RESUMO
The Golgi apparatus is part of the secretory pathway and of central importance for modification, transport and sorting of proteins and lipids. ADP-ribosylation factors, whose activation can be blocked by brefeldin A (BFA), play a major role in functioning of the Golgi network and regulation of membrane traffic and are also involved in proliferation and migration of cancer cells. Due to high cytotoxicity and poor bioavailability, BFA has not passed the preclinical stage of drug development. Recently, AMF-26 and golgicide A have been described as novel inhibitors of the Golgi system with antitumor or bactericidal properties. We provide here further evidence that AMF-26 closely mirrors the mode of action of BFA but is less potent. Using several human cancer cell lines, we studied the effects of AMF-26, BFA and golgicide A on cell homeostasis including Golgi structure, endoplasmic reticulum (ER) stress markers, secretion and viability, and found overall a significant correlation between these parameters. Furthermore, modulation of ADP-ribosylation factor expression has a profound impact on Golgi organization and survival in response to Golgi stress inducers.
Assuntos
Sobrevivência Celular , Complexo de Golgi/metabolismo , Estresse Fisiológico , Fatores de Ribosilação do ADP/efeitos dos fármacos , Fatores de Ribosilação do ADP/metabolismo , Brefeldina A/farmacologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Naftóis/farmacologia , Piridinas/farmacologia , Quinolinas/farmacologiaRESUMO
Tether complexes play important roles in endocytic and exocytic trafficking of lipids and proteins. In yeast, the multisubunit transport protein particle (TRAPP) tether regulates endoplasmic reticulum (ER)-to-Golgi and intra-Golgi transport and is also implicated in autophagy. In addition, the TRAPP complex acts as a guanine nucleotide exchange factor (GEF) for Ypt1, which is homologous to human Rab1a and Rab1b. Here, we show that human TRAPPC13 and other TRAPP subunits are critically involved in the survival response to several Golgi-disrupting agents. Loss of TRAPPC13 partially preserves the secretory pathway and viability in response to brefeldin A, in a manner that is dependent on ARF1 and the large GEF GBF1, and concomitant with reduced caspase activation and ER stress marker induction. TRAPPC13 depletion reduces Rab1a and Rab1b activity, impairs autophagy and leads to increased infectivity to the pathogenic bacterium Shigella flexneri in response to brefeldin A. Thus, our results lend support for the existence of a mammalian TRAPPIII complex containing TRAPPC13, which is important for autophagic flux under certain stress conditions.
Assuntos
Antígenos de Neoplasias/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Células A549 , Fator 1 de Ribosilação do ADP/metabolismo , Antibacterianos/farmacologia , Antígenos de Neoplasias/efeitos dos fármacos , Autofagia/fisiologia , Brefeldina A/farmacologia , Disenteria Bacilar/tratamento farmacológico , Disenteria Bacilar/metabolismo , Técnicas de Silenciamento de Genes , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Células HT29 , Células HeLa , Humanos , Shigella flexneri/efeitos dos fármacos , Proteínas de Transporte Vesicular/antagonistas & inibidores , Proteínas de Transporte Vesicular/efeitos dos fármacosRESUMO
In oncology, the "Warburg effect" describes the elevated production of energy by glycolysis in cancer cells. The ubiquitous and hypoxia-induced 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) plays a noteworthy role in the regulation of glycolysis by producing fructose-2,6-biphosphate (F-2,6-BP), a potent activator of the glycolysis rate-limiting phosphofructokinase PFK-1. Series of amides and sulfonamides derivatives based on a N-aryl 6-aminoquinoxaline scaffold were synthesized and tested for their inhibition of PFKFB3 in vitro in a biochemical assay as well as in HCT116 cells. The carboxamide series displayed satisfactory kinetic solubility and metabolic stability, and within this class, potent lead compounds with low nanomolar activity have been identified with a suitable profile for further in vivo evaluation.
