RESUMO
We show in this study that endogenous NEP and PTEN associate in cells directly through electrostatic interactions between a highly basic residue stretch in the intracellular domain of NEP and the major phosphorylation site in PTEN's tail. NEP binds and engages in higher order complexes both phosphorylated and unphosphorylated PTEN. NEP recruits PTEN to the plasma membrane and enhances its stability and phosphatase activity. As a result, an enzymatically inactive NEP mutant preserves the ability to bind PTEN, inactivates the Akt/PKB kinase, and partially suppresses the growth of PC cells. This study demonstrates a molecular cooperation between NEP and PTEN tumor suppressors in which NEP constitutively recruits and activates PTEN to inhibit the PI3K/Akt oncogenic pathway.
Assuntos
Neprilisina/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Serina-Treonina Quinases , Sequência de Aminoácidos , Membrana Celular/enzimologia , Movimento Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Neprilisina/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/fisiopatologia , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , RNA Interferente Pequeno/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Células Tumorais CultivadasRESUMO
Neutral endopeptidase (NEP) is a cell-surface peptidase normally expressed by prostate epithelial cells and lost in ~50% of primary prostate cancers. NEP directly associates with multiple proteins at the cell surface including Ezrin/Radixin/Moesin (ERM) proteins and the PTEN tumor suppressor protein. Analysis of the N-terminal sequence of the NEP cytosolic domain (N-terminal MGKSESQMDI TDINTPKPKK KQRWTR) identified a myristoylation consensus site. Mutation of Gly-2 to Arg significantly decreased (3)H-myristoylation activity, and correlated with translocation of NEP from the plasma membrane to a perinuclear domain as demonstrated by immunofluorescence staining and Western blotting with an NEP-specific antibody. Removal of this myristoylation residue did not affect NEP enzymatic specific activity. Myristoylated NEP recruited more PTEN protein to the cell membrane fraction than unmyristoylated NEP. These data demonstrate that NEP is myristoylated at Gly-2 and that this modification is an intrinsic signal for membrane targeting.
Assuntos
Neprilisina/metabolismo , Sequência de Aminoácidos , Humanos , Microscopia de Fluorescência , Dados de Sequência Molecular , Neprilisina/análise , Neprilisina/química , PTEN Fosfo-Hidrolase/metabolismo , TransfecçãoRESUMO
Increasing evidence suggests that androgen independent prostate cancer (PC) maintains a functional androgen receptor (AR) pathway despite the low levels of circulating androgen following androgen withdrawal, the molecular mechanisms of which are not well defined yet. To address this question, we investigated the effects of endothelin-1 (ET-1) on AR expression. Western analysis and RT-PCR revealed that in the presence of ET-1, levels of AR significantly increased in a time- and dose-dependent manner in LNCaP cells. Pretreatments with inhibitors of Src and phosphoinositide kinase 3 (PI-3K) suppressed ET-1-induced AR expression. As ET-1 was reported to cause a transient increase in c-myc mRNA levels, we examined the involvement of c-myc in ET-1-mediated AR expression. Transient transfection of c-myc siRNA neutralized ET-1-induced AR expression, suggesting that AR induction by ET-1 is c-myc dependent. AR can regulate the transcription of its own gene via a mechanism in which c-myc plays a crucial role. Therefore, we assessed if ET-1-induced-c-myc leads to the enhancement of AR transcription. Reporter gene assays using the previously identified AR gene enhancer containing a c-myc binding site were conducted in LNCaP cells. We found that ET-1 induced reporter gene activity from the construct containing the wild-type but not mutant c-myc binding site. Chromatin immunoprecipitation assays confirmed that ET-1 increased interaction between c-myc and c-myc binding sites in AR enhancer, suggesting that ET-1-induced AR transcription occurs via c-myc-mediated AR transcription. Together, these data support the notion that ET-1, via Src/PI-3K signaling, augments c-myc expression leading to enhanced AR expression in PC.
