RESUMEN
Lung cancer, the leading cause of cancer mortality, exhibits heterogeneity that enables adaptability, limits therapeutic success, and remains incompletely understood. Single-cell RNA sequencing (scRNA-seq) of metastatic lung cancer was performed using 49 clinical biopsies obtained from 30 patients before and during targeted therapy. Over 20,000 cancer and tumor microenvironment (TME) single-cell profiles exposed a rich and dynamic tumor ecosystem. scRNA-seq of cancer cells illuminated targetable oncogenes beyond those detected clinically. Cancer cells surviving therapy as residual disease (RD) expressed an alveolar-regenerative cell signature suggesting a therapy-induced primitive cell-state transition, whereas those present at on-therapy progressive disease (PD) upregulated kynurenine, plasminogen, and gap-junction pathways. Active T-lymphocytes and decreased macrophages were present at RD and immunosuppressive cell states characterized PD. Biological features revealed by scRNA-seq were biomarkers of clinical outcomes in independent cohorts. This study highlights how therapy-induced adaptation of the multi-cellular ecosystem of metastatic cancer shapes clinical outcomes.
Asunto(s)
Neoplasias Pulmonares/genética , Biomarcadores de Tumor/genética , Línea Celular , Ecosistema , Humanos , Neoplasias Pulmonares/patología , Macrófagos/patología , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Linfocitos T/patología , Microambiente Tumoral/genéticaRESUMEN
Human cerebral cortex size and complexity has increased greatly during evolution. While increased progenitor diversity and enhanced proliferative potential play important roles in human neurogenesis and gray matter expansion, the mechanisms of human oligodendrogenesis and white matter expansion remain largely unknown. Here, we identify EGFR-expressing "Pre-OPCs" that originate from outer radial glial cells (oRGs) and undergo mitotic somal translocation (MST) during division. oRG-derived Pre-OPCs provide an additional source of human cortical oligodendrocyte precursor cells (OPCs) and define a lineage trajectory. We further show that human OPCs undergo consecutive symmetric divisions to exponentially increase the progenitor pool size. Additionally, we find that the OPC-enriched gene, PCDH15, mediates daughter cell repulsion and facilitates proliferation. These findings indicate properties of OPC derivation, proliferation, and dispersion important for human white matter expansion and myelination.
Asunto(s)
Cadherinas/metabolismo , Corteza Cerebral/citología , Células Ependimogliales/metabolismo , Neurogénesis/genética , Células Precursoras de Oligodendrocitos/metabolismo , Proteínas Relacionadas con las Cadherinas , Cadherinas/genética , Proliferación Celular/genética , Células Cultivadas , Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Células Ependimogliales/citología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Inmunohistoquímica , Células Precursoras de Oligodendrocitos/citología , ARN Interferente Pequeño , RNA-Seq , Análisis de la Célula Individual , Sustancia Blanca/citología , Sustancia Blanca/embriología , Sustancia Blanca/metabolismoRESUMEN
Non-coding regions amplified beyond oncogene borders have largely been ignored. Using a computational approach, we find signatures of significant co-amplification of non-coding DNA beyond the boundaries of amplified oncogenes across five cancer types. In glioblastoma, EGFR is preferentially co-amplified with its two endogenous enhancer elements active in the cell type of origin. These regulatory elements, their contacts, and their contribution to cell fitness are preserved on high-level circular extrachromosomal DNA amplifications. Interrogating the locus with a CRISPR interference screening approach reveals a diversity of additional elements that impact cell fitness. The pattern of fitness dependencies mirrors the rearrangement of regulatory elements and accompanying rewiring of the chromatin topology on the extrachromosomal amplicon. Our studies indicate that oncogene amplifications are shaped by regulatory dependencies in the non-coding genome.
