Asunto(s)
Fosfatidilinositol 3-Quinasas , Transducción de Señal , Fosforilación , Transporte Activo de Núcleo Celular , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Proteína de Unión al GTP ran/genética , Proteína de Unión al GTP ran/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Aberrant signaling by the mammalian target of rapamycin (mTOR) contributes to the devastating features of cancer cells. Thus, mTOR is a critical therapeutic target and catalytic inhibitors are being investigated as anti-cancer drugs. Although mTOR inhibitors initially block cell proliferation, cell viability and migration in some cancer cells are quickly restored. Despite sustained inhibition of mTORC1/2 signaling, Akt, a kinase regulating cell survival and migration, regains phosphorylation at its regulatory sites. Mechanistically, mTORC1/2 inhibition promotes reorganization of integrin/focal adhesion kinase-mediated adhesomes, induction of IGFR/IR-dependent PI3K activation, and Akt phosphorylation via an integrin/FAK/IGFR-dependent process. This resistance mechanism contributes to xenograft tumor cell growth, which is prevented with mTOR plus IGFR inhibitors, supporting this combination as a therapeutic approach for cancers.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Movimiento Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Quinasa 1 de Adhesión Focal/metabolismo , Melanoma/tratamiento farmacológico , Complejos Multiproteicos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Somatomedina/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Quinasa 1 de Adhesión Focal/genética , Humanos , Integrina alfa2/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Melanoma/enzimología , Melanoma/patología , Ratones Desnudos , Complejos Multiproteicos/metabolismo , Invasividad Neoplásica , Fosfatidilinositol 3-Quinasa/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Receptor IGF Tipo 1 , Receptores de Somatomedina/genética , Receptores de Somatomedina/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Serina-Treonina Quinasas TOR/metabolismo , Factores de Tiempo , Transfección , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Insufficient nutrients disrupt physiological homeostasis, resulting in diseases and even death. Considering the physiological and pathological consequences of this metabolic stress, the adaptive responses that cells utilize under this condition are of great interest. We show that under low-glucose conditions, cells initiate adaptation followed by apoptosis responses using PERK/Akt and MEK1/ERK2 signaling, respectively. For adaptation, cells engage the ER stress-induced unfolded protein response, which results in PERK/Akt activation and cell survival. Sustained and extreme energetic stress promotes a switch to isoform-specific MEK1/ERK2 signaling, induction of GCN2/eIF2α phosphorylation, and ATF4 expression, which overrides PERK/Akt-mediated adaptation and induces apoptosis through ATF4-dependent expression of pro-apoptotic factors including Bid and Trb3. ERK2 activation during metabolic stress contributes to changes in TCA cycle and amino acid metabolism, and cell death, which is suppressed by glutamate and α-ketoglutarate supplementation. Taken together, our results reveal promising targets to protect cells or tissues from metabolic stress.
Asunto(s)
Glucosa/farmacología , Ácido Glutámico/farmacología , Ácidos Cetoglutáricos/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Transducción de Señal/efectos de los fármacos , Apoptosis , Supervivencia Celular/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Proteína Quinasa 1 Activada por Mitógenos/genética , Estrés Fisiológico/efectos de los fármacosRESUMEN
ERK is a key coordinator of the epithelial-to-mesenchymal transition (EMT) in that a variety of EMT-inducing factors activate signaling pathways that converge on ERK to regulate EMT transcription programs. However, the mechanisms by which ERK controls the EMT program are not well understood. Through an analysis of the global changes of gene expression mediated by ERK2, we identified the transcription factor FoxO1 as a potential mediator of ERK2-induced EMT, and thus we investigated the mechanism by which ERK2 regulates FoxO1. Additionally, our analysis revealed that ERK2 induced the expression of Dock10, a Rac1/Cdc42 GEF, during EMT. We demonstrate that the activation of the Rac1/JNK signaling axis downstream of Dock10 leads to an increase in FoxO1 expression and EMT. Taken together, our study uncovers mechanisms by which epithelial cells acquire less proliferative but more migratory mesenchymal properties and reveals potential therapeutic targets for cancers evolving into a metastatic disease state.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Proteína Forkhead Box O1/genética , Factores de Intercambio de Guanina Nucleótido/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Línea Celular Tumoral , Regulación de la Expresión Génica/genética , Humanos , Sistema de Señalización de MAP Quinasas/genética , Activación Transcripcional/genética , Proteína de Unión al GTP rac1/genéticaRESUMEN
Hyperactivation of Ras-ERK1/2 signaling is critical to the development of many human malignancies, but little is known regarding the specific contribution of ERK1 or ERK2 to oncogenic processes. We demonstrate that ERK2 but not ERK1 signaling is necessary for Ras-induced epithelial-to-mesenchymal transformation (EMT). Further, ERK2 but not ERK1 overexpression is sufficient to induce EMT. Many ERK1/2-interacting proteins contain amino acid motifs, e.g., DEF or D-motifs, which regulate docking with ERK1/2. Remarkably, ERK2 signaling to DEF motif-containing targets is required to induce EMT and correlates with increased migration, invasion, and survival. Importantly, the late-response gene product Fra1 is necessary for Ras- and ERK2-induced EMT through upregulation of ZEB1/2 proteins. Thus, an apparent critical role for ERK2 DEF motif signaling during tumorigenesis is the regulation of Fra1 and the subsequent induction of ZEB1/2, suggesting a potential therapeutic target for Ras-regulated tumorigenesis.
