RESUMO
The transcriptional coactivator YAP1 (yes-associated protein 1) regulates cell proliferation, cell-cell interactions, organ size, and tumorigenesis. Post-transcriptional modifications and nuclear translocation of YAP1 are crucial for its nuclear activity. The objective of this study was to elucidate the mechanism by which the steroid hormone androgen regulates YAP1 nuclear entry and functions in several human prostate cancer cell lines. We demonstrate that androgen exposure suppresses the inactivating post-translational modification phospho-Ser-127 in YAP1, coinciding with increased YAP1 nuclear accumulation and activity. Pharmacological and genetic experiments revealed that intact androgen receptor signaling is necessary for androgen's inactivating effect on phospho-Ser-127 levels and increased YAP1 nuclear entry. We also found that androgen exposure antagonizes Ser/Thr kinase 4 (STK4/MST1) signaling, stimulates the activity of protein phosphatase 2A, and thereby attenuates the phospho-Ser-127 modification and promotes YAP1 nuclear localization. Results from quantitative RT-PCR and CRISPR/Cas9-aided gene knockout experiments indicated that androgen differentially regulates YAP1-dependent gene expression. Furthermore, an unbiased computational analysis of the prostate cancer data from The Cancer Genome Atlas revealed that YAP1 and androgen receptor transcript levels correlate with each other in prostate cancer tissues. These findings indicate that androgen regulates YAP1 nuclear localization and its transcriptional activity through the androgen receptor-STK4/MST1-protein phosphatase 2A axis, which may have important implications for human diseases such as prostate cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Androgênios/farmacologia , Núcleo Celular/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Linhagem Celular Tumoral , Bases de Dados Genéticas , Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas de Sinalização YAPRESUMO
Androgen receptor (AR) signaling is vital to the viability of all forms of prostate cancer (PCa). With the goal of investigating the effect of simultaneous inhibition and depletion of AR on viability of PCa cells, we designed, synthesized and characterized the bioactivities of bifunctional agents which incorporate the independent cancer killing properties of an antiandrogen and genistein, and the AR downregulation effect of genistein within a single molecular template. We observed that a representative conjugate, 9b, is much more cytotoxic to both LNCaP and DU145 cells relative to the antiandrogen and genistein building blocks as single agents or their combination. Moreover, conjugate 9b more effectively down regulates cellular AR protein levels relative to genistein and induces S phase cell cycle arrest. The promising bioactivities of these conjugates warrant further investigation.
Assuntos
Antagonistas de Androgênios/farmacologia , Antineoplásicos/farmacologia , Desenho de Fármacos , Genisteína/farmacologia , Hidantoínas/farmacologia , Antagonistas de Androgênios/síntese química , Antagonistas de Androgênios/química , Antineoplásicos/síntese química , Antineoplásicos/química , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Genisteína/síntese química , Genisteína/química , Humanos , Hidantoínas/química , Estrutura Molecular , Receptores Androgênicos/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
BACKGROUND: This study aimed to evaluate the clinical and radiological features of the patella fixation technique using Toggleloc suspension system in a single ellipsoidal blind patellar tunnel during medial patellofemoral ligament (MPFL) reconstruction. METHODS: This study included 52 patients (25 men, 27 women) who underwent MPFL reconstruction using a semitendinosus tendon graft. The graft was fixed to the ellipsoidal single blind tunnel opened on the medial side of the patella with an endobutton and was fixed to the femoral tunnel by using bioabsorbable screw. Clinical scores (Kujala score, Lysholm score, Tegner activity score and the visual analog scale [VAS] score) were evaluated preoperatively and at the end-follow up. Preoperative and postoperative radiological measurements (trochlea depth, sulcus angle, patellar height, patellar congruence angle, patellar tilt angle and lateral patellofemoral angle) were evaluated with X-ray (Merchant X-ray, anteroposterior and lateral radiography) and computed tomography (CT) of the knee. RESULTS: Postoperative patellar redislocation or subluxation was not observed in any patient. Patellar congruence angle, patellar tilt angle and lateral patellofemoral angle mean values were found to return to normal values in the postoperative period and the results were statistically significant. Also statistically significant improvement in all clinical scores postoperatively. According to the Insall-Salvati index (ISI) and Caton-Deschamps index (CDI) on lateral radiography of the knee at 30° flexion, patellar height decreased in the postoperative period statistically significant. The CDI was above 1.3 in 17 (%32) of our patients. Thirteen of these values decreased to normal values. No radiological progression of patellofemoral osteoarthritis was observed in all patients at the final follow-up evaluation. CONCLUSION: In cases of patellofemoral instability, fixation of the tendon graft in blind ellipsoid tunnel using the Toggleloc suspension system provides satisfactory patellar graft fixation strength, significant functional improvement and a low failure rate.