Asunto(s)
Cromosomas Humanos/genética , Elementos de Facilitación Genéticos , Amplificación de Genes , Oncogenes , Acetilación , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Cromatina/metabolismo , ADN de Neoplasias/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Genes Relacionados con las Neoplasias , Sitios Genéticos , Glioblastoma/genética , Glioblastoma/patología , Histonas/metabolismo , Humanos , Neuroglía/metabolismoRESUMEN
The NRF2 transcription factor controls a cell stress program that is implicated in cancer and there is great interest in targeting NRF2 for therapy. We show that NRF2 activity depends on Fructosamine-3-kinase (FN3K)-a kinase that triggers protein de-glycation. In its absence, NRF2 is extensively glycated, unstable, and defective at binding to small MAF proteins and transcriptional activation. Moreover, the development of hepatocellular carcinoma triggered by MYC and Keap1 inactivation depends on FN3K in vivo. N-acetyl cysteine treatment partially rescues the effects of FN3K loss on NRF2 driven tumor phenotypes indicating a key role for NRF2-mediated redox balance. Mass spectrometry reveals that other proteins undergo FN3K-sensitive glycation, including translation factors, heat shock proteins, and histones. How glycation affects their functions remains to be defined. In summary, our study reveals a surprising role for the glycation of cellular proteins and implicates FN3K as targetable modulator of NRF2 activity in cancer.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Carcinoma Hepatocelular/patología , Femenino , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Glicosilación , Células HEK293 , Células Hep G2 , Xenoinjertos , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción GenéticaRESUMEN
Tyrosine phosphorylation regulates multi-layered signaling networks with broad implications in (patho)physiology, but high-throughput methods for functional annotation of phosphotyrosine sites are lacking. To decipher phosphotyrosine signaling directly in tissue samples, we developed a mass-spectrometry-based interaction proteomics approach. We measured the in vivo EGF-dependent signaling network in lung tissue quantifying >1,000 phosphotyrosine sites. To assign function to all EGF-regulated sites, we determined their recruited protein signaling complexes in lung tissue by interaction proteomics. We demonstrated how mutations near tyrosine residues introduce molecular switches that rewire cancer signaling networks, and we revealed oncogenic properties of such a lung cancer EGFR mutant. To demonstrate the scalability of the approach, we performed >1,000 phosphopeptide pulldowns and analyzed them by rapid mass spectrometric analysis, revealing tissue-specific differences in interactors. Our approach is a general strategy for functional annotation of phosphorylation sites in tissues, enabling in-depth mechanistic insights into oncogenic rewiring of signaling networks.
Asunto(s)
Carcinogénesis/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Fosfotirosina/metabolismo , Células A549 , Animales , Humanos , Espectrometría de Masas/métodos , Mutación , Fosfoproteínas/metabolismo , Fosforilación , Proteómica , Ratas , Ratas Sprague-Dawley , Pez CebraRESUMEN
Diverse genetic, epigenetic, and developmental programs drive glioblastoma, an incurable and poorly understood tumor, but their precise characterization remains challenging. Here, we use an integrative approach spanning single-cell RNA-sequencing of 28 tumors, bulk genetic and expression analysis of 401 specimens from the The Cancer Genome Atlas (TCGA), functional approaches, and single-cell lineage tracing to derive a unified model of cellular states and genetic diversity in glioblastoma. We find that malignant cells in glioblastoma exist in four main cellular states that recapitulate distinct neural cell types, are influenced by the tumor microenvironment, and exhibit plasticity. The relative frequency of cells in each state varies between glioblastoma samples and is influenced by copy number amplifications of the CDK4, EGFR, and PDGFRA loci and by mutations in the NF1 locus, which each favor a defined state. Our work provides a blueprint for glioblastoma, integrating the malignant cell programs, their plasticity, and their modulation by genetic drivers.
Asunto(s)
Neoplasias Encefálicas/genética , Plasticidad de la Célula/genética , Glioblastoma/genética , Adolescente , Anciano , Animales , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Linaje de la Célula/genética , Niño , Estudios de Cohortes , Modelos Animales de Enfermedad , Femenino , Heterogeneidad Genética , Glioblastoma/patología , Xenoinjertos , Humanos , Lactante , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Persona de Mediana Edad , Mutación , RNA-Seq , Análisis de la Célula Individual/métodos , Microambiente Tumoral/genéticaRESUMEN
The posttranslational modifier ubiquitin regulates most cellular processes. Its ability to form polymeric chains of distinct linkages is key to its diverse functionality. Yet, we still lack the experimental tools to induce linkage-specific polyubiquitylation of a protein of interest in cells. Here, we introduce a set of engineered ubiquitin protein ligases and matching ubiquitin acceptor tags for the rapid, inducible linear (M1-), K48-, or K63-linked polyubiquitylation of proteins in yeast and mammalian cells. By applying the so-called "Ubiquiton" system to proteasomal targeting and the endocytic pathway, we validate this tool for soluble cytoplasmic and nuclear as well as chromatin-associated and integral membrane proteins and demonstrate how it can be used to control the localization and stability of its targets. We expect that the Ubiquiton system will serve as a versatile, broadly applicable research tool to explore the signaling functions of polyubiquitin chains in many biological contexts.
