RESUMO
Understanding the functional role of mutated genes in cancer is required to translate the findings of cancer genomics into therapeutic improvement. BTG1 is recurrently mutated in the MCD/C5 subtype of diffuse large B-cell lymphoma (DLBCL), which is associated with extranodal dissemination. Here, we provide evidence that Btg1 knock out accelerates the development of a lethal lymphoproliferative disease driven by Bcl2 overexpression. Furthermore, we show that the scaffolding protein BCAR1 is a BTG1 partner. Moreover, after BTG1 deletion or expression of BTG1 mutations observed in patients with DLBCL, the overactivation of the BCAR1-RAC1 pathway confers increased migration ability in vitro and in vivo. These modifications are targetable with the SRC inhibitor dasatinib, which opens novel therapeutic opportunities in BTG1 mutated DLBCL.
Assuntos
Linfoma Difuso de Grandes Células B , Humanos , Linfoma Difuso de Grandes Células B/patologia , Mutação , Genes cdc , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína Substrato Associada a Crk/genética , Proteína Substrato Associada a Crk/metabolismoRESUMO
AIMS: The adapter protein p130Cas, encoded by the Bcar1 gene, is a key regulator of cell movement, adhesion, and cell cycle control in diverse cell types. Bcar1 constitutive knockout mice are embryonic lethal by embryonic days (E) 11.5-12.5, but the role of Bcar1 in embryonic development remains unclear. Here, we investigated the role of Bcar1 specifically in cardiovascular development and defined the cellular and molecular mechanisms disrupted following targeted Bcar1 deletions. METHODS AND RESULTS: We crossed Bcar1 floxed mice with Cre transgenic lines allowing for cell-specific knockout either in smooth muscle and early cardiac tissues (SM22-Cre), mature smooth muscle cells (smMHC-Cre), endothelial cells (Tie2-Cre), second heart field cells (Mef2c-Cre), or neural crest cells (NCC) (Pax3-Cre) and characterized these conditional knock outs using a combination of histological and molecular biology techniques. Conditional knockout of Bcar1 in SM22-expressing smooth muscle cells and cardiac tissues (Bcar1SM22KO) was embryonically lethal from E14.5-15.5 due to severe cardiovascular defects, including abnormal ventricular development and failure of outflow tract (OFT) septation leading to a single outflow vessel reminiscent of persistent truncus arteriosus. SM22-restricted loss of Bcar1 was associated with failure of OFT cushion cells to undergo differentiation to septal mesenchymal cells positive for SMC-specific α-actin, and disrupted expression of proteins and transcription factors involved in epithelial-to-mesenchymal transformation (EMT). Furthermore, knockout of Bcar1 specifically in NCC (Bcar1PAX3KO) recapitulated part of the OFT septation and aortic sac defects seen in the Bcar1SM22KO mutants, indicating a cell-specific requirement for Bcar1 in NCC essential for OFT septation. In contrast, conditional knockouts of Bcar1 in differentiated smooth muscle, endothelial cells, and second heart field cells survived to term and were phenotypically normal at birth and postnatally. CONCLUSION: Our work reveals a cell-specific requirement for Bcar1 in NCC, early myogenic and cardiac cells, essential for OFT septation, myocardialization and EMT/cell cycle regulation and differentiation to myogenic lineages.
Assuntos
Proteína Substrato Associada a Crk , Cardiopatias Congênitas , Crista Neural , Animais , Proteína Substrato Associada a Crk/genética , Células Endoteliais/patologia , Coração , Cardiopatias Congênitas/patologia , Camundongos , Camundongos Knockout , Crista Neural/patologia , Fatores de TranscriçãoRESUMO
BACKGROUND: ASF1B is a member of the histone H3-H4 chaperone antisilencing feature 1 (ASF1). ASF1B reportedly acts as an oncogene in several cancers including, breast cancer and cervical cancer. To date, the role of ASF1B in lung adenocarcinoma (LUAD) is not elucidated. METHODS: The TCGA database, containing data for 33 cancer types, was used to explore the dysregulation and prognostic value of the ASF1B gene in pan-cancer data. R software packages and public databases/webservers were applied for bioinformatics and statistical analyses. Using in vitro models, immunoprecipitation and immunofluorescence were utilized to investigate if BCAR1 interacted with ASF1B in LUAD. Further, transfection experiments were performed to validate the expression pattern of ASF1B in LUAD and examine its regulating role in tumor-associated processes including tumor cell proliferation and migration. RESULTS: ASF1B was found to be significantly elevated in LUAD and the majority of cancer types, except PCPG (pheochromocytoma and paraganglioma). The overexpression of ASF1B was associated with worse prognostic outcomes in most cancer types including LUAD. ASF1B was associated with lymph node metastasis, and in vitro, it promoted the proliferation and migration of LUAD cells. ASF1B knockdown suppressed LUAD cell proliferation and migration and also diminished the expression of cell cycle, metastasis, and EMT signaling-associated proteins. BCAR1 was found positively correlated and interacting with ASF1B, and BCAR1 overexpression reversed the effects of ASF1B knockdown in LUAD cells. CONCLUSION: These findings indicated that ASF1B plays a significant role in the tumor progression of LUAD and BCAR1 mediates the tumor-promotive effects of ASF1B, acting as an intermediate protein. Therefore, the ASF1B/BCAR1 axis might be regarded as a putative therapeutic target for LUAD.