Assuntos
Patela , Articulação Patelofemoral , Humanos , Feminino , Masculino , Adulto , Articulação Patelofemoral/cirurgia , Articulação Patelofemoral/diagnóstico por imagem , Seguimentos , Patela/cirurgia , Procedimentos de Cirurgia Plástica/métodos , Adulto Jovem , Ligamentos Articulares/cirurgia , Resultado do Tratamento , AdolescenteRESUMO
Mst1/Stk4, a hippo-like serine-threonine kinase, is implicated in many cancers, including prostate cancer. However, the mechanisms regulating Mst1 remain obscure. Here, we characterized the effects of phospho-Thr-120 on Mst1 in prostate cancer cells. We demonstrated that phospho-Thr-120 did not alter the nuclear localization or cleavage of Mst1 in a LNCaP or castration-resistant C4-2 prostate tumor cell model, as revealed by a mutagenesis approach. Phospho-Thr-120 appeared to be specific to cancer cells and predominantly localized in the nucleus. In contrast, phospho-Thr-183, a critical regulator of Mst1 cell death, was exclusively found in the cytoplasm. As assessed by immunohistochemistry, a similar distribution of phospho-Mst1-Thr-120/Thr-183 was also observed in a prostate cancer specimen. In addition, the blockade of PI3K signaling by a small molecule inhibitor, LY294002, increased cytoplasmic phospho-Mst1-Thr-183 without having a significant effect on nuclear phospho-Mst1-Thr-120. However, the attenuation of mammalian target of rapamycin (mTOR) activity by a selective pharmacologic inhibitor, Ku0063794 or CCI-779, caused the up-regulation of nuclear phospho-Mst1-Thr-120 without affecting cytoplasmic phospho-Mst1-Thr-183. This suggests that PI3K and mTOR pathway signaling differentially regulate phospho-Mst1-Thr-120/Thr-183. Moreover, mutagenesis and RNAi data revealed that phospho-Thr-120 resulted in C4-2 cell resistance to mTOR inhibition and reduced the Mst1 suppression of cell growth and androgen receptor-driven gene expression. Collectively, these findings indicate that phospho-Thr-120 leads to the loss of Mst1 functions, supporting cancer cell growth and survival.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Treonina/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromonas/farmacologia , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Nus , Morfolinas/farmacologia , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Serina-Treonina Quinases/genética , Pirimidinas/farmacologia , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Transplante Heterólogo , Carga TumoralRESUMO
TOK-001 and abiraterone are potent 17-heteroarylsteroid (17-HAS) inhibitors of Cyp17, one of the rate-limiting enzymes in the biosynthesis of testosterone from cholesterol in prostate cancer cells. Nevertheless, the molecular mechanism underlying the prevention of prostate cell growth by 17-HASs still remains elusive. Here, we assess the effects of 17-HASs on androgen receptor (AR) activity in LNCaP and LAPC-4 cells. We demonstrate that both TOK-001 and abiraterone reduced AR protein and mRNA expression, and antagonized AR-dependent promoter activation induced by androgen. TOK-001, but not abiraterone, is an effective apparent competitor of the radioligand [(3)H]R1881 for binding to the wild type and various mutant AR (W741C, W741L) proteins. In agreement with these data, TOK-001 is a consistently superior inhibitor than abiraterone of R1881-induced transcriptional activity of both wild type and mutant AR. However, neither agent was able to trans-activate the AR in the absence of R1881. Our data demonstrate that phospho-4EBP1 levels are significantly reduced by TOK-001 and to a lesser extent by abiraterone alcohol, and suggest a mechanism by which cap-dependent translation is suppressed by blocking assembly of the eIF4F and eIF4G complex to the mRNA 5' cap. Thus, the effects of these 17-HASs on AR signaling are complex, ranging from a decrease in testosterone production through the inhibition of Cyp17 as previously described, to directly reducing both AR protein expression and R1881-induced AR trans-activation.