Asunto(s)
Ubiquitina-Proteína Ligasas , Ubiquitina , Animales , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Poliubiquitina/genética , Poliubiquitina/metabolismo , Transducción de Señal , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitinación , Mamíferos/metabolismoRESUMEN
The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase.
Asunto(s)
Receptores ErbB/química , Receptores ErbB/metabolismo , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/aislamiento & purificación , Humanos , Membranas Intracelulares/química , Resonancia Magnética Nuclear Biomolecular , Multimerización de Proteína , Termodinámica , Vesículas Transportadoras/químicaRESUMEN
Through a network of progressively maturing vesicles, the endosomal system connects the cell's interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle "cloud" and a highly dynamic peripheral contingent. How this spatiotemporal organization is achieved and what function(s) it curates is unclear. Here, we reveal the endoplasmic reticulum (ER)-located ubiquitin ligase Ring finger protein 26 (RNF26) as the global architect of the entire endosomal system, including the trans-Golgi network (TGN). To specify perinuclear vesicle coordinates, catalytically competent RNF26 recruits and ubiquitinates the scaffold p62/sequestosome 1 (p62/SQSTM1), in turn attracting ubiquitin-binding domains (UBDs) of various vesicle adaptors. Consequently, RNF26 restrains fast transport of diverse vesicles through a common molecular mechanism operating at the ER membrane, until the deubiquitinating enzyme USP15 opposes RNF26 activity to allow vesicle release into the cell's periphery. By drawing the endosomal system's architecture, RNF26 orchestrates endosomal maturation and trafficking of cargoes, including signaling receptors, in space and time.
Asunto(s)
Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Neoplasias/metabolismo , Línea Celular Tumoral , Células Dendríticas/citología , Células Dendríticas/metabolismo , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Proteína Sequestosoma-1/metabolismo , Vesículas Transportadoras/metabolismo , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
Growth factor receptors rank among the most important oncogenic pathways, but pharmacologic inhibitors often demonstrate limited benefit as monotherapy. Here, we show that epidermal growth factor receptor (EGFR) signaling repressed N6-methyladenosine (m6A) levels in glioblastoma stem cells (GSCs), whereas genetic or pharmacologic EGFR targeting elevated m6A levels. Activated EGFR induced non-receptor tyrosine kinase SRC to phosphorylate the m6A demethylase, AlkB homolog 5 (ALKBH5), thereby inhibiting chromosomal maintenance 1 (CRM1)-mediated nuclear export of ALKBH5 to permit sustained mRNA m6A demethylation in the nucleus. ALKBH5 critically regulated ferroptosis through m6A modulation and YTH N6-methyladenosine RNA binding protein (YTHDF2)-mediated decay of the glutamate-cysteine ligase modifier subunit (GCLM). Pharmacologic targeting of ALKBH5 augmented the anti-tumor efficacy of EGFR and GCLM inhibitors, supporting an EGFR-ALKBH5-GCLM oncogenic axis. Collectively, EGFR reprograms the epitranscriptomic landscape through nuclear retention of the ALKBH5 demethylase to protect against ferroptosis, offering therapeutic paradigms for the treatment of lethal cancers.
Asunto(s)
Desmetilasa de ARN, Homólogo 5 de AlkB , Receptores ErbB , Ferroptosis , Glioblastoma , Humanos , Adenosina/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Receptores ErbB/genética , Ferroptosis/genética , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , ARN Mensajero/genéticaRESUMEN
Eukaryotic cells learn and adapt via unknown network architectures. Recent work demonstrated a circuit of two GTPases used by cells to overcome growth factor scarcity, encouraging our view that artificial and biological intelligence share strikingly similar design principles and that cells function as deep reinforcement learning (RL) agents in uncertain environments.