Assuntos
Endotelina-1/fisiologia , Genes myc , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Primers do DNA , Humanos , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Receptores Androgênicos/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Although glucocorticoids are frequently administered to patients with hormone refractory prostate cancer, their therapeutic effectiveness is limited by the development of glucocorticoid resistance. The molecular mechanisms of glucocorticoid resistance are unknown but are believed to involve neuropeptide growth factors and cytokines. We examined the functional interaction between bombesin and dexamethasone in PC-3 cells and found that bombesin could act as a survival factor by interfering with dexamethasone-mediated growth inhibition. Because glucocorticoids exert their effects through glucocorticoid receptors (GRs), we measured the expression of GR alpha and GR beta isoforms in the presence of bombesin. Western blotting and real time PCR revealed bombesin induced expression of GR beta, but not GR alpha. Because GR isoforms are generated by alternative splicing of a common GR gene, we examined the expression of serine-arginine (SR) proteins involved in alternative splicing, and found that the expression of SRp30 was induced by bombesin in PC-3 cells. To characterize the role of SRp30 in splicing of GR isoforms, siRNAs specific to various SRp30 isoforms were transfected into PC-3 cells. We found that suppression of SRp30c expression by siRNA specifically antagonized bombesin's effect on glucocorticoid-mediated inhibition of PC cells, suggesting that bombesin-induced expression of SRp30c affects GR pre-mRNA splicing, leading to increased GR beta expression and contributing to glucocorticoid resistance in PC cells.
Assuntos
Bombesina/metabolismo , Neurotransmissores/metabolismo , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Receptores de Glucocorticoides/metabolismo , Bombesina/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Neurotransmissores/genética , Proteínas Nucleares/genética , Neoplasias da Próstata , Isoformas de Proteínas/genética , Precursores de RNA/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Receptores de Glucocorticoides/genética , Fatores de Processamento de Serina-ArgininaRESUMO
Multiple studies indicate that neuroendocrine (NE) differentiation in prostate cancer (PC) contributes to androgen-independent progression. Levels of chromogranin A (CgA), which is produced by NE cells, are increased in advanced PC. However, the mechanism by which high levels of circulating CgA contribute to PC progression is unknown. To examine the effects of CgA on PC cells, we first performed proliferation assays in the presence of recombinant CgA (rCgA) in LNCaP and C4-2 PC cells, and found that rCgA increased cell proliferation in a dose and time-dependent manner. NE differentiated PC cells, also overexpress the antiapoptosis protein survivin. Therefore, we examined survivin expression in the presence of CgA in PC cells. Western blot analysis showed that survivin was significantly increased by rCgA and inhibited by an anti-CgA antibody in both LNCaP and C4-2 cells. Survivin expression is believed to be regulated by PI3K/Akt pathway. We next assessed the phosphorylation status of Akt and found that Akt phosphorylation was increased by treatment with rCgA. To determine whether Akt phosphorylation is necessary for rCgA-induced survivin expression, we examined the effects of Akt, MAPK kinase, and protein kinase C inhibitors on CgA-induced survivin expression, and found that survivin expression was reduced in the presence of Akt inhibitors, but not MAPK kinase or protein kinase C inhibitors. Furthermore, in the presence of an Akt inhibitor or small interfering RNA of survivin, CgA-enhanced proliferation of C4-2 and LNCaP cells significantly decreased. Together, our results demonstrate that CgA can increase PC cell survival through Akt-mediated survivin up-regulation.
Assuntos
Apoptose/efeitos dos fármacos , Cromogranina A/farmacologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-akt/genética , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Proteínas Inibidoras de Apoptose , Masculino , Neoplasias da Próstata/patologia , Neoplasias da Próstata/fisiopatologia , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Survivina , Regulação para CimaRESUMO
Neutral endopeptidase 24.11 (NEP) is a 90-110 kDa cell surface cell surface peptidase that is normally expressed by numerous tissues, including prostate, kidney, intestine, endometrium, adrenal glands and lung. This enzyme cleaves peptide bonds on the amino side of hydrophobic amino acids and inactivates a variety of physiologically active peptides, including atrial natriuretic factor, substance P, bradykinin, oxytocin, Leu- and Met-enkephalins, neurotensin, bombesin, endothelin-1, and bombesin-like peptides. NEP reduces the local concentration of peptide available for receptor binding and signal transduction. Loss or decreases in NEP expression have been reported in a variety of malignancies. Reduced NEP may promote peptide-mediated proliferation by allowing accumulation of higher peptide concentrations at the cell surface, and facilitate the development or progression of neoplasia. We have used prostate cancer as model in which to study the involvement of NEP in malignancy. Using a variety of experimental approaches, including recombinant NEP, cell lines expressing wild-type and mutant NEP protein, and cell lines expressing NEP protein with a mutated cytoplasmic domain, we have examined the effects of NEP on cell migration and cell survival. We have shown that the effects of NEP are mediated by its ability to catalytically inactivate substrates such as bombesin and endothelin-1, but also through direct protein-protein interaction with other protein such as Lyn kinase [which associates with the p85 subunit of phosphatidylinositol 3-kinase (PI3-K) resulting in NEP-Lyn-PI3-K protein complex], ezrin/radixin/moesin (ERM) proteins, and the PTEN tumor suppressor protein. We review the mechanisms of NEP's tumor suppressive action and how NEP loss contributes to tumor progression.