Assuntos
Androstadienos/farmacologia , Androstenóis/farmacologia , Benzimidazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Esteroide 17-alfa-Hidroxilase/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Substituição de Aminoácidos , Androstenos , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Mutação de Sentido Incorreto , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Ligação Proteica , Receptores Androgênicos/genética , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismoRESUMO
OBJECTIVES: This study aims to compare the effects of teriparatide, zoledronic acid, and their combination therapy with vitamin K on osteoporotic rats. MATERIALS AND METHODS: We divided a total of 50 female Sprague-Dawley rats into five groups: A (the control group), B and D (the teriparatide group), and C and E (the zoledronic acid group). Following ovariectomy and subcutaneous heparin administration at a dose of 2 IU/kg for four weeks, osteoporosis was created. Groups A, B, and C were fed with standard feed, while Groups D and E were fed with vitamin K-rich feed. After four weeks of treatment, sacrification was performed. The right and left femurs were separated for histopathological and biomechanical evaluation, respectively. For histopathological evaluation, the femurs were decalcified, and the sections were stained with hematoxylin-eosin and evaluated under a light microscope. Fracture healing was evaluated using the classification system as described previously. For biomechanical evaluation, the 3-point stress test and torsion stress test were applied to 10 femurs from each group. RESULTS: Groups B-E were histopathologically and biomechanically superior to Group A in fracture healing of osteoporotic rats; however, it was not statistically significant (p>0.05). The group that received additional vitamin K was histopathologically and biomechanically superior to the group which was fed with standard feed, although it was not statistically significant (p>0.05). CONCLUSION: Our study results indicated that both teriparatide and zoledronic acid had beneficial effects on osteoporotic fractures with comparable histological and biochemical results. Vitamin K promoted teriparatide and zoledronic acid treatment on osteoporotic fracture healing. Based on these findings, combination therapies may yield the most optimal results in biomechanical and histological examinations.
Assuntos
Conservadores da Densidade Óssea , Osteoporose , Fraturas por Osteoporose , Ratos , Feminino , Animais , Ácido Zoledrônico/farmacologia , Ácido Zoledrônico/uso terapêutico , Teriparatida/farmacologia , Teriparatida/uso terapêutico , Conservadores da Densidade Óssea/farmacologia , Conservadores da Densidade Óssea/uso terapêutico , Vitamina K/farmacologia , Vitamina K/uso terapêutico , Ratos Sprague-Dawley , Osteoporose/tratamento farmacológico , Consolidação da FraturaRESUMO
The Polycomb group protein SCML2 and the transcriptional cofactor YAP1 regulate diverse cellular biology, including stem cell maintenance, developmental processes, and gene regulation in mammals and flies. However, their molecular and functional interactions are unknown. Here, we show that SCML2 interacts with YAP1, as revealed by immunological assays and mass spectroscopy. We have demonstrated that the steroid hormone androgen regulates the interaction of SCML2 with YAP1 in human tumor cell models. Our proximity ligation assay and GST pulldown showed that SCML2 and YAP1 physically interacted with each other. Silencing SCML2 by RNAi changed the growth behaviors of cells in response to androgen signaling. Mechanistically, this phenomenon is attributed to the interplay between distinct chromatin modifications and transcriptional programs, likely coordinated by the opposing SCML2 and YAP1 activity. These findings suggest that YAP1 and SCML2 cooperate to regulate cell growth, cell survival, and tumor biology downstream of steroid hormones.
