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1.
J Clin Pediatr Dent ; 44(4): 262-267, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33167018

RESUMO

OBJECTIVE: To evaluate orodental, facial, clinical and molecular characteristics of the patients with Noonan Syndrome (NS). STUDY DESIGN: The orodental, clinical and molecular characteristics of 29 mutation-positive patients with NS were recorded. Orodental examination was performed in 17 patients. All exons and exonintron boundries of PTPN11 and SOS1 genes were analyzed by Sanger sequencing. RESULTS: A total of 29 patients with NS from 27 unrelated families were included in the study. Seventeen patients were examined by a specialist in oral medicine. The most common orodental findings were high-arched palate (n=13), gingivitis (n=6) and severe caries (n=6). Anterior open bite, posterior cross bite, Class II malocclusion, hypodontia, prognathism (maxillary or mandibular), macroglossia and gingival hyperplasia were also detected. Thirteen different mutations were observed in PTPN11 gene and exon 3 was the hotspot region. Hypodontia was detected in two patients who had the same mutation in PTPN11 gene, c.181G>A, p.D61N. CONCLUSION: This study indicated a high prevalance of orodental problems including high-arched palate, severe dental caries and gingivitis in patients with mutation-positive NS. The mutation in PTPN11 gene, c.181G>A, p.D61N, may be associated with hypodontia in patients with NS.


Assuntos
Cárie Dentária , Síndrome de Noonan , Mordida Aberta , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Humanos , Mutação , Síndrome de Noonan/genética , Fenótipo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética
2.
J Pharmacol Sci ; 144(3): 139-146, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32921395

RESUMO

SHP2 is a non-receptor protein tyrosine phosphatase encoded by the PTPN11 gene in human. Clinically, SHP2 has been identified as a causal factor of several diseases, such as Noonan syndrome, LEOPARD syndrome as well as myeloid malignancies. Interestingly, both loss-of-function and gain-of-function mutations occur in the PTPN11 gene. Analyses by biochemical and cell biological means as well as probing with small molecule compounds have demonstrated that SHP2 has both phosphatase-dependent and independent functions. In comparison with its phosphatase activity, the non-phosphatase-like function of SHP2 has not been well introduced or summarized. This review mainly focuses on the phosphatase-independent functions and its regulation by small molecule compounds as well as their use for disease therapy.


Assuntos
Monoéster Fosfórico Hidrolases , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/fisiologia , Cerebrosídeos , Depsipeptídeos , Mutação com Ganho de Função , Humanos , Síndrome LEOPARD/genética , Mutação com Perda de Função , Terapia de Alvo Molecular , Síndrome de Noonan/genética , Monoéster Fosfórico Hidrolases/metabolismo , Piperidinas , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Pirimidinas , Transdução de Sinais/genética
3.
J Vis Exp ; (161)2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32744526

RESUMO

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase (RTK)-driven cell signaling, promoting cell survival and proliferation. In addition, SHP2 is recruited by immune check point receptors to inhibit B and T cell activation. Aberrant SHP2 function has been implicated in the development, progression, and metastasis of many cancers. Indeed, small molecule SHP2 inhibitors have recently entered clinical trials for the treatment of solid tumors with Ras/Raf/ERK pathway activation, including tumors with some oncogenic Ras mutations. However, the current class of SHP2 inhibitors is not effective against the SHP2 oncogenic variants that occur frequently in leukemias, and the development of specific small molecules that target oncogenic SHP2 is the subject of current research. A common problem with most drug discovery campaigns involving cytosolic proteins like SHP2 is that the primary assays that drive chemical discovery are often in vitro assays that do not report the cellular target engagement of candidate compounds. To provide a platform for measuring cellular target engagement, we developed both wild-type and mutant SHP2 cellular thermal shift assays. These assays reliably detect target engagement of SHP2 inhibitors in cells. Here, we provide a comprehensive protocol of this assay, which provides a valuable tool for the assessment and characterization of SHP2 inhibitors.