Asunto(s)
Secuencias de Aminoácidos , Diferenciación Celular/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Transducción de Señal/fisiología , Biomarcadores/metabolismo , Línea Celular Tumoral , Movimiento Celular/fisiología , Células Epiteliales/citología , Células Epiteliales/fisiología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Mutación , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Interferencia de ARN , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc , Homeobox 1 de Unión a la E-Box con Dedos de Zinc , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
The p70 ribosomal protein S6 kinase 1 (S6K1) plays a key role in cell growth and proliferation by regulating insulin sensitivity, metabolism, protein synthesis, and cell cycle. Thus, deregulation of S6K contributes to the progression of type 2 diabetes, obesity, aging, and cancer. Considering the biological and clinical importance of S6K1, a complete understanding of its regulation is critical. One of the key motifs in the activation of S6K1 is a turn motif, but its regulation is not well understood. Here we provide evidence for two mechanisms of modulating turn motif phosphorylation and S6K1 activity. First, mammalian target of rapamycin regulates turn motif phosphorylation by inhibiting its dephosphorylation. Second, we unexpectedly found that glycogen synthase kinase (GSK)-3 promotes turn motif phosphorylation. Our studies show that mammalian target of rapamycin and GSK-3 cooperate to control the activity of S6K1, an important regulator of cell proliferation and growth. Our unexpected results provide a clear rationale for the development and use of drugs targeting GSK-3 to treat diseases such as diabetes, cancer, and age-related diseases that are linked to improper regulation of S6K1.
Asunto(s)
Proliferación Celular , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Línea Celular Tumoral , Cromatografía Liquida , Técnicas de Silenciamiento del Gen , Humanos , Immunoblotting , Inmunoprecipitación , Fosforilación , Espectrometría de Masas en Tándem , TransfecciónRESUMEN
Cells maintain and dynamically change their proteomes according to the environment and their needs. Mechanistic target of rapamycin (mTOR) is a key regulator of proteostasis, homeostasis of the proteome. Thus, dysregulation of mTOR leads to changes in proteostasis and the consequent progression of diseases, including cancer. Based on the physiological and clinical importance of mTOR signaling, we investigated mTOR feedback signaling, proteostasis, and cell fate. Here, we reveal that mTOR targeting inhibits eIF4E-mediated cap-dependent translation, but feedback signaling activates a translation initiation factor, eukaryotic translation initiation factor 3D (eIF3D), to sustain alternative non-canonical translation mechanisms. Importantly, eIF3D-mediated protein synthesis enables cell phenotype switching from proliferative to more migratory. eIF3D cooperates with mRNA-binding proteins such as heterogeneous nuclear ribonucleoprotein F (hnRNPF), heterogeneous nuclear ribonucleoprotein K (hnRNPK), and Sjogren syndrome antigen B (SSB) to support selective mRNA translation following mTOR inhibition, which upregulates and activates proteins involved in insulin receptor (INSR)/insulin-like growth factor 1 receptor (IGF1R)/insulin receptor substrate (IRS) and interleukin 6 signal transducer (IL-6ST)/Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. Our study highlights the mechanisms by which cells establish the dynamic change of proteostasis and the resulting phenotype switch.
