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1.
Mol Cell Proteomics ; 22(9): 100630, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37562535

RESUMEN

Thermal proteome profiling (TPP) is an invaluable tool for functional proteomics studies that has been shown to discover changes associated with protein-ligand, protein-protein, and protein-RNA interaction dynamics along with changes in protein stability resulting from cellular signaling. The increasing number of reports employing this assay has not been met concomitantly with new approaches leading to advancements in the quality and sensitivity of the corresponding data analysis. The gap between data acquisition and data analysis tools is important to fill as TPP findings have reported subtle melt shift changes related to signaling events such as protein posttranslational modifications. In this study, we have improved the Inflect data analysis pipeline (now referred to as InflectSSP, available at https://CRAN.R-project.org/package=InflectSSP) to increase the sensitivity of detection for both large and subtle changes in the proteome as measured by TPP. Specifically, InflectSSP now has integrated statistical and bioinformatic functions to improve objective functional proteomics findings from the quantitative results obtained from TPP studies through increasing both the sensitivity and specificity of the data analysis pipeline. InflectSSP incorporates calculation of a "melt coefficient" into the pipeline with production of average melt curves for biological replicate studies to aid in identification of proteins with significant melts. To benchmark InflectSSP, we have reanalyzed two previously reported datasets to demonstrate the performance of our publicly available R-based program for TPP data analysis. We report new findings following temporal treatment of human cells with the small molecule thapsigargin that induces the unfolded protein response as a consequence of inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase 2A. InflectSSP analysis of our unfolded protein response study revealed highly reproducible and statistically significant target engagement over a time course of treatment while simultaneously providing new insights into the possible mechanisms of action of the small molecule thapsigargin.


Asunto(s)
Proteoma , Proteómica , Humanos , Proteoma/metabolismo , Tapsigargina/farmacología , Proteómica/métodos
2.
J Biol Chem ; 299(4): 104595, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36898579

RESUMEN

The integrated stress response (ISR) is an important mechanism by which cells confer protection against environmental stresses. Central to the ISR is a collection of related protein kinases that monitor stress conditions, such as Gcn2 (EIF2AK4) that recognizes nutrient limitations, inducing phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Gcn2 phosphorylation of eIF2 lowers bulk protein synthesis, conserving energy and nutrients, coincident with preferential translation of stress-adaptive gene transcripts, such as that encoding the Atf4 transcriptional regulator. While Gcn2 is central for cell protection to nutrient stress and its depletion in humans leads to pulmonary disorders, Gcn2 can also contribute to the progression of cancers and facilitate neurological disorders during chronic stress. Consequently, specific ATP-competitive inhibitors of Gcn2 protein kinase have been developed. In this study, we report that one such Gcn2 inhibitor, Gcn2iB, can activate Gcn2, and we probe the mechanism by which this activation occurs. Low concentrations of Gcn2iB increase Gcn2 phosphorylation of eIF2 and enhance Atf4 expression and activity. Of importance, Gcn2iB can activate Gcn2 mutants devoid of functional regulatory domains or with certain kinase domain substitutions derived from Gcn2-deficient human patients. Other ATP-competitive inhibitors can also activate Gcn2, although there are differences in their mechanisms of activation. These results provide a cautionary note about the pharmacodynamics of eIF2 kinase inhibitors in therapeutic applications. Compounds designed to be kinase inhibitors that instead directly activate Gcn2, even loss of function variants, may provide tools to alleviate deficiencies in Gcn2 and other regulators of the ISR.


Asunto(s)
Factor 2 Eucariótico de Iniciación , Proteínas Serina-Treonina Quinasas , Humanos , Adenosina Trifosfato/metabolismo , Activación Enzimática/efectos de los fármacos , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo
3.
Am J Physiol Endocrinol Metab ; 325(5): E624-E637, 2023 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-37792040

RESUMEN

Nonshivering thermogenesis in rodents requires macronutrients to fuel the generation of heat during hypothermic conditions. In this study, we examined the role of the nutrient sensing kinase, general control nonderepressible 2 (GCN2) in directing adaptive thermogenesis during acute cold exposure in mice. We hypothesized that GCN2 is required for adaptation to acute cold stress via activation of the integrated stress response (ISR) resulting in liver production of FGF21 and increased amino acid transport to support nonshivering thermogenesis. In alignment with our hypothesis, female and male mice lacking GCN2 failed to adequately increase energy expenditure and veered into torpor. Mice administered a small molecule inhibitor of GCN2 were also profoundly intolerant to acute cold stress. Gcn2 deletion also impeded liver-derived FGF21 but in males only. Within the brown adipose tissue (BAT), acute cold exposure increased ISR activation and its transcriptional execution in males and females. RNA sequencing in BAT identified transcripts that encode actomyosin mechanics and transmembrane transport as requiring GCN2 during cold exposure. These transcripts included class II myosin heavy chain and amino acid transporters, critical for maximal thermogenesis during cold stress. Importantly, Gcn2 deletion corresponded with higher circulating amino acids and lower intracellular amino acids in the BAT during cold stress. In conclusion, we identify a sex-independent role for GCN2 activation to support adaptive thermogenesis via uptake of amino acids into brown adipose.NEW & NOTEWORTHY This paper details the discovery that GCN2 activation is required in both male and female mice to maintain core body temperature during acute cold exposure. The results point to a novel role for GCN2 in supporting adaptive thermogenesis via amino acid transport and actomyosin mechanics in brown adipose tissue.


