RESUMEN
Protein S (PROS1) is a vitamin K-dependent anticoagulant factor, which also acts as an agonist for the TYRO3, AXL, and MERTK (TAM) tyrosine kinase receptors. PROS1 is produced by the endothelium which also expresses TAM receptors, but little is known about its effects on vascular function and permeability. Transwell permeability assays as well as Western blotting and immunostaining analysis were used to monitor the possible effects of PROS1 on both endothelial cell permeability and on the phosphorylation state of specific signaling proteins. We show that human PROS1, at its circulating concentrations, substantially increases both the basal and VEGFA-induced permeability of endothelial cell (EC) monolayers. PROS1 induces p38 MAPK (Mitogen Activated Protein Kinase), Rho/ROCK (Rho-associated protein kinase) pathway activation, and actin filament remodeling, as well as substantial changes in Vascular Endothelial Cadherin (VEC) distribution and its phosphorylation on Ser665 and Tyr685. It also mediates c-Src and PAK-1 (p21-activated kinase 1) phosphorylation on Tyr416 and Ser144, respectively. Exposure of EC to human PROS1 induces VEC internalization as well as its cleavage into a released fragment of 100 kDa and an intracellular fragment of 35 kDa. Using anti-TAM neutralizing antibodies, we demonstrate that PROS1-induced VEC and c-Src phosphorylation are mediated by both the MERTK and TYRO3 receptors but do not involve the AXL receptor. MERTK and TYRO3 receptors are also responsible for mediating PROS1-induced MLC (Myosin Light Chain) phosphorylation on a site targeted by the Rho/ROCK pathway. Our report provides evidence for the activation of the c-Src/VEC and Rho/ROCK/MLC pathways by PROS1 for the first time and points to a new role for PROS1 as an endogenous vascular permeabilizing factor.
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Tanshinone IIA (T2A) is a bioactive compound that provides promise in the treatment of glioblastoma multiforme (GBM), with a range of molecular mechanisms including the inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) and the induction of autophagy. Recently, T2A has been demonstrated to function through sestrin 2 (SESN) to inhibit mTORC1 activity, but its possible impact on autophagy through this pathway has not been investigated. Here, the model system Dictyostelium discoideum and GBM cell lines were employed to investigate the cellular role of T2A in regulating SESN to inhibit mTORC1 and activate autophagy through a GATOR2 component MIOS. In D. discoideum, T2A treatment induced autophagy and inhibited mTORC1 activity, with both effects lost upon the ablation of SESN (sesn-) or MIOS (mios-). We further investigated the targeting of MIOS to reproduce this effect of T2A, where computational analysis identified 25 novel compounds predicted to strongly bind the human MIOS protein, with one compound (MIOS inhibitor 3; Mi3) reducing cell proliferation in two GBM cells. Furthermore, Mi3 specificity was demonstrated through the loss of potency in the D. discoideum mios- cells regarding cell proliferation and the induction of autophagy. In GBM cells, Mi3 treatment also reduced mTORC1 activity and induced autophagy. Thus, a potential T2A mimetic showing the inhibition of mTORC1 and induction of autophagy in GBM cells was identified.
Asunto(s)
Abietanos , Autofagia , Dictyostelium , Glioblastoma , Diana Mecanicista del Complejo 1 de la Rapamicina , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patología , Abietanos/farmacología , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Dictyostelium/efectos de los fármacos , Dictyostelium/metabolismo , Proliferación Celular/efectos de los fármacos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/antagonistas & inhibidores , SestrinasRESUMEN
Ribosomal protein S6 kinases belong to a family of highly conserved enzymes in eukaryotes that regulate cell growth, proliferation, survival, and the stress response. It is well established that the activation and downstream signalling of p70S6Ks involve multiple phosphorylation events by key regulators of cell growth, survival, and energy metabolism. Here, we report for the first time the covalent modification of p70S6K1 by coenzyme A (CoA) in response to oxidative stress, which regulates its kinase activity. The site of CoA binding (CoAlation) was mapped by mass spectrometry to cysteine 217 (Cys217), located in the kinase activation loop and only one amino acid away from the tripeptide DFG motif, which facilitates ATP-binding. The CoAlation of recombinant p70S6K1 was demonstrated in vitro and was shown to inhibit its kinase activity. Our molecular docking and dynamics analysis revealed the most likely mode for CoA binding to p70S6K1. This mechanism involves the non-covalent binding of the CoA ADP moiety to the p70S6K1 nucleotide-binding pocket, positioning the CoA thiol group in close proximity to form a covalent bond with the surface-exposed Cys217 residue. These findings support a "dual anchor" mechanism for protein kinase inhibition by CoAlation in cellular response to oxidative stress. Furthermore, the inhibition of S6K1 by CoAlation may open new avenues for developing novel inhibitors.
