RESUMEN
The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not entirely understood. The most prevalent post-translational modification within the GTPase core domain of NRAS and KRAS is ubiquitination at lysine 128 (K128), which is significantly decreased in cancer samples compared to normal tissue. Here, we found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAPs), NF1 and RASA1, thus increasing RAS binding to GAP proteins and promoting GAP-mediated GTP hydrolysis. Stimulation of cultured cancer cells with growth factors or cytokines transiently induces K128 ubiquitination and restricts the extent of wild-type RAS activation in a GAP-dependent manner. In KRAS mutant cells, K128 ubiquitination limits tumor growth by restricting RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Reduction of K128 ubiquitination activates both wild-type and mutant RAS signaling and elicits a senescence-associated secretory phenotype, promoting RAS-driven pancreatic tumorigenesis.
Asunto(s)
Unión Proteica , Proteínas Proto-Oncogénicas p21(ras) , Ubiquitinación , Humanos , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Animales , Proteína Activadora de GTPasa p120/metabolismo , Proteína Activadora de GTPasa p120/genética , Ratones , Línea Celular Tumoral , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/genética , Lisina/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas ras/metabolismo , Proteínas ras/genética , Neurofibromina 1RESUMEN
RATIONALE: Noonan syndrome (NS) is one of the most frequent genetic disorders. Bleeding problems are among the most common, yet poorly defined complications associated with NS. A lack of consensus on the management of bleeding complications in patients with NS indicates an urgent need for new therapeutic approaches. OBJECTIVE: Bleeding disorders have recently been described in patients with NS harboring mutations of LZTR1 (leucine zipper-like transcription regulator 1), an adaptor for CUL3 (CULLIN3) ubiquitin ligase complex. Here, we assessed the pathobiology of LZTR1-mediated bleeding disorders. METHODS AND RESULTS: Whole-body and vascular specific knockout of Lztr1 results in perinatal lethality due to cardiovascular dysfunction. Lztr1 deletion in blood vessels of adult mice leads to abnormal vascular leakage. We found that defective adherent and tight junctions in Lztr1-depleted endothelial cells are caused by dysregulation of vesicular trafficking. LZTR1 affects the dynamics of fusion and fission of recycling endosomes by controlling ubiquitination of the ESCRT-III (endosomal sorting complex required for transport III) component CHMP1B (charged multivesicular protein 1B), whereas NS-associated LZTR1 mutations diminish CHMP1B ubiquitination. LZTR1-mediated dysregulation of CHMP1B ubiquitination triggers endosomal accumulation and subsequent activation of VEGFR2 (vascular endothelial growth factor receptor 2) and decreases blood levels of soluble VEGFR2 in Lztr1 haploinsufficient mice. Inhibition of VEGFR2 activity by cediranib rescues vascular abnormalities observed in Lztr1 knockout mice Conclusions: Lztr1 deletion phenotypically overlaps with bleeding diathesis observed in patients with NS. ELISA screening of soluble VEGFR2 in the blood of LZTR1-mutated patients with NS may predict both the severity of NS phenotypes and potential responders to anti-VEGF therapy. VEGFR inhibitors could be beneficial for the treatment of bleeding disorders in patients with NS.