Asunto(s)
Proteostasis , Receptor de Insulina , ARN Mensajero/metabolismo , Receptor de Insulina/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Sirolimus , Biosíntesis de ProteínasRESUMEN
Vascular morphogenesis is a key process for development, reproduction, and pathogenesis. Thus understanding the mechanisms of this process is of pathophysiological importance. Despite the fact that collagen I is the most abundant and potent promorphogenic molecule known, the molecular mechanisms by which this protein regulates endothelial cell tube morphogenesis are still unclear. Here we provide strong evidence that collagen I induces tube morphogenesis by inhibiting glycogen synthase kinase 3ß (GSK3ß). Further mechanistic studies revealed that GSK3ß activity is regulated by protein kinase D (PKD). PKD inhibited GSK3ß activity, which was required for collagen I-induced endothelial tube morphogenesis. We also found that GSK3ß regulated trafficking of integrin α(2)ß(1) in a Rab11-dependent manner. Taken together, our studies highlight the important role of PKD in the regulation of collagen I-induced vascular morphogenesis and show that it is mediated by the modulation of GSK3ß activity and integrin α(2)ß(1) trafficking.
Asunto(s)
Células Endoteliales/enzimología , Glucógeno Sintasa Quinasa 3/fisiología , Integrina alfa2beta1/fisiología , Morfogénesis/fisiología , Proteína Quinasa C/fisiología , Glucógeno Sintasa Quinasa 3 beta , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Transporte de Proteínas/fisiología , Transducción de SeñalRESUMEN
DNA damage and subsequent cellular response are the basis for many cancer treatments. In this issue of Science Signaling, Liu et al. elucidate a mechanism by which cancer cells survive DNA damage induced by radiation and chemotherapy.
Asunto(s)
Daño del ADN , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
Metastasis is the leading cause of cancer mortality. Chromatin remodeling provides the foundation for the cellular reprogramming necessary to drive metastasis. However, little is known about the nature of this remodeling and its regulation. Here, we show that metastasis-inducing pathways regulate histone chaperones to reduce canonical histone incorporation into chromatin, triggering deposition of H3.3 variant at the promoters of poor-prognosis genes and metastasis-inducing transcription factors. This specific incorporation of H3.3 into chromatin is both necessary and sufficient for the induction of aggressive traits that allow for metastasis formation. Together, our data clearly show incorporation of histone variant H3.3 into chromatin as a major regulator of cell fate during tumorigenesis, and histone chaperones as valuable therapeutic targets for invasive carcinomas.
Asunto(s)
Carcinoma/patología , Cromatina/metabolismo , Regulación Neoplásica de la Expresión Génica , Histonas/metabolismo , Metástasis de la Neoplasia/genética , Animales , Carcinogénesis/genética , Carcinoma/genética , Línea Celular Tumoral , Cromatina/genética , Factor 1 de Ensamblaje de la Cromatina/genética , Factor 1 de Ensamblaje de la Cromatina/metabolismo , Progresión de la Enfermedad , Epigénesis Genética , Transición Epitelial-Mesenquimal/genética , Femenino , Histonas/genética , Humanos , Masculino , Ratones , Regiones Promotoras Genéticas/genética , RNA-Seq , Factores de Transcripción/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
ErbB2 (HER2, Neu) and Ras play key roles in tumor invasion and metastasis. We identified a novel mechanism by which integrin alpha(6)beta(4) regulates ErbB2 expression, Ras activation, and the invasion of breast carcinoma cells. Here we show that integrin alpha(6)beta(4) regulates Ras activity especially in serum-depleted condition. Down-regulation of beta(4) integrin by beta(4) short hairpin RNA (shRNA) decreased Ras activity and carcinoma invasion whereas reexpression of this integrin restored Ras activity. ErbB2, a binding partner of epidermal growth factor receptor (EGFR), and EGFR modulated Ras activity, and integrin alpha(6)beta(4) regulated phospho-EGFR level without affecting EGFR expression. We also found that integrin alpha(6)beta(4) is involved in ErbB2 expression. Depletion of beta(4) by shRNA reduced ErbB2 protein level without affecting ErbB2 mRNA level and reexpression of beta(4) increased ErbB2 protein level. Reduction of eukaryotic initiation factor 4E, a rate-limiting factor for cap-dependent translation, decreased ErbB2 protein level, and beta(4) shRNA cells exhibited a shift in ErbB2 mRNA to light polysomes compared with control cells. These results show that integrin alpha(6)beta(4) regulates ErbB2 through translational control. In summary, we propose a novel mechanism for ErbB2 up-regulation and Ras activation in serum-depleted breast cancer cells; integrin alpha(6)beta(4) regulates the expression of ErbB2 and the subsequent phosphorylation of EGFR and activation of Ras. These findings provide a mechanism that substantiates the reported role of alpha(6)beta(4) in carcinoma invasion.
