RESUMEN
In glioblastoma (GBM), heterogeneous expression of amplified and mutated epidermal growth factor receptor (EGFR) presents a substantial challenge for the effective use of EGFR-directed therapeutics. Here we demonstrate that heterogeneous expression of the wild-type receptor and its constitutively active mutant form, EGFRvIII, limits sensitivity to these therapies through an interclonal communication mechanism mediated by interleukin-6 (IL-6) cytokine secreted from EGFRvIII-positive tumor cells. IL-6 activates a NF-κB signaling axis in a paracrine and autocrine manner, leading to bromodomain protein 4 (BRD4)-dependent expression of the prosurvival protein survivin (BIRC5) and attenuation of sensitivity to EGFR tyrosine kinase inhibitors (TKIs). NF-κB and survivin are coordinately up-regulated in GBM patient tumors, and functional inhibition of either protein or BRD4 in in vitro and in vivo models restores sensitivity to EGFR TKIs. These results provide a rationale for improving anti-EGFR therapeutic efficacy through pharmacological uncoupling of a convergence point of NF-κB-mediated survival that is leveraged by an interclonal circuitry mechanism established by intratumoral mutational heterogeneity.
Asunto(s)
Resistencia a Antineoplásicos/genética , Glioblastoma/fisiopatología , FN-kappa B/genética , FN-kappa B/metabolismo , Transducción de Señal/genética , Animales , Comunicación Celular , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Interleucina-6/metabolismo , Ratones , Ratones Desnudos , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Hepatitis delta virus (HDV) is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in E. coli of His-tagged small antigen of HDV (S-HDAg), its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose) resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 µg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 µg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 107. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy.