RESUMO
UNLABELLED: Transcription factors of the far-upstream element-binding protein (FBP) family represent cellular pathway hubs, and their overexpression in liver cancer (hepatocellular carcinoma [HCC]) stimulates tumor cell proliferation and correlates with poor prognosis. Here we determine the mode of oncogenic FBP overexpression in HCC cells. Using perturbation approaches (kinase inhibitors, small interfering RNAs) and a novel system for rapalog-dependent activation of AKT isoforms, we demonstrate that activity of the phosphatidylinositol-4,5-biphosphate 3-kinase/AKT pathway is involved in the enrichment of nuclear FBP1 and FBP2 in liver cancer cells. In human HCC tissues, phospho-AKT significantly correlates with nuclear FBP1/2 accumulation and expression of the proliferation marker KI67. Mechanistic target of rapamycin (mTOR) inhibition or blockade of its downstream effector eukaryotic translation initiation factor 4E activity equally reduced FBP1/2 concentrations. The mTORC1 inhibitor rapamycin diminishes FBP enrichment in liver tumors after hydrodynamic gene delivery of AKT plasmids. In addition, the multikinase inhibitor sorafenib significantly reduces FBP levels in HCC cells and in multidrug resistance 2-deficient mice that develop HCC due to severe inflammation. Both FBP1/2 messenger RNAs are highly stable, with FBP2 being more stable than FBP1. Importantly, inhibition of phosphatidylinositol-4,5-biphosphate 3-kinase/AKT/mTOR signaling significantly diminishes FBP1/2 protein stability in a caspase-3/-7-dependent manner. CONCLUSION: These data provide insight into a transcription-independent mechanism of FBP protein enrichment in liver cancer; further studies will have to show whether this previously unknown interaction between phosphatidylinositol-4,5-biphosphate 3-kinase/AKT/mTOR pathway activity and caspase-mediated FBP stabilization allows the establishment of interventional strategies in FBP-positive HCCs.
Assuntos
Carcinoma Hepatocelular/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias Hepáticas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Caspase 3/metabolismo , Caspase 7/metabolismo , Feminino , Humanos , Masculino , Estabilidade Proteica , Proteínas de Ligação a RNARESUMO
Transcription factors integrate a variety of oncogenic input information, facilitate tumour growth and cell dissemination, and therefore represent promising therapeutic target structures. Because over-expression of DNA-interacting far upstream element binding protein (FBP) supports non-small cell lung cancer (NSCLC) migration, we asked whether its repressor, FBP-interacting repressor (FIR) is functionally inactivated and how FIR might affect NSCLC cell biology. Different FIR splice variants were highly expressed in the majority of NSCLCs, with the highest levels in tumours carrying genomic gains of chromosome 8q24.3, which contained the FIR gene locus. Nuclear FIR expression was significantly enriched at the invasion front of primary NSCLCs, but this did not correlate with tumour cell proliferation. FIR accumulation was associated with worse patient survival and tumour recurrence; in addition, FIR over-expression significantly correlated with lymph node metastasis in squamous cell carcinomas (SCCs). In vitro, we applied newly developed methods and modelling approaches for the quantitative and time-resolved description of the pro-migratory and pro-invasive capacities of SCC cells. siRNA-mediated silencing of all FIR variants significantly reduced the speed and directional movement of tumour cells in all phases of migration. Furthermore, sprouting efficiency and single cell invasiveness were diminished following FIR inhibition. Interestingly, the silencing of FIR isoforms lacking exon 2 (FIR(Δexon2)) alone was sufficient to reduce lateral migration and invasion. In summary, by using scale-spanning data derived from primary human tissues, quantitative cellular analyses and mathematical modelling, we have demonstrated that concomitant over-expression of FIR and its splice variants drives NSCLC migration and dissemination.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas de Transporte/metabolismo , Movimento Celular , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias Pulmonares/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proliferação de Células , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/terapia , Metástase Linfática , Microscopia de Vídeo , Invasividade Neoplásica , Recidiva Local de Neoplasia , Prognóstico , Isoformas de Proteínas , Interferência de RNA , Fatores de Processamento de RNA , Proteínas de Ligação a RNA , Proteínas Repressoras , Transdução de Sinais , Fatores de Tempo , Imagem com Lapso de Tempo , TransfecçãoRESUMO