Asunto(s)
Vasos Sanguíneos/metabolismo , Endosomas/metabolismo , Células Endoteliales/metabolismo , Hemorragia/metabolismo , Síndrome de Noonan/metabolismo , Factores de Transcripción/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Malformaciones Vasculares/metabolismo , Animales , Vasos Sanguíneos/anomalías , Vasos Sanguíneos/efectos de los fármacos , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , Modelos Animales de Enfermedad , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/genética , Endosomas/patología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Haploinsuficiencia , Células HeLa , Hemorragia/genética , Hemorragia/patología , Hemorragia/prevención & control , Humanos , Linfocinas/genética , Linfocinas/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica , Síndrome de Noonan/tratamiento farmacológico , Síndrome de Noonan/genética , Síndrome de Noonan/patología , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas , Quinazolinas/farmacología , Transducción de Señal , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Ubiquitinación , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Malformaciones Vasculares/tratamiento farmacológico , Malformaciones Vasculares/genética , Malformaciones Vasculares/patologíaRESUMEN
Quiescence (G0) allows cycling cells to reversibly cease proliferation. A decision to enter quiescence is suspected of occurring early in G1, before the restriction point (R). Surprisingly, we have identified G2 as an interval during which inhibition of the protein phosphatase PP2A results in failure to exhibit stable quiescence. This effect is accompanied by shortening of the ensuing G1. The PP2A subcomplex required for stable G0 contains the B56γ B subunit. After PP2A inhibition in G2, aberrant overexpression of cyclin E occurs during mitosis and is responsible for overriding quiescence. Strikingly, suppression of Ras signaling re-establishes normal cyclin E levels during M and restores G0. These data point to PP2A-B56γ-driven Ras signaling modulation in G2 as essential for suppressing aberrant cyclin E expression during mitosis and thereby achieving normal G0 control. Thus, G2 is an interval during which the length and growth factor dependence of the next G1 interval are established.
Asunto(s)
Fase G1/genética , Fase G2/genética , Proteína Oncogénica p21(ras)/genética , Proteína Fosfatasa 2/genética , Fase de Descanso del Ciclo Celular/fisiología , Línea Celular , Ciclina E/biosíntesis , Humanos , Células MCF-7 , Mitosis/genética , Subunidades de Proteína/genética , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal/genéticaRESUMEN
Dysregulated splicing is a common event in cancer even in the absence of mutations in the core splicing machinery. The aberrant long non-coding transcriptome constitutes an uncharacterized level of regulation of post-transcriptional events in cancer. Here, we found that the stress-induced long non-coding RNA (lncRNA), LINC02657 or LASTR (lncRNA associated with SART3 regulation of splicing), is upregulated in hypoxic breast cancer and is essential for the growth of LASTR-positive triple-negative breast tumors. LASTR is upregulated in several types of epithelial cancers due to the activation of the stress-induced JNK/c-JUN pathway. Using a mass-spectrometry based approach, we identified the RNA-splicing factor SART3 as a LASTR-interacting partner. We found that LASTR promotes splicing efficiency by controlling SART3 association with the U4 and U6 small nuclear ribonucleoproteins (snRNP) during spliceosome recycling. Intron retention induced by LASTR depletion downregulates expression of essential genes, ultimately decreasing the fitness of cancer cells.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Neoplasias/genética , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Estrés Fisiológico , Animales , Hipoxia de la Célula , Línea Celular Tumoral , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación Neoplásica de la Expresión Génica , Genes Esenciales , Humanos , Intrones/genética , Sistema de Señalización de MAP Quinasas , Ratones Desnudos , Empalme del ARN/genética , ARN Largo no Codificante/genética , Regulación hacia Arriba/genéticaRESUMEN
Radiotherapy is one of the most used treatment approaches for head and neck squamous cell carcinoma (HNSCC). Targeted inhibition of DNA repair machinery has the potential to improve treatment response by tailoring treatment to cancer cells lacking specific DNA repair pathways. Human papillomavirus (HPV)-negative and HPV-positive HNSCCs respond differently to radiotherapy treatment, suggesting that different approaches of DNA repair inhibition should be employed for these HNSCC groups. Here, we searched for optimal radiosensitization approaches for HPV-positive and HPV-negative HNSCCs by performing a targeted CRISPR-Cas9 screen. We found that inhibition of base excision repair resulted in a better radiotherapy response in HPV-positive HNSCC, which is correlated with upregulation of genes involved in base excision repair. In contrast, inhibition of nonhomologous end-joining and mismatch repair showed strong effects in both HNSCC groups. We validated the screen results by combining radiotherapy with targeted inhibition of DNA repair in several preclinical models including primary and recurrent patient-derived HNSCC xenografts. These findings underline the importance of stratifying HNSCC patients for combination treatments.
