RESUMEN
Aberrant promoter DNA hypermethylation is a hallmark of cancer; however, whether this is sufficient to drive cellular transformation is not clear. To investigate this question, we use a CRISPR-dCas9 epigenetic editing tool, where an inactive form of Cas9 is fused to DNA methyltransferase effectors. Using this system, here we show simultaneous de novo DNA methylation of genes commonly methylated in cancer, CDKN2A, RASSF1, HIC1 and PTEN in primary breast cells isolated from healthy human breast tissue. We find that promoter methylation is maintained in this system, even in the absence of the fusion construct, and this prevents cells from engaging senescence arrest. Our data show that the key driver of this phenotype is repression of CDKN2A transcript p16 where myoepithelial cells harbour cancer-like gene expression but do not exhibit anchorage-independent growth. This work demonstrates that hit-and-run epigenetic events can prevent senescence entry, which may facilitate tumour initiation.
Asunto(s)
Mama/citología , Transformación Celular Neoplásica/genética , Senescencia Celular/genética , Metilación de ADN/genética , Edición Génica/métodos , Mama/metabolismo , Sistemas CRISPR-Cas/genética , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Metiltransferasa 3A , Epigenómica , Femenino , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Fosfohidrolasa PTEN/genética , Regiones Promotoras Genéticas/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
The Hippo tumor suppressor pathway is essential for development and tissue growth control, encompassing a core cassette consisting of the Hippo (MST1/2), Warts (LATS1/2), and Tricornered (NDR1/2) kinases together with MOB1 as an important signaling adaptor. However, it remains unclear which regulatory interactions between MOB1 and the different Hippo core kinases coordinate development, tissue growth, and tumor suppression. Here, we report the crystal structure of the MOB1/NDR2 complex and define key MOB1 residues mediating MOB1's differential binding to Hippo core kinases, thereby establishing MOB1 variants with selective loss-of-interaction. By studying these variants in human cancer cells and Drosophila, we uncovered that MOB1/Warts binding is essential for tumor suppression, tissue growth control, and development, while stable MOB1/Hippo binding is dispensable and MOB1/Trc binding alone is insufficient. Collectively, we decrypt molecularly, cell biologically, and genetically the importance of the diverse interactions of Hippo core kinases with the pivotal MOB1 signal transducer.The Hippo tumor suppressor pathway is essential for development and tissue growth control. Here the authors employ a multi-disciplinary approach to characterize the interactions of the three Hippo kinases with the signaling adaptor MOB1 and show how they differently affect development, tissue growth and tumor suppression.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Animales Modificados Genéticamente , Línea Celular , Línea Celular Tumoral , Drosophila melanogaster/genética , Vía de Señalización Hippo , Humanos , Quinasas Quinasa Quinasa PAM/genética , Modelos Moleculares , Conformación Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
By controlling the YAP1 proto-oncoprotein Hippo signalling plays important roles in cancer-associated processes. Current evidence suggests that the Hippo kinases MST1/2 together with the MOB1 scaffold protein promote the formation of active MOB1/LATS complexes which phosphorylate and thereby inhibit YAP1. However, the regulatory mechanisms of MST1/2-MOB1-LATS signalling are currently underinvestigated. Therefore, we studied LATS2 variants carrying specific modifications that mimic gain or loss of phosphorylation and/or abolish MOB1/LATS2 interactions. We discovered that Ser872 T-loop and Thr1041 hydrophobic motif (HM) phosphorylation of LATS2 is essential for LATS2 activation. MST1/2 phosphorylate LATS2 on Thr1041, but not Ser872, while MOB1 binding to LATS2 supports