Assuntos
Amidas/química , Fosfofrutoquinase-2/antagonistas & inibidores , Quinoxalinas/química , Quinoxalinas/farmacologia , Sulfonamidas/química , Células HCT116 , Humanos , Cinética , SolubilidadeRESUMO
Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors induced in diverse pathophysiological settings. Inhibition of HIF-2α has become a strategy for cancer treatment since the discovery that small molecules, upon binding into a small cavity of the HIF-2α PAS B domain, can alter its conformation and disturb the activity of the HIF dimer complex. Herein, the design, synthesis, and systematic SAR exploration of cycloalkyl[c]thiophenes as novel HIF-2α inhibitors are described, providing the first chemotype featuring an alkoxy-aryl scaffold. X-ray data confirmed the ability of these inhibitors to induce perturbation of key amino acids by appropriately presenting key pharmacophoric elements in the hydrophobic cavity. Selected compounds showed inhibition of VEGF-A secretion in cancer cells and prevention of Arg1 expression and activity in IL4-stimulated macrophages. Moreover, in vivo target gene modulation was demonstrated with compound 35r. Thus, the disclosed HIF-2α inhibitors represent valuable tools for investigating selective HIF-2α inhibition and its effect on tumor biology.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Tiofenos , Humanos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Tiofenos/farmacologia , Fatores de Transcrição , Hipóxia , Subunidade alfa do Fator 1 Induzível por HipóxiaRESUMO
Metastatic clear cell renal cell carcinomas (ccRCCs) are resistant to DNA-damaging chemotherapies, limiting therapeutic options for patients whose tumors are resistant to tyrosine kinase inhibitors and/or immune checkpoint therapies. Here we show that mouse and human ccRCCs were frequently characterized by high levels of endogenous DNA damage and that cultured ccRCC cells exhibited intact cellular responses to chemotherapy-induced DNA damage. We identify that pharmacological inhibition of the DNA damage-sensing kinase ataxia telangiectasia and Rad3-related protein (ATR) with the orally administered, potent, and selective drug M4344 (gartisertib) induced antiproliferative effects in ccRCC cells. This effect was due to replication stress and accumulation of DNA damage in S phase. In some cells, DNA damage persisted into subsequent G2/M and G1 phases, leading to the frequent accumulation of micronuclei. Daily single-agent treatment with M4344 inhibited the growth of ccRCC xenograft tumors. M4344 synergized with chemotherapeutic drugs including cisplatin and carboplatin and the poly(ADP-ribose) polymerase inhibitor olaparib in mouse and human ccRCC cells. Weekly M4344 plus cisplatin treatment showed therapeutic synergy in ccRCC xenografts and was efficacious in an autochthonous mouse ccRCC model. These studies identify ATR inhibition as a potential novel therapeutic option for ccRCC.
Assuntos
Antineoplásicos , Carcinoma de Células Renais , Humanos , Animais , Camundongos , Carcinoma de Células Renais/tratamento farmacológico , Cisplatino , Proteínas Mutadas de Ataxia Telangiectasia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêuticoRESUMO
The apoptotic and therapeutic activities of the histone deacetylase inhibitor (HDACi) vorinostat are blocked by overexpression of Bcl-2 or Bcl-X(L). Herein, we used the small molecule inhibitor ABT-737 to restore sensitivity of Emu-myc lymphomas overexpressing Bcl-2 or Bcl-X(L) to vorinostat and valproic acid (VPA). Combining low-dose ABT-737 with vorinostat or VPA resulted in synergistic apoptosis of these cells. ABT-737 was ineffective against Emu-myc/Mcl-1 and Emu-myc/A1 cells either as a single agent or in combination with HDACi. However, in contrast to the reported binding specificity data, Emu-myc/Bcl-w lymphomas were insensitive to ABT-737 used alone or in combination with HDACi, indicating that the regulatory activity of ABT-737 is restricted to Bcl-2 and Bcl-X(L). Emu-myc lymphomas that expressed Bcl-2 throughout the tumorigenesis process were especially sensitive to ABT-737, while those forced to overexpress Mcl-1 were not. This supports the notion that tumor cells "addicted" to ABT-737 target proteins (ie, Bcl-2 or Bcl-X(L)) are likely to be the most sensitive target cell population. Our studies provide important preclinical data on the binding specificity of ABT-737 and its usefulness against primary hematologic malignancies when used as a single agent and in combination with HDACi.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Compostos de Bifenilo/farmacologia , Inibidores Enzimáticos/administração & dosagem , Inibidores de Histona Desacetilases , Linfoma/tratamento farmacológico , Nitrofenóis/farmacologia , Sulfonamidas/farmacologia , Animais , Compostos de Bifenilo/administração & dosagem , Sobrevivência Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Genes bcl-2 , Genes myc , Ácidos Hidroxâmicos/administração & dosagem , Linfoma/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nitrofenóis/administração & dosagem , Piperazinas/administração & dosagem , Piperazinas/farmacologia , Especificidade por Substrato , Sulfonamidas/administração & dosagem , VorinostatRESUMO
