ABSTRACT
BACKGROUND: TWIST1 plays a key role in EMT-mediated tumor invasion and metastasis. Since bone metastasis is a hallmark of advanced prostate cancer and is detected in at least 85% of patients who die of this disease, it is of great importance to understand the regulation of the cellular signaling pathways involved in the metastatic process. METHODS: Prostatic cell lines were analyzed using real time RT-PCR, chromatin immunoprecipitations (ChIP) and transfection of siRNA's and reporter constructs. RESULTS: We report in this paper that TWIST1 is an androgen-regulated gene under tight regulation of NKX3-1. Androgens repress the expression of TWIST1 via NKX3-1, which is a prostate-specific tumor suppressor that is down-regulated in the majority of metastatic prostate tumors. We show that NKX3-1 binds to the TWIST1 promoter and that NKX3-1 over-expression reduces the activity of a TWIST1 promoter reporter construct, whereas NKX3-1 siRNA up-regulates endogenous TWIST1 mRNA in prostate cancer cells. CONCLUSION: Our finding that NKX3-1 represses TWIST1 expression emphasizes the functional importance of NKX3-1 in regulating TWIST1 expression during prostate cancer progression to metastatic disease.
ABSTRACT
BACKGROUND: Androgen deprivation is the only effective systemic therapy available for patients with prostatic carcinoma, but is associated with a gradual transition to a hormone-refractory prostate cancer (HRCAP) in which ligand-independent activation of the androgen receptor has been implicated. The beta(2)-adrenergic receptor (beta(2)-AR) is a well-known activator of the androgen receptor. METHODS: Prostatic cell lines were analyzed using cDNA micro-array, real time RT-PCR, radioligand binding assay, cAMP measurements, transfection and thymidine incorporation assay. Clinical specimens were studied by immunohistochemistry and Affymetrix microarrays. RESULTS: Here, we show that beta(2)-AR was transiently down-regulated both at mRNA- and protein levels when hormone-sensitive prostate cancer cells, LNCaP, were cultured in steroid stripped medium (charcoal-stripped fetal calf serum) or when the cells were treated with the anti-androgen, bicalutamide (Casodex). The number of beta-adrenergic receptors was modestly up-regulated in androgen independent cell lines (LNCaP-C4, LNCaP-C4-2 and DU145) compared to LNCaP. Triiodothyronine (T3) increased the level of beta(2)-AR and the effect of T3 was inhibited by bicalutamide. Immunohistochemical staining of human prostate specimens showed high expression of beta(2)-AR in glandular, epithelial cells and increased expression in malignant cells compared to benign hyperplasia and normal tissue. Interestingly, beta(2)-AR mRNA was strongly down-regulated by androgen ablation therapy of prostate cancer patients. CONCLUSION: The level of beta(2)-AR was increased by T3 in prostatic adenocarcinoma cells and reduced in prostate cancer patients who had received androgen ablation therapy for 3 months.
Subject(s)
Adenocarcinoma/physiopathology , Prostatic Neoplasms/physiopathology , Receptors, Adrenergic, beta-2/genetics , Receptors, Adrenergic, beta-2/metabolism , Adenocarcinoma/drug therapy , Adenocarcinoma/pathology , Androgen Antagonists/pharmacology , Androgen Antagonists/therapeutic use , Androgens/metabolism , Anilides/pharmacology , Antineoplastic Agents/pharmacology , Biopsy , Cell Line, Tumor , Down-Regulation , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/physiology , Humans , Immunohistochemistry , Male , Nitriles/pharmacology , Oligonucleotide Array Sequence Analysis , Prostate/metabolism , Prostate/pathology , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Receptors, Androgen/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , Tosyl Compounds/pharmacology , Triiodothyronine/pharmacology , Up-RegulationABSTRACT
Neuroendocrine (NE) cells may play a role in prostate cancer progression. Both androgen deprivation and cAMP are well known inducers of NE differentiation (NED) in the prostate. Gene-expression profiling of LNCaP cells, incubated in androgen stripped medium, showed that the Cbeta isoform of PKA is up-regulated during NE differentiation. Furthermore, by using semi-quantitative RT-PCR and immunoblotting analysis, we observed that the Cbeta splice variants are differentially regulated during this process. Whereas the Cbeta2 splice variant is down-regulated in growth arrested LNCaP cells, the Cbeta1, Cbeta3 and Cbeta4 variants, as well as the RIIbeta subunit of PKA, are induced in NE-like LNCaP cells. The opposite effect of Cbeta expression could be mimicked by androgen stimulation, implying the Cbeta gene of PKA as a putative new target gene for the androgen receptor in prostate cancer. Moreover, to investigate expression of PKA subunits during prostate cancer progression, we did immunoblotting of several prostatic cell lines and normal and tumor tissue from prostate cancer patients. Interestingly, multiple Cbeta subunits were also observed in human prostate specimens, and the Cbeta2 variant was up-regulated in tumor cells. In conclusion, it seems that the Cbeta isoforms play different roles in proliferation and differentiation and could therefore be potential markers for prostate cancer progression.
