RESUMEN
Selpercatinib is a small molecule that binds at the RET kinase active site. It inhibits activity of constitutively dimerized RET fusion proteins and activated point mutants, thereby blocking downstream signals for proliferation and survival. It is the first selective RET inhibitor to be FDA approved for tumor agnostic targeting of oncogenic RET fusion proteins. To view this Bench to Bedside, open or download the PDF.
Asunto(s)
Neoplasias , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas c-ret , Humanos , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-ret/antagonistas & inhibidores , Piridinas , Aprobación de DrogasRESUMEN
PURPOSE: Breast cancer (BC) is considered a heterogeneous disease composed of distinct subtypes with diverse clinical outcomes. Luminal subtype tumors have the best prognosis, and patients benefit from endocrine therapy. However, resistance to endocrine therapies in BC is an obstacle to successful treatment, and novel biomarkers are needed to understand and overcome this mechanism. The RET, BCAR1, and BCAR3 genes may be associated with BC progression and endocrine resistance. METHODS: Aiming to evaluate the expression profile and prognostic value of RET, BCAR1, and BCAR3, we performed immunohistochemistry on tissue microarrays (TMAs) containing a cohort of 361 Luminal subtype BC. RESULTS: Low expression levels of these three proteins were predominantly observed. BCAR1 expression was correlated with nuclear grade (p = 0.057), and BCAR3 expression was correlated with lymph node status (p = 0.011) and response to hormonal therapy (p = 0.021). Further, low expression of either BCAR1 or BCAR3 was significantly associated with poor prognosis (p = 0.005; p = 0.042). Pairwise analysis showed that patients with tumors with low BCAR1/low BCAR3 expression had a poorer overall survival (p = 0.013), and the low BCAR3 expression had the worst prognosis with RET high expression stratifying these patients into two different groups. Regarding the response to hormonal therapy, non-responder patients presented lower expression of RET in comparison to the responder group (p = 0.035). Additionally, the low BCAR1 expression patients had poorer outcomes than BCAR1 high (p = 0.015). CONCLUSION: Our findings suggest RET, BCAR1, and BCAR3 as potential candidate markers for endocrine therapy resistance in Luminal BC.
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Neoplasias de la Mama , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Biomarcadores de Tumor/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Proteína Sustrato Asociada a CrK , Femenino , Factores de Intercambio de Guanina Nucleótido , Humanos , Inmunohistoquímica , Pronóstico , Proteínas Proto-Oncogénicas c-retAsunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas c-ret , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Inhibidores de Proteínas Quinasas/efectos adversos , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-ret/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-ret/genéticaRESUMEN
The RET receptor tyrosine kinase is implicated in normal development and cancer. RET is expressed as two isoforms, RET9 and RET51, with unique C-terminal tail sequences that recruit distinct protein complexes to mediate signals. Upon activation, RET isoforms are internalized with distinct kinetics, suggesting differences in regulation. Here, we demonstrate that RET9 and RET51 differ in their abilities to recruit E3 ubiquitin ligases to their unique C-termini. RET51, but not RET9, interacts with, and is ubiquitylated by CBL, which is recruited through interactions with the GRB2 adaptor protein. RET51 internalization was not affected by CBL knockout but was delayed in GRB2-depleted cells. In contrast, RET9 ubiquitylation requires phosphorylation-dependent changes in accessibility of key RET9 C-terminal binding motifs that facilitate interactions with multiple adaptor proteins, including GRB10 and SHANK2, to recruit the NEDD4 ubiquitin ligase. We showed that NEDD4-mediated ubiquitylation is required for RET9 localization to clathrin-coated pits and subsequent internalization. Our data establish differences in the mechanisms of RET9 and RET51 ubiquitylation and internalization that may influence the strength and duration of RET isoform signals and cellular outputs.This article has an associated First Person interview with the first authors of the paper.
