RESUMEN
The Epstein-Barr virus (EBV) is one of the predominant tumor viruses in humans, but so far no therapeutic or prophylactic vaccination against this transforming pathogen is available. We demonstrated that heterologous prime-boost vaccination with the nuclear antigen 1 of EBV (EBNA1), either targeted to the DEC205 receptor on DCs or expressed from a recombinant modified vaccinia virus Ankara (MVA) vector, improved priming of antigen-specific CD4+ T cell help. This help supported the expansion and maintenance of EBNA1-specific CD8+ T cells that are most efficiently primed by recombinant adenoviruses that encode EBNA1. These combined CD4+ and CD8+ T cell responses protected against EBNA1-expressing T and B cell lymphomas, including lymphoproliferations that emerged spontaneously after EBNA1 expression. In particular, the heterologous EBNA1-expressing adenovirus, boosted by EBNA1-encoding MVA vaccination, demonstrated protection as a prophylactic and therapeutic treatment for the respective lymphoma challenges. Our study shows that such heterologous prime-boost vaccinations against EBV-associated malignancies as well as symptomatic primary EBV infection should be further explored for clinical development.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Linfoma/terapia , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Línea Celular Tumoral , Infecciones por Virus de Epstein-Barr/complicaciones , Vectores Genéticos , Células HEK293 , Herpesvirus Humano 4 , Humanos , Inmunoglobulina G/química , Interferón gamma/inmunología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Linfoma/inmunología , Linfoma/virología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ratas , Resultado del Tratamiento , Vacunación , Virus Vaccinia/inmunologíaRESUMEN
The bidirectional regulation of epithelial-mesenchymal transitions (EMT) is key in tumorigenesis. Rho GTPases regulate this process via canonical pathways that impinge on the stability of cell-to-cell contacts, cytoskeletal dynamics, and cell invasiveness. Here, we report that the Rho GTPase activators Vav2 and Vav3 utilize a new Rac1-dependent and miR-200c-dependent mechanism that maintains the epithelial state by limiting the abundance of the Zeb2 transcriptional repressor in breast cancer cells. In parallel, Vav proteins engage a mir-200c-independent expression prometastatic program that maintains epithelial cell traits only under 3D culture conditions. Consistent with this, the depletion of endogenous Vav proteins triggers mesenchymal features in epithelioid breast cancer cells. Conversely, the ectopic expression of an active version of Vav2 promotes mesenchymal-epithelial transitions using E-cadherin-dependent and independent mechanisms depending on the mesenchymal breast cancer cell line used. In silico analyses suggest that the negative Vav anti-EMT pathway is operative in luminal breast tumors. Gene signatures from the Vav-associated proepithelial and prometastatic programs have prognostic value in breast cancer patients.
Asunto(s)
Neoplasias de la Mama/patología , MicroARNs/genética , Proteínas Proto-Oncogénicas c-vav/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Xenoinjertos , Humanos , Ratones , Proteínas Proto-Oncogénicas c-vav/genéticaRESUMEN
Phosphatidylinositol-5 phosphate (PI5P) and other mono-phosphoinositides (mono-PIs) play second messenger roles in both physiological and pathological conditions. Despite this, their intracellular targets and mechanisms of action remain poorly characterized. Here, we show that Vav1, a protein that exhibits both Rac1 GDP/GTP exchange and adaptor activities, is positively modulated by PI5P and, possibly, other mono-PIs. Unlike other phospholipid-protein complexes, the affinity and specificity of the Vav1-lipid interaction entail a new structural solution that involves the synergistic action of the Vav1 C1 domain and an adjacent polybasic tail. This new regulatory layer, which is not conserved in the Vav family paralogs, favors the engagement of optimal Vav1 signaling outputs in lymphocytes.
Asunto(s)
Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Animales , Humanos , Células Jurkat , Ratones , Fosfatidilinositoles/metabolismo , Fosforilación , Unión Proteica/fisiología , Proteínas Proto-Oncogénicas c-vav/genética , Transducción de Señal/fisiología , Linfocitos T/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The guanosine triphosphatases of the Rho and Rac subfamilies regulate protumorigenic pathways and are activated by guanine nucleotide exchange factors (Rho GEFs), which could be potential targets for anticancer therapies. We report that two Rho GEFs, Vav2 and Vav3, play synergistic roles in breast cancer by sustaining tumor growth, neoangiogenesis, and many of the steps involved in lung-specific metastasis. The involvement of Vav proteins in these processes did not correlate with Rac1 and RhoA activity or cell migration, implying the presence of additional biological programs. Microarray analyses revealed that Vav2 and Vav3 controlled a vast transcriptional program in breast cancer cells through mechanisms that were shared between the two proteins, isoform-specific or synergistic. Furthermore, the abundance of Vav-regulated transcripts was modulated by Rac1-dependent and Rac1-independent pathways. This transcriptome encoded therapeutically targetable proteins that played nonredundant roles in primary tumorigenesis and lung-specific metastasis, such as integrin-linked kinase (Ilk), the transforming growth factor-ß family ligand inhibin ßA, cyclooxygenase-2, and the epithelial cell adhesion molecule Tacstd2. It also contained gene signatures that predicted disease outcome in breast cancer patients. These results identify possible targets for treating breast cancer and lung metastases and provide a potential diagnostic tool for clinical use.
