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To compare cell adhesion molecules levels in cerebrospinal fluid (CSF) between Zika virus (ZIKV)-exposed neonates with/without microcephaly (cases) and controls, 16 neonates (cases), 8 (50%) with and 8 (50%) without microcephaly, who underwent lumbar puncture (LP) during the ZIKV epidemic (2015-2016) were included. All mothers reported ZIKV clinical symptoms during gestation, all neonates presented with congenital infection findings, and other congenital infections were ruled out. Fourteen control neonates underwent LP in the same laboratory (2017-2018). Five cell adhesion proteins were measured in the CSF using mass spectrometry. Neurexin-1 (3.50 [2.00-4.00] vs. 7.5 [5.00-10.25], P = 0.001), neurexin-3 (0.00 [0.00-0.00] vs. 3.00 [1.50-4.00], P = 0.001) and neural cell adhesion molecule 2 (NCAM2) (0.00 [0.00-0.75] vs. 1.00 [1.00-2.00], P = 0.001) were significantly lower in microcephalic and non-microcephalic cases than in controls. When these two sub-groups of prenatally ZIKA-exposed children were compared to controls separately, the same results were found. When cases with and without microcephaly were compared, no difference was found. Neurexin-3 (18.8% vs. 78.6%, P = 0.001) and NCAM2 (25.0% vs. 85.7%, P = 0.001) were less frequently found among the cases. A positive correlation was found between cephalic perimeter and levels of these two proteins. Neurexin-2 and neurexin-2b presented no significant differences. Levels of three cell adhesion proteins were significantly lower in CSF of neonates exposed to ZIKV before birth than in controls, irrespective of presence of congenital microcephaly. Moreover, the smaller the cephalic perimeter, the lower CSF cell adhesion protein levels. These findings suggest that low CSF levels of neurexin-1, neurexin-3 and NCAM2 may reflect the effects of ZIKV on foetal brain development.
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Microcefalia , Complicações Infecciosas na Gravidez , Infecção por Zika virus , Zika virus , Recém-Nascido , Gravidez , Feminino , Criança , Humanos , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/epidemiologia , Microcefalia/epidemiologia , Estudos de Casos e Controles , Adesão Celular , Complicações Infecciosas na Gravidez/epidemiologia , Moléculas de Adesão Celular , Moléculas de Adesão de Célula NervosaRESUMO
We investigated the feasibility of detecting the presence of specific autoantibodies against potential tumor-associated peptide antigens by enriching these antibody-peptide complexes using Melon Gel resin and mass spectrometry. Our goal was to find tumor-associated phospho-sites that trigger immunoreactions and raise autoantibodies that are detectable in plasma of glioma patients. Such immunoglobulins can potentially be used as targets in immunotherapy. To that aim, we describe a method to detect the presence of antibodies in biological samples that are specific to selected clinically relevant peptides. The method is based on the formation of antibody-peptide complexes by mixing patient plasma with a glioblastoma multiforme (GBM) derived peptide library, enrichment of antibodies and antibody-peptide complexes, the separation of peptides after they are released from immunoglobulins by molecular weight filtration and finally mass spectrometric quantification of these peptides. As proof of concept, we successfully applied the method to dinitrophenyl (DNP)-labeled α-casein peptides mixed with anti-DNP. Further, we incubated human plasma with a phospho-peptide library and conducted targeted analysis on EGFR and GFAP phospho-peptides. As a result, immunoaffinity against phospho-peptide GSHQIS[+80]LDNPDYQQDFFPK (EGFR phospho-site S1166) was detected in high-grade glioma (HGG) patient plasma but not in healthy donor plasma. For the GFAP phospho-sites selected, such immunoaffinity was not observed.
