EGFR feedback-inhibition by Ran-binding protein 6 is disrupted in cancer

Transport of macromolecules through the nuclear pore by importins and exportins plays a critical role in the spatial regulation of protein activity. How cancer cells co-opt this process to promote tumorigenesis remains unclear. The epidermal growth factor receptor (EGFR) plays a critical role in normal development and in human cancer. Here we describe a mechanism of EGFR regulation through the importin β family member RAN-binding protein 6 (RanBP6), a protein of hitherto unknown functions. We show that RanBP6 silencing impairs nuclear translocation of signal transducer and activator of transcription 3 (STAT3), reduces STAT3 binding to the EGFR promoter, results in transcriptional derepression of EGFR, and increased EGFR pathway output. Focal deletions of the RanBP6 locus on chromosome 9p were found in a subset of glioblastoma (GBM) and silencing of RanBP6 promoted glioma growth in vivo. Our results provide an example of EGFR deregulation in cancer through silencing of components of the nuclear import pathway.This research was supported by the National Brain Tumor Society (I.K.M.), the National Institutes of Health grants 1R01NS080944-01 (I.K.M.), 1 R35 NS105109 01 (I.K.M.), and P30CA008748 (MSKCC Core Grant), the Geoffrey Beene Cancer Research Foundation (I.K.M.), the Cycle of Survival (I.K.M.), and the Seve Ballesteros Foundation (M.S.). B.O. was supported by an American–Italian Cancer Foundation fellowship and a MSKCC Brain Tumor Center grant. W.-Y.H. is the recipient of a FY15 Horizon Award from the U.S. Department of Defense (W81XWH-15-PRCRP-HA). A.C.-G. is the recipient of the Severo-Ochoa PhD fellowship. Further support was provided by the Sontag Foundation (B.S.T.). We thank all members of the Mellinghoff laboratory for helpful suggestions. We thank Dr. Fiona Ginty (Diagnostic Imaging and Biomedical Technologies, GE Global Research Center, Niskayuna, New York, USA) for assistance with multiplexed immunofluorescence. We thank A.J. Schuhmacher and C.S. Clemente-Troncone for assistance with the in vivo experiments, M. Kaufmann for assistance in the luciferase assays and N. Yannuzzi for assistance in cloning.S

Understanding the mechanisms of chemoresistance and modeling tumor growth in glioblastoma

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 22-03-2019Esta tesis tiene embargado el acceso al texto completo hasta el 22-09-2020Glioblastoma is the most common and malignant brain tumor. Standard of care for GBM includes resection of the tumor mass, followed by concurrent radiotherapy and chemotherapy with the alkylating agent Temozolomide (TMZ) However, TMZ is not effective in long-term and patients develop resistance and, consequently, recurrence in the disease. In order to identify genes that modulate TMZ resistance, we have performed a forward genetic screen using the PiggyBac transposon system for an insertional mutagenesis screen in human haploid cells (Hap1). We were able to identify and validate the mismatch repair (MMR) component MSH6, which is known to be related to resistance to TMZ. MSH6 inactivating mutations are found in 20% of the patients resulting in chemotherapy failure. We then performed a compound screening with 120 drugs either FDA-approved or under clinical trials looking for MMR synthetic lethality. After a pilot study, we were not able to validate any candidate compound and further experiments will be needed in order to identify possible drugs that sensitize the MMR-deficient cells. Drug resistance and the ability of the drug to cross the blood brain barrier are the main limitations of treating GBM patients. It is also important the development and/or testing of new compounds that prolong the survival. We have tested the efficacy of a byalkykating agent Val-083, currently in clinical trials. Preliminary data show better efficacy as compared to TMZ in GBM cells (even in those cells that are resistant to TMZ). Moreover, we observed an additive effect in combination with TMZ both in vitro and ex vivo. Finally, the identification of the molecular alterations that occur in GBM opens a new window for the study of the disease as well as for the validation of novel therapies. Consequently, animal models will be needed to recreate these alterations that occur in patients. One of the most widely mouse model used for the study of gliomas is the RCAS-TVA-based somatic gene transfer system. We have developed a new RCAS/Tva-CRISPR/Cas9 mouse model combining the advantages (feasible, versatile, resemble human GBM features) of the RCAS/Tva system and the genome editing capacity of the CRISPR/Cas9. We have been able to recreate, in a time-controlled manner, different genetic alterations of tumor suppressor genes (TSGs) that have a known role in gliomagenesis. As a result, we have developed a powerful tool that can recapitulate molecular features found in GBM patients.El glioblastoma (GBM) es tumor cerebral más común y maligno. Su tratamiento incluye cirugía, radio y quimioterapia utilizando un agente alquilante llamado Temozolomida (TMZ). Sin embargo, la TMZ muestra una baja eficiencia a largo plazo y los pacientes desarrollan resistencia y un relapso de la enfermedad. Para poder identificar genes que puedan estar modulando la respuesta a TMZ, realizamos un “screening” genético mediante el sistema de transposones “PiggyBac” en células haploides humanas. Validamos el gen MSH6, un gen relacionado con la ruta de reparación de los errores de apareamiento de bases durante la replicación del ADN (genes MMR). Es bien conocido el papel de MSH6 con la resistencia a TMZ. Se ha visto que los genes MMR se encuentran alterados en un 20% de los pacientes con GBM que desarrollaron resistencia a TMZ, indicando la importancia de desarrollar terapias alternativas a las actuales. Realizamos otro “screening” con 120 compuestos aprobados por la FDA o en ensayos clínicos en busca de letalidad sintética con células deficientes de la vía MMR. Estudios preliminares mostraron que ningún compuesto candidato era más eficaz en estas células y nuevos experimentos serán necesarios para poder identificar compuestos que generen letalidad sintética en estás células. La resistencia al tratamiento convencional y la capacidad de los compuestos en atravesar la barrera hematoencefálica son factores limitantes para los pacientes con GBM. Uno de los objetivos actuales es el desarrollo de nuevos compuestos que prologuen su supervivencia. Así, hemos testado la eficacia de otro agente alquilante, denominado Val-083, observando mayor eficacia que la TMZ en células de GBM, y un efecto sinérgico en combinación con TMZ in vivo y ex vivo. La identificación de alteraciones moleculares en gliomas abre nueva ventana en el estudio de esta enfermedad. Así, modelos animales precisos que recreen las alteraciones descritas en pacientes se antojan necesarios. Uno de los modelos murinos más usados para el estudio de gliomas es el sistema de transferencia génica RCAS/Tva. Desarrollamos un nuevo modelo RCAS/Tva-CRISPR/Cas9 que combina las ventajas del sistema RCAS/Tva (versátil, recapitulación de las características de los GBM humanos) con el sistema de edición génica CRISPR/Cas9. Generamos gliomas deficientes en genes supresores de tumores que se sabe tienen un papel relevante en el desarrollo de gliomas. De esta forma, tenemos una potente herramienta que nos puede permitir recrear de manera eficaz diferentes alteraciones encontradas en pacientes con GBM.This work was supported by the following grant: - Severo Ochoa PhD Fellowship by the Ministry of Economy and Competitiveness 201