RESUMEN

Rationale: Chronic obstructive pulmonary disease is an independent risk factor for lung cancer, but the underlying molecular mechanisms are unknown. We hypothesized that lung stromal cells activate pathological gene expression programs that support oncogenesis.Objectives: To identify molecular mechanisms operating in the lung stroma that support the development of lung cancer.Methods: The study included subjects with and without lung cancer across a spectrum of lung-function values. We conducted a multiomics analysis of nonmalignant lung tissue to quantify the transcriptome, translatome, and proteome.Measurements and Main Results: Cancer-associated gene expression changes predominantly manifested as alterations in the efficiency of mRNA translation modulating protein levels in the absence of corresponding changes in mRNA levels. The molecular mechanisms that drove these cancer-associated translation programs differed based on lung function. In subjects with normal to mildly impaired lung function, the mammalian target of rapamycin (mTOR) pathway served as an upstream driver, whereas in subjects with severe airflow obstruction, pathways downstream of pathological extracellular matrix emerged. Consistent with a role during cancer initiation, both the mTOR and extracellular matrix gene expression programs paralleled the activation of previously identified procancer secretomes. Furthermore, an in situ examination of lung tissue showed that stromal fibroblasts expressed cancer-associated proteins from two procancer secretomes: one that included IL-6 (in cases of mild or no airflow obstruction), and one that included BMP1 (in cases of severe airflow obstruction).Conclusions: Two distinct stromal gene expression programs that promote cancer initiation are activated in patients with lung cancer depending on lung function. Our work has implications both for screening strategies and for personalized approaches to cancer treatment.

Asunto(s)

Neoplasias Pulmonares/etiología , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Células del Estroma/patología , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Volumen Espiratorio Forzado , Humanos , Masculino , Persona de Mediana Edad , Proteoma , Enfermedad Pulmonar Obstructiva Crónica/patología , Transcriptoma

RESUMEN

BACKGROUND: Mutation of HNF1B, the gene encoding transcription factor HNF-1ß, is one cause of autosomal dominant tubulointerstitial kidney disease, a syndrome characterized by tubular cysts, renal fibrosis, and progressive decline in renal function. HNF-1ß has also been implicated in epithelial-mesenchymal transition (EMT) pathways, and sustained EMT is associated with tissue fibrosis. The mechanism whereby mutated HNF1B leads to tubulointerstitial fibrosis is not known. METHODS: To explore the mechanism of fibrosis, we created HNF-1ß-deficient mIMCD3 renal epithelial cells, used RNA-sequencing analysis to reveal differentially expressed genes in wild-type and HNF-1ß-deficient mIMCD3 cells, and performed cell lineage analysis in HNF-1ß mutant mice. RESULTS: The HNF-1ß-deficient cells exhibited properties characteristic of mesenchymal cells such as fibroblasts, including spindle-shaped morphology, loss of contact inhibition, and increased cell migration. These cells also showed upregulation of fibrosis and EMT pathways, including upregulation of Twist2, Snail1, Snail2, and Zeb2, which are key EMT transcription factors. Mechanistically, HNF-1ß directly represses Twist2, and ablation of Twist2 partially rescued the fibroblastic phenotype of HNF-1ß mutant cells. Kidneys from HNF-1ß mutant mice showed increased expression of Twist2 and its downstream target Snai2. Cell lineage analysis indicated that HNF-1ß mutant epithelial cells do not transdifferentiate into kidney myofibroblasts. Rather, HNF-1ß mutant epithelial cells secrete high levels of TGF-ß ligands that activate downstream Smad transcription factors in renal interstitial cells. CONCLUSIONS: Ablation of HNF-1ß in renal epithelial cells leads to the activation of a Twist2-dependent transcriptional network that induces EMT and aberrant TGF-ß signaling, resulting in renal fibrosis through a cell-nonautonomous mechanism.

