RESUMO
Glioblastoma (GBM) is currently the most aggressive form of brain tumor identified, and STAT3 is known to play an important role in gliomagenesis. Moreover, while several studies have used pharmacological approaches to modulate STAT3 activity, the results have been contradictory. In this study, expressions of STAT3, pSTAT3 (Y705), and pSTAT3 (S727) were evaluated using immunohistochemistry assays of tissue microarrays containing non-neoplastic tissue (NN, n=12), grade II astrocytomas (n=33), grade III astrocytomas (n=12), and GBM (n=85) specimens. In GBM specimens, STAT3 was overexpressed and exhibited greater nuclear localization compared with lower grade astrocytomas and NN. Conversely, nuclear localization of pSTAT3 (Y705) and pSTAT3 (S727) exhibited a similar phenotype in both GBMs and NNs. MET was also detected as a non-canonical pathway marker for STAT3. For tumors with higher levels of STAT3 nuclear localization, and not pSTAT3 (Y705) and pSTAT3 (S727), these specimens exhibited increased levels of MET expression. Thus, a non-canonical pathway may mediate a proportion of the STAT3 that translocates to the nucleus. Moreover, tumors which exhibited greater nuclear localization of STAT3 corresponded with patients that presented with lower rates of recurrence-free survival and overall survival. In contrast, the phosphorylated forms of STAT3 did not correlate with patient survival. These findings suggest that phosphorylation-independent mechanisms may mediate the nuclear translocation and activation of STAT3. Further studies are needed to identify the mechanisms involved, especially those that provide targets to achieve efficient inhibition and control of GBM progression.
Assuntos
Neoplasias Encefálicas/metabolismo , Núcleo Celular/metabolismo , Glioblastoma/metabolismo , Fator de Transcrição STAT3/metabolismo , Adulto , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Fosforilação , Prognóstico , Taxa de SobrevidaRESUMO
In recent years, prion protein (PrP(C)) has been considered as a promising target molecule for cancer therapies, due its direct or indirect participation in tumor growth, metastasis, and resistance to cell death induced by chemotherapy. PrP(C) functions as a scaffold protein, forming multiprotein complexes on the plasma membrane, which elicits distinct signaling pathways involved in diverse biological phenomena and could be modulated depending on the cell type, complex composition, and organization. In addition, PrP(C) and its partners participate in self-renewal of embryonic, tissue-specific stem cells and cancer stem cells, which are suggested to be responsible for the origin, maintenance, relapse, and dissemination of tumors. Interference with protein-protein interaction has been recognized as an important therapeutic strategy in cancer; indeed, the possible interference in PrP(C) engagement with specific partners is a novel strategy. Recently, our group successfully used that approach to interfere with the interaction between PrP(C) and HSP-90/70 organizing protein (HOP, also known as stress-inducible protein 1 - STI1) to control the growth of human glioblastoma in animal models. Thus, PrP(C)-organized multicomplexes have emerged as feasible candidates for anti-tumor therapy, warranting further exploration.
Assuntos
Neoplasias/metabolismo , Príons/metabolismo , Animais , Humanos , Ligantes , Células-Tronco Neoplásicas/metabolismo , Ligação Proteica , Transdução de SinaisRESUMO
Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation. Using yeast two-hybrid screenings we identified several nuclear proteins as STI1 interactors. Overexpression of one of these interactors, PIAS1, seems to be specifically involved in STI1 nuclear retention and in directing STI1 and Hsp90 to specific sub-nuclear regions. PIAS1 and STI1 co-immunoprecipitate and PIAS1 can function as an E3 SUMO ligase for STI. Using mass spectrometry we identified five SUMOylation sites in STI1. A STI1 mutant lacking these five sites is not SUMOylated, but still accumulates in the nucleus in response to increased expression of PIAS1, suggesting the possibility that a direct interaction with PIAS1 could be responsible for STI1 nuclear retention. To test this possibility, we mapped the interaction sites between PIAS1 and STI1 using yeast-two hybrid assays and surface plasmon resonance and found that a large domain in the N-terminal region of STI1 interacts with high affinity with amino acids 450-480 of PIAS1. Knockdown of PIAS1 in astrocytes impairs the accumulation of nuclear STI1 in response to irradiation. Moreover, a PIAS1 mutant lacking the STI1 binding site is unable to increase STI1 nuclear retention. Interestingly, in human glioblastoma multiforme PIAS1 expression is increased and we found a significant correlation between increased PIAS1 expression and STI1 nuclear localization. These experiments provide evidence that direct interaction between STI1 and PIAS1 is involved in the accumulation of nuclear STI1. This retention mechanism could facilitate nuclear chaperone activity.