UNLABELLED: The far upstream element binding protein (FBP) and the FBP-interacting repressor (FIR) represent molecular tools for transcriptional fine tuning of target genes. Strong overexpression of FBP in human hepatocellular carcinoma (HCC) supports tumor growth and correlates with poor patient prognosis. However, the role of the transcriptional repressor FIR in hepatocarcinogenesis remains poorly delineated. We show that overexpression of FIR correlates with tumor dedifferentiation and tumor cell proliferation in about 60% of primary HCCs. Elevated FIR levels are associated with genomic gains of the FIR gene locus at chromosome 8q24.3 in human HCC specimens. In vitro, nuclear enrichment of FIR supports HCC cell proliferation and migration. Expression profiling of HCC cells after small interfering RNA (siRNA)-mediated silencing of FIR identified the transcription factor DP-1 (TFDP1) as a transcriptional target of FIR. Surprisingly, FIR stimulates the expression of FBP in a TFDP1/E2F1-dependent manner. FIR splice variants lacking or containing exon 2 and/or exon 5 are expressed in the majority of HCCs but not in normal hepatocytes. Specific inhibition of FIR isoforms with and without exon 2 revealed that both groups of FIR splice variants facilitate tumor-supporting effects. This finding was confirmed in xenograft transplantation experiments with lentiviral-infected short hairpin RNA (shRNA) targeting all FIR variants as well as FIR with and without exon 2. CONCLUSION: High-level nuclear FIR does not facilitate repressor properties but supports tumor growth in HCC cells. Thus, the pharmacological inhibition of FIR might represent a promising therapeutic strategy for HCC patients with elevated FIR expression.
Assuntos
Carcinoma Hepatocelular/fisiopatologia , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , DNA Helicases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Neoplasias Hepáticas/fisiopatologia , Proteínas de Ligação a RNA/fisiologia , Proteínas Repressoras/fisiologia , Animais , Carcinoma Hepatocelular/patologia , DNA Helicases/efeitos dos fármacos , DNA Helicases/genética , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Éxons/genética , Humanos , Técnicas In Vitro , Neoplasias Hepáticas/patologia , Camundongos SCID , Camundongos Transgênicos , Isoformas de Proteínas/genética , Fatores de Processamento de RNA , RNA Interferente Pequeno/farmacologia , Proteínas de Ligação a RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/efeitos dos fármacos , Proteínas Repressoras/genética , Fator de Transcrição DP1/fisiologia , Transplante HeterólogoRESUMO
BACKGROUND & AIMS: Cancer cells often lose contact inhibition to undergo anchorage-independent proliferation and become resistant to apoptosis by inactivating the Hippo signaling pathway, resulting in activation of the transcriptional co-activator yes-associated protein (YAP). However, the oncogenic mechanisms of YAP activity are unclear. METHODS: By using cross-species analysis of expression data, the Notch ligand Jagged-1 (Jag-1) was identified as a downstream target of YAP in hepatocytes and hepatocellular carcinoma (HCC) cells. We analyzed the functions of YAP in HCC cells via overexpression and RNA silencing experiments. We used transgenic mice that overexpressed a constitutively activated form of YAP (YAP(S127A)), and measured protein levels in HCC, colorectal and pancreatic tumor samples from patients. RESULTS: Human HCC cell lines and mouse hepatocytes that overexpress YAP(S127A) up-regulated Jag-1, leading to activation of the Notch pathway and increased proliferation. Induction of Jag-1, activation of Notch, and cell proliferation required binding of YAP to its transcriptional partner TEA domain family member 4 (TEAD4); TEAD4 binding required the Mst1/2 but not ß-catenin signaling. Levels of YAP correlated with Jag-1 expression and Notch signaling in human tumor samples and correlated with shorter survival times of patients with HCC or colorectal cancer. CONCLUSIONS: The transcriptional regulator YAP up-regulates Jag-1 to activate Notch signaling in HCC cells and mouse hepatocytes. YAP-dependent activity of Jag-1 and Notch correlate in human HCC and colorectal tumor samples with patient survival times, suggesting the use of YAP and Notch inhibitors as therapeutics for gastrointestinal cancer. Transcript profiling: microarray information was deposited at the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jxepvsumwosqkve&acc=GSE35004).
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Carcinoma Hepatocelular/genética , Proteínas de Ligação a DNA/fisiologia , Hepatócitos/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Neoplasias Hepáticas/genética , Proteínas de Membrana/fisiologia , Proteínas Musculares/fisiologia , Fosfoproteínas/fisiologia , Receptores Notch/fisiologia , Fatores de Transcrição/fisiologia , Animais , Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Proteína Jagged-1 , Neoplasias Hepáticas/metabolismo , Camundongos , Proteínas Serrate-Jagged , Fatores de Transcrição de Domínio TEA , Regulação para Cima , Proteínas de Sinalização YAPRESUMO
Stability of many tumor-relevant proteins is partly mediated by E3 ligases, which determine substrate specificity within the ubiquitin system. Recent data demonstrated that increased nuclear expression of the E3 ligase seven in absentia homologue (SIAH)-1 in human hepatocarcinogenesis supports tumor cell proliferation and migration. To define whether closely related SIAH-2 synergizes with protumorigenic SIAH-1, we systematically analyzed expression, localization and functional relevance of SIAH-2 in human hepatocellular carcinoma (HCC). Nuclear accumulation of SIAH-2 is detectable in more than 60% of all HCCs and correlates with tumor progression, cell proliferation and distant metastasis. An inverse correlation between nuclear SIAH-1 and SIAH-2 was detected, suggesting independent mechanisms for nuclear enrichment. Inhibition of nuclear SIAH-2 by RNAi in HCC cell lines reduced proliferation as well as lateral tumor cell motility and transmigration; however, combined knock down of both SIAH-1 and SIAH-2 did not further amplify biological effects compared to single gene inhibition. Reduction of SIAH-2 expression sensitizes HCC cells to the treatment with different cytostatic drugs, demonstrating that SIAH-2-targeting approaches may increase the response of HCC cells to conventional chemotherapy. Together, these data show that SIAH-2--as described for SIAH-1--accumulates in nuclei of HCC cells where it supports tumor growth and tumor cell dissemination. Because the nuclear pattern of SIAH-2 differs in HCC tissues from the SIAH-1 pattern and because the inactivation of SIAH-2 is not compensated by SIAH-1, the specific inhibition of SIAH-2 (especially in combination with other drugs) represents a promising therapeutic strategy for HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Movimento Celular , Núcleo Celular/metabolismo , Proliferação de Células , Humanos , Neoplasias Hepáticas/genética , Metástase Neoplásica , Proteínas Nucleares/genética , Interferência de RNA , RNA Interferente Pequeno , Ubiquitina-Proteína Ligases/genéticaRESUMO
Hepatocellular carcinoma (HCC) is one of the most frequent human malignancies with poor prognosis and increasing incidence in the Western world. Only for a minority of HCC patients, surgical treatment options offer potential cure and therapeutic success of pharmacological approaches is limited. Highly specific approaches (e.g., kinase inhibitors) did not significantly improve the situation so far, possibly due to functional compensation, genetic heterogeneity of HCC, and development of resistance under selective pressure. In contrast, transcriptional regulators (especially transcription factors and co-factors) may integrate and process input signals of different (oncogenic) pathways and therefore represent cellular bottlenecks that regulate tumor cell biology. In this review, we want to summarize the current knowledge about central transcriptional regulators in human hepatocarcinogenesis and their potential as therapeutic target structures. Genomic and transcriptomic data of primary human HCC revealed that many of these factors showed up in subgroups of HCCs with a more aggressive phenotype, suggesting that aberrant activity of transcriptional regulators collect input information to promote tumor initiation and progression. Therefore, expression and dysfunction of transcription factors and co-factors may gain relevance for diagnostics and therapy of HCC.
