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Leucine-rich repeat kinase-2 (LRRK2), a gene mutated in familial and sporadic Parkinson's disease (PD), controls multiple cellular processes important for GLIA physiology. Interestingly, emerging studies report that LRRK2 is highly expressed in oligodendrocyte precursor cells (OPCs) compared to the pathophysiology of other brain cells and oligodendrocytes (OLs) in PD. Altogether, these observations suggest crucial function(s) of LRRK2 in OPCs/Ols, which would be interesting to explore. In this study, we investigated the role of LRRK2 in OLs. We showed that LRRK2 knock-out (KO) OPC cultures displayed defects in the transition of OPCs into OLs, suggesting a role of LRRK2 in OL differentiation. Consistently, we found an alteration of myelin basic protein (MBP) striosomes in LRRK2 KO mouse brains and reduced levels of oligodendrocyte transcription factor 2 (Olig2) and Mbp in olig2:EGFP and mbp:RFP transgenic zebrafish embryos injected with lrrk2 morpholino (MO). Moreover, lrrk2 knock-down zebrafish exhibited a lower amount of nerve growth factor (Ngf) compared to control embryos, which represents a potent regulator of oligodendrogenesis and myelination. Overall, our findings indicate that LRRK2 controls OL differentiation, affecting the number of mature OLs.
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Diferenciación Celular , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Oligodendroglía , Proteínas de Pez Cebra , Pez Cebra , Animales , Pez Cebra/metabolismo , Pez Cebra/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Oligodendroglía/metabolismo , Oligodendroglía/citología , Diferenciación Celular/genética , Ratones , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Ratones Noqueados , Proteína Básica de Mielina/metabolismo , Proteína Básica de Mielina/genética , Animales Modificados GenéticamenteRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0304172.].
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The activation loop (A-loop) of kinases, a key regulatory region, is recurrently mutated in several kinase proteins in cancer resulting in dysregulated kinase activity and response to kinase inhibitors. FGFR1 receptor tyrosine kinase represents an important oncogene and therapeutic target for solid and hematological tumors. Here we investigate the biochemical and molecular effects of D647N mutation lying in the A-loop of FGFR1. When expressed in normal and tumoral in vitro cell models, FGFR1D647N is phosphorylated also in the absence of ligands, and this is accompanied by the activation of intracellular signaling. The expression of FGFR1D647N significantly increases single and collective migration of cancer cells in vitro and in vivo, when compared to FGFR1WT. FGFR1D647N expression exacerbates the aggressiveness of cancer cells, increasing their invasiveness in vitro and augmenting their pro-angiogenic capacity in vivo. Remarkably, the D647N mutation significantly increases the sensitivity of FGFR1 to the ATP-competitive inhibitor Erdafitinib suggesting the possibility that this mutation could become a specific target for the development of new inhibitors. Although further efforts are warranted for an exhaustive description of the activation mechanisms, for the identification of more specific inhibitors and for confirming the clinical significance of mutated FGFR1D647N, overall our data demonstrate that the D647N substitution of FGFR1 is a novel pro-oncogenic activating mutation of the receptor that, when found in cancer patients, may anticipate good response to erdafitinib treatment.
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Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Transducción de Señal , Humanos , Ligandos , Línea Celular Tumoral , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Fosforilación , MutaciónRESUMEN
BACKGROUND: Cancer stem-like cells (CSCs) are a subpopulation of tumor cells responsible for tumor initiation, metastasis, chemoresistance, and relapse. Recently, CSCs have been identified in Uveal Melanoma (UM), which represents the most common primary tumor of the eye. UM is highly resistant to systemic chemotherapy and effective therapies aimed at improving overall survival of patients are eagerly required. METHODS: Herein, taking advantage from a pan Fibroblast Growth Factor (FGF)-trap molecule, we singled out and analyzed a UM-CSC subset with marked stem-like properties. A hierarchical clustering of gene expression data publicly available on The Cancer Genome Atlas (TCGA) was performed to identify patients' clusters. RESULTS: By disrupting the FGF/FGF receptor (FGFR)-mediated signaling, we unmasked an FGF-sensitive UM population characterized by increased expression of numerous stemness-related transcription factors, enhanced aldehyde dehydrogenase (ALDH) activity, and tumor-sphere formation capacity. Moreover, FGF inhibition deeply affected UM-CSC survival in vivo in a chorioallantoic membrane (CAM) tumor graft assay, resulting in the reduction of tumor growth. At clinical level, hierarchical clustering of TCGA gene expression data revealed a strong correlation between FGFs/FGFRs and stemness-related genes, allowing the identification of three distinct clusters characterized by different clinical outcomes. CONCLUSIONS: Our findings support the evidence that the FGF/FGFR axis represents a master regulator of cancer stemness in primary UM tumors and point to anti-FGF treatments as a novel therapeutic strategy to hit the CSC component in UM.