Asunto(s)
Coenzima A , Simulación del Acoplamiento Molecular , Estrés Oxidativo , Proteínas Quinasas S6 Ribosómicas 70-kDa , Humanos , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Coenzima A/metabolismo , Fosforilación , Unión Proteica , Sitios de Unión , Cisteína/metabolismo , Simulación de Dinámica MolecularRESUMEN
Ribosomal S6 kinases (S6Ks) are critical regulators of cell growth, homeostasis, and survival, with dysregulation of these kinases found to be associated with various malignancies. While S6K1 has been extensively studied, S6K2 has been neglected despite its clear involvement in cancer progression. Protein arginine methylation is a widespread post-translational modification regulating many biological processes in mammalian cells. Here, we report that p54-S6K2 is asymmetrically dimethylated at Arg-475 and Arg-477, two residues conserved amongst mammalian S6K2s and several AT-hook-containing proteins. We demonstrate that this methylation event results from the association of S6K2 with the methyltransferases PRMT1, PRMT3, and PRMT6 in vitro and in vivo and leads to nuclear the localisation of S6K2 that is essential to the pro-survival effects of this kinase to starvation-induced cell death. Taken together, our findings highlight a novel post-translational modification regulating the function of p54-S6K2 that may be particularly relevant to cancer progression where general Arg-methylation is often elevated.
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Fenómenos Biológicos , Proteínas Quinasas S6 Ribosómicas 90-kDa , Animales , Fosforilación , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Mamíferos/metabolismoRESUMEN
While we previously revealed RSK4 as a therapeutic target in lung and bladder cancers, the wider role of this kinase in other cancers remains controversial. Indeed, other reports instead proposed RSK4 as a tumour suppressor in colorectal and gastric cancers and are contradictory in breast malignancies. One explanation for these discrepancies may be the expression of different RSK4 isoforms across cancers. Four RNAs are produced from the RSK4 gene, with two being protein-coding. Here, we analysed the expression of the latter across 30 normal and 33 cancer tissue types from the combined GTEx/TCGA dataset and correlated it with clinical features. This revealed the expression of RSK4 isoforms 1 and 2 to be independent prognostic factors for patient survival, pathological stage, cancer metastasis, recurrence, and immune infiltration in brain, stomach, cervical, and kidney cancers. However, we found that upregulation of either isoform can equally be associated with good or bad prognosis depending on the cancer type, and changes in the expression ratio of isoforms fail to predict clinical outcome. Hence, differential isoform expression alone cannot explain the contradictory roles of RSK4 in cancers, and further research is needed to highlight the underlying mechanisms for the context-dependent function of this kinase.
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Neoplasias de la Mama , Neoplasias Renales , Humanos , Femenino , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Relevancia Clínica , Isoformas de Proteínas/genética , Neoplasias de la Mama/genéticaRESUMEN
Here, we show that miR-515-5p inhibits cancer cell migration and metastasis. RNA-seq analyses of both oestrogen receptor receptor-positive and receptor-negative breast cancer cells overexpressing miR-515-5p reveal down-regulation of NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNAs. We demonstrate that miR-515-5p inhibits MARK4 directly 3' UTR interaction and that MARK4 knock-down mimics the effect of miR-515-5p on breast and lung cancer cell migration. MARK4 overexpression rescues the inhibitory effects of miR-515-5p, suggesting miR-515-5p mediates this process through MARK4 down-regulation. Furthermore, miR-515-5p expression is reduced in metastases compared to primary tumours derived from both in vivo xenografts and samples from patients with breast cancer. Conversely, miR-515-5p overexpression prevents tumour cell dissemination in a mouse metastatic model. Moreover, high miR-515-5p and low MARK4 expression correlate with increased breast and lung cancer patients' survival, respectively. Taken together, these data demonstrate the importance of miR-515-5p/MARK4 regulation in cell migration and metastasis across two common cancers.
