RESUMO
Tumor-initiating cells contained within the aggressive brain tumor glioma (glioma stem cells, GSCs) promote radioresistance and disease recurrence. However, mechanisms of resistance are not well understood. Herein, we show that the proteome-level regulation occurring upon radiation treatment of several patient-derived GSC lines predicts their resistance status, whereas glioma transcriptional subtypes do not. We identify a mechanism of radioresistance mediated by the transfer of the metabolic enzyme NAMPT to radiosensitive cells through microvesicles (NAMPT-high MVs) shed by resistant GSCs. NAMPT-high MVs rescue the proliferation of radiosensitive GSCs and fibroblasts upon irradiation, and upon treatment with a radiomimetic drug or low serum, and increase intracellular NAD(H) levels. Finally, we show that the presence of NAMPT within the MVs and its enzymatic activity in recipient cells are necessary to mediate these effects. Collectively, we demonstrate that the proteome of GSCs provides unique information as it predicts the ability of glioma to resist radiation treatment. Furthermore, we establish NAMPT transfer via MVs as a mechanism for rescuing the proliferation of radiosensitive cells upon irradiation.
Assuntos
Glioma , Proteoma , Humanos , Proteoma/metabolismo , Proteômica , Recidiva Local de Neoplasia , Glioma/radioterapia , Glioma/metabolismo , Células-Tronco Neoplásicas/metabolismoRESUMO
IGF2BP2 binds to a number of RNA transcripts and has been suggested to function as a tumor promoter, although little is known regarding the mechanisms that regulate its roles in RNA metabolism. Here we demonstrate that IGF2BP2 binds to the 3' untranslated region of the transcript encoding ATP6V1A, a catalytic subunit of the vacuolar ATPase (v-ATPase), and serves as a substrate for the NAD+-dependent deacetylase SIRT1, which regulates how IGF2BP2 affects the stability of the ATP6V1A transcript. When sufficient levels of SIRT1 are expressed, it catalyzes the deacetylation of IGF2BP2, which can bind to the ATP6V1A transcript but does not mediate its degradation. However, when SIRT1 expression is low, the acetylated form of IGF2BP2 accumulates, and upon binding to the ATP6V1A transcript recruits the XRN2 nuclease, which catalyzes transcript degradation. Thus, the stability of the ATP6V1A transcript is significantly compromised in breast cancer cells when SIRT1 expression is low or knocked-down. This leads to a reduction in the expression of functional v-ATPase complexes in cancer cells and to an impairment in their lysosomal activity, resulting in the production of a cellular secretome consisting of increased numbers of exosomes enriched in ubiquitinated protein cargo and soluble hydrolases, including cathepsins, that together combine to promote tumor cell survival and invasiveness. These findings describe a previously unrecognized role for IGF2BP2 in mediating the degradation of a messenger RNA transcript essential for lysosomal function and highlight how its sirtuin-regulated acetylation state can have significant biological and disease consequences.
Assuntos
Neoplasias , ATPases Vacuolares Próton-Translocadoras , Humanos , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Sirtuína 1/metabolismo , RNA/metabolismo , Processos Neoplásicos , Lisossomos/genética , Lisossomos/metabolismo , Neoplasias/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The ion pump Na+, K+-ATPase (NKA) is a receptor for the cardiotonic steroid ouabain. Subsaturating concentration of ouabain triggers intracellular calcium oscillations, stimulates cell proliferation and adhesion, and protects from apoptosis. However, it is controversial whether ouabain-bound NKA is considered a signal transducer. To address this question, we performed a global analysis of protein phosphorylation in COS-7 cells, identifying 2580 regulated phosphorylation events on 1242 proteins upon 10- and 20-min treatment with ouabain. Regulated phosphorylated proteins include the inositol triphosphate receptor and stromal interaction molecule, which are essential for initiating calcium oscillations. Hierarchical clustering revealed that ouabain triggers a structured phosphorylation response that occurs in a well-defined, time-dependent manner and affects specific cellular processes, including cell proliferation and cell-cell junctions. We additionally identify regulation of the phosphorylation of several calcium and calmodulin-dependent protein kinases (CAMKs), including 2 sites of CAMK type II-γ (CAMK2G), a protein known to regulate apoptosis. To verify the significance of this result, CAMK2G was knocked down in primary kidney cells. CAMK2G knockdown impaired ouabain-dependent protection from apoptosis upon treatment with high glucose or serum deprivation. In conclusion, we establish NKA as the coordinator of a broad, tightly regulated phosphorylation response in cells and define CAMK2G as a downstream effector of NKA.-Panizza, E., Zhang, L., Fontana, J. M., Hamada, K., Svensson, D., Akkuratov, E. E., Scott, L., Mikoshiba, K., Brismar, H., Lehtiö, J., Aperia, A. Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na+, K+-ATPase control of cell adhesion, proliferation, and survival.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Ouabaína/farmacologia , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/fisiologia , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Células COS , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Chlorocebus aethiops , Regulação para Baixo/efeitos dos fármacos , Glucose/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/enzimologia , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Proteínas Quinases Ativadas por Mitógeno/genética , Modelos Moleculares , Fosforilação , Conformação Proteica , Proteínas Quinases/efeitos dos fármacos , Proteoma , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Ratos , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacosRESUMO
