RESUMEN
Integrins, a family of heterodimeric receptors for extracellular matrix, are promising therapeutic targets for ovarian cancer, particularly high-grade serous-type (HGSOC), as they drive tumor cell attachment, migration, proliferation and survival by activating focal adhesion kinase (FAK)-dependent signaling. Owing to the potential off-target effects of FAK inhibitors, disruption of the integrin signaling axis remains to be a challenge. Here, we tackled this barrier by screening for inhibitors being functionally cooperative with small-molecule VS-6063, a phase II FAK inhibitor. From this screening, JQ1, a potent inhibitor of Myc oncogenic network, emerged as the most robust collaborator. Treatment with a combination of VS-6063 and JQ1 synergistically caused an arrest of tumor cells at the G2/M phase and a decrease in the XIAP-linked cell survival. Our subsequent mechanistic analyses indicate that this functional cooperation was strongly associated with the concomitant disruption of activation or expression of FAK and c-Myc as well as their downstream signaling through the PI3K/Akt pathway. In line with these observations, we detected a strong co-amplification or upregulation at genomic or protein level for FAK and c-Myc in a large portion of primary tumors in the TCGA or a local HGSOC patient cohort. Taken together, our results suggest that the integrin-FAK signaling axis and c-Myc synergistically drive cell proliferation, survival and oncogenic potential in HGSOC. As such, our study provides key genetic, functional and signaling bases for the small-molecule-based co-targeting of these two distinct oncogenic drivers as a new line of targeted therapy against human ovarian cancer.
RESUMEN
Expression of E-cadherin, a hallmark of epithelial-mesenchymal transition (EMT), is often lost due to promoter DNA methylation in basal-like breast cancer (BLBC), which contributes to the metastatic advantage of this disease; however, the underlying mechanism remains unclear. Here, we identified that Snail interacted with Suv39H1 (suppressor of variegation 3-9 homolog 1), a major methyltransferase responsible for H3K9me3 that intimately links to DNA methylation. We demonstrated that the SNAG domain of Snail and the SET domain of Suv39H1 were required for their mutual interactions. We found that H3K9me3 and DNA methylation on the E-cadherin promoter were higher in BLBC cell lines. We showed that Snail interacted with Suv39H1 and recruited it to the E-cadherin promoter for transcriptional repression. Knockdown of Suv39H1 restored E-cadherin expression by blocking H3K9me3 and DNA methylation and resulted in the inhibition of cell migration, invasion and metastasis of BLBC. Our study not only reveals a critical mechanism underlying the epigenetic regulation of EMT, but also paves a way for the development of new treatment strategies against this disease.
Asunto(s)
Neoplasias de la Mama/genética , Cadherinas/genética , Carcinoma/genética , Represión Epigenética , Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Células Cultivadas , Represión Epigenética/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Metiltransferasas/química , Metiltransferasas/genética , Metiltransferasas/fisiología , Modelos Moleculares , Unión Proteica/genética , Unión Proteica/fisiología , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Factores de Transcripción de la Familia Snail , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
Tumour necrosis factor-alpha (TNF-alpha) is an important inflammatory factor that acts as a master switch in establishing an intricate link between inflammation and cancer. A wide variety of evidence has pointed to a critical role of TNF-alpha in tumour proliferation, migration, invasion and angiogenesis. The function of TNF-alpha as a key regulator of the tumour microenvironment is well recognised. We will emphasise the contribution of TNF-alpha and the nuclear factor-kappaB pathway on tumour cell invasion and metastasis. Understanding the mechanisms underlying inflammation-mediated metastasis will reveal new therapeutic targets for cancer prevention and treatment.