RESUMO
The EPHA3 protein tyrosine kinase, a member of the ephrin receptor family, regulates cell fate, cell motility, and cell-cell interaction. These cellular events are critical for tissue development, immunological responses, and the processes of tumorigenesis. Earlier studies revealed that signaling via the STK4-encoded MST1 serine-threonine protein kinase, a core component of the Hippo pathway, attenuated EPHA3 expression. Here, we investigated the mechanism by which MST1 regulates EPHA3. Our findings have revealed that the transcriptional regulators YAP1 and TEAD1 are crucial activators of EPHA3 transcription. Silencing YAP1 and TEAD1 suppressed the EPHA3 protein and mRNA levels. In addition, we identified putative TEAD enhancers in the distal EPHA3 promoter, where YAP1 and TEAD1 bind and promote EPHA3 expression. Furthermore, EPHA3 knockout by CRISPR/Cas9 technology reduced cell-cell interaction and cell motility. These findings demonstrate that EPHA3 is transcriptionally regulated by YAP1/TEAD1 of the Hippo pathway, suggesting that it is sensitive to cell contact-dependent interactions.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Sinalização YAP , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Serina-Treonina Quinases/genética , Receptor EphA3/genética , Receptor EphA3/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVE: The aim of this study was to compare clinical and histopathological effects of oral versus intraarticular corticosteroid application in a rat model of frozen shoulder. METHODS: In this study, eighty adult Sprague-Dawley rats were used. The animals were divided into 5 equal groups. The frozen shoulder model was created by immobilizing animals' shoulders with internal fixation with sutures for 8 weeks. At the 8th week, sham (n: 16) and control (n: 16) groups were sacrificed to collect data for healthy and affected shoulders. Also, at the 8th week, 50 mg/ kg methylprednisolone was started for the oral treatment group, and a single dose of 0.5 mg/kg triamcinolone acetonide was injected for the intraarticular treatment group. The effect of additional steroid treatment was expected for 2 weeks, then all remaining treatment and natural course groups were sacrificed on the 10th week. RESULTS: After sacrification, specimens taken as "en bloc" scapulothoracic disarticulation were randomly divided into two groups for a range of motion measurement and histopathological examination. The control (frozen shoulder model) group's shoulder range of motion in all directions was lower than the sham (healthy) group (P < 0.01). Natural course and intraarticular steroid groups, compared to the frozen shoulder model showed a significant increase in the direction of abduction (P < 0.05). Also, it was found for treatment groups that in all directions the range of motion was not as good as the healthy values (P < 0.01). The intraarticular treatment group showed higher degrees of abduction compared to the natural course and oral steroid treatment groups (P < 0.01). Oral steroid treatment group's range of motion was not significantly better than the disease model and had no superiority to the natural course group (P > 0.05). Histopathologically, no statistically significant difference was observed between the groups for signs of frozen shoulder which was found in the immobilized group (P > 0.05). Histopathologically, immobilization was found to cause thickening of the capsule that cannot be resolved by treatment. (P < 0.05). CONCLUSION: In frozen shoulder disease, intraarticular steroid injection seems to be superior in increasing the range of motion than oral steroid treatment.
