RESUMO
PURPOSE: Currently, there are no accurate markers for predicting potentially lethal prostate cancer (PC) before biopsy. This study aimed to develop urine tests to predict clinically significant PC (sPC) in men at risk. METHODS: Urine samples from 928 men, namely, 660 PC patients and 268 benign subjects, were analyzed by gas chromatography/quadrupole time-of-flight mass spectrophotometry (GC/Q-TOF MS) metabolomic profiling to construct four predictive models. Model I discriminated between PC and benign cases. Models II, III, and GS, respectively, predicted sPC in those classified as having favorable intermediate risk or higher, unfavorable intermediate risk or higher (according to the National Comprehensive Cancer Network risk groupings), and a Gleason sum (GS) of ≥ 7. Multivariable logistic regression was used to evaluate the area under the receiver operating characteristic curves (AUC). RESULTS: In Models I, II, III, and GS, the best AUCs (0.94, 0.85, 0.82, and 0.80, respectively; training cohort, N = 603) involved 26, 24, 26, and 22 metabolites, respectively. The addition of five clinical risk factors (serum prostate-specific antigen, patient age, previous negative biopsy, digital rectal examination, and family history) significantly improved the AUCs of the models (0.95, 0.92, 0.92, and 0.87, respectively). At 90% sensitivity, 48%, 47%, 50%, and 36% of unnecessary biopsies could be avoided. These models were successfully validated against an independent validation cohort (N = 325). Decision curve analysis showed a significant clinical net benefit with each combined model at low threshold probabilities. Models II and III were more robust and clinically relevant than Model GS. CONCLUSION: This urine test, which combines urine metabolic markers and clinical factors, may be used to predict sPC and thereby inform the necessity of biopsy in men with an elevated PC risk.
Assuntos
Metaboloma , Neoplasias da Próstata , Humanos , Masculino , Biópsia , Gradação de Tumores , Antígeno Prostático Específico , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/urina , Fatores de Risco , Detecção Precoce de Câncer/métodos , Urinálise/métodos , Urina/químicaRESUMO
BACKGROUND: Dysregulation of pericellular proteolysis usually accounts for cancer cell invasion and metastasis. Isolation of a cell-surface protease system for lung cancer metastasis is an important issue for mechanistic studies and therapeutic target identification. METHODS: Immunohistochemistry of a tissue array (n = 64) and TCGA database (n = 255) were employed to assess the correlation between serine protease inhibitors (SPIs) and lung adenocarcinoma progression. The role of SPI in cell motility was examined using transwell assays. Pulldown and LC/MS/MS were performed to identify the SPI-modulated novel protease(s). A xenografted mouse model was harnessed to demonstrate the role of the SPI in lung cancer metastasis. RESULTS: Hepatocyte growth factor activator inhibitor-2 (HAI-2) was identified to be downregulated following lung cancer progression, which was related to poor survival and tumour invasion. We further isolated a serum-derived serine protease, plasmin, to be a novel target of HAI-2. Downregulation of HAI-2 promotes cell surface plasmin activity, EMT, and cell motility. HAI-2 can suppress plasmin-mediated activations of HGF and TGF-ß1, EMT and cell invasion. In addition, downregulated HAI-2 increased metastasis of lung adenocarcinoma via upregulating plasmin activity. CONCLUSION: HAI-2 functions as a novel inhibitor of plasmin to suppress lung cancer cell motility, EMT and metastasis.
Assuntos
Adenocarcinoma de Pulmão/metabolismo , Fibrinolisina/metabolismo , Neoplasias Pulmonares/metabolismo , Glicoproteínas de Membrana/metabolismo , Células A549 , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/secundário , Animais , Linhagem Celular Tumoral , Movimento Celular , Progressão da Doença , Transição Epitelial-Mesenquimal , Fibrinolisina/antagonistas & inibidores , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Fator de Crescimento Transformador beta1/metabolismoRESUMO
In prostate cancer (PCa), similar to many other cancers, distant organ metastasis symbolizes the beginning of the end disease, which eventually leads to cancer death. Many mechanisms have been identified in this process that can be rationalized into targeted therapy. Among them, epithelial-to-mesenchymal transition (EMT) is originally characterized as a critical step for cell trans-differentiation during embryo development and now recognized in promoting cancer cells invasiveness because of high mobility and migratory abilities of mesenchymal cells once converted from carcinoma cells. Nevertheless, the underlying pathways leading to EMT appear to be very diverse in different cancer types, which certainly represent a challenge for developing effective intervention. In this article, we have carefully reviewed the key factors involved in EMT of PCa with clinical correlation in hope to facilitate the development of new therapeutic strategy that is expected to reduce the disease mortality.