LAQ824 and LBH589 (panobinostat) are histone deacetylase inhibitors (HDACi) developed as cancer therapeutics and we have used the Emu-myc lymphoma model to identify the molecular events required for their antitumor effects. Induction of tumor cell death was necessary for these agents to mediate therapeutic responses in vivo and both HDACi engaged the intrinsic apoptotic cascade that did not require p53. Death receptor pathway blockade had no effect on the therapeutic activities of LAQ824 and LBH589; however, overexpression of Bcl-2 or Bcl-X(L) protected lymphoma cells from HDACi-induced killing and suppressed their therapeutic activities. Deletion of Apaf-1 or Caspase-9 delayed HDACi-induced lymphoma killing in vitro and in vivo, associated with suppression of many biochemical indicators of apoptosis, but did not provide long-term resistance to these agents and failed to inhibit their therapeutic activities. Emu-myc lymphomas lacking a functional apoptosome displayed morphologic and biochemical features of autophagy after treatment with LAQ824 and LBH589, indicating that, in the absence of a complete intrinsic apoptosis pathway involving apoptosome formation, these HDACi can still mediate a therapeutic response. Our data indicate that damage to the mitochondria is the key event necessary for LAQ824 and LBH589 to mediate tumor cell death and a robust therapeutic response.
Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Linfoma/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Histona Desacetilases/metabolismo , Humanos , Indóis , Linfoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Transplante de Neoplasias , Panobinostat , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Morte Celular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Taxa de Sobrevida , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Histone deacetylase inhibitors (HDACi) and agents such as recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TRAIL receptor (TRAIL-R) antibodies are anticancer agents that have shown promise in preclinical settings and in early phase clinical trials as monotherapies. Although HDACi and activators of the TRAIL pathway have different molecular targets and mechanisms of action, they share the ability to induce tumor cell-selective apoptosis. The ability of HDACi to induce expression of TRAIL-R death receptors 4 and 5 (DR4/DR5), and induce tumor cell death via the intrinsic apoptotic pathway provides a molecular rationale to combine these agents with activators of the TRAIL pathway that activate the alternative (death receptor) apoptotic pathway. Herein, we demonstrate that the HDACi vorinostat synergizes with the mouse DR5-specific monoclonal antibody MD5-1 to induce rapid and robust tumor cell apoptosis in vitro and in vivo. Importantly, using a preclinical mouse breast cancer model, we show that the combination of vorinostat and MD5-1 is safe and induces regression of established tumors, whereas single agent treatment had little or no effect. Functional analyses revealed that rather than mediating enhanced tumor cell apoptosis via the simultaneous activation of the intrinsic and extrinsic apoptotic pathways, vorinostat augmented MD5-1-induced apoptosis concomitant with down-regulation of the intracellular apoptosis inhibitor cellular-FLIP (c-FLIP). These data demonstrate that combination therapies involving HDACi and activators of the TRAIL pathway can be efficacious for the treatment of cancer in experimental mouse models.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Inibidores de Histona Desacetilases , Neoplasias Mamárias Experimentais/tratamento farmacológico , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/antagonistas & inibidores , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Animais , Anticorpos Monoclonais/farmacologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/biossíntese , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Proteínas de Neoplasias/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , VorinostatRESUMO
Tumor cells typically enhance their metabolic capacity to sustain their higher rate of growth and proliferation. One way to elevate the nutrient intake into cancer cells is to increase the expression of genes encoding amino acid transporters, which may represent targetable vulnerabilities. Here, we study the regulation and function of the broad amino acid transporter SLC6A14 in combination with metabolic stress, providing insights into an uncharacterized aspect of the transporter activity. We analyze the pattern of transcriptional changes in a panel of breast cancer cell lines upon metabolic stress and found that SLC6A14 expression levels are increased in the absence of methionine. Methionine deprivation, which can be achieved via modulation of dietary methionine intake in tumor cells, in turn leads to a heightened activation of the AMP-activated kinase (AMPK) in SLC6A14-deficient cells. While SLC6A14 genetic deficiency does not have a major impact on cell proliferation, combined depletion of AMPK and SLC6A14 leads to an increase in apoptosis upon methionine starvation, suggesting that combined targeting of SLC6A14 and AMPK can be exploited as a therapeutic approach to starve tumor cells.