Assuntos
Adenocarcinoma de Pulmão/genética , Proteínas de Ciclo Celular/genética , Neoplasias Pulmonares/genética , Células A549 , Adenocarcinoma de Pulmão/etiologia , Adenocarcinoma de Pulmão/metabolismo , Idoso , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Biologia Computacional , Simulação por Computador , Proteína Substrato Associada a Crk/genética , Proteína Substrato Associada a Crk/metabolismo , Bases de Dados Genéticas , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Técnicas In Vitro , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Regulação para CimaRESUMO
p130 Crk-associated substrate (p130Cas) is associated with poor prognosis and treatment resistance in breast and lung cancers. To elucidate p130Cas functional and clinical role in colorectal cancer (CRC) progression/therapy resistance, we performed cell culture experiments and bioinformatic/statistical analyses of clinical data sets. p130Cas expression was associated with poor survival in the cancer genome atlas (TCGA) data set. Knockdown/reconstitution experiments showed that p130Cas drives migration but, unexpectedly, inhibits proliferation in CRC cells. TCGA data analyses identified the growth factor epiregulin (EREG) as inversely correlated with p130Cas. p130Cas knockdown and simultaneous EREG treatment further enhanced proliferation. RNA interference and EREG treatment experiments suggested that p130Cas/EREG limit each other's expression/activity. Inverse p130Cas/EREG Spearman correlations were prominent in right-sided and earlier stage CRC. p130Cas was inducible by 5-fluorouracil (5-FU) and FOLFIRI (folinic acid, 5-FU, irinotecan), and p130Cas and EREG were upregulated in distant metastases (GSE121418). Positive p130Cas/EREG correlations were observed in metastases, preferentially in post-treatment samples (especially pulmonary metastases). p130Cas knockdown sensitized CRC cells to FOLFIRI independent of EREG treatment. RNA sequencing and gene ontology analyses revealed that p130Cas is involved in cytochrome P450 drug metabolism and epithelial-mesenchymal transition. p130Cas expression was associated with poor survival in right-sided, stage I/II, MSS (microsatellite stable), or BRAF-mutated CRC. In summary, p130Cas represents a prognostic factor and potential therapeutic target in CRC.
Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias Colorretais/diagnóstico , Proteína Substrato Associada a Crk/genética , Epirregulina/genética , Transição Epitelial-Mesenquimal/genética , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Atlas como Assunto , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Camptotecina/análogos & derivados , Camptotecina/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Proteína Substrato Associada a Crk/antagonistas & inibidores , Proteína Substrato Associada a Crk/metabolismo , Epirregulina/metabolismo , Feminino , Fluoruracila/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Humanos , Leucovorina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Anotação de Sequência Molecular , Mutação , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de SobrevidaRESUMO
Background: We investigated the roles of breast cancer anti-estrogen resistance 1 (BCAR1/p130Cas) in the formation and immunoevasion of invasive circulating tumor cells (CTCs) in lung adenocarcinoma (LUAD). Methods: Biomarkers of CTCs including BCAR1 and CD274, were evaluated by the CanPatrol method. Proteomics analysis of LUAD cells and exosomes after BCAR1 overexpression (BCAR1-OE) was performed by mass spectrometry. Cell functions and relevant signaling pathways were investigated after BCAR1 knockdown (BCAR1-KO) or BCAR1-OE in LUAD cells. Lastly, in vitro and in vivo experiments were performed to confirm the roles of BCAR1 in the formation and immunoevasion of CTCs. Results: High expression of BCAR1 by CTCs correlated with CD274 expression and epithelial-to-mesenchymal transition (EMT). RAC1, together with BCAR1, was found to play an important role in the carcinogenesis of LUAD. RAC1 functioned with BCAR1 to induce EMT and to enhance cell proliferation, colony formation, cell invasion and migration, and anoikis resistance in LUAD cells. BCAR1 up-regulated CD274 expression probably by shuttling the short isoform of BRD4 (BRD4-S) into the nucleus. CTCs, as well as tumor formation, were prohibited in nude mice xenografted with BCAR1-KO cells. The co-expression of BCAR1/RAC1 and BCAR1/CD274 was confirmed in LUAD. BCAR1 expression in LUAD is an indicator of poor prognosis, and it associates with immunoevasion. Conclusion: BCAR1, as a new target for the treatment of LUAD, plays roles in the formation and immunoevasion of invasive CTCs. The mechanism includes triggering EMT via RAC1 signaling and up-regulating CD274 expression by shuttling BRD4-S into the nucleus.