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Anticorpos , Receptores ErbB , Glioma , Peptídeos , Anticorpos/química , Autoanticorpos , Bioensaio , Receptores ErbB/química , Receptores ErbB/metabolismo , Glioma/imunologia , Glioma/metabolismo , Humanos , Imunoglobulinas/química , Imunoglobulinas/metabolismo , Biblioteca de Peptídeos , Peptídeos/química , Fosfopeptídeos/química , Ligação ProteicaRESUMO
Formalin-fixed paraffin-embedded (FFPE) tissues are routinely prepared and collected for diagnostics in pathology departments. These are, therefore, the most accessible research sources in pathology archives. In this study we investigated whether we can apply a targeted and quantitative parallel reaction monitoring (PRM) method for FFPE tissue samples in a sensitive and reproducible way. The feasibility of this technical approach was demonstrated for normal brain and glioblastoma multiforme tissues. Two methods were used: PRM measurement of a tryptic digest without phosphopeptide enrichment (Direct-PRM) and after Fe-NTA phosphopeptide enrichment (Fe-NTA-PRM). With these two methods, the phosphorylation ratio could be determined for four selected peptide pairs that originate from neuroblast differentiation-associated protein (AHNAK S5448-p), calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D T337-p), eukaryotic translation initiation factor 4B (EIF4B S93-p), and epidermal growth factor receptor (EGFR S1166-p). In normal brain FFPE tissues, the Fe-NTA-PRM method enabled the quantification of targeted phosphorylated peptides with high reproducibility (CV < 14%). Our results indicate that formalin fixation does not impede relative quantification of a phospho-site and its phosphorylation ratio in FFPE tissues. The developed workflow combining these methods opens ways to study archival FFPE tissues for phosphorylation ratio determination in proteins.
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Formaldeído , Proteômica , Espectrometria de Massas , Inclusão em Parafina , Fosforilação , Reprodutibilidade dos Testes , Fixação de TecidosRESUMO
Introduction: The goal of this review is to survey proteomics and next generation sequencing (NGS) approaches to discover phosphopeptide neoantigens that can be used as targets for individualized cancer therapy and diagnostics. Neoantigens are peptides or proteins that can be derived from tumor-specific or viral alterations containing mutated sequences including different post-translational modifications that will not be found in normal human cells. These neoantigens can be highly specific for the individual affected tissue. Mass spectrometry combined with NGS allow identifying autoantibodies specific for neoantigens, including neoantigens that contain phosphorylated moieties. Areas covered: We discuss recognition of neoantigens through the application of phosphoproteomics. We focus on phosphoprotein modifications that can be accurately identified with mass spectrometry and can serve as rarely described targets for immunotherapy and diagnosis in cancer and autoimmune diseases. Identification of these phospho-moiety containing neoantigens gives the possibility to search for corresponding autoantibodies, especially if NGS and mass spectrometry are combined. Expert commentary: Identification of post-translational modifications with mass spectrometry gives possibilities to define modifications that are specific for cancer and autoimmune diseases. This technology develops rapidly, and gives opportunities to select neoantigens that are highly specific for cancer and immune diseases and to sequence autoantibodies involved.
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Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteômica/métodos , Autoanticorpos/metabolismo , Humanos , Imunoterapia/métodos , Fosfopeptídeos/metabolismoRESUMO
We show that parallel reaction monitoring (PRM) can be used for exact quantification of phosphorylation ratios of proteins using stable-isotope-labeled peptides. We have compared two different PRM approaches on a digest of a U87 cell culture, namely, direct-PRM (tryptic digest measured by PRM without any further sample preparation) and TiO2-PRM (tryptic digest enriched with TiO2 cartridges, followed by PRM measurement); these approaches are compared for the following phosphorylation sites: neuroblast differentiation-associated protein (AHNAK S5480-p), calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D T337-p), and epidermal growth factor receptor (EGFR S1166-p). A reproducible percentage of phosphorylation could be determined (CV 6-13%) using direct-PRM or TiO2-PRM. In addition, we tested the approaches in a cell culture experiment in which U87 cells were deprived of serum. As a "gold standard" we included immune precipitation of EGFR followed by PRM (IP-PRM). For EGFR (S1166) and AHNAK (S5480) a statistical significant change in the percentage of phosphorylation could be observed as a result of serum deprivation; for EGFR (S1166) this change was observed for both TiO2-PRM and IP-PRM. The presented approach has the potential to multiplex and to quantify the ratio of phosphorylation in a single analysis.