Assuntos
Bursite , Articulação do Ombro , Corticosteroides/uso terapêutico , Animais , Bursite/tratamento farmacológico , Injeções Intra-Articulares , Amplitude de Movimento Articular , Ratos , Ratos Sprague-Dawley , Resultado do Tratamento , Triancinolona Acetonida/farmacologia , Triancinolona Acetonida/uso terapêuticoRESUMO
Akt kinases mediate cell growth and survival. Here, we report that a pro-apoptotic kinase, Mst1/STK4, is a physiological Akt1 interaction partner. Mst1 was identified as a component of an Akt1 multiprotein complex isolated from lipid raft-enriched fractions of LNCaP human prostate cancer cells. Endogenous Mst1, along with its paralog, Mst2, acted as inhibitors of endogenous Akt1. Surprisingly, mature Mst1 as well as both of its caspase cleavage products, which localize to distinct subcellular compartments and are not structurally homologous, complexed with and inhibited Akt1. cRNAs encoding full-length Mst1, and N- and C-terminal caspase Mst1 cleavage products, reverted an early lethal phenotype in zebrafish development induced by expression of membrane-targeted Akt1. Mst1 and Akt1 localized to identical subcellular sites in human prostate tumors. Mst1 levels declined with progression from clinically localized to hormone refractory disease, coinciding with an increase in Akt activation with transition from hormone naïve to hormone-resistant metastases. These results position Mst1/2 within a novel branch of the phosphoinositide 3-kinase/Akt pathway and suggest an important role in cancer progression.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/fisiologia , Animais , Apoptose , Caspases/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Microdomínios da Membrana/química , Modelos Biológicos , Fenótipo , Estrutura Terciária de Proteína , Peixe-ZebraRESUMO
OBJECTIVES: This study aims to compare clinical results of repair using two versus three double-loaded suture anchors in arthroscopic Bankart repair. PATIENTS AND METHODS: Between July 2012 and December 2017, a total of 40 patients (38 males, 2 females; mean age: 31.6±8.1; range: 17 to 47 years) who underwent Bankart arthroscopic surgery and were followed for minimum two years were retrospectively analyzed. Group 1 (n=17) underwent arthroscopic Bankart repair with two double-loaded suture anchors, while Group 2 (n=23) underwent repair with three double-loaded suture anchors. Clinical outcomes of the patients and recurrences were compared. RESULTS: At the final postoperative follow-up, a significant improvement was observed in the functional outcomes in all patients. No statistically significant difference was found (p>0.05) in the mean clinical scores of the Constant Shoulder Score between Group 1 (94.2±7.8) and Group 2 (95.4±4.1). There was no significant difference in the mean Rowe scores (Group 1: 95.6±4.6 vs. Group 2: 96.3±3.8, respectively) and external rotation loss (at neutral Group 1: 1.9° vs. Group 2: 2.2°, respectively). Three of our patients had recurrent dislocation during a major traumatic event (n=2 in Group 1 and n=1 in Group 2). CONCLUSION: Our study results suggest that stability is not correlated with the use of either two versus three double-loaded suture anchors in arthroscopic Bankart repairs.
Assuntos
Artroplastia/instrumentação , Artroscopia/instrumentação , Instabilidade Articular/cirurgia , Luxação do Ombro/cirurgia , Âncoras de Sutura , Adolescente , Adulto , Artroplastia/métodos , Artroscopia/métodos , Feminino , Humanos , Instabilidade Articular/etiologia , Masculino , Amplitude de Movimento Articular , Recidiva , Estudos Retrospectivos , Luxação do Ombro/complicações , Luxação do Ombro/fisiopatologia , Resultado do Tratamento , Adulto JovemRESUMO
The Hippo pathway controls several biological processes, including cell growth, differentiation, motility, stemness, cell contact, immune cell maturation, organ size, and tumorigenesis. The Hippo pathway core kinases MST1/2 and LATS1/2 in mammals phosphorylate and inactivate YAP1 signaling. Increasing evidence indicates that loss of MST1/2 and LATS1/2 function is linked to the biology of many cancer types with poorer outcomes, likely due to the activation of oncogenic YAP1/TEAD signaling. Therefore, there is a renewed interest in blocking the YAP1/TEAD functions to prevent cancer growth. This review introduces the Hippo pathway components and examines their role and therapeutic potentials in prostate, kidney, and bladder cancer.
RESUMO
The varus ankle deformity can lead to osteoarthritis; therefore, numerous supramalleolar tibia osteotomy techniques are described to correct this deformity. Many of these techniques are more suitable for uniplanar ankle deformity. Particularly, if there are multiplane ankle deformities, the use of the six-axis deformity correction system may be successful in solving the problems which may occur during the correction. In this article, we report two cases of three plane deformities of ankle joint due to trauma sequelae, which were treated with supramalleolar osteotomy using a hexapod fixator which is called the Smart Correction Frame®.