Asunto(s)
Actomiosina , Temperatura Corporal , Ratones , Masculino , Femenino , Animales , Actomiosina/metabolismo , Termogénesis/genética , Hígado/metabolismo , Frío , Tejido Adiposo Pardo/metabolismo , Aminoácidos/metabolismo , Ratones Endogámicos C57BL
4.
PLoS Genet ; 12(12): e1006518, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27977682

RESUMEN

The unfolded protein response (UPR) regulates cell fate following exposure of cells to endoplasmic reticulum stresses. PERK, a UPR protein kinase, regulates protein synthesis and while linked with cell survival, exhibits activities associated with both tumor progression and tumor suppression. For example, while cells lacking PERK are sensitive to UPR-dependent cell death, acute activation of PERK triggers both apoptosis and cell cycle arrest, which would be expected to contribute tumor suppressive activity. We have evaluated these activities in the BRAF-dependent melanoma and provide evidence revealing a complex role for PERK in melanoma where a 50% reduction is permissive for BrafV600E-dependent transformation, while complete inhibition is tumor suppressive. Consistently, PERK mutants identified in human melanoma are hypomorphic with dominant inhibitory function. Strikingly, we demonstrate that small molecule PERK inhibitors exhibit single agent efficacy against BrafV600E-dependent tumors highlighting the clinical value of targeting PERK.


Asunto(s)
Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Supresoras de Tumor/genética , eIF-2 Quinasa/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Transformación Celular Neoplásica/efectos de los fármacos , Retículo Endoplásmico/genética , Retículo Endoplásmico/patología , Dosificación de Gen/genética , Haploinsuficiencia/genética , Humanos , Melanoma/tratamiento farmacológico , Melanoma/patología , Mutación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Proteínas Supresoras de Tumor/biosíntesis , Respuesta de Proteína Desplegada/genética , eIF-2 Quinasa/antagonistas & inhibidores , eIF-2 Quinasa/biosíntesis
5.
Artículo en Inglés | MEDLINE | ID: mdl-30181373

RESUMEN

Toxoplasma gondii is an obligate intracellular parasite that has infected one-third of the population. Upon infection of warm-blooded vertebrates, the replicating form of the parasite (tachyzoite) converts into a latent form (bradyzoite) present in tissue cysts. During immune deficiency, bradyzoites can reconvert into tachyzoites and cause life-threatening toxoplasmosis. We previously reported that translational control through phosphorylation of the α subunit of T. gondii eukaryotic initiation factor 2 (eIF2α) (TgIF2α) is a critical component of the parasite stress response. Diverse stresses can induce the conversion of tachyzoites to bradyzoites, including those disrupting the parasite's endoplasmic reticulum (ER) (ER stress). Toxoplasma possesses four eIF2α kinases, one of which (TgIF2K-A) localizes to the parasite ER analogously to protein kinase R-like endoplasmic reticulum kinase (PERK), the eIF2α kinase that responds to ER stress in mammalian cells. Here, we investigated the effects of a PERK inhibitor (PERKi) on Toxoplasma Our results show that the PERKi GSK2606414 blocks the enzymatic activity of TgIF2K-A and reduces TgIF2α phosphorylation specifically in response to ER stress. PERKi also significantly impeded multiple steps of the tachyzoite lytic cycle and sharply lowered the frequency of bradyzoite differentiation in vitro Pretreatment of host cells with PERKi prior to infection did not affect parasite infectivity, and PERKi still impaired parasite replication in host cells lacking PERK. In mice, PERKi conferred modest protection from a lethal dose of Toxoplasma Our findings represent the first pharmacological evidence supporting TgIF2K-A as an attractive new target for the treatment of toxoplasmosis.


Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Toxoplasma/efectos de los fármacos , Toxoplasmosis/tratamiento farmacológico , eIF-2 Quinasa/antagonistas & inhibidores , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/parasitología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Parásitos/efectos de los fármacos , Parásitos/metabolismo , Fosforilación/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Toxoplasmosis/parasitología
6.
Cancer Res ; 84(8): 1286-1302, 2024 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-38266162

RESUMEN

TFE3 is a member of the basic helix-loop-helix leucine zipper MiT transcription factor family, and its chimeric proteins are associated with translocation renal cell carcinoma (tRCC). Despite the variety of gene fusions, most TFE3 fusion partner genes are related to spliceosome machinery. Dissecting the function of TFE3 fused to spliceosome machinery factors (TFE3-SF) could direct the development of effective therapies for this lethal disease, which is refractory to standard treatments for kidney cancer. Here, by using a combination of in silico structure prediction, transcriptome profiling, molecular characterization, and high-throughput high-content screening (HTHCS), we interrogated a number of oncogenic mechanisms of TFE3-SF fusions. TFE3-SF fusions drove the transformation of kidney cells and promoted distinct oncogenic phenotypes in a fusion partner-dependent manner, differentially altering the transcriptome and RNA splicing landscape and activating different oncogenic pathways. Inhibiting TFE3-SF dimerization reversed its oncogenic activity and represented a potential target for therapeutic intervention. Screening the FDA-approved drugs library LOPAC and a small-molecule library (Microsource) using HTHCS combined with FRET technology identified compounds that inhibit TFE3-SF dimerization. Hit compounds were validated in 2D and 3D patient-derived xenograft models expressing TFE3-SF. The antihistamine terfenadine decreased cell proliferation and reduced in vivo tumor growth of tRCC. Overall, these results unmask therapeutic strategies to target TFE3-SF dimerization for treating patients with tRCC. SIGNIFICANCE: TFE3-splicing factor fusions possess both transcription and splicing factor functions that remodel the transcriptome and spliceosome and can be targeted with dimerization inhibitors to suppress the growth of translocation renal cell carcinoma.


Asunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Humanos , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Factores de Empalme de ARN/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Neoplasias Renales/patología , Fusión Génica , Translocación Genética , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo
7.
J Clin Invest ; 134(16)2024 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-38889047

RESUMEN

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eukaryotic translation initiation factor-2α (eIF2α). In T1D, maladaptive unfolded protein response (UPR) in insulin-producing ß cells renders these cells susceptible to autoimmunity. We found that inhibition of the eIF2α kinase PKR-like ER kinase (PERK), a common component of the UPR and ISR, reversed the mRNA translation block in stressed human islets and delayed the onset of diabetes, reduced islet inflammation, and preserved ß cell mass in T1D-susceptible mice. Single-cell RNA-Seq of islets from PERK-inhibited mice showed reductions in the UPR and PERK signaling pathways and alterations in antigen-processing and presentation pathways in ß cells. Spatial proteomics of islets from these mice showed an increase in the immune checkpoint protein programmed death-ligand 1 (PD-L1) in ß cells. Golgi membrane protein 1, whose levels increased following PERK inhibition in human islets and EndoC-ßH1 human ß cells, interacted with and stabilized PD-L1. Collectively, our studies show that PERK activity enhances ß cell immunogenicity and that inhibition of PERK may offer a strategy for preventing or delaying the development of T1D.


Asunto(s)
Diabetes Mellitus Tipo 1 , eIF-2 Quinasa , Animales , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismo , eIF-2 Quinasa/antagonistas & inhibidores , eIF-2 Quinasa/inmunología , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/prevención & control , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/enzimología , Ratones , Humanos , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/patología , Células Secretoras de Insulina/metabolismo , Ratones Endogámicos NOD , Respuesta de Proteína Desplegada , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 2 Eucariótico de Iniciación/genética
8.
bioRxiv ; 2024 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-38895427

RESUMEN

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eIF2α. In T1D, maladaptive unfolded protein response (UPR) in insulin-producing ß cells renders these cells susceptible to autoimmunity. We show that inhibition of the eIF2α kinase PERK, a common component of the UPR and ISR, reverses the mRNA translation block in stressed human islets and delays the onset of diabetes, reduces islet inflammation, and preserves ß cell mass in T1D-susceptible mice. Single-cell RNA sequencing of islets from PERK-inhibited mice shows reductions in the UPR and PERK signaling pathways and alterations in antigen processing and presentation pathways in ß cells. Spatial proteomics of islets from these mice shows an increase in the immune checkpoint protein PD-L1 in ß cells. Golgi membrane protein 1, whose levels increase following PERK inhibition in human islets and EndoC-ßH1 human ß cells, interacts with and stabilizes PD-L1. Collectively, our studies show that PERK activity enhances ß cell immunogenicity, and inhibition of PERK may offer a strategy to prevent or delay the development of T1D.