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In vertebrates, two homologous heterotetrameric AP1 complexes regulate the intracellular protein sorting via vesicles. AP-1 complexes are ubiquitously expressed and are composed of four different subunits: γ, ß1, µ1 and σ1. Two different complexes are present in eukaryotic cells, AP1G1 (contains γ1 subunit) and AP1G2 (contains γ2 subunit); both are indispensable for development. One additional tissue-specific isoform exists for µ1A, the polarized epithelial cells specific to µ1B; two additional tissue-specific isoforms exist for σ1A: σ1B and σ1C. Both AP1 complexes fulfil specific functions at the trans-Golgi network and endosomes. The use of different animal models demonstrated their crucial role in the development of multicellular organisms and the specification of neuronal and epithelial cells. Ap1g1 (γ1) knockout mice cease development at the blastocyst stage, while Ap1m1 (µ1A) knockouts cease during mid-organogenesis. A growing number of human diseases have been associated with mutations in genes encoding for the subunits of adaptor protein complexes. Recently, a new class of neurocutaneous and neurometabolic disorders affecting intracellular vesicular traffic have been referred to as adaptinopathies. To better understand the functional role of AP1G1 in adaptinopathies, we generated a zebrafish ap1g1 knockout using CRISPR/Cas9 genome editing. Zebrafish ap1g1 knockout embryos cease their development at the blastula stage. Interestingly, heterozygous females and males have reduced fertility and showed morphological alterations in the brain, gonads and intestinal epithelium. An analysis of mRNA profiles of different marker proteins and altered tissue morphologies revealed dysregulated cadherin-mediated cell adhesion. These data demonstrate that the zebrafish model organism enables us to study the molecular details of adaptinopathies and thus also develop treatment strategies.
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Trastornos del Neurodesarrollo , Factor de Transcripción AP-1 , Proteínas de Pez Cebra , Pez Cebra , Animales , Femenino , Humanos , Masculino , Ratones , Endosomas/metabolismo , Células Epiteliales/metabolismo , Isoformas de Proteínas/metabolismo , Red trans-Golgi/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Trastornos del Neurodesarrollo/genética , Factor de Transcripción AP-1/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Severe COVID-19 is characterized by angiogenic features, such as intussusceptive angiogenesis, endothelialitis, and activation of procoagulant pathways. This pathological state can be ascribed to a direct SARS-CoV-2 infection of human lung ECs. Recently, we showed the capability of SARS-CoV-2 to infect ACE2-negative primary human lung microvascular endothelial cells (HL-mECs). This occurred through the interaction of an Arg-Gly-Asp (RGD) motif, endowed on the Spike protein at position 403-405, with αvß3 integrin expressed on HL-mECs. HL-mEC infection promoted the remodeling of cells toward a pro-inflammatory and pro-angiogenic phenotype. The RGD motif is distinctive of SARS-CoV-2 Spike proteins up to the Omicron BA.1 subvariant. Suddenly, a dominant D405N mutation was expressed on the Spike of the most recently emerged Omicron BA.2, BA.4, and BA.5 subvariants. Here we demonstrate that the D405N mutation inhibits Omicron BA.5 infection of HL-mECs and their dysfunction because of the lack of Spike/integrins interaction. The key role of ECs in SARS-CoV-2 pathogenesis has been definitively proven. Evidence of mutations retrieving the capability of SARS-CoV-2 to infect HL-mECs highlights a new scenario for patients infected with the newly emerged SARS-CoV-2 Omicron subvariants, suggesting that they may display less severe disease manifestations than those observed with previous variants.