Assuntos
Inibidores Enzimáticos/uso terapêutico , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Inibidores Enzimáticos/farmacologia , Humanos , Transdução de Sinais
4.
Am J Hum Genet ; 107(3): 499-513, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32721402

RESUMO

Signal transduction through the RAF-MEK-ERK pathway, the first described mitogen-associated protein kinase (MAPK) cascade, mediates multiple cellular processes and participates in early and late developmental programs. Aberrant signaling through this cascade contributes to oncogenesis and underlies the RASopathies, a family of cancer-prone disorders. Here, we report that de novo missense variants in MAPK1, encoding the mitogen-activated protein kinase 1 (i.e., extracellular signal-regulated protein kinase 2, ERK2), cause a neurodevelopmental disease within the RASopathy phenotypic spectrum, reminiscent of Noonan syndrome in some subjects. Pathogenic variants promote increased phosphorylation of the kinase, which enhances translocation to the nucleus and boosts MAPK signaling in vitro and in vivo. Two variant classes are identified, one of which directly disrupts binding to MKP3, a dual-specificity protein phosphatase negatively regulating ERK function. Importantly, signal dysregulation driven by pathogenic MAPK1 variants is stimulus reliant and retains dependence on MEK activity. Our data support a model in which the identified pathogenic variants operate with counteracting effects on MAPK1 function by differentially impacting the ability of the kinase to interact with regulators and substrates, which likely explains the minor role of these variants as driver events contributing to oncogenesis. After nearly 20 years from the discovery of the first gene implicated in Noonan syndrome, PTPN11, the last tier of the MAPK cascade joins the group of genes mutated in RASopathies.


Assuntos
Carcinogênese/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Transtornos do Neurodesenvolvimento/genética , Síndrome de Noonan/genética , Pré-Escolar , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Mutação de Sentido Incorreto/genética , Transtornos do Neurodesenvolvimento/patologia , Síndrome de Noonan/fisiopatologia , Fenótipo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Transdução de Sinais , Sequenciamento Completo do Exoma , Proteínas ras/genética
6.
Cancer Res ; 80(13): 2889-2902, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32350067

RESUMO

The protein tyrosine phosphatase SHP2 binds to phosphorylated signaling motifs on regulatory immunoreceptors including PD-1, but its functional role in tumor immunity is unclear. Using preclinical models, we show that RMC-4550, an allosteric inhibitor of SHP2, induces antitumor immunity, with effects equivalent to or greater than those resulting from checkpoint blockade. In the tumor microenvironment, inhibition of SHP2 modulated T-cell infiltrates similar to checkpoint blockade. In addition, RMC-4550 drove direct, selective depletion of protumorigenic M2 macrophages via attenuation of CSF1 receptor signaling and increased M1 macrophages via a mechanism independent of CD8+ T cells or IFNγ. These dramatic shifts in polarized macrophage populations in favor of antitumor immunity were not seen with checkpoint blockade. Consistent with a pleiotropic mechanism of action, RMC-4550 in combination with either checkpoint or CSF1R blockade caused additive antitumor activity with complete tumor regressions in some mice; tumors intrinsically sensitive to SHP2 inhibition or checkpoint blockade were particularly susceptible. Our preclinical findings demonstrate that SHP2 thus plays a multifaceted role in inducing immune suppression in the tumor microenvironment, through both targeted inhibition of RAS pathway-dependent tumor growth and liberation of antitumor immune responses. Furthermore, these data suggest that inhibition of SHP2 is a promising investigational therapeutic approach. SIGNIFICANCE: Inhibition of SHP2 causes direct and selective depletion of protumorigenic M2 macrophages and promotes antitumor immunity, highlighting an investigational therapeutic approach for some RAS pathway-driven cancers.


Assuntos
Neoplasias da Mama/imunologia , Imunossupressores/farmacologia , Macrófagos/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Microambiente Tumoral/imunologia , Regulação Alostérica , Animais , Apoptose , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/prevenção & controle , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
7.
BMC Med Genet ; 21(1): 50, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32164556