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Espectrometria de Massas/métodos , Fosforilação , Linhagem Celular , Receptores ErbB/metabolismo , Humanos , Marcação por Isótopo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , PeptídeosRESUMO
The discovery of genes and molecular pathways involved in the formation of brain metastasis would direct the development of therapeutic strategies to prevent this deadly complication of cancer. By comparing gene expression profiles of Estrogen Receptor negative (ER-) primary breast tumors between patients who developed metastasis to brain and to organs other than brain, we found that T lymphocytes promote the formation of brain metastases. To functionally test the ability of T cells to promote brain metastasis, we used an in vitro blood-brain barrier (BBB) model. By co-culturing T lymphocytes with breast cancer cells, we confirmed that T cells increase the ability of breast cancer cells to cross the BBB. Proteomics analysis of the tumor cells revealed Guanylate-Binding Protein 1 (GBP1) as a key T lymphocyte-induced protein that enables breast cancer cells to cross the BBB. The GBP1 gene appeared to be up-regulated in breast cancer of patients who developed brain metastasis. Silencing of GBP1 reduced the ability of breast cancer cells to cross the in vitro BBB model. In addition, the findings were confirmed in vivo in an immunocompetent syngeneic mouse model. Co-culturing of ErbB2 tumor cells with activated T cells induced a significant increase in Gbp1 expression by the cancer cells. Intracardial inoculation of the co-cultured tumor cells resulted in preferential seeding to brain. Moreover, intracerebral outgrowth of the tumor cells was demonstrated. The findings point to a role of T cells in the formation of brain metastases in ER- breast cancers, and provide potential targets for intervention to prevent the development of cerebral metastases.
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Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundário , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ligação ao GTP/metabolismo , Linfócitos T/metabolismo , Adulto , Idoso , Animais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Células Cultivadas , Técnicas de Cocultura , Feminino , Proteínas de Ligação ao GTP/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Pessoa de Meia-Idade , Metástase Neoplásica/fisiopatologia , Transplante de Neoplasias , Proteoma , RNA Mensageiro/metabolismoRESUMO
Multiple sclerosis (MScl) frequently is remitted during the third trimester of pregnancy but exacerbated in the first postpartum period. In this context, we investigated protein identification, its abundance, and its change in urine related to these two periods. Using mass spectrometry (LTQ Orbitrap), we identified 1699 tryptic peptides (related to 402 proteins) in urine from 31 MScl and 8 control at these two periods. Pregnancy-related peptides were significantly elevated (p < 0.01) in MScl patients compared with controls (Analysis 1: 531 peptides in MScl and 36 peptides in controls higher abundant in the third trimester compared to postpartum). When comparing the longitudinal differences (Analysis 2), we identified 43 (related to 35 proteins) MScl disease-associated peptides (p < 0.01) with increased or decreased difference ratio in MScl compared with controls. The most discriminating peptides identified were trefoil factor 3 and lysosomal-associated membrane protein 2. Both proteins have a role in the innate immune system. Three proteins with a significant decreased ratio were plasma glutamate carboxypeptidase, Ig mu chain C region, and osteoclast associated immune like receptor. Our results indicate that the protein expression pattern in urine of MScl patients contains information about remote CNS and brain disease processes.
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Esclerose Múltipla/urina , Fragmentos de Peptídeos/urina , Período Pós-Parto/urina , Terceiro Trimestre da Gravidez/urina , Proteoma/isolamento & purificação , Adulto , Carboxipeptidases/genética , Carboxipeptidases/urina , Cromatografia Líquida , Feminino , Expressão Gênica , Humanos , Cadeias mu de Imunoglobulina/genética , Cadeias mu de Imunoglobulina/urina , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteína 2 de Membrana Associada ao Lisossomo/urina , Esclerose Múltipla/patologia , Peptídeos/genética , Peptídeos/urina , Gravidez , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Espectrometria de Massas em Tandem , Fator Trefoil-3 , Tripsina/química , UrináliseRESUMO
The recent accurate and precise determination of the electron affinity (EA) of the astatine atom At0 warrants a re-investigation of the estimated thermodynamic properties of At0 and astatine containing molecules as this EA was found to be much lower (by 0.4 eV) than previous estimated values. In this contribution we estimate, from available data sources, the following thermodynamic and physicochemical properties of the alkali astatides (MAt, M = Li, Na, K, Rb, Cs): their solid and gaseous heats of formation, lattice and gas-phase binding enthalpies, sublimation energies and melting temperatures. Gas-phase charge-transfer dissociation energies for the alkali astatides (the energy requirement for M+ At- â M0 + At0 ) have been obtained and are compared with those for the other alkali halides. Use of Born-Haber cycles together with the new AE (At0 ) value allows the re-evaluation of ΔHf (At0 )g (=56 ± 5 kJ/mol); it is concluded that (At2 )g is a weakly bonded species (bond strength <50 kJ/mol), significantly weaker bonded than previously estimated (116 kJ/mol) and much weaker bonded than I2 (148 kJ/mol), but in agreement with the finding from theory that spin-orbit coupling considerably reduces the bond strength in At2 . The hydration enthalpy (ΔHaq ) of At- is estimated to be -230 ± 2 kJ/mol (using ΔHaq [H+ ] = -1150.1 kJ/mol), in good agreement with molecular dynamics calculations. Arguments are presented that the largest alkali halide, CsAt, like the smallest, LiF, will be only sparingly soluble in water, following the generalization from hard/soft acid/base principles that "small likes small" and "large likes large."
