tosyl Compounds/therapeutic use , Androgens/metabolism , CDC2 Protein Kinase/antagonists & inhibitors , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Guanine/therapeutic use , Humans , Male , Phosphorylation/drug effects , Retinoblastoma Protein/metabolismABSTRACT
Abnormal differentiation in epithelial stem cells or their immediate proliferative progeny, the transiently amplifying population (TAP), may explain malignant pathogenesis in the human prostate. These models are of particular importance as differing sensitivities to androgen among epithelial cell subpopulations during differentiation are recognised and may account for progression to androgen independent prostate cancer. Androgens are crucial in driving terminal differentiation and their indirect effects via growth factors from adjacent androgen responsive stroma are becoming better characterised. However, direct effects of androgen on immature cells in the context of a prostate stem cell model have not been investigated in detail and are studied in this work. In alpha2beta1hi stem cell enriched basal cells, androgen analogue R1881 directly promoted differentiation by the induction of differentiation-specific markers CK18, androgen receptor (AR), PSA and PAP. Furthermore, treatment with androgen down-regulated alpha2beta1 integrin expression, which is implicated in the maintenance of the immature basal cell phenotype. The alpha2beta1hi cells were previously demonstrated to lack AR expression and the direct effects of androgen were confirmed by inhibition using the anti-androgen bicalutamide. AR protein expression in alpha2beta1hi cells became detectable when its degradation was repressed by the proteosomal inhibitor MG132. Stratifying the alpha2beta1hi cells into stem (CD133(+)) and transient amplifying population (TAP) (CD133(-)) subpopulations, AR mRNA expression was found to be restricted to the CD133(-) (TAP) cells. The presence of a functional AR in the TAP, an androgen independent subpopulation for survival, may have particular clinical significance in hormone resistant prostate cancer, where both the selection of immature cells and functioning AR regulated pathways are involved.
Subject(s)
Cell Differentiation/drug effects , Cell Proliferation/drug effects , Epithelial Cells/drug effects , Metribolone/pharmacology , Neoplastic Stem Cells/drug effects , Prostatic Neoplasms/metabolism , Receptors, Androgen/drug effects , Testosterone Congeners/pharmacology , AC133 Antigen , Acid Phosphatase , Aged , Aged, 80 and over , Androgen Antagonists/pharmacology , Anilides/pharmacology , Antigens, CD/analysis , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/metabolism , Cells, Cultured , Cysteine Proteinase Inhibitors/pharmacology , Dose-Response Relationship, Drug , Epithelial Cells/metabolism , Epithelial Cells/pathology , Fibroblast Growth Factor 7/metabolism , Glycoproteins/analysis , Humans , Integrin alpha2beta1/metabolism , Keratin-18/biosynthesis , Leupeptins/pharmacology , Male , Middle Aged , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Nitriles/pharmacology , Peptides/analysis , Phenotype , Prostate-Specific Antigen/biosynthesis , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors , Protein Tyrosine Phosphatases/biosynthesis , RNA, Messenger/biosynthesis , Receptors, Androgen/biosynthesis , Receptors, Androgen/genetics , Signal Transduction/drug effects , Tosyl Compounds/pharmacologyABSTRACT
Fibroblast growth factors (FGF), and in particular FGF8, have been strongly implicated in prostate carcinogenesis. This study investigated the expression of Sef, a key inhibitory regulator of FGF signalling, in prostate cancer. In a panel of cell lines, hSef was detected in both androgen-dependent and independent cells but was significantly reduced in highly metastatic derivative clones. hSef expression was not influenced by androgenic stimulation. Forced downregulation of hSef by siRNA increased FGF8b induced cell migration (P=0.02) and invasion (P=0.007). Reduced hSef levels also enhanced FGF8b stimulated expression of MMP9 (P=0.005). mRNA in situ hybridization revealed hSef expression in 80% (8/10) of benign biopsies but in only 69% (23/33) of Gleason sum 4-7 and 35% (10/28) of Gleason sum 8-10 cancer biopsies (P=0.004). Quantitative PCR of microdissected glands confirmed this trend (P=0.001). hSef was expressed in 69% (27/39) of non-metastatic tumours but in only 18% (2/11) of metastatic tumours (P=0.004, n=50). hSef expression was next correlated with earlier data on FGF8b expression in a subgroup of cancers. In this cohort, 86% (19/22) of high-grade cancers expressed FGF8 but only 31% (7/22) expressed hSef. Positive FGF8 expression but a loss of hSef was observed in 88% (7/8) of metastatic tumours. In contrast, metastasis was evident in only 10% (1/10) of tumours, which co-expressed both FGF8 and hSef (P<0.001). These results suggest evidence that hSef is downregulated in advanced prostate cancer and might facilitate an enhanced tumorigenic response to FGFs. Further research into the role of hSef in cancer cell signalling and the mechanism of its downregulation may contribute to more effective targeting of growth factors in prostate cancer.