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COVID-19 , Virosis , Humanos , Células Endoteliales , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genética , Integrinas , MutaciónRESUMEN
Adipose tissue (AT) is a highly active and plastic endocrine organ. It secretes numerous soluble molecules known as adipokines, which act locally to AT control the remodel and homeostasis or exert pleiotropic functions in different peripheral organs. Aberrant production or loss of certain adipokines contributes to AT dysfunction associated with metabolic disorders, including obesity. The AT plasticity is strictly related to tissue vascularization. Angiogenesis supports the AT expansion, while regression of blood vessels is associated with AT hypoxia, which in turn mediates tissue inflammation, fibrosis and metabolic dysfunction. Several adipokines can regulate endothelial cell functions and are endowed with either pro- or anti-angiogenic properties. Here we address the role of adipokines in the regulation of angiogenesis. A better understanding of the link between adipokines and angiogenesis will open the way for novel therapeutic approaches to treat obesity and metabolic diseases.
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Adipoquinas , Tejido Adiposo , Enfermedades Metabólicas , Humanos , Adipoquinas/metabolismo , Tejido Adiposo/irrigación sanguínea , Tejido Adiposo/metabolismo , Inflamación/metabolismo , Enfermedades Metabólicas/metabolismo , Obesidad/metabolismo , Neovascularización Fisiológica/fisiologíaRESUMEN
Gremlin-1 is a secreted bone morphogenetic protein (BMP) antagonist playing a pivotal role in the regulation of tissue formation and embryonic development. Since its first identification in 1997, gremlin-1 has been shown to be a multifunctional factor involved in wound healing, inflammation, cancer and tissue fibrosis. Among others, the activity of gremlin-1 is mediated by its interaction with BMPs or with membrane receptors such as the vascular endothelial growth factor receptor 2 (VEGFR2) or heparan sulfate proteoglycans (HSPGs). Growing evidence has highlighted a central role of gremlin-1 in the homeostasis of the adipose tissue (AT). Of note, gremlin-1 is involved in AT dysfunction during type 2 diabetes, obesity and non-alcoholic fatty liver disease (NAFLD) metabolic disorders. In this review we discuss recent findings on gremlin-1 involvement in AT biology, with particular attention to its role in metabolic diseases, to highlight its potential as a prognostic marker and therapeutic target.
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Diabetes Mellitus Tipo 2 , Factor A de Crecimiento Endotelial Vascular , Humanos , Proteínas Morfogenéticas ÓseasRESUMEN
Gremlin-1 is a secreted cystine-knot protein that acts as an antagonist of bone morphogenetic proteins (BMPs), and as a ligand of heparin and the vascular endothelial growth factor receptor 2 (VEGFR2), thus regulating several physiological and pathological processes, including embryonic development, tissue fibrosis and cancer. Gremlin-1 exerts all these biological activities only in its homodimeric form. Here, we propose a multi-step approach for the expression and purification of homodimeric, fully active, histidine-tagged recombinant gremlin-1, using mammalian HEK293T cells. Ion metal affinity chromatography (IMAC) of crude supernatant followed by heparin-affinity chromatography enables obtaining a highly pure recombinant dimeric gremlin-1 protein, exhibiting both BMP antagonist and potent VEGFR2 agonist activities.
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Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Cromatografía de Afinidad/métodos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Recombinantes/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/agonistas , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/aislamiento & purificación , Proteínas Recombinantes/genéticaRESUMEN
BACKGROUND: Adipose tissue plays a pivotal role in the development and progression of the metabolic syndrome which along with its complications is an epidemic of the 21st century. Irisin is an adipo-myokine secreted mainly by skeletal muscle and targeting, among others, adipose tissue. In brown adipose tissue it upregulates uncoupling protein-1 (UCP1) which is responsible for mitochondrial non-shivering thermogenesis. METHODS: Here we analyzed the effects of irisin on the metabolic activity of 3T3-L1 derived adipocytes through a mitochondrial flux assay. We also assessed the effects of irisin on the intracellular signaling through Western Blot. Finally, the gene expression of ucp1 and lipolytic genes was examined through RT-qPCR. RESULTS: Irisin affects mitochondrial respiration and lipolysis in a time-dependent manner through the regulation of PI3K-AKT pathway. Irisin also induces the expression of UCP1 and the regulation of NF-κB, and CREB and ERK pathways. CONCLUSION: Our data supports the role of irisin in the induction of non-shivering thermogenesis, the regulation of energy expenditure and lipolysis in adipocytes. GENERAL SIGNIFICANCE: Irisin may be an attractive therapeutic target in the treatment of obesity and related metabolic disorders.