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Paraneoplastic neurological syndromes (PNS) are severe immune mediated effects of cancer. The presence of IgG autoantibodies against onconeural antigens in serum is a hallmark of the disease. Multiple paraneoplastic antibodies have been described, including antibodies against HuD, Yo, amphiphysin and CV2. In this study, we test the hypothesis that primary amino-acid structures of the antigen binding part of antibodies from various individuals share common sequences that are specific for each auto-antigen. We selected 60 patients with PNS, associated with antibodies against HuD, Yo, Amp or CV2. Affinity purified IgG was separated using SDS-PAGE and IgG heavy chains were excised, trypsinized and subjected to tandem mass spectrometry. We selected masses that uniquely identified a PNS autoantibody group, and used MS/MS fragmentation spectra to obtain information on peptide sequences. Out of 19,173 unique masses, 28 immunoglobulin-derived peptides were found exclusively in samples from a single autoantibody defined PNS group. Our results confirm that specific peptide structures exist in the antigen binding site of IgG that are shared between individuals harboring autoantibodies against the same onconeural antigen. Thus, the immune response in these patients followed converging paths during the rearrangement, selection and maturation of immunoglobulin sequences. The identified peptides can be applied in the diagnosis of PNS, but these data also indicate that a similar approach in a variety of other diseases involving an immune response would have an appealing outlook.
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Antígenos/imunologia , Imunoglobulinas/sangue , Síndromes Paraneoplásicas do Sistema Nervoso/diagnóstico , Síndromes Paraneoplásicas do Sistema Nervoso/imunologia , Peptídeos/sangue , Espectrometria de Massas em Tandem , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/química , Imunoglobulina G/imunologia , Imunoglobulinas/química , Imunoglobulinas/imunologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologiaRESUMO
Autoantibodies are increasingly used as biomarkers in the detection of autoimmune disorders and cancer. Disease specific antibodies are generally detected by their binding to specific antigens. As an alternative approach, we propose to identify specific complementarity determining regions (CDR) of IgG that relate to an autoimmune disorder or cancer instead of the specific antigen(s). In this manuscript, we tested the technical feasibility to detect and identify CDRs of specific antibodies by mass spectrometry. We used a commercial pooled IgG preparation as well as purified serum IgG fractions that were spiked with different amounts of a fully human monoclonal antibody (adalimumab). These samples were enzymatically digested and analyzed by nanoLC Orbitrap mass spectrometry. In these samples, we were able to identify peptides derived from the CDRs of adalimumab. These peptides could be detected at an amount of 110 attomole, 5 orders of magnitude lower than the total IgG concentration in these samples. Using higher energy collision induced dissociation (HCD) fragmentation and subsequent de novo sequencing, we could successfully identify 50% of the detectable CDR peptides of adalimumab. In addition, we demonstrated that an affinity purification with anti-dinitrophenol (DNP) monoclonal antibody enhanced anti-DNP derived CDR detection in a serum IgG background. In conclusion, specific CDR peptides could be detected and sequenced at relatively low levels (attomole-femtomole range) which should allow the detection of clinically relevant CDR peptides in patient samples.