RESUMO
Histone deacetylase inhibitors (HDACi) are compounds that target the epigenome and cause tumor cell-selective apoptosis. A large number of these agents that have different chemical structures and can target multiple HDACs are being testing in clinical trials and vorinostat is now an approved drug for the treatment of cutaneous T-cell lymphoma. Although these agents are showing promise for the treatment of hematologic malignancies, it is possible that different drugs may have different mechanistic, biological, and therapeutic activities. When comparing an HDACi belonging to the hydroxamic acid class of compounds (vorinostat) with a cyclic tetrapeptide (romidepsin), we showed that these agents regulate the expression of a common set of cellular genes, but certain genes specifically responded to each agent. Using the Emu-myc mouse model of B-cell lymphoma, we showed previously that overexpression of the prosurvival proteins Bcl-2 and Bcl-XL inhibited the apoptotic and therapeutic activities of the vorinostat. Herein, we compared and contrasted the apoptotic-inducing activities of the hydroxamic acid oxamflatin with romidepsin. Like vorinostat, oxamflatin was unable to kill lymphomas overexpressing Bcl-2 and Bcl-XL, indicating that these proteins can generally protect cells against this class of HDACi. In contrast, romidepsin was able to induce apoptosis in lymphomas overexpressing Bcl-2 with delayed kinetics of cell death and could mediate therapeutic responses against these lymphomas. However, romidepsin was inactive when Bcl-XL was overexpressed. These data provide strong support that HDACi of different chemical classes may have subtle yet potentially important differences in their molecular and biological activities.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose , Depsipeptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Animais , Antibióticos Antineoplásicos/uso terapêutico , Depsipeptídeos/uso terapêutico , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/uso terapêutico , Ácidos Hidroxâmicos/farmacologia , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
Energy and biomass production in cancer cells are largely supported by aerobic glycolysis in what is called the Warburg effect. The process is regulated by key enzymes, among which phosphofructokinase PFK-2 plays a significant role by producing fructose-2,6-biphosphate; the most potent activator of the glycolysis rate-limiting step performed by phosphofructokinase PFK-1. Herein, the synthesis, biological evaluation and structure-activity relationship of novel inhibitors of 6-phosphofructo-2-kinase/fructose-2,6-biphosphataseâ 3 (PFKFB3), which is the ubiquitous and hypoxia-induced isoform of PFK-2, are reported. X-ray crystallography and docking were instrumental in the design and optimisation of a series of N-aryl 6-aminoquinoxalines. The most potent representative, N-(4-methanesulfonylpyridin-3-yl)-8-(3-methyl-1-benzothiophen-5-yl)quinoxalin-6-amine, displayed an IC50 of 14â nm for the target and an IC50 of 0.49â µm for fructose-2,6-biphosphate production in human colon carcinoma HCT116 cells. This work provides a new entry in the field of PFKFB3 inhibitors with potential for development in oncology.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fosfofrutoquinase-2/antagonistas & inibidores , Quinoxalinas/química , Quinoxalinas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Células HCT116 , Humanos , Ácido Láctico/antagonistas & inibidores , Ácido Láctico/biossíntese , Modelos Moleculares , Estrutura Molecular , Fosfofrutoquinase-2/metabolismo , Quinoxalinas/síntese química , Relação Estrutura-AtividadeRESUMO
Cytotoxic activities of several Golgi-dispersing compounds including AMF-26/M-COPA, brefeldin A and golgicide A have previously been shown to induce autophagy or apoptosis. Here, we demonstrate that these Golgi disruptors also trigger ferroptosis, a non-apoptotic form of cell death characterized by iron-dependent oxidative degradation of lipids. Inhibitors of ferroptosis not only counteract cell death, but they also protect from Golgi dispersal and inhibition of protein secretion in response to several Golgi stress agents. Furthermore, the application of sublethal doses of ferroptosis-inducers such as erastin and sorafenib, low cystine growth conditions, or genetic knockdown of SLC7A11 and GPX4 all similarly protect cells from Golgi stress and lead to modulation of ACSL4, SLC7A5, SLC7A11 or GPX4 levels. Collectively, this study suggests a previously unrecognized function of the Golgi apparatus, which involves cellular redox control and prevents ferroptotic cell death.