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Fibronectinas , Lipólisis , Termogénesis , Células 3T3-L1 , Adipocitos/metabolismo , Tejido Adiposo Pardo/metabolismo , Animales , Fibronectinas/genética , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Termogénesis/genéticaRESUMEN
Fibroblast growth factor receptors (FGFRs) are recurrently altered by single nucleotide variants (SNVs) in many human cancers. The prevalence of SNVs in FGFRs depends on the cancer type. In some tumors, such as the urothelial carcinoma, mutations of FGFRs occur at very high frequency (up to 60%). Many characterized mutations occur in the extracellular or transmembrane domains, while fewer known mutations are found in the kinase domain. In this study, we performed a bioinformatics analysis to identify novel putative cancer driver or therapeutically actionable mutations of the kinase domain of FGFRs. To pinpoint those mutations that may be clinically relevant, we exploited the recurrence of alterations on analogous amino acid residues within the kinase domain (PK_Tyr_Ser-Thr) of different kinases as a predictor of functional impact. By exploiting MutationAligner and LowMACA bioinformatics resources, we highlighted novel uncharacterized mutations of FGFRs which recur in other protein kinases. By revealing unanticipated correspondence with known variants, we were able to infer their functional effects, as alterations clustering on similar residues in analogous proteins have a high probability to elicit similar effects. As FGFRs represent an important class of oncogenes and drug targets, our study opens the way for further studies to validate their driver and/or actionable nature and, in the long term, for a more efficacious application of precision oncology.
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Carcinogénesis/patología , Mutación , Neoplasias/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factores de Crecimiento de Fibroblastos/genética , Secuencia de Aminoácidos , Carcinogénesis/genética , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Fosforilación , Dominios ProteicosRESUMEN
The bioimaging of cancer cells by the specific targeting of overexpressed biomarkers is an approach that holds great promise in the identification of selective diagnostic tools. Tumor-associated human carbonic anhydrase (hCA) isoforms IX and XII have been considered so far as well-defined biomarkers, with their expression correlating with cancer progression and aggressiveness. Therefore, the availability of highly performant fluorescent tools tailored for their targeting and able to efficiently visualize such key targets is in high demand. We report here on the design and synthesis of a kind of quantum dot (QD)-based fluorescent glyconanoprobe coated with a binary mixture of ligands, which, according to the structure of the terminal domains, impart specific property sets to the fluorescent probe. Specifically, monosaccharide residues ensured the dispersibility in the biological medium, CA inhibitor residues provided specific targeting of membrane-anchored hCA IX overexpressed on bladder cancer cells, and the quantum dots imparted the optical/fluorescence properties.
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The tremendous number of cancer variants that can be detected by NGS analyses has required the development of computational approaches to prioritize mutations on the basis of their biological and clinical significance. Standard strategies take a gene-centric approach to the problem, allowing exclusively the identification of highly frequent variants. On the contrary, protein domain (PD)-based approaches allow to identify functionally relevant low frequency variants by searching for mutations that recur on analogous residues across homologous proteins (i.e. containing the same PD). Such approaches enable to transfer information about the effects and druggability from one known mutation to unknown ones. Here we describe how PD-based strategies work, and discuss how they could be exploited for mutation prioritization. The principle that mutations clustered on specific residues of PDs have the same functional consequences and are therapeutically actionable in a similar manner could help the choice of patient-specific targeted drugs, eventually improving the management of cancer patients.
