RESUMEN
Bortezomib (bort) has improved overall survival in patients with multiple myeloma (MM), but the majority of them develop drug resistance. In this study, we demonstrate that bone marrow (BM) fibroblasts (cancer-associated fibroblasts; CAFs) from bort-resistant patients are insensitive to bort and protect the RPMI8226 and patients' plasma cells against bort-induced apoptosis. Bort triggers CAFs to produce high levels of interleukin (IL)-6, IL-8, insulin-like growth factor (IGF)-1 and transforming growth factor (TGF) ß. Proteomic studies on CAFs demonstrate that bort resistance parallels activation of oxidative stress and pro-survival autophagy. Indeed, bort induces reactive oxygen species in bort-resistant CAFs and activates autophagy by increasing light chain 3 protein (LC3)-II and inhibiting p62 and phospho-mammalian target of rapamycin. The small-interfering RNA knockdown of Atg7, and treatment with 3-methyladenine, restores bort sensitivity in bort-resistant CAFs and produces cytotoxicity in plasma cells co-cultured with CAFs. In the syngeneic 5T33 MM model, bort-treatment induces the expansion of LC3-II(+) CAFs. TGFß mediates bort-induced autophagy, and its blockade by LY2109761, a selective TßRI/II inhibitor, reduces the expression of p-Smad2/3 and LC3-II and induces apoptosis in bort-resistant CAFs. A combination of bort and LY2109761 synergistically induces apoptosis of RPMI8226 co-cultured with bort-resistant CAFs. These data define a key role for CAFs in bort resistance of plasma cells and provide the basis for a novel targeted therapeutic approach.
Asunto(s)
Antineoplásicos/farmacología , Bortezomib/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Mieloma Múltiple/tratamiento farmacológico , Pirazoles/farmacología , Pirroles/farmacología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Anciano , Anciano de 80 o más Años , Animales , Autofagia/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Combinación de Medicamentos , Resistencia a Antineoplásicos/genética , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Mieloma Múltiple/genética , Mieloma Múltiple/mortalidad , Mieloma Múltiple/patología , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/metabolismo , Células Plasmáticas/patología , Cultivo Primario de Células , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Análisis de Supervivencia , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Increased angiogenesis and an altered blood-brain barrier have been reported in the brain of dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy. To further elucidate the mechanisms underlying angiogenesis in Duchenne muscular dystrophy, in this study we evaluated whether nerve growth factor (NGF) and nerve growth factor receptors (NGFRs) are involved, then correlated NGF-NGFRs expression with vascular endothelial growth factor (VEGF) and its receptor-2 (VEGFR-2) content and matrix metalloproteinases-2 and -9 (MMP-2 and -9) activity, by confocal laser microscopy and immunohistochemistry. Results showed that neurons, astrocytes and ependymal cells were strongly labeled by NGF in mdx brain, expressing NGFRs on glial and endothelial cells. In controls, NGF faintly labeled neurons and astrocytes, whereas endothelial cells were negative for NGFRs. Immunogold electron microscopy demonstrated NGFR gold particles on endothelial cells in mdx brain, while in controls few particles were recognizable only on glial end feet. Western blotting and real time polymerase chain reaction (RT-PCR) demonstrated a higher expression of NGF and NGFR mRNA and protein in mdx brain as compared to controls, and increase of VEGF-VEGFR-2 and active MMP-2 and -9 content. Overall, these data suggest that in the brain of mdx mice, an upregulation of the NGF-NGFRs system might be involved directly, or indirectly through the activation of VEGF-VEGFR-2 and MMP-2 and -9, in the angiogenic response taking place in this pathological condition.
