RESUMEN
Mutations to fibroblast growth factor receptor 3 (FGFR3) and phosphatase and tensin homologue (PTEN) signalling pathway components (for example, PTEN loss, PIK3CA, AKT1, TSC1/2) are common in bladder cancer, yet small-molecule inhibitors of these nodes (FGFR/PTENi) show only modest activity in preclinical models. As activation of autophagy is proposed to promote survival under FGFR/PTENi, we have investigated this relationship in a panel of 18 genetically diverse bladder cell lines. We found that autophagy inhibition does not sensitise bladder cell lines to FGFR/PTENi, but newly identify an autophagy-independent cell death synergy in FGFR3-mutant cell lines between mTOR (mammalian target of rapamycin) pathway inhibitors and chloroquine (CQ)-an anti-malarial drug used as a cancer therapy adjuvant in over 30 clinical trials. The mechanism of synergy is consistent with lysosomal cell death (LCD), including cathepsin-driven caspase activation, and correlates with suppression of cSREBP1 and cholesterol biosynthesis in sensitive cell lines. Remarkably, loss of viability can be rescued by saturating cellular membranes with cholesterol or recapitulated by statin-mediated inhibition, or small interfering RNA knockdown, of enzymes regulating cholesterol metabolism. Modulation of CQ-induced cell death by atorvastatin and cholesterol is reproduced across numerous cell lines, confirming a novel and fundamental role for cholesterol biosynthesis in regulating LCD. Thus, we have catalogued the molecular events underlying cell death induced by CQ in combination with an anticancer therapeutic. Moreover, by revealing a hitherto unknown aspect of lysosomal biology under stress, we propose that suppression of cholesterol metabolism in cancer cells should elicit synergy with CQ and define a novel approach to future cancer treatments.
Asunto(s)
Cloroquina/farmacología , Colesterol/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Atorvastatina/farmacología , Autofagia , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Lisosomas/metabolismo , Fosfohidrolasa PTEN/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/fisiología , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
BACKGROUND: Activation of wild-type p53 with the small molecule sirtuin inhibitor Tenovin-6 (Tnv-6) induces p53-dependent apoptosis in many malignant cells. In contrast, Tnv-6 reduces chronic lymphocytic leukaemia (CLL) cell viability with dysregulation of autophagy, without increasing p53-pathway activity. METHODS: Here, we have investigated whether a quiescent phenotype (unique to CLL) determines the Tnv-6 response, by comparing the effects of Tnv-6 on activated and proliferating CLL. We further studied if these responses are p53-dependent. RESULTS: Unlike quiescent cells, cell death in activated cultures treated with Tnv-6 was consistently associated with p53 upregulation. However, p53 acetylation remained unchanged, without caspase-3 cleavage or apoptosis on electron microscopy. Instead, cellular ultrastructure and protein profiles indicated autophagy inhibition, with reduced ubiquitin-proteasome activity. In specimens with mutant TP53 cultured with Tnv-6, changes in the autophagy-associated protein LC3 occurred independently of p53. Cells treated with Tnv-6 analogues lacking sirtuin inhibitory activity had attenuated LC3 lipidation compared with Tnv-6 (Pîº0.01), suggesting that autophagy dysregulation occurs predominantly through an effect on sirtuins. CONCLUSION: These cell cycle and p53-independent anti-leukaemic mechanisms potentially offer novel therapeutic approaches to target leukaemia-sustaining cells in CLL, including in disease with p53-pathway dysfunction. Whether targets in addition to sirtuins contribute to autophagy dysregulation by Tnv-6, requires further investigation.
Asunto(s)
Autofagia/fisiología , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Proteína p53 Supresora de Tumor/metabolismo , Anciano , Anciano de 80 o más Años , Apoptosis/efectos de los fármacos , Apoptosis/genética , Autofagia/efectos de los fármacos , Autofagia/genética , Benzamidas/farmacología , Caspasa 3/genética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Ciclo Celular/fisiología , Proliferación Celular/efectos de los fármacos , Humanos , Interleucina-2/farmacología , Leucemia Linfocítica Crónica de Células B/genética , Proteínas Asociadas a Microtúbulos/genética , Persona de Mediana Edad , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína p53 Supresora de Tumor/genética , Ubiquitina/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Phospholipase D (PLD)1 was phosphorylated in vivo and by an associated kinase in vitro following immunoprecipitation. Both phosphorylation events were greatly reduced in a catalytically inactive point mutant in which the serine residue at position 911 was converted into alanine (S911A). The kinase could be enriched from detergent-extracted brain membranes and bind and phosphorylate PLD1 that was immunoprecipitated from COS-7 cells. Using in-gel kinase assays we determined that the size of the kinase is approximately 40 kDa and that PLD1 is more effective than S911A in binding the kinase. Preliminary analysis of the phosphorylation sites on PLD1 suggested that the kinase belongs to the casein kinase 2 (CK2) family. Consistent with this, we found that the kinase could utilize GTP, and could be inhibited by heparin and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Membrane fractions from Chinese hamster ovary (CHO) cell lines that inducibly express PLD1 contained an endogenous kinase activity that phosphorylated PLD1 using GTP and was inhibited by DRB. Direct evidence that the kinase is CK2 came from observations that immunoprecipitates using PLD1 antibodies contained immunoreactive CK2alpha, and immunoprecipitates using CK2alpha antibodies contained immunoreactive PLD1. Co-expression of PLD1 in COS-7 cells with the two recombinant CK2 subunits, alpha or beta, suggests that the association of PLD1 with the kinase is through the beta subunit. Supporting this, phosphorylation of PLD1 by purified recombinant CK2alpha was enhanced by purified recombinant CK2beta. Assays measuring PLD1 catalytic activity following phosphorylation by CK2 suggest that this phosphorylation event does not influence PLD1-mediated hydrolysis of phosphatidylcholine in vitro.