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Movimiento Celular , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Metástasis de la Neoplasia , Proteínas Serina-Treonina Quinasas/genética , Células A549 , Animales , Apoptosis , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular/genética , Regulación hacia Abajo , Femenino , Humanos , Neoplasias Pulmonares/genética , Células MCF-7 , Ratones , Invasividad Neoplásica , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , ARN MensajeroRESUMEN
The increased cap-independent translation of anti-apoptotic proteins is involved in the development of drug resistance in lung cancer but signalling events regulating this are poorly understood. Fibroblast growth factor 2 (FGF-2) signalling-induced S6 kinase 2 (S6K2) activation is necessary, but the downstream mediator(s) coupling this kinase to the translational response is unknown. Here, we show that S6K2 binds and phosphorylates hnRNPA1 on novel Ser4/6 sites, increasing its association with BCL-XL and XIAP mRNAs to promote their nuclear export. In the cytoplasm, phosphoS4/6-hnRNPA1 dissociates from these mRNAs de-repressing their IRES-mediated translation. This correlates with the phosphorylation-dependent association of hnRNPA1 with 14-3-3 leading to hnRNPA1 sumoylation on K183 and its re-import into the nucleus. A non-phosphorylatible, S4/6A mutant prevented these processes, hindering the pro-survival activity of FGF-2/S6K2 signalling. Interestingly, immunohistochemical staining of lung and breast cancer tissue samples demonstrated that increased S6K2 expression correlates with decreased cytoplasmic hnRNPA1 and increased BCL-XL expression. In short, phosphorylation on novel N-term sites of hnRNPA1 promotes translation of anti-apoptotic proteins and is indispensable for the pro-survival effects of FGF-2.
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Núcleo Celular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Proteínas 14-3-3/metabolismo , Transporte Activo de Núcleo Celular , Línea Celular , Células HEK293 , Ribonucleoproteína Nuclear Heterogénea A1 , Humanos , Transducción de Señal , Sumoilación , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
CUTL1, also known as CDP, Cut, or Cux-1, is a homeodomain transcriptional regulator known to be involved in development and cell cycle progression. Here we report that CUTL1 activity is associated with increased migration and invasiveness in numerous tumor cell lines, both in vitro and in vivo. Furthermore, we identify CUTL1 as a transcriptional target of transforming growth factor beta and a mediator of its promigratory effects. CUTL1 activates a transcriptional program regulating genes involved in cell motility, invasion, and extracellular matrix composition. CUTL1 expression is significantly increased in high-grade carcinomas and is inversely correlated with survival in breast cancer. This suggests that CUTL1 plays a central role in coordinating a gene expression program associated with cell motility and tumor progression.
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Movimiento Celular/fisiología , Invasividad Neoplásica/patología , Neoplasias/patología , Proteínas Nucleares/fisiología , Proteínas Represoras/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Supervivencia sin Enfermedad , Regulación hacia Abajo/genética , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio , Humanos , Ratones , Ratones Desnudos , Células 3T3 NIH , Invasividad Neoplásica/genética , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , ARN Bicatenario/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Proteína Smad4 , Transactivadores/metabolismo , Factores de Transcripción , Transcripción Genética/efectos de los fármacos , Transfección , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia Arriba/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Glioblastomas are a highly aggressive cancer type which respond poorly to current pharmaceutical treatments, thus novel therapeutic approaches need to be investigated. One such approach involves the use of the bioactive natural product Tanshinone IIA (T2A) derived from the Chinese herb Danshen, where mechanistic insight for this anti-cancer agent is needed to validate its use. Here, we employ a tractable model system, Dictyostelium discoideum, to provide this insight. T2A potently inhibits cellular proliferation of Dictyostelium, suggesting molecular targets in this model. We show that T2A rapidly reduces phosphoinositide 3 kinase (PI3K) and protein kinase B (PKB) activity, but surprisingly, the downstream complex mechanistic target of rapamycin complex 1 (mTORC1) is only inhibited following chronic treatment. Investigating regulators of mTORC1, including PKB, tuberous sclerosis complex (TSC), and AMP-activated protein kinase (AMPK), suggests these enzymes were not responsible for this effect, implicating an additional molecular mechanism of T2A. We identify this mechanism as the increased expression of sestrin, a negative regulator of mTORC1. We further show that combinatory treatment using a PI3K inhibitor and T2A gives rise to a synergistic inhibition of cell proliferation. We then translate our findings to human and mouse-derived glioblastoma cell lines, where both a PI3K inhibitor (Paxalisib) and T2A reduces glioblastoma proliferation in monolayer cultures and in spheroid expansion, with combinatory treatment significantly enhancing this effect. Thus, we propose a new approach for cancer treatment, including glioblastomas, through combinatory treatment with PI3K inhibitors and T2A.