Assuntos
Articulação do Tornozelo , Fixadores Externos , Deformidades Adquiridas do Pé , Osteoartrite/prevenção & controle , Osteotomia , Adolescente , Adulto , Traumatismos do Tornozelo/complicações , Articulação do Tornozelo/diagnóstico por imagem , Articulação do Tornozelo/patologia , Articulação do Tornozelo/cirurgia , Desenho Assistido por Computador , Feminino , Deformidades Adquiridas do Pé/complicações , Deformidades Adquiridas do Pé/fisiopatologia , Deformidades Adquiridas do Pé/cirurgia , Humanos , Masculino , Osteoartrite/etiologia , Osteotomia/instrumentação , Osteotomia/métodos , Radiografia/métodos , Recuperação de Função Fisiológica , Resultado do TratamentoRESUMO
The serine-threonine kinase, Akt, has been linked to cholesterol-sensitive signaling mechanisms, suggesting a possible means whereby cholesterol might affect tumor cell growth and survival. However, it has not been shown whether Akt itself, as distinct from upstream components of the pathway (e.g., membrane phosphoinositides), can be directly responsible for cholesterol-mediated effects. Consistent with this possibility, we identified an Akt1 subpopulation in cholesterol-rich lipid raft fractions prepared from LNCaP human prostate cancer cells. Phosphorylation of this Akt subspecies was ablated with methyl-beta-cyclodextrin, a cholesterol-binding compound, under conditions where nonlipid raft-resident Akt was unaffected. A myristoylated Akt1 (MyrAkt1) fusion protein expressed in LNCaP cells was found to be highly enriched in lipid rafts, indicating that oncogenic Akt is overrepresented in cholesterol-rich membranes compared with wild-type Akt. Notably, lipid raft-resident MyrAkt1 exhibited a markedly distinct substrate preference compared with MyrAkt1 immunoprecipitated from cytosol and nonraft membrane fractions, suggesting a redirection of signal transduction when the protein is present in cholesterol-rich membranes. Expression of MyrAkt1 in LNCaP cells overcame their characteristic dependence on constitutive signaling through the phosphoinositide 3'-kinase pathway. This protective effect was substantially diminished with cyclodextrin treatment. Phosphorylation of Akt substrates in lipid raft fractions, but not in cytosol/nonraft membrane fractions, was ablated with cyclodextrin. In addition, in control (LacZ transfected) cells, lipid raft fractions were relatively enriched in phosphorylated Akt substrates. Collectively, these data show that a subpopulation of Akt is cholesterol sensitive and that the oncogenic effects conferred by myristoylation arise, in part, from the tendency of the membrane-targeted form of the protein to reside in cholesterol-rich membrane microdomains.
Assuntos
Colesterol/metabolismo , Ácido Mirístico/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Ciclodextrinas/farmacologia , Humanos , Masculino , Microdomínios da Membrana/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Fosforilação , Neoplasias da Próstata/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
Androgen receptor (AR) plays an important role in normal prostate function as well as in the etiology of prostate cancer. Activation of AR is dictated by hormone binding and by interactions with coregulators. Several of these coregulators are known targets of Ras-related signals. Recent evidence suggests that Ras activation may play a causal role in the progression of prostate cancer toward a more malignant and hormone-insensitive phenotype. In the present study, we used a transcription factor-transcription factor interaction array method to identify the zinc finger protein Ras-responsive element binding protein (RREB-1) as a partner and coregulator of AR. In LNCaP prostate cancer cells, RREB-1 was found to be present in a complex with endogenous AR as determined by coimmunoprecipitation, glutathione S-transferase pull down, and immunofluorescence analyses. RREB-1 bound to the prostate-specific antigen (PSA) promoter as assessed by chromatin immunoprecipitation. Transient expression of RREB-1 down-regulated AR-mediated promoter activity and suppressed expression of PSA protein. The repressor activity of RREB-1 was significantly attenuated by cotransfection of activated Ras. Moreover, expression of the dominant-negative N-17-Ras or, alternatively, use of the MAPK kinase inhibitor PD98059 [2-(2-amino-3-methyoxyphenyl)-4H-1-benzopyran-4-one] abolished the effect of Ras in attenuating RREB-1-mediated repression. Furthermore, inhibition of RREB-1 expression by RNA interference enhanced the effect of Ras on PSA promoter activity and PSA expression. In addition, activation of the Ras pathway depleted AR from the RREB-1/AR complex. Collectively, our data for the first time identify RREB-1 as a repressor of AR and further implicate the Ras/MAPK kinase pathway as a likely antagonist of the inhibitory effects of RREB-1 on androgenic signaling.