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Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/imunologia , Adalimumab , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais Humanizados , Biomarcadores/sangue , Regiões Determinantes de Complementaridade/sangue , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/química , Imunoglobulina G/imunologia , Espectrometria de Massas , Camundongos , Peptídeos/sangue , Peptídeos/química , Peptídeos/imunologia , Valores de ReferênciaRESUMO
Severe acute respiratory virus-2 (SARS-CoV-2) has spread globally leading to a devastating loss of life. Large registry studies have begun to shed light on the epidemiological and clinical vulnerabilities of cancer patients who succumb to or endure poor outcomes of SARS-CoV-2. Specific treatment for COVID-19 infections in cancer patients is lacking while the demand for treatment is increasing. Therefore, we explored the effect of Rintatolimod (Ampligen®) (AIM ImmunoTech, Ocala, FL, USA), a Toll-like receptor 3 (TLR3) agonist, to treat uninfected human pancreatic cancer cells (HPACs). The direct effect of Rintatolimod was measured by targeted gene expression profiling and by proteomics measurements. Our results show that Rintatolimod induces an antiviral effect in HPACs by inducing RNase-L-dependent and independent pathways of the innate immune system. Treatment with Rintatolimod activated the interferon signaling pathway, leading to the overexpression of several cytokines and chemokines in epithelial cells. Furthermore, Rintatolimod treatment increased the expression of angiogenesis-related genes without promoting fibrosis, which is the main cause of death in patients with COVID-19. We conclude that Rintatolimod could be considered an early additional treatment option for cancer patients who are infected with SARS-CoV-2 to prevent the complicated severity of the disease.
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OBJECTIVE: To compare immunoglobulin levels in cerebrospinal fluid (CSF) of neonates exposed to Zika virus (ZIKV) during foetal life (cases) with levels in CSF of control neonates. METHODS: We identified 16 neonates who underwent lumbar puncture (LP), during the ZIKV epidemic (December/2015 to March/2016) whose mothers reported ZIKV clinical symptoms during gestation (cases). Congenital microcephaly was defined as head circumference ≤31.9â¯cm (boys) and ≤31.5â¯cm (girls) for term neonates, or ≤2 standard deviations below the mean for premature (<37 weeks) neonates. Subsequently, we identified neonates who underwent LP in the same lab and fulfilled criteria to be controls: age ≤4 days, CSF white blood cell count ≤8/mm3, CSF protein ≤132â¯mg/dL, CSF red blood cell count ≤1,000/mm3, neither central nervous system illness, nor congenital infection, nor microcephaly. CSF immunoglobulin concentrations were measured by mass spectrometry. RESULTS: 13 controls were included. IgM, IgA, IgG, IgK, and IgL were significantly higher among cases (p < 0.001). Eight (50%) ZIKV exposed infants had congenital microcephaly. These showed the strongest immunoglobulin elevation of the IgM and IgA classes. CONCLUSION: Neonates exposed to ZIKV infection during gestation present with elevated distinct immunoglobulins in CSF, both in cases that developed microcephaly and in cases that did not.
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Epidemias , Microcefalia , Complicações Infecciosas na Gravidez , Infecção por Zika virus , Zika virus , Feminino , Humanos , Imunoglobulinas , Lactente , Recém-Nascido , Masculino , Microcefalia/epidemiologia , Gravidez , Complicações Infecciosas na Gravidez/epidemiologia , Infecção por Zika virus/epidemiologiaRESUMO
The goal of this manuscript is to explore the role of clinical proteomics for detecting mutations in chronic obstructive pulmonary disease (COPD) and lung cancer by mass spectrometry-based technology. COPD and lung cancer caused by smoke inhalation are most likely linked by challenging the immune system via partly shared pathways. Genome-wide association studies have identified several single nucleotide polymorphisms which predispose an increased susceptibility to COPD and lung cancer. In lung cancer, this leads to coding mutations in the affected tissues, development of neoantigens, and different functionality and abundance of proteins in specific pathways. If a similar reasoning can also be applied in COPD will be discussed. The technology of mass spectrometry has developed into an advanced technology for proteome research detecting mutated peptides or proteins and finding relevant molecular mechanisms that will enable predicting the response to immunotherapy in COPD and lung cancer patients.