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Mesothelioma is an aggressive cancer of the mesothelial layer associated with an extensive fibrotic response. The latter is in large part mediated by cancer-associated fibroblasts which mediate tumour progression and poor prognosis. However, understanding of the crosstalk between cancer cells and fibroblasts in this disease is mostly lacking. Here, using co-cultures of patient-derived mesothelioma cell lines and lung fibroblasts, we demonstrate that fibroblast activation is a self-propagated process producing a fibrotic extracellular matrix (ECM) and triggering drug resistance in mesothelioma cells. Following characterisation of mesothelioma cells/fibroblasts signalling crosstalk, we identify several FDA-approved targeted therapies as far more potent than standard-of-care Cisplatin/Pemetrexed in ECM-embedded co-culture spheroid models. In particular, the SRC family kinase inhibitor, Saracatinib, extends overall survival well beyond standard-of-care in a mesothelioma genetically-engineered mouse model. In short, we lay the foundation for the rational design of novel therapeutic strategies targeting mesothelioma/fibroblast communication for the treatment of mesothelioma patients.
Asunto(s)
Fibroblastos Asociados al Cáncer , Mesotelioma Maligno , Mesotelioma , Animales , Ratones , Humanos , Mesotelioma/tratamiento farmacológico , Mesotelioma/genética , Fibroblastos , PulmónRESUMEN
Low-risk gestational trophoblastic neoplasia including choriocarcinoma is often effectively treated with Methotrexate (MTX) as a first line therapy. However, MTX resistance (MTX-R) occurs in at least ≈33% of cases. This can sometimes be salvaged with actinomycin-D but often requires more toxic combination chemotherapy. Moreover, additional therapy may be needed and, for high-risk patients, 5% still die from the multidrug-resistant disease. Consequently, new treatments that are less toxic and could reverse MTX-R are needed. Here, we compared the proteome/phosphoproteome of MTX-resistant and sensitive choriocarcinoma cells using quantitative mass-spectrometry to identify therapeutically actionable molecular changes associated with MTX-R. Bioinformatics analysis of the proteomic data identified cell cycle and DNA damage repair as major pathways associated with MTX-R. MTX-R choriocarcinoma cells undergo cell cycle delay in G1 phase that enables them to repair DNA damage more efficiently through non-homologous end joining in an ATR-dependent manner. Increased expression of cyclin-dependent kinase 4 (CDK4) and loss of p16Ink4a in resistant cells suggested that CDK4 inhibition may be a strategy to treat MTX-R choriocarcinoma. Indeed, inhibition of CDK4/6 using genetic silencing or the clinically relevant inhibitor, Palbociclib, induced growth inhibition both in vitro and in an orthotopic in vivo mouse model. Finally, targeting the ATR pathway, genetically or pharmacologically, re-sensitised resistant cells to MTX in vitro and potently prevented the growth of MTX-R tumours in vivo. In short, we identified two novel therapeutic strategies to tackle MTX-R choriocarcinoma that could rapidly be translated into the clinic.