Subject(s)
Androgens/pharmacology , Cyclic AMP-Dependent Protein Kinases/metabolism , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Prostatic Neoplasms/metabolism , Cell Differentiation , Cell Line, Tumor , Cyclic AMP-Dependent Protein Kinases/genetics , Gene Expression Profiling , Humans , Isoenzymes/chemistry , Male , Neoplasm Staging , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Protein IsoformsABSTRACT
BACKGROUND: Prostate cancer is the most frequent new cancer diagnosis in the western world today. There is an urgent need to obtain validated molecular markers that can identify clinically significant prostate cancer. MATERIAL AND METHODS: The article represents our view of the current status of molecular markers in diagnosis of prostate cancer based on literature searches (PubMed). RESULTS AND INTERPRETATION: Prostate specific antigen (PSA) is a sensitive serum marker for pathology in the prostate (cancer, infection, benign hyperplasia). The level of PSA, however, is poorly correlated with grade and stage of prostate cancer. Genomic and proteomic methodology has recently been used to discover more then 200 putative new markers for prostate cancer like alpha-methylacyl CoA racemase (AMACR), hepsin, glutathione S-transferase pi, EZH2 and DD3(PCA3). To date, none of these markers have been adequately validated for clinical use. Knowledge about the role of these candidates in prostate cancer biology and evaluation of their correlation to clinical parameters will be of importance in the validation process.
Subject(s)
Biomarkers, Tumor/analysis , Prostatic Neoplasms/diagnosis , Genetic Predisposition to Disease , Humans , Male , Molecular Diagnostic Techniques , Predictive Value of Tests , Prognosis , Prostate-Specific Antigen/blood , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Sensitivity and SpecificityABSTRACT
Protein kinase A (PKA)-anchoring protein AKAP95 is localized to the nucleus in interphase, where it primarily associates with the nuclear matrix. A yeast two-hybrid screen for AKAP95 interaction partners identified the minichromosome maintenance (MCM) 2 protein, a component of the pre-replication complex. AKAP95-MCM2 interaction was mapped to residues 1-195 of AKAP95 and corroborated by glutathione S-transferase precipitation and immunoprecipitation from chromatin. Disruption of AKAP95-MCM2 interaction with an AKAP95-(1-195) peptide within HeLa cell nuclei abolishes initiation of DNA replication in G1 phase and the elongation phase of replication in vitro without affecting global nuclear organization or import. Disruption of the C-terminal zinc finger of AKAP95 reduces efficiency of replication initiation. Disruption of the PKA-binding domain does not impair replication in G1- or S-phase nuclei, whereas a PKA inhibitor affects the initiation but not the elongation phase of replication. Depleting AKAP95 from nuclei partially depletes MCM2 and abolishes replication. Recombinant AKAP95 restores intranuclear MCM2 and replication in a dose-dependent manner. Our results suggest a role of AKAP95 in DNA replication by providing a scaffold for MCM2.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , DNA Replication/physiology , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Binding Sites , Cell Nucleus/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , DNA-Binding Proteins/chemistry , Enzyme Inhibitors/pharmacology , G1 Phase , HeLa Cells , Humans , In Vitro Techniques , Intracellular Signaling Peptides and Proteins , Minichromosome Maintenance Complex Component 2 , Nuclear Proteins/chemistry , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Two-Hybrid System TechniquesABSTRACT
A-kinase (or PKA)-anchoring protein AKAP95 is a zinc-finger protein implicated in mitotic chromosome condensation by acting as a targeting molecule for the condensin complex. We have identified determinants of chromatin-binding, condensin-targeting and chromosome-condensation activities of AKAP95. Binding of AKAP95 to chromatin is conferred by residues 387-450 and requires zinc finger ZF1. Residues 525-569 are essential for condensation of AKAP95-free chromatin and condensin recruitment to chromosomes. Mutation of either zinc finger of AKAP95 abolishes condensation. However, ZF1 is dispensable for condensin targeting, whereas the C-terminal ZF2 is required. AKAP95 interacts with Xenopus XCAP-H condensin subunit in vitro and in vivo but not with the human hCAP-D2 subunit. The data illustrate the involvement of overlapping, but distinct, domains of AKAP95 for condensin recruitment and chromosome condensation and argue for a key role of ZF1 in chromosome condensation and ZF2 in condensin targeting. Moreover, condensin recruitment to chromatin is not sufficient to promote condensation.
