RESUMEN
Melanoma progression is associated with increased invasion and, often, decreased levels of microphthalmia-associated transcription factor (MITF). Accordingly, downregulation of MITF induces invasion in melanoma cells; however, little is known about the underlying mechanisms. Here, we report for the first time that depletion of MITF results in elevation of intracellular GTP levels and increased amounts of active (GTP-bound) RAC1, RHO-A and RHO-C. Concomitantly, MITF-depleted cells display larger number of invadopodia and increased invasion. We further demonstrate that the gene for guanosine monophosphate reductase (GMPR) is a direct MITF target, and that the partial repression of GMPR accounts mostly for the above phenotypes in MITF-depleted cells. Reciprocally, transactivation of GMPR is required for MITF-dependent suppression of melanoma cell invasion, tumorigenicity and lung colonization. Moreover, loss of GMPR accompanies downregulation of MITF in vemurafenib-resistant BRAFV600E-melanoma cells and underlies the increased invasion in these cells. Our data uncover novel mechanisms linking MITF-dependent inhibition of invasion to suppression of guanylate metabolism.
Asunto(s)
Guanosina Trifosfato/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Expresión Génica Ectópica , Matriz Extracelular/metabolismo , Femenino , GMP-Reductasa/genética , GMP-Reductasa/metabolismo , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Espacio Intracelular/metabolismo , Melanocitos/metabolismo , Melanoma/metabolismo , Melanoma/patología , Melanoma Experimental , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
It is generally accepted that intracellular oxidative stress induced by proteasome inhibitors is a byproduct of endoplasmic reticulum (ER) stress. Here we report a mechanism underlying the ability of proteasome inhibitors bortezomib (BTZ) and carfilzomib (CFZ) to directly induce oxidative and ER stresses in multiple myeloma (MM) cells via transcriptional repression of a gene encoding mitochondrial thioredoxin reductase (TXNRD2). TXNRD2 is critical for maintenance of intracellular red-ox status and detoxification of reactive oxygen species. Depletion of TXNRD2 to the levels detected in BTZ- or CFZ-treated cells causes oxidative stress, ER stress and death similar to those induced by proteasome inhibitors. Reciprocally, restoration of near-wildtype TXNRD2 amounts in MM cells treated with proteasome inhibitors reduces oxidative stress, ER stress and cell death by ~46%, ~35% and ~50%, respectively, compared with cells with unrestored TXNRD2 levels. Moreover, cells from three MM cell lines selected for resistance to BTZ demonstrate elevated levels of TXNRD2, indirectly confirming its functional role in BTZ resistance. Accordingly, ectopic expression of TXNRD2 in MM cell xenografts in immunocompromised mice blunts therapeutic effects of BTZ. Our data identify TXNRD2 as a potentially clinically relevant target, inhibition of which is critical for proteasome inhibitor-dependent cytotoxicity, oxidative stress and ER stress.
Asunto(s)
Mieloma Múltiple/tratamiento farmacológico , Inhibidores de Proteasoma/uso terapéutico , Tiorredoxina Reductasa 2/fisiología , Animales , Apoptosis/efectos de los fármacos , Bortezomib/farmacología , Línea Celular Tumoral , Estrés del Retículo Endoplásmico/efectos de los fármacos , Femenino , Humanos , Ratones , Mieloma Múltiple/enzimología , Mieloma Múltiple/patología , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Malignant melanoma possesses one of the highest metastatic potentials among human cancers. Acquisition of invasive phenotypes is a prerequisite for melanoma metastases. Elucidation of the molecular mechanisms underlying melanoma invasion will greatly enhance the design of novel agents for melanoma therapeutic intervention. Here, we report that guanosine monophosphate synthase (GMPS), an enzyme required for the de novo biosynthesis of GMP, has a major role in invasion and tumorigenicity of cells derived from either BRAF(V600E) or NRAS(Q61R) human metastatic melanomas. Moreover, GMPS levels are increased in metastatic human melanoma specimens compared with primary melanomas arguing that GMPS is an attractive candidate for anti-melanoma therapy. Accordingly, for the first time we demonstrate that angustmycin A, a nucleoside-analog inhibitor of GMPS produced by Streptomyces hygroscopius efficiently suppresses melanoma cell invasion in vitro and tumorigenicity in immunocompromised mice. Our data identify GMPS as a powerful driver of melanoma cell invasion and warrant further investigation of angustmycin A as a novel anti-melanoma agent.
Asunto(s)
Guanosina Monofosfato/metabolismo , Melanoma/enzimología , Nucleotidiltransferasas/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacología , Animales , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Immunoblotting , Inmunohistoquímica , Melanoma/patología , Ratones , Ratones SCID , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/genética , Neoplasias Cutáneas , Melanoma Cutáneo MalignoRESUMEN
Cancer progression, response to therapy and metastasis depend on tumor microenvironment. Integrins are cell-adhesion receptors that mediate interactions of cells with extracellular matrix. The αv-ß-family of integrins contributes to tumorigenesis, response to therapy and cancer stem cell biology. Thus, understanding the function of specific integrins in cancer is critical for the development of therapeutic approaches targeting integrins. The study investigated the role of integrin ß5 in breast carcinomas by depleting integrin ß5 using RNA interference and reexpression of integrin ß5. Depletion of integrin ß5 in triple-negative breast carcinoma cells markedly reduced tumor take, growth and tumor angiogenesis, whereas reexpression of integrin ß5 rescued this phenotype. Reduction in tumor angiogenesis is associated with lower expression of vascular endothelial growth factor-A in integrin ß5-depleted tumors. Tumor cells deficient in integrin ß5 have lower migration and proliferative capacities. Biochemical assays revealed that integrin ß5 mediates the Src-focal adhesion kinase and MEK-extracellular signal-regulated kinase signaling events that operate independently, and inhibition of these pathways phenocopies integrin ß5 deficiency. Breast carcinoma cells express high levels of integrin ß5, whereas expression of integrin ß3 is limited to stromal compartments and integrin ß6 is lost in metastatic cells. Together, these findings show a critical role for integrin ß5 in the tumorigenic potential of breast carcinoma cells and therapeutic targeting of integrin ß5 is especially attractive for triple-negative breast carcinomas, which are refractory to most of the current therapies.