RESUMO
The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of both NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor.
Assuntos
Núcleo Celular/metabolismo , Receptor EphB4/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Linhagem Celular Tumoral , DNA/metabolismo , Expressão Gênica , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Masculino , Dados de Sequência Molecular , Sinais de Localização Nuclear , Neoplasias da Próstata , Ligação Proteica , Receptor EphB4/química , alfa Carioferinas/metabolismoRESUMO
BACKGROUND: The EphB4 receptor tyrosine kinase is overexpressed in many cancers including prostate cancer. The molecular mechanisms by which this ephrin receptor influences cancer progression are complex as there are tumor-promoting ligand-independent mechanisms in place as well as ligand-dependent tumor suppressive pathways. METHODS: We employed transient knockdown of EPHB4 in prostate cancer cells, coupled with gene microarray analysis, to identify genes that were regulated by EPHB4 and may represent linked tumor-promoting factors. We validated target genes using qRT-PCR and employed functional assays to determine their role in prostate cancer migration and invasion. RESULTS: We discovered that over 500 genes were deregulated upon EPHB4 siRNA knockdown, with integrin ß8 (ITGB8) being the top hit (29-fold down-regulated compared to negative non-silencing siRNA). Gene ontology analysis found that the process of cell adhesion was highly deregulated and two other integrin genes, ITGA3 and ITGA10, were also differentially expressed. In parallel, we also discovered that over-expression of EPHB4 led to a concomitant increase in ITGB8 expression. In silico analysis of a prostate cancer progression microarray publically available in the Oncomine database showed that both EPHB4 and ITGB8 are highly expressed in prostatic intraepithelial neoplasia, the precursor to prostate cancer. Knockdown of ITGB8 in PC-3 and 22Rv1 prostate cancer cells in vitro resulted in significant reduction of cell migration and invasion. CONCLUSIONS: These results reveal that EphB4 regulates integrin ß8 expression and that integrin ß8 plays a hitherto unrecognized role in the motility of prostate cancer cells and thus targeting integrin ß8 may be a new treatment strategy for prostate cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica , Cadeias beta de Integrinas/biossíntese , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptor EphB4/fisiologia , Linhagem Celular Tumoral , Humanos , Masculino , Receptores Proteína Tirosina Quinases/fisiologiaRESUMO
Although at present, there is a high incidence of prostate cancer, particularly in the Western world, mortality from this disease is declining and occurs primarily only from clinically significant late stage tumors with a poor prognosis. A major current focus of this field is the identification of new biomarkers which can detect earlier, and more effectively, clinically significant tumors from those deemed "low risk", as well as predict the prognostic course of a particular cancer. This strategy can in turn offer novel avenues for targeted therapies. The large family of Receptor Tyrosine Kinases, the Ephs, and their binding partners, the ephrins, has been implicated in many cancers of epithelial origin through stimulation of oncogenic transformation, tumor angiogenesis, and promotion of increased cell survival, invasion and migration. They also show promise as both biomarkers of diagnostic and prognostic value and as targeted therapies in cancer. This review will briefly discuss the complex roles and biological mechanisms of action of these receptors and ligands and, with regard to prostate cancer, highlight their potential as biomarkers for both diagnosis and prognosis, their application as imaging agents, and current approaches to assessing them as therapeutic targets. This review demonstrates the need for future studies into those particular family members that will prove helpful in understanding the biology and potential as targets for treatment of prostate cancer.
Assuntos
Efrinas/fisiologia , Neoplasias da Próstata/tratamento farmacológico , Receptor EphA1/fisiologia , Biomarcadores , Efrinas/análise , Humanos , Masculino , Células Neoplásicas Circulantes/química , Neoplasias da Próstata/etiologia , Receptor EphA1/análise , Receptor EphA1/antagonistas & inibidores , Transdução de SinaisRESUMO
Overexpression of the receptor tyrosine kinase EphB4 is common in epithelial cancers and linked to tumor progression by promoting angiogenesis, increasing survival and facilitating invasion and migration. However, other studies have reported loss of EphB4 suggesting a tumor suppressor function in some cancers. These opposing roles may be regulated by (i) the presence of the primary ligand ephrin-B2 that regulates pathways involved in tumor suppression or (ii) the absence of ephrin-B2 that allows EphB4 signaling via ligand-independent pathways that contribute to tumor promotion. To explore this theory, EphB4 was overexpressed in the prostate cancer cell line 22Rv1 and the mammary epithelial cell line MCF-10A. Overexpressed EphB4 localized to lipid-rich regions of the plasma membrane and confirmed to be ligand-responsive as demonstrated by increased phosphorylation of ERK1/2 and internalization. EphB4 overexpressing cells demonstrated enhanced anchorage-independent growth, migration and invasion, all characteristics associated with an aggressive phenotype, and therefore supporting the hypothesis that overexpressed EphB4 facilitates tumor promotion. Importantly, these effects were reversed in the presence of ephrin-B2 which led to a reduction in EphB4 protein levels, demonstrating that ligand-dependent signaling is tumor suppressive. Furthermore, extended ligand stimulation caused a significant decrease in proliferation that correlated with a rise in caspase-3/7 and -8 activities. Together, these results demonstrate that overexpression of EphB4 confers a transformed phenotype in the case of MCF-10A cells and an increased metastatic phenotype in the case of 22Rv1 cancer cells and that both phenotypes can be restrained by stimulation with ephrin-B2, in part by reducing EphB4 levels.
