RESUMO
The proteasome is a validated target of cancer therapeutics. Inhibition of proteasome activity results in the activation of the unfolded protein response (UPR) characterized by phosphorylation of eukaryotic initiation factor 2α (eIF2α), global translational arrest, and increased expression of the proapoptotic CHOP (C/EBP homologous protein) protein. Defects in the UPR response has been reported to result in altered sensitivity of tumor cells to proteasome inhibitors. Here, we characterized the effects of the deubiquitinase (DUB) inhibitor VLX1570 on protein homeostasis, both at the level of the UPR and on protein translation, in acute lymphoblastic leukemia (ALL). Similar to the 20S inhibitor bortezomib, VLX1570 induced accumulation of polyubiquitinated proteins and increased expression of the chaperone Grp78/Bip in ALL cells. Both compounds induced cleavage of PARP (Poly (ADP-ribose) polymerase) in ALL cells, consistent with induction of apoptosis. However, and in contrast to bortezomib, VLX1570 treatment resulted in limited induction of the proapoptotic CHOP protein. Translational inhibition was observed by both bortezomib and VLX1570. We report that in distinction to bortezomib, suppression of translation by VXL1570 occurred at the level of elongation. Increased levels of Hsc70/Hsp70 proteins were observed on polysomes following exposure to VLX1570, possibly suggesting defects in nascent protein folding. Our findings demonstrate apoptosis induction in ALL cells that appears to be uncoupled from CHOP induction, and show that VLX1570 suppresses protein translation by a mechanism distinct from that of bortezomib.
Assuntos
Azepinas/farmacologia , Compostos de Benzilideno/farmacologia , Enzimas Desubiquitinantes/antagonistas & inibidores , Enzimas Desubiquitinantes/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Animais , Apoptose/efeitos dos fármacos , Bortezomib/farmacologia , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Dobramento de Proteína/efeitos dos fármacos , Fator de Transcrição CHOP/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Peixe-ZebraRESUMO
Although more traditionally associated with degradation and maintenance of protein homeostasis, the ubiquitin-proteasome system (UPS) has emerged as a critical component in the regulation of cancer cell growth and survival. The development of inhibitors that block the proteolytic activities of the proteasome have highlighted its suitability as a bona fide anti-cancer drug target. However, key determinants including the development of drug resistance and dose-limiting toxicity call for the identification of alternative components of the UPS for novel drug targeting. Recently the deubiquitinases (DUBs), a diverse family of enzymes that catalyze ubiquitin removal, have attracted significant interest as targets for the development of next generation UPS inhibitors. In particular, pharmacological inhibition of the proteasomal cysteine DUBs (i.e., USP14 and UCHL5) has been shown to be particularly cytotoxic to cancer cells and inhibit tumour growth in several in vivo models. In the current review we focus on the modes of action of proteasome DUB inhibitors and discus the potential of DUB inhibitors to circumvent acquired drug resistance and provide a therapeutic option for the treatment of cancer.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Inibidores de Proteassoma/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Humanos , Neoplasias/enzimologia , Inibidores de Proteassoma/administração & dosagem , Inibidores de Proteassoma/efeitos adversos , Proteases Específicas de Ubiquitina/antagonistas & inibidoresRESUMO
The proteasome-associated deubiquitinase USP14 is a potential drug target. Using an inducible USP14 knockout system in colon cancer cells, we found that USP14 depletion impedes cellular proliferation, induces cell cycle arrest, and leads to a senescence-like phenotype. Transcriptomic analysis revealed altered gene expression related to cell division and cellular differentiation. USP14 knockout cells also exhibited changes in morphology, actin distribution, and expression of actin cytoskeletal components. Increased ubiquitin turnover was observed, offset by upregulation of polyubiquitin genes UBB and UBC. Pharmacological inhibition of USP14 with IU1 increased ubiquitin turnover but did not affect cellular growth or morphology. BioGRID data identified USP14 interactors linked to actin cytoskeleton remodeling, DNA damage repair, mRNA splicing, and translation. In conclusion, USP14 loss in colon cancer cells induces a transient quiescent cancer phenotype not replicated by pharmacologic inhibition of its deubiquitinating activity.