Subject(s)
Down-Regulation/genetics , Gene Expression Regulation, Neoplastic/genetics , Prostatic Neoplasms/genetics , Receptors, Interleukin/genetics , Signal Transduction/genetics , Androgens/metabolism , Cell Line, Tumor , Fibroblast Growth Factor 8/biosynthesis , Fibroblast Growth Factor 8/genetics , Gene Expression Profiling , Humans , Male , Neoplasm Metastasis/genetics , Neoplasm Metastasis/pathology , Polymerase Chain Reaction , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Receptors, Interleukin/metabolismABSTRACT
PURPOSE: We determined whether the nature of any protective barrier in the bladder is composed of a secreted mucous gel layer. MATERIALS AND METHODS: We collected 24-hour urine samples for analysis from 8 healthy 22 to 49-year-old volunteers and 5, 19 to 59-year-old patients treated with bladder reconstruction, in addition to scrapings from 100 freshly slaughtered pig bladders. Samples were subjected to homogenization, dialysis, freeze-drying, papain digestion, gel chromatography, equilibrium density gradient centrifugation, periodic acid-Schiff assay and amino acid analysis. Normal human bladder, pig bladder, normal ileum and transposed intestinal segments were studied for the presence of a mucous layer using a new method of histological analysis. RESULTS: Mucin content in normal urine is 2.7 mg/24 hours, meaning that less than 0.6% of nondialyzable material in normal urine is mucin. The mucin content of urine from reconstructed bladders amounted to 86 mg/24 hours (5.2% of nondialyzable material). We observed that glycosaminoglycans accounted for 41% of the peak total elution volume of PAS positive material in normal urine. Mucin estimation in urine can be grossly overestimated if contaminating glycoconjugates are not removed. Biochemical analysis of material scraped off the pig bladder surface demonstrated that the maximum thickness of a continuous layer that could be achieved was 13.6 mum. While we could visualize an obvious mucous layer on control ileal samples and biopsies of transposed ileal segments from patients with bladder reconstruction, we were unable to note a distinct, measurable mucous layer lining the bladder surface in humans or pigs. CONCLUSIONS: Mucin levels in normal human and pig urine would be enough for slow turnover of a thin barrier but the large increase in mucin in the urine of patients with transposed intestinal segments demonstrates that any layer in normal bladder is much different than that lining the transposed intestinal segment. The most likely constituents of this barrier are membrane bound rather than secreted mucins along with the proteoglycan components of the glycocalix.
Subject(s)
Mucins/urine , Mucus/metabolism , Urinary Bladder/pathology , Urinary Diversion , Urothelium/metabolism , Adult , Amino Acids/analysis , Animals , Biopsy , Female , Gels , Humans , Ileum/pathology , Intestinal Mucosa/pathology , Male , Middle Aged , Postoperative Complications/urine , Reference Values , Swine , Urinary Bladder/chemistryABSTRACT
Gonadotropin-releasing hormone analogue (GnRHa) therapy is an established method of androgen withdrawal in the treatment of prostate cancer. The present study investigated if the expression of prostate GnRH receptors (GnRHRs) might influence the response to GnRHa. GnRHR protein expression was first studied in a panel of prostate cancer cell lines. In androgen-dependent cells, GnRHR expression was unchanged following acute or chronic androgen withdrawal. In these cells, GnRHa significantly inhibited androgen-induced cell proliferation (p = 0.01). In contrast, GnRHa was unable to further suppress basal levels of cell proliferation induced by androgen withdrawal. In androgen-independent prostate cancer cells, variable levels of GnRHR expression were observed. In these cells, GnRHa treatment blocked cell proliferation (p = 0.001) and invasion (up to 70%) induced by fibroblast growth factor stimulation. Crucially, this effect was only evident in cells that expressed high levels of the GnRHR. GnRHa treatment also significantly inhibited the ability of these cells to recover from a cytotoxic insult (50% inhibition). The clinical significance of prostate GnRHR was tested by immunohistochemistry in a preliminary cohort of patients treated with GnRHa or surgical castration. There was no association between GnRHR expression and pathological grade, clinical stage, time to PSA nadir (p = 0.82) (n = 35) or progression to hormone refractory disease (p = 0.22) (n = 21), irrespective of the treatment method. GnRHa therapy in the presence of high GnRHR expression however, was found to be associated with longer disease-specific survival (mean survival 85 months, p = 0.002). In contrast, high GnRHR expression was not associated with survival among surgically castrated patients (mean survival 50 months, p = 0.7). Taken together, these data support the notion of a functional interaction between GnRHa and the GnRHR, which results in an anti-tumourigenic effect on prostate cancer cells. Findings from this report have direct implications for the use of GnRHR as a novel therapeutic target in hormone refractory prostate cancer.