Assuntos
Transição Epitelial-Mesenquimal , Próstata/patologia , Neoplasias da Próstata/patologia , Animais , Progressão da Doença , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , MicroRNAs/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Próstata/metabolismo , Neoplasias da Próstata/genética , RNA Longo não Codificante/genéticaRESUMO
Ketamine, a dissociative anesthetic, is misused and abused worldwide as an illegal recreational drug. In addition to its neuropathic toxicity, ketamine abuse has numerous effects, including renal failure; however, the underlying mechanism is poorly understood. The process called epithelial phenotypic changes (EPCs) causes the loss of cell-cell adhesion and cell polarity in renal diseases, as well as the acquisition of migratory and invasive properties. Madin-Darby canine kidney cells, an in vitro cell model, were subjected to experimental manipulation to investigate whether ketamine could promote EPCs. Our data showed that ketamine dramatically decreased transepithelial electrical resistance and increased paracellular permeability and junction disruption, which were coupled to decreased levels of apical junctional proteins (ZO-1, occludin, and E-cadherin). Consistent with the downregulation of epithelial markers, the mesenchymal markers N-cadherin, fibronectin, and vimentin were markedly upregulated following ketamine stimulation. Of the E-cadherin repressor complexes tested, the mRNA levels of Snail, Slug, Twist, and ZEB1 were elevated. Moreover, ketamine significantly enhanced migration and invasion. Ketamine-mediated changes were at least partly caused by the inhibition of GSK-3ß activity through Ser-9 phosphorylation by the PI3K/Akt pathway. Inhibiting PI3K/Akt with LY294002 reactivated GSK-3ß and suppressed ketamine-enhanced permeability, EPCs, and motility. These findings were recapitulated by the inactivation of GSK-3ß using the inhibitor 3F8. Taken together, these results provide evidence that ketamine induces renal distal tubular EPCs through the downregulation of several junction proteins, the upregulation of mesenchymal markers, the activation of Akt, and the inactivation of GSK-3ß.
Assuntos
Analgésicos/farmacologia , Membrana Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/genética , Ketamina/farmacologia , Animais , Caderinas/genética , Caderinas/metabolismo , Adesão Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Cães , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Junções Intercelulares/efeitos dos fármacos , Células Madin Darby de Rim Canino , Ocludina/genética , Ocludina/metabolismo , Fenótipo , Transdução de Sinais , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Vimentina/genética , Vimentina/metabolismo , Dedos de Zinco/genética , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Although classical protein tyrosine phosphatase (PTP) superfamily members are cysteine-dependent, emerging evidence shows that many acid phosphatases (AcPs) function as histidine-dependent PTPs in vivo. These AcPs dephosphorylate phospho-tyrosine substrates intracellularly and could have roles in development and disease. In contrast to cysteine-dependent PTPs, they utilize histidine, rather than cysteine, for substrate dephosphorylation. Structural analyses reveal that active site histidine, but not cysteine, faces towards the substrate and functions as the phosphate acceptor. Nonetheless, during dephosphorylation, both histidine-dependent and cysteine-dependent PTPs use their active site arginine and aspartate for substrate binding and proton donation, respectively. Thus, we propose that they should be referred to as a distinct group of 'histidine-dependent PTPs' within the PTP superfamily.