Assuntos
Apoptose , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Efrina-B2/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptor EphB4/metabolismo , Western Blotting , Adesão Celular , Movimento Celular , Proliferação de Células , Feminino , Imunofluorescência , Humanos , Imunoprecipitação , Ligantes , Masculino , Microdomínios da Membrana , Fosforilação , Transdução de Sinais , Células Tumorais CultivadasRESUMO
EphB4 is a membrane-bound receptor tyrosine kinase (RTK) commonly over-produced by many epithelial cancers but with low to no expression in most normal adult tissues. EphB4 over-production promotes ligand-independent signaling pathways that increase cancer cell viability and stimulate migration and invasion. Several studies have shown that normal ligand-dependent signaling is tumour suppressive and therefore novel therapeutics which block the tumour promoting ligand-independent signaling and/or stimulate tumour suppressive ligand-dependent signaling will find application in the treatment of cancer. An EphB4-specific polyclonal antibody, targeting a region of 200 amino acids in the extracellular portion of EphB4, showed potent in vitro anti-cancer effects measured by an increase in apoptosis and a decrease in anchorage independent growth. Peptide exclusion was used to identify the epitope targeted by this antibody within the cysteine-rich region of the EphB4 protein, a sequence defined as a potential ligand interacting interface. Addition of antibody to cancer cells resulted in phosphorylation and subsequent degradation of the EphB4 protein, suggesting a mechanism that is ligand mimetic and tumour suppressive. A monoclonal antibody which specifically targets this identified extracellular epitope of EphB4 significantly reduced breast cancer xenograft growth in vivo confirming that EphB4 is a useful target for ligand-mimicking antibody-based anti-cancer therapies.
Assuntos
Anticorpos Monoclonais/genética , Cisteína/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptor EphB4/metabolismo , Anticorpos Monoclonais/farmacologia , Apoptose , Proliferação de Células , Humanos , Transdução de SinaisRESUMO
Ovarian cancer is the most lethal of all gynecologic malignancies, although its aetiology remains poorly understood. A role for the gonadotropins, follicle-stimulating hormone (FSH) and luteinising hormone (LH), has been implicated in a variety of different aspects of ovarian cancer tumorigenesis, including cellular proliferation, migration and invasion. This review focuses on the latest advances in knowledge concerning signalling pathways and functional consequences of gonadotropin action, including changes in protein-, miRNA- and gene expression, in epithelial ovarian cancer cells.
Assuntos
Gonadotropinas/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/metabolismo , Transdução de Sinais , Animais , Antineoplásicos Hormonais/metabolismo , Antineoplásicos Hormonais/uso terapêutico , Carcinoma Epitelial do Ovário , Feminino , Hormônio Foliculoestimulante/metabolismo , Gonadotropinas/genética , Humanos , Ligantes , Hormônio Luteinizante/metabolismo , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Receptores da Gonadotropina/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The gonadotropin hypothesis proposes that elevated serum gonadotropin levels may increase the risk of epithelial ovarian cancer (EOC). We have studied the effect of treating EOC cell lines (OV207 and OVCAR-3) with FSH or LH. Both gonadotropins activated the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and increased cell migration that was inhibited by the MAPK 1 inhibitor PD98059. Both extra- and intracellular calcium ion signalling were implicated in gonadotropin-induced ERK1/2 activation as treatment with either the calcium chelator EGTA or an inhibitor of intracellular calcium release, dantrolene, inhibited gonadotropin-induced ERK1/2 activation. Verapamil was also inhibitory, indicating that gonadotropins activate calcium influx via L-type voltage-dependent calcium channels. The cAMP/protein kinase A (PKA) pathway was not involved in the mediation of gonadotropin action in these cells as gonadotropins did not increase intracellular cAMP formation and inhibition of PKA did not affect gonadotropin-induced phosphorylation of ERK1/2. Activation of ERK1/2 was inhibited by the protein kinase C (PKC) inhibitor GF 109203X as well as by the PKCdelta inhibitor rottlerin, and downregulation of PKCdelta was inhibited by small interfering RNA (siRNA), highlighting the importance of PKCdelta in the gonadotropin signalling cascade. Furthermore, in addition to inhibition by PD98059, gonadotropin-induced ovarian cancer cell migration was also inhibited by verapamil, GF 109203X and rottlerin. Similarly, gonadotropin-induced proliferation was inhibited by PD98059, verapamil, GF 109203X and PKCdelta siRNA. Taken together, these results demonstrate that gonadotropins induce both ovarian cancer cell migration and proliferation by activation of ERK1/2 signalling in a calcium- and PKCdelta-dependent manner.