Assuntos
Invasividade Neoplásica/fisiopatologia , Neprilisina/fisiologia , Neoplasias da Próstata/fisiopatologia , Apoptose/efeitos dos fármacos , Proteína Tirosina Quinase CSK , Movimento Celular/efeitos dos fármacos , Ativação Enzimática , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Adesões Focais/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Masculino , Neurofibromina 2/metabolismo , PTEN Fosfo-Hidrolase , Inibidores de Fosfoinositídeo-3 Quinase , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Acetato de Tetradecanoilforbol/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Quinases da Família srcRESUMO
The neutral endopeptidase (NEP) gene is transcriptionally regulated by androgen in prostate cancer cells. We previously identified in the NEP gene an androgen responsive element (NEP-ARE) and an androgen responsive region (NEP-ARR) that together conveyed only moderate androgen-inducibility [Mol. Cell. Endocrinol. 170 (2000) 131]. Therefore, we characterized the entire genomic structure of the NEP gene and identified ARE1 (ACTCAACAttgTGTCCTTT) and ARE2 (CAGGACAtttTGTCCC), which are located in the 3'-untranslated region and in intron 17, respectively. Steroid-dependent enhancement of transcription was assayed by transfecting the pGL-3-luciferase reporter plasmid containing three copies of ARE1 or ARE2 into PC-3 cells. Luciferase activities were increased 3.6-fold (ARE1) and 5-fold (ARE2) by androgen (AR), 4.2-fold (ARE1) and 8.2-fold (ARE2) by dexamethasone, and 3-fold (ARE1) and 4.1-fold (ARE2) by progesterone. Mutation of the ARE1 and ARE2 sequences completely abrogated androgen-inducibility. We next showed that both ARE1 and ARE2 are involved in the transcriptional regulation of the NEP gene, demonstrating in vitro and in vivo binding with AR as determined by electrophoretic mobility gel shift and chromatin immunoprecipitation (ChIP) assays, Furthermore, ARE1 and ARE2 mediate coordinated androgen-inducibility in both an SV40 promoter and the native NEP type 1 promoter. These data indicate the newly identified ARE1 and ARE2 together with the previously identified NEP-ARE function as androgen response elements, and that androgen regulation of the NEP gene is regulated by the coordinated action of multiple AREs in prostate cancer cells.
Assuntos
Androgênios/metabolismo , Neprilisina/genética , Neprilisina/metabolismo , Elementos de Resposta/fisiologia , Androgênios/fisiologia , Sequência de Bases , Extratos Celulares/farmacologia , Núcleo Celular/química , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Éxons , Glucocorticoides/farmacologia , Humanos , Íntrons , Dados de Sequência Molecular , Progesterona/farmacologia , Regiões Promotoras Genéticas/fisiologia , Ligação Proteica/efeitos dos fármacos , Receptores Androgênicos/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
In order to identify gene products associated with the development of acquired therapeutic resistance by prostate cancer cells, we created two novel apoptosis-resistant prostate cancer cell lines, LNCaP-TR (phorbol-ester [TPA]-Resistant) and LNCaP-SSR (Serum Starvation-Resistant) by repeated transient exposure of cultured human LNCaP cells to apoptotic stimuli followed by expansion of surviving cell populations. These cell lines were found to be cross-resistant to the alternative selective agent and also hormone-resistant when xenografted into castrated male immunodeficient mice. RNA from the LNCaP-TR line was comparatively screened using a subtractive hybridization-PCR procedure. This allowed us to identify a 249 bp cDNA fragment that hybridized to a 4.8 kb mRNA preferentially expressed by the apoptosis-resistant cells. Using RACE procedures, we cloned and sequenced the complete 4.8 kb cDNA. It is an unusual member of the protocadherin gene family containing two large overlapping open reading frames encoding homologous polypeptides, one having a signal sequence and the other lacking a signal sequence and we refer to it as protocadherin-PC. LNCaP cells directly transformed with protocadherin-PC cDNA were comparatively resistant to phorbol-ester induced apoptosis. Antibody recognition studies demonstrating the cytoplasmic nature of the protcadherin-PC translation product and its propensity to bind beta-catenin suggest that it might influence the apoptotic sensitivity of prostate cancer cells through a unique mechanism.