Assuntos
Androgênios/fisiologia , Proteínas de Ligação a DNA/fisiologia , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/fisiologia , Dedos de Zinco/fisiologia , Anticorpos/fisiologia , Linhagem Celular , Humanos , Masculino , Receptores Androgênicos/imunologiaRESUMO
OBJECTIVES: The aim of this study was to evaluate early results of proximal humerus fractures treated with the PHILOS locking plate. METHODS: Proximal humerus fractures of 22 patients (13 males, 9 females; mean age 57 years; range 35 to 83 years) were treated with the PHILOS locking plate. According to the Neer classification, 13 patients had comminuted fractures, four patients had valgus impact injuries, two patients had fracture-dislocations, and three patients had fractures involving the proximal 1/3 of the humerus. The fractures were reduced by the transdeltoid lateral approach (n=8) using minimally invasive surgery, and by the anterior deltopectoral approach (n=14) using open surgery. Passive and active exercises were initiated on the second postoperative day and after 4 to 6 weeks, respectively. The results were assessed using the Constant-Murley shoulder scoring system. The mean follow up was 14 months (range 12 to 19 months). RESULTS: Radiographically, union was observed in 20 patients at the end of 10 weeks. In one patient, time to union was 16 weeks. One patient underwent autogenous bone grafting because of nonunion after 16 weeks. The mean Constant-Murley score was 75.5 (range 51 to 93). There was no significant difference between Constant-Murley scores of patients undergoing the transdeltoid lateral and anterior deltopectoral approaches (p>0.05). Plate fixation was associated with minimal varus deformity in two patients, and subacromial impingement in one patient. Implant failure did not occur. Reflex sympathetic dystrophy and avascular necrosis were observed in two patients, respectively. CONCLUSION: Fixation with the PHILOS plate is a near-ideal technique with a high union rate in the treatment of proximal humeral fractures.
Assuntos
Placas Ósseas , Fixação Interna de Fraturas/instrumentação , Fixação Interna de Fraturas/métodos , Luxação do Ombro/cirurgia , Fraturas do Ombro/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Consolidação da Fratura/fisiologia , Fraturas Cominutivas/complicações , Fraturas Cominutivas/diagnóstico por imagem , Fraturas Cominutivas/patologia , Fraturas Cominutivas/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Procedimentos Cirúrgicos Minimamente Invasivos/métodos , Radiografia , Luxação do Ombro/complicações , Luxação do Ombro/diagnóstico por imagem , Luxação do Ombro/patologia , Fraturas do Ombro/diagnóstico por imagem , Fraturas do Ombro/patologia , Fatores de Tempo , Resultado do TratamentoRESUMO
The functional consequences of changes in membrane lipid composition that coincide with malignant growth are poorly understood. Sufficient data have been acquired from studies of lipid binding proteins, post-translational modifications of signaling proteins, and biochemical inhibition of lipidogenic pathways to indicate that growth and survival pathways might be substantially re-directed by alterations in the lipid content of membranes. Cholesterol and glycosphingolipids segregate into membrane patches that exhibit a liquid-ordered state in comparison to membrane domains containing relatively lower amounts of these classes of lipids. These "lipid raft" structures, which may vary in size and stability in different cell types, both accumulate and exclude signaling proteins and have been implicated in signal transduction through a number of cancer-relevant pathways. In prostate cancer cells, signaling from epidermal growth factor receptor (EGFR) to the serine-threonine kinase Akt1, as well as from IL-6 to STAT3, have been demonstrated to be influenced by experimental interventions that target cholesterol homeostasis. The recent finding that classical steroid hormone receptors also reside in these microdomains, and thus may function within these structures in a signaling capacity independent of their role as nuclear factors, suggests a novel means of cross-talk between receptor tyrosine kinase-derived and steroidogenic signals. Potential points of intersection between components of the EGFR family of receptor tyrosine kinases and androgen receptor signaling pathways, which may be sensitive to disruptions in cholesterol metabolism, are discussed. Understanding the manner in which these pathways converge within cholesterol-rich membranes may present new avenues for therapeutic intervention in hormone-dependent cancers.
Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Receptores ErbB/fisiologia , Hormônios/fisiologia , Transdução de Sinais/fisiologia , Colesterol/fisiologia , Microdomínios da Membrana/fisiologia , Receptores Androgênicos/fisiologiaRESUMO
Heparin-binding epidermal growth factor-like growth factor (HB-EGF), an ErbB1 ligand and prostate stromal growth factor, is an antagonist of androgen receptor (AR) function. In the LNCaP prostate cancer model, HB-EGF reduced AR protein levels and AR transactivation without affecting AR mRNA level or protein turnover. The signal to attenuate AR was mediated by the mammalian target of rapamycin, as shown by genetic and pharmacologic methods, and was independent of ErbB2/HER-2, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase pathways. Additional evidence suggests that AR protein levels are highly sensitive to regulation by cap-dependent mRNA translation. These findings reveal a novel mechanism for regulation of AR by a classic growth factor system and indicate that a rapamycin-sensitive post-transcriptional pathway can attenuate or possibly bypass AR-mediated signaling.
Assuntos
Proteínas Quinases/fisiologia , Receptores Androgênicos/fisiologia , Antagonistas de Receptores de Andrógenos , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Regulação para Baixo , Ativação Enzimática , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Biossíntese de Proteínas , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR , Transcrição Gênica , Ativação Transcricional , TransfecçãoRESUMO
Mechanisms of castration-resistant prostate cancer (CRPC) are not well understood, thus hindering rational-based drug design. Activation of STAT3/5A, key components of the JAK/STAT pathway, is implicated in aggressive PC, yet their clinical relevance in CRPC remains elusive. Here, we evaluated the possible role of STAT3/5A in CRPC using immunological, quantitative mRNA expression profiling, and pharmacological methods. We observed a strong nuclear immunoreactivity for STAT3 and STAT5A in 93% (n=14/15) and 80% (n=12/15) of CRPC cases, respectively, compared with benign prostatic hyperplasia (BPH). We demonstrated that PC cells express varying levels of STAT3 and STAT5A transcripts. In addition, we demonstrate that pimozide, a psychotropic drug and an indirect inhibitor of STAT5, attenuated PC cells growth, and induced apoptosis in a dose-dependent manner. Furthermore, our analysis of the PC public data revealed that the STAT3/5A genes were frequently amplified in metastatic CRPC. These findings suggest that STAT3/5A potentially serves as a predictive biomarker to evaluate the therapeutic efficacy of a cancer drug targeting the JAK/STAT pathway. Since the JAK/STAT and AR pathways are suggested to be functionally synergistic, inhibition of the JAK/STAT signaling alone or together with AR may lead to a novel treatment modality for patients with advanced PC.
RESUMO
Dysregulation of MST1/STK4, a key kinase component of the Hippo-YAP pathway, is linked to the etiology of many cancers with poor prognosis. However, how STK4 restricts the emergence of aggressive cancer remains elusive. Here, we investigated the effects of STK4, primarily localized in the cytoplasm, lipid raft, and nucleus, on cell growth and gene expression in aggressive prostate cancer. We demonstrated that lipid raft and nuclear STK4 had superior suppressive effects on cell growth in vitro and in vivo compared with cytoplasmic STK4. Using RNA sequencing and bioinformatics analysis, we identified several differentially expressed (DE) genes that responded to ectopic STK4 in all three subcellular compartments. We noted that the number of DE genes observed in lipid raft and nuclear STK4 cells were much greater than cytoplasmic STK4. Our functional annotation clustering showed that these DE genes were commonly associated with oncogenic pathways such as AR, PI3K/AKT, BMP/SMAD, GPCR, WNT, and RAS as well as unique pathways such as JAK/STAT, which emerged only in nuclear STK4 cells. These findings indicate that MST1/STK4/Hippo signaling restricts aggressive tumor cell growth by intersecting with multiple molecular pathways, suggesting that targeting of the STK4/Hippo pathway may have important therapeutic implications for cancer.