Asunto(s)
Neoplasias de la Mama/metabolismo , Receptores ErbB/metabolismo , Integrina alfa6beta4/fisiología , Receptor ErbB-2/metabolismo , Proteínas ras/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/biosíntesis , Receptores ErbB/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Humanos , Integrina alfa6beta4/biosíntesis , Integrina alfa6beta4/deficiencia , Integrina alfa6beta4/genética , Invasividad Neoplásica , Fosforilación , Biosíntesis de Proteínas , Quinazolinas , ARN Interferente Pequeño/genética , Receptor ErbB-2/biosíntesis , Receptor ErbB-2/genética , Transducción de Señal , Activación Transcripcional , Transfección , Tirfostinos/farmacologíaRESUMEN
Active Ras proteins contribute to breast carcinogenesis and progression. Here, we provide evidence that active H-Ras regulates the expression and activity of the E2F family of transcription factors in SUM-159 breast carcinoma cells. In addition, we show by using a DNA-binding mutant of E2F, as well as expression of specific E2Fs that are transcriptionally active, that the active E2Fs1-3 can mediate the H-Ras-dependent invasion of SUM-159 cells. The inhibitory E2Fs4-5, in contrast, do not influence invasion. One mechanism by which the active E2Fs promote H-Ras-dependent invasion seems to be their ability to increase expression of the beta4 integrin subunit, a component of the alpha6beta4 integrin that is known to enhance carcinoma invasion. Specifically, expression of E2Fs1-3 increased beta4 mRNA, protein, and cell surface expression. The active E2Fs were unable to stimulate invasion in cells that expressed a beta4 short hairpin RNA. This effect of the active E2Fs on beta4 expression does not seem to result from E2F-mediated beta4 transcription because the beta4 promoter lacks known E2F binding motifs. In summary, the data reported here indicate a novel mechanism by which H-Ras can promote the invasion of breast carcinoma cells. This mechanism links active H-Ras, transcriptionally active E2F, and the alpha6beta4 integrin in a common pathway that culminates in enhanced alpha6beta4-dependent invasion.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Factores de Transcripción E2F/metabolismo , Integrina alfa6beta4/biosíntesis , Proteínas ras/metabolismo , Animales , Neoplasias de la Mama/genética , Adhesión Celular/fisiología , Línea Celular Tumoral , Colágeno , Combinación de Medicamentos , Humanos , Integrina alfa6beta4/genética , Laminina/metabolismo , Ratones , Células 3T3 NIH , Invasividad Neoplásica , Proteoglicanos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transfección , Proteínas ras/genéticaRESUMEN
Metabolic reprogramming is a hallmark of cancer that includes increased glucose uptake and accelerated aerobic glycolysis. This phenotype is required to fulfill anabolic demands associated with aberrant cell proliferation and is often mediated by oncogenic drivers such as activated BRAF. In this study, we show that the MAPK-activated p90 ribosomal S6 kinase (RSK) is necessary to maintain glycolytic metabolism in BRAF-mutated melanoma cells. RSK directly phosphorylated the regulatory domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2), an enzyme that catalyzes the synthesis of fructose-2,6-bisphosphate during glycolysis. Inhibition of RSK reduced PFKFB2 activity and glycolytic flux in melanoma cells, suggesting an important role for RSK in BRAF-mediated metabolic rewiring. Consistent with this, expression of a phosphorylation-deficient mutant of PFKFB2 decreased aerobic glycolysis and reduced the growth of melanoma in mice. Together, these results indicate that RSK-mediated phosphorylation of PFKFB2 plays a key role in the metabolism and growth of BRAF-mutated melanomas.Significance: RSK promotes glycolytic metabolism and the growth of BRAF-mutated melanoma by driving phosphorylation of an important glycolytic enzyme. Cancer Res; 78(9); 2191-204. ©2018 AACR.