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Neoplasias de Cabeza y Cuello/terapia , Recurrencia Local de Neoplasia/terapia , Infecciones por Papillomavirus/terapia , Fármacos Sensibilizantes a Radiaciones/administración & dosificación , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Animales , Bencimidazoles/administración & dosificación , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Quimioradioterapia/métodos , Cromonas/administración & dosificación , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Femenino , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/virología , Humanos , Masculino , Ratones , Persona de Mediana Edad , Morfolinas/administración & dosificación , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/virología , Papillomaviridae/efectos de los fármacos , Papillomaviridae/genética , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Dosificación Radioterapéutica , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/virología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The RASopathies are a group of genetic disorders that result from germline pathogenic variants affecting RAS-mitogen activated protein kinase (MAPK) pathway genes. RASopathies share RAS/MAPK pathway dysregulation and share phenotypic manifestations affecting numerous organ systems, causing lifelong and at times life-limiting medical complications. RASopathies may benefit from precision medicine approaches. For this reason, the Sixth International RASopathies Symposium focused on exploring precision medicine. This meeting brought together basic science researchers, clinicians, clinician scientists, patient advocates, and representatives from pharmaceutical companies and the National Institutes of Health. Novel RASopathy genes, variants, and animal models were discussed in the context of medication trials and drug development. Attempts to define and measure meaningful endpoints for treatment trials were discussed, as was drug availability to patients after trial completion.
Asunto(s)
Enfermedades Genéticas Congénitas/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas ras/genética , Enfermedades Genéticas Congénitas/patología , Mutación de Línea Germinal/genética , Humanos , Transducción de Señal/genéticaRESUMEN
Ubiquitination is a versatile and dynamic post-translational modification in which single ubiquitin molecules or polyubiquitin chains are attached to target proteins, giving rise to mono- or poly-ubiquitination, respectively. The majority of research in the ubiquitin field focused on degradative polyubiquitination, whereas more recent studies uncovered the role of single ubiquitin modification in important physiological processes. Monoubiquitination can modulate the stability, subcellular localization, binding properties, and activity of the target proteins. Understanding the function of monoubiquitination in normal physiology and pathology has important therapeutic implications, as alterations in the monoubiquitin pathway are found in a broad range of genetic diseases. This review highlights a link between monoubiquitin signaling and the pathogenesis of genetic disorders.
Asunto(s)
Enzimas Desubicuitinizantes/metabolismo , Enfermedades Genéticas Congénitas/metabolismo , Procesamiento Proteico-Postraduccional , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/biosíntesis , Ubiquitinación , Enfermedades Genéticas Congénitas/genética , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Estabilidad Proteica , Transporte de Proteínas , Proteolisis , Fracciones Subcelulares/metabolismo , Especificidad por SustratoRESUMEN
Protein-protein interactions (PPIs) are central in cell metabolism but research tools for the structural and functional characterization of these PPIs are often missing. Here we introduce broadly applicable immunization (Cross-link PPIs and immunize llamas, ChILL) and selection strategies (Display and co-selection, DisCO) for the discovery of diverse nanobodies that either stabilize or disrupt PPIs in a single experiment. We apply ChILL and DisCO to identify competitive, connective, or fully allosteric nanobodies that inhibit or facilitate the formation of the SOS1â¢RAS complex and modulate the nucleotide exchange rate on this pivotal GTPase in vitro as well as RAS signalling in cellulo. One of these connective nanobodies fills a cavity that was previously identified as the binding pocket for a series of therapeutic lead compounds. The long complementarity-determining region (CDR3) that penetrates this binding pocket serves as pharmacophore for extending the repertoire of potential leads.