Assuntos
Proliferação de Células , Senescência Celular , Neoplasias Colorretais , Ubiquitina Tiolesterase , Humanos , Senescência Celular/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Linhagem Celular Tumoral , Fenótipo , Complexo de Endopeptidases do Proteassoma/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Ubiquitina/metabolismoRESUMO
Dienone compounds have been demonstrated to display tumor-selective anti-cancer activity independently of the mutational status of TP53. Previous studies have shown that cell death elicited by this class of compounds is associated with inhibition of the ubiquitin-proteasome system (UPS). Here we extend previous findings by showing that the dienone compound b-AP15 inhibits proteasomal degradation of long-lived proteins. We show that exposure to b-AP15 results in increased association of the chaperones VCP/p97/Cdc48 and BAG6 with proteasomes. Comparisons between the gene expression profile generated by b-AP15 to those elicited by siRNA showed that knock-down of the proteasome-associated deubiquitinase (DUB) USP14 is the closest related to drug response. USP14 is a validated target for b-AP15 and we show that b-AP15 binds covalently to two cysteines, Cys203 and Cys257, in the ubiquitin-binding pocket of the enzyme. Consistent with this, deletion of USP14 resulted in decreased sensitivity to b-AP15. Targeting of USP14 was, however, found to not fully account for the observed proteasome inhibition. In search for additional targets, we utilized genome-wide CRISPR/Cas9 library screening and Proteome Integral Solubility Alteration (PISA) to identify mechanistically essential genes and b-AP15 interacting proteins respectively. Deletion of genes encoding mitochondrial proteins decreased the sensitivity to b-AP15, suggesting that mitochondrial dysfunction is coupled to cell death induced by b-AP15. Enzymes known to be involved in Phase II detoxification such as aldo-ketoreductases and glutathione-S-transferases were identified as b-AP15-targets using PISA. The finding that different exploratory approaches yielded different results may be explained in terms of a "target" not necessarily connected to the "mechanism of action" thus highlighting the importance of a holistic approach in the identification of drug targets. We conclude that b-AP15, and likely also other dienone compounds of the same class, affect protein degradation and proteasome function at more than one level.
RESUMO
Dienone compounds with a 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore have been widely reported to show tumor cell selectivity. These compounds target the ubiquitin-proteasome system (UPS), known to be essential for the viability of tumor cells. The induction of oxidative stress, depletion of glutathione, and induction of high-molecular-weight (HMW) complexes have also been reported. We here examined the response of acute myeloid leukemia (AML) cells to the dienone compound VLX1570. AML cells have relatively high protein turnover rates and have also been reported to be sensitive to depletion of reduced glutathione. We found AML cells of diverse cytogenetic backgrounds to be sensitive to VLX1570, with drug exposure resulting in an accumulation of ubiquitin complexes, induction of ER stress, and the loss of cell viability in a dose-dependent manner. Caspase activation was observed but was not required for the loss of cell viability. Glutathione depletion was also observed but did not correlate to VLX1570 sensitivity. Formation of HMW complexes occurred at higher concentrations of VLX1570 than those required for the loss of cell viability and was not enhanced by glutathione depletion. To study the effect of VLX1570 we developed a zebrafish PDX model of AML and confirmed antigrowth activity in vivo. Our results show that VLX1570 induces UPS inhibition in AML cells and encourage further work in developing compounds useful for cancer therapeutics.
Assuntos
Azepinas/farmacologia , Compostos de Benzilideno/farmacologia , Leucemia Mieloide Aguda/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/antagonistas & inibidores , Animais , Azepinas/química , Compostos de Benzilideno/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Curcumina/farmacologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glutationa/metabolismo , Heme Oxigenase-1/metabolismo , Humanos , Peso Molecular , Poliubiquitina/metabolismo , Fatores de Tempo , Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos , Peixe-Zebra/embriologiaRESUMO
Inhibitors of the 20S proteasome such as bortezomib (Velcade®) and carfilzomib (Kypriolis®) are in clinical use for the treatment of patients with multiple myeloma and mantle cell lymphoma. In an attempt to identify novel inhibitors of the ubiquitin-proteasome system (UPS) we used the connectivity map (CMap) resource, based on alterations of gene expression profiles by perturbagens, and performed COMPARE analyses of drug sensitivity patterns in the NCI60 panel. Cmap analysis identified a large number of small molecules with strong connectivity to proteasome inhibition, including both well characterized inhibitors of the 20S proteasome and molecules previously not described to inhibit the UPS. A number of these compounds have been reported to be cytotoxic to tumor cells and were tested for their ability to decrease processing of proteasome substrates. The antibiotic thiostrepton and the natural products celastrol and curcumin induced strong accumulation of polyubiquitinated proteasome substrates in exposed cells. Other compounds elicited modest increases of proteasome substrates, including the protein phosphatase inhibitor BCI-Cl and the farnesyltransferase inhibitor manumycin A, suggesting that these compounds inhibit proteasome function. Induction of chaperone expression in the absence of proteasome inhibition was observed by a number of compounds, suggesting other effects on the UPS. We conclude that the combination of bioinformatic analyses and cellular assays resulted in the identification of compounds with potential to inhibit the UPS.