Asunto(s)
GTP Fosfohidrolasas , Transducción de Señal , GTP Fosfohidrolasas/metabolismoRESUMEN
Apical-basal polarity is maintained by distinct protein complexes that reside in membrane junctions, and polarity loss in monolayered epithelial cells can lead to formation of multilayers, cell extrusion, and/or malignant overgrowth. Yet, how polarity loss cooperates with intrinsic signals to control directional invasion toward neighboring epithelial cells remains elusive. Using the Drosophila ovarian follicular epithelium as a model, we found that posterior follicle cells with loss of lethal giant larvae (lgl) or Discs large (Dlg) accumulate apically toward germline cells, whereas cells with loss of Bazooka (Baz) or atypical protein kinase C (aPKC) expand toward the basal side of wildtype neighbors. Further studies revealed that these distinct multilayering patterns in the follicular epithelium were determined by epidermal growth factor receptor (EGFR) signaling and its downstream target Pointed, a zinc-finger transcription factor. Additionally, we identified Rho kinase as a Pointed target that regulates formation of distinct multilayering patterns. These findings provide insight into how cell polarity genes and receptor tyrosine kinase signaling interact to govern epithelial cell organization and directional growth that contribute to epithelial tumor formation.
Asunto(s)
Polaridad Celular , Proteínas de Drosophila , Receptores ErbB , Animales , Polaridad Celular/fisiología , Drosophila melanogaster , Proteínas de Drosophila/metabolismo , Células Epiteliales/metabolismo , Epitelio/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismoRESUMEN
Spatially organized reaction dynamics between proto-oncogenic epidermal growth factor receptor (EGFR) and protein tyrosine phosphatases determine EGFR phosphorylation dynamics in response to growth factors and thereby cellular behavior within developing tissues. We show that the reaction dynamics of mutual inhibition between RPTPγ phosphatase and autocatalytic ligandless EGFR phosphorylation enable highly sensitive promigratory EGFR signaling responses to subnanomolar EGF levels, when < 5% receptors are occupied by EGF. EGF thereby triggers an autocatalytic phospho-EGFR reaction by the initial production of small amounts of phospho-EGFR through transient, asymmetric EGF-EGFR2 dimers. Single cell RPTPγ oxidation imaging revealed that phospho-EGFR induces activation of NADPH oxidase, which in turn inhibits RPTPγ-mediated dephosphorylation of EGFR, tilting the autocatalytic RPTPγ/EGFR toggle switch reaction towards ligandless phosphorylated EGFR. Reversibility of this reaction to EGF is maintained by the constitutive phosphatase activity of endoplasmic reticulum-associated TCPTP. This RPTPγ/EGFR reaction at the plasma membrane causes promigratory signaling that is separated from proliferative signaling induced by accumulated, liganded, phosphorylated EGF-EGFR in endosomes. Accordingly, loss of RPTPγ results in constitutive promigratory signaling from phosphorylated EGFR monomers. RPTPγ is thus a suppressor of promigratory oncogenic but not of proliferative EGFR signaling.
Asunto(s)
Factor de Crecimiento Epidérmico , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo , Receptores ErbB/metabolismo , Transducción de Señal , Fosforilación , Oxidación-ReducciónRESUMEN
Memory T cells provide long-lasting protective immunity, and distinct subpopulations of memory T cells drive chronic inflammatory diseases such as asthma. Asthma is a chronic allergic inflammatory disease with airway remodeling including fibrotic changes. The immunological mechanisms that induce airway fibrotic changes remain unknown. We found that interleukin-33 (IL-33) enhanced amphiregulin production by the IL-33 receptor, ST2hi memory T helper 2 (Th2) cells. Amphiregulin-epidermal growth factor receptor (EGFR)-mediated signaling directly reprogramed eosinophils to an inflammatory state with enhanced production of osteopontin, a key profibrotic immunomodulatory protein. IL-5-producing memory Th2 cells and amphiregulin-producing memory Th2 cells appeared to cooperate to establish lung fibrosis. The analysis of polyps from patients with eosinophilic chronic rhinosinusitis revealed fibrosis with accumulation of amphiregulin-producing CRTH2hiCD161hiCD45RO+CD4+ Th2 cells and osteopontin-producing eosinophils. Thus, the IL-33-amphiregulin-osteopontin axis directs fibrotic responses in eosinophilic airway inflammation and is a potential target for the treatment of fibrosis induced by chronic allergic disorders.
