RESUMEN
Astrocytes respond and contribute to neuroinflammation by adopting inflammatory reactive states. Although recent efforts have characterized the gene expression signatures associated with these reactive states, the cell biology underlying inflammatory reactive astrocyte phenotypes remains under-explored. Here, we used CRISPR-based screening in human iPSC-derived astrocytes to identify mTOR activation a driver of cytokine-induced endolysosomal system remodeling, manifesting as alkalinization of endolysosomal compartments, decreased autophagic flux, and increased exocytosis of certain endolysosomal cargos. Through endolysosomal proteomics, we identified and focused on one such cargo-IL-32, a disease-associated pro-inflammatory cytokine not present in rodents, whose secretion mechanism is not well understood. We found that IL-32 was partially secreted in extracellular vesicles likely to be exosomes. Furthermore, we found that IL-32 was involved in the polarization of inflammatory reactive astrocyte states and was upregulated in astrocytes in multiple sclerosis lesions. We believe that our results advance our understanding of cell biological pathways underlying inflammatory reactive astrocyte phenotypes and identify potential therapeutic targets.
Asunto(s)
Astrocitos , Exosomas , Interleucinas , Lisosomas , Serina-Treonina Quinasas TOR , Astrocitos/metabolismo , Humanos , Exosomas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Lisosomas/metabolismo , Interleucinas/metabolismo , Endosomas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Células Cultivadas , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/patología , Inflamación/metabolismo , Inflamación/patologíaRESUMEN
Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant persister cells (DTPs) which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Resistencia a Antineoplásicos , Neoplasias Pulmonares , Transducción de Señal , Factores de Transcripción , Proteínas Señalizadoras YAP , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas Señalizadoras YAP/metabolismo , Línea Celular Tumoral , Animales , Resistencia a Antineoplásicos/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasia Residual , Ratones , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 1 de Adhesión Focal/genética , Receptores ErbB/metabolismo , Receptores ErbB/genética , Quinasa de Linfoma Anaplásico/metabolismo , Quinasa de Linfoma Anaplásico/genética , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Antineoplásicos/uso terapéutico , Antineoplásicos/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cancers have been associated with a diverse array of genomic alterations. To help mechanistically understand such alterations in breast-invasive carcinoma, we applied affinity purificationmass spectrometry to delineate comprehensive biophysical interaction networks for 40 frequently altered breast cancer (BC) proteins, with and without relevant mutations, across three human breast cell lines. These networks identify cancer-specific protein-protein interactions (PPIs), interconnected and enriched for common and rare cancer mutations, that are substantially rewired by the introduction of key BC mutations. Our analysis identified BPIFA1 and SCGB2A1 as PIK3CA-interacting proteins, which repress PI3K-AKT signaling, and uncovered USP28 and UBE2N as functionally relevant interactors of BRCA1. We also show that the protein phosphatase 1 regulatory subunit spinophilin interacts with and regulates dephosphorylation of BRCA1 to promote DNA double-strand break repair. Thus, PPI landscapes provide a powerful framework for mechanistically interpreting disease genomic data and can identify valuable therapeutic targets.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Neoplasias/metabolismo , Mapas de Interacción de Proteínas , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Humanos , Espectrometría de Masas , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/aislamiento & purificación , Purificación por Afinidad en TándemRESUMEN
A major goal of cancer research is to understand how mutations distributed across diverse genes affect common cellular systems, including multiprotein complexes and assemblies. Two challengeshow to comprehensively map such systems and how to identify which are under mutational selectionhave hindered this understanding. Accordingly, we created a comprehensive map of cancer protein systems integrating both new and published multi-omic interaction data at multiple scales of analysis. We then developed a unified statistical model that pinpoints 395 specific systems under mutational selection across 13 cancer types. This map, called NeST (Nested Systems in Tumors), incorporates canonical processes and notable discoveries, including a PIK3CA-actomyosin complex that inhibits phosphatidylinositol 3-kinase signaling and recurrent mutations in collagen complexes that promote tumor proliferation. These systems can be used as clinical biomarkers and implicate a total of 548 genes in cancer evolution and progression. This work shows how disparate tumor mutations converge on protein assemblies at different scales.
Asunto(s)
Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Mapas de Interacción de Proteínas/genética , Genes Relacionados con las Neoplasias , Humanos , Mutación , Mapeo de Interacción de Proteínas/métodosRESUMEN
We outline a framework for elucidating tumor genetic complexity through multidimensional protein-protein interaction maps and apply it to enhancing our understanding of head and neck squamous cell carcinoma. This network uncovers 771 interactions from cancer and noncancerous cell states, including WT and mutant protein isoforms. Prioritization of cancer-enriched interactions reveals a previously unidentified association of the fibroblast growth factor receptor tyrosine kinase 3 with Daple, a guanine-nucleotide exchange factor, resulting in activation of Gαi- and p21-activated protein kinase 1/2 to promote cancer cell migration. Additionally, we observe mutation-enriched interactions between the human epidermal growth factor receptor 3 (HER3) receptor tyrosine kinase and PIK3CA (the alpha catalytic subunit of phosphatidylinositol 3-kinase) that can inform the response to HER3 inhibition in vivo. We anticipate that the application of this framework will be valuable for translating genetic alterations into a molecular and clinical understanding of the underlying biology of many disease areas.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Resistencia a Antineoplásicos/genética , Neoplasias de Cabeza y Cuello/metabolismo , Mapas de Interacción de Proteínas , Animales , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Movimiento Celular , Femenino , Neoplasias de Cabeza y Cuello/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Desnudos , Proteínas de Microfilamentos/metabolismo , Mutación , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The ability of the small GTPase Cdc42 to regulate diverse cellular processes depends on tight spatial control of its activity. Cdc42 function is best understood at the plasma membrane (PM), where it regulates cytoskeletal organization and cell polarization. Active Cdc42 has also been detected at the Golgi, but its role and regulation at this organelle are only partially understood. Here we analyze the spatial distribution of Cdc42 activity by moni-toring the dynamics of the Cdc42 FLARE biosensor using the phasor approach to FLIM-FRET. Phasor analysis revealed that Cdc42 is active at all Golgi cisternae and that this activity is controlled by Tuba and ARHGAP10, two Golgi-associated Cdc42 regulators. To our surprise, FGD1, another Cdc42 GEF at the Golgi, was not required for Cdc42 regulation at the Golgi, although its depletion decreased Cdc42 activity at the PM. Similarly, changes in Golgi morphology did not affect Cdc42 activity at the Golgi but were associated with a substantial reduction in PM-associated Cdc42 activity. Of interest, cells with reduced Cdc42 activity at the PM displayed altered centrosome morphology, suggesting that centrosome regulation may be mediated by active Cdc42 at the PM. Our study describes a novel quantitative approach to determine Cdc42 activity at specific subcellular locations and reveals new regulatory principles and functions of this small GTPase.