RESUMO

BACKGROUND: Noonan syndrome (NS), an autosomal dominant developmental genetic disorder, is caused by germline mutations in genes associated with the RAS / mitogen-activated protein kinase (MAPK) pathway. In several studies PTPN11 is one of the genes with a significant number of pathogenic variants in NS-affected patients. Therefore, clinically diagnosed NS individuals are initially tested for pathogenic variants in PTPN11 gene to confirm the relationship before studying genotype-phenotype correlation. METHODS: Individuals (363) with clinically diagnosed NS from four hospitals in South India were recruited and the exons of PTPN11 gene were sequenced. RESULTS: Thirty-two previously described pathogenic variants in eight different exons in PTPN11 gene were detected in 107 patients, of whom 10 were familial cases. Exons 3, 8 and 13 had the highest number of pathogenic variants. The most commonly identified pathogenic variants in this series were in exon 8 (c.922A > G, c.923A > G), observed in 22 of the affected. Congenital cardiac anomalies were present in 84% of the mutation-positive cohort, the majority being defects in the right side of the heart. The most common facial features were downward-slanting palpebral fissures, hypertelorism and low-set posteriorly rotated ears. Other clinical features included short stature (40%), pectus excavatum (54%) and, in males, unilateral or bilateral cryptorchidism (44%). CONCLUSION: The clinical features and mutational spectrum observed in our cohort are similar to those reported in other large studies done worldwide. This is the largest case series of NS-affected individuals with PTPN11 mutations described till date from India.


Assuntos
Síndrome de Noonan/genética , Polimorfismo de Nucleotídeo Único , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Adolescente , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Análise Mutacional de DNA , Família , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Cardiopatias Congênitas/epidemiologia , Cardiopatias Congênitas/genética , Humanos , Índia/epidemiologia , Lactente , Recém-Nascido , Masculino , Síndrome de Noonan/epidemiologia , Fenótipo , Adulto Jovem
8.
BMC Gastroenterol ; 20(1): 34, 2020 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054441

RESUMO

BACKGROUND: Noonan syndrome is an autosomal dominant, variably expressed multisystem disorder characterized by specific facial and cardiac defects, delayed growth, ectodermal abnormalities, and lymphatic dysplasias. Lymphedema and chylous pleural effusions are common in Noonan syndrome, but protein-losing enteropathy (PLE) has only rarely been described in the condition and little is known about its genetic associations. CASE PRESENTATION: We report the case of a 30-year-old Chinese woman who developed severe recurrent edema and hypoproteinemia. Gastroduodenoscopy showed a "snowflake" appearance of lymphangiectasia in the duodenum, and CT reconstruction of the small intestine showed segmental thickening of the intestinal wall with localized stenosis. Whole exome sequencing revealed that the patient harbored a pathogenic variant of PTPN11 (c.A922G p.N308D), which was unfortunately inherited by her 2.5-year-old daughter who had short stature and atrial septal defect but no hypoproteinemia. CONCLUSIONS: This case of Noonan syndrome with PLE was associated with a PTPN11 mutation. A comprehensive review of PLE in Noonan syndrome revealed that PLE often presents late in this context but there is no clear genotype-phenotype correlation. Genetic evaluation with next-generation sequencing can be useful for securing the diagnosis and planning early intervention and management.


Assuntos
Síndrome de Noonan/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Enteropatias Perdedoras de Proteínas/genética , Adulto , Edema , Feminino , Estudos de Associação Genética , Humanos , Hipocalcemia , Hipoproteinemia , Mutação , Linhagem , Sequenciamento Completo do Exoma
9.
Sci Rep ; 10(1): 2725, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066785

RESUMO

Superparamagnetic iron oxide nanoparticles (SPIONs) have been investigated for wide variety of applications. Their unique properties render them highly applicable as MRI contrast agents, in magnetic hyperthermia or targeted drug delivery. SPIONs surface properties affect a whole array of parameters such as: solubility, toxicity, stability, biodistribution etc. Therefore, progress in the field of SPIONs surface functionalization is crucial for further development of therapeutic or diagnostic agents. In this study, SPIONs were synthesized by thermal decomposition of iron (III) acetylacetonate Fe(acac)3 and functionalized with dihexadecyl phosphate (DHP) via phase transfer. Bioactivity of the SPION-DHP was assessed on SW1353 and TCam-2 cancer derived cell lines. The following test were conducted: cytotoxicity and proliferation assay, reactive oxygen species (ROS) assay, SPIONs uptake (via Iron Staining and ICP-MS), expression analysis of the following genes: alkaline phosphatase (ALPL); ferritin light chain (FTL); serine/threonine protein phosphatase 2A (PP2A); protein tyrosine phosphatase non-receptor type 11 (PTPN11); transferrin receptor 1 (TFRC) via RT-qPCR. SPION-DHP nanoparticles were successfully obtained and did not reveal significant cytotoxicity in the range of tested concentrations. ROS generation was elevated, however not correlated with the concentrations. Gene expression profile was slightly altered only in SW1353 cells.