Asunto(s)
Melanoma/genética , Fosfofructoquinasa-2/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proliferación Celular/genética , Reprogramación Celular/genética , Glucosa/metabolismo , Glucólisis/genética , Células HeLa , Humanos , Melanoma/metabolismo , Melanoma/patología , FosforilaciónRESUMEN
Hypoxia plays a key role in tumor cell survival, invasion, and metastasis. Here we show that hypoxia increases tumor cell invasion by the modulation of Rab11, an important molecule for vesicular trafficking, especially membrane protein recycling and translocation of proteins from trans-Golgi network to plasma membrane. Dominant-negative Rab11 dramatically decreased hypoxia-induced invasion of MDA-MB-231 breast carcinoma cells without affecting cell apoptosis. Hypoxia-induced Rab11 trafficking is regulated by microtubule stability, as evidenced by the findings that hypoxia increases Glu tubulin and that colchicine blocks Rab11 trafficking and invasion. Inhibition of GSK-3beta activity by hypoxia seems to be central to microtubule stabilization and invasion. In fact, expression of a dominant-negative GSK-3beta was sufficient to stimulate invasion in normoxia. One target of Rab11-mediated trafficking that contributes to invasion is the integrin alpha6beta4. Hypoxia induced a significant increase in alpha6beta4 surface expression but it had no effect on the surface expression of alpha3beta1. This increase is dependent on Rab11 and stable microtubules. In summary, we identify vesicle trafficking as a novel target of hypoxic stimulation that is important for tumor invasion.
Asunto(s)
Neoplasias de la Mama/patología , Carcinoma/patología , Integrina alfa6beta4/metabolismo , Microtúbulos/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Carcinoma/genética , Carcinoma/metabolismo , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Activación Enzimática , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Humanos , Integrina alfa6beta4/biosíntesis , Ratones , Células 3T3 NIH , Invasividad Neoplásica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Transfección , Proteínas de Unión al GTP rab/biosíntesis , Proteínas de Unión al GTP rab/genéticaRESUMEN
Matrix metalloproteinase (MMP)-9 plays a key role in tumor invasion. Inhibitors of MMP-9 were screened from Metasequoia glyptostroboides (Dawn redwood) and one potent inhibitor, isoginkgetin, a biflavonoid, was identified. Noncytotoxic levels of isoginkgetin decreased MMP-9 production profoundly, but up-regulated the level of tissue inhibitor of metalloproteinase (TIMP)-1, an inhibitor of MMP-9, in HT1080 human fibrosarcoma cells. The major mechanism of Ras-dependent MMP-9 production in HT1080 cells was phosphatidylinositol 3-kinase (PI3K)/Akt/nuclear factor-kappaB (NF-kappaB) activation. Expression of dominant-active H-Ras and p85 (a subunit of PI3K) increased MMP-9 activity, whereas dominant-negative forms of these molecules decreased the level of MMP-9. H-Ras did not increase MMP-9 in the presence of a PI3K inhibitor, LY294002, and a NF-kappaB inhibitor, SN50. Further studies showed that isoginkgetin regulated MMP-9 production via PI3K/Akt/NF-kappaB pathway, as evidenced by the findings that isoginkgetin inhibited activities of both Akt and NF-kappaB. PI3K/Akt is a well-known key pathway for cell invasion, and isoginkgetin inhibited HT1080 tumor cell invasion substantially. Isoginkgetin was also quite effective in inhibiting the activities of Akt and MMP-9 in MDA-MB-231 breast carcinomas and B16F10 melanoma. Moreover, isoginkgetin treatment resulted in marked decrease in invasion of these cells. In summary, PI3K/Akt is a major pathway for MMP-9 expression and isoginkgetin markedly decreased MMP-9 expression and invasion through inhibition of this pathway. This suggests that isoginkgetin could be a potential candidate as a therapeutic agent against tumor invasion.
Asunto(s)
Biflavonoides/farmacología , Flavonoides/farmacología , Metaloproteinasa 9 de la Matriz/metabolismo , Neoplasias/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Fibrosarcoma/metabolismo , Humanos , Inhibidores de la Metaloproteinasa de la Matriz , Invasividad Neoplásica , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Células Tumorales Cultivadas , Quinasa de Factor Nuclear kappa BRESUMEN
mTORC1 is a central signaling node in controlling cell growth, proliferation, and metabolism that is aberrantly activated in cancers and certain cancer-associated genetic disorders, such as tuberous sclerosis complex (TSC) and sporadic lymphangioleiomyomatosis. However, while mTORC1-inhibitory compounds (rapamycin and rapalogs) attracted interest as candidate therapeutics, clinical trials have not replicated the promising findings in preclinical models, perhaps because these compounds tend to limit cell proliferation without inducing cell death. In seeking to address this issue, we performed a high-throughput screen for small molecules that could heighten the cytotoxicity of mTORC1 inhibitors. Here we report the discovery that combining inhibitors of mTORC1 and glutamate cysteine ligase (GCLC) can selectively and efficiently trigger apoptosis in Tsc2-deficient cells but not wild-type cells. Mechanistic investigations revealed that coinhibition of mTORC1 and GCLC decreased the level of the intracellular thiol antioxidant glutathione (GSH), thereby increasing levels of reactive oxygen species, which we determined to mediate cell death in Tsc2-deficient cells. Our findings offer preclinical proof of concept for a strategy to selectively increase the cytotoxicity of mTORC1 inhibitors as a therapy to eradicate tumor cells marked by high mTORC1 signaling, based on cotargeting a GSH-controlled oxidative stress pathway. Cancer Res; 76(16); 4816-27. ©2016 AACR.