RESUMO
The secretory pathway is a major determinant of cellular homoeostasis. While research into secretory stress signaling has so far mostly focused on the endoplasmic reticulum (ER), emerging data suggest that the Golgi itself serves as an important signaling hub capable of initiating stress responses. To systematically identify novel Golgi stress mediators, we performed a transcriptomic analysis of cells exposed to three different pharmacological compounds known to elicit Golgi fragmentation: brefeldin A, golgicide A, and monensin. Subsequent gene-set enrichment analysis revealed a significant contribution of the ETS family transcription factors ELK1, GABPA/B, and ETS1 to the control of gene expression following compound treatment. Induction of Golgi stress leads to a late activation of the ETS upstream kinases MEK1/2 and ERK1/2, resulting in enhanced ETS factor activity and the transcription of ETS family target genes related to spliceosome function and cell death induction via alternate MCL1 splicing. Further genetic analyses using loss-of-function and gain-of-function experiments suggest that these transcription factors operate in parallel.
Assuntos
Processamento Alternativo/genética , Complexo de Golgi/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Estresse Fisiológico , Transcrição Gênica , Células A549 , Processamento Alternativo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Brefeldina A/farmacologia , Citoproteção/efeitos dos fármacos , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Complexo de Golgi/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monensin/farmacologia , Piridinas/farmacologia , Quinolinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Spliceossomos/efeitos dos fármacos , Spliceossomos/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Disruption of the Golgi apparatus can induce a distinct form of programmed cell death that has not been thoroughly characterized. We found that pharmacological application of Golgi stress leads to induction of death receptors (DRs) 4 and 5. DR4 appears to be primarily responsible for the initiation of cell death downstream of Golgi stress, whereas DR5 seems to be more important for cell death triggered by endoplasmic reticulum (ER) stress in specific cancer cell lines. DR induction downstream of either Golgi or ER stress mainly causes intracellular accumulation of DR4 presumably at the Golgi, rather than increased expression on the cell surface. Nevertheless, cells treated with secretory pathway stressors displayed an increased susceptibility to TRAIL (tumor necrosis factor related apoptosis inducing ligand), the endogenous ligand of DR4/5, probably due to intracellular sequestration of the caspase-8 regulator CFLAR (caspase-8 and FADD-like apoptosis regulator). These findings have implications for the treatment of cancer with DR agonists and our general understanding of DR signaling while highlighting the role of the Golgi apparatus as a cell death signaling platform.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Apoptose/efeitos dos fármacos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Caspase 8/genética , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/genética , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/genética , Humanos , Neoplasias/genética , Neoplasias/patologiaRESUMO
The Golgi apparatus is increasingly recognized as a major hub for cellular signaling and is involved in numerous pathologies, including neurodegenerative diseases and cancer. The study of Golgi stress-induced signaling pathways relies on the selectivity of the available tool compounds of which currently only a few are known. To discover novel Golgi-fragmenting agents, transcriptomic profiles of cells treated with brefeldin A, golgicide A, or monensin were generated and compared with a database of gene expression profiles from cells treated with other bioactive small molecules. In parallel, a phenotypic screen was performed for compounds that alter normal Golgi structure. Histone deacetylase (HDAC) inhibitors and DNA-damaging agents were identified as novel Golgi disruptors. Further analysis identified HDAC1/HDAC9 as well as BRD8 and DNA-PK as important regulators of Golgi breakdown mediated by HDAC inhibition. We provide evidence that combinatorial HDACi/(+)-JQ1 treatment spurs synergistic Golgi dispersal in several cancer cell lines, pinpointing a possible link between drug-induced toxicity and Golgi morphology alterations.