Assuntos
Adenocarcinoma de Pulmão/genética , Proteína Substrato Associada a Crk/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Células Neoplásicas Circulantes/patologia , Adenocarcinoma de Pulmão/mortalidade , Adenocarcinoma de Pulmão/patologia , Animais , Antígeno B7-H1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteína Substrato Associada a Crk/metabolismo , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Integrin adhesion complexes regulate cytoskeletal dynamics during cell migration. Adhesion activates phosphorylation of integrin-associated signaling proteins, including Cas (p130Cas, BCAR1), by Src-family kinases. Cas regulates leading-edge protrusion and migration in cooperation with its binding partner, BCAR3. However, it has been unclear how Cas and BCAR3 cooperate. Here, using normal epithelial cells, we find that BCAR3 localization to integrin adhesions requires Cas. In return, Cas phosphorylation, as well as lamellipodia dynamics and cell migration, requires BCAR3. These functions require the BCAR3 SH2 domain and a specific phosphorylation site, Tyr 117, that is also required for BCAR3 downregulation by the ubiquitin-proteasome system. These findings place BCAR3 in a co-regulatory positive-feedback circuit with Cas, with BCAR3 requiring Cas for localization and Cas requiring BCAR3 for activation and downstream signaling. The use of a single phosphorylation site in BCAR3 for activation and degradation ensures reliable negative feedback by the ubiquitin-proteasome system.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína Substrato Associada a Crk/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Pseudópodes/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adesão Celular , Linhagem Celular , Proteína Substrato Associada a Crk/metabolismo , Células Epiteliais , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Integrinas/metabolismo , Fosforilação , Domínios de Homologia de srcRESUMO
The High-Risk Human Papillomaviruses (HR-HPVs) 16 and 18 are known to cause cervical cancer, which is primarily attributed to E6 and E7 oncoproteins. In addition, recent studies have focused on the vital role of the p130 pocket protein as an oncosuppressor to limit the expression of E2F transcription factors required for cell cycle progression. In view of this, the current study was conducted to investigate the mechanism by which transfection with HPV16/18 E7 leads to the deregulation of the host cell cycle, altering the localisation of p130, and expression of differentiation genes in Human Keratinocytes (HaCaT) cells. Co-immunoprecipitation, Western blot analysis, immunofluorescence microscopy, flow cytometry, quantitative-Polymerase Chain Reaction (qPCR), and the inhibition of p130 by MG132 inhibitor were employed to investigate the loss of p130 and its disruption in HPV 16/18 E7-transfected HaCaT cells. The HPV16- and HPV18-transformed cells, known as CaSki and HeLa, respectively, were also used to complement the ectopic expressions of E7 in HaCaT cells. Normal keratinocytes displayed higher level of p130 expression than HPV-transformed cells. In addition, the immunofluorescence analysis revealed that both HPV 16/18 E7-transfected HaCaT and HPV-transformed cells exhibited higher level of cytoplasmic p130 compared to nuclear p130. A significant increase in the number of S/G2 phase cells in HPV-transformed cells was also recorded since E7 has been shown to stimulate proliferation through the deactivation of Retinoblastoma Protein (pRB)-dependent G1/S checkpoint. Furthermore, the findings recorded the down-regulation of keratinocyte differentiation markers, namely p130, keratin10, and involucrin. The proteasomal degradation of the exported p130 confirmed the cellular localisation pattern of p130, which was commonly observed in cancerous cells. The findings provide strong evidence that the localisation of nuclear p130 nuclear was disrupted by HPV16/18 E7 led to the deregulation of the cell cycle and the impairment of cellular differentiation ultimately lead to cellular transformation.