Asunto(s)
Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas c-vav/genética , Transcriptoma , Animales , Antígenos de Neoplasias/genética , Apoptosis/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Ciclooxigenasa 2/genética , Femenino , Humanos , Subunidades beta de Inhibinas/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-vav/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Proteína de Unión al GTP rac1/genéticaRESUMEN
Guanine nucleotide-exchange factors (GEFs) activate ADP-ribosylation factor (ARF) GTPases that recruit coat proteins to membranes to initiate transport vesicle formation. Three mammalian GEFs are inhibited by brefeldin A (BFA). GBF1, predominantly associated with cis-Golgi membranes, functions early in the secretory pathway, whereas BIG1 and BIG2 act in trans-Golgi or later sites. Perturbation of endoplasmic reticulum (ER) functions can result in accumulation of unfolded or misfolded proteins that causes ER stress and unfolded protein response (UPR), with accumulation of ER stress response element (ERSE) gene products. BFA treatment of cells causes accumulation of proteins in the ER, ER stress, and ultimately apoptosis. To assess involvement of BFA-sensitive GEFs in the damage resulting from prolonged BFA treatment, HepG2 cells were selectively depleted of BIG1, BIG2, or GBF1 by using specific siRNA. Only GBF1 siRNA dramatically slowed cell growth, led to cell-cycle arrest in G(0)/G(1) phase, and caused dispersion of Golgi markers beta-COP and GM130, whereas ER structure appeared intact. GBF1 depletion also significantly increased levels of ER proteins calreticulin and protein disulfide isomerase (PDI). Proteomic analysis identified ER chaperones involved in the UPR that were significantly increased in amounts in GBF1-depleted cells. Upon ER stress, transcription factor ATF6 translocates from the ER to Golgi, where it is sequentially cleaved by site 1 and site 2 proteases, S1P and S2P, to a 50-kDa form that activates transcription of ERSE genes. Depletion of GBF1, but not BIG1 or BIG2, induced relocation of S2P from Golgi to ER with proteolysis of ATF6 followed by up-regulation of ER chaperones, mimicking a UPR response.
Asunto(s)
Apoptosis/genética , Brefeldino A/farmacología , Factores de Intercambio de Guanina Nucleótido/deficiencia , Pliegue de Proteína , Factor de Transcripción Activador 6/metabolismo , Western Blotting , Línea Celular , Citometría de Flujo , Aparato de Golgi/metabolismo , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Microscopía Fluorescente , Transporte de Proteínas , Interferencia de ARN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Brefeldin A-inhibited guanine nucleotide-exchange proteins, BIG1 and BIG2, are activators of ADP-ribosylation factor GTPases that are essential for regulating vesicular traffic among intracellular organelles. Biochemical analyses and immunofluorescence microscopy demonstrated BIG1 in nuclei as well as membranes and cytosol of serum-starved HepG2 cells. Within 20 min after addition of 8-Br-cAMP, BIG1 accumulated in nuclei, and this effect was blocked by protein kinase A (PKA) inhibitors H-89 and PKI, suggesting a dependence on PKA-catalyzed phosphorylation. BIG2 localization was not altered by cAMP, nor did BIG2 small interfering RNA influence nuclear accumulation of BIG1 induced by cAMP. Mutant BIG1 (S883A) in which Ala replaced Ser-883, a putative PKA phosphorylation site, did not move to the nucleus with cAMP addition, whereas replacement with Asp (S883D) resulted in nuclear accumulation of BIG1 without or with cAMP exposure, consistent with the mechanistic importance of a negative charge at that site. Mutation (712KPK714) of the nuclear localization signal inhibited BIG1 accumulation in nuclei, and PKA-catalyzed phosphorylation of S883, although necessary, was not sufficient for nuclear accumulation, as shown by the double mutation S883D/nuclear localization signal. A role for microtubules in cAMP-induced translocation of BIG1 is inferred from its inhibition by nocodazole. Thus, two more critical elements of BIG1 molecular structure were identified, as well as the potential function of microtubules in a novel PKA effect on BIG1 translocation.