Asunto(s)
Unión Proteica , Proteína SOS1 , Anticuerpos de Dominio Único , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/metabolismo , Proteína SOS1/metabolismo , Proteína SOS1/química , Proteína SOS1/genética , Proteína SOS1/inmunología , Humanos , Animales , Regulación Alostérica , Proteínas ras/metabolismo , Proteínas ras/química , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/inmunología , Sitios de Unión , Camélidos del Nuevo Mundo/inmunología , Inmunización , Transducción de Señal , Modelos MolecularesRESUMEN
Aerobic glycolysis is a hallmark of cancer development, but this dogma has been challenged by reports showing a key role of oxidative phosphorylation (OXPHOS) in cancer cell survival. It has been proposed that increased levels of intramitochondrial proteins in cancer cells are associated with high OXPHOS activity and increased sensitivity to OXPHOS inhibitors. However, the molecular mechanisms leading to the high expression of OXPHOS proteins in cancer cells remain unknown. Multiple proteomics studies have detected the ubiquitination of intramitochondrial proteins, suggesting the contribution of the ubiquitin system to the proteostatic regulation of OXPHOS proteins. Here, we identified the ubiquitin hydrolase OTUB1 as a regulator of the mitochondrial metabolic machinery essential for lung cancer cell survival. Mitochondria-localized OTUB1 modulates respiration by inhibiting K48-linked ubiquitination and turnover of OXPHOS proteins. An increase in OTUB1 expression is commonly observed in one-third of non-small-cell lung carcinomas and is associated with high OXPHOS signatures. Moreover, OTUB1 expression highly correlates with the sensitivity of lung cancer cells to mitochondrial inhibitors.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Fosforilación Oxidativa , Neoplasias Pulmonares/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Proteostasis , Ubiquitina/metabolismo , Enzimas Desubicuitinizantes/metabolismoRESUMEN
Blood flow produces shear stress exerted on the endothelial layer of the vessels. Spatial characterization of the endothelial proteome is required to uncover the mechanisms of endothelial activation by shear stress, as blood flow varies in the vasculature. An integrative ubiquitinome and proteome analysis of shear-stressed endothelial cells demonstrated that the non-degradative ubiquitination of several GTPases is regulated by mechano-signaling. Spatial analysis reveals increased ubiquitination of the small GTPase RAP1 in the descending aorta, a region exposed to laminar shear stress. The ubiquitin ligase WWP2 is identified as a novel regulator of RAP1 ubiquitination during shear stress response. Non-degradative ubiquitination fine-tunes the function of GTPases by modifying their interacting network. Specifically, WWP2-mediated RAP1 ubiquitination at lysine 31 switches the balance from the RAP1/ Talin 1 (TLN1) toward RAP1/ Afadin (AFDN) or RAP1/ RAS Interacting Protein 1 (RASIP1) complex formation, which is essential to suppress shear stress-induced reactive oxygen species (ROS) production and maintain endothelial barrier integrity. Increased ROS production in endothelial cells in the descending aorta of endothelial-specific Wwp2-knockout mice leads to increased levels of oxidized lipids and inflammation. These results highlight the importance of the spatially regulated non-degradative ubiquitination of GTPases in endothelial mechano-activation.
Asunto(s)
Células Endoteliales , GTP Fosfohidrolasas , Animales , Ratones , Células Endoteliales/metabolismo , GTP Fosfohidrolasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteoma/metabolismo , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismo , Ratones Noqueados , UbiquitinaciónRESUMEN
PURPOSE: TIPRL1 (target of rapamycin signaling pathway regulator-like 1) is a known interactor and inhibitor of protein phosphatases PP2A, PP4 and PP6 - all pleiotropic modulators of the DNA Damage Response (DDR). Here, we investigated the role of TIPRL1 in the radiotherapy (RT) response of Head and Neck Squamous Cell Carcinoma (HNSCC). METHODS: TIPRL1 mRNA (cBioportal) and protein expression (immunohistochemistry) in HNSCC samples were linked with clinical patient data. TIPRL1-depleted HNSCC cells were generated by CRISPR/Cas9 editing, and effects on colony growth, micronuclei formation (microscopy), cell cycle (flow cytometry), DDR signaling (immunoblots) and proteome (mass spectrometry) following RT were assessed. Mass spectrometry was used for TIPRL1 phosphorylation and interactomics analysis in irradiated cells. RESULTS: TIPRL1 expression was increased in tumor versus non-tumor tissue, with high tumoral TIPRL1 expression associating with lower locoregional control and decreased survival of RT-treated patients. TIPRL1 deletion in HNSCC cells resulted in increased RT sensitivity, a faster but prolonged cell cycle arrest, increased micronuclei formation and an altered proteome-wide DDR. Upon irradiation, ATM phosphorylates TIPRL1 at Ser265. A non-phospho Ser265Ala mutant could not rescue the increased radiosensitivity phenotype of TIPRL1-depleted cells. While binding to PP2A-like phosphatases was confirmed, DNA-dependent protein kinase (DNA-PKcs), RAD51 recombinase and nucleosomal histones were identified as novel TIPRL1 interactors. Histone binding, although stimulated by RT, was adversely affected by TIPRL1 Ser265 phosphorylation. CONCLUSIONS: Our findings underscore a clinically relevant role for TIPRL1 and its ATM-dependent phosphorylation in RT resistance through modulation of the DDR, highlighting its potential as a new HNSCC predictive marker and therapeutic target.