Assuntos
Proteína Substrato Associada a Crk/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Alphapapillomavirus/genética , Alphapapillomavirus/patogenicidade , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular , Proteína Substrato Associada a Crk/genética , Proteínas de Ligação a DNA/genética , Feminino , Células HeLa , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 16/patogenicidade , Papillomavirus Humano 18/metabolismo , Papillomavirus Humano 18/patogenicidade , Humanos , Queratinócitos/metabolismo , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/genética , Proteínas Repressoras/genética , Proteína p130 Retinoblastoma-Like/genética , Transfecção , Neoplasias do Colo do Útero/metabolismoRESUMO
Cells have developed a unique set of molecular mechanisms that allows them to probe mechanical properties of the surrounding environment. These systems are based on deformable primary mechanosensors coupled to tension transmitting proteins and enzymes generating biochemical signals. This modular setup enables to transform a mechanical load into more versatile biochemical information. Src kinase appears to be one of the central components of the mechanotransduction network mediating force-induced signalling across multiple cellular contexts. In tight cooperation with primary sensors and the cytoskeleton, Src functions as an effector molecule necessary for transformation of mechanical stimuli into biochemical outputs executing cellular response and adaptation to mechanical cues.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína Substrato Associada a Crk/genética , Citoesqueleto/metabolismo , Mecanotransdução Celular/genética , Neoplasias/metabolismo , Fatores de Transcrição/genética , Quinases da Família src/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteína Substrato Associada a Crk/metabolismo , Citoesqueleto/patologia , Citoesqueleto/ultraestrutura , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Matriz Extracelular/ultraestrutura , Regulação da Expressão Gênica , Via de Sinalização Hippo , Humanos , Integrinas/genética , Integrinas/metabolismo , Neoplasias/genética , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Estresse Mecânico , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP , Quinases da Família src/metabolismoRESUMO
OBJECTIVE: To clarify the clinical significance of breast cancer anti-estrogen resistance protein 1 (BCAR1) expression in circulating tumor cells (CTCs) in the peripheral blood and tumor tissues in patients with early stage lung adenocarcinoma (ES-LUAD). METHODS: The study cohort included 60 patients with stage I LUAD (50 IA and 10 IB) who underwent surgery from November 2015 to November 2018 and 31 healthy controls. The expression levels of BCAR1 and markers of epithelial-mesenchymal transition (EMT) in peripheral blood CTCs were detected using CanPatrolTM technology before surgery, and immunohistochemical analysis was used to detect BCAR1 expression in tumor tissues collected from 40 patients. The predictive power of BCAR1 expression in CTCs and tumor tissues on disease-free survival (DFS) was analyzed. The Cancer Genome Atlas (TCGA) database was used to study BCAR1 expression and overall survival as validation. The Gene Expression Profiling Interactive Analysis online tool was used to analyze the correlations between the expression levels of BCAR1 and EMT molecular markers. RESULTS: Both the number and detection rates of BCAR1-negative CTCs and BCAR1-positive CTCs in peripheral blood of lung cancer patients were significantly higher as compared with healthy controls (p < 0.05). BCAR1-positive CTCs more commonly co-expressed both epithelial and mesenchymal markers. Kaplan-Meier analysis demonstrated that patients with BCAR1(++) CTCs in peripheral blood before surgery were more prone to recurrence or metastasis after 2 years. COX analysis showed that patients with higher abundance of BCAR1(++) CTCs had a poorer prognosis (hazard ratio [HR] = 1.712, 95% confidence interval [CI] = 1.077-2.272, p = 0.023). Furthermore, high BCAR1 expression in tumor tissues was predictive of a poor prognosis (HR = 2.654, 95% CI = 1.239-5.686, p = 0.012), as validated by TCGA database (HR = 2.217, 95% CI = 1.069-4.595, p = 0.032). In addition, BCAR1 expression in LUAD tissues from TCGA was significantly positively correlated with the expression of both epithelial markers (e.g., ck8/18/19) and mesenchymal markers (e.g., vimentin and twist). CONCLUSION: BCAR1 may have a "dual impact" on EMT markers in tumor tissues and CTCs due to micro-environmental disparities, resulting in important clinical significance, which can potentially guide accurate treatment of LUAD.