9.
Antioxid Redox Signal ; 39(4-6): 351-373, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-36943285

RESUMEN

Significance: Organisms adapt to changing environments by engaging cellular stress response pathways that serve to restore proteostasis and enhance survival. A primary adaptive mechanism is the integrated stress response (ISR), which features phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2). Four eIF2α kinases respond to different stresses, enabling cells to rapidly control translation to optimize management of resources and reprogram gene expression for stress adaptation. Phosphorylation of eIF2 blocks its guanine nucleotide exchange factor, eIF2B, thus lowering the levels of eIF2 bound to GTP that is required to deliver initiator transfer RNA (tRNA) to ribosomes. While bulk messenger RNA (mRNA) translation can be sharply lowered by heightened phosphorylation of eIF2α, there are other gene transcripts whose translation is unchanged or preferentially translated. Among the preferentially translated genes is ATF4, which directs transcription of adaptive genes in the ISR. Recent Advances and Critical Issues: This review focuses on how eIF2α kinases function as first responders of stress, the mechanisms by which eIF2α phosphorylation and other stress signals regulate the exchange activity of eIF2B, and the processes by which the ISR triggers differential mRNA translation. To illustrate the synergy between stress pathways, we describe the mechanisms and functional significance of communication between the ISR and another key regulator of translation, mammalian/mechanistic target of rapamycin complex 1 (mTORC1), during acute and chronic amino acid insufficiency. Finally, we discuss the pathological conditions that stem from aberrant regulation of the ISR, as well as therapeutic strategies targeting the ISR to alleviate disease. Future Directions: Important topics for future ISR research are strategies for modulating this stress pathway in disease conditions and drug development, molecular processes for differential translation and the coordinate regulation of GCN2 and other stress pathways during physiological and pathological conditions. Antioxid. Redox Signal. 39, 351-373.


Asunto(s)
Factor 2B Eucariótico de Iniciación , Factor 2 Eucariótico de Iniciación , Animales , Factor 2B Eucariótico de Iniciación/química , Factor 2B Eucariótico de Iniciación/genética , Factor 2B Eucariótico de Iniciación/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Fosforilación , Regulación de la Expresión Génica , Estrés Fisiológico , Mamíferos/metabolismo
10.
Front Med (Lausanne) ; 10: 1146115, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37181357

RESUMEN

Pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC) is characterized by a profound inflammatory tumor microenvironment (TME) with high heterogeneity, metastatic propensity, and extreme hypoxia. The integrated stress response (ISR) pathway features a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) and regulate translation in response to diverse stress conditions, including hypoxia. We previously demonstrated that eIF2 signaling pathways were profoundly affected in response to Redox factor-1 (Ref-1) knockdown in human PDAC cells. Ref-1 is a dual function enzyme with activities of DNA repair and redox signaling, responds to cellular stress, and regulates survival pathways. The redox function of Ref-1 directly regulates multiple transcription factors including HIF-1α, STAT3, and NF-κB, which are highly active in the PDAC TME. However, the mechanistic details of the crosstalk between Ref-1 redox signaling and activation of ISR pathways are unclear. Following Ref-1 knockdown, induction of ISR was observed under normoxic conditions, while hypoxic conditions were sufficient to activate ISR irrespective of Ref-1 levels. Inhibition of Ref-1 redox activity increased expression of p-eIF2 and ATF4 transcriptional activity in a concentration-dependent manner in multiple human PDAC cell lines, and the effect on eIF2 phosphorylation was PERK-dependent. Treatment with PERK inhibitor, AMG-44 at high concentrations resulted in activation of the alternative ISR kinase, GCN2 and induced levels of p-eIF2 and ATF4 in both tumor cells and cancer-associated fibroblasts (CAFs). Combination treatment with inhibitors of Ref-1 and PERK enhanced cell killing effects in both human pancreatic cancer lines and CAFs in 3D co-culture, but only at high doses of PERK inhibitors. This effect was completely abrogated when Ref-1 inhibitors were used in combination with GCN2 inhibitor, GCN2iB. We demonstrate that targeting of Ref-1 redox signaling activates the ISR in multiple PDAC lines and that this activation of ISR is critical for inhibition of the growth of co-culture spheroids. Combination effects were only observed in physiologically relevant 3D co-cultures, suggesting that the model system utilized can greatly affect the outcome of these targeted agents. Inhibition of Ref-1 signaling induces cell death through ISR signaling pathways, and combination of Ref-1 redox signaling blockade with ISR activation could be a novel therapeutic strategy for PDAC treatment.