Asunto(s)
Movimiento Celular/genética , FN-kappa B/fisiología , Neoplasias/patología , Factores de Transcripción/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Humanos , Modelos Biológicos , FN-kappa B/genética , Invasividad Neoplásica , Neoplasias/genética , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
HER-2/neu amplification or overexpression can make cancer cells resistant to apoptosis and promotes their growth. p53 is crucial in regulating cell growth and apoptosis, and is often mutated or deleted in many types of tumour. Moreover, many tumours with a wild-type gene for p53 do not have normal p53 function, suggesting that some oncogenic signals suppress the function of p53. In this study, we show that HER-2/neu-mediated resistance to DNA-damaging agents requires the activation of Akt, which enhances MDM2-mediated ubiquitination and degradation of p53. Akt physically associates with MDM2 and phosphorylates it at Ser166 and Ser186. Phosphorylation of MDM2 enhances its nuclear localization and its interaction with p300, and inhibits its interaction with p19ARF, thus increasing p53 degradation. Our study indicates that blocking the Akt pathway mediated by HER-2/neu would increase the cytotoxic effect of DNA-damaging drugs in tumour cells with wild-type p53.
Asunto(s)
Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Receptor ErbB-2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitinas/metabolismo , Células 3T3 , Animales , Línea Celular Transformada , Núcleo Celular/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Daño del ADN , Proteína p300 Asociada a E1A , Etopósido/farmacología , Humanos , Ratones , Proteínas Nucleares/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-mdm2 , Receptor ErbB-2/genética , Transactivadores/metabolismo , Proteína p14ARF Supresora de Tumor/metabolismoRESUMEN
Amplification or overexpression of HER-2/neu in cancer cells confers resistance to apoptosis and promotes cell growth. The cellular localization of p21Cip1/WAF1 has been proposed to be critical either in promoting cell survival or in inhibiting cell growth. Here we show that HER-2/neu-mediated cell growth requires the activation of Akt, which associates with p21Cip1/WAF1 and phosphorylates it at threonine 145, resulting in cytoplasmic localization of p21Cip1/WAF1. Furthermore, blocking the Akt pathway with a dominant-negative Akt mutant restores the nuclear localization and cell-growth-inhibiting activity of p21Cip1/WAF1. Our results indicate that HER-2/neu induces cytoplasmic localization of p21Cip1/WAF1 through activation of Akt to promote cell growth, which may have implications for the oncogenic activity of HER-2/neu and Akt.
Asunto(s)
División Celular/fisiología , Ciclinas/metabolismo , Citoplasma/metabolismo , Inhibidores Enzimáticos/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Receptor ErbB-2/metabolismo , Células 3T3 , Adenocarcinoma/genética , Adenocarcinoma/patología , Secuencias de Aminoácidos , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , División Celular/genética , Fraccionamiento Celular , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Femenino , Fibroblastos , Regulación de la Expresión Génica , Ratones , Microscopía Fluorescente , Fosforilación , Transporte de Proteínas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , Receptor ErbB-2/genética , Transducción de Señal/fisiología , TransfecciónRESUMEN
Overexpression and activation of HER-2/neu, a proto-oncogene, play a pivotal role in cancer formation. Strong expression of HER-2/neu in cancers has been associated with poor prognosis. Reduced expression of p27(Kip1), a cyclin-dependent kinase inhibitor, correlates with poor clinical outcome in many types of carcinomas. Because many cancers with the overexpression of HER-2/neu overlap with those affected by reduced p27 expression, we studied the link between HER-2/neu oncogenic signals and p27 regulation. We found that down-regulation of p27 correlates with HER-2/neu overexpression. To address the molecular mechanism of this inverse correlation, we found that reduction of p27 is caused by enhanced ubiquitin-mediated degradation, and the HER-2/Grb2/MAPK pathway is involved in the decrease of p27 stability. Also, HER-2/neu activity causes mislocation of p27 and Jun activation domain-binding protein 1 (JAB1), an exporter of p27, into the cytoplasm, thereby facilitating p27 degradation. These results reveal that HER-2/neu signals reduce p27 stability and thus present potential points for therapeutic intervention in HER-2/neu-associated cancers.