Asunto(s)
Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Proteínas Proto-Oncogénicas c-ret/metabolismo , Ubiquitinación , Secuencias de Aminoácidos , Proteína Adaptadora GRB10/genética , Proteína Adaptadora GRB10/metabolismo , Células HEK293 , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-ret/genéticaRESUMEN
Familial medullary thyroid cancer (MTC) and its precursor, C cell hyperplasia (CCH), is associated with germline RET mutations causing multiple endocrine neoplasia type 2. However, some rare families with apparent MTC/CCH predisposition do not have a detectable RET mutation. To identify novel MTC/CCH predisposition genes we undertook exome resequencing studies in a family with apparent predisposition to MTC/CCH and no identifiable RET mutation. We identified a novel ESR2 frameshift mutation, c.948delT, which segregated with histological diagnosis following thyroid surgery in family members and demonstrated loss of ESR2-encoded ERß expression in the MTC tumour. ERα and ERß form heterodimers binding DNA at specific oestrogen-responsive elements (EREs) to regulate gene transcription. ERß represses ERα-mediated activation of the ERE and the RET promoter contains three EREs. In vitro, we showed that ESR2 c.948delT results in unopposed ERα mediated increased cellular proliferation, activation of the ERE and increased RET expression. In vivo, immunostaining of CCH and MTC using an anti-RET antibody demonstrated increased RET expression. Together these findings identify germline ESR2 mutation as a novel cause of familial MTC/CCH and provide important insights into a novel mechanism causing increased RET expression in tumourigenesis.
Asunto(s)
Carcinoma Medular/congénito , Receptor beta de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica , Mutación de Línea Germinal/genética , Neoplasia Endocrina Múltiple Tipo 2a/genética , Neoplasia Endocrina Múltiple Tipo 2a/metabolismo , Proteínas Proto-Oncogénicas c-ret/metabolismo , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Adulto , Carcinoma Medular/genética , Carcinoma Medular/metabolismo , Carcinoma Medular/patología , Proliferación Celular , Susceptibilidad a Enfermedades , Genotipo , Humanos , Masculino , Neoplasia Endocrina Múltiple Tipo 2a/patología , Linaje , Proteínas Proto-Oncogénicas c-ret/genética , Neoplasias de la Tiroides/patología , Células Tumorales Cultivadas , Regulación hacia Arriba , Adulto JovenRESUMEN
The RET receptor tyrosine kinase (RTK) contributes to kidney and nervous system development, and is implicated in a number of human cancers. RET is expressed as two protein isoforms, RET9 and RET51, with distinct interactions and signaling properties that contribute to these processes. RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line-derived neurotropic factor (GDNF) ligand stimulation but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence (TIRF) microscopy to demonstrate that RET internalization occurs primarily through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin-associated adaptor protein complex 2 (AP2) interacts directly with each RET isoform through its AP2 µ subunit, and is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin-mediated endocytosis but that RET9 and RET51 have distinct internalization kinetics that may contribute to differences in their biological functions.
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Complejo 2 de Proteína Adaptadora/metabolismo , Membrana Celular/metabolismo , Endocitosis/fisiología , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endosomas/metabolismo , Humanos , Isoformas de Proteínas/metabolismo , Transporte de Proteínas/fisiología , Transducción de Señal/fisiologíaRESUMEN
Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTK) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumour pheochromocytoma (PCC) can be caused by activating mutations of the RET receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumour suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability, and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.
RESUMEN
Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.
Asunto(s)
Membrana Celular , Proteínas de la Membrana , Proteínas Proto-Oncogénicas c-ret , Proteínas Proto-Oncogénicas c-ret/metabolismo , Proteínas Proto-Oncogénicas c-ret/genética , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Membrana Celular/metabolismo , Transducción de Señal , Transporte de Proteínas , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Proliferación Celular , Neoplasias de las Glándulas Suprarrenales/genética , Neoplasias de las Glándulas Suprarrenales/metabolismo , Neoplasias de las Glándulas Suprarrenales/patologíaRESUMEN
The REarranged during Transfection (RET) gene encodes a receptor tyrosine kinase required for maturation of the enteric nervous system. RET sequence variants occur in the congenital abnormality Hirschsprung disease (HSCR), characterized by absence of ganglia in the intestinal tract. Although HSCR-RET variants are predicted to inactivate RET, the molecular mechanisms of these events are not well characterized. Using structure-based models of RET, we predicted the molecular consequences of 23 HSCR-associated missense variants and how they lead to receptor dysfunction. We validated our predictions in biochemical and cell-based assays to explore mutational effects on RET protein functions. We found a minority of HSCR-RET variants abrogated RET kinase function, while the remaining mutants were phosphorylated and transduced intracellular signals. HSCR-RET sequence variants also impacted on maturation, stability, and degradation of RET proteins. We showed that each variant conferred a unique combination of effects that together impaired RET protein activity. However, all tested variants impaired RET-mediated cellular functions, including cell transformation and migration. Our data indicate that the molecular mechanisms of impaired RET function in HSCR are highly variable. Although a subset of variants cause loss of RET kinase activity and downstream signaling, enzymatic inactivation is not the sole mechanism at play in HSCR.