Assuntos
Adenocarcinoma/patologia , Androgênios , Apoptose/genética , Caderinas/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Neoplasias Hormônio-Dependentes/patologia , Peptídeos/genética , Neoplasias da Próstata/patologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Caderinas/biossíntese , Caderinas/química , Caderinas/imunologia , Clonagem Molecular , Meios de Cultura Livres de Soro/farmacologia , Citoplasma/metabolismo , DNA Complementar/genética , Resistência a Medicamentos , Amplificação de Genes , Genes , Humanos , Masculino , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/química , Proteínas de Neoplasias/imunologia , Transplante de Neoplasias , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Fases de Leitura Aberta , Peptídeos/química , Peptídeos/imunologia , Fenótipo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Biossíntese de Proteínas , Sinais Direcionadores de Proteínas/genética , Protocaderinas , Técnica de Subtração , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
PURPOSE: Neuropeptide growth factors such as bombesinare implicated in progression to androgen-independent prostate cancer (PC). We examined the impact of bombesin on androgen receptor (AR)-mediated gene expression. EXPERIMENTAL DESIGN: The AR together with the AR-responsive probasin ARR(3)tk-luc or PSA-pPUE-ELB-luc promoter was cotransfected into Swiss 3T3 and PC-3 cells, both of which express high-affinity bombesin receptors; the cells were incubated with bombesin (0-50 nM) and dihydrotestosterone (DHT; 0-10 nM), and luciferase activities were measured. DHT increased transcription approximately 40-fold at doses of 1 and 10 nM but had no effect at 10 pM. Bombesin alone, or with 1 or 10 nM DHT, did not further increase transcription. However, 5 nM bombesin and 10 pM DHT, doses that by themselves had no effect, resulted in a approximately 20 fold increase in transcription (P < 0.005). This synergistic effect was blocked by bombesin receptor antagonists and recombinant neutral endopeptidase, which hydrolyzes bombesin. Bombesin and DHT together also increased binding of nuclear extracts from PC-3 cells transfected with AR to a consensus androgen response element in mobility shift assays and increased the level of secreted prostate-specific antigen in LNCaP cell supernatant compared with DHT or bombesin alone. Immunoprecipitation of AR from (32)P-labeled LNCaP cells revealed that 5 nM bombesin + 10 pM DHT induced AR phosphorylation comparable with 1 nM DHT, whereas bombesin or 10 pM DHT alone did not. CONCLUSIONS: These data indicate that bombesin can synergize with low (castrate) levels of DHT to induce AR-mediated transcription and suggest that neuropeptides promote AR-mediated signaling in androgen-independent prostate cancer.
Assuntos
Bombesina/farmacologia , Di-Hidrotestosterona/farmacologia , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Sinergismo Farmacológico , Ensaio de Desvio de Mobilidade Eletroforética , Expressão Gênica/efeitos dos fármacos , Humanos , Luciferases/metabolismo , Masculino , Fosforilação/efeitos dos fármacos , Testes de Precipitina , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Receptores da Bombesina/genética , Receptores da Bombesina/metabolismo , Elementos de Resposta , Transcrição Gênica , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
BACKGROUND: Neutral endopeptidase 24.11 (NEP) is a cell-surface peptidase that inactivates a variety of neuropeptide substrates. In addition to catalytic activity, NEP can exert biological effects through protein-protein interactions. We previously reported that NEP directly associated with tyrosine-phosphorylated Lyn kinase, and with the p85 subunit of the phosphatidylinositol 3-kinase (PI3 kinase) resulting in an NEP-Lyn-PI3 kinase protein complex. MATERIALS AND METHODS: In this report, we investigated the association of NEP with cytoplasmic proteins using ProteinChip Array, surface enhanced laser desorption/ionization (SELDI) technology combined with time-of-flight mass spectrometry, as well as immunoprecipitation and Western blottings. RESULTS: Using immunocapture on the ProteinChip surface, we identified a 122 kDa protein which associates with NEP derived from LNCaP cell lysates which had the identical molecular weight as the beta-subunit of p110 subunit of phosphatidylinositol 3-kinase. The identity of the p110 beta was confirmed by Western blot analysis of NEP and p110 beta immunoprecipitates using monoclonal antibodies specific for NEP and p110 beta. CONCLUSION: These data confirm the association of phosphatidylinositol 3-kinase (consisting of the p85 adaptor and p110 beta-subunit) with NEP. Furthermore, this work demonstrates the ability of mass spectrometry to identify proteins interacting with NEP and potentially other cell-surface peptidases.