Asunto(s)
Antineoplásicos/farmacología , Complejos Multiproteicos/antagonistas & inhibidores , Estrés Oxidativo/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Ensayos Analíticos de Alto Rendimiento , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones SCID , Microscopía Confocal , Microscopía Electrónica de Transmisión , Reacción en Cadena de la Polimerasa , Especies Reactivas de Oxígeno , Sirolimus/farmacología , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Δ(5)-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ(5)-3-ketosteroid to its conjugated Δ(4)-isomers at a rate that approaches the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 Oη and C3-O of equilenin, an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with equilenin, suggesting that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-equilenin complex revealed that the distance between Tyr14 Oη and C3-O of the bound steroid was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1-11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1.7-2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI.
Asunto(s)
Equilenina/metabolismo , Pseudomonas putida/enzimología , Esteroide Isomerasas/química , Esteroide Isomerasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Equilenina/química , Enlace de Hidrógeno , Modelos Moleculares , Mutación , Unión Proteica , Espectroscopía de Protones por Resonancia Magnética , Pseudomonas putida/genética , Esteroide Isomerasas/metabolismo , Especificidad por SustratoRESUMEN
Deregulation of translation initiation factors contributes to many pathogenic conditions, including cancer. Here, we report the definition of a novel regulatory pathway for translational initiation with possible therapeutic import in cancer. Specifically, we found that casein kinase 1ε (CK1ε) is highly expressed in breast tumors and plays a critical role in cancer cell proliferation by controlling mRNA translation. Eukaryotic translation initiation factor eIF4E, an essential component of the translation initiation complex eIF4F, is downregulated by binding the negative-acting factor 4E-BP1. We found that genetic or pharmacologic inhibition of CK1ε attenuated 4E-BP1 phosphorylation, thereby increasing 4E-BP1 binding to eIF4E and inhibiting mRNA translation. Mechanistic investigations showed that CK1ε interacted with and phosphorylated 4E-BP1 at two novel sites T41 and T50, which were essential for 4E-BP1 inactivation along with increased mRNA translation and cell proliferation. In summary, our work identified CK1ε as a pivotal regulator of mRNA translation and cell proliferation that acts by inhibiting 4E-BP1 function. As CK1ε is highly expressed in breast tumors, these findings offer an initial rationale to explore CK1ε blockade as a therapeutic strategy to treat cancers driven by deregulated mRNA translation.
Asunto(s)
Neoplasias de la Mama/enzimología , Caseína Cinasa 1 épsilon/metabolismo , ARN Mensajero/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Muerte Celular/genética , Muerte Celular/fisiología , Procesos de Crecimiento Celular/genética , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Factores Eucarióticos de Iniciación , Femenino , Células HEK293 , Humanos , Ratones , Ratones Desnudos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , ARN Mensajero/genéticaRESUMEN
Integrins are major mediators of cancer cell adhesion to extracellular matrix. Through this interaction, integrins play critical roles in cell migration, invasion, metastasis, and resistance to apoptosis during tumor progression. Recent studies highlight the importance of integrin trafficking, endocytosis and recycling, for the functions of integrins in cancer cells. Understanding the molecular mechanisms of integrin trafficking is pivotal for understanding tumor progression and for the development of anticancer drugs.
RESUMEN
The major participants of the Ras/ERK and PI3-kinase (PI3K) pathways are well characterized. The cellular response to activation of these pathways, however, can vary dramatically. How differences in signal strength, timing, spatial location, and cellular context promote specific cell-fate decisions remains unclear. Nuclear transport processes can have a major impact on the determination of cell fate; however, little is known regarding how nuclear transport is regulated by or regulates these pathways. Here we show that RSK and Akt, which are activated downstream of Ras/ERK and PI3K, respectively, modulate the Ran gradient and nuclear transport by interacting with, phosphorylating, and regulating Ran-binding protein 3 (RanBP3) function. Our findings highlight an important link between two major cell-fate determinants: nuclear transport and the Ras/ERK/RSK and PI3K/Akt signaling pathways.