RESUMEN
Upregulation of the ERK1 and ERK2 (ERK1/2) MAP kinase (MAPK) cascade occurs in >30% of cancers, often through mutational activation of receptor tyrosine kinases or other upstream genes, including KRAS and BRAF. Efforts to target endogenous MAPKs are challenged by the fact that these kinases are required for viability in mammals. Additionally, the effectiveness of new inhibitors of mutant BRAF has been diminished by acquired tumor resistance through selection for BRAF-independent mechanisms of ERK1/2 induction. Furthermore, recently identified ERK1/2-inducing mutations in MEK1 and MEK2 (MEK1/2) MAPK genes in melanoma confer resistance to emerging therapeutic MEK inhibitors, underscoring the challenges facing direct kinase inhibition in cancer. MAPK scaffolds, such as IQ motif-containing GTPase activating protein 1 (IQGAP1), assemble pathway kinases to affect signal transmission, and disrupting scaffold function therefore offers an orthogonal approach to MAPK cascade inhibition. Consistent with this, we found a requirement for IQGAP1 in RAS-driven tumorigenesis in mouse and human tissue. In addition, the ERK1/2-binding IQGAP1 WW domain peptide disrupted IQGAP1-ERK1/2 interactions, inhibited RAS- and RAF-driven tumorigenesis, bypassed acquired resistance to the BRAF inhibitor vemurafenib (PLX-4032) and acted as a systemically deliverable therapeutic to significantly increase the lifespan of tumor-bearing mice. Scaffold-kinase interaction blockade acts by a mechanism distinct from direct kinase inhibition and may be a strategy to target overactive oncogenic kinase cascades in cancer.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Neoplasias/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas ras/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Análisis Mutacional de ADN , Resistencia a Antineoplásicos , Humanos , Indoles/uso terapéutico , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Mutación , Trasplante de Neoplasias , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas B-raf/genética , Homología de Secuencia de Aminoácido , Sulfonamidas/uso terapéutico , Vemurafenib , Cicatrización de HeridasRESUMEN
More than 100 human genetic skin diseases, impacting over 20% of the population, are characterized by disrupted epidermal differentiation. A significant proportion of the 90 genes identified in these disorders to date are concentrated within several functional pathways, suggesting the emergence of organizing themes in epidermal differentiation. Among these are the Notch, transforming growth factor ß (TGFß), IκB kinase (IKK), Ras/mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), p63, and Wnt signaling pathways, as well as core biological processes mediating calcium homeostasis, tissue integrity, cornification, and lipid biogenesis. Here, we review recent results supporting the central role of these pathways in epidermal differentiation, highlighting the integration of genetic information with functional studies to illuminate the biological actions of these pathways in humans as well as to guide development of future therapeutics to correct their dysfunction.
Asunto(s)
Diferenciación Celular/genética , Epidermis/fisiología , Transducción de Señal/genética , Enfermedades de la Piel/genética , Enfermedades de la Piel/fisiopatología , Animales , Epidermis/metabolismo , Redes Reguladoras de Genes/fisiología , Humanos , Modelos Biológicos , Enfermedades de la Piel/etiologíaRESUMEN
Regions of the budding yeast G1 cyclin Cln3 were characterized using mutational analysis and viability assays to identify functionally relevant and novel mutant alleles of CLN3. Cyclin proteins are conserved, and Cln3 contains a region with homology to the cyclin box, which is thought to mediate physical interactions with the cyclin-dependent kinase. CLN3 was found to have characteristics similar to the conserved cyclin fold found in higher eukaryotic cyclin boxes, which consist of five alpha-helices. Peptide linker sequences inserted within helices 1, 2, 3 and 5 resulted in a loss of Cln3 function, showing cyclin fold structure similar to that previously observed for the G1 cyclin Cln2. A clustered-charge-to-alanine scan mutagenesis revealed two regions of Cln3 important for Cln3-dependent viability. The first region encompasses the conserved cyclin box. The second region is identified with alanine substitutions located well past the cyclin box, just prior to the C-terminal region of Cln3 important for protein stability. Cln3 with mutational changes in each of these regions are expressed at steady-state levels higher than wild-type Cln3, and show some defect in binding to Cdc28. The conserved hydrophobic patch domain (HPD) of cyclins is present within the first helix of the cyclin box. Alanine substitutions introduced into the HPD of Cln3 and Cln2 show functional defects while maintaining physical interaction with Cdc28 as measured by co-immunoprecipitation assay.