RESUMEN
Endothelial cells (ECs) grant access of disseminated cancer cells to distant organs. However, the molecular players regulating the activation of quiescent ECs at the premetastatic niche (PMN) remain elusive. Here, we find that ECs at the PMN coexpress tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its cognate death receptor 5 (DR5). Unexpectedly, endothelial TRAIL interacts intracellularly with DR5 to prevent its signaling and preserve a quiescent vascular phenotype. In absence of endothelial TRAIL, DR5 activation induces EC death and nuclear factor κB/p38-dependent EC stickiness, compromising vascular integrity and promoting myeloid cell infiltration, breast cancer cell adhesion, and metastasis. Consistently, both down-regulation of endothelial TRAIL at the PMN by proangiogenic tumor-secreted factors and the presence of the endogenous TRAIL inhibitors decoy receptor 1 (DcR1) and DcR2 favor metastasis. This study discloses an intracrine mechanism whereby TRAIL blocks DR5 signaling in quiescent endothelia, acting as gatekeeper of the vascular barrier that is corrupted by the tumor during cancer cell dissemination.
Asunto(s)
Neoplasias de la Mama , Células Endoteliales , Humanos , Femenino , Células Endoteliales/metabolismo , Ligandos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF , Apoptosis/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
It is widely accepted that the p53 tumor suppressor restricts abnormal cells by induction of growth arrest or by triggering apoptosis. Here we show that, in addition, p53 protects the genome from oxidation by reactive oxygen species (ROS), a major cause of DNA damage and genetic instability. In the absence of severe stresses, relatively low levels of p53 are sufficient for upregulation of several genes with antioxidant products, which is associated with a decrease in intracellular ROS. Downregulation of p53 results in excessive oxidation of DNA, increased mutation rate and karyotype instability, which are prevented by incubation with the antioxidant N-acetylcysteine (NAC). Dietary supplementation with NAC prevented frequent lymphomas characteristic of Trp53-knockout mice, and slowed the growth of lung cancer xenografts deficient in p53. Our results provide a new paradigm for a nonrestrictive tumor suppressor function of p53 and highlight the potential importance of antioxidants in the prophylaxis and treatment of cancer.
Asunto(s)
Apoptosis/fisiología , Daño del ADN , Regulación de la Expresión Génica/fisiología , Modelos Biológicos , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Acetilcisteína/farmacología , Animales , Northern Blotting , Western Blotting , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Cartilla de ADN , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Vectores Genéticos , Inestabilidad Genómica/efectos de los fármacos , Humanos , Cariotipificación , Lentivirus , Ratones , Mutagénesis , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The conjugation of a single ubiquitin or monoubiquitination acts as a versatile signal that can have both degradative and non-degradative functions. The latter is of particular interest as emerging evidence indicates that ubiquitin-driven alterations of the protein interaction landscape play a key role in multiple signaling pathways. Whereas early studies were focused on how monoubiquitination alters the interactions of proteins containing ubiquitin-binding domains, more recent reports demonstrate that ubiquitin conjugation can also affect the binding mode by changing the surface of the ubiquitinated substrate. Furthermore, monoubiquitination modulates the interactions with other macromolecules, such as DNA or lipids, underscoring the diverse role of monoubiquitination in cellular processes. In this review, we discussed how monoubiquitination achieves its function by modulating the interaction landscape.