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Variación Genética/genética , Neoplasias/genética , Dominios Proteicos/genética , HumanosRESUMEN
HIV-1 matrix protein p17 variants (vp17s) derived from non-Hodgkin's lymphoma (NHL) tissues of HIV-1-seropositive (HIV+) patients promote B-cell growth by activating the Akt signaling pathway. It is fundamental to understand the role played by vp17s in producing a microenvironment that fosters lymphoma development and progression. Therefore, we asked whether vp17s could be secreted from infected cells in their biologically active form. In this study, we show that two B-cell growth-promoting vp17s, NHL-a101 and NHL-a102, characterized by amino acid insertions at position 117 to 118 (Ala-Ala) or 125 to 126 (Gly-Asn), respectively, are secreted from HIV-1-infected Jurkat T cells during the active phase of viral replication. Secretion of biologically active vp17s also occurred in HeLa cells nucleofected with a plasmid expressing the entire Gag gene, following proteolytic cleavage of the Gag precursor polyprotein (Pr55Gag) by cellular aspartyl proteases. Binding of Pr55Gag to phosphatidylinositol-(4,5)-bisphosphate was indispensable for allowing the unconventional secretion of both wildtype p17 and vp17s. Indeed, here we demonstrate that inhibition of Pr55Gag binding to phosphatidylinositol-(4,5)-bisphosphate by using neomycin, or its enzymatic depletion achieved by overexpression of 5ptaseIV, significantly impair the secretion of p17s. We also demonstrated that heparan sulfate proteoglycans were involved in tethering p17s at the cell surface. This finding opens up an interesting way for investigating whether tethered p17s on the surface of HIV-1 reservoirs may represent a likely target for immune-mediated killing.
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Linfocitos B , Antígenos VIH , VIH-1 , Productos del Gen gag del Virus de la Inmunodeficiencia Humana , Membrana Celular/metabolismo , Células HeLa , Humanos , Unión Proteica , Proteolisis , Transducción de Señal , Replicación ViralRESUMEN
During multiple myeloma (MM) progression the activation of the angiogenic process represents a key step for the formation of the vascular niche, where different stromal components and neoplastic cells collaborate and foster tumor growth. Among the different pro-angiogenic players, Fibroblast Growth Factor 2 (FGF2) plays a pivotal role in BM vascularization occurring during MM progression. Long Pentraxin 3 (PTX3), a natural FGF antagonist, is able to reduce the activation of stromal components promoted by FGF2 in various in vitro models. An increased FGF/PTX3 ratio has also been found to occur during MM evolution, suggesting that restoring the "physiological" FGF/PTX3 ratio in plasma cells and BM stromal cells (BMSCs) might impact MM. In this work, taking advantage of PTX3-inducible human MM models, we show that PTX3 produced by tumor cells is able to restore a balanced FGF/PTX3 ratio sufficient to prevent the activation of the FGF/FGFR system in endothelial cells and to reduce the angiogenic capacity of MM cells in different in vivo models. As a result of this anti-angiogenic activity, PTX3 overexpression causes a significant reduction of the tumor burden in both subcutaneously grafted and systemic MM models. These data pave the way for the exploitation of PTX3-derived anti-angiogenic approaches in MM.
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Alterations in KRAS have been identified as the most recurring somatic variants in the multiple myeloma (MM) mutational landscape. Combining DNA and RNA sequencing, we studied 756 patients and observed KRAS as the most frequently mutated gene in patients at diagnosis; in addition, we demonstrated the persistence or de novo occurrence of the KRAS aberration at disease relapse. Small-molecule inhibitors targeting KRAS have been developed; however, they are selective for tumors carrying the KRASG12C mutation. Therefore, there is still a need to develop novel therapeutic approaches to target the KRAS mutational events found in other tumor types, including MM. We used AZD4785, a potent and selective antisense oligonucleotide that selectively targets and downregulates all KRAS isoforms, as a tool to dissect the functional sequelae secondary to KRAS silencing in MM within the context of the bone marrow niche and demonstrated its ability to significantly silence KRAS, leading to inhibition of MM tumor growth, both in vitro and in vivo, and confirming KRAS as a driver and therapeutic target in MM.
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Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Mutación/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Humanos , Ratones SCID , Terapia Molecular Dirigida , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Oligonucleótidos Antisentido/uso terapéutico , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéuticoRESUMEN
Vascular endothelial growth factor receptor 2 (VEGFR2) activating mutations are emerging as important oncogenic driver events. Understanding the biological implications of such mutations may help to pinpoint novel therapeutic targets. Here we show that activated VEGFR2 via the pro-oncogenic R1051Q mutation induces relevant metabolic changes in melanoma cells. The expression of VEGFR2R1051Q leads to higher energy metabolism and ATP production compared to control cells expressing VEGFR2WT. Furthermore, activated VEGFR2R1051Q augments the dependence on glutamine (Gln) of melanoma cells, thus increasing Gln uptake and their sensitivity to Gln deprivation and to inhibitors of glutaminase, the enzyme initiating Gln metabolism by cells. Overall, these results highlight Gln addiction as a metabolic vulnerability of tumors harboring the activating VEGFR2R1051Q mutation and suggest novel therapeutic approaches for those patients harboring activating mutations of VEGFR2.