Assuntos
Carcinoma Hepatocelular/genética , Transformação Celular Neoplásica/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Hepáticas/genética , Carcinoma Hepatocelular/fisiopatologia , Humanos , Neoplasias Hepáticas/fisiopatologia , Ativação Transcricional/fisiologiaRESUMO
BACKGROUND & AIMS: Differential expression of tumor-relevant proteins based on aberrant proteasomal degradation may contribute to human (hepato)carcinogenesis. Recently, we identified the E3 ubiquitin ligase seven in absentia homolog (SIAH)-1 as frequently dysregulated in human hepatocellular carcinoma (HCC). We therefore systematically analyzed the expression, functional relevance, as well as possible downstream effectors of SIAH-1 in human liver carcinogenesis. METHODS: SIAH-1 expression was analyzed at the transcript and protein levels in human hepatocarcinogenesis and in HCC cells. Proliferation, apoptosis, and migration of different HCC cell lines were examined after siRNA-mediated inhibition of SIAH-1. In order to identify downstream effectors that mediate SIAH-1 effects, correlative analyses of protein expression profiles were performed. RESULTS: In HCC tissues both reduction of cytoplasmic SIAH-1 and especially its nuclear accumulation positively correlated with HCC progression. RNA interference revealed that nuclear expression of SIAH-1 predominantly supported HCC cell proliferation and migration while only moderately affecting anti-apoptosis. In de-differentiated human HCCs, nuclear SIAH-1 accumulation significantly correlated with the expression of the transcription factor far-upstream element (FUSE)-binding protein (FBP)-3. In vitro, SIAH-1 positively and indirectly regulated FBP-3 which itself primarily supported HCC cell proliferation. Indeed, high level expression of FBP-3 in human HCCs significantly correlated with reduced overall survival of patients. CONCLUSIONS: Nuclear accumulation of the E3 ubiquitin ligase SIAH-1 supports different pro-tumorigenic cellular processes associated with tumor growth and tumor cell dissemination in human hepatocarcinogenesis. It promotes HCC cell proliferation by at least partly employing the transcription factor FBP-3. Therefore, interference with SIAH-1 activity represents a promising approach to suppress HCC growth.
Assuntos
Carcinoma Hepatocelular/genética , Transformação Celular Neoplásica/metabolismo , Neoplasias Hepáticas/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Apoptose , Carcinoma Hepatocelular/enzimologia , Linhagem Celular Tumoral , Movimento Celular , Núcleo Celular/metabolismo , Proliferação de Células , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/enzimologia , Proteínas Nucleares/antagonistas & inibidores , RNA Interferente Pequeno/genética , Estatísticas não Paramétricas , Fatores de Transcrição/metabolismo , Transfecção , Ubiquitina-Proteína Ligases/antagonistas & inibidoresRESUMO
UNLABELLED: Microtubule-dependent effects are partly regulated by factors that coordinate polymer dynamics such as the microtubule-destabilizing protein stathmin (oncoprotein 18). In cancer cells, increased microtubule turnover affects cell morphology and cellular processes that rely on microtubule dynamics such as mitosis and migration. However, the molecular mechanisms deregulating modifiers of microtubule activity in human hepatocarcinogenesis are poorly understood. Based on profiling data of human hepatocellular carcinoma (HCC), we identified far upstream element binding proteins (FBPs) as significantly coregulated with stathmin. Coordinated overexpression of two FBP family members (FBP-1 and FBP-2) in >70% of all analyzed human HCCs significantly correlated with poor patient survival. In vitro, FBP-1 predominantly induced tumor cell proliferation, while FBP-2 primarily supported migration in different HCC cell lines. Surprisingly, reduction of FBP-2 levels was associated with elevated FBP-1 expression, suggesting a regulatory interplay of FBP family members that functionally discriminate between cell division and mobility. Expression of FBP-1 correlated with stathmin expression in HCC tissues and inhibition of FBP-1 but not of FBP-2 drastically reduced stathmin at the transcript and protein levels. In contrast, further overexpression of FBP-1 did not affect stathmin bioavailability. Accordingly, analyzing nuclear and cytoplasmic areas of HCC cells revealed that reduced FBP-1 levels affected cell morphology and were associated with a less malignant phenotype. CONCLUSION: The coordinated activation of FBP-1 and FBP-2 represents a novel and frequent pro-tumorigenic mechanism promoting proliferation (tumor growth) and motility (dissemination) of human liver cancer cells. FBPs promote tumor-relevant functions by at least partly employing the microtubule-destabilizing factor stathmin and represent a new potential target structure for HCC treatment.
Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Movimento Celular/fisiologia , Proliferação de Células , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Apoptose/fisiologia , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Humanos , Microtúbulos/metabolismo , Proteínas de Ligação a RNA , Estatmina/metabolismoRESUMO
UNLABELLED: The inhibition of heat shock protein 90 (Hsp90) has emerged as a promising antineoplastic strategy in diverse human malignancies. Hsp90 has been predicted to be involved in hepatocellular carcinoma (HCC) development; however, its role in hepatocarcinogenesis remains elusive. Using chemically distinctive Hsp90 inhibitors, we show that Hsp90 capacitates the aberrant expression and activity of crucial hepatocarcinogenesis-driving factors (e.g., insulin-like growth factor receptor 1, hepatocyte growth factor receptor, protein kinase B, v-raf-1 murine leukemia viral oncogene homolog 1, and cyclin-dependent kinase 4). In vitro, Hsp90 inhibition with both geldanamycin analogs (17-allylamino-17-desmethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-desmethoxygeldanamycin (17-DMAG)) and the non-quinone compound 8-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine (PU-H71) reduced the viability of various HCC cell lines, induced the simultaneous degradation of numerous hepatocarcinogenic factors, and caused substantial cell cycle arrest and apoptosis. In contrast, nontumorigenic hepatocytes were less susceptible to Hsp90 inhibition. Because conventional geldanamycin-derivate Hsp90 inhibitors induce dose-limiting liver toxicity, we tested whether novel Hsp90 inhibitors lacking the benzoquinone moiety, which has been deemed responsible for hepatotoxicity, can elicit antineoplastic activity without causing significant liver damage. In HCC xenograft mouse models, PU-H71 was retained in tumors at pharmacologically relevant concentrations while being rapidly cleared from nontumorous liver. PU-H71 showed potent and prolonged in vivo Hsp90 inhibitory activity and reduced tumor growth without causing toxicity. CONCLUSION: Hsp90 constitutes a promising therapeutic target in HCC. Non-quinone Hsp90 inhibitors exhibit tumor-specific accumulation and exert potent antineoplastic activity without causing significant hepatotoxicity.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias Hepáticas/tratamento farmacológico , Animais , Humanos , Camundongos , Quinonas , Células Tumorais CultivadasRESUMO
The discovery of isozyme-selective histone deacetylase (HDAC) inhibitors is critical for understanding the biological functions of individual HDACs and for validating HDACs as drug targets. The isozyme HDAC10 contributes to chemotherapy resistance and has recently been described to be a polyamine deacetylase, but no studies toward selective HDAC10 inhibitors have been published. Using two complementary assays, we found Tubastatin A, an HDAC6 inhibitor, to potently bind HDAC10. We synthesized Tubastatin A derivatives and found that a basic amine in the cap group was required for strong HDAC10 binding. HDAC10 inhibitors mimicked knockdown by causing dose-dependent accumulation of acidic vesicles in a neuroblastoma cell line. Furthermore, docking into human HDAC10 homology models indicated that a hydrogen bond between a cap group nitrogen and the gatekeeper residue Glu272 was responsible for potent HDAC10 binding. Taken together, our data provide an optimal platform for the development of HDAC10-selective inhibitors, as exemplified with the Tubastatin A scaffold.
Assuntos
Benzamidas/metabolismo , Ácido Glutâmico/química , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/metabolismo , Animais , Benzamidas/síntese química , Benzamidas/química , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Desacetilase 6 de Histona/química , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Ligantes , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Peixe-ZebraRESUMO
Methionine aminopeptidases (MetAPs) are responsible for the cotranslational cleavage of initiator methionines from nascent proteins. The MetAP2 subtype is up-regulated in many cancers, and selective inhibition of MetAP2 suppresses both vascularization and growth of tumors in animal models. The natural product fumagillin is a selective and potent irreversible inhibitor of MetAP2, and semisynthetic derivatives of fumagillin have shown promise in clinical studies for the treatment of cancer, and, more recently, for obesity. Further development of fumagillin derivatives has been complicated, however, by their generally poor pharmacokinetics. In an attempt to overcome these limitations, we developed an easily diversifiable synthesis of a novel class of MetAP2 inhibitors that were designed to mimic fumagillin's molecular scaffold but have improved pharmacological profiles. These substances were found to be potent and selective inhibitors of MetAP2, as demonstrated in biochemical enzymatic assays against three MetAP isoforms. Inhibitors with the same relative and absolute stereoconfiguration as fumagillin displayed significantly higher activity than their diastereomeric and enantiomeric isomers. X-ray crystallographic analysis revealed that the inhibitors covalently modify His231 in the MetAP2 active site via ring-opening of a spiroepoxide. Biochemically active substances inhibited the growth of endothelial cells and a MetAP2-sensitive cancer cell line, while closely related inactive isomers had little effect on the proliferation of either cell type. These effects correlated with altered N-terminal processing of the protein 14-3-3-γ. Finally, selected substances were found to have improved stabilities in mouse plasma and microsomes relative to the clinically investigated fumagillin derivative beloranib.
