Activation Of mTORC1 Signaling Research Articles

Metabolic support by macrophages sustains colonic epithelial homeostasis

The intestinal epithelium has a high turnover rate and constantly renews itself through proliferation of intestinal crypt cells, which depends on insufficiently characterized signals from the microenvironment. Here, we showed that colonic macrophages were located directly adjacent to epithelial crypt cells in mice, where they metabolically supported epithelial cell proliferation in an mTORC1-dependent manner. Specifically, deletion of tuberous sclerosis complex 2 (Tsc2) in macrophages activated mTORC1 signaling that protected against colitis-induced intestinal damage and induced the synthesis of the polyamines spermidine and spermine. Epithelial cells ingested these polyamines and rewired their cellular metabolism to optimize proliferation and defense. Notably, spermine directly stimulated proliferation of colon epithelial cells and colon organoids. Genetic interference with polyamine production in macrophages altered global polyamine levels in the colon and modified epithelial cell proliferation. Our results suggest that macrophages act as "commensals" that provide metabolic support to promote efficient self-renewal of the colon epithelium.

Long Noncoding RNA Gpr137b-ps Promotes Advanced Atherosclerosis via the Regulation of Autophagy in Macrophages.

Current therapies cannot completely reverse advanced atherosclerosis. High levels of amino acids, induced by Western diet, stimulate mTORC1 (mammalian target of rapamycin complex 1)-autophagy defects in macrophages, accelerating atherosclerotic plaque progression. In addition, autophagy-lysosomal dysfunction contributes to plaque necrotic core enlargement and lipid accumulation. Therefore, it is essential to investigate the novel mechanism and molecules to reverse amino acid-mTORC1-autophagy signaling dysfunction in macrophages of patients with advanced atherosclerosis. We observed that Gpr137b-ps (G-protein-coupled receptor 137B, pseudogene) was upregulated in advanced atherosclerotic plaques. The effect of Gpr137b-ps on the progression of atherosclerosis was studied by generating advanced plaques in ApoE-/- mice with cardiac-specific knockout of Gpr137b-ps. Bone marrow-derived macrophages and mouse mononuclear macrophage cell line RAW264.7 cells were subjected to starvation or amino acid stimulation to study amino acid-mTORC1-autophagy signaling. Using both gain- and loss-of-function approaches, we explored the mechanism of Gpr137b-ps-regulated autophagy. Our results demonstrated that Gpr137b-ps deficiency led to enhanced autophagy in macrophages and reduced atherosclerotic lesions, characterized by fewer necrotic cores and less lipid accumulation. Knockdown of Gpr137b-ps increased autophagy and prevented amino acid-induced mTORC1 signaling activation. As the downstream binding protein of Gpr137b-ps, HSC70 (heat shock cognate 70) rescued the impaired autophagy induced by Gpr137b-ps. Furthermore, Gpr137b-ps interfered with the HSC70 binding to G3BP (Ras GTPase-activating protein-binding protein), which tethers the TSC (tuberous sclerosis complex) complex to lysosomes and suppresses mTORC1 signaling. In addition to verifying that the NTF2 (nuclear transport factor 2) domain of G3BP binds to HSC70 by in vitro protein synthesis, we further demonstrated that HSC70 binds to the NTF2 domain of G3BP through its W90-F92 motif by using computational modeling. These findings reveal that Gpr137b-ps plays an essential role in the regulation of macrophage autophagy, which is crucial for the progression of advanced atherosclerosis. Gpr137b-ps impairs the interaction of HSC70 with G3BP to regulate amino acid-mTORC1-autophagy signaling, and these results provide a new potential therapeutic direction for the treatment of advanced atherosclerosis.

Acute effect of electrical stimulation on muscle protein synthesis and break-down in the soleus muscle of hindlimb unloaded rats.

Electrical stimulation (ES) is effective for disuse-induced muscle atrophy. However, the acute effect of ES on muscle protein synthesis (MPS) and muscle protein breakdown (MPB) remains unclear. We investigated the effect of a single-session ES treatment on mTORC1 signaling, MPS, and MPB in the soleus muscle of 2-week hindlimb unloaded rats. Sprague Dawley rats (n = 12 male) were randomly divided into control (CON) and hindlimb unloaded (HU) groups. After 2 weeks, the right soleus muscle was percutaneously stimulated and underwent supramaximal isometric contractions. The left soleus muscle served as an internal control. We collected soleus muscle samples 6 h after ES. Two weeks of HU decreased p70S6K and S6rp activation, downstream factors for mTORC1 signaling, and SUnSET method-assessed MPS, but increased the LC3-II/I ratio, an indicator of autophagy. ES on disused muscle successfully activated mTORC1 signaling but did not affect MPS. Contrary, ES decreased ubiquitinated proteins expression and LC3B-II/I ratio. HU might affect mTORC1 activation and MPS differently in response to acute ES possibly due to excessive ROS production caused by ES. Our findings suggest that ES applied to disused skeletal muscles may suppress MPB, but its effect on MPS appears to be attenuated.

An mTORC1-Dependent Mouse Model for Cardiac Sarcoidosis.