Assuntos
Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Biomarcadores Tumorais , Proteína Substrato Associada a Crk/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Células Neoplásicas Circulantes/patologia , Adenocarcinoma de Pulmão/mortalidade , Idoso , Linhagem Celular Tumoral , Proteína Substrato Associada a Crk/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , PrognósticoRESUMO
Mechanical loading plays an important role in bone homeostasis. However, molecular mechanisms behind the mechanical regulation of bone homeostasis are poorly understood. We previously reported p130Cas (Cas) as a key molecule in cellular mechanosensing at focal adhesions. Here, we demonstrate that Cas is distributed in the nucleus and supports mechanical loading-mediated bone homeostasis by alleviating NF-κB activity, which would otherwise prompt inflammatory processes. Mechanical unloading modulates Cas distribution and NF-κB activity in osteocytes, the mechanosensory cells in bones. Cas deficiency in osteocytes increases osteoclastic bone resorption associated with NF-κB-mediated RANKL expression, leading to osteopenia. Upon shear stress application on cultured osteocytes, Cas translocates into the nucleus and down-regulates NF-κB activity. Collectively, fluid shear stress-dependent Cas-mediated alleviation of NF-κB activity supports bone homeostasis. Given the ubiquitous expression of Cas and NF-κB together with systemic distribution of interstitial fluid, the Cas-NF-κB interplay may also underpin regulatory mechanisms in other tissues and organs.
Assuntos
Osso e Ossos/metabolismo , Proteína Substrato Associada a Crk/metabolismo , Homeostase , NF-kappa B/metabolismo , Transdução de Sinais , Estresse Mecânico , Animais , Biomarcadores , Reabsorção Óssea , Osso e Ossos/diagnóstico por imagem , Proteína Substrato Associada a Crk/genética , Expressão Gênica , Camundongos , Camundongos Knockout , Osteoclastos/metabolismo , Osteócitos/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , Microtomografia por Raio-XRESUMO
BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used to treat sports-related muscle injuries. However, NSAIDs were recently shown to impede the muscle healing process after acute injury. Migration of skeletal muscle cells is a crucial step during the muscle healing process. The present study was performed to investigate the effect and molecular mechanisms of action of ibuprofen, a commonly used NSAID, on the migration of skeletal muscle cells. METHODS: Skeletal muscle cells isolated from the gastrocnemius muscle of Sprague-Dawley rats were treated with ibuprofen. MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was used to evaluate cell viability, and cell apoptosis was evaluated by TUNEL assay, after ibuprofen treatment. Skeletal muscle cell migration and spreading were evaluated using the transwell filter migration assay and F-actin staining, respectively. The protein expression of p130cas and CrkII, which are cell migration facilitating genes, was determined by western blot analysis. The overexpression of p130cas of muscle cells was achieved by p130cas vector transfection. RESULTS: The results demonstrated that ibuprofen did not have a significant negative effect on cell viability and apoptosis. Ibuprofen inhibited the migration and spreading of skeletal muscle cells in a dose-dependent manner. Ibuprofen also dose-dependently decreased the protein expression of p130cas and CrkII. Furthermore, overexpression of p130cas resulted in the promotion of cell migration and spreading and counteracted ibuprofen-mediated inhibition. CONCLUSION: This study suggested that ibuprofen exerts a potentially adverse effect on the migration of skeletal muscle cells by downregulating protein expression of p130cas and CrkII. These results indicate a possible mechanism underlying the possible negative effect of NSAIDs on muscle regeneration.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Proteína Substrato Associada a Crk/metabolismo , Ibuprofeno/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/fisiologia , Proteínas Proto-Oncogênicas c-crk/metabolismo , Animais , Anti-Inflamatórios não Esteroides/efeitos adversos , Traumatismos em Atletas/tratamento farmacológico , Traumatismos em Atletas/patologia , Traumatismos em Atletas/fisiopatologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Proteína Substrato Associada a Crk/genética , Regulação para Baixo/efeitos dos fármacos , Humanos , Ibuprofeno/efeitos adversos , Músculo Esquelético/lesões , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Proteínas Proto-Oncogênicas c-crk/genética , Ratos , Ratos Sprague-Dawley , Regeneração/efeitos dos fármacos , Regeneração/fisiologia , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologiaRESUMO
Most human cancers acquire mutations causing defects in the p53 signaling pathway. The tumor suppressor p53 becomes activated in response to genotoxic stress and is essential for arresting the cell cycle to facilitate DNA repair or to initiate apoptosis. p53-induced cell cycle-arrest is mediated by expression of the CDK inhibitor p21WAF1/Cip1, which prevents phosphorylation and inactivation of the pocket proteins RB, p130, and p107. In a hypophosphorylated state, pocket proteins bind to E2F factors forming RB-E2F and DREAM transcriptional repressor complexes. Here, we analyze the influence of RB and DREAM on p53-induced gene repression and cell-cycle arrest. We show that abrogation of DREAM function by knockout of the DREAM component LIN37 results in a reduced repression of cell-cycle genes. We identify the genes repressed by the p53-DREAM pathway and describe a set of genes that is downregulated by p53 independent of LIN37/DREAM. Most strikingly, p53-dependent repression of cell-cycle genes is completely abrogated in LIN37-/-;RB-/- cells leading to a loss of the G1/S checkpoint. Taken together, we show that DREAM and RB are key factors in the p53 signaling pathway to downregulate a large number of cell-cycle genes and to arrest the cell cycle at the G1/S transition.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , Regulação da Expressão Gênica , Proteínas Interatuantes com Canais de Kv/metabolismo , Proteínas Repressoras/metabolismo , Proteína do Retinoblastoma/genética , Transativadores/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Células Cultivadas , Proteína Substrato Associada a Crk/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Fibroblastos/metabolismo , Genes cdc , Células HCT116 , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Camundongos , Proteínas Repressoras/genética , Proteína do Retinoblastoma/metabolismo , Proteína p107 Retinoblastoma-Like/genética , Transativadores/genética , Transativadores/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb's tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms.