Asunto(s)

Gota/genética , Gota/patología , Factor Nuclear 1-beta del Hepatocito/genética , Hiperuricemia/genética , Hiperuricemia/patología , Enfermedades Renales/genética , Enfermedades Renales/patología , Animales , Línea Celular , Linaje de la Célula/genética , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal/genética , Femenino , Fibrosis , Genes Dominantes , Gota/metabolismo , Factor Nuclear 1-beta del Hepatocito/deficiencia , Factor Nuclear 1-beta del Hepatocito/metabolismo , Humanos , Hiperuricemia/metabolismo , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/metabolismo , Masculino , Ratones , Ratones Transgénicos , Mutación , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Proteína 1 Relacionada con Twist/deficiencia , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismo

RESUMEN

Autosomal dominant polycystic kidney disease (ADPKD) is a debilitating disease that is characterized by the accumulation of numerous fluid-filled cysts in the kidney. ADPKD is primarily caused by mutations in two genes, PKD1 and PKD2 Long noncoding RNAs (lncRNA), defined by a length >200 nucleotides and absence of a long ORF, have recently emerged as epigenetic regulators of development and disease; however, their involvement in PKD has not been explored previously. Here, we performed deep RNA-Seq to identify lncRNAs that are dysregulated in two orthologous mouse models of ADPKD (kidney-specific Pkd1 and Pkd2 mutant mice). We identified a kidney-specific, evolutionarily conserved lncRNA called Hoxb3os that was down-regulated in cystic kidneys from Pkd1 and Pkd2 mutant mice. The human ortholog HOXB3-AS1 was down-regulated in cystic kidneys from ADPKD patients. Hoxb3os was highly expressed in renal tubules in adult WT mice, whereas its expression was lost in the cyst epithelium of mutant mice. To investigate the function of Hoxb3os, we utilized CRISPR/Cas9 to knock out its expression in mIMCD3 cells. Deletion of Hoxb3os resulted in increased phosphorylation of mTOR and its downstream targets, including p70 S6 kinase, ribosomal protein S6, and the translation repressor 4E-BP1. Consistent with activation of mTORC1 signaling, Hoxb3os mutant cells displayed increased mitochondrial respiration. The Hoxb3os mutant phenotype was partially rescued upon re-expression of Hoxb3os in knockout cells. These findings identify Hoxb3os as a novel lncRNA that is down-regulated in ADPKD and regulates mTOR signaling and mitochondrial respiration.

Asunto(s)

Riñón Poliquístico Autosómico Dominante/genética , ARN Largo no Codificante/genética , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Animales , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación , Riñón Poliquístico Autosómico Dominante/metabolismo , Canales Catiónicos TRPP/genética

RESUMEN

Chronic obstructive pulmonary disease (COPD) is a known risk factor for developing lung cancer but the underlying mechanisms remain unknown. We hypothesise that the COPD stroma contains molecular mechanisms supporting tumourigenesis.We conducted an unbiased multi-omic analysis to identify gene expression patterns that distinguish COPD stroma in patients with or without lung cancer. We obtained lung tissue from patients with COPD and lung cancer (tumour and adjacent non-malignant tissue) and those with COPD without lung cancer for profiling of proteomic and mRNA (both cytoplasmic and polyribosomal). We used the Joint and Individual Variation Explained (JIVE) method to integrate and analyse across the three datasets.JIVE identified eight latent patterns that robustly distinguished and separated the three groups of tissue samples (tumour, adjacent and control). Predictive variables that associated with the tumour, compared to adjacent stroma, were mainly represented in the transcriptomic data, whereas predictive variables associated with adjacent tissue, compared to controls, were represented at the translatomic level. Pathway analysis revealed extracellular matrix and phosphatidylinositol-4,5-bisphosphate 3-kinase-protein kinase B signalling pathways as important signals in the tumour adjacent stroma.The multi-omic approach distinguishes tumour adjacent stroma in lung cancer and reveals two stromal expression patterns associated with cancer.