Assuntos
Aminopeptidases/antagonistas & inibidores , Cicloexanos/farmacologia , Ácidos Graxos Insaturados/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Triazóis/farmacologia , Linhagem Celular , Cristalografia por Raios X , Humanos , Sesquiterpenos/farmacologiaRESUMO
Newly made proteins must achieve a functional shape, the native configuration, before they can play their physiological roles in the cell. Proteins must also travel to the locale (e.g., the mitochondrion) in the cell where their functions are required. In these processes of folding into the native configuration and translocation to the place of work, proteins may be assisted by molecules called molecular chaperones. Stressors can unfold (denature) proteins, and genetic defects can cause misfolding and, in addition, both abnormalities can lead to polypeptide aggregation. Chaperones play a role in assisting refolding of partially denatured or misfolded proteins, thus preventing aggregation. Clearly, molecular chaperones are key cell components under normal, physiological circumstances, as well as in potentially harmful situations resulting from environmental or inherited factors. Hence, molecular chaperones constitute attractive targets for a variety of efforts aiming at improving the cell's performance, particularly under stress, to prevent disease, or at least to slow down its progression and to contain the deleterious effects of stress. In our efforts in this direction, we have undertaken to investigate the chaperoning systems of cells belonging to the phylogenetic domain Archaea. The findings reported here pertain to the distribution of the molecular chaperone machine, the chaperonins, and the prefoldins, among archaea. The genes hsp70(dnaK), hsp40(dnaJ), and grpE encoding the components of the molecular chaperone machine were present only in some archeaeal species: this contrasts with bacteria and eucarya, which do have the genes with no known exception. The group I, or bacterial, chaperonin-genes groEL and groES occured in the genomes of Methanosarcina species but were not found in any of the other archaea whose genomes have been sequenced. While all the archaea studied had between one and three chaperonins of group II (thermosome subunits), Methanosarcina acetivorans was exceptional since it had five of these chaperonins. This is the largest number of group II chaperonins ever found in a prokaryote. Furthermore, two of the M. acetivorans chaperonins were different from, albeit related to, the other known archaeal and eucaryal chaperonins of group II. Prefoldins were found in all archaea examined. Overall, the results provide clues to the evolution of the chaperoning systems, which must have played a critical role in survival since life started. Also, the data suggest new avenues of research for elucidating the evolution of assisted protein folding and for uncovering roles and interactions not yet described for these molecules.
Assuntos
Archaea/classificação , Proteínas Arqueais/classificação , Chaperonas Moleculares/classificação , Filogenia , Bactérias/classificação , Evolução Biológica , Chaperonina 10/classificação , Chaperonina 60/classificação , Dobramento de ProteínaRESUMO
Pancreatic endocrine tumors (PET) represent a heterogenous group of neoplasms. Although surgical resection is considered a safe and effective treatment for many PET, therapeutic options for inoperable and progressive PET are limited. The expression of heat-shock protein (HSP) 90 was investigated in 120 clinically and pathomorphologically well-characterized PET from 84 patients using immunohistochemistry. In addition, in 19 snap-frozen PET and in three healthy pancreatic tissues, we performed immunoblot analyses, and in 15 snap-frozen PET and in three healthy pancreatic tissues, we investigated the expression of HSP90 isoforms by means of semiquantitative RT-PCR. Functional tests were conducted using the human pancreas carcinoid cell line BON and the mouse insulinoma cell line ß-TC-3. HSP90 was expressed in 95% of the PET patients. The transcript levels of the HSP90 isoforms HSP90α, HSP90ß, glucose-related protein 94, and TNF receptor-associated protein 1 were significantly increased in PET compared with non-neoplastic pancreatic tissues. The treatment of the cell lines BON and ß-TC-3 with the HSP90 inhibitors 17-allylamino-17-demethoxygeldanamycin and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin resulted in significant, dose-dependent reduction of cell viability, cell cycle arrest, and increased apoptosis. Furthermore, HSP90 inhibition induced the degradation and inactivation of several oncogenetic HSP90 client proteins in a time- and dose-dependent manner. HSP90 inhibitors increased the therapeutic effects of doxorubicin and 5-fluorucacil in BON and ß-TC-3 cells. HSP90 is expressed in the vast majority of PET and its inhibition reveals significant treatment effects in vitro. Thus, HSP90 qualifies as a promising new target.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Neoplasias Pancreáticas/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antibióticos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzoquinonas/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Feminino , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Humanos , Lactamas Macrocíclicas/farmacologia , Masculino , Camundongos , Pessoa de Meia-Idade , Neoplasia Endócrina Múltipla Tipo 1/tratamento farmacológico , Neoplasia Endócrina Múltipla Tipo 1/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , RNA Mensageiro/metabolismo , Adulto JovemRESUMO