Assuntos
Fosfatase Ácida/fisiologia , Histidina/química , Proteínas Tirosina Fosfatases/fisiologia , Fosfatase Ácida/metabolismo , Animais , Domínio Catalítico , Cisteína/química , Humanos , Modelos Biológicos , Neurônios/metabolismo , Fosfatos/química , Filogenia , Plantas/metabolismo , Conformação Proteica , Proteínas Tirosina Fosfatases/metabolismo , Especificidade da EspécieRESUMO
PURPOSE: Biomarkers predicting clinically significant prostate cancer (sPC) before biopsy are currently lacking. This study aimed to develop a non-invasive urine test to predict sPC in at-risk men using urinary metabolomic profiles. MATERIALS AND METHODS: Urine samples from 934 at-risk subjects and 268 treatment-naïve PC patients were subjected to liquid chromatography/mass spectrophotometry (LC-MS)-based metabolomics profiling using both C18 and hydrophilic interaction liquid chromatography (HILIC) column analyses. Four models were constructed (training cohort [n=647]) and validated (validation cohort [n=344]) for different purposes. Model I differentiates PC from benign cases. Models II, III, and a Gleason score model (model GS) predict sPC that is defined as National Comprehensive Cancer Network (NCCN)-categorized favorable-intermediate risk group or higher (Model II), unfavorable-intermediate risk group or higher (Model III), and GS ≥7 PC (model GS), respectively. The metabolomic panels and predicting models were constructed using logistic regression and Akaike information criterion. RESULTS: The best metabolomic panels from the HILIC column include 25, 27, 28 and 26 metabolites in Models I, II, III, and GS, respectively, with area under the curve (AUC) values ranging between 0.82 and 0.91 in the training cohort and between 0.77 and 0.86 in the validation cohort. The combination of the metabolomic panels and five baseline clinical factors that include serum prostate-specific antigen, age, family history of PC, previously negative biopsy, and abnormal digital rectal examination results significantly increased AUCs (range 0.88-0.91). At 90% sensitivity (validation cohort), 33%, 34%, 41%, and 36% of unnecessary biopsies were avoided in Models I, II, III, and GS, respectively. The above results were successfully validated using LC-MS with the C18 column. CONCLUSIONS: Urinary metabolomic profiles with baseline clinical factors may accurately predict sPC in men with elevated risk before biopsy.
RESUMO
Prostate cancer is one of the most frequently diagnosed cancers in males, and PC-3 is a cell model popularly used for investigating the behavior of late stage prostate cancer. Lysophosphatidic acid (LPA) is a lysophospholipid that mediates multiple behaviors in cancer cells, such as proliferation, migration and adhesion. We have previously demonstrated that LPA enhances vascular endothelial growth factor (VEGF)-C expression in PC-3 cells by activating the generation of reactive oxygen species (ROS), which is known to be an important mediator in cancer progression. Using flow cytometry, we showed that LPA triggers ROS generation within 10min and that the generated ROS can be suppressed by pretreatment with the NADPH oxidase (Nox) inhibitor diphenylene iodonium. In addition, transfection with LPA1 and LPA3 siRNA efficiently blocked LPA-induced ROS production, suggesting that both receptors are involved in this pathway. Using specific inhibitors and siRNA, phospholipase C (PLC) and protein kinase C (PKC) were also suggested to participate in LPA-induced ROS generation. Overall, we demonstrated that LPA induces ROS generation in PC-3 prostate cancer cells and this is mediated through the PLC/PKC/Nox pathway.
Assuntos
Lisofosfolipídeos/fisiologia , Neoplasias da Próstata/metabolismo , Proteína Quinase C/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Humanos , Lisofosfolipídeos/farmacologia , Masculino , Neoplasias da Próstata/enzimologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Fosfolipases Tipo C/biossínteseRESUMO
UNLABELLED: Alteration of cell surface proteolysis has been proposed to play a role in liver fibrosis, a grave complication of biliary atresia (BA). In this study we investigated the roles of hepatocyte growth factor activator inhibitor (HAI)-1 and -2 in the progression of BA. The expression levels of HAI-1 and -2 were significantly increased in BA livers compared with those in neonatal hepatitis and correlated with disease progression. In BA livers, HAI-1 and -2 were coexpressed in cells involved in ductular reactions. In other selective cholangiopathies, ductular cells positive for HAI-1 or HAI-2 also increased in number. Inflammatory cytokines, growth factors, and bile acids differentially up-regulated expression of HAI-1 and -2 transcripts in fetal liver cells and this induction could be antagonized by a cyclooxygenase-2 inhibitor. Conditioned media from cell lines stably overexpressing HAI-1 or HAI-2 enhanced the fibrogenic activity of portal fibroblasts and stellate cells, suggesting that both proteins might be involved in liver fibrosis. Because HAI-1 and -2 colocalized in ductular reactions sharing similar features to those observed during normal liver development, we sought to investigate the role of HAI-1 and -2 in cholangiopathies by exploring their functions in fetal liver cells. Knockdown of HAI-1 or HAI-2 promoted bidirectional differentiation of hepatoblast-derived cells. In addition, we showed that the hepatocyte growth factor activator, mitogen-activated protein kinase kinase 1, and phosphatidylinositol 3-kinase signaling pathways were involved in hepatic differentiation enhanced by HAI-2 knockdown. CONCLUSION: HAI-1 and -2 are overexpressed in the liver in cholangiopathies with ductular reactions and are possibly involved in liver fibrosis and hepatic differentiation; they could be investigated as disease markers and potential therapeutic targets.