Asunto(s)

Neoplasias Pulmonares/genética , Proteoma/genética , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Transducción de Señal , Transcriptoma/genética , Anciano , Estudios de Casos y Controles , Femenino , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , ARN Mensajero/genética

RESUMEN

While selective estrogen receptor modulators, such as tamoxifen, have contributed to increased survival in patients with hormone receptor-positive breast cancer, the development of resistance to these therapies has led to the need to investigate other targetable pathways involved in oncogenic signaling. Approval of the mTOR inhibitor everolimus in the therapy of secondary endocrine resistance demonstrates the validity of this approach. Importantly, mTOR activation regulates eukaryotic messenger RNA translation. Eukaryotic translation initiation factor 4E (eIF4E), a component of the cap-dependent translation complex eIF4F, confers resistance to drug-induced apoptosis when overexpressed in multiple cell types. The eIF4F complex is downstream of multiple oncogenic pathways, including mTOR, making it an appealing drug target. Here, we show that the eIF4F translation pathway was hyperactive in tamoxifen-resistant (TamR) MCF-7L breast cancer cells. While overexpression of eIF4E was not sufficient to confer resistance to tamoxifen in MCF-7L cells, its function was necessary to maintain resistance in TamR cells. Targeting the eIF4E subunit of the eIF4F complex through its degradation using an antisense oligonucleotide (ASO) or via sequestration using a mutant 4E-BP1 inhibited the proliferation and colony formation of TamR cells and partially restored sensitivity to tamoxifen. Further, the use of these agents also resulted in cell cycle arrest and induction of apoptosis in TamR cells. Finally, the use of a pharmacologic agent which inhibited the eIF4E-eIF4G interaction also decreased the proliferation and anchorage-dependent colony formation in TamR cells. These results highlight the eIF4F complex as a promising target for patients with acquired resistance to tamoxifen and, potentially, other endocrine therapies.

Asunto(s)

Resistencia a Antineoplásicos/genética , Factor 4F Eucariótico de Iniciación/metabolismo , Biosíntesis de Proteínas , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Factor 4F Eucariótico de Iniciación/genética , Femenino , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Células MCF-7 , Oligorribonucleótidos Antisentido/genética , Fosforilación , Polirribosomas , Unión Proteica , Transducción de Señal/efectos de los fármacos

RESUMEN

The transcription factor hepatocyte nuclear factor-1ß (HNF-1ß) is essential for normal kidney development and function. Inactivation of HNF-1ß in mouse kidney tubules leads to early-onset cyst formation and postnatal lethality. Here, we used Pkhd1/Cre mice to delete HNF-1ß specifically in renal collecting ducts (CDs). CD-specific HNF-1ß mutant mice survived long term and developed slowly progressive cystic kidney disease, renal fibrosis, and hydronephrosis. Compared with wild-type littermates, HNF-1ß mutant mice exhibited polyuria and polydipsia. Before the development of significant renal structural abnormalities, mutant mice exhibited low urine osmolality at baseline and after water restriction and administration of desmopressin. However, mutant and wild-type mice had similar plasma vasopressin and solute excretion levels. HNF-1ß mutant kidneys showed increased expression of aquaporin-2 mRNA but mislocalized expression of aquaporin-2 protein in the cytoplasm of CD cells. Mutant kidneys also had decreased expression of the UT-A urea transporter and collectrin, which is involved in apical membrane vesicle trafficking. Treatment of HNF-1ß mutant mIMCD3 cells with hypertonic NaCl inhibited the induction of osmoregulated genes, including Nr1h4, which encodes the transcription factor FXR that is required for maximal urinary concentration. Chromatin immunoprecipitation and sequencing experiments revealed HNF-1ß binding to the Nr1h4 promoter in wild-type kidneys, and immunoblot analysis revealed downregulated expression of FXR in HNF-1ß mutant kidneys. These findings reveal a novel role of HNF-1ß in osmoregulation and identify multiple mechanisms, whereby mutations of HNF-1ß produce defects in urinary concentration.