Asunto(s)
Enfermedad de Hirschsprung/genética , Mutación , Proteínas Proto-Oncogénicas c-ret/genética , Sitios de Unión/genética , Western Blotting , Movimiento Celular/genética , Células HEK293 , Enfermedad de Hirschsprung/metabolismo , Humanos , Modelos Moleculares , Fosforilación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-ret/química , Proteínas Proto-Oncogénicas c-ret/metabolismo , Estabilidad del ARN/genética , Transducción de Señal/genética , TransfecciónRESUMEN
The TMEM127 gene encodes a transmembrane protein of poorly known function that is mutated in pheochromocytomas, neural crest-derived tumors of adrenomedullary cells. Here, we report that, at single-nucleus resolution, TMEM127-mutant tumors share precursor cells and transcription regulatory elements with pheochromocytomas carrying mutations of the tyrosine kinase receptor RET. Additionally, TMEM127-mutant pheochromocytomas, human cells, and mouse knockout models of TMEM127 accumulate RET and increase its signaling. TMEM127 contributes to RET cellular positioning, trafficking, and lysosome-mediated degradation. Mechanistically, TMEM127 binds to RET and recruits the NEDD4 E3 ubiquitin ligase for RET ubiquitination and degradation via TMEM127 C-terminal PxxY motifs. Lastly, increased cell proliferation and tumor burden after TMEM127 loss can be reversed by selective RET inhibitors in vitro and in vivo. Our results define TMEM127 as a component of the ubiquitin system and identify aberrant RET stabilization as a likely mechanism through which TMEM127 loss-of-function mutations cause pheochromocytoma.
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Neoplasias de las Glándulas Suprarrenales , Feocromocitoma , Humanos , Animales , Ratones , Feocromocitoma/genética , Feocromocitoma/metabolismo , Feocromocitoma/patología , Mutación de Línea Germinal , Neoplasias de las Glándulas Suprarrenales/genética , Neoplasias de las Glándulas Suprarrenales/metabolismo , Neoplasias de las Glándulas Suprarrenales/patología , Mutación/genética , Ubiquitinación , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Proto-Oncogénicas c-ret/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismoRESUMEN
Protein translocation to the cell membrane and transport through intracellular compartments are dynamic processes frequently altered in cancer cells. Abnormal protein localization can affect key cell functions, including transduction of extracellular signals and organization of the cytoskeleton, significantly affecting oncogenicity and therapeutic responses. In this chapter, we describe a surface protein biotinylation method that allows the study of membrane localization and endosomal transport of membrane-associated proteins. Surface biotinylation can be used to evaluate baseline protein levels at the membrane, and other processes such as internalization, recycling, and degradation of proteins in response to different treatments or as a consequence of oncogenic mutations. Further, the combination of this technique with other strategies, such as treatments with transport inhibitors, allows investigation of specific steps of protein trafficking through the cell.
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Citoesqueleto , Endosomas , Transporte Biológico , Biotinilación , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Endosomas/metabolismo , Transporte de ProteínasAsunto(s)
Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-ret/antagonistas & inhibidores , Antineoplásicos/farmacología , Humanos , Terapia Molecular Dirigida , Neoplasias/enzimología , Medicina de Precisión , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-ret/metabolismoRESUMEN
Internalization and intracellular trafficking of membrane proteins are now recognized as essential mechanisms that contribute to a number of cellular processes. Current methods lack the ability to specifically label the plasma membrane of a live cell, follow internalization of labeled membrane molecules, and conclusively differentiate newly formed membrane-derived vesicles from pre-existing endocytic or secretory structures in the cytoplasm. Here, we detail a visualization method for surface biotinylation of plasma membrane-derived vesicles that allows us to follow their progress from membrane to cytosol at specific time points. Using the transmembrane receptor RET as a model, we demonstrate how this method can be applied to identify plasma membrane-derived vesicle maturation, determine RET's presence within these structures, and monitor RET's recycling to the cell surface. This method improves on static and less discriminatory methods, providing a tool for analysis of real-time vesicle trafficking that is applicable to many systems.
