MTORC1 and -2 Coordinate Transcriptional and Translational Reprogramming in Resistance to DNA Damage and Replicative Stress in Breast Cancer Cells
ABSTRACT: mTOR coordinates growth signals with metabolic pathways and protein synthesis and is hyperactivated in many human cancers. We have characterized the coordinated mTORC1 and -2 transcription and translation response using genome-wide translatome and transcriptome profiling on cells inhibited in mTORC1/2 with PP242 or only mTORC1 with RAD001, with or without concurrent IR. In this dataset, we include the expression data obtained from SUM149 cells treated with RAD001 + IR, RAD001, PP242+IR, PP242, IR, DMSO. We include both total (transcriptional) data as well as polysome arrays. These data are used to obtain differentially expressed genes in all treatments compared to DMSO in both polysome and transcriptional data. Overall design: 35 samples were analyzed. We examined the following comparisons using Limma with a p-value < 0.05, log fold-change > 1.5: PP242 vs. DMSO, PP242+IR vs. DMSO, RAD001 vs DMSO, RAD001 + IR vs DMSO, IR vs DMSO in polysome samples, total sampes, and using translational efficiency.
INSTRUMENT(S): [HTA-2_0] Affymetrix Human Transcriptome Array 2.0 [transcript (gene) version]
Project description:Rapalogs, inhibitors of mTORC1 (mammalian target of rapamycin complex 1), increase life span and delay age-related phenotypes in many species. However, the molecular mechanisms have not been fully elucidated. We determined gene expression changes comparing 6- and 24-month-old rats in the kidney, liver, and skeletal muscle, and asked which of these changes were counter-regulated by a clinically-translatable (short-term and low-concentration) treatment, with a rapalog (RAD001). Surprisingly, RAD001 had a more pronounced effect on the kidney under this regimen in comparison to the liver or skeletal muscle. Histologic evaluation of kidneys revealed that the severity of chronic progressive nephropathy lesions was lower in kidneys from 24-month-old rats treated with RAD001 compared with vehicle. In addition to other gene expression changes, c-Myc, which has been shown to regulate aging, was induced by aging in the kidney and counter-regulated by RAD001. RAD001 caused a decrease in c-Myc protein, which could be rescued by a proteasome inhibitor. These findings point to settings for use of mTORC1 inhibitors to treat age-related disorders, and highlight c-Myc regulation as one of the potential mechanisms by which mTORC1 inhibition is perturbing age-related phenotypes. Overall design: Transcriptional profiling was performed in kidney, liver and gastrocnemius muscles from three experimental groups of male Sprague Dawley rats. Rats aged 4.5 month (m) and 22.5 m were treated with vehicle and rats aged 22.5 m were treated with RAD001 for 6 weeks, with a read-out at 6 and 24 months.
Project description:Gp130 receptor engagement on neoplastic cells provides a link by which an inflammatory microenvironment facilitates tumour promotion. Although hyperactivation of the gp130-dependent Stat3 signalling node is commonly observed in solid tumours, Stat3 remains a challenging therapeutic target. To mimic excessive Stat3 signalling, we molecularly validate the gp130FF mouse as a preclinical model for inflammation-associated intestinal-type gastric cancer (IGC), with aberrant mammalian target of rapamycin (mTOR) pathway activity as shared feature. Accordingly, administration of the mTorc1 inhibitor RAD001 reversibly reduced IGC burden in gp130FF mice and suppressed colitis-associated cancer in wild-type mice. Since the therapeutic effect of RAD001 occurs independently of Stat3 hyperactivation, which is also dispensable for gp130-dependent engagement of the PI3K/Akt/mTorc1 pathway, we conclude that mTorc1 signalling limits tumour promoting Stat3 activity The mouse whole-genome gene expression profiling was performed on Illumina's MouseWG-6 v2.0 Expression BeadChips for 24 mice, with 8 mice in each group (gp130WT antral tissue, gp130FF unaffected antral tissue and gp130FF tumour tissue).