BACKGROUND: Tartrate-resistant acid phosphatase (TRAP/ACP5), a metalloenzyme that is characteristic for its expression in activated osteoclasts and in macrophages, has recently gained considerable focus as a driver of metastasis and was associated with clinically relevant parameters of cancer progression and cancer aggressiveness. METHODS: MDA-MB-231 breast cancer cells with different TRAP expression levels (overexpression and knockdown) were generated and characterized for protein expression and activity levels. Functional cell experiments, such as proliferation, migration and invasion assays were performed as well as global phosphoproteomic and proteomic analysis was conducted to connect molecular perturbations to the phenotypic changes. RESULTS: We identified an association between metastasis-related properties of TRAP-overexpressing MDA-MB-231 breast cancer cells and a TRAP-dependent regulation of Transforming growth factor (TGFß) pathway proteins and Cluster of differentiation 44 (CD44). Overexpression of TRAP increased anchorage-independent and anchorage-dependent cell growth and proliferation, induced a more elongated cellular morphology and promoted cell migration and invasion. Migration was increased in the presence of the extracellular matrix (ECM) proteins osteopontin and fibronectin and the basement membrane proteins collagen IV and laminin I. TRAP-induced properties were reverted upon shRNA-mediated knockdown of TRAP or treatment with the small molecule TRAP inhibitor 5-PNA. Global phosphoproteomics and proteomics analyses identified possible substrates of TRAP phosphatase activity or signaling intermediates and outlined a TRAP-dependent regulation of proteins involved in cell adhesion and ECM organization. Upregulation of TGFß isoform 2 (TGFß2), TGFß receptor type 1 (TßR1) and Mothers against decapentaplegic homolog 2 (SMAD2), as well as increased intracellular phosphorylation of CD44 were identified upon TRAP perturbation. Functional antibody-mediated blocking and chemical inhibition demonstrated that TRAP-dependent migration and proliferation is regulated via TGFß2/TßR, whereas proliferation beyond basal levels is regulated through CD44. CONCLUSION: Altogether, TRAP promotes metastasis-related cell properties in MDA-MB-231 breast cancer cells via TGFß2/TßR and CD44, thereby identifying a potential signaling mechanism associated to TRAP action in breast cancer cells.
Assuntos
Receptores de Hialuronatos/metabolismo , Fosfatase Ácida Resistente a Tartarato/fisiologia , Fator de Crescimento Transformador beta2/fisiologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Forma Celular , Feminino , Humanos , Fosforilação , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Transdução de SinaisRESUMO
A subset of Non-Small Cell Lung Carcinoma (NSCLC) carries chromosomal rearrangements involving the Anaplastic Lymphoma Kinase (ALK) gene. ALK-rearranged NSCLC are typically adenocarcinoma characterized by a solid signet-ring cell pattern that is frequently associated with a metastatic phenotype. Recent reports linked the presence of ALK rearrangement to an epithelial-mesenchymal transition (EMT) phenotype in NSCLC, but the extent and the mechanisms of an ALK-mediated EMT in ALK-rearranged NSCLC are largely unknown. We found that the ALK-rearranged H2228 and DFCI032, but not the H3122, cell lines displayed a mesenchymal phenotype. In these cell lines, oncogenic ALK activity dictated an EMT phenotype by directly suppressing E-cadherin and up-regulating vimentin expression, as well as expression of other genes involved in EMT. We found that the epithelial splicing regulatory protein 1 (ESRP1), a key regulator of the splicing switch during EMT, was repressed by EML4-ALK activity. The treatment of NSCLC cells with ALK tyrosine kinase inhibitors (TKIs) led to up-regulation of ESRP1 and E-cadherin, thus reverting the phenotype from mesenchymal to epithelial (MET). Consistently, ESRP1 knock-down impaired E-cadherin up-regulation upon ALK inhibition, whereas enforced expression of ESRP1 was sufficient to increase E-cadherin expression. These findings demonstrate an ALK oncogenic activity in the regulation of an EMT phenotype in a subset of NSCLC with potential implications for the biology of ALK-rearranged NSCLC in terms of metastatic propensity and resistance to therapy.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/enzimologia , Proteínas de Ligação a RNA/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Quinase do Linfoma Anaplásico , Animais , Antígenos CD , Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Caderinas/genética , Caderinas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Proteínas de Ligação a RNA/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Vimentina/genética , Vimentina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Non-small cell lung cancer (NSCLC) harboring chromosomal rearrangements of the anaplastic lymphoma kinase (ALK) gene is treated with ALK tyrosine kinase inhibitors (TKI), but the treatment is successful for only a limited amount of time; most patients experience a relapse due to the development of drug resistance. Here, we show that a vaccine against ALK induced a strong and specific immune response that both prophylactically and therapeutically impaired the growth of ALK-positive lung tumors in mouse models. The ALK vaccine was efficacious also in combination with ALK TKI treatment and significantly delayed tumor relapses after TKI suspension. We found that lung tumors containing ALK rearrangements induced an immunosuppressive microenvironment, regulating the expression of PD-L1 on the surface of lung tumor cells. High PD-L1 expression reduced ALK vaccine efficacy, which could be restored by administration of anti-PD-1 immunotherapy. Thus, combinations of ALK vaccine with TKIs and immune checkpoint blockade therapies might represent a powerful strategy for the treatment of ALK-driven NSCLC.