Assuntos
Proliferação de Células/genética , Ciclina D/genética , Mapas de Interação de Proteínas/genética , Proteína do Retinoblastoma/genética , Ciclo Celular/genética , Proteína Substrato Associada a Crk/genética , Ciclina D/química , Quinase 4 Dependente de Ciclina/química , Quinase 4 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/química , Quinase 6 Dependente de Ciclina/genética , Ciclinas/genética , Fase G1/genética , Humanos , Simulação de Acoplamento Molecular , Fosforilação/genética , Ligação Proteica/genética , Conformação Proteica em alfa-Hélice/genética , Proteína do Retinoblastoma/química , Proteína p107 Retinoblastoma-Like/genética , Fase S/genéticaRESUMO
Few therapy options exist for patients with advanced papillary and anaplastic thyroid cancer. We and others have previously identified c-Src as a key mediator of thyroid cancer pro-tumorigenic processes and a promising therapeutic target for thyroid cancer. To increase the efficacy of targeting Src in the clinic, we sought to define mechanisms of resistance to the Src inhibitor, dasatinib, to identify key pathways to target in combination. Using a panel of thyroid cancer cell lines expressing clinically relevant mutations in BRAF or RAS, which were previously developed to be resistant to dasatinib, we identified a switch to a more invasive phenotype in the BRAF-mutant cells as a potential therapy escape mechanism. This phenotype switch is driven by FAK kinase activity, and signaling through the p130Cas>c-Jun signaling axis. We have further shown this more invasive phenotype is accompanied by alterations in the secretome through the increased expression of pro-inflammatory cytokines, including IL-1ß, and the pro-invasive metalloprotease, MMP-9. Furthermore, IL-1ß signals via a feedforward autocrine loop to promote invasion through a FAK>p130Cas>c-Jun>MMP-9 signaling axis. We further demonstrate that upfront combined inhibition of FAK and Src synergistically inhibits growth and invasion, and induces apoptosis in a panel of BRAF- and RAS-mutant thyroid cancer cell lines. Together our data demonstrate that acquired resistance to single-agent Src inhibition promotes a more invasive phenotype through an IL-1ß>FAK>p130Cas>c-Jun >MMP signaling axis, and that combined inhibition of FAK and Src has the potential to block this inhibitor-induced phenotype switch.
Assuntos
Proteína Substrato Associada a Crk/genética , Resistencia a Medicamentos Antineoplásicos/genética , Quinase 1 de Adesão Focal/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-jun/genética , Neoplasias da Glândula Tireoide/genética , Quinases da Família src/genética , Apoptose/genética , Linhagem Celular Tumoral , Dasatinibe/farmacologia , Humanos , Mutação/genética , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/genética , Neoplasias da Glândula Tireoide/tratamento farmacológicoRESUMO
BACKGROUND: p130 Crk-associated substrate (p130CAS; also known as BCAR1) is a scaffold protein that modulates many essential cellular processes such as cell adhesion, proliferation, survival, cell migration, and intracellular signaling. p130Cas has been shown to be highly expressed in a variety of human cancers of epithelial origin. However, few data are available regarding the role of p130Cas during normal epithelial development and homeostasis. METHODS: To this end, we have generated a genetically modified mouse in which p130Cas protein was specifically ablated in the epidermal tissue. RESULTS: By using this murine model, we show that p130Cas loss results in increased cell proliferation and reduction of cell adhesion to extracellular matrix. In addition, epidermal deletion of p130Cas protein leads to premature expression of "late" epidermal differentiation markers, altered membrane E-cadherin/catenin proteins localization and aberrant tyrosine phosphorylation of E-cadherin/catenin complexes. Interestingly, these alterations in adhesive properties in absence of p130Cas correlate with abnormalities in progenitor cells balance resulting in the amplification of a more committed cell population. CONCLUSION: Altogether, these results provide evidence that p130Cas is an important regulator of epidermal cell fate and homeostasis.