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Metabolismo Energético , Mutación con Ganancia de Función , Glutamina/metabolismo , Melanoma/metabolismo , Neoplasias Cutáneas/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Adenosina Trifosfato/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Metabolismo Energético/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glutaminasa/antagonistas & inhibidores , Glutaminasa/metabolismo , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Transducción de Señal , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Tumor neovascularization may occur via both angiogenic and vasculogenic events. In order to investigate the vessel formation during tumor growth, we developed a novel experimental model that takes into account the differentiative and tumorigenic properties of Embryonic Stem cells (ESCs). Leukemia Inhibitory Factor-deprived murine ESCs were grafted on the top of the chick embryo chorionallantoic membrane (CAM) in ovo. Cell grafts progressively grew, forming a vascularized mass within 10 days. At this stage, the grafts are formed by cells with differentiative features representative of all three germ layers, thus originating teratomas, a germinal cell tumor. In addition, ESC supports neovascular events by recruiting host capillaries from surrounding tissue that infiltrates the tumor mass. Moreover, immunofluorescence studies demonstrate that perfused active blood vessels within the tumor are of both avian and murine origin because of the simultaneous occurrence of angiogenic and vasculogenic events. In conclusion, the chick embryo ESC/CAM-derived teratoma model may represent a useful approach to investigate both vasculogenic and angiogenic events during tumor growth and for the study of natural and synthetic modulators of the two processes.
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Diferenciación Celular , Células Madre Embrionarias/patología , Neovascularización Patológica , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/fisiología , Teratoma/irrigación sanguínea , Teratoma/patología , Animales , Embrión de Pollo , Membrana Corioalantoides , Células Madre Embrionarias/metabolismo , Ratones , Ratones Noqueados , Teratoma/metabolismoRESUMEN
In cancer genomics, recurrence of mutations in gene families that share homologous domains has recently emerged as a reliable indicator of functional impact and can be exploited to reveal the pro-oncogenic effect of previously uncharacterized variants. Pan-cancer analyses of mutation hotspots in the catalytic domain of a subset of tyrosine kinase receptors revealed that two infrequent mutations of VEGFR2 (R1051Q and D1052N) recur in analogous proteins and correlate with reduced patient survival. Functional validation showed that both R1051Q and D1052N mutations increase the enzymatic activity of VEGFR2. The expression of VEGFR2R1051Q potentiates the PI3K/Akt signaling axis in cancer cells, increasing their tumorigenic potential in vitro and in vivo. In addition, it confers to cancer cells an increased sensitivity to the VEGFR2-targeted tyrosine kinase inhibitor Linifanib. In the context of an efficacious application of anti-cancer targeted therapies, these findings indicate that the screening for uncharacterized mutations, like VEGFR2R1051Q, may help to predict patient prognosis and drug response, with significant clinical implications.
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Biomarcadores de Tumor/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/secundario , Melanoma/patología , Mutación , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Movimiento Celular , Proliferación Celular , Perfilación de la Expresión Génica , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fosforilación , Pronóstico , Inhibidores de Proteínas Quinasas/uso terapéutico , Tasa de Supervivencia , Células Tumorales Cultivadas , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The chick embryo chorioallantoic membrane (CAM) represents a powerful in vivo model to study several physiological and pathological processes including inflammation and tumor progression. Nevertheless, the possibility of deepening the molecular processes in the CAM system is biased by the absence/scarcity of chemical and biological reagents, designed explicitly for avian species. This is particularly true for transcriptional factors, proteinaceous molecules that regulate various cellular responses, including proliferation, survival, and differentiation. Here, we propose a detailed antibody-independent protocol to visualize the activation and nuclear translocation of transcriptional factors in cells or in tissues of different animal species. As a proof of concept, DNA/cAMP response element-binding protein (CREB) interaction was characterized on the CAM tissue using oligonucleotides containing the palindromic binding sequence of CREB. Scrambled oligonucleotides were used as controls. In situ DNA/protein interaction protocol is a versatile method that is useful for the study of transcription factors in the cell and tissue of different origins.