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Coriocarcinoma , Quinasa 6 Dependiente de la Ciclina/metabolismo , Metotrexato , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Coriocarcinoma/tratamiento farmacológico , Coriocarcinoma/genética , Coriocarcinoma/metabolismo , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Dactinomicina , Femenino , Humanos , Metotrexato/farmacología , Ratones , Embarazo , ProteómicaRESUMEN
The S6 kinases (S6Ks) have been linked to a number of cellular processes, including translation, insulin metabolism, cell survival, and RNA splicing. Signaling via the phosphotidylinositol 3-kinase and mammalian target of rapamycin (mTOR) pathways is critical in regulating the activity and subcellular localization of S6Ks. To date, nuclear functions of both S6K isoforms, S6K1 and S6K2, are not well understood. To better understand S6K nuclear roles, we employed affinity purification of S6Ks from nuclear preparations followed by mass spectrometry analysis for the identification of novel binding partners. In this study, we report that in contrast to S6K1, the S6K2 isoform specifically associates with a number of RNA-binding proteins, including heterogeneous ribonucleoproteins (hnRNPs). We focused on studying the mechanism and physiological relevance of the S6K2 interaction with hnRNP F/H. Interestingly, the S6K2-hnRNP F/H interaction was not affected by mitogenic stimulation, whereas mTOR binding to hnRNP F/H was induced by serum stimulation. In addition, we define a new role of hnRNP F in driving cell proliferation, which could be partially attenuated by rapamycin treatment. S6K2-driven cell proliferation, on the other hand, could be blocked by small interfering RNA-mediated down-regulation of hnRNP F. These results demonstrate that the specific interaction between mTOR and S6K2 with hnRNPs is implicated in the regulation of cell proliferation.
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Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/química , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Línea Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Humanos , Mitógenos/química , Modelos Biológicos , Unión Proteica , Isoformas de Proteínas , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Sirolimus/químicaRESUMEN
Lung and bladder cancers are mostly incurable because of the early development of drug resistance and metastatic dissemination. Hence, improved therapies that tackle these two processes are urgently needed to improve clinical outcome. We have identified RSK4 as a promoter of drug resistance and metastasis in lung and bladder cancer cells. Silencing this kinase, through either RNA interference or CRISPR, sensitized tumor cells to chemotherapy and hindered metastasis in vitro and in vivo in a tail vein injection model. Drug screening revealed several floxacin antibiotics as potent RSK4 activation inhibitors, and trovafloxacin reproduced all effects of RSK4 silencing in vitro and in/ex vivo using lung cancer xenograft and genetically engineered mouse models and bladder tumor explants. Through x-ray structure determination and Markov transient and Deuterium exchange analyses, we identified the allosteric binding site and revealed how this compound blocks RSK4 kinase activation through binding to an allosteric site and mimicking a kinase autoinhibitory mechanism involving the RSK4's hydrophobic motif. Last, we show that patients undergoing chemotherapy and adhering to prophylactic levofloxacin in the large placebo-controlled randomized phase 3 SIGNIFICANT trial had significantly increased (P = 0.048) long-term overall survival times. Hence, we suggest that RSK4 inhibition may represent an effective therapeutic strategy for treating lung and bladder cancer.
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Neoplasias Pulmonares , Neoplasias de la Vejiga Urinaria , Animales , Línea Celular Tumoral , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Humanos , Pulmón/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Ratones , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/genéticaRESUMEN
PURPOSE: The Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancers and plays an important role in small cell lung cancer (SCLC) biology. We investigated the potential of targeting mTOR signaling as a novel antitumor approach in SCLC. EXPERIMENTAL DESIGN: The expression of mTOR in patient specimens and in a panel of SCLC cell lines was analyzed. The effects on SCLC cell survival and downstream signaling were determined following mTOR inhibition by the rapamycin derivative RAD001 (Everolimus) or down-regulation by small interfering RNA. RESULTS: We found elevated expression of mTOR in patient specimens and SCLC cell lines, compared with normal lung tissue and normal lung epithelial cells. RAD001 treatment impaired basal and growth factor-stimulated cell growth in a panel of SCLC cell lines. Cells with increased Akt pathway activation were more sensitive to RAD001. Accordingly, a constitutive activation of the Akt/mTOR pathway was sufficient to sensitize resistant SCLC cells to the cytotoxic effect of RAD001. In the sensitive cells, RAD001 showed a strong additive effect to the proapoptotic action of the chemotherapeutic agent etoposide. Intriguingly, we observed low Bcl-2 family proteins levels in the SCLC cells with a constitutive Akt pathway activation, whereas an increased expression was detected in the RAD001-resistant SCLC cells. An antisense construct targeting Bcl-2 or a Bcl-2-specific inhibitor was able to sensitize resistant SCLC cells to RAD001. Moreover, SCLC tumor growth in vivo was significantly inhibited by RAD001. CONCLUSION: Together, our data show that inhibiting mTOR signaling with RAD001 potently disrupts growth and survival signaling in human SCLC cells.
