RESUMO
Pancreatic ductal adenoma carcinoma (PDAC) is considered one of the deadliest solid cancers as it is usually diagnosed in advanced stages and has a poor response to treatment. The enormous effort made in the last 2 decades in the oncology field has not led to significant progress in improving early diagnosis or therapy for PDAC. The stroma of PDAC plays an active role in tumour initiation and progression and includes immune cells and stromal cells. We previously reported that Bcl2-associated athanogene (BAG3) secreted by PDAC cells activates tumour-associated macrophages to promote tumour growth. The disruption of this tumour-stroma axis by the anti-BAG3 H2L4 therapeutic antibody is sufficient to delay tumour growth and limit metastatic spreading in different PDAC preclinical models. In the present study, we examined the role of BAG3 to activate human fibroblasts (HF) in releasing cytokines capable of supporting tumour progression. Treatment of fibroblasts with recombinant BAG3 induced important changes in the organisation of the cytoskeleton of these cells and stimulated the production of interleukin-6, monocyte chemoattractant protein-1/C-C motif chemokine ligand 2, and hepatocyte growth factor. Specifically, we observed that BAG3 triggered a depolymerisation of microtubules at the periphery of the cell while they were conserved in the perinuclear area. Conversely, the vimentin-based intermediate filaments increased and spread to the edges of the cells. Finally, the conditioned medium (CM) collected from BAG3-treated HF promoted the survival, proliferation, and migration of the PDAC cells. Blocking of the PDAC-fibroblast axis by the H2L4 therapeutic anti-BAG3 antibody, resulted in inhibition of cytokine release and, consequently, the inhibition of the migratory phenotype conferred by the CM to PDAC cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/farmacologia , Proteínas Reguladoras de Apoptose/farmacologia , Carcinoma Ductal Pancreático/metabolismo , Movimento Celular/efeitos dos fármacos , Citocinas/metabolismo , Citocinas/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Humanos , Neoplasias Pancreáticas/patologia , Proteínas Recombinantes/farmacologia , Células Sf9 , SpodopteraRESUMO
Chaperone-assisted selective autophagy (CASA) is a newly-described selective tension-induced macroautophagy pathway mediated by Bag3 that is believed to be essential for mechanotransduction in skeletal muscle and to be an important regulator of the immune system. We investigated CASA machinery both in healthy and in fifteen papillomavirus-associated neoplastic bovine urothelium. The components of CASA complex, that comprises the molecular chaperones HspA8/Hsc70 and Hsp8B/Hsp22 and the cochaperones Bag3 and STUB1/CHIP, were studied by molecular, microscopic and submicroscopic investigations. CASA complex was found to be constitutively expressed in healthy bovine urothelium; its expression increased in urothelial cancers of cattle, namely thirteen papillary carcinomas and two papillary urothelial neoplasm of low malignant potential (PUNLMPs). We suggest that basal levels of CASA are important in the healthy urothelium which interfaces with the community of urinary microbiota thus representing an important epithelial cell-autonomous mechanism of antibacterial defense. Co-immunoprecipitation studies using an antibody against bovine papillomavirus E5 protein revealed that the oncoprotein co-localized with CASA complex in urothelial cancer cells. This suggests that infection by BPV E5 could influence cell behaviour by interfering with basal autophagy processes although this study did not conclusively show that this interaction increased the expression of CASA proteins. In neoplastic urothelium, CASA could be involved in regulating fundamental cellular processes such adhesion, migration, and proliferation and so might influence the biological behaviour of urothelial tumors in cattle.
Assuntos
Autofagia/fisiologia , Doenças dos Bovinos/metabolismo , Chaperonas Moleculares/metabolismo , Infecções por Papillomavirus/veterinária , Neoplasias da Bexiga Urinária/veterinária , Urotélio/virologia , Animais , Bovinos , Doenças dos Bovinos/patologia , Regulação da Expressão Gênica , Papillomaviridae/classificação , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/virologia , Urotélio/patologiaRESUMO
Bcl2-associated athanogene 3 (BAG3), is constitutively expressed in a few normal cell types, including myocytes, peripheral nerves and in the brain, and is also expressed in certain tumors. To date, the main studies about the role of BAG3 are focused on its pro-survival effect in tumors through various mechanisms that vary according to cellular type. Recently, elevated concentrations of a soluble form of BAG3 were described in patients affected by advanced stage of heart failure (HF), identifying BAG3 as a potentially useful biomarker in monitoring HF progression. Despite the finding of high levels of BAG3 in the sera of HF patients, there are no data on its possible role on the modulation of vascular tone and blood pressure levels. The aim of this study was to investigate the possible hemodynamic effects of BAG3 performing both in vitro and in vivo experiments. Through vascular reactivity studies, we demonstrate that BAG3 is capable of evoking dose-dependent vasorelaxation. Of note, BAG3 exerts its vasorelaxant effect on resistance vessels, typically involved in the blood pressure regulation. Our data further show that the molecular mechanism through which BAG3 exerts this effect is the activation of the PI3K/Akt signalling pathway leading to nitric oxide release by endothelial cells. Finally, we show that in vivo BAG3 administration is capable of regulating blood pressure and that this is dependent on eNOS regulation since this ability is lost in eNOS KO animals.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Vasos Sanguíneos/fisiologia , Homeostase , Animais , Pressão Sanguínea/efeitos dos fármacos , Vasos Sanguíneos/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Muscular/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Fenilefrina/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sístole/efeitos dos fármacos , Vasodilatação/efeitos dos fármacosRESUMO
