RESUMO
The breadth and depth at which cancer models are interrogated contribute to the successful clinical translation of drug discovery efforts. In colorectal cancer (CRC), model availability is limited by a dearth of large-scale collections of patient-derived xenografts (PDXs) and paired tumoroids from metastatic disease, where experimental therapies are typically tested. Here we introduce XENTURION, an open-science resource offering a platform of 128 PDX models from patients with metastatic CRC, along with matched PDX-derived tumoroids. Multidimensional omics analyses indicate that tumoroids retain extensive molecular fidelity with parental PDXs. A tumoroid-based trial with the anti-EGFR antibody cetuximab reveals variable sensitivities that are consistent with clinical response biomarkers, mirror tumor growth changes in matched PDXs, and recapitulate EGFR genetic deletion outcomes. Inhibition of adaptive signals upregulated by EGFR blockade increases the magnitude of cetuximab response. These findings illustrate the potential of large living biobanks, providing avenues for molecularly informed preclinical research in oncology.
Assuntos
Cetuximab , Neoplasias Colorretais , Receptores ErbB , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/tratamento farmacológico , Animais , Cetuximab/uso terapêutico , Cetuximab/farmacologia , Receptores ErbB/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Camundongos , Feminino , Metástase Neoplásica , MasculinoRESUMO
The activation of the majority of AGC kinases is regulated by two phosphorylation events on two conserved serine/threonine residues located on the activation loop and on the hydrophobic motif, respectively. In AGC kinase family, phosphomimetic substitutions with aspartate or glutamate, leading to constitutive activation, have frequently occurred at the hydrophobic motif site. On the contrary, phosphomimetic substitutions in the activation loop are absent across the evolution of AGC kinases. This observation is explained by the failure of aspartate and glutamate to mimic phosphorylatable serine/threonine in this regulatory site. By detailed 3D structural simulations of RSK2 and further biochemical evaluation in cells, we show that the phosphomimetic residue on the activation loop fails to form a critical salt bridge with R114, necessary to reorient the αC-helix and to activate the protein. By a phylogenetic analysis, we point at a possible coevolution of a phosphorylatable activation loop and the presence of a conserved positively charged amino acid on the αC-helix. In sum, our analysis leads to the unfeasibility of phosphomimetic substitution in the activation loop of RSK and, at the same time, highlights the peculiar structural role of activation loop phosphorylation.
Assuntos
Substituição de Aminoácidos , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Motivos de Aminoácidos , Ativação Enzimática , Evolução Molecular , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Mimetismo Molecular , Fosforilação , Filogenia , Estrutura Secundária de Proteína , Proteínas Quinases S6 Ribossômicas 90-kDa/genéticaRESUMO
Somatic activating mutations within the PIK3CA gene have been recently detected in sporadic lymphatic and venous malformations, and in vascular malformations (VM) associated to overgrowth syndromes, such as CLOVES and Klippel-Trenaunay syndrome. Although VM are often limited to specific tissue areas and can be well treated, in extended or recurrent lesions novel therapeutic approaches are needed. We generated a mouse model of VM by local expression of PIK3CA-activating mutation in endothelial cells. PIK3CA-driven lesions are characterized by large areas of hemorrhage, hyperplastic vessels, infiltrates of inflammatory cells, and elevated endothelial cell density. Such vascular lesions are ameliorated by administration of dual PI3K/mTOR inhibitor, BEZ235, and mTOR inhibitor, Everolimus. Unexpectedly, the expression of PIK3CA-activating mutations in human endothelial cells results in both increased proliferation rates and senescence. Moreover, active forms of PIK3CA strongly promote the angiogenic sprouting. Treatment with PI3K/mTOR inhibitors restores normal endothelial cell proliferation rate and reduces the amount of senescent cells, whereas treatment with Akt inhibitor is less effective. Our findings reveal that PIK3CA mutations have a key role in the pathogenesis of VM and PIK3CA-driven experimental lesions can be effectively treated by PI3K/mTOR inhibitors.
Assuntos
Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Serina-Treonina Quinases TOR/antagonistas & inibidores , Malformações Vasculares/genética , Animais , Bovinos , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Embrião de Mamíferos , Células Endoteliais , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Cordão Umbilical , Malformações Vasculares/metabolismo , Malformações Vasculares/patologiaRESUMO
Extrusion of apoptotic cells from epithelial tissues requires orchestrated morphological rearrangements of the apoptotic cell and its neighbors. However, the connections between the apoptotic cascade and events leading to extrusion are not fully understood. Here, we characterize an apoptotic extrusion apical actin ring (EAAR) that is assembled within the apoptotic cell and drives epithelial extrusion. Caspase-mediated cleavage of myotonic dystrophy kinase-related CDC42-binding kinase-α (MRCKα) triggers a signaling pathway that leads to the assembly of EAAR that pulls actin bundles, resulting in the compaction and removal of the cell body. We provide a detailed portrait of the EAAR including F-actin flow, the contribution of myosin contraction, and actin polymerization at bundles' terminals when the product of MRCKα cleavage is expressed. These results add to our understanding of the mechanisms controlling the process of epithelial extrusion by establishing a causal relationship between the triggering events of apoptosis, the activation of MRCKα, and its subsequent effects on the dynamics of actomyosin cytoskeleton rearrangement.
Assuntos
Actomiosina/metabolismo , Apoptose/fisiologia , Caspases/metabolismo , Células Epiteliais/metabolismo , Miotonina Proteína Quinase/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Células CACO-2 , Miosinas Cardíacas/metabolismo , Linhagem Celular , Cães , Células HEK293 , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Centro Organizador dos Microtúbulos/fisiologia , Cadeias Leves de Miosina/metabolismo , Miosinas/metabolismo , Transdução de Sinais/fisiologia , Quinases Associadas a rho/metabolismoRESUMO
Cellular protrusions are highly dynamic structures involved in fundamental processes, including cell migration and invasion. For a cell to migrate, its leading edge must form protrusions, and then adhere or retract. The spatial and temporal coordination of protrusions and retraction is yet to be fully understood. The study of protrusion dynamics mainly relies on live-microscopy often coupled to fluorescent labeling. Here we report the use of an alternative, label-free, quantitative and rapid assay to analyze protrusion dynamics in a cell population based on the real-time recording of cell activity by means of electronic sensors. Cells are seeded on a plate covered with electrodes and their shape changes map into measured impedance variations. Upon growth factor stimulation the impedance increases due to protrusive activity and decreases following retraction. Compared to microscopy-based methods, impedance measurements are suitable to high-throughput studies on different cell lines, growth factors and chemical compounds. We present data indicating that this assay lends itself to dissect the biochemical signaling pathways controlling adhesive protrusions. Indeed, we show that the protrusion phase is sustained by actin polymerization, directly driven by growth factor stimulation. Contraction instead mainly relies on myosin action, pointing at a pivotal role of myosin in lamellipodia retraction.