Assuntos
Descoberta de Drogas , Perfilação da Expressão Gênica , Neoplasias/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Transcriptoma , Linhagem Celular Tumoral , Bases de Dados Genéticas , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Mapas de Interação de Proteínas , Transdução de SinaisRESUMO
Auranofin is a gold (I)-containing compound used for the treatment of rheumatic arthritis. Auranofin has anticancer activity in animal models and is approved for clinical trials for lung and ovarian carcinomas. Both the cytosolic and mitochondrial forms of the selenoprotein thioredoxin reductase (TrxR) are well documented targets of auranofin. Auranofin was recently reported to also inhibit proteasome activity at the level of the proteasome-associated deubiquitinases (DUBs) UCHL5 and USP14. We here set out to re-examine the molecular mechanism underlying auranofin cytotoxicity towards cultured cancer cells. The effects of auranofin on the proteasome were examined in cells and in vitro, effects on DUB activity were assessed using different substrates. The cellular response to auranofin was compared to that of the 20S proteasome inhibitor bortezomib and the 19S DUB inhibitor b-AP15 using proteomics. Auranofin was found to inhibit mitochondrial activity and to an induce oxidative stress response at IC50 doses. At 2-3-fold higher doses, auranofin inhibits proteasome processing in cells. At such supra-pharmacological concentrations USP14 activity was inhibited. Analysis of protein expression profiles in drug-exposed tumor cells showed that auranofin induces a response distinct from that of the 20S proteasome inhibitor bortezomib and the DUB inhibitor b-AP15, both of which induced similar responses. Our results support the notion that the primary mechanism of action of auranofin is TrxR inhibition and suggest that proteasome DUB inhibition is an off-target effect. Whether proteasome inhibition will contribute to the antineoplastic effect of auranofin in treated patients is unclear but remains a possibility.
Assuntos
Antineoplásicos/farmacologia , Auranofina/farmacologia , Reposicionamento de Medicamentos , Selenoproteínas/antagonistas & inibidores , Tiorredoxina Redutase 1/antagonistas & inibidores , Tiorredoxina Redutase 2/antagonistas & inibidores , Bortezomib/farmacologia , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Piperidonas/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Tiorredoxina Redutase 2/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidoresRESUMO
BACKGROUND: b-AP15/VLX1570 are small molecule inhibitors of the ubiquitin specific peptidase 14 (USP14) and ubiquitin carboxyl-terminal hydrolase 5 (UCHL5) deubiquitinases (DUBs) of the 19S proteasome. b-AP15/VLX1570 have been shown to be cytotoxic to cells resistant to bortezomib, raising the possibility that this class of drugs can be used as a second-line therapy for treatment-resistant multiple myeloma. Limited information is available with regard to potential resistance mechanisms to b-AP15/VLX1570. RESULTS: We found that b-AP15-induced cell death is cell-cycle dependent and that non-cycling tumor cells may evade b-AP15-induced cell death. Such non-cycling cells may re-enter the proliferative state to form colonies of drug-sensitive cells. Long-term selection of cells with b-AP15 resulted in limited drug resistance (~2-fold) that could be reversed by buthionine sulphoximine, implying altered glutathione (GSH) metabolism as a resistance mechanism. In contrast, drug uptake and overexpression of drug efflux transporters were found not to be associated with b-AP15 resistance. CONCLUSIONS: The proteasome DUB inhibitors b-AP15/VLX1570 are cell cycle-active. The slow and incomplete development of resistance towards these compounds is an attractive feature in view of future clinical use.
Assuntos
Apoptose/efeitos dos fármacos , Azepinas/farmacologia , Compostos de Benzilideno/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/patologia , Resistencia a Medicamentos Antineoplásicos , Inibidores de Proteassoma/farmacologia , Antineoplásicos/farmacologia , Bortezomib/farmacologia , Neoplasias do Colo/tratamento farmacológico , Glutationa/metabolismo , Humanos , Células Tumorais CultivadasRESUMO
Proteasome inhibitors have been shown to induce cell death in cancer cells by triggering an acute proteotoxic stress response characterized by accumulation of poly-ubiquitinated proteins, ER stress and the production of reactive oxygen species. The aggresome pathway has been described as an escape mechanism from proteotoxicity by sequestering toxic cellular aggregates. Here we show that b-AP15, a small-molecule inhibitor of proteasomal deubiquitinase activity, induces poly-ubiquitin accumulation in absence of aggresome formation. b-AP15 was found to affect organelle transport in treated cells, raising the possibility that microtubule-transport of toxic protein aggregates is inhibited, leading to enhanced cytotoxicity. In contrast to the antiproliferative effects of the clinically used proteasome inhibitor bortezomib, the effects of b-AP15 are not further enhanced by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). Our results suggest an inhibitory effect of b-AP15 on the transport of misfolded proteins, resulting in a lack of aggresome formation, and a strong proteotoxic stress response.