Assuntos
Neprilisina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Western Blotting , Humanos , Masculino , Espectrometria de Massas , Melanoma/enzimologia , Testes de Precipitina , Neoplasias da Próstata/enzimologia , Análise Serial de Proteínas , Estrutura Terciária de Proteína , Células Tumorais CultivadasRESUMO
Neutral endopeptidase (NEP) is a cell-surface peptidase that inhibits prostate cancer cell growth partly via inhibition of Akt kinase. We investigated the antitumor effects of an adenovirus gene delivery system (AdNEP) to restore NEP expression in DU145 prostate cancer cells in combination with paclitaxel chemotherapy. DU145 cells were infected with adenovirus expressing NEP or LacZ, treated with paclitaxel, and assessed for cell viability, Akt activation and induction of apoptosis. Athymic mice with established DU145 xenografts were injected intratumorally with AdNEP or AdLacZ and intraperitoneally with paclitaxel and monitored for tumor growth over 28 days. Compared to AdLacZ plus paclitaxel, AdNEP plus paclitaxel significantly inhibited DU145 cell growth and increased apoptosis as determined by increased caspase-3 and PARP-1 proteolytic fragments. In a xenograft model, tumor volume was reduced in mice treated with AdNEP plus paclitaxel (122.85±89.5 mm3; P<0.01) compared with mice treated with AdNEP plus saline (653.9±230.3 mm3), AdLacZ plus paclitaxel (575.9±176.6 mm3) or AdLacZ plus saline (920.2±238.2 mm3). In conclusion, these data suggest that NEP can augment taxane-induced apoptosis through inhibition of Akt/Bad signaling, and that the combination of NEP plus paclitaxel may be an effective strategy to inhibit castration-resistant prostate cancer growth.
Assuntos
Técnicas de Transferência de Genes , Neprilisina/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/terapia , Adenoviridae/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Camundongos , Neprilisina/uso terapêutico , Paclitaxel/administração & dosagem , Neoplasias da Próstata/patologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Prostate cancer progression requires active androgen receptor (AR) signaling which occurs following translocation of AR from the cytoplasm to the nucleus. Chemotherapy with taxanes improves survival in patients with castrate resistant prostate cancer (CRPC). Taxanes induce microtubule stabilization, mitotic arrest, and apoptotic cell death, but recent data suggest that taxanes can also affect AR signaling. Here, we report that taxanes inhibit ligand-induced AR nuclear translocation and downstream transcriptional activation of AR target genes such as prostate-specific antigen. AR nuclear translocation was not inhibited in cells with acquired ß-tubulin mutations that prevent taxane-induced microtubule stabilization, confirming a role for microtubules in AR trafficking. Upon ligand activation, AR associated with the minus-end-microtubule motor dynein, thereby trafficking on microtubules to translocate to the nucleus. Analysis of circulating tumor cells (CTC) isolated from the peripheral blood of CRPC patients receiving taxane chemotherapy revealed a significant correlation between AR cytoplasmic sequestration and clinical response to therapy. These results indicate that taxanes act in CRPC patients at least in part by inhibiting AR nuclear transport and signaling. Further, they suggest that monitoring AR subcellular localization in the CTCs of CRPC patients might predict clinical responses to taxane chemotherapy.
Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Antineoplásicos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/metabolismo , Taxoides/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Resistencia a Medicamentos Antineoplásicos , Dineínas/metabolismo , Feminino , Humanos , Masculino , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Metástase Neoplásica , Células Neoplásicas Circulantes/efeitos dos fármacos , Células Neoplásicas Circulantes/metabolismo , Neoplasias Ovarianas/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Neutral endopeptidase (NEP) is a 90- to 110-kDa cell-surface peptidase that is normally expressed by numerous tissues but whose expression is lost or reduced in a variety of malignancies. The anti-tumorigenic function of NEP is mediated not only by its catalytic activity but also through direct protein-protein interactions of its cytosolic region with several binding partners, including Lyn kinase, PTEN, and ezrin/radixin/moesin (ERM) proteins. We have previously shown that mutation of the K(19)K(20)K(21) basic cluster in NEPs' cytosolic region to residues QNI disrupts binding to the ERM proteins. Here we show that the ERM-related protein merlin (NF2) does not bind NEP or its cytosolic region. Using experimental data, threading, and sequence analysis, we predicted the involvement of moesin residues E(159)Q(160) in binding to the NEP cytosolic domain. Mutation of these residues to NL (to mimic the corresponding N(159)L(160) residues in the nonbinder merlin) disrupted moesin binding to NEP. Mutation of residues N(159)L(160)Y(161)K(162)M(163) in merlin to the corresponding moesin residues resulted in NEP binding to merlin. This engineered NEP peptide-merlin interaction was diminished by the QNI mutation in NEP, supporting the role of the NEP basic cluster in binding. We thus identified the region of interaction between NEP and moesin, and engineered merlin into a NEP-binding protein. These data form the basis for further exploration of the details of NEP-ERM binding and function.
Assuntos
Proteínas dos Microfilamentos/metabolismo , Neprilisina/genética , Neurofibromina 2/metabolismo , Domínios e Motivos de Interação entre Proteínas/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Simulação por Computador , Humanos , Camundongos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Neprilisina/química , Neprilisina/metabolismo , Neurofibromina 2/química , Neurofibromina 2/genética , Peptídeos/genética , Peptídeos/metabolismo , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
BACKGROUND: The neurofibromatosis 2 (NF2) tumor suppressor gene product merlin is an important regulator of contact-dependent cell proliferation. Phosphorylation of merlin at serine 518 (Ser518) by the Rac effector p21-activated kinase (PAK) inactivates merlin's growth suppressing function, and is regulated by cell-culture conditions, including cell density, cell/substrate attachment, and growth factor availability. We examined the regulation of merlin expression and merlin phosphorylation in prostate cancer cells. METHODS: Phosphorylation of merlin in five prostate cancer cell lines (LNCaP, DU145, PC3, 22RV1, and LAPC-4) was examined by Western blotting using anti-phospho-merlin (Ser518) antibody. The activity of PAK, an upstream regulator of merlin phosphorylation, was measured by Western blotting using phospho-PAK (Ser141) antibody. The effects of various cell-culture conditions on the phosphorylation levels of merlin and PAK were analyzed. RESULTS: Both merlin expression and phosphorylation were low in LNCaP, PC3, 22RV1, and LAPC-4 prostate cancer cells. In DU145 cells, total and phosphorylated merlin were abundant, but phosphorylation was not inhibited by high cell density, serum withdrawal, the addition of hyaluronic acid or inhibition of CD44 expression, all of which are reported to inhibit merlin phosphorylation in non-neoplastic cells. PAK activation was elevated in DU145 cells and the addition of a PAK-specific inhibitor peptide but not the Rac1-specific inhibitor NSC23766 inhibited both PAK and merlin phosphorylation. CONCLUSIONS: Merlin is inactivated in DU145 prostate cancer cells by PAK-mediated constitutive phosphorylation, identifying a novel mechanism of merlin inactivation in neoplastic cells.
Assuntos
Neurofibromina 2/metabolismo , Neoplasias da Próstata/metabolismo , Western Blotting , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Masculino , Microscopia de Fluorescência , Neurofibromina 2/biossíntese , Neurofibromina 2/genética , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , RNA Interferente Pequeno/genética , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/metabolismoRESUMO
BACKGROUND: Neutral endopeptidase (NEP) is a cell-surface peptidase that can regulate the activation of Akt kinase through catalytic-dependent and independent mechanisms. NEP expression is absent in approximately 50% of prostate cancers. The authors investigated whether NEP loss in vivo would result in Akt phosphorylation and potentially contribute to prostate cancer progression by examining the interaction of NEP, Akt, and phosphatase and tensin homolog (PTEN) in a prostate xenograft model and in clinical specimens from patients with prostate cancer. METHODS: Using a tetracycline-repressible expression system to express NEP in a tumor animal xenograft model, the effects of NEP were tested on tumor growth, Akt phosphorylation, and PTEN expression. The clinical relevance of NEP, phosphorylated Akt, and PTEN protein expression also was investigated in 204 patients who had undergone radical prostatectomy. RESULTS: The results indicated that the induction of NEP expression inhibited established xenograft tumor growth, diminished Akt phosphorylation, and increased PTEN protein levels. In humans, prostate cancers with complete loss of NEP expression were significantly more likely to express phosphorylated Akt (P = .02). Moreover, patients who had prostate cancers with concomitant loss of NEP and expression of phosphorylated Akt had an increased, independent risk of prostate-specific antigen (PSA) recurrence (P = .03). In the study cohort, loss of PTEN protein expression did not correlated significantly with phosphorylated Akt or with patients' clinical outcome. CONCLUSIONS: The findings from this investigation demonstrated that NEP loss leads to Akt activation and contributes to the clinical progression of prostate cancer.
