RESUMEN
The evolutionarily conserved Hippo inhibitory pathway plays critical roles in tissue homeostasis and organ size control, while mutations affecting certain core components contribute to tumorigenesis. Here we demonstrate that proliferation of Hippo pathway mutant human tumor cells exhibiting high constitutive TEAD transcriptional activity was markedly inhibited by dominant negative TEAD4, which did not inhibit the growth of Hippo wild-type cells with low levels of regulatable TEAD-mediated transcription. The tankyrase inhibitor, XAV939, identified in a screen for inhibitors of TEAD transcriptional activity, phenocopied these effects independently of its other known functions by stabilizing angiomotin and sequestering YAP in the cytosol. We also identified one intrinsically XAV939 resistant Hippo mutant tumor line exhibiting lower and less durable angiomotin stabilization. Thus, angiomotin stabilization provides a new mechanism for targeting tumors with mutations in Hippo pathway core components as well as a biomarker for sensitivity to such therapy.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica/fisiología , Compuestos Heterocíclicos con 3 Anillos/farmacología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Factores de Transcripción/metabolismo , Angiomotinas , Línea Celular Tumoral , Proliferación Celular/fisiología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Vía de Señalización Hippo , Humanos , Proteínas de Microfilamentos , Mutación , Factores de Transcripción de Dominio TEA , Tanquirasas/antagonistas & inhibidoresRESUMEN
Loss of primary cilia is a key feature of von Hippel-Lindau tumor suppressor (VHL)-associated pathology. Although VHL-deficiency predisposes cells to precipitous cilia disassembly in response to growth factor cues, it does not affect ciliogenesis. Here, using a siRNA-based screen to find genes that are essential for ciliogenesis only in the presence of the VHL tumor suppressor gene product pVHL, we identify ubiquitin-specific protease (USP)8. The pVHL-dependency of USP8 for ciliogenesis is directly linked to its function as a HIF1α deubiquitinating enzyme. By counteracting pVHL-mediated ubiquitination of HIF1α, USP8 maintains a basal expression of HIF1α and HIF transcriptional output in normoxia, including the repression of Rabaptin5, which is essential for endosome trafficking-mediated ciliogenesis.
Asunto(s)
Cilios/fisiología , Endopeptidasas/fisiología , Complejos de Clasificación Endosomal Requeridos para el Transporte/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ubiquitina Tiolesterasa/fisiología , Ubiquitinación , Animales , Hipoxia de la Célula , Endocitosis , Células HEK293 , Humanos , Ratones , Oxígeno/metabolismo , Unión Proteica , Estabilidad Proteica , Transporte de Proteínas , Proteínas de Transporte Vesicular/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
The generation of novel genes and proteins throughout evolution has been proposed to occur as a result of whole genome and gene duplications, exon shuffling, and retrotransposition events. The analysis of such genes might thus shed light into the functional complexity associated with highly evolved species. One such case is represented by TBC1D3, a primate-specific gene, harboring a TBC domain. Because TBC domains encode Rab-specific GAP activities, TBC-containing proteins are predicted to play a major role in endocytosis and intracellular traffic. Here, we show that the TBC1D3 gene originated late in evolution, likely through a duplication of the RNTRE locus, and underwent gene amplification during primate speciation. Despite possessing a TBC domain, TBC1D3 is apparently devoid of Rab-GAP activity. However, TBC1D3 regulates the optimal rate of epidermal growth factor-mediated macropinocytosis by participating in a novel pathway involving ARF6 and RAB5. In addition, TBC1D3 binds and colocalize to GGA3, an ARF6-effector, in an ARF6-dependent manner, and synergize with it in promoting macropinocytosis, suggesting that the two proteins act together in this process. Accordingly, GGA3 siRNA-mediated ablation impaired TBC1D3-induced macropinocytosis. We thus uncover a novel signaling pathway that appeared after primate speciation. Within this pathway, a TBC1D3:GGA3 complex contributes to optimal propagation of signals, ultimately facilitating the macropinocytic process.