Assuntos
Astrócitos/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Animais , Astrócitos/citologia , Astrócitos/efeitos da radiação , Morte Celular/genética , Morte Celular/fisiologia , Morte Celular/efeitos da radiação , Núcleo Celular/metabolismo , Células Cultivadas , Dano ao DNA , Raios gama , Técnicas de Silenciamento de Genes , Células HEK293 , Haploinsuficiência , Proteínas de Choque Térmico/deficiência , Proteínas de Choque Térmico/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Inibidoras de STAT Ativados/antagonistas & inibidores , Proteínas Inibidoras de STAT Ativados/genética , Mapas de Interação de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Estresse Fisiológico , Sumoilação , Técnicas do Sistema de Duplo-HíbridoRESUMO
Prion protein (PrP) can be considered a pivotal molecule because it interacts with several partners to perform a diverse range of critical biological functions that might differ in embryonic and adult cells. In recent years, there have been major advances in elucidating the putative role of PrP in the basic biology of stem cells in many different systems. Here, we review the evidence indicating that PrP is a key molecule involved in driving different aspects of the potency of embryonic and tissue-specific stem cells in self-perpetuation and differentiation in many cell types. It has been shown that PrP is involved in stem cell self-renewal, controlling pluripotency gene expression, proliferation, and neural and cardiomyocyte differentiation. PrP also has essential roles in distinct processes that regulate tissue-specific stem cell biology in nervous and hematopoietic systems and during muscle regeneration. Results from our own investigations have shown that PrP is able to modulate self-renewal and proliferation in neural stem cells, processes that are enhanced by PrP interactions with stress inducible protein 1 (STI1). Thus, the available data reveal the influence of PrP in acting upon the maintenance of pluripotent status or the differentiation of stem cells from the early embryogenesis through adulthood.
Assuntos
Príons/metabolismo , Células-Tronco/metabolismo , Animais , Biologia Celular , Desenvolvimento Embrionário , Humanos , Camundongos , Células-Tronco/patologiaRESUMO
ADAMs are transmembrane proteins implicated in several biological functions, including cytokine and growth factor shedding, fertilization, muscle and nervous system development. Here, we show for the first time that ADAM23, which is predominantly expressed in the central nervous system, co-localizes with cellular prion protein (PrP(C)) at plasma membrane of mouse hippocampal neurons and neuroblastoma cells. Co-immunoprecipitation and pull-down assay showed a physical interaction between ADAM23 and both recombinant and endogenous PrP(C). Glycosylation seems to be not relevant to the observed interaction since both ADAM23 and PrP(C) recombinant proteins expressed in bacteria or extracted from eukaryotic cells treated with tunicamycin are still able to bind each other. In vitro binding assays also suggested that the disintegrin domain of ADAM23 is able to interact directly with PrP(C). Taken together, these findings point out PrP(C) as a novel molecular partner for ADAM23 in the nervous systems.
Assuntos
Proteínas ADAM/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas PrPC/metabolismo , Proteínas ADAM/genética , Animais , Membrana Celular/metabolismo , Células Cultivadas , Glicosilação , Hipocampo/citologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso/genética , Neuroblastoma , Neurônios/metabolismo , Proteínas PrPC/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Prion protein (PrP(C)) interaction with stress inducible protein 1 (STI1) mediates neuronal survival and differentiation. However, the function of PrP(C) in astrocytes has not been approached. In this study, we show that STI1 prevents cell death in wild-type astrocytes in a protein kinase A-dependent manner, whereas PrP(C)-null astrocytes were not affected by STI1 treatment. At embryonic day 17, cultured astrocytes and brain extracts derived from PrP(C)-null mice showed a reduced expression of glial fibrillary acidic protein (GFAP) and increased vimentin and nestin expression when compared with wild-type, suggesting a slower rate of astrocyte maturation in PrP(C)-null animals. Furthermore, PrP(C)-null astrocytes treated with STI1 did not differentiate from a flat to a process-bearing morphology, as did wild-type astrocytes. Remarkably, STI1 inhibited proliferation of both wild-type and PrP(C)-null astrocytes in a protein kinase C-dependent manner. Taken together, our data show that PrP(C) and STI1 are essential to astrocyte development and act through distinct signaling pathways.
Assuntos
Astrócitos/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células , Proteínas de Choque Térmico/metabolismo , Proteínas PrPC/metabolismo , Animais , Astrócitos/citologia , Encéfalo/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Neurológicos , Proteínas do Tecido Nervoso/metabolismo , Nestina , Proteínas PrPC/genética , Proteína Quinase C/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Vimentina/metabolismoRESUMO
The secreted cochaperone STI1 triggers activation of protein kinase A (PKA) and ERK1/2 signaling by interacting with the cellular prion (PrP(C)) at the cell surface, resulting in neuroprotection and increased neuritogenesis. Here, we investigated whether STI1 triggers PrP(C) trafficking and tested whether this process controls PrP(C)-dependent signaling. We found that STI1, but not a STI1 mutant unable to bind PrP(C), induced PrP(C) endocytosis. STI1-induced signaling did not occur in cells devoid of endogenous PrP(C); however, heterologous expression of PrP(C) reconstituted both PKA and ERK1/2 activation. In contrast, a PrP(C) mutant lacking endocytic activity was unable to promote ERK1/2 activation induced by STI1, whereas it reconstituted PKA activity in the same condition, suggesting a key role of endocytosis in the former process. The activation of ERK1/2 by STI1 was transient and appeared to depend on the interaction of the two proteins at the cell surface or shortly after internalization. Moreover, inhibition of dynamin activity by expression of a dominant-negative mutant caused the accumulation and colocalization of these proteins at the plasma membrane, suggesting that both proteins use a dynamin-dependent internalization pathway. These results show that PrP(C) endocytosis is a necessary step to modulate STI1-dependent ERK1/2 signaling involved in neuritogenesis.