Assuntos
Piperidonas/farmacologia , Inibidores de Proteassoma/farmacologia , Proteólise/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Bortezomib/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Piperidonas/uso terapêutico , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Ubiquitina/metabolismoRESUMO
A large number of natural products have been advocated as anticancer agents. Many of these compounds contain functional groups characterized by chemical reactivity. It is not clear whether distinct mechanisms of action can be attributed to such compounds. We used a chemical library screening approach to demonstrate that a substantial fraction (~20%) of cytotoxic synthetic compounds containing Michael acceptor groups inhibit proteasome substrate processing and induce a cellular response characteristic of proteasome inhibition. Biochemical and structural analyses showed binding to and inhibition of proteasome-associated cysteine deubiquitinases, in particular ubiquitin specific peptidase 14 (USP14). The results suggested that compounds bind to a crevice close to the USP14 active site with modest affinity, followed by covalent binding. A subset of compounds was identified where cell death induction was closely associated with proteasome inhibition and that showed significant antineoplastic activity in a zebrafish embryo model. These findings suggest that proteasome inhibition is a relatively common mode of action by cytotoxic compounds containing Michael acceptor groups and help to explain previous reports on the antineoplastic effects of natural products containing such functional groups.
Assuntos
Antineoplásicos/administração & dosagem , Inibidores de Proteassoma/administração & dosagem , Bibliotecas de Moléculas Pequenas/administração & dosagem , Ubiquitina Tiolesterase/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Domínio Catalítico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Ligação Proteica , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina Tiolesterase/química , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The non-genotoxic nature of proteasome inhibition makes it an attractive therapeutic option for the treatment of pediatric malignancies. We recently described the small molecule VLX1570 as an inhibitor of proteasome deubiquitinase (DUB) activity that induces proteotoxic stress and apoptosis in cancer cells. Here we show that acute lymphoblastic leukemia (ALL) cells are highly sensitive to treatment with VLX1570, resulting in the accumulation of polyubiquitinated proteasome substrates and loss of cell viability. VLX1570 treatment increased the levels of a number of proteins, including the chaperone HSP70B', the oxidative stress marker heme oxygenase-1 (HO-1) and the cell cycle regulator p21Cip1. Unexpectedly, polybiquitin accumulation was found to be uncoupled from ER stress in ALL cells. Thus, increased phosphorylation of eIF2α occurred only at supra-pharmacological VLX1570 concentrations and did not correlate with polybiquitin accumulation. Total cellular protein synthesis was found to decrease in the absence of eIF2α phosphorylation. Furthermore, ISRIB (Integrated Stress Response inhibitor) did not overcome the inhibition of protein synthesis. We finally show that VLX1570 can be combined with L-asparaginase for additive or synergistic antiproliferative effects on ALL cells. We conclude that ALL cells are highly sensitive to the proteasome DUB inhibitor VLX1570 suggesting a novel therapeutic option for this disease.
Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Compostos de Benzilideno/farmacologia , Enzimas Desubiquitinantes/antagonistas & inibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Homeostase/fisiologia , Humanos , Reação em Cadeia da Polimerase , Inibidores de Proteassoma/farmacologia , Proteínas/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacosRESUMO
Inhibition of deubiquitinase (DUB) activity is a promising strategy for cancer therapy. VLX1570 is an inhibitor of proteasome DUB activity currently in clinical trials for relapsed multiple myeloma. Here we show that VLX1570 binds to and inhibits the activity of ubiquitin-specific protease-14 (USP14) in vitro, with comparatively weaker inhibitory activity towards UCHL5 (ubiquitin-C-terminal hydrolase-5). Exposure of multiple myeloma cells to VLX1570 resulted in thermostabilization of USP14 at therapeutically relevant concentrations. Transient knockdown of USP14 or UCHL5 expression by electroporation of siRNA reduced the viability of multiple myeloma cells. Treatment of multiple myeloma cells with VLX1570 induced the accumulation of proteasome-bound high molecular weight polyubiquitin conjugates and an apoptotic response. Sensitivity to VLX1570 was moderately affected by altered drug uptake, but was unaffected by overexpression of BCL2-family proteins or inhibitors of caspase activity. Finally, treatment with VLX1570 was found to lead to extended survival in xenograft models of multiple myeloma. Our findings demonstrate promising antiproliferative activity of VLX1570 in multiple myeloma, primarily associated with inhibition of USP14 activity.