Asunto(s)
Mapas de Interacción de Proteínas , Ubiquitina , Unión Proteica , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
KRAS-mutant lung adenocarcinomas represent the largest molecular subgroup of non-small cell lung cancers (NSCLC) and are notorious for their dismal survival perspectives. To gain more insights in etiology and therapeutic response, we focused on the tumor suppressor Protein Phosphatase 2A (PP2A) as a player in KRAS oncogenic signaling. We report that the PP2A activator PTPA (encoded by PPP2R4) is commonly affected in NSCLC by heterozygous loss and low-frequent loss-of-function mutation, and this is specifically associated with poorer overall survival of KRAS-mutant lung adenocarcinoma patients. Reduced or mutant PPP2R4 expression in A549 cells increased anchorage-independent growth in vitro and xenograft growth in vivo, correlating with increased Ki67 and c-MYC expression. Moreover, KrasG12D-induced lung tumorigenesis was significantly accelerated in Ppp2r4 gene trapped mice as compared to Ppp2r4 wild-type. A confined kinase inhibitor screen revealed that PPP2R4-depletion induced resistance against selumetinib (MEK inhibitor), but unexpectedly sensitized cells for temsirolimus (mTOR inhibitor), in vitro and in vivo. Our findings underscore a clinically relevant role for PTPA loss-of-function in KRAS-mutant NSCLC etiology and kinase inhibitor response.
Asunto(s)
Adenocarcinoma del Pulmón/tratamiento farmacológico , Fosfoproteínas Fosfatasas/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/genética , Células A549 , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Animales , Bencimidazoles/farmacología , Línea Celular Tumoral , Humanos , Antígeno Ki-67/genética , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteína Fosfatasa 2/genética , Proteínas Proto-Oncogénicas c-myc/genética , Sirolimus/análogos & derivados , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Loss of chromosome 9p21 is observed in one-thirds of clear-cell renal cell carcinoma (ccRCC) and is associated with poorer patient survival. Unexpectedly, 9p21 LOH does not lead to decreased expression of the 9p21 tumor suppressor genes, CDKN2A and CDKN2B, suggesting alternative mechanisms of 9p-mediated tumorigenesis. Concordantly, CRISPR-mediated 9p21 deletion promotes growth of immortalized human embryonic kidney epithelial cells independently of the CDKN2A/B pathway inactivation. The 9p21 locus has a highly accessible chromatin structure, suggesting that 9p21 loss might contribute to kidney cancer progression by dysregulating genes distal to the 9p21 locus. We identified several 9p21 regulatory hubs by assessing which of the 9p21-interacting genes are dysregulated in 9p21-deleted kidney cells and ccRCCs. By focusing on the analysis of the homeobox gene 13 (HOXB13) locus, we found that 9p21 loss relieves the HOXB13 locus, decreasing HOXB13 methylation and promoting its expression. Upregulation of HOXB13 facilitates cell growth and is associated with poorer survival of patients with ccRCC. IMPLICATIONS: The results of our study propose a novel tumor suppressive mechanism on the basis of coordinated expression of physically associated genes, providing a better understanding of the role of chromosomal deletions in cancer.