11.
Clin Cancer Res ; 29(23): 4870-4882, 2023 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-37733811

RESUMEN

PURPOSE: Tumors activate protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK, also called EIF2AK3) in response to hypoxia and nutrient deprivation as a stress-mitigation strategy. Here, we tested the hypothesis that inhibiting PERK with HC-5404 enhances the antitumor efficacy of standard-of-care VEGF receptor tyrosine kinase inhibitors (VEGFR-TKI). EXPERIMENTAL DESIGN: HC-5404 was characterized as a potent and selective PERK inhibitor, with favorable in vivo properties. Multiple renal cell carcinoma (RCC) tumor models were then cotreated with both HC-5404 and VEGFR-TKI in vivo, measuring tumor volume across time and evaluating tumor response by protein analysis and IHC. RESULTS: VEGFR-TKI including axitinib, cabozantinib, lenvatinib, and sunitinib induce PERK activation in 786-O RCC xenografts. Cotreatment with HC-5404 inhibited PERK in tumors and significantly increased antitumor effects of VEGFR-TKI across multiple RCC models, resulting in tumor stasis or regression. Analysis of tumor sections revealed that HC-5404 enhanced the antiangiogenic effects of axitinib and lenvatinib by inhibiting both new vasculature and mature tumor blood vessels. Xenografts that progress on axitinib monotherapy remain sensitive to the combination treatment, resulting in ∼20% tumor regression in the combination group. When tested across a panel of 18 RCC patient-derived xenograft (PDX) models, the combination induced greater antitumor effects relative to monotherapies. In this single animal study, nine out of 18 models responded with ≥50% tumor regression from baseline in the combination group. CONCLUSIONS: By disrupting an adaptive stress response evoked by VEGFR-TKI, HC-5404 presents a clinical opportunity to improve the antitumor effects of well-established standard-of-care therapies in RCC.


Asunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Animales , Humanos , Carcinoma de Células Renales/patología , Axitinib/farmacología , Axitinib/uso terapéutico , Neoplasias Renales/patología , Inhibidores de Proteínas Quinasas/uso terapéutico
12.
Clin Cancer Res ; 29(24): 5155-5172, 2023 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-37982738

RESUMEN

PURPOSE: The integrated stress response (ISR) kinase PERK serves as a survival factor for both proliferative and dormant cancer cells. We aim to validate PERK inhibition as a new strategy to specifically eliminate solitary disseminated cancer cells (DCC) in secondary sites that eventually reawake and originate metastasis. EXPERIMENTAL DESIGN: A novel clinical-grade PERK inhibitor (HC4) was tested in mouse syngeneic and PDX models that present quiescent/dormant DCCs or growth-arrested cancer cells in micro-metastatic lesions that upregulate ISR. RESULTS: HC4 significantly blocks metastasis, by killing quiescent/slow-cycling ISRhigh, but not proliferative ISRlow DCCs. HC4 blocked expansion of established micro-metastasis that contained ISRhigh slow-cycling cells. Single-cell gene expression profiling and imaging revealed that a significant proportion of solitary DCCs in lungs were indeed dormant and displayed an unresolved ER stress as revealed by high expression of a PERK-regulated signature. In human breast cancer metastasis biopsies, GADD34 expression (PERK-regulated gene) and quiescence were positively correlated. HC4 effectively eradicated dormant bone marrow DCCs, which usually persist after rounds of therapies. Importantly, treatment with CDK4/6 inhibitors (to force a quiescent state) followed by HC4 further reduced metastatic burden. In HNSCC and HER2+ cancers HC4 caused cell death in dormant DCCs. In HER2+ tumors, PERK inhibition caused killing by reducing HER2 activity because of sub-optimal HER2 trafficking and phosphorylation in response to EGF. CONCLUSIONS: Our data identify PERK as a unique vulnerability in quiescent or slow-cycling ISRhigh DCCs. The use of PERK inhibitors may allow targeting of pre-existing or therapy-induced growth arrested "persister" cells that escape anti-proliferative therapies.


Asunto(s)
Neoplasias de la Mama , Humanos , Animales , Ratones , Femenino , Línea Celular Tumoral , Ciclo Celular , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proliferación Celular , Muerte Celular , eIF-2 Quinasa/genética
13.
Oncogene ; 41(44): 4855-4865, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-36182969

RESUMEN

Amino acid restriction has recently emerged as a compelling strategy to inhibit tumor growth. Recent work suggests that amino acids can regulate cellular signaling in addition to their role as biosynthetic substrates. Using lymphoid cancer cells as a model, we found that asparagine depletion acutely reduces the expression of c-MYC protein without changing its mRNA expression. Furthermore, asparagine depletion inhibits the translation of MYC mRNA without altering the rate of MYC protein degradation. Of interest, the inhibitory effect on MYC mRNA translation during asparagine depletion is not due to the activation of the general controlled nonderepressible 2 (GCN2) pathway and is not a consequence of the inhibition of global protein synthesis. In addition, both the 5' and 3' untranslated regions (UTRs) of MYC mRNA are not required for this inhibitory effect. Finally, using a MYC-driven mouse B cell lymphoma model, we found that shRNA inhibition of asparagine synthetase (ASNS) or pharmacological inhibition of asparagine production can significantly reduce the MYC protein expression and tumor growth when environmental asparagine becomes limiting. Since MYC is a critical oncogene, our results uncover a molecular connection between MYC mRNA translation and asparagine bioavailability and shed light on a potential to target MYC oncogene post-transcriptionally through asparagine restriction.