Assuntos
Colestase/patologia , Hepatite/patologia , Cirrose Hepática/patologia , Glicoproteínas de Membrana/genética , Proteínas Secretadas Inibidoras de Proteinases/genética , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Colestase/fisiopatologia , Feminino , Fibroblastos/citologia , Células Estreladas do Fígado/citologia , Hepatite/congênito , Hepatite/fisiopatologia , Hepatócitos/citologia , Humanos , Lactente , Recém-Nascido , Cirrose Hepática/congênito , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Ratos , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
Mammalian DNA methyltransferases (DNMTs), including DNMT1, DNMT3A, and DNMT3B, are key DNA methylation enzymes and play important roles in gene expression regulation. Dysregulation of DNMTs is linked to various diseases and carcinogenesis, and therefore except for the two approved anticancer azanucleoside drugs, various non-nucleoside DNMT inhibitors have been identified and reported. However, the underlying mechanisms for the inhibitory activity of these non-nucleoside inhibitors still remain largely unknown. Here, we systematically tested and compared the inhibition activities of five non-nucleoside inhibitors toward the three human DNMTs. We found that harmine and nanaomycin A blocked the methyltransferase activity of DNMT3A and DNMT3B more efficiently than resveratrol, EGCG, and RG108. We further determined the crystal structure of harmine in complex with the catalytic domain of the DNMT3B-DNMT3L tetramer revealing that harmine binds at the adenine cavity of the SAM-binding pocket in DNMT3B. Our kinetics assays confirm that harmine competes with SAM to competitively inhibit DNMT3B-3L activity with a Ki of 6.6 µM. Cell-based studies further show that harmine treatment inhibits castration-resistant prostate cancer cell (CRPC) proliferation with an IC50 of â¼14 µM. The CPRC cells treated with harmine resulted in reactivating silenced hypermethylated genes compared to the untreated cells, and harmine cooperated with an androgen antagonist, bicalutamide, to effectively inhibit the proliferation of CRPC cells. Our study thus reveals, for the first time, the inhibitory mechanism of harmine on DNMTs and highlights new strategies for developing novel DNMT inhibitors for cancer treatment.
Assuntos
Antineoplásicos , Neoplasias de Próstata Resistentes à Castração , Masculino , Animais , Humanos , Metilação de DNA , Harmina/farmacologia , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação da Expressão Gênica , Transformação Celular Neoplásica/genética , Inibidores Enzimáticos/farmacologia , Antineoplásicos/farmacologia , DNA/metabolismo , Mamíferos/genética , Mamíferos/metabolismoRESUMO
BACKGROUND: Neuroendocrine prostate cancer (NEPC) is often diagnosed as a sub-type from the castration-resistant prostate cancer (CRPC) recurred from the second generation of anti-androgen treatment and is a rapidly progressive fatal disease. The molecular mechanisms underlying the trans-differentiation from CRPC to NEPC are not fully characterized, which hampers the development of effective targeted therapy. METHODS: Bioinformatic analyses were conducted to determine the clinical correlation of sphingosine kinase 1 (SphK1) in CRPC progression. To investigate the transcriptional regulation SphK1 and neuroendocrine (NE) transcription factor genes, both chromosome immunoprecipitation and luciferase reporter gene assays were performed. To demonstrate the role of SphK1 in NEPC development, neurosphere assay was carried out along with several biomarkers determined by quantitative PCR and western blot. Furthermore, in vivo NEPC xenograft models and patient-derived xenograft (PDX) model were employed to determine the effect of SphK1 inhibitors and target validation. RESULTS: Significant prevalence of SphK1 in NEPC development is observed from clinical datasets. SphK1 is transcriptionally repressed by androgen receptor-RE1-silencing transcription factor (REST) complex. Furthermore, sphingosine 1-phosphate produced by SphK1 can modulate REST protein turnover via MAPK signaling pathway. Also, decreased REST protein levels enhance the expression of NE markers in CRPC, enabling the transition to NEPC. Finally, specific SphK1 inhibitors can effectively inhibit the growth of NEPC tumors and block the REST protein degradation in PDX. CONCLUSIONS: SphK1 plays a central role in NEPC development, which offers a new target for this lethal cancer using clinically approved SphK1 inhibitors.