Asunto(s)
Carcinoma de Células Renales/genética , Deleción Cromosómica , Cromosomas Humanos Par 9/genética , Proteínas de Homeodominio/genética , Neoplasias Renales/genética , Regulación hacia Arriba , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Proteínas de Homeodominio/metabolismo , Humanos , Estimación de Kaplan-Meier , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Pérdida de Heterocigocidad , ARN Largo no Codificante/genética , RNA-Seq/métodosRESUMEN
Meningiomas are the most common benign brain tumors. Mutations of the E3 ubiquitin ligase TRAF7 occur in 25% of meningiomas and commonly cooccur with mutations in KLF4, yet the functional link between TRAF7 and KLF4 mutations remains unclear. By generating an in vitro meningioma model derived from primary meningeal cells, we elucidated the cooperative interactions that promote meningioma development. By integrating TRAF7-driven ubiquitinome and proteome alterations in meningeal cells and the TRAF7 interactome, we identified TRAF7 as a proteostatic regulator of RAS-related small GTPases. Meningioma-associated TRAF7 mutations disrupted either its catalytic activity or its interaction with RAS GTPases. TRAF7 loss in meningeal cells altered actin dynamics and promoted anchorage-independent growth by inducing CDC42 and RAS signaling. TRAF deficiency-driven activation of the RAS/MAPK pathway promoted KLF4-dependent transcription that led to upregulation of the tumor-suppressive Semaphorin pathway, a negative regulator of small GTPases. KLF4 loss of function disrupted this negative feedback loop and enhanced mutant TRAF7-mediated cell transformation. Overall, this study provides new mechanistic insights into meningioma development, which could lead to novel treatment strategies. SIGNIFICANCE: The intricate molecular cross-talk between the ubiquitin ligase TRAF7 and the transcription factor KLF4 provides a first step toward the identification of new therapies for patients with meningioma.
Asunto(s)
Neoplasias Encefálicas/genética , Regulación Neoplásica de la Expresión Génica , Meningioma/genética , Mutación , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Proteínas ras/genética , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Biología Computacional , Células HEK293 , Humanos , Factor 4 Similar a Kruppel/genética , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteoma , Semaforinas/metabolismo , Análisis de Secuencia de ADN , Transducción de Señal , Activación Transcripcional , Ubiquitina/química , Proteína de Unión al GTP cdc42/genética , Proteínas ras/metabolismoRESUMEN
Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, lacking effective therapy. Many TNBCs show remarkable response to carboplatin-based chemotherapy, but often develop resistance over time. With increasing use of carboplatin in the clinic, there is a pressing need to identify vulnerabilities of carboplatin-resistant tumors. In this study, we generated carboplatin-resistant TNBC MDA-MB-468 cell line and patient derived TNBC xenograft models. Mass spectrometry-based proteome profiling demonstrated that carboplatin resistance in TNBC is linked to drastic metabolism rewiring and upregulation of anti-oxidative response that supports cell replication by maintaining low levels of DNA damage in the presence of carboplatin. Carboplatin-resistant cells also exhibited dysregulation of the mitotic checkpoint. A kinome shRNA screen revealed that carboplatin-resistant cells are vulnerable to the depletion of the mitotic checkpoint regulators, whereas the checkpoint kinases CHEK1 and WEE1 are indispensable for the survival of carboplatin-resistant cells in the presence of carboplatin. We confirmed that pharmacological inhibition of CHEK1 by prexasertib in the presence of carboplatin is well tolerated by mice and suppresses the growth of carboplatin-resistant TNBC xenografts. Thus, abrogation of the mitotic checkpoint by CHEK1 inhibition re-sensitizes carboplatin-resistant TNBCs to carboplatin and represents a potential strategy for the treatment of carboplatin-resistant TNBCs.
Asunto(s)
Carboplatino/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas Tirosina Quinasas/genética , Pirazinas/farmacología , Pirazoles/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica , Puntos de Control del Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Daño del ADN , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Proteínas de Neoplasias/clasificación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteoma/clasificación , Proteoma/genética , Proteoma/metabolismo , Transducción de Señal , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The SV40 small t antigen (ST) is a potent oncoprotein that perturbs the function of protein phosphatase 2A (PP2A). ST directly interacts with the PP2A scaffolding A subunit and alters PP2A activity by displacing regulatory B subunits from the A subunit. We have determined the crystal structure of full-length ST in complex with PP2A A subunit at 3.1 A resolution. ST consists of an N-terminal J domain and a C-terminal unique domain that contains two zinc-binding motifs. Both the J domain and second zinc-binding motif interact with the intra-HEAT-repeat loops of HEAT repeats 3-7 of the A subunit, which overlaps with the binding site of the PP2A B56 subunit. Intriguingly, the first zinc-binding motif is in a position that may allow it to directly interact with and inhibit the phosphatase activity of the PP2A catalytic C subunit. These observations provide a structural basis for understanding the oncogenic functions of ST.