JC virus, JCV, is a human neurotropic polyomavirus whose replication in glial cells causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). In addition, JCV possesses oncogenic activity and expression of its transforming protein, large T-antigen (T-Ag), in several experimental animals induces tumors of neural origin. Further, the presence of JCV DNA and T-Ag have been repeatedly observed in several human malignant tissues including primitive neuroectodermal tumors and glioblastomas. Earlier studies have demonstrated that Bag3, a member of the Bcl-2-associated athanogene (Bag) family of proteins, which is implicated in autophagy and apoptosis, is downregulated upon JCV infection of glial cells and that JCV T-Ag is responsible for suppressing the activity of the BAG3 promoter. Here, we investigated the possible impact of Bag3 on T-Ag expression in JCV-infected human primary glial cells as well as in cells derived from T-Ag-induced medulloblastoma in transgenic animals. Results from these studies revealed that overexpression of Bag3 drastically decreases the level of T-Ag expression by inducing the autophagic degradation of the viral protein. Interestingly, this event leads to the inhibition of JCV infection of glial cells, suggesting that the reduced levels of T-antigen seen upon the overexpression of Bag3 has a biological impact on the viral lytic cycle. Results from protein-protein interaction studies showed that T-Ag and Bag3 physically interact with each other through the zinc-finger of T-Ag and the proline rich domains of Bag3, and this interaction is important for the autophagic degradation of T-Ag. Our observations open a new avenue of research for better understanding of virus-host interaction by investigating the interplay between T-Ag and Bag3, and their impact on the development of JCV-associated diseases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos Transformantes de Poliomavirus/metabolismo , Autofagia , Vírus JC/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antígenos Transformantes de Poliomavirus/genética , Proteínas Reguladoras de Apoptose , Astrócitos/citologia , Astrócitos/metabolismo , Astrócitos/virologia , Sítios de Ligação , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , DNA Viral/genética , Dosagem de Genes , Interações Hospedeiro-Patógeno , Humanos , Vírus JC/genética , Vírus JC/fisiologia , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Ligação Proteica , Proteólise , Interferência de RNA , Células Tumorais CultivadasAssuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Neoplasias Cutâneas/genética , Adulto , Apoptose/genética , Proteínas Reguladoras de Apoptose , Feminino , Humanos , Masculino , Melanoma/fisiopatologia , Melanoma/secundário , Pessoa de Meia-Idade , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/fisiopatologiaRESUMO
Human exposures to environmental toxicants have been associated with etiology of many diseases including inflammation, cancer, and cardiovascular and neurodegenerative disorders. To counteract the detrimental effect of environmental insults, mammalian cells have evolved a hierarchy of sophisticated sensing and signaling mechanisms to turn on or off endogenous antioxidant responses accordingly. One of the major cellular antioxidant responses is the induction of antioxidative and carcinogen-detoxification enzymes through the cytoplasmic oxidative stress system (Nrf2-Keap1) activated by a variety of natural and synthetic chemopreventive agents. Under normal conditions, Keap1 anchors the Nrf2 transcription factor within the cytoplasm targeting it for ubiquitination and proteasomal degradation to maintain low levels of Nrf2 that mediate the constitutive expression of Nrf2 downstream genes. When cells are exposed to chemopreventive agents and oxidative stress, a signal involving phosphorylation and/or redox modification of critical cysteine residues in Keap1 inhibits the enzymatic activity of the Keap1-Cul3-Rbx1 E3 ubiquitin ligase complex, resulting in decreased Nrf2 ubiquitination and degradation. As a consequence, free Nrf2 translocates into the nucleus and in combination with other transcription factors (e.g., sMaf, ATF4, JunD, PMF-1) transactivates the antioxidant response elements (AREs)/electrophile response elements (EpREs) of many cytoprotective genes, as well as Nrf2 itself. Upon recovery of cellular redox homeostasis, Keap1 travels into the nucleus to dissociate Nrf2 from the ARE. Subsequently, the Nrf2-Keap1 complex is exported out of the nucleus by the nuclear export sequence (NES) in Keap1. Once in the cytoplasm, the Nrf2-Keap1 complex associates with the Cul3-Rbx1 core ubiquitin machinery, resulting in degradation of Nrf2 and termination of the Nrf2/ARE signaling pathway. The discovery of multiple nuclear localization signals (NLSs) and nuclear export signals (NESs) in Nrf2 also suggests that the nucleocytoplasm translocation of transcription factors is the consequence of a dynamic equilibrium of multivalent NLSs and NESs. On the other hand, Keap1 may provide an additional regulation of the quantity of Nrf2 both in basal and inducible conditions. This chapter summarizes the current body of knowledge regarding the molecular mechanisms through which ARE inducers (chemopreventive agents) regulate the coordinated transcriptional induction of genes encoding phase II and antioxidant enzymes as well as other defensive proteins, via the nuclear factor-erythroid 2 (NF-E2-p45)-related factor 2(Nrf2)/(ARE) signaling pathway.