Asunto(s)
Membrana Celular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Proteínas de la Membrana/metabolismo , Imagen Molecular/métodos , Biotinilación , Citosol/metabolismo , Endocitosis , Células HeLa , Humanos , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-ret/metabolismo , Vesículas Secretoras/metabolismo , Coloración y EtiquetadoRESUMEN
The inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2) is caused by mutations of the RET receptor tyrosine kinase and is characterized by medullary thyroid carcinoma. MEN 2 subtypes have distinct mutational spectrums and vary in severity. The most severe disease subtype, MEN 2B, is associated with a specific RET mutation (M918T) that has been predicted to alter downstream signaling and target gene expression patterns. We used gene expression microarray analysis to identify target genes modulated by RET. We compared two oncogenic RET mutants, associated with MEN 2A (2ARET) or MEN 2B (2BRET) disease subtypes, that are predicted to have distinct downstream target genes. We showed that overall, 2ARET and 2BRET modulated genes with similar functional ontologies. Further, when we validated our microarray data by quantitative real time PCR, we did not detect major differences in gene expression associated with these mutants when differences in receptor activity levels were considered. We did, however, detect differences in gene expression induced by two RET COOH-terminal isoforms, RET9 and RET51, irrespective of the RET form present (wildtype, 2ARET, or 2BRET). Our data suggest that similar transcriptional programs contribute to all forms of MEN 2 but that differences in target gene expression may contribute to developmental pattern differences observed between RET isoforms.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasia Endocrina Múltiple Tipo 2a/genética , Neoplasia Endocrina Múltiple Tipo 2b/genética , Proteínas Proto-Oncogénicas c-ret/genética , Humanos , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-ret/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal , Neoplasias de la Tiroides/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a therapeutically challenging disease with poor survival rates, owing to late diagnosis and early dissemination. These tumors frequently undergo perineural invasion, spreading along nerves regionally and to distant sites. The RET receptor tyrosine kinase is implicated in increased aggressiveness, local invasion, and metastasis in multiple cancers, including PDAC. RET mediates directional motility and invasion towards sources of its neurotrophic factor ligands, suggesting that it may enhance perineural invasion of tumor cells towards nerves. RET is expressed as two main isoforms, RET9 and RET51, which differ in their protein interactions and oncogenic potentials, however, the contributions of RET isoforms to neural invasion have not been investigated. In this study, we generated total RET and isoform-specific knockdown PDAC cell lines and assessed the contributions of RET isoforms to PDAC invasive spread. Our data show that RET activity induces cell polarization and actin remodeling through activation of CDC42 and RHOA GTPases to promote directional motility in PDAC cells. Further, we show that RET interacts with the adaptor protein TKS5 to induce invadopodia formation, enhance matrix degradation and promote tumor cell invasion through a SRC and GRB2-dependent mechanism. Finally, we show that RET51 is the predominant isoform contributing to these RET-mediated invasive processes in PDAC. Together, our work suggests that RET expression in pancreatic cancers may enhance tumor aggressiveness by promoting perineural invasion, and that RET expression may be a valuable marker of invasiveness, and a potential therapeutic target in the treatment of these cancers.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas c-ret/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Línea Celular Tumoral , Movimiento Celular , Polaridad Celular , Proliferación Celular , Técnicas de Cocultivo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Invasividad Neoplásica/patología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-ret/genética , Esferoides Celulares , Proteínas de Unión al GTP rho/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