Project description:mRNA translation plays a major role in homeostasis, whereas its dysregulation underpins a variety of pathological states including cancer, metabolic syndrome and neurological disorders. Ternary complex (TC) and eIF4F complex assembly are two major rate-limiting steps in translation initiation that are thought to be regulated by eIF2α phosphorylation, and the mTOR/4E-BP pathway, respectively2. However, how TC and eIF4F assembly are coordinated remains largely unknown. Using polysome-profiling, we show that on a genome-wide scale mTOR suppresses translation of mRNAs, which are translationally activated under short-term ER stress when TC recycling is attenuated by eIF2α phosphorylation. During acute nutrient or growth factor stimulation, mTORC1 induces eIF2β phosphorylation, which increases recruitment of NCK1 to eIF2, decreases eIF2α phosphorylation and bolsters TC recycling. Accordingly, eIF2β appears to act as a previously unidentified mediator of mTORC1 on protein synthesis and proliferation. In addition, we demonstrate a formerly undocumented role for CK2 in regulation of translation initiation, whereby CK2 stimulates phosphorylation of eIF2β and simultaneously bolsters eIF4F complex assembly via the mTORC1/4E-BP pathway. These findings imply a previously unrecognized mode of translation regulation whereby mTORC1 and CK2 coordinate TC and eIF4F complex assembly to stimulate cell proliferation. Overall design: Comparisons of polysome-associated and cytoplasmic mRNA from starved cells or cells stimulated with insulin in the presence or absence of Torin-1.
Project description:Inhibition of deregulated protein kinases by small molecule drugs has evolved into a major therapeutic strategy for the treatment of human malignancies. Imatinib mesylate has emerged as the leading compound to treat chronic myeloid leukemia (CML), through its inhibition of Bcr- Abl tyrosine kinases, and other cancers. However, resistance to imatinib develops frequently, particularly in late-stage disease and has necessitated the development of new BCR-ABL inhibitors. The synthesis of a new series of phenylaminopyrimidines, structurally related to imatinib showed large interest since the introduction of the nilotibin. To identify the cellular pathways affected by new synthesized compounds, we applied mass spectrometry together with stable isotope labeling by amino acids in cell culture (SILAC) for the comparative study of protein expression in K562 cells that were untreated or treated with imatinib and imatinib derivates. Further, the global proteome of the K562 cells treated with imatinib were quantitatively compared with the cells treated with the new compounds. This study enriched our knowledge about direct cellular targets of kinase selective drugs. Further the results offered important new knowledge for gaining insights into the structural effects of action of the new compounds. Samples were analyzed on a longer column (30cm) and a longer gradient (180min). Raw data files were processed with Mascot distiller 2.3. The mgf files were searched with Mascot daemon 2.3. The quantification was also done by Mascot Distiller. All data was stored in ms_lims. The manual validation of false peptide ratios was done with Rover (part of ms_lims). Fixed modifications: none. Variable modifications: acetylation of peptide N-terminus, pyroglutamate formation of N-terminal glutamine, methionine oxidation. Enzyme: trypsine with one missed cleavage allowed. Precursor mass tolerance: 10 ppm. Peptide fragment mass tolerance: 0.5 Da Quantitation method: SILAC arginine and lysine +6 Da. Overview of the 17 different analyses: B SK23 vs DMSO C Y22 vs DMSO D SK20 vs DMSO E Y18 vs DMSO I SK20 vs DMSO K Y18 vs DMSO O Y22 vs DMSO R Imatinib vs Water Z Imatinib vs Water J SK20 vs Imatinib M SK23 vs Imatinib N Y22 vs Imatinib P SK23 vs Imatinib Q Y18 vs Imatinib S Y22 vs Imatinib T SK20 vs Imatinib Y Y18 vs Imatinib
Project description:Microarray analysis in the mouse metastatic tumor after ɣ-irradiation(ɣ-IR): non-irradiated primary tumor vs. radiated primary tumor vs. metastatic tumor after ɣ-irradiation Metastatic tumors in C6-L (rat glioma cells ) xenografted mice were studied after local treatment with fractionated γ-IR. To accurately detect the metastatic nodules after γ-IR, we observed the effect of γ-IR on distant metastatic tumor growth. Metastatic nodules after γ-IR indicated extensive colonization of C6-L cells in the lungs within 6 weeks after γ-IR. Identified and described the molecular events occurring after γ-IR through gene expression profiling to elucidate genetic changes (differentially expressed genes between the γ-IR primary tumors vs. non-γ-IR primary tumors and metastatic lung nodules vs. γ-IR primary tumors). We investigated the change of gene expression profile in the γ-IR primary tumors vs. non-γ-IR primary tumors and metastatic lung nodules vs. γ-IR primary tumors in rat glioma (C6-L cell) xenograft model.