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Antineoplásicos/farmacología , Carcinoma de Células Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Sirolimus/análogos & derivados , Animales , Carcinoma de Células Pequeñas/patología , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Resistencia a Antineoplásicos , Etopósido/farmacología , Everolimus , Humanos , Neoplasias Pulmonares/patología , Ratones , Proteínas Quinasas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Transducción de Señal/fisiología , Sirolimus/farmacología , Factor de Células Madre/farmacología , Serina-Treonina Quinasas TORRESUMEN
Receptor-linked class I phosphoinositide 3-kinases (PI3Ks) induce assembly of signal transduction complexes through protein-protein and protein-lipid interactions that mediate cell proliferation, survival, and migration. Although class II PI3Ks have the potential to make the same phosphoinositides as class I PI3Ks, their precise cellular role is currently unclear. In this report, we demonstrate that class II phosphoinositide 3-kinase C2beta (PI3KC2beta) associates with the Eps8/Abi1/Sos1 complex and is recruited to the EGF receptor as part of a multiprotein signaling complex also involving Shc and Grb2. Increased expression of PI3KC2beta stimulated Rac activity in A-431 epidermoid carcinoma cells, resulting in enhanced membrane ruffling and migration speed of the cells. Conversely, expression of dominant negative PI3KC2beta reduced Rac activity, membrane ruffling, and cell migration. Moreover, PI3KC2beta-overexpressing cells were protected from anoikis and displayed enhanced proliferation, independently of Rac function. Taken together, these findings suggest that PI3KC2beta regulates the migration and survival of human tumor cells by distinct molecular mechanisms.
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Movimiento Celular , Citoesqueleto/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Uniones Adherentes/metabolismo , Anoicis/fisiología , Cadherinas/metabolismo , Proliferación Celular , Fosfatidilinositol 3-Quinasas Clase II , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/citología , Proteína Adaptadora GRB2/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Complejos Multiproteicos/metabolismo , Unión Proteica , Proteína SOS1/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , TransfecciónRESUMEN
Glycogen synthase kinase-3 (GSK3) is over-expressed and hyperactivated in non-small cell lung carcinoma (NSCLC) and plays a role in ensuring the correct alignment of chromosomes on the metaphase plate during mitosis through regulation of microtubule stability. This makes the enzyme an attractive target for cancer therapy. We examined the effects of a selective cell-permeant GSK3 inhibitor (CHIR99021), used alone or in combination with paclitaxel, using an in vitro cell growth assay, a quantitative chromosome alignment assay, and a tumor xenograft model. CHIR99021 inhibits the growth of human H1975 and H1299 NSCLC cell lines in a synergistic manner with paclitaxel. CHIR99021 and paclitaxel promoted a synergistic defect in chromosomal alignment when compared to each compound administered as monotherapy. Furthermore, we corroborated our in vitro findings in a mouse tumor xenograft model. Our results demonstrate that a GSK3 inhibitor and paclitaxel act synergistically to inhibit the growth of NSCLC cells in vitro and in vivo via a mechanism that may involve converging modes of action on microtubule spindle stability and thus chromosomal alignment during metaphase. Our findings provide novel support for the use of the GSK3 inhibitor, CHIR99021, alongside taxol-based chemotherapy in the treatment of human lung cancer.
