RESUMO
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.
Assuntos
Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras) , Ubiquitinação , Humanos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Animais , Proteína p120 Ativadora de GTPase/metabolismo , Proteína p120 Ativadora de GTPase/genética , Camundongos , Linhagem Celular Tumoral , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , Lisina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas ras/metabolismo , Proteínas ras/genética , Neurofibromina 1RESUMO
Genome engineering technologies are powerful tools in cell-based immunotherapy to optimize or fine-tune cell functionalities. However, their use for multiple gene edits poses relevant biological and technical challenges. Short hairpin RNA (shRNA)-based cell engineering bypasses these criticalities and represents a valid alternative to CRISPR-based gene editing. Here, we describe a microRNA (miRNA)-based multiplex shRNA platform obtained by combining highly efficient miRNA scaffolds into a chimeric cluster, to deliver up to four shRNA-like sequences. Thanks to its limited size, our cassette could be deployed in a one-step process along with all the CAR components, streamlining the generation of engineered CAR T cells. The plug-and-play design of the shRNA platform allowed us to swap each shRNA-derived guide sequence without affecting the system performance. Appropriately choosing the target sequences, we were able to either achieve a functional KO, or fine-tune the expression levels of the target genes, all without the need for gene editing. Through our strategy we achieved easy, safe, efficient, and tunable modulation of multiple target genes simultaneously. This approach allows for the effective introduction of multiple functionally relevant tweaks in the transcriptome of the engineered cells, which may lead to increased performance in challenging environments, e.g., solid tumors.
RESUMO
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.
Assuntos
Neoplasias Encefálicas/genética , Regulação Neoplásica da Expressão Gênica , Meningioma/genética , Mutação , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Proteínas ras/genética , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Biologia Computacional , Células HEK293 , Humanos , Fator 4 Semelhante a Kruppel/genética , Camundongos , Camundongos Nus , Transplante de Neoplasias , Proteoma , Semaforinas/metabolismo , Análise de Sequência de DNA , Transdução de Sinais , Ativação Transcricional , Ubiquitina/química , Proteína cdc42 de Ligação ao GTP/genética , Proteínas ras/metabolismoRESUMO
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.
Assuntos
Vasos Sanguíneos/metabolismo , Endossomos/metabolismo , Células Endoteliais/metabolismo , Hemorragia/metabolismo , Síndrome de Noonan/metabolismo , Fatores de Transcrição/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Malformações Vasculares/metabolismo , Animais , Vasos Sanguíneos/anormalidades , Vasos Sanguíneos/efeitos dos fármacos , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Modelos Animais de Doenças , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/genética , Endossomos/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Haploinsuficiência , Células HeLa , Hemorragia/genética , Hemorragia/patologia , Hemorragia/prevenção & controle , Humanos , Linfocinas/genética , Linfocinas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica , Síndrome de Noonan/tratamento farmacológico , Síndrome de Noonan/genética , Síndrome de Noonan/patologia , Fosforilação , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico , Quinazolinas/farmacologia , Transdução de Sinais , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Ubiquitinação , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Malformações Vasculares/tratamento farmacológico , Malformações Vasculares/genética , Malformações Vasculares/patologiaRESUMO
Chromosome segregation during mitosis is antagonistically regulated by the Aurora-B kinase and RepoMan (recruits PP1 onto mitotic chromatin at anaphase)-associated phosphatases PP1/PP2A. Aurora B is overexpressed in many cancers but, surprisingly, this only rarely causes lethal aneuploidy. Here we show that RepoMan abundance is regulated by the same mechanisms that control Aurora B, including FOXM1-regulated expression and proteasomal degradation following ubiquitination by APC/C-CDH1 or SCFFBXW7. The deregulation of these mechanisms can account for the balanced co-overexpression of Aurora B and RepoMan in many cancers, which limits chromosome segregation errors. In addition, Aurora B and RepoMan independently promote cancer cell proliferation by reducing checkpoint--induced cell-cycle arrest during interphase. The co-up-regulation of RepoMan and Aurora B in tumors is inversely correlated with patient survival, underscoring its potential importance for tumor progression. Finally, we demonstrate that high RepoMan levels sensitize cancer cells to Aurora-B inhibitors. Hence, the co-up-regulation of RepoMan and Aurora B is associated with tumor aggressiveness but also exposes a vulnerable target for therapeutic intervention.
Assuntos
Aurora Quinase B/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Proteína Forkhead Box M1/metabolismo , Células HEK293 , Humanos , Interfase , Mitose , Modelos Biológicos , Neoplasias/metabolismo , Neoplasias/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteólise , Resultado do TratamentoRESUMO
Activation of the RAS oncogenic pathway, frequently ensuing from mutations in RAS genes, is a common event in human cancer. Recent reports demonstrate that reversible ubiquitination of RAS GTPases dramatically affects their activity, suggesting that enzymes involved in regulating RAS ubiquitination may contribute to malignant transformation. Here, we identified the de-ubiquitinase OTUB1 as a negative regulator of RAS mono- and di-ubiquitination. OTUB1 inhibits RAS ubiquitination independently of its catalytic activity resulting in sequestration of RAS on the plasma membrane. OTUB1 promotes RAS activation and tumorigenesis in wild-type RAS cells. An increase of OTUB1 expression is commonly observed in non-small-cell lung carcinomas harboring wild-type KRAS and is associated with increased levels of ERK1/2 phosphorylation, high Ki67 score, and poorer patient survival. Our results strongly indicate that dysregulation of RAS ubiquitination represents an alternative mechanism of RAS activation during lung cancer development.