Assuntos
Neprilisina/deficiência , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Progressão da Doença , Ativação Enzimática , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Recidiva Local de Neoplasia/enzimologia , Recidiva Local de Neoplasia/patologia , Transplante de Neoplasias , Neprilisina/biossíntese , Neprilisina/genética , Neprilisina/metabolismo , Fosforilação , Estudos Prospectivos , Antígeno Prostático Específico , Prostatectomia , Neoplasias da Próstata/genética , Neoplasias da Próstata/cirurgia , Transplante HeterólogoRESUMO
Neprilysin is a cell surface peptidase that catalytically inactivates neuropeptide substrates and functions as a tumor suppressor via its enzymatic function and multiple protein-protein interactions. We investigated whether neutral endopeptidase could inhibit angiogenesis in vivo utilizing a murine corneal pocket angiogenesis model and found that it reduced fibroblast growth factor-2-induced angiogenesis by 85% (p < 0.01) but had no effect on that of vascular endothelial growth factor. Treatment with recombinant neprilysin, but not enzymatically inactive neprilysin, resulted in a slight increase in basic fibroblast growth factor electrophoretic mobility from proteolytic cleavage between amino acids Leu-135 and Gly-136, which was inhibited by the neutral endopeptidase inhibitor CGS24592 and heparin. Cleavage kinetics were rapid, comparable with that of other known neprilysin substrates. Functional studies involving neprilysin-expressing vascular endothelial cells demonstrated that neutral endopeptidase inhibition significantly enhanced fibroblast growth factor-mediated endothelial cell growth, capillary array formation, and signaling, whereas exogenous recombinant neprilysin inhibited signaling. Recombinant constructs confirmed that cleavage products neither promoted capillary array formation nor induced signaling. Moreover, mutation of the cleavage site resulted in concomitant loss of cleavage and increased the potency of fibroblast growth factor-2 to induce capillary array formation. These data indicate that neprilysin proteolytically inactivates fibroblast growth factor-2, resulting in negative regulation of angiogenesis.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Neovascularização Fisiológica , Neprilisina/metabolismo , Linhagem Celular , Células Endoteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/genética , Glicina/metabolismo , Humanos , Leucina/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Mutação/genética , Neprilisina/genética , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
Neutral endopeptidase 24.11 (NEP) is a cell surface peptidase expressed by numerous tissues including prostatic epithelial cells. We reported that NEP inhibits prostate cancer cell proliferation and cell migration by enzymatic inactivation of neuropeptide substrates and through protein-protein interaction independent of catalytic function. The cytoplasmic domain of NEP contains a positively charged amino acid cluster, previously identified as a binding site for ezrin/radixin/moesin (ERM) proteins. We report here that NEP co-immunoprecipitates with ERM proteins in NEP-expressing LNCaP prostate cancer cells and MeWo melanoma cells. Co-immunoprecipitation showed that ERM proteins associate with wild-type NEP protein but not NEP protein containing a truncated cytoplasmic domain or point mutations replacing the positively charged amino acid cluster. In vitro binding assays showed that NEP binds directly to recombinant N terminus fragments of ERM proteins at the positively charged amino acid cluster within the NEP cytoplasmic domain. Binding of ERM proteins to NEP results in decreased binding of ERM proteins to the hyaluronan receptor CD44, a main binding partner of ERM proteins. Moreover, cells expressing wild-type NEP demonstrate decreased adhesion to hyaluronic acid and cell migration. These data suggest that NEP can affect cell adhesion and migration through direct binding to ERM proteins.