Asunto(s)
Anfirregulina/inmunología , Eosinófilos/inmunología , Osteopontina/metabolismo , Fibrosis Pulmonar/inmunología , Transducción de Señal/inmunología , Células Th2/inmunología , Anfirregulina/biosíntesis , Anfirregulina/metabolismo , Anfirregulina/farmacología , Animales , Modelos Animales de Enfermedad , Receptores ErbB/metabolismo , Femenino , Memoria Inmunológica/inmunología , Inmunomodulación , Interleucina-33/metabolismo , Ratones , Rinitis/inmunología , Rinitis/patología , Sinusitis/inmunología , Sinusitis/patología , Transcripción Genética/efectos de los fármacosRESUMEN
FBXW7 is an E3 ubiquitin ligase that targets proteins for proteasome-mediated degradation and is mutated in various cancer types. Here, we use CRISPR base editors to introduce different FBXW7 hotspot mutations in human colon organoids. Functionally, FBXW7 mutation reduces EGF dependency of organoid growth by ~10,000-fold. Combined transcriptomic and proteomic analyses revealed increased EGFR protein stability in FBXW7 mutants. Two distinct phosphodegron motifs reside in the cytoplasmic tail of EGFR. Mutations in these phosphodegron motifs occur in human cancer. CRISPR-mediated disruption of the phosphodegron motif at T693 reduced EGFR degradation and EGF growth factor dependency. FBXW7 mutant organoids showed reduced sensitivity to EGFR-MAPK inhibitors. These observations were further strengthened in CRC-derived organoid lines and validated in a cohort of patients treated with panitumumab. Our data imply that FBXW7 mutations reduce EGF dependency by disabling EGFR turnover.
Asunto(s)
Proteínas F-Box , Neoplasias , Humanos , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/farmacología , Factor de Crecimiento Epidérmico/metabolismo , Proteómica , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Proteínas F-Box/genéticaRESUMEN
The tumor suppressor LKB1 is a serine/threonine protein kinase that is frequently mutated in human lung adenocarcinoma (LUAD). LKB1 regulates a complex signaling network that is known to control cell polarity and metabolism; however, the pathways that mediate the tumor-suppressive activity of LKB1 are incompletely defined. To identify mechanisms of LKB1-mediated growth suppression, we developed a spheroid-based cell culture assay to study LKB1-dependent growth. We then performed genome-wide CRISPR screens in spheroidal culture and found that LKB1 suppresses growth, in part, by activating the PIKFYVE lipid kinase. Finally, we used chemical inhibitors and a pH-sensitive reporter to determine that LKB1 impairs growth by promoting the internalization of wild-type EGFR in a PIKFYVE-dependent manner.
Asunto(s)
Quinasas de la Proteína-Quinasa Activada por el AMP , Fosfatidilinositol 3-Quinasas , Proteínas Serina-Treonina Quinasas , Esferoides Celulares , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Quinasas de la Proteína-Quinasa Activada por el AMP/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP/genética , Esferoides Celulares/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Proliferación Celular , Línea Celular Tumoral , Sistemas CRISPR-Cas , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genéticaRESUMEN
Non-small cell lung cancer (NSCLC), a major life-threatening disease accounting for 85% of all lung cancer cases, has been treated with tyrosine kinase inhibitors (TKIs), but often resulted in drug resistance, and approximately 60% of TKI-resistant cases are due to acquired secondary (epithelial growth factor receptor) EGFR-T790M mutation. To identify alternative targets for TKI-resistant NSCLC with EGFR-T790M mutation, we found that the three globo-series glycosphingolipids are increasingly expressed on this type of NSCLC cell lines, and among them, the increase of stage-specific embryonic antigen-4 (SSEA-4) expression is the most significant. Compared to TKI-sensitive cell lines, SSEA-4 and the key enzyme ß3GalT5 responsible for the synthesis of SSEA3 are more expressed in TKI-resistant NSCLC cell lines with EGFR-T790M mutation, and the expression levels strongly correlate with poor survival in patients with EGFR mutation. In addition, we demonstrated that a SSEA-4 targeted monoclonal antibody, especially the homogeneous glycoform with well-defined Fc glycan designed to improve effective functions, is highly effective against this subpopulation of NSCLC in cell-based and animal studies. These findings provide a direction for the prediction of tumor recurrence and treatment of TKI-resistant NSCLC with EGFR-T790M mutation.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Antígenos Embrionarios Específico de Estadio , Animales , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Receptores ErbB/genética , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Recurrencia Local de NeoplasiaRESUMEN
SNX32 is a member of the evolutionarily conserved Phox (PX) homology domain- and Bin/Amphiphysin/Rvs (BAR) domain- containing sorting nexin (SNX-BAR) family of proteins, which play important roles in sorting and membrane trafficking of endosomal cargoes. Although SNX32 shares the highest amino acid sequence homology with SNX6, and has been believed to function redundantly with SNX5 and SNX6 in retrieval of the cation-independent mannose-6-phosphate receptor (CI-MPR) from endosomes to the trans-Golgi network (TGN), its role(s) in intracellular protein trafficking remains largely unexplored. Here, we report that it functions in parallel with SNX1 in mediating epidermal growth factor (EGF)-stimulated postendocytic trafficking of the epidermal growth factor receptor (EGFR). Moreover, SNX32 interacts directly with EGFR, and recruits SNX5 to promote sorting of EGF-EGFR into multivesicular bodies (MVBs) for lysosomal degradation. Thus, SNX32 functions distinctively from other SNX-BAR proteins to mediate signaling-coupled endolysosomal trafficking of EGFR.