Asunto(s)
Proteínas de Ciclo Celular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Genes erbB-2 , Proteínas Asociadas a Microtúbulos/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas Supresoras de Tumor , Células 3T3 , Animales , Línea Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Humanos , Cinética , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , Proteínas Quinasas/metabolismo , Proto-Oncogenes Mas , Proteínas Recombinantes/metabolismo , Transducción de Señal , Transfección , Ubiquitinas/metabolismoRESUMEN
Overexpression of HER-2/neu correlates with poor survival of breast and ovarian cancer patients and induces resistance to tumor necrosis factor (TNF), which causes cancer cells to escape from host immune defenses. The mechanism of HER-2/neu-induced TNF resistance is unknown. Here we report that HER-2/neu activates Akt and NF-kappaB without extracellular stimulation. Blocking of the Akt pathway by a dominant-negative Akt sensitizes the HER-2/neu-overexpressing cells to TNF-induced apoptosis and inhibits IkappaB kinases, IkappaB phosphorylation, and NF-kappaB activation. Our results suggested that HER-2/neu constitutively activates the Akt/NF-kappaB anti-apoptotic cascade to confer resistance to TNF on cancer cells and reduce host defenses against neoplasia.
Asunto(s)
Apoptosis , Neoplasias de la Mama/metabolismo , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptor ErbB-2/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Células 3T3 , Animales , Neoplasias de la Mama/mortalidad , Resistencia a Medicamentos , Activación Enzimática , Humanos , Quinasa I-kappa B , Ratones , Pronóstico , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptor ErbB-2/genética , Transducción de SeñalRESUMEN
The adenovirus E1A protein has been implicated in increasing cellular susceptibility to apoptosis induced by tumor necrosis factor (TNF); however, its mechanism of action is still unknown. Since activation of nuclear factor kappaB (NF-kappaB) has been shown to play an anti-apoptotic role in TNF-induced apoptosis, we examined apoptotic susceptibility and NF-kappaB activation induced by TNF in the E1A transfectants and their parental cells. Here, we reported that E1A inhibited activation of NF-kappaB and rendered cells more sensitive to TNF-induced apoptosis. We further showed that this inhibition was through suppression of IkappaB kinase (IKK) activity and IkappaB phosphorylation. Moreover, deletion of the p300 and Rb binding domains of E1A abolished its function in blocking IKK activity and IkappaB phosphorylation, suggesting that these domains are essential for the E1A function in down-regulating IKK activity and NF-kappaB signaling. However, the role of E1A in inhibiting IKK activity might be indirect. Nevertheless, our results suggest that inhibition of IKK activity by E1A is an important mechanism for the E1A-mediated sensitization of TNF-induced apoptosis.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Proteínas E1A de Adenovirus/genética , Apoptosis , Femenino , Humanos , Quinasa I-kappa B , Cinética , Masculino , FN-kappa B/antagonistas & inhibidores , Neoplasias Ováricas , Neoplasias de la Próstata , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Transfección , Células Tumorales CultivadasRESUMEN
Monoamine oxidase B (MAO B) catalyzes the oxidative deamination of biogenic and xenobiotic amines. The oxidative step is coupled to the reduction of an obligatory cofactor, FAD, which is covalently linked to the apoenzyme at Cys397. Our previous studies identified two noncovalent flavin-binding regions in MAO B (residues 6-34 and 39-46) (Kwan, S.-W., Lewis, D. A., Zhou, B. P., and Abell, C. W. (1995) Arch. Biochem. Biophys. 316, 385-391; Zhou, B. P., Lewis, D. A., Kwan, S.-W., Kirksey, T. J., and Abell, C. W. (1995) Biochemistry 34, 9526-9531). In these regions, Glu34 and Tyr44 were found to be required for the initial binding of FAD. By comparing sequences with enzymes in the oxidoreductase family, we now have found an additional FAD-binding site in MAO B (residues 222-227), which is highly conserved across species (human, bovine, and rat). This conserved sequence contains adjacent glycine and aspartate residues (Gly226 and Asp227). Based on the x-ray crystal structures of several oxidoreductases (Eggink, G., Engel, H., Vriend, G., Terpstra, P., and Witholt, B. (1990) J. Mol. Biol. 212, 135-142; Van Driessche, G., Kol, M., Chen, Z.-W., Mathews, F. S., Meyer, T. E., Bartsch, R. G., Cusanovich, M. A., and Van Beeumen, J. J. (1996) Protein Sci. 5, 1753-1764), the Gly residue at the end of a beta-strand facilitates a sharp turn and extends the beta-carbonyl group of Asp to interact with the 3'-hydroxyl group of the ribityl chain of FAD. To assess the hypothesis that Gly226 and Asp227 are involved in FAD binding in MAO B, site-specific mutants that encode substitutions at these positions were prepared and expressed in mammalian COS-7 cells. Our results indicate that Gly226 and the beta-carbonyl group of Asp227 are required for covalent flavinylation and catalytic activity of MAO B, but not for noncovalent binding of FAD. Our studies also reveal that mutagenesis at Glu34 and Tyr44 not only interferes with covalent flavinylation and catalytic activity of MAO B, but also with noncovalent binding of FAD. Based on these collective results, we propose that the coupling of FAD to the MAO B apoenzyme is a multistep process.