Assuntos
Cisteína Endopeptidases/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/fisiopatologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Linhagem Celular Tumoral , Enzimas Desubiquitinantes , Modelos Animais de Doenças , Humanos , Camundongos Nus , UbiquitinaçãoRESUMO
Cytokinin receptors play a key role in cytokinin-dependent processes regulating plant growth, development, and adaptation; therefore, the functional properties of these receptors are of great importance. Previously the properties of cytokinin receptors were investigated in heterologous assay systems using unicellular microorganisms, mainly bacteria, expressing receptor proteins. However, within microorganisms receptors reside in an alien environment that might distort the receptor properties. Therefore, a new assay system has been developed allowing studies of individual receptors within plant membranes (i.e. closer to their natural environment). The main ligand-binding characteristics of receptors from Arabidopsis [AHK2, AHK3, and AHK4] and maize (ZmHK1) were refined in this new system, and the properties of full-length Arabidopsis receptor AHK2 were characterized for the first time. Ligand specificity profiles of receptors towards cytokinin bases were comparable with the profiles retrieved in bacterial assay systems. In contrast, cytokinin-9-ribosides displayed a strongly reduced affinity for receptors in the plant assay system, indicating that ribosides as the common transport form of cytokinins have no or very weak cytokinin activity. This underpins the central role of free bases as the sole biologically active cytokinin compounds. According to molecular modelling and docking studies, N (9)-ribosylation alters the bonding pattern in cytokinin-receptor interaction and prevents ß6-ß7 loop movement important for tight hormone binding. A common feature of all receptors was a greatly reduced ligand binding at low (5.0-5.5) pH. The particularly high sensitivity of ZmHK1 to pH changes leads to the suggestion that some cytokinin receptors may play an additional role as pH sensors in the lumen of the endoplasmic reticulum.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Citocininas/metabolismo , Modelos Moleculares , Proteínas Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Expressão Gênica , Histidina Quinase , Concentração de Íons de Hidrogênio , Ligantes , Simulação de Acoplamento Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Proteínas Quinases/genética , Receptores de Superfície Celular/genética , Transdução de Sinais , Nicotiana/genética , Nicotiana/metabolismo , Zea mays/genética , Zea mays/metabolismoRESUMO
Inappropriate activation of epidermal growth factor receptor (EGFR) plays a causal role in many cancers including colon cancer. The activation of EGFR by phosphorylation is balanced by receptor kinase and protein tyrosine phosphatase activities. However, the mechanisms of negative EGFR regulation by tyrosine phosphatases remain largely unexplored. Our previous results indicate that protein tyrosine phosphatase receptor type O (PTPRO) is down-regulated in a subset of colorectal cancer (CRC) patients with a poor prognosis. Here we identified PTPRO as a phosphatase that negatively regulates SRC by directly dephosphorylating Y416 phosphorylation site. SRC activation triggered by PTPRO down-regulation induces phosphorylation of both EGFR at Y845 and the c-CBL ubiquitin ligase at Y731. Increased EGFR phosphorylation at Y845 promotes its receptor activity, whereas enhanced phosphorylation of c-CBL triggers its degradation promoting EGFR stability. Importantly, hyperactivation of SRC/EGFR signaling triggered by loss of PTPRO leads to high resistance of colon cancer to EGFR inhibitors. Our results not only highlight the PTPRO contribution in negative regulation of SRC/EGFR signaling but also suggest that tumors with low PTPRO expression may be therapeutically targetable by anti-SRC therapies.
Assuntos
Neoplasias do Colo/enzimologia , Receptores ErbB/antagonistas & inibidores , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Quinases da Família src/metabolismo , Células CACO-2 , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Gefitinibe , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Sistema de Sinalização das MAP Quinases , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Quinazolinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/biossíntese , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Transdução de SinaisRESUMO
Cytokinins are ubiquitous plant hormones; their signal is perceived by sensor histidine kinases-cytokinin receptors. This review focuses on recent advances on cytokinin receptor structure, in particular sensing module and adjacent domains which play an important role in hormone recognition, signal transduction and receptor subcellular localization. Principles of cytokinin binding site organization and point mutations affecting signaling are discussed. To date, more than 100 putative cytokinin receptor genes from different plant species were revealed due to the total genome sequencing. This allowed us to employ an evolutionary and bioinformatics approaches to clarify some new aspects of receptor structure and function. Non-transmembrane areas adjacent to the ligand-binding CHASE domain were characterized in detail and new conserved protein motifs were recovered. Putative mechanisms for cytokinin-triggered receptor activation were suggested.
Assuntos
Arabidopsis/genética , Citocininas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Plantas/genética , Receptores de Superfície Celular/química , Transdução de Sinais , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/metabolismo , Biologia Computacional , Evolução Molecular , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Receptores de Superfície Celular/genéticaRESUMO
Bromination of 6-benzylaminopurine (1) with Br(2) in AcOH in the presence of AcONa afforded 6-benzylamino-8-bromopurine (2) in 59% yield. The position of bromination was confirmed by direct transformation of bromide 2 by reaction with NaN(3) in dimethyl sulfoxide to 8-azido-6-benzylaminopurine (3) in a yield of 70% and comparison of its properties with the known compound 2-azido-6-benzylaminopurine (11). Compounds 3 and 11 were checked for their biological activity in specific biotests based on the primary cytokinin effects in living plants. Both synthesized compounds displayed effects similar to the typical cytokinin 6-benzylaminopurine (1).