Asunto(s)
Asparagina , Neoplasias , Ratones , Animales , Asparagina/genética , Asparagina/metabolismo , Disponibilidad Biológica , Genes myc , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Neoplasias/genética , Aminoácidos/metabolismo , Regiones no Traducidas 3'/genética
14.
Elife ; 112022 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-36107759

RESUMEN

A stress adaptation pathway termed the integrated stress response has been suggested to be active in many cancers including prostate cancer (PCa). Here, we demonstrate that the eIF2 kinase GCN2 is required for sustained growth in androgen-sensitive and castration-resistant models of PCa both in vitro and in vivo, and is active in PCa patient samples. Using RNA-seq transcriptome analysis and a CRISPR-based phenotypic screen, GCN2 was shown to regulate expression of over 60 solute-carrier (SLC) genes, including those involved in amino acid transport and loss of GCN2 function reduces amino acid import and levels. Addition of essential amino acids or expression of 4F2 (SLC3A2) partially restored growth following loss of GCN2, suggesting that GCN2 targeting of SLC transporters is required for amino acid homeostasis needed to sustain tumor growth. A small molecule inhibitor of GCN2 showed robust in vivo efficacy in androgen-sensitive and castration-resistant mouse models of PCa, supporting its therapeutic potential for the treatment of PCa.


Prostate cancer is the fourth most common cancer worldwide, affecting over a million people each year. Existing drug treatments work by blocking the effects or reducing the levels of the hormone testosterone. However, these drug regimens are not always effective, so finding alternative treatments is an important area of research. One option is to target the 'integrated stress response', a pathway that acts as a genetic switch, turning on a group of genes that counteract cellular stress and are essential for the survival of cancer cells. The reason cancer cells are under stress is because they are hungry. They need to make a lot of proteins and other metabolic intermediates to grow and divide, which means they need plenty of amino acids, the building blocks that make up proteins and fuel metabolism. Amino acids enter cells through molecular gates called amino acid transporters, and scientists think the integrated stress response might play a role in this process. One of the integrated stress response components is a protein called General Control Nonderepressible 2, or GCN2 for short. In healthy cells, this protein helps to boost amino acid levels when supplies start to run low. Cordova et al. examined human prostate cancer cells to find out what role GCN2 plays in this cancer. In both lab-grown cells and tissue from patients, GCN2 was active and played a critical role in prostate tumor growth by turning on the genes for amino acid transporters to increase the levels of amino acids entering the cancer cells. Deleting the gene for GCN2, or blocking its effects with an experimental drug, slowed the growth of cultured prostate cancer cells and reduced tumor growth in mice. In these early experiments, Cordova et al. did not notice any toxic side effects to healthy tissues. If GCN2 works in the same way in humans as it does in mice, blocking it might help to control prostate cancer growth. The integrated stress response is also active in other cancer types, so the same logic might apply to different tumors. However, before GCN2 blockers can become treatments, researchers need a more complete understanding of their molecular effects.


Asunto(s)
Neoplasias de la Próstata , eIF-2 Quinasa , Animales , Humanos , Masculino , Ratones , Aminoácidos/metabolismo , Aminoácidos Esenciales , Andrógenos , eIF-2 Quinasa/metabolismo , Homeostasis , Ratones Endogámicos C57BL , Neoplasias de la Próstata/genética
15.
J Biol Chem ; 285(22): 16893-911, 2010 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-20233714

RESUMEN

Two important nutrient-sensing and regulatory pathways, the general amino acid control (GAAC) and the target of rapamycin (TOR), participate in the control of yeast growth and metabolism during changes in nutrient availability. Amino acid starvation activates the GAAC through Gcn2p phosphorylation of translation factor eIF2 and preferential translation of GCN4, a transcription activator. TOR senses nitrogen availability and regulates transcription factors such as Gln3p. We used microarray analyses to address the integration of the GAAC and TOR pathways in directing the yeast transcriptome during amino acid starvation and rapamycin treatment. We found that GAAC is a major effector of the TOR pathway, with Gcn4p and Gln3p each inducing a similar number of genes during rapamycin treatment. Although Gcn4p activates a common core of 57 genes, the GAAC directs significant variations in the transcriptome during different stresses. In addition to inducing amino acid biosynthetic genes, Gcn4p in conjunction with Gln3p activates genes required for the assimilation of secondary nitrogen sources such as gamma-aminobutyric acid (GABA). Gcn2p activation upon shifting to secondary nitrogen sources is suggested to occur by means of a dual mechanism. First, Gcn2p is induced by the release of TOR repression through a mechanism involving Sit4p protein phosphatase. Second, this eIF2 kinase is activated by select uncharged tRNAs, which were shown to accumulate during the shift to the GABA medium. This study highlights the mechanisms by which the GAAC and TOR pathways are integrated to recognize changing nitrogen availability and direct the transcriptome for optimal growth adaptation.