Assuntos
Carcinoma Neuroendócrino/etiologia , Fosfotransferases (Aceptor do Grupo Álcool)/efeitos adversos , Neoplasias da Próstata/etiologia , Carcinoma Neuroendócrino/genética , Humanos , Masculino , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/estatística & dados numéricos , Sistemas Neurossecretores/anormalidades , Sistemas Neurossecretores/fisiopatologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologiaRESUMO
Dysregulation of pericellular proteolysis is strongly implicated in cancer metastasis through alteration of cell invasion and the microenvironment. Matriptase-2 (MT-2) is a membrane-anchored serine protease which can suppress prostate cancer (PCa) cell invasion. In this study, we showed that MT-2 was down-regulated in PCa and could suppress PCa cell motility, tumor growth, and metastasis. Using microarray and biochemical analysis, we found that MT-2 shifted TGF-ß action towards its tumor suppressor function by repressing epithelial-to-mesenchymal transition (EMT) and promoting Smad2 phosphorylation and nuclear accumulation to upregulate two TGF-ß1 downstream effectors (p21 and PAI-1), culminating in hindrance of PCa cell motility and malignant growth. Mechanistically, MT-2 could dramatically up-regulate the expression of nuclear receptor NR4A3 via iron metabolism in PCa cells. MT-2-induced NR4A3 further coactivated Smad2 to activate p21 and PAI-1 expression. In addition, NR4A3 functioned as a suppressor of PCa and mediated MT-2 signaling to inhibit PCa tumorigenesis and metastasis. These results together indicate that NR4A3 sustains MT-2 signaling to suppress PCa cell invasion, tumor growth, and metastasis, and serves as a contextual factor for the TGF-ß/Smad2 signaling pathway in favor of tumor suppression via promoting p21 and PAI-1 expression.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Membrana , Neoplasias da Próstata , Receptores de Esteroides , Receptores dos Hormônios Tireóideos , Serina Endopeptidases , Linhagem Celular Tumoral , Movimento Celular , Proteínas de Ligação a DNA/metabolismo , Transição Epitelial-Mesenquimal , Humanos , Masculino , Proteínas de Membrana/metabolismo , Invasividade Neoplásica , Inibidor 1 de Ativador de Plasminogênio , Próstata/patologia , Neoplasias da Próstata/patologia , Receptores de Esteroides/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Serina Endopeptidases/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Microambiente TumoralRESUMO
Antithrombin, a major anticoagulant, is robustly transported into extravascular compartments where its target proteases are largely unknown. This serpin was previously detected in human milk as complexes with matriptase, a membrane-bound serine protease broadly expressed in epithelial and carcinoma cells, and under tight regulation by hepatocyte growth factor activator inhibitor (HAI)-1, a transmembrane Kunitz-type serine protease inhibitor that forms heat-sensitive complexes with active matriptase. In the current study, we detect, in addition to matriptase-HAI-1 complexes, heat-resistant matriptase complexes generated by nontransformed mammary, prostate, and epidermal epithelial cells that we show to be matriptase-antithrombin complexes. These findings suggest that in addition to HAI-1, interstitial antithrombin participates in the regulation of matriptase activity in epithelial cells. This physiological mechanism appears, however, to largely be lost in cancer cells since matriptase-antithrombin complexes were not detected in all but two of a panel of seven breast, prostate, and ovarian cancer cell lines. Using purified active matriptase, we further characterize the formation of matriptase-antithrombin complex and show that heparin can significantly potentiate the inhibitory potency of antithrombin against matriptase. Second-order rate constants for the inhibition were determined to be 3.9 × 10(3) M(-1)s(-1) in the absence of heparin and 1.2 × 10(5) M(-1)s(-1) in the presence of heparin, a 30-fold increase, consistent with the established role of heparin in activating antithrombin function. Taken together these data suggest that normal epithelial cells employ a dual mechanism involving HAI-1 and antithrombin to control matriptase and that the antithrombin-based mechanism appears lost in the majority of carcinoma cells.