Assuntos
Condrócitos/efeitos dos fármacos , Compostos Férricos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Nanopartículas de Magnetita/química , Organofosfatos/química , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Apoferritinas/genética , Apoferritinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Compostos Férricos/química , Humanos , Hidroxibutiratos/química , Pentanonas/química , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Succímero/química
10.
Yi Chuan ; 42(2): 183-193, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-32102775

RESUMO

The protein tyrosine phosphatase SHP2 of higher vertebrates, encoded by ptpn11 gene, catalyzes the dephosphorylation of tyrosine phosphorylation site, and plays regulatory roles in various signaling pathways by cooperating with other protein tyrosine kinase. Previous studies have shown that SHP2 plays an important role in the activation and signal transduction of T and B cells in higher vertebrates. To study the role of a SHP2 homologous molecule of lampreys (Lja-SHP2) in immune response, we cloned and expressed the open reading frame sequence of Lja-SHP2 gene in prokaryotic expression vector pET-32a. The recombinant protein was successfully expressed in E. coli and the rabbit-derived polyclonal antibody was prepared. Lampetra japonica were immunized with mixed bacteria, and the mRNA and protein of Lja-SHP2 in immune-related cells and tissues were detected by real-time quantitative PCR and Western blotting after immunization. The Lja-SHP2 mRNA and protein were not significantly affected in leukocytes and supraneural myeloid bodies, but up-regulated significantly in gill tissues (P<0.05) after challenged by mixed bacteria, which indicated that Lja-SHP2 mainly participates in the immune response of gill tissues after mixed bacteria stimulation. To further investigate whether Lja-SHP2 level was affected in three lymphocyte subsets, the B-cell mitogen lipopolysaccharide (LPS) and T-cell mitogen phytohaemagglutinin (PHA) were employed to boost the immune response in L. japonica. LPS immune stimulation increased Lja-SHP2 in leucocytes significantly compared with the control group, and but had a marginal effect on Lja-SHP2 expression in gills and supraneural myeloid bodies. PHA immune stimulation could up-regulate Lja-SHP2 level in leukocytes, gill tissues and supraneural myeloid bodies. The change of Lja-SHP2 was especially dramatical in leukocytes, which was about 2.5 times higher than that in the control group, suggesting that Lja-SHP2 is involved in the lamprey immune response mediated by PHA. Consistent with the previous finding that PHA could induce the activation of VLRA+ lymphocytes, our results showed that Lja-SHP2 might be included in the immune response of VLRA+ lymphocytes mediated by PHA in gills. This research will benefit exploring the functions of Lja-SHP2 in the immune response of lamprey and will provide clues for understanding the phylogenesis of SHP2 molecular family, and its roles in the early occurrence and evolution of adaptive immune system in higher vertebrates.


Assuntos
Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Lampreias/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/imunologia , Animais , Lampreias/imunologia , Linfócitos/imunologia , Filogenia , Proteínas Recombinantes
11.
Biochemistry (Mosc) ; 85(1): 108-118, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32079522

RESUMO

The MAPK (RAS/BRAF/MEK/ERK) signaling pathway is a kinase cascade involved in the regulation of cell proliferation, differentiation, and survival in response to external stimuli. The V600E mutation in the BRAF gene has been detected in various tumors, resulting in a 500-fold increase in BRAF kinase activity. However, monotherapy with selective BRAF V600E inhibitors often leads to reactivation of MAPK signaling cascade and emergence of drug resistance. Therefore, new targets are being developed for the inhibition of components of the aberrantly activated cascade. It was recently discovered that resistance to BRAF V600E inhibitors may be associated with the activity of the tyrosine phosphatase SHP-2 encoded by the PTPN11 gene. In this paper, we analyzed transcriptional effects of PTPN11 gene knockdown and selective suppression of BRAF V600E in a model of thyroid follicular epithelium. We found that the siRNA-mediated knockdown of PTPN11 after vemurafenib treatment prevented an increase in the expression CCNA1 and NOTCH4 genes involved in the formation of drug resistance of tumors. On the other hand, downregulation of PTPN11 expression blocked the transcriptional activation of genes (p21, p15, p16, RB1, and IGFBP7) involved in cell cycle regulation and oncogene-induced senescence in response to BRAF V600E expression. Therefore, it can be assumed that SHP-2 participates not only in emergence of drug resistance in cancer cells, but also in oncogene-induced cell senescence.


Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 11/fisiologia , Proteínas Proto-Oncogênicas B-raf/metabolismo , Células Epiteliais da Tireoide , Neoplasias da Glândula Tireoide/metabolismo , Ciclo Celular , Linhagem Celular , Senescência Celular , Resistencia a Medicamentos Antineoplásicos/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Células Epiteliais da Tireoide/citologia , Células Epiteliais da Tireoide/metabolismo , Glândula Tireoide/citologia , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Vemurafenib/uso terapêutico
12.
Int J Mol Sci ; 21(2)2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31936901

RESUMO

Noonan syndrome (NS) is a genetic disorder caused by the hyperactivation of the RAS-MAPK molecular pathway. About 50% of NS cases are caused by mutations affecting the SHP2 protein, a multi-domain phosphatase with a fundamental role in the regulation of the RAS-MAPK pathway. Most NS-causing mutations influence the stability of the inactive form of SHP2. However, one NS-causing mutation, namely T42A, occurs in the binding pocket of the N-SH2 domain of the protein. Here, we present a quantitative characterization of the effect of the T42A mutation on the binding of the N-terminal SH2 domain of SHP2 with a peptide mimicking Gab2, a fundamental interaction that triggers the activation of the phosphatase in the cellular environment. Our results show that whilst the T42A mutation does not affect the association rate constant with the ligand, it causes a dramatic increase of the affinity for Gab2. This effect is due to a remarkable decrease of the microscopic dissociation rate constant of over two orders of magnitudes. In an effort to investigate the molecular basis of the T42A mutation in causing Noonan syndrome, we also compare the experimental results with a more conservative variant, T42S. Our findings are discussed in the context of the structural data available on SHP2.


Assuntos
Predisposição Genética para Doença/genética , Mutação , Síndrome de Noonan/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas Adaptadoras de Transdução de Sinal , Humanos , Cinética , Modelos Moleculares , Mutagênese , Ligação Proteica , Conformação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Domínios de Homologia de src
13.
J Biol Chem ; 295(9): 2601-2613, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31953320

RESUMO

The nonreceptor protein-tyrosine phosphatase (PTP) SHP2 is encoded by the proto-oncogene PTPN11 and is a ubiquitously expressed key regulator of cell signaling, acting on a number of cellular processes and components, including the Ras/Raf/Erk, PI3K/Akt, and JAK/STAT pathways and immune checkpoint receptors. Aberrant SHP2 activity has been implicated in all phases of tumor initiation, progression, and metastasis. Gain-of-function PTPN11 mutations drive oncogenesis in several leukemias and cause developmental disorders with increased risk of malignancy such as Noonan syndrome. Until recently, small molecule-based targeting of SHP2 was hampered by the failure of orthosteric active-site inhibitors to achieve selectivity and potency within a useful therapeutic window. However, new SHP2 allosteric inhibitors with excellent potency and selectivity have sparked renewed interest in the selective targeting of SHP2 and other PTP family members. Crucially, drug discovery campaigns focusing on SHP2 would greatly benefit from the ability to validate the cellular target engagement of candidate inhibitors. Here, we report a cellular thermal shift assay that reliably detects target engagement of SHP2 inhibitors. Using this assay, based on the DiscoverX InCell Pulse enzyme complementation technology, we characterized the binding of several SHP2 allosteric inhibitors in intact cells. Moreover, we demonstrate the robustness and reliability of a 384-well miniaturized version of the assay for the screening of SHP2 inhibitors targeting either WT SHP2 or its oncogenic E76K variant. Finally, we provide an example of the assay's ability to identify and characterize novel compounds with specific cellular potency for either WT or mutant SHP2.


Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Animais , Carcinogênese/genética , Linhagem Celular , Mutação com Ganho de Função , Humanos , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo
14.
Fitoterapia ; 141: 104484, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31954180

RESUMO

The Src-homology 2 domain-containing phosphatase 2 (SHP2), encoded by PTPN11, has been reported oncogenic tyrosine phosphatase associated with various tumors and played critical roles in many cell signaling events. Targeting SHP2 by small molecules may be a promising way for cancer therapy. Herein, a new abietane diterpenoid, named 3-acetoxylteuvincenone G (3-AG), was isolated from the whole plants of Ajuga ovalifolia var. calantha. The structure of the new compound was elucidated by means of extensive spectroscopic analyses. Using recombinant enzyme activity assay and cellular thermal shift assay, we found that 3-AG was a selective inhibitor of SHP2. Molecular docking suggested 3-AG displayed an orientation favorable to nucleophilic attack in the catalytic domain of SHP2. 3-AG suppressed A549 cell proliferation (IC50 = 10.79 ± 0.14 µM), invasion and induced cell apoptosis through SHP2/ERK1/2 and SHP2/AKT pathways. In summary, 3-AG, a potent, selective, and efficacious SHP2 inhibitor, may be a promising small molecule to treat human lung epithelial cancer.


Assuntos
Abietanos/farmacologia , Apoptose/efeitos dos fármacos , Diterpenos/química , Diterpenos/farmacologia , Lamiaceae/química , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Células A549 , Abietanos/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética
15.
Oncogene ; 39(1): 50-63, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31462705

RESUMO

Resistance of breast cancer to human epidermal growth factor receptor 2 (HER2) inhibitors involves reprogramming of the kinome through HER2/HER3 signaling via the activation of multiple tyrosine kinases and transcriptional upregulation. The heterogeneity of induced kinases prevents kinase targeting by a single kinase inhibitor and presents a major challenge to the treatment of therapeutically recalcitrant HER2-positive breast cancers (HER2+ BCs). As a result, there is a critical need for effective treatment that attacks the aberrant kinome activation associated with resistance to HER2-targeted therapy. Here, we describe a novel treatment strategy that targets cyclin-dependent kinase 7 (CDK7) in HER2 inhibitor-resistant (HER2iR) breast cancer. We show that both HER2 inhibitor-sensitive (HER2iS) and HER2iR breast cancer cell lines exhibit high sensitivity to THZ1, a newly identified covalent inhibitor of the transcription regulatory kinase CDK7. CDK7 promotes cell cycle progression through inhibition of transcription, rather than via direct phosphorylation of classical CDK targets. The transcriptional kinase activity of CDK7 is regulated by HER2, and by the receptor tyrosine kinases activated in response to HER2 inhibition, as well as by the downstream SHP2 and PI3K/AKT pathways. A low dose of THZ1 displayed potent synergy with the HER2 inhibitor lapatinib in HER2iR BC cells in vitro. Dual HER2 and CDK7 inhibition induced tumor regression in two HER2iR BC xenograft models in vivo. Our data support the utilization of CDK7 inhibition as an additional therapeutic avenue that blocks the activation of genes engaged by multiple HER2iR kinases.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Quinases Ciclina-Dependentes/genética , Fenilenodiaminas/farmacologia , Pirimidinas/farmacologia , Receptor ErbB-2/genética , Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lapatinib/farmacologia , Proteína Oncogênica v-akt/genética , Fosfatidilinositol 3-Quinases/genética , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Receptor ErbB-2/antagonistas & inibidores , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética
16.
Int J Cancer ; 146(8): 2243-2254, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-31525277

RESUMO

Constitutive activation of FGFR1, as a result of diverse chromosome translocations, is the hallmark of stem cell leukemia/lymphoma syndrome. The BCR-FGFR1 variant is unique in that the BCR component contributes a serine-threonine kinase (STK) to the N-terminal end of the chimeric FGFR1 kinase. We have deleted the STK domain and mutated the critical Y177 residue and demonstrate that the transforming activity of these mutated genes is reduced compared to the BCR-FGFR1 parental kinase. In addition, we demonstrate that deletion of the FGFR1 tyrosine kinase domain abrogates transforming ability, which is not compensated for by BCR STK activity. Unbiased screening for proteins that are inactivated as a result of loss of the BCR STK identified activated S6 kinase and SHP2 kinase. Genetic and pharmacological inhibition of SHP2 function in SCLL cells expressing BCR-FGFR1 in vitro leads to reduced viability and increased apoptosis. In vivo treatment of SCLL in mice with SHP099 leads to suppression of leukemogenesis, supporting an important role for SHP2 in FGFR1-driven leukemogenesis. In combination with the BGJ398 FGFR1 inhibitor, cell viability in vitro is further suppressed and acts synergistically with SHP099 in vivo suggesting a potential combined targeted therapy option in this subtype of SCLL disease.


