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1.
Nat Commun ; 15(1): 2130, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38503739

RESUMO

The Epidermal Growth Factor Receptor (EGFR) is frequently found to be mutated in non-small cell lung cancer. Oncogenic EGFR has been successfully targeted by tyrosine kinase inhibitors, but acquired drug resistance eventually overcomes the efficacy of these treatments. Attempts to surmount this therapeutic challenge are hindered by a poor understanding of how and why cancer mutations specifically amplify ligand-independent EGFR auto-phosphorylation signals to enhance cell survival and how this amplification is related to ligand-dependent cell proliferation. Here we show that drug-resistant EGFR mutations manipulate the assembly of ligand-free, kinase-active oligomers to promote and stabilize the assembly of oligomer-obligate active dimer sub-units and circumvent the need for ligand binding. We reveal the structure and assembly mechanisms of these ligand-free, kinase-active oligomers, uncovering oncogenic functions for hitherto orphan transmembrane and kinase interfaces, and for the ectodomain tethered conformation of EGFR. Importantly, we find that the active dimer sub-units within ligand-free oligomers are the high affinity binding sites competent to bind physiological ligand concentrations and thus drive tumor growth, revealing a link with tumor proliferation. Our findings provide a framework for future drug discovery directed at tackling oncogenic EGFR mutations by disabling oligomer-assembling interactions.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Ligantes , Receptores ErbB/metabolismo , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética
2.
Cells ; 9(12)2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33302515

RESUMO

Epidermal growth factor receptor (EGFR) takes centre stage in carcinogenesis throughout its entire cellular trafficking odyssey. When loaded in extracellular vesicles (EVs), EGFR is one of the key proteins involved in the transfer of information between parental cancer and bystander cells in the tumour microenvironment. To hijack EVs, EGFR needs to play multiple signalling roles in the life cycle of EVs. The receptor is involved in the biogenesis of specific EV subpopulations, it signals as an active cargo, and it can influence the uptake of EVs by recipient cells. EGFR regulates its own inclusion in EVs through feedback loops during disease progression and in response to challenges such as hypoxia, epithelial-to-mesenchymal transition and drugs. Here, we highlight how the spatiotemporal rules that regulate EGFR intracellular function intersect with and influence different EV biogenesis pathways and discuss key regulatory features and interactions of this interplay. We also elaborate on outstanding questions relating to EGFR-driven EV biogenesis and available methods to explore them. This mechanistic understanding will be key to unravelling the functional consequences of direct anti-EGFR targeted and indirect EGFR-impacting cancer therapies on the secretion of pro-tumoural EVs and on their effects on drug resistance and microenvironment subversion.


Assuntos
Vesículas Extracelulares/metabolismo , Neoplasias/metabolismo , Progressão da Doença , Endocitose , Transição Epitelial-Mesenquimal , Receptores ErbB/química , Receptores ErbB/metabolismo , Humanos , Neoplasias/patologia , Transdução de Sinais , Tetraspaninas/metabolismo , Microambiente Tumoral
3.
Sci Rep ; 7(1): 3598, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28620180

RESUMO

The PIM1 serine/threonine protein kinase mediates growth factor and survival signalling, and cooperates potently with c-MYC during tumorigenesis. PIM1 is overexpressed in many human cancers and is a promising target for drug development. PIM1 levels are regulated mainly through cytokine-induced transcription and protein degradation, but mechanisms regulating its activity and levels remain largely unexplored. Here, we show that PIM1 is modified in vitro and in cultured cells by the Small ubiquitin-like modifier (SUMO) on two independent sites: K169, within a consensus SUMOylation motif (IK169DE171) in the active site of PIM1, and also at a second promiscuous site. Alanine substitution of E171 (within the consensus motif) abolished SUMOylation, significantly increased the half-life of PIM1, and markedly reduced its ubiquitylation. Mechanistically, SUMOylation promoted ubiquitin-mediated degradation of PIM1 via recruitment of the SUMO-targeted ubiquitin ligase, RNF4. Additionally, SUMOylated PIM1 showed enhanced protein kinase activity in vitro. Interestingly, the E171A mutant was active in vitro but displayed altered substrate specificity in cultured cells, consistent with the idea that SUMOylation may govern PIM1 substrate specificity under certain contexts. Taken together, these data demonstrate that the protein kinase activity and levels of PIM1 can be regulated by a covalent post-translational modification.


Assuntos
Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Proteína SUMO-1/metabolismo , Fatores de Transcrição/metabolismo , Substituição de Aminoácidos , Domínio Catalítico , Análise Mutacional de DNA , Humanos , Ligação Proteica , Proteólise , Proteínas Proto-Oncogênicas c-pim-1/genética , Sumoilação , Ubiquitinação
4.
Cell Cycle ; 13(9): 1413-23, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24626184

RESUMO

p68 (DDX5) acts both as an ATP-dependent RNA helicase and as a transcriptional co-activator of several cancer-associated transcription factors, including the p53 tumor suppressor. p68 is aberrantly expressed in a high proportion of cancers, but the oncogenic drive for, or the consequences of, these expression changes remain unclear. Here we show that elevated p68 expression in a cohort of human breast cancers is associated significantly with elevated levels of the oncogenic protein kinase, Polo-like kinase-1 (PLK1). Patients expressing detectable levels of both p68 and PLK1 have a poor prognosis, but only if they also have mutation in the TP53 gene (encoding p53), suggesting that p68 can regulate PLK1 levels in a manner that is suppressed by p53. In support of this hypothesis, we show that p68 stimulates expression from the PLK1 promoter, and that silencing of endogenous p68 expression downregulates endogenous PLK1 gene expression. In the absence of functional p53, p68 stimulates the expression of PLK1 both at basal levels and in response to the clinically relevant drug, etoposide. In keeping with a role as a transcriptional activator/co-activator, chromatin immuno-precipitation analysis shows that p68 is associated with the PLK1 promoter, irrespective of the p53 status. However, its recruitment is stimulated by etoposide in cells lacking p53, suggesting that p53 can oppose association of p68 with the PLK1 promoter. These data provide a model in which p68 and p53 interplay regulates PLK1 expression, and which describes the behavior of these molecules, and the outcome of their interaction, in human breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/genética , RNA Helicases DEAD-box/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Adenosina Trifosfatases/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Fitogênicos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Etoposídeo/farmacologia , Feminino , Humanos , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Quinase 1 Polo-Like
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