Assuntos
Antitrombina III/metabolismo , Carcinoma/enzimologia , Células Epiteliais/enzimologia , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Antígenos Nucleares/metabolismo , Neoplasias da Mama/enzimologia , Linhagem Celular Tumoral , Epiderme/enzimologia , Feminino , Heparina/farmacologia , Humanos , Masculino , Glândulas Mamárias Humanas/enzimologia , Neoplasias Ovarianas/enzimologia , Próstata/enzimologia , Neoplasias da Próstata/enzimologia , Fatores de Transcrição/metabolismoRESUMO
Deregulation of both ErbB-2 signaling and matriptase activity has been associated with human prostate cancer (PCa) progression. In this communication, we investigated the roles of both ErbB-2 signaling in matriptase zymogen activation and matriptase in ErbB-2-induced PCa malignancy. In a human PCa cell progression model, we observed that advanced PCa C-81 LNCaP cells exhibited an aggressive phenotype with increased cell migration and invasion capacity; these cells concurrently showed both enhanced ErbB-2 phosphorylation and increased matriptase zymogen activation compared with parental C-33 LNCaP cells. Moreover, ErbB2 activation, both ligand-dependent (eg, epidermal growth factor treatment) and ligand-independent (eg, overexpression), was able to induce matriptase zymogen activation in this cell line. Inhibition of ErbB-2 activity by either the specific inhibitor, AG825, in epidermal growth factor-treated C-33 LNCaP cells or ErbB-2 knockdown in C-81 LNCaP cells, reduced matriptase activation. These observations were confirmed by similar studies using both DU145 and PC3 cells. Together, these data suggest that ErbB-2 signaling plays an important role in matriptase zymogen activation. ErbB-2-enhanced matriptase activation was suppressed by a phosphatidylinositol 3-kinase inhibitor (ie, LY294002) but not by a MEK inhibitor (ie, PD98059). Suppression of matriptase expression by small hairpin RNA knockdown in ErbB-2-overexpressing LNCaP cells dramatically suppressed cancer cell invasion. In summary, our data indicate that ErbB-2 signaling via the phosphatidylinositol 3-kinase pathway results in up-regulated matriptase zymogen activity, which contributes to PCa cell invasion.
Assuntos
Carcinoma/patologia , Genes erbB-2/fisiologia , Neoplasias da Próstata/patologia , Serina Endopeptidases/fisiologia , Androgênios/farmacologia , Carcinoma/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Cultivadas , Progressão da Doença , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/genética , Ativação Enzimática/genética , Técnicas de Silenciamento de Genes , Humanos , Masculino , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Neoplasias da Próstata/genética , RNA Interferente Pequeno/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
TMPRSS2, a type II transmembrane serine protease, is highly expressed by the epithelium of the human prostate gland. To explore the regulation and function of TMPRSS2 in the prostate, a panel of monoclonal antibodies with high sensitivity and specificity were generated. Immunodetection showed TMPRSS2 on the apical plasma membrane of the prostate luminal cells and demonstrated its release into semen as a component of prostasomes, organelle-like vesicles that may facilitate sperm function and enhance male reproduction. In prostate cancer cells, TMPRSS2 expression was increased and the protein mislocalized over the entire tumor cell membrane. In both LNCaP prostate cancer cells and human semen, TMPRSS2 protein was detected predominantly as inactive zymogen forms as part of an array of multiple noncovalent and disulfide-linked complexes, suggesting that TMPRSS2 activity may be regulated by unconventional mechanisms. Our data suggested that TMPRSS2, an apical surface serine protease, may have a normal role in male reproduction as a component of prostasomes. The aberrant cellular localization, and increased expression of the protease seen in cancer, may contribute to prostate tumorigenesis by providing access of the enzyme to nonphysiological substrates and binding-proteins.