Asunto(s)
Aminoácidos/química , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/fisiología , Nitrógeno/química , Proteínas Serina-Treonina Quinasas/fisiología , Saccharomyces cerevisiae/metabolismo , Perfilación de la Expresión Génica , Modelos Biológicos , Fosforilación , Biosíntesis de Proteínas , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN de Transferencia/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad de la Especie , Serina-Treonina Quinasas TOR , Transcripción Genética , Ácido gamma-Aminobutírico/metabolismo
16.
J Immunol ; 183(1): 568-77, 2009 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-19542468

RESUMEN

Both IL-23- and IL-1-mediated signaling pathways play important roles in Th17 cell differentiation, cytokine production, and autoimmune diseases. The IL-1R-associated kinase 4 (IRAK4) is critical for IL-1/TLR signaling. We show here that inactivation of IRAK4 kinase in mice (IRAK4 KI) results in significant resistance to experimental autoimmune encephalomyelitis due to a reduction in infiltrating inflammatory cells into the CNS and reduced Ag-specific CD4(+) T cell-mediated IL-17 production. Adoptive transfer of myelin oligodendrocyte glycoprotein 35-55-specific IRAK4 KI Th17 cells failed to induce experimental autoimmune encephalomyelitis in either wild-type or IRAK4 KI recipient mice, indicating the lack of autoantigen-specific Th17 cell activities in the absence of IRAK4 kinase activity. Furthermore, the absence of IRAK4 kinase activity blocked induction of IL-23R expression, STAT3 activation by IL-23, and Th17 cytokine expression in differentiated Th17 cells. Importantly, blockade of IL-1 signaling by IL-1RA inhibited Th17 differentiation and IL-23-induced cytokine expression in differentiated Th17 cells. The results of these studies demonstrate that IL-1-mediated IRAK4 kinase activity in T cells is essential for induction of IL-23R expression, Th17 differentiation, and autoimmune disease.


Asunto(s)
Diferenciación Celular/inmunología , Encefalomielitis Autoinmune Experimental/enzimología , Encefalomielitis Autoinmune Experimental/inmunología , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/fisiología , Interleucina-17/fisiología , Linfocitos T Colaboradores-Inductores/enzimología , Linfocitos T Colaboradores-Inductores/inmunología , Secuencia de Aminoácidos , Animales , Diferenciación Celular/genética , Inhibición de Migración Celular/genética , Inhibición de Migración Celular/inmunología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/prevención & control , Activación Enzimática/genética , Activación Enzimática/inmunología , Femenino , Técnicas de Sustitución del Gen , Glicoproteínas/administración & dosificación , Glicoproteínas/antagonistas & inhibidores , Inmunidad Innata/genética , Quinasas Asociadas a Receptores de Interleucina-1/deficiencia , Quinasas Asociadas a Receptores de Interleucina-1/genética , Interleucina-17/antagonistas & inhibidores , Interleucina-17/biosíntesis , Leucocitos Mononucleares/enzimología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/patología , Ratones , Datos de Secuencia Molecular , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/antagonistas & inhibidores , Transducción de Señal/genética , Transducción de Señal/inmunología , Médula Espinal/inmunología , Médula Espinal/patología , Linfocitos T Colaboradores-Inductores/patología
18.
Cell Death Dis ; 9(3): 254, 2018 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-29449538

RESUMEN

Oxidative stress determines cell fate through several mechanisms, among which regulation of mRNA translation by the phosphorylation of the alpha (α) subunit of the translation initiation factor eIF2α at serine 51 (eIF2αP) plays a prominent role. Increased eIF2αP can contribute to tumor progression as well as tumor suppression. While eIF2αP is increased in most cells to promote survival and adaptation to different forms of stress, we demonstrate that eIF2αP is reduced in tuberous sclerosis complex 2 (TSC2)-deficient cells subjected to oxidative insults. Decreased eIF2αP in TSC2-deficient cells depends on reactive oxygen species (ROS) production and is associated with a reduced activity of the endoplasmic reticulum (ER)-resident kinase PERK owing to the hyper-activation of the mammalian target of rapamycin complex 1 (mTORC1). Downregulation of PERK activity and eIF2αP is accompanied by increased ROS production and enhanced susceptibility of TSC2-deficient cells to extrinsic pro-oxidant stress. The decreased levels of eIF2αP delay tumor formation of TSC2-deficient cells in immune deficient mice, an effect that is significantly alleviated in mice subjected to an anti-oxidant diet. Our findings reveal a previously unidentified connection between mTORC1 and eIF2αP in TSC2-deficient cells with potential implications in tumor suppression in response to oxidative insults.


Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Fibroblastos/enzimología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Esclerosis Tuberosa/enzimología , eIF-2 Quinasa/metabolismo , Animales , Antioxidantes/farmacología , Muerte Celular , Células Cultivadas , Regulación hacia Abajo , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Humanos , Ratones , Ratones SCID , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Neoplasias/prevención & control , Estrés Oxidativo/efectos de los fármacos , Fosforilación , Serina , Transducción de Señal , Factores de Tiempo , Esclerosis Tuberosa/genética , Esclerosis Tuberosa/patología , Proteína 2 del Complejo de la Esclerosis Tuberosa/deficiencia , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Carga Tumoral
19.
Cell Rep ; 22(11): 2978-2994, 2018 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-29539425

RESUMEN

Abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6), has recently been approved for the treatment of hormone receptor-positive breast cancer. In this study, we use murine syngeneic tumor models and in vitro assays to investigate the impact of abemaciclib on T cells, the tumor immune microenvironment and the ability to combine with anti-PD-L1 blockade. Abemaciclib monotherapy resulted in tumor growth delay that was associated with an increased T cell inflammatory signature in tumors. Combination with anti-PD-L1 therapy led to complete tumor regressions and immunological memory, accompanied by enhanced antigen presentation, a T cell inflamed phenotype, and enhanced cell cycle control. In vitro, treatment with abemaciclib resulted in increased activation of human T cells and upregulated expression of antigen presentation genes in MCF-7 breast cancer cells. These data collectively support the clinical investigation of the combination of abemaciclib with agents such as anti-PD-L1 that modulate T cell anti-tumor immunity.


Asunto(s)
Aminopiridinas/uso terapéutico , Bencimidazoles/uso terapéutico , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/uso terapéutico , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/uso terapéutico , Receptor de Muerte Celular Programada 1/metabolismo , Aminopiridinas/farmacología , Bencimidazoles/farmacología , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/farmacología , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/farmacología , Humanos , Microambiente Tumoral
20.
Mol Cancer Res ; 13(10): 1377-88, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26130148

RESUMEN

UNLABELLED: The mTOR nucleates two complexes, namely mTOR complex 1 and 2 (mTORC1 and mTORC2), which are implicated in cell growth, survival, metabolism, and cancer. Phosphorylation of the α-subunit of translation initiation factor eIF2 at serine 51 (eIF2αS51P) is a key event of mRNA translation initiation and a master regulator of cell fate during cellular stress. Recent studies have implicated mTOR signaling in the stress response, but its connection to eIF2αS51P has remained unclear. Herein, we report that genetic as well as catalytic inhibition of mTORC2 induces eIF2αS51P. On the other hand, the allosteric inhibitor rapamycin induces eIF2αS51P through pathways that are independent of mTORC1 inactivation. Increased eIF2αS51P by impaired mTORC2 depends on the inactivation of AKT, which primes the activation of the endoplasmic reticulum (ER)-resident kinase PERK/PEK. The biologic function of eIF2αS51P was characterized in tuberous sclerosis complex (TSC)-mutant cells, which are defective in mTORC2 and AKT activity. TSC-mutant cells exhibit increased PERK activity, which is downregulated by the reconstitution of the cells with an activated form of AKT1. Also, TSC-mutant cells are increasingly susceptible to ER stress, which is reversed by AKT1 reconstitution. The susceptibility of TSC-mutant cells to ER stress is further enhanced by the pharmacologic inhibition of PERK or genetic inactivation of eIF2αS51P. Thus, the PERK/eIF2αS51P arm is an important compensatory prosurvival mechanism, which substitutes for the loss of AKT under ER stress. IMPLICATIONS: A novel mechanistic link between mTOR function and protein synthesis is identified in TSC-null tumor cells under stress and reveals potential for the development of antitumor treatments with stress-inducing chemotherapeutics.


Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Línea Celular Tumoral , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/fisiología , Fibrosarcoma/tratamiento farmacológico , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Humanos , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Complejos Multiproteicos/antagonistas & inhibidores , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina/metabolismo , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Transfección , Regulación hacia Arriba , eIF-2 Quinasa/metabolismo
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