both phosphorylation events. Significantly, LATS2-PIF, a LATS2 variant containing the PRK2 HM, acts as a hyperactive LATS2 kinase that efficiently phosphorylates YAP1 and inhibits the transcriptional co-activity of YAP1. This inhibitory function of LATS2-PIF is dependent on LATS2 kinase activity, while MOB1/LATS2 and YAP1/LATS2 complex formation is dispensable, suggesting that elevated LATS2 kinase activity can be sufficient to oppose YAP1. Taken together, our characterisation of LATS2 variants uncovers novel insights into the regulation of LATS kinases in Hippo signalling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Mutación , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Serina-Treonina Quinasa 3 , Transducción de Señal , Factores de Transcripción , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Señalizadoras YAPRESUMEN
Inactivation of the tumor suppressor gene RASSF1A by promoter hypermethylation represents a key event underlying the initiation and progression of lung cancer. RASSF1A inactivation is also associated with poor prognosis and may promote metastatic spread. In this study, we investigated how RASSF1A inactivation conferred invasive phenotypes to human bronchial cells. RNAi-mediated silencing of RASSF1A induced epithelial-to-mesenchymal transition (EMT), fomenting a motile and invasive cellular phenotype in vitro and increased metastatic prowess in vivo. Mechanistic investigations revealed that RASSF1A blocked tumor growth by stimulating cofilin/PP2A-mediated dephosphorylation of the guanine nucleotide exchange factor GEF-H1, thereby stimulating its ability to activate the antimetastatic small GTPase RhoB. Furthermore, RASSF1A reduced nuclear accumulation of the Hippo pathway transcriptional cofactor Yes-associated protein (YAP), which was reinforced by RhoB activation. Collectively, our results indicated that RASSF1 acts to restrict EMT and invasion by indirectly controlling YAP nuclear shuttling and activation through a RhoB-regulated cytoskeletal remodeling process, with potential implications to delay the progression of RASSF1-hypermethylated lung tumors.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Invasividad Neoplásica/genética , Metástasis de la Neoplasia/genética , Fosfoproteínas/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Proteínas Supresoras de Tumor/genética , Proteína de Unión al GTP rhoB/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Metilación de ADN/genética , Regulación Neoplásica de la Expresión Génica/genética , Silenciador del Gen/fisiología , Genes Supresores de Tumor/fisiología , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Invasividad Neoplásica/patología , Metástasis de la Neoplasia/patología , Regiones Promotoras Genéticas/genética , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
Mps one binder proteins (MOBs) are conserved regulators of essential signalling pathways. Biochemically, human MOB2 (hMOB2) can inhibit NDR kinases by competing with hMOB1 for binding to NDRs. However, biological roles of hMOB2 have remained enigmatic. Here, we describe novel functions of hMOB2 in the DNA damage response (DDR) and cell cycle regulation. hMOB2 promotes DDR signalling, cell survival and cell cycle arrest after exogenously induced DNA damage. Under normal growth conditions in the absence of exogenously induced DNA damage hMOB2 plays a role in preventing the accumulation of endogenous DNA damage and a subsequent p53/p21-dependent G1/S cell cycle arrest. Unexpectedly, these molecular and cellular phenotypes are not observed upon NDR manipulations, indicating that hMOB2 performs these functions independent of NDR signalling. Thus, to gain mechanistic insight, we screened for novel binding partners of hMOB2, revealing that hMOB2 interacts with RAD50, facilitating the recruitment of the MRE11-RAD50-NBS1 (MRN) DNA damage sensor complex and activated ATM to DNA damaged chromatin. Taken together, we conclude that hMOB2 supports the DDR and cell cycle progression.
