RESUMO
An inverted pH gradient across the cell membranes is a typical feature of malignant cancer cells that are characterized by extracellular acidosis and cytosol alkalization. These dysregulations are able to create a unique milieu that favors tumor progression, metastasis and chemo/immune-resistance traits of solid tumors. A key event mediating tumor cell pH alterations is an aberrant activation of ion channels and proton pumps such as (H+)-vacuolar-ATPase (V-ATPase). TM9SF4 is a poorly characterized transmembrane protein that we have recently shown to be related to cannibal behavior of metastatic melanoma cells. Here, we demonstrate that TM9SF4 represents a novel V-ATPase-associated protein involved in V-ATPase activation. We have observed in HCT116 and SW480 colon cancer cell lines that TM9SF4 interacts with the ATP6V1H subunit of the V-ATPase V1 sector. Suppression of TM9SF4 with small interfering RNAs strongly reduces assembly of V-ATPase V0/V1 sectors, thus reversing tumor pH gradient with a decrease of cytosolic pH, alkalization of intracellular vesicles and a reduction of extracellular acidity. Such effects are associated with a significant inhibition of the invasive behavior of colon cancer cells and with an increased sensitivity to the cytotoxic effects of 5-fluorouracil. Our study shows for the first time the important role of TM9SF4 in the aberrant constitutive activation of the V-ATPase, and the development of a malignant phenotype, supporting the potential use of TM9SF4 as a target for future anticancer therapies.
Assuntos
Neoplasias do Colo/patologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Proteínas de Membrana/metabolismo , Invasividade Neoplásica/patologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Western Blotting , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Concentração de Íons de Hidrogênio , Imunoprecipitação , Microscopia Confocal , Reação em Cadeia da Polimerase , TransfecçãoRESUMO
TNF-related apoptosis-inducing ligand or Apo2L (Apo2L/TRAIL) is a promising anti-cancer drug owing to its ability to trigger apoptosis by binding to TRAIL-R1 or TRAIL-R2, two membrane-bound receptors that are often expressed by tumor cells. TRAIL can also bind non-functional receptors such as TRAIL-R4, but controversies still exist regarding their potential to inhibit TRAIL-induced apoptosis. We show here that TRAIL-R4, expressed either endogenously or ectopically, inhibits TRAIL-induced apoptosis. Interestingly, the combination of chemotherapeutic drugs with TRAIL restores tumor cell sensitivity to apoptosis in TRAIL-R4-expressing cells. This sensitization, which mainly occurs at the death-inducing signaling complex (DISC) level, through enhanced caspase-8 recruitment and activation, is compromised by c-FLIP expression and is independent of the mitochondria. Importantly, TRAIL-R4 expression prevents TRAIL-induced tumor regression in nude mice, but tumor regression induced by TRAIL can be restored with chemotherapy. Our results clearly support a negative regulatory function for TRAIL-R4 in controlling TRAIL signaling, and unveil the ability of TRAIL-R4 to cooperate with c-FLIP to inhibit TRAIL-induced cell death.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo , Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Proteínas Ligadas por GPI/metabolismo , Humanos , Modelos Biológicos , Neoplasias/tratamento farmacológico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Membro 10c de Receptores do Fator de Necrose Tumoral , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagem , Receptores Chamariz do Fator de Necrose Tumoral/antagonistas & inibidores , Receptores Chamariz do Fator de Necrose Tumoral/genéticaRESUMO
CD95(APO-1/Fas)-mediated apoptosis of bystander uninfected T cells exerts a major role in the HIV-1-mediated CD4+ T-cell depletion. HIV-1 gp120 has a key role in the induction of sensitivity of human lymphocytes to CD95-mediated apoptosis through its interaction with the CD4 receptor. Recently, we have shown the importance of CD95/ezrin/actin association in CD95-mediated apoptosis. In this study, we explored the hypothesis that the gp120-mediated CD4 engagement could be involved in the induction of susceptibility of primary human T lymphocytes to CD95-mediated apoptosis through ezrin phosphorylation and ezrin-to-CD95 association. Here, we show that gp120/IL-2 combined stimuli, as well as the direct CD4 triggering, on human primary CD4(+)T lymphocytes induced an early and stable ezrin activation through phosphorylation, consistent with the induction of ezrin/CD95 association and susceptibility to CD95-mediated apoptosis. Our results provide a new mechanism through which HIV-1-gp120 may predispose resting CD4(+)T cell to bystander CD95-mediated apoptosis and support the key role of ezrin/CD95 linkage in regulating susceptibility to CD95-mediated apoptosis.