Assuntos
Células Epiteliais , Próstata/citologia , Próstata/enzimologia , Neoplasias da Próstata/enzimologia , Sêmen/enzimologia , Serina Endopeptidases/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Masculino , Camundongos , Análise em Microsséries , Pessoa de Meia-Idade , Próstata/patologia , Neoplasias da Próstata/patologia , Serina Endopeptidases/genéticaRESUMO
Current clinical trials of combined EGFR-tyrosine kinase inhibitors (TKI) and immune checkpoint blockade (ICB) therapies show no additional effect. This raises questions regarding whether EGFR-TKIs attenuate ICB-enhanced CD8+ T lymphocyte function. Here we show that the EGFR-TKI afatinib suppresses CD8+ T lymphocyte proliferation, and we identify CAD, a key enzyme of de novo pyrimidine biosynthesis, to be a novel afatinib target. Afatinib reduced tumor-infiltrating lymphocyte numbers in Lewis lung carcinoma (LLC)-bearing mice. Early afatinib treatment inhibited CD8+ T lymphocyte proliferation in patients with non-small cell lung cancer, but their proliferation unexpectedly rebounded following long-term treatment. This suggests a transient immunomodulatory effect of afatinib on CD8+ T lymphocytes. Sequential treatment of afatinib with anti-PD1 immunotherapy substantially enhanced therapeutic efficacy in MC38 and LLC-bearing mice, while simultaneous combination therapy showed only marginal improvement over each single treatment. These results suggest that afatinib can suppress CD8+ T lymphocyte proliferation by targeting CAD, proposing a timing window for combined therapy that may prevent the dampening of ICB efficacy by EGFR-TKIs. SIGNIFICANCE: This study elucidates a mechanism of afatinib-mediated immunosuppression and provides new insights into treatment timing for combined targeted therapy and immunotherapy. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3270/F1.large.jpg.
Assuntos
Afatinib/farmacologia , Antineoplásicos Imunológicos/farmacologia , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Desoxirribonucleases/antagonistas & inibidores , Agentes de Imunomodulação/farmacologia , Pirimidinas/biossíntese , Animais , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Quimioterapia Combinada , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/antagonistas & inibidoresRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
TMPRSS2 is an important membrane-anchored serine protease involved in human prostate cancer progression and metastasis. A serine protease physiologically often comes together with a cognate inhibitor for execution of proteolytically biologic function; however, TMPRSS2's cognate inhibitor is still elusive. To identify the cognate inhibitor of TMPRSS2, in this study, we applied co-immunoprecipitation and LC/MS/MS analysis and isolated hepatocyte growth factor activator inhibitors (HAIs) to be potential inhibitor candidates for TMPRSS2. Moreover, the recombinant HAI-2 proteins exhibited a better inhibitory effect on TMPRSS2 proteolytic activity than HAI-1, and recombinant HAI-2 proteins had a high affinity to form a complex with TMPRSS2. The immunofluorescence images further showed that TMPRSS2 was co-localized to HAI-2. Both KD1 and KD2 domain of HAI-2 showed comparable inhibitory effects on TMPRSS2 proteolytic activity. In addition, HAI-2 overexpression could suppress the induction effect of TMPRSS2 on pro-HGF activation, extracellular matrix degradation and prostate cancer cell invasion. We further determined that the expression levels of TMPRSS2 were inversely correlated with HAI-2 levels during prostate cancer progression. In orthotopic xenograft animal model, TMPRSS2 overexpression promoted prostate cancer metastasis, and HAI-2 overexpression efficiently blocked TMPRSS2-induced metastasis. In summary, the results together indicate that HAI-2 can function as a cognate inhibitor for TMPRSS2 in human prostate cancer cells and may serve as a potential factor to suppress TMPRSS2-mediated malignancy.
Assuntos
Glicoproteínas de Membrana/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Serina Endopeptidases/metabolismo , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Xenoenxertos , Humanos , Masculino , Glicoproteínas de Membrana/química , Invasividade Neoplásica , Neoplasias da Próstata/etiologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , ProteóliseRESUMO
Matriptase, a transmembrane serine protease, is broadly expressed by, and crucial for the integrity of, the epithelium. Matriptase is synthesized as a zymogen and undergoes autoactivation to become an active protease that is immediately inhibited by, and forms complexes with, hepatocyte growth factor activator inhibitor (HAI-1). To investigate where matriptase is activated and how it is secreted in vivo, we determined the expression and activation status of matriptase in seminal fluid and urine and the distribution and subcellular localization of the protease in the prostate and kidney. The in vivo studies revealed that while the latent matriptase is localized at the basolateral surface of the ductal epithelial cells of both organs, only matriptase-HAI-1 complexes and not latent matriptase are detected in the body fluids, suggesting that activation, inhibition, and transcytosis of matriptase would have to occur for the secretion of matriptase. These complicated processes involved in the in vivo secretion were also observed in polarized Caco-2 intestinal epithelial cells. The cells target latent matriptase to the basolateral plasma membrane where activation, inhibition, and secretion of matriptase appear to take place. However, a proportion of matriptase-HAI-1 complexes, but not the latent matriptase, appears to undergo transcytosis to the apical plasma membrane for secretion. When epithelial cells lose their polarity, they secrete both latent and activated matriptase. Although most epithelial cells retain very low levels of matriptase-HAI-1 complex by rapidly secreting the complex, gastric chief cells may activate matriptase and store matriptase-HAI-1 complexes in the pepsinogen-secretory granules, suggesting an intracellular activation and regulated secretion in these cells. Taken together, while zymogen activation and closely coupled HAI-1-mediated inhibition are common features for matriptase regulation, the cellular location of matriptase activation and inhibition, and the secretory route for matriptase-HAI-1 complex may vary along with the functional divergence of different epithelial cells.