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Antineoplásicos Fitogénicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Glucógeno Sintasa Quinasa 3/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Paclitaxel/uso terapéutico , Piridinas/uso terapéutico , Pirimidinas/uso terapéutico , Animales , Antineoplásicos Fitogénicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Aberraciones Cromosómicas/efectos de los fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Humanos , Masculino , Ratones , Ratones Desnudos , Paclitaxel/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
Lung cancer is the main cancer killer in both men and women, mostly due to the rapid development of drug resistant metastatic disease. Here, we evaluate the potential involvement of SRC family kinases (SFK) in lung cancer biology and assess the possible benefits of their inhibition as a therapeutic approach. We demonstrated that various SRC family members, including LYN and LCK, normally expressed solely in hematopoietic cells and neural tissues, are overexpressed and activated in a panel of SCLC and NSCLC cell lines. This was clinically relevant as LYN and FYN are also overexpressed in lung cancer clinical specimens. Moreover, LYN overexpression correlated with decreased patient survival on univariate and multivariate analysis. Dasatinib (BMS-354825), a SRC/ABL inhibitor, effectively blocked SFK activation at nanomolar concentrations which correlated with a significant decrease in cell numbers of multiple lung cancer cell lines. This effect was matched by a decrease in DNA synthesis, but only moderate induction of apoptosis. Indeed, dasatinib as well as PP2, another SFK inhibitor, strongly induced autophagy that likely prevented apoptosis. However, inhibition of this autophagic response induced robust apoptosis and sensitised lung cancer cells to dasatinib in vitro and in vivo. Our results provide an explanation for why dasatinib failed in NSCLC clinical trials. Furthermore, our data suggest that combining SFK inhibitors with autophagy inhibitors could provide a novel therapeutic approach in this disease.
RESUMEN
The mitochondrial release of cytochrome c and Smac/DIABLO has been implicated in the activation of apoptosis in response to cell stress. Smac promotes cytochrome c-induced activation of caspases by sequestering the inhibitor of apoptosis protein (IAP) family of potent caspase suppressors. Differential release from mitochondria of cytochrome c and Smac can occur, but the underlying mechanism and physiological significance of this are unclear. Here we show that the mechanism by which fibroblast growth factor 2 (FGF-2) protects small cell lung cancer (SCLC) cells from etoposide-induced cell death involves inhibition of Smac release but not of cytochrome c release. This process is MEK dependent and correlates with an increased expression of XIAP and cellular IAP-1, mediated principally through translational regulation. Exogenous expression of XIAP is sufficient to inhibit caspase 9 activation, Smac release, and cell death induced by etoposide. Prevention of the FGF-2-promoted increase in levels of functional IAPs by RNA interference or the cell-permeant Smac amino-terminal peptide blocked FGF-2-induced protection. FGF-2 can thus protect SCLC cells from chemotherapeutic drugs by modulating IAP levels via posttranscriptional regulation, providing a mechanism for postmitochondrial survival signaling by the MEK/mitogen-activated protein kinase pathway.
Asunto(s)
Apoptosis/fisiología , Carcinoma de Células Pequeñas/metabolismo , Proteínas Portadoras/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Biosíntesis de Proteínas , Proteínas/metabolismo , Proteínas Reguladoras de la Apoptosis , Carcinoma de Células Pequeñas/patología , Caspasas/metabolismo , Muerte Celular/fisiología , Línea Celular Tumoral , Medio de Cultivo Libre de Suero , Citocromos c/metabolismo , Activación Enzimática , Etopósido/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Inhibidoras de la Apoptosis , Péptidos y Proteínas de Señalización Intracelular , MAP Quinasa Quinasa 1 , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/genética , Proteína Inhibidora de la Apoptosis Ligada a XRESUMEN
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA-binding proteins associated with complex and diverse biological processes such as processing of heterogeneous nuclear RNAs (hnRNAs) into mature mRNAs, RNA splicing, transactivation of gene expression, and modulation of protein translation. hnRNPA1 is the most abundant and ubiquitously expressed member of this protein family and has been shown to be involved in multiple molecular events driving malignant transformation. In addition to selective mRNA splicing events promoting expression of specific protein variants, hnRNPA1 regulates the gene expression and translation of several key players associated with tumorigenesis and cancer progression. Here, we will summarize our current knowledge of the involvement of hnRNPA1 in cancer, including its roles in regulating cell proliferation, invasiveness, metabolism, adaptation to stress and immortalization. WIREs RNA 2017, 8:e1431. doi: 10.1002/wrna.1431 For further resources related to this article, please visit the WIREs website.