Asunto(s)

Factor Nuclear 1-beta del Hepatocito/fisiología , Túbulos Renales Colectores/fisiología , Animales , Línea Celular , Femenino , Regulación de la Expresión Génica , Masculino , Ratones Transgénicos , Poliuria/genética , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Orina

RESUMEN

BACKGROUND: Control of mRNA translation is fundamentally altered in cancer. Insulin-like growth factor-I (IGF-I) signaling regulates key translation mediators to modulate protein synthesis (e.g. eIF4E, 4E-BP1, mTOR, and S6K1). Importantly the Amplified in Breast Cancer (AIB1) oncogene regulates transcription and is also a downstream mediator of IGF-I signaling. MATERIALS AND METHODS: To determine if AIB1 also affects mRNA translation, we conducted gain and loss of AIB1 function experiments in estrogen receptor alpha (ERα)(+) (MCF-7L) and ERα(-) (MDA-MB-231, MDA-MB-435 and LCC6) breast cancer cells. RESULTS: AIB1 positively regulated IGF-I-induced mRNA translation in both ERα(+) and ERα(-) cells. Formation of the eIF4E-4E-BP1 translational complex was altered in the AIB1 ERα(+) and ERα(-) knockdown cells, leading to a reduction in the eIF4E/4E-BP1 and eIF4G/4E-BP1 ratios. In basal and IGF-I stimulated MCF-7 and LCC6 cells, knockdown of AIB1 decreased the integrity of the cap-binding complex, reduced global IGF-I stimulated polyribosomal mRNA recruitment with a concomitant decrease in ten of the thirteen genes tested in polysome-bound mRNAs mapping to proliferation, cell cycle, survival, transcription, translation and ribosome biogenesis ontologies. Specifically, knockdown of AIB1 decreased ribosome-bound mRNA and steady-state protein levels of the transcription factors ERα and E2F1 in addition to reduced ribosome-bound mRNA of the ribosome biogenesis factor BYSL in a cell-line specific manner to regulate mRNA translation. CONCLUSION: The oncogenic transcription factor AIB1 has a novel role in the regulation of polyribosome recruitment and formation of the translational complex. Combinatorial therapies targeting IGF signaling and mRNA translation in AIB1 expressing breast cancers may have clinical benefit and warrants further investigation.

Asunto(s)

Neoplasias de la Mama/genética , Factor I del Crecimiento Similar a la Insulina/genética , Coactivador 3 de Receptor Nuclear/genética , Biosíntesis de Proteínas , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular , Receptor alfa de Estrógeno/genética , Factor 4E Eucariótico de Iniciación/biosíntesis , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Células MCF-7 , Fosfoproteínas/biosíntesis , Fosforilación , Proteínas Quinasas S6 Ribosómicas 70-kDa/biosíntesis , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/biosíntesis

RESUMEN

Translation initiation factor eIF4E mediates normal cell proliferation, yet induces tumorigenesis when overexpressed. The mechanisms by which eIF4E directs such distinct biologic outputs remain unknown. We found that mouse mammary morphogenesis during pregnancy and lactation is accompanied by increased cap-binding capability of eIF4E and activation of the eIF4E-dependent translational apparatus, but only subtle oscillations in eIF4E abundance. Using a transgenic mouse model engineered so that lactogenic hormones stimulate a sustained increase in eIF4E abundance in stem/progenitor cells of lactogenic mammary epithelium during successive pregnancy/lactation cycles, eIF4E overexpression increased self-renewal, triggered DNA replication stress, and induced formation of premalignant and malignant lesions. Using complementary in vivo and ex vivo approaches, we found that increasing eIF4E levels rescued cells harboring oncogenic c-Myc or H-RasV12 from DNA replication stress and oncogene-induced replication catastrophe. Our findings indicate that distinct threshold levels of eIF4E govern its biologic output in lactating mammary glands and that eIF4E overexpression in the context of stem/progenitor cell population expansion can initiate malignant transformation by enabling cells to evade DNA damage checkpoints activated by oncogenic stimuli. Maintaining eIF4E levels below its proneoplastic threshold is an important anticancer defense in normal cells, with important implications for understanding pregnancy-associated breast cancer.