Assuntos
Azepinas/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Histona Desacetilase 1/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Triazóis/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Perfilação da Expressão Gênica/métodos , Complexo de Golgi/efeitos dos fármacos , Histona Desacetilase 1/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , HumanosRESUMO
PKCalpha and Ets1 are both associated with breast cancer progression. Our previous studies suggested that these proteins are likely to functionally interact with one another. Here, we show that attenuation of endogenous PKCalpha expression (siPalpha) by RNA interference leads to reduced Ets1 protein expression in a variety of cancer cells. Pulse-chase experiments and treatment with proteasome inhibitor MG-132 revealed that siPalpha interferes with both Ets1 protein synthesis and stability. The effect of siPalpha on Ets1 expression could be partially prevented by KN-93, suggesting that calcium/calmodulin-dependent kinase II (CaMKII), a modulator of Ets1 activity, may play a role in PKCalpha-dependent Ets1 regulation. In contrast, Ets1-regulating kinases ERK1/2 were not found to be involved in this process. To assess the importance of the PKCalpha/Ets1 interaction, we compared the biological responses of MDA-MB-231 cells to PKCalpha- and Ets1-specific siRNAs (siE1). While only siPalpha induced changes in cellular morphology and anchorage-independent growth, both siRNAs similarly affected cellular responses to the antitumor drug mithramycin A and to UV light. Microarray analyses further showed that the expression of a certain set of genes was equally affected by siPalpha and siE1. The data suggest that Ets1 serves as an effector for PKCalpha to fulfil certain functions in cancer cells.
Assuntos
Neoplasias/metabolismo , Plicamicina/análogos & derivados , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Forma Celular , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Plicamicina/farmacologia , Proteína Quinase C/genética , Proteína Quinase C-alfa , Proteína Proto-Oncogênica c-ets-1 , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ets , Interferência de RNA , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Raios UltravioletaRESUMO
BACKGROUND: Transforming growth factor-beta (TGFbeta) plays an important role in late-stage carcinogenesis by stimulating invasive behavior of cancer cells, promoting neo-angiogenesis and by helping cancer cells to escape surveillance by the immune system. It also supports colonization of the bone by metastatic breast cancer cells by increasing expression of osteolytic parathyroid hormone-related protein (PTHrP). Interfering with TGFbeta signalling may thus weaken the malignant properties of cancer cells. We investigated to what extent two inhibitors, SB-202190 and SB-203580, interfere with TGFbeta-signalling in invasive MDA-MB-231 breast cancer cells. These compounds, formerly used as p38-MAPK-specific inhibitors, were recently also demonstrated to inhibit TGFbeta type I receptor kinase. RESULTS: Our results show that these inhibitors delay the onset of TGFbeta-induced nuclear accumulation of Smad3 and reduces its amplitude. This effect was accompanied by a strong reduction in TGFbeta-responsivess of the slow-responder genes pthrp, pai-1 and upa, while the reactivity of the fast-responder gene smad7 to TGFbeta remained almost unchanged. Neither was the TGFbeta response of the fast-responder ese-1/esx gene, whose expression we found to be strongly downregulated by TGFbeta, affected by the inhibitors. CONCLUSION: The data show that SB-202190 and SB-203580 suppress TGFbeta-dependent activation of genes that are important for the acquisition of invasive behavior, while having no effect on the expression of the natural TGFbeta inhibitor Smad7. This suggests that these compounds are potent inhibitors of malignant behavior of cancer cells.
Assuntos
Núcleo Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Transativadores/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Humanos , Imidazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Smad3 , Transativadores/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Parathyroid hormone-related protein (PTHrP) is produced by many tumors not associated with humoral hypercalcemia, including breast cancers. In this study, we used three human immortalized mammary epithelial cell lines that differ in tumorigenicity and PTHrP expression. Using RT-PCR we investigated 5' and 3' alternative splicing of PTHrP transcripts and promoter usage in the lines. Increased levels of P3-derived transcripts and the 1-139 mRNA isoform were observed in the most tumorigenic cell line. Transient transfection experiments identified elements close to P3 promoter that appeared to account for a portion of differential PTHrP expression among the three cell lines. Using site-directed mutagenesis, a previously described Ets-1/Sp1 binding site upstream of P3 was determined to be crucial for full activity of this promoter. RT-PCR and western blot evaluation of Ets family member expression found that Ese-1 was present in all three lines, but that appreciable levels of Ets-1 protein were present exclusively in the most tumorigenic line. Cotransfection of Ets-1 expression vectors activated PTHrP reporter constructs in the most tumorigenic line but not in the other cell lines. These findings suggest a potential mechanism by which PTHrP transcription may be regulated as a consequence of events that promote tumorigenic behavior in breast epithelial cells.