Assuntos
Vacinas Anticâncer/imunologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/terapia , Inibidores de Proteínas Quinases/uso terapêutico , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/imunologia , Quinase do Linfoma Anaplásico , Animais , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Crizotinibe , Humanos , Neoplasias Pulmonares/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/imunologia , Pirazóis/imunologia , Pirazóis/uso terapêutico , Piridinas/imunologia , Piridinas/uso terapêutico , Microambiente Tumoral/imunologia , Vacinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
About one-third of oestrogen receptor alpha-positive breast cancer patients treated with tamoxifen relapse. Here we identify the nuclear receptor retinoic acid receptor alpha as a marker of tamoxifen resistance. Using quantitative mass spectrometry-based proteomics, we show that retinoic acid receptor alpha protein networks and levels differ in a tamoxifen-sensitive (MCF7) and a tamoxifen-resistant (LCC2) cell line. High intratumoural retinoic acid receptor alpha protein levels also correlate with reduced relapse-free survival in oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen solely. A similar retinoic acid receptor alpha expression pattern is seen in a comparable independent patient cohort. An oestrogen receptor alpha and retinoic acid receptor alpha ligand screening reveals that tamoxifen-resistant LCC2 cells have increased sensitivity to retinoic acid receptor alpha ligands and are less sensitive to oestrogen receptor alpha ligands compared with MCF7 cells. Our data indicate that retinoic acid receptor alpha may be a novel therapeutic target and a predictive factor for oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Recidiva Local de Neoplasia , Receptores do Ácido Retinoico/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Hormonais/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Receptor alfa de Estrogênio/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Especificidade de Órgãos , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Análise de Sobrevida , Tamoxifeno/uso terapêuticoRESUMO
SDH genes, encoding succinate dehydrogenase, act as tumour suppressor genes, linking mitochondrial dysfunction with tumourigenesis. Heterozygous germline mutations in SDHA, SDHB, SDHC, SDHD and in the assembly factor encoding gene SDHAF2 have all been shown to predispose to heritable endocrine neoplasias such as pheochromocytomas (PHEO) and paragangliomas (PGLs) called 'PHEO-PGL syndrome'. SDH genes mutations, in addition to deletions or truncations which are most likely pathogenic, often include missense substitutions which can be of uncertain significance. Unclassified missense substitutions may be difficult to interpret unless the cause-effect link between mutation and the disease is established by functional and in silico studies or by the familial segregation with the phenotype. Using the yeast model, here, we report functional investigations on several missense SDH mutations found in patients affected by pheochromocytomas or paragangliomas. The aim of this study was to evaluate whether and to which extent the yeast model may be useful for establishing the pathological significance of missense SDH mutations in humans. The results of our study demonstrate that the yeast is a good functional model to validate the pathogenic significance of SDHB missense mutations while, for missense mutations in SDHC and SDHD genes, the model can be informative only when the variation involves a conserved residue in a conserved domain.
Assuntos
Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Paraganglioma/genética , Feocromocitoma/genética , Succinato Desidrogenase/genética , Teste de Complementação Genética , Humanos , Fosforilação Oxidativa , Estresse Oxidativo , Paraganglioma/enzimologia , Fenótipo , Feocromocitoma/enzimologia , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , SíndromeRESUMO
Mutations of succinate dehydrogenase (SDH) subunits B, C and D are associated to pheochromocytoma/paraganglioma (PGL) development. The mechanisms linking SDH mutations to tumorigenesis are currently unknown. We report a novel germline missense SDHB mutation (C191Y) in a patient affected by a glomus tumor. The missense mutation hits an amino acid residue conserved from mammals to the yeast Saccharomyces cerevisiae. The pathogenic significance of the human mutation was validated in a yeast model. SDH2(C184Y) mutant allele equivalent to human SDHB(C191Y) did not restore the OXPHOS phenotype of the Deltasdh2 null mutant. In the mutant, SDH activity was also abolished along with a reduction in respiration. Sensitivity to oxidative stress was increased in the mutant, as revealed by reduced growth in the presence of menadione. Remarkably, the frequency of petite colony formation was increased in the mutant yeast strain, indicating an increased mtDNA mutability. Histochemistry demonstrates that SDH activity was selectively absent in the patient tumor tissue. Overall, our results demonstrate that the C191Y SDHB mutation suppresses SDH enzyme activity leading to increased ROS formation and mtDNA mutability in our yeast model. These findings further our understanding of the mechanisms underlying PGL development and point to the yeast model as a valid tool to investigate on the possible pathogenic relevance of SDH novel mutations and/or rare polymorphism.