Asunto(s)

Neoplasias de la Mama/genética , Carcinogénesis/genética , Factor 4E Eucariótico de Iniciación/biosíntesis , Glándulas Mamarias Humanas/metabolismo , Animales , Neoplasias de la Mama/patología , Proliferación Celular/genética , Replicación del ADN/genética , Factor 4E Eucariótico de Iniciación/genética , Femenino , Humanos , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Glándulas Mamarias Humanas/patología , Ratones , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Embarazo , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Proteínas ras/biosíntesis

RESUMEN

Normal growth and development depends upon high fidelity regulation of cap-dependent translation initiation, a process that is usurped and redirected in cancer to mediate acquisition of malignant properties. The epithelial-to-mesenchymal transition (EMT) is a key translationally regulated step in the development of epithelial cancers and pathological tissue fibrosis. To date, no compounds targeting EMT have been developed. Here we report the synthesis of a novel class of histidine triad nucleotide binding protein (HINT)-dependent pronucleotides that interdict EMT by negatively regulating the association of eIF4E with the mRNA cap. Compound eIF4E inhibitor-1 potently inhibited cap-dependent translation in a dose-dependent manner in zebrafish embryos without causing developmental abnormalities and prevented eIF4E from triggering EMT in zebrafish ectoderm explants without toxicity. Metabolism studies with whole cell lysates demonstrated that the prodrug was rapidly converted into 7-BnGMP. Thus we have successfully developed the first nontoxic small molecule able to inhibit EMT, a key process in the development of epithelial cancer and tissue fibrosis, by targeting the interaction of eIF4E with the mRNA cap and demonstrated the tractability of zebrafish as a model organism for studying agents that modulate EMT. Our work provides strong motivation for the continued development of compounds designed to normalize cap-dependent translation as novel chemo-preventive agents and therapeutics for cancer and fibrosis.

Asunto(s)

Sistemas de Liberación de Medicamentos , Epitelio/embriología , Mesodermo/embriología , Complejo Proteico Nuclear de Unión a la Caperuza/antagonistas & inhibidores , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Pez Cebra/embriología , Amidas/síntesis química , Amidas/química , Amidas/farmacología , Animales , Secuencia de Bases , Embrión no Mamífero/embriología , Factor 4E Eucariótico de Iniciación/antagonistas & inhibidores , Humanos , Concentración 50 Inhibidora , Datos de Secuencia Molecular , Neoplasias/metabolismo , Ácidos Fosfóricos/síntesis química , Ácidos Fosfóricos/química , Ácidos Fosfóricos/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive lung disease in which fibroblasts accumulate in the alveolar wall within a type I collagen-rich matrix. Although lung fibroblasts derived from patients with IPF display durable pathological alterations in proliferative function, the molecular mechanisms differentiating IPF fibroblasts from their normal counterparts remain unknown. Polymerized type I collagen normally inhibits fibroblast proliferation, providing a physiological mechanism to limit fibroproliferation after tissue injury. We demonstrate that beta1 integrin interaction with polymerized collagen inhibits normal fibroblast proliferation by suppression of the phosphoinositide 3-kinase (PI3K)-Akt-S6K1 signal pathway due to maintenance of high phosphatase activity of the tumor suppressor phosphatase and tensin homologue (PTEN). In contrast, IPF fibroblasts eluded this restraint, displaying a pathological pattern of beta1 integrin signaling in response to polymerized collagen that leads to aberrant activation of the PI3K-Akt-S6K1 signal pathway caused by inappropriately low PTEN activity. Mice deficient in PTEN showed a prolonged fibroproliferative response after tissue injury, and immunohistochemical analysis of IPF lung tissue demonstrates activation of Akt in cells within fibrotic foci. These results provide direct evidence for defective negative regulation of the proliferative pathway in IPF fibroblasts and support the theory that the pathogenesis of IPF involves an intrinsic fibroblast defect.