Assuntos
Leucemia/metabolismo , Linfoma/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas c-bcr/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Transformação Celular Neoplásica , Sinergismo Farmacológico , Feminino , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Leucemia/tratamento farmacológico , Leucemia/genética , Leucemia/patologia , Linfoma/tratamento farmacológico , Linfoma/genética , Linfoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Fusão Oncogênica/genética , Compostos de Fenilureia/administração & dosagem , Compostos de Fenilureia/farmacologia , Piperidinas/administração & dosagem , Piperidinas/farmacologia , Domínios Proteicos , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogênicas c-bcr/biossíntese , Proteínas Proto-Oncogênicas c-bcr/genética , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/biossíntese , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética
17.
Leukemia ; 34(3): 799-810, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31628430

RESUMO

RAS-pathway mutations are recurrent events in myeloid malignancies. However, there is limited data on the significance of RAS-pathway mutations in patients with myelofibrosis (MF). We analyzed next-generation sequencing data of 16 genes, including RAS-pathway genes, from 723 patients with primary and secondary MF across three international centers and evaluated their significance. N/KRAS variants were present in 6% of patients and were typically sub-clonal (median VAF = 20%) relative to other genes variants. RAS variants were associated with advanced MF features including leukocytosis (p = 0.02), high somatic mutation burden (p < 0.01) and the presence of established "molecular high-risk" (MHR) mutations. MF patients with N/KRAS mutations had shorter 3-year overall survival (OS) (34% vs 58%, p < 0.001) and higher incidence of acute myeloid leukemia at 3 years (18% vs 11%, p = 0.03). In a multivariate Cox model, RAS mutations were associated with decreased OS (HR 1.93, p < 0.001). We created a novel score to predict OS incorporating RAS mutations, and it predicted OS across training and validation cohorts. Patients with intermediate risk/high-risk DIPSS with RAS mutations who received ruxolitinib had a nonsignificant longer 2-year OS relative to those who did not receive ruxolitinib. These data demonstrate the importance of identifying RAS mutations in MF patients.


Assuntos
Genes ras , Mutação , Mielofibrose Primária/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA , Feminino , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Policitemia Vera/genética , Mielofibrose Primária/diagnóstico , Prognóstico , Modelos de Riscos Proporcionais , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirazóis/farmacologia , Estudos Retrospectivos , Risco , Trombocitemia Essencial/genética , Resultado do Tratamento , Adulto Jovem , Tirosina Quinase 3 Semelhante a fms/genética
18.
Lab Invest ; 100(4): 630-642, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31653968

RESUMO

Corneal nerve fibers serving sensory, reflex, and neurotrophic functions sustain corneal homeostasis and transparency to promote normal visual function. It is not known whether corneal epithelium is also important for the corneal innervation. Herein, we generated a compound transgenic mouse strain, K14rtTA;tetO-Cre (TC);Shp2flox/flox, in which Shp2 was conditionally knocked out from K14-positive cells including corneal epithelium (Shp2K14ce-cko) upon doxycycline (dox) administration. Our data reveal that Shp2K14ce-cko caused corneal denervation. More specifically, corneal epithelium thickness and corneal sensitivity reduced dramatically in Shp2K14ce-cko mice. In addition, corneal epithelial wound healing after debridement was delayed substantially in the mutant mice. These defects manifested in Shp2K14ce-cko mice resemble the symptoms of human neurotrophic keratopathy. Our in vitro study shows that neurite outgrowth of the mouse primary trigeminal ganglion cells (TGCs) was inhibited when as cocultured with mouse corneal epithelial cells (TKE2) transfected by Shp2-, Mek1/2-, or ∆Np63-targeted siRNA but not by Akt1/2-targeted siRNA. Furthermore, ∆Np63 RNA interference downregulated Ngf expression in TKE2 cells. Cotransfection experiments reveal that Shp2 tightly monitored ΔNp63 protein levels in HEK293 and TKE2 cells. Taken together, our data suggest that the Shp2-mediated MAPK pathway regulated ΔNp63, which in turn positively regulated Ngf in epithelium to promote corneal innervation and epithelial homeostasis.