Assuntos
Encéfalo/metabolismo , Endocitose/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Choque Térmico/metabolismo , Neurônios/metabolismo , Proteínas PrPC/metabolismo , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinaminas/metabolismo , Ativação Enzimática/fisiologia , Proteínas de Choque Térmico/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Técnicas de Cultura de Órgãos , Proteínas PrPC/genética , Transporte Proteico/fisiologiaRESUMO
Cellular prion protein (PrPc) has a pivotal role in prion diseases. PrPc is a specific receptor for laminin (LN) gamma1 peptide and several lines of evidence indicate that it is also involved in neural plasticity. Here we investigated whether the interaction between PrPc and LN plays a role in rat memory formation. We found that post-training intrahippocampal infusion of PrPc-derived peptides that contain the LN binding site (PrPc163-182 and PrPc173-192) or of anti-PrPc or anti-LN antibodies that inhibit PrPc-LN interaction impaired inhibitory avoidance memory retention. The amnesic effect of anti-PrPc antibodies and PrPc173-192 peptide was reversed by co-infusion of a LN gamma1 chain-derived peptide containing the PrPc-binding site, suggesting that PrPc-LN interaction is indeed crucial for memory consolidation. In addition, PrPc173-192 peptide and anti-PrPc or anti-LN antibodies also inhibited the activation of hippocampal cAMP-dependent protein kinase A (PKA) and extracellular regulated kinase (ERK1/2), two kinases that mediate the up-regulation of signaling pathways needed for consolidation of inhibitory avoidance memory. Our findings show that, through its interaction with LN, hippocampal PrPc plays a critical role in memory processing and suggest that this role is mediated by activation of both PKA and ERK1/2 signaling pathways.
Assuntos
Hipocampo/metabolismo , Laminina/metabolismo , Aprendizagem/fisiologia , Memória/fisiologia , Proteínas PrPC/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Sítios de Ligação/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Laminina/antagonistas & inibidores , Laminina/imunologia , Masculino , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Proteínas PrPC/química , Estrutura Terciária de Proteína/fisiologia , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Cancer/testis (CT) antigens are immunogenic proteins expressed in normal gametogenic tissues and in different types of tumors. CT antigens are promising candidates for cancer immunotherapy, and the identification of novel CT antigens is a prerequisite for the development of cancer vaccines. We have identified a CT antigen, named CTSP-1, with partial similarity to the breast differentiation antigen NY-BR-1. CTSP-1 presents several splicing and polyadenylation variants and has a very restricted expression pattern among normal tissues. CTSP-1 is exclusively expressed in normal testis and is aberrantly expressed in 47.6% (10 of 21) of tumor cell lines and in 44.4% (75 of 169) of tumors from different histological types. The highest percentages of positive expression were observed in melanomas (59.0%) followed by prostate (58.0%) and lung (57.0%) tumors. CTSP-1 is part of a highly conserved gene family, and members of this family also have a restricted expression pattern and similar protein structure. Antibodies against members of this gene family were detected in 10% (14 of 141) of plasma samples from patients with a wide spectrum of tumors. The highest percentages of antibody response were observed in patients with prostate (20.8%), thyroid (20.0%), and breast (16.6%) tumors. Because of its very restricted expression pattern in normal tissues and immunogenicity in different types of tumors, CTSP-1 should be considered a promising candidate for cancer immunotherapy.
Assuntos
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Neoplasias/genética , Neoplasias/imunologia , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Processamento Alternativo/genética , Animais , Antígenos de Neoplasias/classificação , Antígenos de Neoplasias/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Variação Genética/genética , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Neoplasias/classificação , Neoplasias/metabolismo , Proteínas Nucleares/classificação , Proteínas Nucleares/metabolismo , Poliadenilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Prions are composed of an isoform of a normal sialoglycoprotein called PrP(c), whose physiological role has been under investigation, with focus on the screening for ligands. Our group described a membrane 66 kDa PrP(c)-binding protein with the aid of antibodies against a peptide deduced by complementary hydropathy. Using these antibodies in western blots from two-dimensional protein gels followed by sequencing the specific spot, we have now identified the molecule as stress-inducible protein 1 (STI1). We show that this protein is also found at the cell membrane besides the cytoplasm. Both proteins interact in a specific and high affinity manner with a K(d) of 10(-7) M. The interaction sites were mapped to amino acids 113-128 from PrP(c) and 230-245 from STI1. Cell surface binding and pull-down experiments showed that recombinant PrP(c) binds to cellular STI1, and co-immunoprecipitation assays strongly suggest that both proteins are associated in vivo. Moreover, PrP(c) interaction with either STI1 or with the peptide we found that represents the binding domain in STI1 induce neuroprotective signals that rescue cells from apoptosis.