Asunto(s)

Proliferación Celular , Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Integrina beta1/metabolismo , Alveolos Pulmonares/metabolismo , Fibrosis Pulmonar/metabolismo , Transducción de Señal , Animales , Línea Celular , Activación Enzimática/genética , Femenino , Fibroblastos/patología , Humanos , Masculino , Ratones , Ratones Noqueados , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Alveolos Pulmonares/patología , Fibrosis Pulmonar/patología , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal/genética

RESUMEN

One of the earliest steps in translation initiation is recognition of the mRNA cap structure (m7GpppX) by the initiation factor eIF4E. Studies of interactions between purified eIF4E and its binding partners provide important information for understanding mechanisms underlying translational control in normal and cancer cells. Numerous impediments of the available methods used for eIF4E purification led us to develop a novel methodology for obtaining fractions of eIF4E free from undesired by-products. Herein we report methods for bacterial expression of eIF4E tagged with mutant dihydrofolate reductase (DHFR) followed by isolation and purification of the DHFR-eIF4E protein by using affinity and anion exchange chromatography. Fluorescence quenching experiments indicated the cap-analog, 7MeGTP, bound to DHFR-eIF4E and eIF4E with a dissociation constant (K(d)) of 6+/-5 and 10+/-3 nM, respectively. Recombinant eIF4E and DHFR-eIF4E were both shown to significantly enhance in vitro translation in dose dependent manner by 75% at 0.5 microM. Nevertheless increased concentrations of eIF4E and DHFR-eIF4E significantly inhibited translation in a dose dependent manner by a maximum at 2 microM of 60% and 90%, respectively. Thus, we have demonstrated that we have developed an expression system for fully functional recombinant eIF4E. We have also shown that the fusion protein DHFR-eIF4E is functional and thus may be useful for cell based affinity tag studies with fluorescently labeled trimethoprim analogs.

Asunto(s)

Factor 4E Eucariótico de Iniciación/genética , Tetrahidrofolato Deshidrogenasa/genética , Animales , Secuencia de Bases , Cromatografía Liquida/métodos , Cartilla de ADN , Fluorescencia , Ratones , Proteínas Recombinantes de Fusión/genética

RESUMEN

Pathologic redirection of translational control by constitutive activation of eukaryotic translation initiation factor 4F (eIF4F), the cap-dependent translation initiation apparatus, is an obligatory step in oncogenesis; however, its mechanism remains undefined. Here, we simulate this pro-oncogenic state by overexpressing eIF4E, the rate-limiting component of eIF4F, in primary human mammary epithelial cells (HMECs) and examine the resultant changes in cell biology and gene expression profiles of total and polyribosome-bound mRNA genome wide. Overexpressed eIF4E rescues primary HMECs from telomere-independent growth arrest and disables checkpoints governing S-phase entry as well as apoptosis in HMECs immortalized by telomerase, imparting cells with proliferative and survival autonomy. Although the transcriptional response to increased eIF4E was modest, the translational response was large, selective, and bidirectional. In addition to translational activation of known and novel eIF4E-responsive oncogenic drivers regulating cell growth and survival, our data unveil previously unrecognized cellular defenses including translational activation of tumor suppressors, translational repression of transcripts enriched with miRNA target sites, and translational modulation of genes governing translation itself. These findings provide insight into the proneoplastic and compensatory mechanisms embedded in the oncogenic translational program. They support a model whereby deregulated eIF4E moves human epithelial cells along the cancer pathway by profoundly altering ribosomal recruitment to cancer-related transcripts, and eIF4E-modified cells counter these potentially oncogenic alterations with a compensatory translational mechanism that mitigates acquisition of malignancy.

Asunto(s)

Mama/metabolismo , Epitelio/metabolismo , Factor 4E Eucariótico de Iniciación/fisiología , Apoptosis , Línea Celular , Proliferación Celular , Supervivencia Celular , Progresión de la Enfermedad , Factor 4E Eucariótico de Iniciación/metabolismo , Humanos , MicroARNs/metabolismo , Biosíntesis de Proteínas , Fase de Descanso del Ciclo Celular , Fase S , Telomerasa/metabolismo , Telómero/ultraestructura