Asunto(s)
Daño del ADN , Proteínas del Tejido Nervioso/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Células COS , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Chlorocebus aethiops , Proteínas Cromosómicas no Histona/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Doxorrubicina/farmacología , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de la radiación , Humanos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , ARN Interferente Pequeño/metabolismo , Radiación Ionizante , Puntos de Control de la Fase S del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase S del Ciclo Celular/efectos de la radiación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Autophagy plays key roles in development, oncogenesis, cardiovascular, metabolic, and neurodegenerative diseases. Hence, understanding how autophagy is regulated can reveal opportunities to modify autophagy in a disease-relevant manner. Ideally, one would want to functionally define autophagy regulators whose enzymatic activity can potentially be modulated. Here, we describe the STK38 protein kinase (also termed NDR1) as a conserved regulator of autophagy. Using STK38 as bait in yeast-two-hybrid screens, we discovered STK38 as a novel binding partner of Beclin1, a key regulator of autophagy. By combining molecular, cell biological, and genetic approaches, we show that STK38 promotes autophagosome formation in human cells and in Drosophila. Upon autophagy induction, STK38-depleted cells display impaired LC3B-II conversion; reduced ATG14L, ATG12, and WIPI-1 puncta formation; and significantly decreased Vps34 activity, as judged by PI3P formation. Furthermore, we observed that STK38 supports the interaction of the exocyst component Exo84 with Beclin1 and RalB, which is required to initiate autophagosome formation. Upon studying the activation of STK38 during autophagy induction, we found that STK38 is stimulated in a MOB1- and exocyst-dependent manner. In contrast, RalB depletion triggers hyperactivation of STK38, resulting in STK38-dependent apoptosis under prolonged autophagy conditions. Together, our data establish STK38 as a conserved regulator of autophagy in human cells and flies. We also provide evidence demonstrating that STK38 and RalB assist the coordination between autophagic and apoptotic events upon autophagy induction, hence further proposing a role for STK38 in determining cellular fate in response to autophagic conditions.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/fisiología , Proteínas de la Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Apoptosis/fisiología , Beclina-1 , Línea Celular Tumoral , Células Cultivadas , Drosophila , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación , Unión Proteica , Técnicas del Sistema de Dos HíbridosRESUMEN
The human MST1/hMOB1/NDR1 tumour suppressor cascade regulates important cellular processes, such as centrosome duplication. hMOB1/NDR1 complex formation appears to be essential for NDR1 activation by autophosphorylation on Ser281 and hydrophobic motif (HM) phosphorylation at Thr444 by MST1. To dissect these mechanistic relationships in MST1/hMOB1/NDR signalling, we designed NDR1 variants carrying modifications that mimic HM phosphorylation and/or abolish hMOB1/NDR1 interactions. Significantly, the analyses of these variants revealed that NDR1-PIF, an NDR1 variant containing the PRK2 hydrophobic motif, remains hyperactive independent of hMOB1/NDR1-PIF complex formation. In contrast, as reported for the T444A phospho-acceptor mutant, NDR1 versions carrying single phospho-mimicking mutations at the HM phosphorylation site, namely T444D or T444E, do not display increased kinase activities. Collectively, these observations suggest that in cells Thr444 phosphorylation by MST1 depends on the hMOB1/NDR1 association, while Ser281 autophosphorylation of NDR1 can occur independently. By testing centrosome-targeted NDR1 variants in NDR1- or MST1-depleted cells, we further observed that centrosome-enriched NDR1-PIF requires neither hMOB1 binding nor MST1 signalling to function in centrosome overduplication. Taken together, our biochemical and cell biological characterisation of NDR1 versions provides novel unexpected insights into the regulatory mechanisms of NDR1 and NDR1's role in centrosome duplication.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteoglicanos/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Células COS , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular , Centrosoma/metabolismo , Chlorocebus aethiops , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
The RecQ4 protein shows homology to both the S.cerevisiae DNA replication protein Sld2 and the DNA repair related RecQ helicases. Experimental data also suggest replication and repair functions for RecQ4, but the precise details of its involvement remain to be clarified.Here we show that depletion of DmRecQ4 by dsRNA interference in S2 cells causes defects consistent with a replication function for the protein. The cells show reduced proliferation associated with an S phase block, reduced BrdU incorporation, and an increase in cells with a subG1 DNA content. At the molecular level we observe reduced chromatin association of DNA polymerase-alpha and PCNA. We also observe increased chromatin association of phosphorylated H2AvD--consistent with the presence of DNA damage and increased apoptosis.Analysis of DmRecQ4 repair function suggests a direct role in NER, as the protein shows rapid but transient nuclear localisation after UV treatment. Re-localisation is not observed after etoposide or H2O2 treatment, indicating that the involvement of DmRecQ4 in repair is likely to be pathway specific.Deletion analysis of DmRecQ4 suggests that the SLD2 domain was essential, but not sufficient, for replication function. In addition a DmRecQ4 N-terminal deletion could efficiently re-localise on UV treatment, suggesting that the determinants for this response are contained in the C terminus of the protein. Finally several deletions show differential rescue of dsRNA generated replication and proliferation phenotypes. These will be useful for a molecular analysis of the specific role of DmRecQ4 in different cellular pathways.