OBJECTIVES: Increased expression of REarranged during Transfection (RET) kinase is reported in 10-20 % of lung adenocarcinomas (LUAD) and is associated with metastasis and reduced survival. Ezrin is a scaffold protein that promotes protein interactions with the actin cytoskeleton to regulate cell migration and is also associated with invasion and metastasis in cancers. RET isoforms interact with unique combinations of scaffold proteins to promote distinct signaling pathways. We hypothesized that RET isoforms associate distinctly with Ezrin for cytoskeletal reorganization and LUAD cell migration processes. METHODS: HCC1833 and A549 LUAD, SH-SY5Y neuroblastoma or HEK-293 cells expressing RET and Ezrin were stimulated with the RET ligand glial cell line-derived neurotrophic factor (GDNF) and treated with RET, Ezrin or Src inhibitors. Co-immunoprecipitation or pull-down assays coupled to immunoblotting were used to investigate protein activation and interactions. Immunofluorescence confocal microscopy assessed LUAD cytoskeletal reorganization and colocalization of RET and Ezrin. Live-cell fluorescence imaging was used to measure cell migration and chemotaxis. RESULTS: GDNF promoted activation, interaction and colocalization of RET51 isoform and Ezrin. Inhibition of RET or Src impaired Ezrin interactions with RET and Src. GDNF stimulation enhanced the formation of actin-rich filopodia, in which both RET and Ezrin were enriched, and promoted chemotaxis in LUAD cells. However, inhibition of RET, Src or Ezrin suppressed filopodia formation, reduced colocalization of Ezrin with RET, and impaired cell migration and/ or chemotaxis. We further showed that GDNF-mediated activation of RET and Ezrin promoted RhoA-GTPase activity and signaling of ROCK1 and ROCK2 in LUAD cells. CONCLUSIONS: Expression and activation of RET51 mediates unique protein interactions with Ezrin to promote LUAD cell chemotaxis for cancer cell dissemination, which may have implications in LUAD metastatic progression.
Asunto(s)
Adenocarcinoma del Pulmón/patología , Movimiento Celular , Quimiotaxis , Proteínas del Citoesqueleto/metabolismo , Neoplasias Pulmonares/patología , Neuroblastoma/patología , Proteínas Proto-Oncogénicas c-ret/metabolismo , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/metabolismo , Apoptosis , Proliferación Celular , Proteínas del Citoesqueleto/genética , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas , Proteínas Proto-Oncogénicas c-ret/genética , Células Tumorales CultivadasRESUMEN
The Hippo pathway has emerged as a key signaling pathway that regulates various biological functions. Dysregulation of the Hippo pathway has been implicated in a broad range of human cancer types. While a number of stimuli affecting the Hippo pathway have been reported, its upstream kinase and extracellular regulators remain largely unknown. Here we performed the first comprehensive gain-of-functional screen for receptor tyrosine kinases (RTKs) regulating the Hippo pathway using an RTK overexpression library and a Hippo signaling activity biosensor. Surprisingly, we found that the majority of RTKs could regulate the Hippo signaling activity. We further characterized several of these novel relationships [TAM family members (TYRO3, AXL, METRK), RET, and FGFR family members (FGFR1 and FGFR2)] and found that the Hippo effectors YAP/TAZ are central mediators of the tumorigenic phenotypes (e.g., increased cell proliferation, transformation, increased cell motility, and angiogenesis) induced by these RTKs and their extracellular ligands (Gas6, GDNF, and FGF) through either PI3K or MAPK signaling pathway. Significantly, we identify FGFR, RET, and MERTK as the first RTKs that can directly interact with and phosphorylate YAP/TAZ at multiple tyrosine residues independent of upstream Hippo signaling, thereby activating their functions in tumorigenesis. In conclusion, we have identified several novel kinases and extracellular stimuli regulating the Hippo pathway. Our findings also highlight the pivotal role of the Hippo pathway in mediating Gas6/GDNF/FGF-TAM/RET/FGFR-MAPK/PI3K signaling during tumorigenesis and provide a compelling rationale for targeting YAP/TAZ in RTK-driven cancers.