Assuntos
Adesão Celular , Diferenciação Celular , Proteína Substrato Associada a Crk/deficiência , Proteína Substrato Associada a Crk/genética , Epiderme/metabolismo , Deleção de Genes , Homeostase/genética , Animais , Proliferação de Células , Matriz Extracelular/metabolismo , Queratinócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , FenótipoRESUMO
Two recent studies found that RBMS1 gene rs7593730 and BCAR1 gene rs7202877 are related to type 2 diabetes. However, the association of these loci with type 2 diabetes mellitus (T2DM) has not been examined in Chinese. We performed a replication study to investigate the association of the 2 susceptibility loci with T2DM in the Chinese population. We genotyped 1961 Chinese participants (991 with T2DM and 970 controls) for each of the 2 single nucleotide polymorphisms (SNPs) rs7593730 in RBMS1 and rs7202877 near BCAR1 using SNPscan and examined their association with T2DM using logistic regression analysis. We also analyzed the correlation of the SNP alleles and clinical phenotypes. In additive model, genotype association analysis of BCAR1 rs7202877 loci revealed that the homozygous of rs7202877 GG carriers had significantly decreased T2DM risk compared to homozygous carriers of TT (P=0.038, OR 0.44, 95% CI 0.20-0.96). In the recessive model, the GG genotype GG had significantly decreased T2DM risk compared to GT+TT (P=0.043, OR 0.67, 95% CI 0.46-0.99). Allele G was statistically significantly correlated with TC (mmol/L) (P=0.036) and LDL-C (mmol/L) (P=0.007). As for rs7593730, the carriers of CT and TT genotype had significantly decreased T2DM risk compared to the carriers of CC genotype (CT: CC P=0.038, OR 0.71, 95% CI 0.51-0.98; TT: CC P=0.010, OR 0.32, 95% CI 0.13-0.76). In a dominant model, TT+CT: CC (P=0.013, OR 0.673, 95% CI 0.49-0.92) and in a recessive model, TT: CT+CC (P=0.019, OR 0.59, 95% CI 0.39-0.92). The T allele carriers had significantly decreased T2DM risk compared to the carriers of C (P=0.002, OR 0.65, 95% CI 0.50-0.86). Allele T was statistically correlated with FINS (P=0.010). In conclusion, our study showed that RBMS1 gene rs7593730 and BCAR1 gene rs7202877 were significantly associated with type 2 diabetes in the Chinese population.
Assuntos
Povo Asiático/genética , Proteína Substrato Associada a Crk/genética , Proteínas de Ligação a DNA/genética , Diabetes Mellitus Tipo 2/etnologia , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Proteínas de Ligação a RNA/genética , Adulto , Alelos , Estudos de Casos e Controles , China , Feminino , Triagem de Portadores Genéticos , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Bone-marrow-derived mesenchymal stem cells (MSCs) have great potential in transplantation medicine due to their multiple advantages. However, the controlled differentiation of MSCs is one of the key aspects of effective clinical transplantation. Growing evidence suggests that the cell cycle plays an important role in regulating differentiation, while p130 and E2F4 are key to cell cycle checkpoints. The aim of the study is to evaluate the effects and mechanism of p130/E2F4 on the multidifferentiation of MSCs. Our data showed that the transduction efficiencies of p130 or E2F4 mediated by lentiviral vectors were 80.3%-84.4%. p130 and E2F4 mRNA expression was significantly higher in MSC-p130 and MSC-E2F4 cells than in MSC normal control (NC) cells. Similar results were also observed for p130 and E2F4 protein expression. After osteogenic or adipogenic differentiation, the G1 phase was significantly delayed in the MSC-p130 and MSC-E2F4 groups compared with that in the MSC-NC group. However, the G1 phase in the MSC-p130 and MSC-E2F4 groups did the opposite after chondrogenic differentiation. Moreover, overexpressing p130 or E2F4 significantly improved osteogenic differentiation while inhibiting adipogenic and chondrogenic differentiation of mouse MSCs (mMSCs). Moreover, overexpressing p130 or E2F4 significantly improved migration but not proliferation of mMSCs. Our data suggest that cell cycle regulation may be involved in p130/E2F4-mediated changes in the multipotential abilities of bone-marrow-derived mMSCs.