Asunto(s)
Flavina-Adenina Dinucleótido/metabolismo , Monoaminooxidasa/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Células COS , Catálisis , Secuencia Conservada/genética , Flavoproteínas/química , Expresión Génica/genética , Humanos , Datos de Secuencia Molecular , Monoaminooxidasa/genética , Mutagénesis Sitio-Dirigida/genética , Unión Proteica/fisiología , Homología de Secuencia de Aminoácido , Transfección/genéticaRESUMEN
The adenoviral E1A protein has been implicated in the potentiation of apoptosis induced by various external stimuli, but the exact mechanism of that potentiation is not clear. In this study, we compared the sensitivity to ionizing gamma-irradiation of E1A transfectants with that of parental cells in a human ovarian cancer cell line (SKOV3.ip1); we found that the E1A transfectants became sensitive to radiation-induced apoptosis. Recently, activation of the transcription factor nuclear factor-kappaB (NF-kappaB) has been shown to play a key role in the anti-apoptotic pathway of radiation-induced apoptosis. In an attempt to determine whether NF-kappaB was involved in the E1A-mediated sensitization of radiation-induced apoptosis, we found that radiation-induced activation of NF-kappaB occurred in the parental cells but was blocked in the E1A transfectants. Furthermore, parental cells cotransfected with NF-kappaB and E1A were better protected from undergoing apoptosis upon irradiation than those transfected with E1A alone. Thus, our results suggest that inhibition of NF-kappaB activation by E1A is a plausible mechanism for E1A-mediated sensitization of radiation-induced apoptosis.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Apoptosis/efectos de la radiación , FN-kappa B/antagonistas & inhibidores , Proteínas E1A de Adenovirus/genética , Línea Celular , Fragmentación del ADN , Citometría de Flujo , Humanos , Transfección , Células Tumorales CultivadasRESUMEN
Monoamine oxidase B (MAO B) catalyzes the oxidative deamination of biogenic and xenobiotic amines. The oxidative step is coupled to the reduction of an obligatory cofactor, FAD, which is covalently linked to the enzyme at Cys397. In this study, we developed a novel riboflavin-depleted (Rib-) COS-7 cell line to study the flavinylation of MAO B. ApoMAO B can be obtained by expressing MAO B cDNA in these cells. We found that MAO B is expressed equally in the presence or absence of FAD and that apoMAO B can be inserted into the outer mitochondrial membrane. Flavinylation of MAO B was achieved by introducing MAO B cDNA and different flavin derivatives simultaneously into Rib- COS-7 cells via electroporation. Since the addition of riboflavin, FMN, or FAD resulted in equal levels of MAO B activity, we conclude that the flavin which initially binds to apoMAO B is FAD. In our previous work, we used site-directed mutagenesis to show that Glu34 in the dinucleotide-binding motif of MAO B is essential for MAO B activity, and we postulated that this residue is involved in FAD binding. In this study, we tested the role of residue 34 in flavin binding by expressing wild-type or mutant MAO B cDNA in Rib- COS-7 cells with the addition of [14C]FAD. We found that Glu34 is essential for both FAD binding and catalytic activity. Thus, FAD binds to MAO B in a dual manner at Glu34 noncovalently and Cys397 covalently. We conclude that Glu34 is critical for the initial non-covalent binding of FAD and is instrumental in delivering FAD to the covalent attachment site at Cys397.