RESUMEN

Aberrant hyperactivation of the cap-dependent protein synthesis apparatus has been documented in a wide range of solid tumors, including epithelial carcinomas, but causal linkage has only been established in breast carcinoma. In this report, we sought to determine if targeted disruption of deregulated cap-dependent translation abrogates tumorigenicity and enhances cell death in non-small cell lung cancer (NSCLC). NSCLC cell lines were stably transfected with either wild-type 4E-BP1 (HA-4E-BP1) or the dominant-active mutant 4E-BP1(A37/A46) (HA-TTAA). Transfected NSCLC cells with enhanced translational repression showed pronounced cell death following treatment with gemcitabine. In addition, transfected HA-TTAA and HA-4E-BP1wt proteins suppressed growth in a cloning efficiency assay. NSCLC cells transduced with HA-TTAA also show decreased tumorigenicity in xenograft models. Xenograft tumors expressing HA-TTAA were significantly smaller than control tumors. This work shows that hyperactivation of the translational machinery is necessary for maintenance of the malignant phenotype in NSCLC, identifies the molecular strategy used to activate translation, and supports the development of lung cancer therapies that directly target the cap-dependent translation initiation complex.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Biosíntesis de Proteínas , Caperuzas de ARN , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas de Ciclo Celular , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones , Fosfoproteínas/genética , Transfección , Gemcitabina

RESUMEN

Common human malignancies acquire derangements of the translation initiation complex, eIF4F, but their functional significance is unknown. Hypophosphorylated 4E-BP proteins negatively regulate eIF4F assembly by sequestering its mRNA cap binding component eIF4E, whereas hyperphosphorylation abrogates this function. We found that breast carcinoma cells harbor increases in the eIF4F constituent eIF4GI and hyperphosphorylation of 4E-BP1 which are two alterations that activate eIF4F assembly. Ectopic expression of eIF4E in human mammary epithelial cells enabled clonal expansion and anchorage-independent growth. Transfer of 4E-BP1 phosphorylation site mutants into breast carcinoma cells suppressed their tumorigenicity, whereas loss of these 4E-BP1 phosphorylation site mutants accompanied spontaneous reversion to a malignant phenotype. Thus, eIF4F activation is an essential component of the malignant phenotype in breast carcinoma.

Asunto(s)

Epitelio/metabolismo , Factor 4F Eucariótico de Iniciación/fisiología , Glándulas Mamarias Humanas/patología , Proteínas Adaptadoras Transductoras de Señales , Apoptosis , Sitios de Unión , Neoplasias de la Mama/patología , Carcinoma/patología , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , División Celular , Línea Celular Tumoral , Supervivencia Celular , Células Cultivadas , ADN/química , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Factor 4F Eucariótico de Iniciación/metabolismo , Citometría de Flujo , Humanos , Immunoblotting , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Antígeno Ki-67/biosíntesis , Mutación , Fenotipo , Fosfoproteínas/metabolismo , Fosforilación , Retroviridae/genética , Factores de Tiempo , Transfección

RESUMEN

Eukaryotic translation initiation factor 4E (eIF4E) is the mRNA cap-binding protein required for translation of cellular mRNAs utilizing the 5' cap structure. The rate-limiting factor for mRNA recruitment to ribosomes, eIF4E is a major target for regulation of translation by growth factors, hormones, and other extracellular stimuli. When overexpressed, eIF4E exerts profound effects on cell growth and survival, leading to suppression of oncogene-dependent apoptosis, causing malignant transformation and conferring tumors with multiple drug resistance. We found previously that overexpressed eIF4E interdicts the apoptotic pathway induced by growth factor withdrawal and cytotoxic drugs by selectively activating the expression of Bcl-X(L), thus preventing mitochondrial release of cytochrome c. In this study, we examined the impact of ectopic eIF4E expression on apoptosis mediated by the endoplasmic reticulum (ER). Here we show that eIF4E rescued cells from the ER stressors brefeldin A, tunicamycin, thapsigargin, and the Ca(2+) ionophore A23187. In addition, we found that cells rescued from Ca(2+) ionophore-triggered apoptosis did not release calcium from their ER nor did they translocate caspase-12 from the ER to the cytoplasm. These data lend strong support to the concept that eIF4E functions as a pleiotropic regulator of cell viability and that integration of critical organelle-mediated checkpoints for apoptosis can be controlled by the cap-dependent translation apparatus.