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Antígenos/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Proteômica , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/imunologia , Sequência de Aminoácidos , Antígenos/química , Antígenos/imunologia , Humanos , Neoplasias Pulmonares/complicações , Neoplasias Pulmonares/mortalidade , Mutação de Sentido Incorreto , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/mortalidadeRESUMO
Although many patients are cured from prostate cancer (PCa) by surgery only, there are still patients who will experience rising prostate-specific antigen (PSA) levels after surgery, a condition known as biochemical recurrence (BCR). Novel protein prognostic markers in PCa tissue might enable finding better treatment for those patients experiencing BCR with a high chance of metastasis. In this study, we aimed to identify altered proteins in prostate cancer tissue, and to evaluate their potential role as prognostic markers. We used two proteomics strategies to analyse 34 prostate tumours (PCa) and 33 normal adjacent prostate (NAP) tissues. An independent cohort of 481 samples was used to evaluate the expression of three proteins: AGR2, FASN and LOX5 as prognostic markers of the disease. Tissue microarray immunohistochemical staining indicated that a low percentage of positive tumour cells for AGR2 (HR (95% CI) = 0.61 (0.43-0.93)), and a low percentage of positive tumour cells for LOX5 expression (HR (95% CI) = 2.53 (1.23-5.22)) are predictors of BCR after RP. In contrast, FASN expression had no prognostic value for PCa.
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Infections with typhoidal Salmonella isolates that are resistant to fluoroquinolone antibiotics have become very common in several Asian countries. In the majority of these cases, resistance to fluoroquinolone-based antibiotics is associated with genetic mutations in the quinolone resistance-determining region (QRDR) of the bacterial DNA gyrase gene gyrA. The objective of this study was to detect these amino acid substitutions by high-resolution mass spectrometry instead of sequencing of the gyrA gene. A liquid chromatography-mass spectrometry (LC-MS) methodology was developed and evaluated for the detection of amino acid substitutions in the GyrA protein of 23 typhoidal Salmonella isolates. These isolates included typhoidal Salmonella that possessed different antibiotic sensitivities to fluoroquinolone antibiotics. The LC-MS methodology correctly identified peptide sequences associated with phenotypic QRDR mutations of the GyrA protein in all 23 phenotypically diverse typhoidal Salmonella isolates tested. In conclusion, a reliable and rapid LC-MS methodology has been developed that is able to identify GyrA QRDR mutations that are involved in the development of fluoroquinolone resistance in typhoidal Salmonella spp. Furthermore, this 'proof of principle' study indicates the potential usefulness of LC-MS in future detection of antibiotic resistance.
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Substituição de Aminoácidos , Antibacterianos/farmacologia , DNA Girase/análise , Fluoroquinolonas/farmacologia , Espectrometria de Massas/métodos , Salmonella typhi/efeitos dos fármacos , Cromatografia Líquida , Humanos , Proteínas Mutantes/análise , Salmonella typhi/química , Salmonella typhi/isolamento & purificação , Fatores de TempoRESUMO
BACKGROUND: Glioblastoma is the most malignant tumor of the central nervous system and still lacks effective treatment. This study explores mutational biomarkers of 11 drugs targeting either the RTK/Ras/PI3K, the p53 or the Rb pathway using 25 patient-derived glioblastoma stem-like cell cultures (GSCs). RESULTS: We found that TP53 mutated GSCs were approximately 3.5 fold more sensitive to dual inhibition of mammalian target of rapamycin complex 1 and 2 (mTORC1/2) compared to wild type GSCs. We identified that Bcl-2(Thr56/Ser70) phosphorylation contributed to the resistance of TP53 wild type GSCs against dual mTORC1/2 inhibition. The Bcl-2 inhibitor ABT-263 (navitoclax) increased sensitivity to the mTORC1/2 inhibitor AZD8055 in TP53 wild type GSCs, while sensitivity to AZD8055 in TP53 mutated GSCs remained unchanged. CONCLUSION: Our data suggest that Bcl-2 confers resistance to mTORC1/2 inhibitors in TP53 wild type GSCs and that combined inhibition of both mTORC1/2 and Bcl-2 is worthwhile to explore further in TP53 wild type glioblastomas, whereas in TP53 mutated glioblastomas dual mTORC1/2 inhibitors should be explored.