PURPOSE: The underlying molecular mechanisms of thymic epithelial malignancies (TEMs) are poorly understood. Consequently, there is a lack of efficacious targeted therapies and patient prognosis remains dismal, particularly for advanced TEMs. We sought to investigate protumorigenic mechanism relevant to this understudied cancer. EXPERIMENTAL DESIGN: Recently established cell lines derived from thymic epithelial tumors were used as a model system. The antitumor activity of specific heat shock protein 90 (Hsp90) inhibitors was investigated by an analysis of cell viability, cell cycle, and apoptosis using MTT-assays and flow cytometry. Western blotting was used to investigate the altered expression of Hsp90 clients. Pharmacological inhibitors against select Hsp90 clients, as well as RNAi, were employed to test the relevance of each client independently. Tissue microarray analysis was performed to match the in vitro findings with observations obtained from patient-derived samples. RESULTS: Hsp90 inhibition significantly reduces cell viability of thymic carcinoma cells, induces cell cycle arrest and apoptosis, and blocks invasiveness. Hsp90 inhibition triggers the degradation of multiple oncogenic clients, for example insulin-like growth factor 1 receptor (IGF-1R), CDK4, and the inactivation of PI3K/Akt and RAF/Erk signaling. Mechanistically, the IGF/IGF-1R-signaling axis contributes to the establishment of the antiapoptotic phenotype of thymic cancer cells. Finally, IGF-1R is overexpressed in advanced TEMs. CONCLUSIONS: We have unraveled a novel protumorigenic mechanism in TEMs, namely Hsp90-capacitated overexpression of IGF-1R, which confers apoptosis evasion in malignant thymic epithelial cells. Our data indicate that Hsp90 inhibition, which simultaneously blocks multiple cancer hallmarks, represents a therapeutic strategy in TEMs that may merit evaluation in clinical trials.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Receptor IGF Tipo 1/metabolismo , Neoplasias do Timo/metabolismo , Idoso , Apoptose/efeitos dos fármacos , Benzoquinonas/farmacologia , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Criança , Pré-Escolar , Relação Dose-Resposta a Droga , Feminino , Gefitinibe , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Lactamas Macrocíclicas/farmacologia , Masculino , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinazolinas/farmacologia , Interferência de RNA , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/genética , Transdução de Sinais/efeitos dos fármacos , Neoplasias do Timo/genética , Neoplasias do Timo/patologiaRESUMO
Dynamic instability of the microtubule network modulates processes such as cell division and motility, as well as cellular morphology. Overexpression of the microtubule-destabilizing phosphoprotein stathmin is frequent in human malignancies and represents a promising therapeutic target. Although stathmin inhibition gives rise to antineoplastic effects, additional and functionally redundant microtubule-interacting proteins may attenuate the efficiency of this therapeutic approach. We have systematically analyzed the expression and potential protumorigenic effects of stathmin family members in human non-small cell lung cancer (NSCLC). Both stathmin and stathmin-like 3 (SCLIP) were overexpressed in adenocarcinoma as well as squamous cell carcinoma (SCC) tissues and induced tumor cell proliferation, migration, and matrix invasion in respective cell lines. Accordingly, reduced stathmin and SCLIP levels affected cell morphology and were associated with a less malignant phenotype. Combined inhibition of both factors caused additive effects on tumor cell motility, indicating partial functional redundancy. Because stathmin and SCLIP expression significantly correlated in NSCLC tissues, we searched for common upstream regulators and identified the far upstream sequence element-binding protein-1 (FBP-1) as a pivotal inducer of several stathmin family members. Our results indicate that the coordinated overexpression of microtubule-destabilizing factors by FBP-1 is a critical step to facilitate microtubule dynamics and subsequently increases proliferation and motility of tumor cells.