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Carcinogénesis/genética , Mutación con Ganancia de Función/genética , Neoplasias/genética , Proteínas Serina-Treonina Quinasas/genética , Aciltransferasas , Animales , Técnicas Biosensibles/métodos , Proliferación Celular/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Vía de Señalización Hippo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Neoplasias/patología , Fosfatidilinositol 3-Quinasas/genética , Fosfoproteínas/genética , Fosforilación , Transducción de Señal/genética , Factores de Transcripción/genéticaRESUMEN
The RET receptor tyrosine kinase plays important roles in regulating cellular proliferation, migration, and survival in the normal development of neural crest derived tissues. However, aberrant activation of RET, through oncogenic mutations or overexpression, can contribute to tumourigenesis, regional invasion, and metastasis of several human cancers. RET is expressed as two main isoforms with unique C-terminal sequences that differ in protein interactions and subcellular trafficking in response to RET activation, and which also have distinct oncogenic potentials. The long isoform, termed RET51, is internalized from the membrane in response to stimulation by its ligand, GDNF, but is known to recycle back to the surface via RAB11 endosomes. However, the mechanisms regulating this process and its cellular effects have not been defined. Here, we show that recycling of RET51 requires a multicomponent complex that includes the endosomal-sorting protein GGA3, which mediates GDNF-dependent slow recycling of RET51 receptors to the plasma membrane. Our data show that the GRB2 adapter associates with RET51 through interactions with its C-terminal sequences, facilitating recruitment of active ARF6 and GGA3 interaction, and that depletion of GGA3 or ARF6 reduced RET51 recycling. Further, GGA3 knockdown accelerated RET51 degradation and also attenuated RET-mediated AKT activation. Finally, we showed that recycling of RET51 to the cell surface through association with GGA3 and ARF6 contributes to RET51-dependent cell motility, migration, and invasion. Our data establish RET recycling as a mechanism coordinating location and duration of RET signals in order to direct cell movement and invasion.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Biomarcadores de Tumor/metabolismo , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Neuroblastoma/patología , Proteínas Proto-Oncogénicas c-ret/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Apoptosis , Biomarcadores de Tumor/genética , Proliferación Celular , Humanos , Invasividad Neoplásica , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas Proto-Oncogénicas c-ret/genética , Células Tumorales CultivadasRESUMEN
Multiple endocrine neoplasia 2B (MEN 2B) is an inherited syndrome of early onset endocrine tumors and developmental anomalies. The disease is caused primarily by a methionine to threonine substitution of residue 918 in the kinase domain of the RET receptor (2B-RET); however, the molecular mechanisms that lead to the disease phenotype are unclear. In this study, we show that the M918T mutation causes a 10-fold increase in ATP binding affinity and leads to a more stable receptor-ATP complex, relative to the wild-type receptor. Further, the M918T mutation alters local protein conformation, correlating with a partial loss of RET kinase autoinhibition. Finally, we show that 2B-RET can dimerize and become autophosphorylated in the absence of ligand stimulation. Our data suggest that multiple distinct but complementary molecular mechanisms underlie the MEN 2B phenotype and provide potential targets for effective therapeutics for this disease.
Asunto(s)
Neoplasia Endocrina Múltiple Tipo 2b/genética , Neoplasia Endocrina Múltiple Tipo 2b/metabolismo , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Proto-Oncogénicas c-ret/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Dimerización , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Neoplasia Endocrina Múltiple Tipo 2b/enzimología , Oncogenes , Fosforilación , Conformación Proteica , Proteínas Proto-Oncogénicas c-ret/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-ret/química , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
The Glial cell line-derived neurotrophic Family Ligands (GFL) are soluble neurotrophic factors that are required for development of multiple human tissues, but which are also important contributors to human cancers. GFL signaling occurs through the transmembrane RET receptor tyrosine kinase, a well-characterized oncogene. GFL-independent RET activation, through rearrangement or point mutations occurs in thyroid and lung cancers. However, GFL-mediated activation of wildtype RET is an increasingly recognized mechanism promoting tumor growth and dissemination of a much broader group of cancers. RET and GFL expression have been implicated in metastasis or invasion in diverse human cancers including breast, pancreatic, and prostate tumors, where they are linked to poorer patient prognosis. In addition to directly inducing tumor growth in these diseases, GFL-RET signaling promotes changes in the tumor microenvironment that alter the surrounding stroma and cellular composition to enhance tumor invasion and metastasis. As such, GFL RET signaling is an important target for novel therapeutic approaches to limit tumor growth and spread and improve disease outcomes.