Assuntos
Neoplasias da Mama/etiologia , Glândulas Mamárias Humanas/patologia , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteínas Proto-Oncogênicas/fisiologia , Fatores de Transcrição/fisiologia , Processamento Alternativo , Neoplasias da Mama/patologia , Transformação Celular Neoplásica/patologia , Células Cultivadas , Células Epiteliais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Regiões Promotoras Genéticas , Isoformas de Proteínas/análise , Proteína Proto-Oncogênica c-ets-1 , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-ets , RNA Mensageiro/análise , Fatores de Transcrição/análiseRESUMO
Transcription factor Ets1 is expressed in invasive breast cancer cells. In T-cells, the splicing variant DeltaVII-Ets1 is naturally produced along with full length Ets1 (fl-Ets1). Though its function is unknown, the lack of important inhibitory domains predicts a regulatory role of DeltaVII-Ets1 in fl-Ets1-controlled transcription. Examining the expression status of DeltaVII-Ets1 in invasive Ets1-producing MDA-MB-231 breast cancer cells, we found that the DeltaVII-Ets1 protein could only be detected when nuclear proteins from these cells were fractionated by ionic exchange chromatography. When overexpressed, DeltaVII-Ets1 was found to be partially degraded in breast cancer cells, but not in Jurkat T-cells or SK-Mel melanoma cells. In contrast, no proteolytic products resulted from ectopic expression of fl-Ets1 suggesting that breast cancer cells are able to specifically cleave DeltaVII-Ets1. Overexpression of DeltaVII-Ets1 reduced survival of MDA-MB-231 cells, but not of MCF-7 cells. A mutant version of DeltaVII-Ets1, lacking first 129 N-terminal amino acids, had no effect. These data suggest that Ets1-producing invasive breast cancer cells specifically downregulate DeltaVII-Ets1, as it may be able to adversely affect the survival of these cells.
Assuntos
Processamento Alternativo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Regulação para Baixo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Aminoácidos/química , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular , Cromatografia por Troca Iônica , Proteínas de Fluorescência Verde , Humanos , Células Jurkat , Proteínas Luminescentes/metabolismo , Mutação , Invasividade Neoplásica , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Proteína Proto-Oncogênica c-ets-1 , Proteínas Proto-Oncogênicas c-ets , Frações Subcelulares , Fatores de Tempo , Transcrição Gênica , TransfecçãoRESUMO
We have previously shown that PKC inhibitors interfere with the Ets1/Smad3-dependent regulation of parathyroid hormone-related protein (PTHrP) P3 promoter activity by TGFbeta in invasive MDA-MB-231 breast cancer cells. By examining PKC expression in a variety of breast cancer cell lines, the protein level of PKCalpha was found to be much higher in Ets1-expressing MDA-MB-231 and MDA-MB-435 breast cancer cells than in Ets1-deficient MCF-7 and SK-BR3 cells. No correlation of Ets1 expression with the expression of other PKC subtypes (PKCbeta1, PKCbeta2, PKCdelta or PKCepsilon) could be observed. In contrast to MDA-MB-231 cells, PKCalpha-deficient MCF-7 cells do not support Ets1-induced activation of the PTHrP P3 promoter suggesting that PKCalpha may be important for Ets1 activity. A constitutively active form of PKCalpha was found to potentiate the P3 promoter activation by Ets1 alone and in synergy with Smad3. PKCalpha, but not PKCepsilon, also induced phosphorylation of the Ets1 protein. Both PKCalpha effects on Ets1 depended on the exon VII domain of Ets1. Using verapamil and ionomycin, we could show that PKCalpha induces Ets1 phosphorylation independent of calcium mobilization. Collectively, our data suggest that PKCalpha may regulate Ets1 activity in invasive breast cancer cells.