Project description:Amino acid availability regulates translation through the action of the GCN2 and mTORC1 pathways. Low amino acids activate the eIF2α kinase GCN2 through binding of uncharged tRNAs to a histidyl-tRNA synthetase−related regulatory domain. Once activated GCN2 phosphorylates eIF2α, inhibiting ternary complex formation and translation initiation. Recent studies show that mTORC1 is particularly sensitive to arginine and leucine status, with a deprivation of these amino acids leading to a strong inhibition of mTORC1 that prevents the phosphorylation and inactivation of the translational repressor 4EBP1. Though amino acids are known regulators of translation, the effects that deficiencies of specific amino acids have on translation have yet to be determined. We demonstrate that deprivation of leucine or methionine results in large inhibitory effects on translation initiation and on polysome formation that are not replicated by overexpressing non-phosphorylatable 4EBP1 or a phosphomimetic eIF2α. Our results demonstrate that a lack of either leucine or methionine has a major impact on mRNA translation, though they act by quite different mechanisms. Leucine deprivation appears to primarily inhibit ribosome loading, whereas methionine deprivation appears to primarily impair start site recognition. These data point to a unique regulatory effect that methionine status has on translation initiation. Overall design: Ribosome profiling of single Amino acid starvation
Project description:The mTOR-allosteric inhibitor, RAD001, in combination with a PI3K/mTOR ATP-site competitive inhibitor, BEZ235, causes gene reprogramming, autophagy and tumor regression, in a mouse model approximating human HCC with poor prognosis, leading to an investigator Phase 1B-2 clinical trial. Comparative study of total RNA obtained from normal and tumor liver tissue under RAD001, BEZ235, or RAD001 + BEZ235.
Project description:The mTOR-allosteric inhibitor, RAD001, in combination with a PI3K/mTOR ATP-site competitive inhibitor, BEZ235, causes gene reprogramming, autophagy and tumor regression, in a mouse model approximating human HCC with poor prognosis, leading to an investigator Phase 1B-2 clinical trial. Overall design: Comparative study of total RNA obtained from normal and tumor liver tissue under RAD001, BEZ235, or RAD001 + BEZ235.
Project description:Reduced cancer incidence has been reported among type II diabetics treated with metformin. Metformin exhibits anti-proliferative and anti-neoplastic effects associated with inhibition of mTORC1, but the mechanisms are poorly understood. We provide the first genome-wide analysis of translational targets of canonical mTOR inhibitors (rapamycin and PP242) and metformin, revealing that metformin controls gene expression at the level of mRNA translation to an extent comparable to that of canonical mTOR inhibitors. Importantly, metformin's anti-proliferative activity can be explained by selective translational suppression of mRNAs encoding cell cycle regulators via the mTORC1/4E-BP pathway. Thus, metformin selectively inhibits mRNA translation of encoded proteins that promote neoplastic proliferation, motivating further studies of this compound and related biguanides in cancer prevention and treatment. MCF7 cells were treated with rapamycin, metformin or PP242 at concentrations that inhibited proliferation to 50% of control. Both cytoplasmic and polysome-associated mRNA was extracted from treatments and a vehicle treated control and probed with microarrays.