Assuntos
Córnea , Sistema de Sinalização das MAP Quinases , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Cicatrização , Animais , Córnea/inervação , Córnea/metabolismo , Córnea/fisiologia , Lesões da Córnea/metabolismo , Epitélio Anterior/metabolismo , Homeostase/genética , Homeostase/fisiologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Transgênicos , Neuritos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Cicatrização/genética , Cicatrização/fisiologia
19.
Eur J Pediatr ; 179(3): 463-472, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31807902

RESUMO

Juvenile myelomonocytic leukemia (JMML) is a heterogeneous childhood leukemia. The management of patients with JMML requires accurate assessment of genetic and clinical features to help in patient risk stratification. This study aimed to investigate the association between genomic alterations and prognosis in children with JMML. Genomic DNA was extracted from a total of 93 patients with JMML for targeted sequencing. Univariable and multivariable analysis were used to evaluate the correlation between gene mutations and prognosis of the patients. Patients with PTPN11 mutation exhibited significantly lower event-free survival (EFS) compared with non-PTPN11 mutations (P = 0.005). Patients without or with one somatic alteration at diagnosis showed significantly better prognosis in comparison with those with more than two alterations (P = 0.009). PTPN11 mutation with additional alterations showed significantly the poorest outcome in comparison with those with only one non-PTPN11 mutation, only one PTPN11 mutation, and combined mutations without PTPN11, respectively (P < 0.0001).Conclusion: Both PTPN11 mutation and the number of somatic alterations detected at diagnosis are likely to be the major determinant of outcome in JMML. The subgroup of patients with PTPN11 mutation showed the shortest survival which was even worsened when a secondary mutation was present.


Assuntos
Leucemia Mielomonocítica Juvenil/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Adolescente , Biomarcadores/análise , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Leucemia Mielomonocítica Juvenil/mortalidade , Masculino , Mutação , Estudos Retrospectivos
20.
Asian J Androl ; 22(1): 79-87, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31210146

RESUMO

The transition from spermatogonia to spermatocytes and the initiation of meiosis are key steps in spermatogenesis and are precisely regulated by a plethora of proteins. However, the underlying molecular mechanism remains largely unknown. Here, we report that Src homology domain tyrosine phosphatase 2 (Shp2; encoded by the protein tyrosine phosphatase, nonreceptor type 11 [Ptpn11] gene) is abundant in spermatogonia but markedly decreases in meiotic spermatocytes. Conditional knockout of Shp2 in spermatogonia in mice using stimulated by retinoic acid gene 8 (Stra8)-cre enhanced spermatogonial differentiation and disturbed the meiotic process. Depletion of Shp2 in spermatogonia caused many meiotic spermatocytes to die; moreover, the surviving spermatocytes reached the leptotene stage early at postnatal day 9 (PN9) and the pachytene stage at PN11-13. In preleptotene spermatocytes, Shp2 deletion disrupted the expression of meiotic genes, such as disrupted meiotic cDNA 1 (Dmc1), DNA repair recombinase rad51 (Rad51), and structural maintenance of chromosome 3 (Smc3), and these deficiencies interrupted spermatocyte meiosis. In GC-1 cells cultured in vitro, Shp2 knockdown suppressed the retinoic acid (RA)-induced phosphorylation of extracellular-regulated protein kinase (Erk) and protein kinase B (Akt/PKB) and the expression of target genes such as synaptonemal complex protein 3 (Sycp3) and Dmc1. Together, these data suggest that Shp2 plays a crucial role in spermatogenesis by governing the transition from spermatogonia to spermatocytes and by mediating meiotic progression through regulating gene transcription, thus providing a potential treatment target for male infertility.


Assuntos
Meiose/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Espermatócitos/metabolismo , Espermatogênese/genética , Espermatogônias/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Sobrevivência Celular , Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas Cromossômicas não Histona/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Infertilidade Masculina , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Ligação a Fosfato/genética , Rad51 Recombinase/genética , Reação em Cadeia da Polimerase em Tempo Real , Espermatócitos/citologia , Espermatogônias/citologia
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