Asunto(s)
Factor de Crecimiento Epidérmico , Receptores ErbB , Lisosomas , Transporte de Proteínas , Nexinas de Clasificación , Nexinas de Clasificación/metabolismo , Nexinas de Clasificación/genética , Receptores ErbB/metabolismo , Lisosomas/metabolismo , Humanos , Transporte de Proteínas/fisiología , Factor de Crecimiento Epidérmico/metabolismo , Células HeLa , Endosomas/metabolismo , Red trans-Golgi/metabolismo , Cuerpos Multivesiculares/metabolismoRESUMEN
The Shoc2 scaffold protein is crucial in transmitting signals within the Epidermal Growth Factor Receptor (EGFR)-mediated Extracellular signal-Regulated Kinase (ERK1/2) pathway. While the significance of Shoc2 in this pathway is well-established, the precise mechanisms through which Shoc2 governs signal transmission remain to be fully elucidated. Hereditary variants in Shoc2 are responsible for Noonan Syndrome with Loose anagen Hair (NSLH). However, due to the absence of known enzymatic activity in Shoc2, directly assessing how these variants affect its function is challenging. ERK1/2 phosphorylation is used as a primary parameter of Shoc2 function, but the impact of Shoc2 mutants on the pathway activation is unclear. This study investigates how the NSLH-associated Shoc2 variants influence EGFR signals in the context of the ERK1/2 and AKT downstream signaling pathways. We show that when the ERK1/2 pathway is a primary signaling pathway activated downstream of EGFR, Shoc2 variants cannot upregulate ERK1/2 phosphorylation to the level of the WT Shoc2. Yet, when the AKT and ERK1/2 pathways were activated, in cells expressing Shoc2 variants, ERK1/2 phosphorylation was higher than in cells expressing WT Shoc2. In cells expressing the Shoc2 NSLH mutants, we found that the AKT signaling pathway triggers the PAK activation, followed by phosphorylation of Raf-1/MEK1/2 and activation of the ERK1/2 signaling axis. Hence, our studies reveal a previously unrecognized feedback regulation downstream of the EGFR and provide additional evidence for the role of Shoc2 as a "gatekeeper" in controlling the selection of downstream effectors within the EGFR signaling network.
RESUMEN
As key oncogenic drivers in non-small-cell lung cancer (NSCLC), various mutations in the epidermal growth factor receptor (EGFR) with variable drug sensitivities have been a major obstacle for precision medicine. To achieve clinical-level drug recommendations, a platform for clinical patient case retrieval and reliable drug sensitivity prediction is highly expected. Therefore, we built a database, D3EGFRdb, with the clinicopathologic characteristics and drug responses of 1339 patients with EGFR mutations via literature mining. On the basis of D3EGFRdb, we developed a deep learning-based prediction model, D3EGFRAI, for drug sensitivity prediction of new EGFR mutation-driven NSCLC. Model validations of D3EGFRAI showed a prediction accuracy of 0.81 and 0.85 for patients from D3EGFRdb and our hospitals, respectively. Furthermore, mutation scanning of the crucial residues inside drug-binding pockets, which may occur in the future, was performed to explore their drug sensitivity changes. D3EGFR is the first platform to achieve clinical-level drug response prediction of all approved small molecule drugs for EGFR mutation-driven lung cancer and is freely accessible at https://www.d3pharma.com/D3EGFR/index.php.