Assuntos
Polaridade Celular , Precursores Enzimáticos/metabolismo , Células Epiteliais/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Serina Endopeptidases/metabolismo , Animais , Líquidos Corporais/enzimologia , Linhagem Celular , Ativação Enzimática , Células Epiteliais/citologia , Feminino , Humanos , Rim/citologia , Rim/enzimologia , Masculino , Próstata/citologia , Próstata/enzimologia , Estômago/citologia , Estômago/enzimologiaRESUMO
Gram-negative bacteria have been found to be a major population in prostatitis and prostate cancer (PCa) tissues. Lipopolysaccharide (LPS), a major compound of Gram-negative bacteria, with stimulatory activities in some cancer types, but has not been fully studied in PCa. In this study, we examined the effect of LPS on the invasion of PCa cells. Interestingly, LPS can enhance the invasiveness of PCa, but had no significant effect on PCa cell viability. Using protease inhibitor screening and biochemical analyses, matriptase, a member of the membrane-anchored serine protease family, is found to play a key role in LPS-induced PCa cell invasion. Mechanistically, Toll-like receptor 4 (TLR4, LPS receptor)-sphingosine kinase 1 (SphK1) signaling underlies LPS-induced matriptase activation and PCa cell invasion. Specifically, LPS induced the S225 phosphorylation of SphK1 and the translocation of SphK1 to plasma membrane, leading to the production of sphingosine 1-phosphate (S1P), ERK1/2 and matriptase activation via S1P receptor 4 (S1PR4). This phenomenon is further validated using the patient-derived explant (PDE) model. Indeed, there is a significant correlation among the elevated SphK1 levels, the Gleason grades of PCa specimens, and the poor survival of PCa patients. Taken together, this study demonstrates a potential impact of LPS on PCa progression. Our results provide not only a new finding of the role of bacterial infection in PCa progression but also potential therapeutic target(s) associated with PCa metastasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Polissacarídeos/farmacologia , Neoplasias da Próstata/patologia , Serina Endopeptidases/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Progressão da Doença , Ativação Enzimática , Humanos , Masculino , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/metabolismoRESUMO
Afatinib, used for the first-line treatment of non-small-cell lung carcinoma (NSCLC) patients with distinct epidermal growth factor receptor (EGFR) mutations, inactivates EGFR by mimicking ATP structure and forming a covalent adduct with EGFR. We developed a method to unravel potential targets of afatinib in NSCLC cells through immunoprecipitation of afatinib-labeling proteins with anti-afatinib antiserum and mass spectrometry analysis. Ribonucleotide reductase (RNR) is one of target proteins of afatinib revealed by this method. Treatment of afatinib at 10-100 nM potently inhibited intracellular RNR activity in an in vitro assay using permeabilized PC-9 cells (formerly known as PC-14). PC-9 cells treated with 10 µM afatinib displayed elevated markers of DNA damage. Long-term treatment of therapeutic concentrations of afatinib in PC-9 cells caused significant decrease in protein levels of RNR subunit M2 at 1-10 nM and RNR subunit M1 at 100 nM. EGFR-null Chinese hamster ovary (CHO) cells treated with afatinib also showed similar effects. Afatinib repressed the upregulation of RNR subunit M2 induced by gemcitabine. Covalent modification with afatinib resulting in inhibition and protein downregulation of RNR underscores the therapeutic and off-target effects of afatinib. Afatinib may serve as a lead compound of chemotherapeutic drugs targeting RNR. This method can be widely used in the identification of potential targets of other covalent drugs.