Asunto(s)

Apoptosis/fisiología , Retículo Endoplásmico/fisiología , Factor 4E Eucariótico de Iniciación/metabolismo , Células 3T3 , Animales , Calcimicina/farmacología , Calcio/fisiología , Señalización del Calcio/fisiología , Caspasa 12 , Inhibidores de Caspasas , Caspasas/metabolismo , Permeabilidad de la Membrana Celular , Cinética , Ratones , Orgánulos/fisiología , Biosíntesis de Proteínas/fisiología

RESUMEN

Eukaryotic translation initiation factor 4E (eIF4E) markedly reduces cellular susceptibility to apoptosis. However, the mechanism by which the translation apparatus operates on the cellular apoptotic machinery remains uncertain. Here we show that eIF4E-mediated rescue from Myc-dependent apoptosis is accompanied by inhibition of mitochondrial cytochrome c release. Experiments achieving gain and loss of function demonstrate that eIF4E-mediated rescue is governed by pretranslational and translational activation of bcl-x as well as by additional intermediates acting directly on, or upstream of, the mitochondria. Thus, our data trace a pathway controlling apoptotic susceptibility that begins with the activity state of the protein synthesis machinery and leads to interdiction of the apoptotic program at the mitochondrial checkpoint.

Asunto(s)

Apoptosis/fisiología , Grupo Citocromo c/metabolismo , Factor 4E Eucariótico de Iniciación/metabolismo , Genes myc , Proteínas Proto-Oncogénicas c-bcl-2/genética , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Embrión de Mamíferos , Fibroblastos/citología , Fibroblastos/fisiología , Cinética , Mitocondrias/fisiología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/genética , Ratas , Transcripción Genética , Proteína bcl-X

RESUMEN

Translational control has been recently added to well-recognized genomic, transcriptional, and posttranslational mechanisms regulating apoptosis. We previously found that overexpressed eukaryotic initiation factor 4E (eIF4E) rescues cells from apoptosis, while ectopic expression of wild-type eIF4E-binding protein 1 (4E-BP1), the most abundant member of the 4E-BP family of eIF4E repressor proteins, activates apoptosis--but only in transformed cells. To test the possibility that nontransformed cells require less cap-dependent translation to suppress apoptosis than do their transformed counterparts, we intensified the level of translational repression in nontransformed fibroblasts. Here, we show that inhibition of 4E-BP1 phosphorylation by rapamycin triggers apoptosis in cells ectopically expressing wild-type 4E-BP1 and that expression of 4E-BP1 phosphorylation site mutants potently activates apoptosis in a phosphorylation site-specific manner. In general, proapoptotic potency paralleled repression of cap-dependent translation. However, this relationship was not a simple monotone. As repression of cap-dependent translation intensified, apoptosis increased to a maximum value. Further repression resulted in less apoptosis--a state associated with activation of translation through internal ribosomal entry sites. These findings show: that phosphorylation events govern the proapoptotic potency of 4E-BP1, that 4E-BP1 is proapoptotic in normal as well as transformed fibroblasts, and that malignant transformation is associated with a higher requirement for cap-dependent translation to inhibit apoptosis. Our results suggest that 4E-BP1-mediated control of apoptosis occurs through qualitative rather than quantitative changes in protein synthesis, mediated by a dynamic interplay between cap-dependent and cap-independent processes.

Asunto(s)

Apoptosis/genética , Apoptosis/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Fosfoproteínas/genética , Fosfoproteínas/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Apoptosis/efectos de los fármacos , Sitios de Unión/genética , Proteínas Portadoras/química , Proteínas de Ciclo Celular , Supervivencia Celular , Células Clonales , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular , Mutagénesis Sitio-Dirigida , Fosfoproteínas/química , Fosforilación , Biosíntesis de Proteínas , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sirolimus/farmacología