Asunto(s)
Flavinas/metabolismo , Monoaminooxidasa/metabolismo , Animales , Apoenzimas/genética , Apoenzimas/metabolismo , Sitios de Unión , Línea Celular , ADN Complementario/genética , Flavina-Adenina Dinucleótido/metabolismo , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Técnicas In Vitro , Mitocondrias/enzimología , Monoaminooxidasa/genética , Mutagénesis Sitio-DirigidaRESUMEN
Monoamine oxidase B (MAO B), an integral protein of the outer mitochondrial membrane, catalyzes the oxidative deamination of various neuroactive and vasoactive amines. A covalently bound FAD cofactor at Cys-397 of human MAO B is required for the oxidation of the amine substrates. In addition to the covalent binding site, MAO B also contains a noncovalent FAD binding region (residues 6-34) known as the dinucleotide binding motif. Previously, we have shown that Glu-34 is required for catalytic activity, presumably by forming a hydrogen bond between the carboxylate group of glutamate and the 2'-hydroxyl group of ribose in the AMP moiety of FAD. In this work, we have identified a third FAD binding site in MAO B (residues 39-46) by sequence comparisons to other flavoenzymes. The conserved sequence contains a tyrosine residue (Tyr-44) which, based on the X-ray crystal structure of ferredoxin-NADP+ reductase, is postulated to participate in FAD binding through van der Waals contact with the isoalloxazine ring and a hydrogen bond to the 3'-hydroxy of the ribityl moiety. To test the postulated role of this tyrosine residue, site-directed mutants that encode substitutions at Tyr-44 were prepared and expressed in mammalian COS-7 cells. Variant MAO B enzymes were then characterized with respect to enzymatic activity and [14C]FAD incorporation. Substitution of tyrosine with phenylalanine had no effect on MAO B activity or the level of [14C]FAD incorporation compared to the wild-type enzyme, indicating that the hydroxyl group of the tyrosine residue was not essential at residue 44.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Monoaminooxidasa/química , Monoaminooxidasa/metabolismo , Mutagénesis Sitio-Dirigida , Tirosina , Secuencia de Aminoácidos , Animales , Arabidopsis/enzimología , Secuencia de Bases , Sitios de Unión , Línea Celular , Chlorocebus aethiops , Secuencia Conservada , Escherichia coli/enzimología , Flavina-Adenina Dinucleótido/metabolismo , Humanos , Riñón , Cinética , Modelos Estructurales , Datos de Secuencia Molecular , Monoaminooxidasa/biosíntesis , Oligodesoxirribonucleótidos , Mutación Puntual , Conformación Proteica , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Spinacia oleracea/enzimología , TransfecciónRESUMEN
Monoamine oxidase B (MAO B), an integral protein of the outer mitochondrial membrane, catalyzes the oxidative deamination of neuroactive and vasoactive amines. The oxidation step is coupled to the reduction of an obligatory FAD cofactor. In this study, we have examined the role of one amino acid (Glu34) in human MAO B that is thought to play a crucial role in binding to the 2'-hydroxy group of ribose in the AMP moiety of FAD. Glu34 is located within a region of the MAO B molecule of high sequence identity to the dinucleotide-binding site in other flavoproteins. In MAO B, this region is postulated to consist of a beta 1-sheet-alpha-helix-beta 2-sheet motif which culminates with a Glu at the C-terminal end of the second beta-sheet. We used site-directed mutagenesis to convert Glu at position 34 to Asp, Gln, and Ala. The wild-type and mutant cDNAs were then transiently transfected into COS-7 cells and assayed for MAO B activity. All three variants exhibited a dramatic decrease in the enzymatic activity as compared to wild-type MAO B, and only the Asp variant retained any detectable activity. Our studies indicate that the Glu34 residue in human MAO B is essential for catalysis. Whether Glu34 is responsible only for alignment of the FAD for participation in the oxidation/reduction cycle or also for the initial binding of FAD to the apoenzyme remains to be determined.