Assuntos
Diferenciação Celular/genética , Proteína Substrato Associada a Crk/genética , Fator de Transcrição E2F4/genética , Células-Tronco Mesenquimais/metabolismo , Adipogenia/genética , Células da Medula Óssea/classificação , Células da Medula Óssea/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Movimento Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Vetores Genéticos , Humanos , Lentivirus/genética , Células-Tronco Mesenquimais/citologia , Osteogênese/genéticaRESUMO
Glioblastoma (GBM) is an invasive brain cancer with tumor cells that disperse from the primary mass, escaping surgical resection and invariably giving rise to lethal recurrent lesions. Here we report that PTP-PEST, a cytoplasmic protein tyrosine phosphatase, controls GBM cell invasion by physically bridging the focal adhesion protein Crk-associated substrate (Cas) to valosin-containing protein (Vcp), an ATP-dependent protein segregase that selectively extracts ubiquitinated proteins from multiprotein complexes and targets them for degradation via the ubiquitin proteasome system. Both Cas and Vcp are substrates for PTP-PEST, with the phosphorylation status of tyrosine 805 (Y805) in Vcp impacting affinity for Cas in focal adhesions and controlling ubiquitination levels and protein stability. Perturbing PTP-PEST-mediated phosphorylation of Cas and Vcp led to alterations in GBM cell-invasive growth in vitro and in preclinical mouse models. Collectively, these data reveal a novel regulatory mechanism involving PTP-PEST, Vcp, and Cas that dynamically balances phosphorylation-dependent ubiquitination of key focal proteins involved in GBM cell invasion.Significance: PTP-PEST balances GBM cell growth and invasion by interacting with the ATP-dependent ubiquitin segregase Vcp/p97 and regulating phosphorylation and stability of the focal adhesion protein p130Cas.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3809/F1.large.jpg Cancer Res; 78(14); 3809-22. ©2018 AACR.
Assuntos
Adesões Focais/genética , Glioblastoma/genética , Fosforilação/genética , Proteína Tirosina Fosfatase não Receptora Tipo 12/genética , Ubiquitinação/genética , Animais , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Proteína Substrato Associada a Crk/genética , Glioblastoma/patologia , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica/genética , Tirosina/genética , Proteína com Valosina/genéticaRESUMO
Development of complex neural circuits like the peripheral somatosensory system requires intricate mechanisms to ensure axons make proper connections. While much is known about ligand-receptor pairs required for dorsal root ganglion (DRG) axon guidance, very little is known about the cytoplasmic effectors that mediate cellular responses triggered by these guidance cues. Here we show that members of the Cas family of cytoplasmic signaling adaptors are highly phosphorylated in central projections of the DRG as they enter the spinal cord. Furthermore, we provide genetic evidence that Cas proteins regulate fasciculation of DRG sensory projections. These data establish an evolutionarily conserved requirement for Cas adaptor proteins during peripheral nervous system axon pathfinding. They also provide insight into the interplay between axonal fasciculation and adhesion to the substrate.
Assuntos
Fasciculação Axônica , Proteína Substrato Associada a Crk/metabolismo , Gânglios Espinais/crescimento & desenvolvimento , Animais , Proteína Substrato Associada a Crk/análise , Proteína Substrato Associada a Crk/genética , Gânglios Espinais/metabolismo , Gânglios Espinais/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Fosforilação , RNA Mensageiro/análise , RNA Mensageiro/genética , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismo , Medula Espinal/ultraestruturaRESUMO
BACKGROUND: Breast cancer anti-estrogen resistance 1 (BCAR1/p130cas) is a hub for diverse oncogenic signaling cascades and promotes tumor development and progression. METHODS: To understand the effect of BCAR1 in prostate cancer, we analyzed its expression on more than 11,000 prostate cancer samples. BCAR1 expression levels were compared with clinical characteristics, PSA recurrence, molecular subtype defined by ERG status and 3p, 5q, 6q and PTEN deletion. RESULTS: BCAR1 staining was barely detectable in normal prostate glands but seen in 77.6% of 9472 interpretable cancers, including strong expression in 38.5%, moderate in 23.2% and weak in 15.9% of cases. BCAR1 up regulation was associated with positive ERG status (p < 0.0001), high Gleason score (p < 0.0001), advanced pathological tumor stage (p = 0.0082), lower preoperative PSA level (p < 0.0001), increased cell proliferation (p < 0.0001), early PSA recurrence (p = 0.0008), and predicted prognosis independently from clinico-pathological parameters available at the time of the initial biopsy. However, subset analyses revealed that the prognostic impact of BCAR1 expression was limited to ERG-negative cancer. That BCAR1 up regulation was linked to almost all analyzed deletions (p < 0.0001 each for PTEN, 5q, 6q deletion) may suggest a functional link to genomic instability. CONCLUSION: The results of our study identify BCAR1 as a prognostic biomarker with potential clinical value for risk stratification of ERG-negative prostate cancer.