Background Sarcoidosis is an inflammatory, granulomatous disease of unknown cause affecting multiple organs, including the heart. Untreated, unresolved granulomatous inflammation can lead to cardiac fibrosis, arrhythmias, and eventually heart failure. Here we characterize the cardiac phenotype of mice with chronic activation of mammalian target of rapamycin (mTOR) complex 1 signaling in myeloid cells known to cause spontaneous pulmonary sarcoid-like granulomas. Methods and Results The cardiac phenotype of mice with conditional deletion of the tuberous sclerosis 2 (TSC2) gene in CD11c+ cells (TSC2fl/flCD11c-Cre; termed TSC2KO) and controls (TSC2fl/fl) was determined by histological and immunological stains. Transthoracic echocardiography and invasive hemodynamic measurements were performed to assess myocardial function. TSC2KO animals were treated with either everolimus, an mTOR inhibitor, or Bay11-7082, a nuclear factor-kB inhibitor. Activation of mTOR signaling was evaluated on myocardial samples from sudden cardiac death victims with a postmortem diagnosis of cardiac sarcoidosis. Chronic activation of mTORC1 signaling in CD11c+ cells was sufficient to initiate progressive accumulation of granulomatous infiltrates in the heart, which was associated with increased fibrosis, impaired cardiac function, decreased plakoglobin expression, and abnormal connexin 43 distribution, a substrate for life-threatening arrhythmias. Mice treated with the mTOR inhibitor everolimus resolved granulomatous infiltrates, prevented fibrosis, and improved cardiac dysfunction. In line, activation of mTOR signaling in CD68+ macrophages was detected in the hearts of sudden cardiac death victims who suffered from cardiac sarcoidosis. Conclusions To our best knowledge this is the first animal model of cardiac sarcoidosis that recapitulates major pathological hallmarks of human disease. mTOR inhibition may be a therapeutic option for patients with cardiac sarcoidosis.

Antidepressant-like effects of tomatidine and tomatine, steroidal alkaloids from unripe tomatoes, via activation of mTORC1 in the medial prefrontal cortex in lipopolysaccharide-induced depression model mice

ABSTRACT Ketamine, an N-methyl-D-aspartate receptor antagonist, produces rapid antidepressant effects in patients with treatment-resistant depression. However, owing to the undesirable adverse effects of ketamine, there is an urgent need for developing safer and more effective prophylactic and therapeutic interventions for depression. Preclinical studies have demonstrated that activation of the mechanistic target of rapamycin complex 1 (mTORC1) in the medial prefrontal cortex (mPFC) mediates the rapid antidepressant effects of ketamine. The steroidal alkaloid tomatidine and its glycoside α-tomatine (tomatine) can activate mTORC1 signaling in peripheral tissues/cells. We examined whether tomatidine and tomatine exerted prophylactic and therapeutic antidepressant-like actions via mPFC mTORC1 activation using a mouse model of lipopolysaccharide (LPS)-induced depression. Male mice were intraperitoneally (i.p.) administered tomatidine/tomatine before and after the LPS challenge to test their prophylactic and therapeutic effects, respectively. LPS-induced depression-like behaviors in the tail suspension test (TST) and forced swim test (FST) were significantly reversed by prophylactic and therapeutic tomatidine/tomatine administration. LPS-induced anhedonia in the female urine sniffing test was reversed by prophylactic, but not therapeutic, injection of tomatidine, and by prophylactic and therapeutic administration of tomatine. Intra-mPFC infusion of rapamycin, an mTORC1 inhibitor, blocked the prophylactic and therapeutic antidepressant-like effects of tomatidine/tomatine in TST and FST. Moreover, both tomatidine and tomatine produced antidepressant-like effects in ovariectomized female mice, a model of menopause-associated depression. These results indicate that tomatidine and tomatine exert prophylactic and therapeutic antidepressant-like effects via mTORC1 activation in the mPFC and suggest these compounds as promising candidates for novel prophylactic and therapeutic agents for depression.

Maternal sodium acetate supplementation promotes lactation performance of sows and their offspring growth performance.

Milk yield and composition are critical determining factors for the early growth and development of neonates. The objective of this experiment was to comprehensively evaluate the effects of dietary sodium acetate (SA) supplementation on the milk yield and composition of sows and the growth performance of their offspring. A total of 80 sows (Landrace×Yorkshire, 3 to 6 parity) were randomly assigned to 2 groups (with or without 0.1% SA) from d 85 of gestation to d 21 of lactation. The result shows that maternal 0.1% SA supplementation significantly increased sows milk yield, milk fat, immunoglobulin A (IgA) and IgG content in milk (P<0.05), with the up-regulation of short-chain fatty acids receptors (GPR41 and GPR43) expression and the activation of mammalian target of rapamycin complex C1 (mTORC1) signaling pathway. Consistently, in our invitro experiment, SA also activated mTORC1 signaling in porcine mammary epithelial cells (P<0.05). Furthermore, the improvement of milk quality and quantity caused by maternal SA supplementation led to the increase in body weight (BW) and average daily weight gain (ADG) of weaning piglets, with the improvement of gut health and colonization of the beneficial bacteria (P<0.05). In conclusion, maternal supplementation of 0.1% SA improved the lactation performance (milk yield and milk fat) of sows, possibly with the activation of GPR41/GPR43-mTORC1 signaling. Furthermore, enhanced milk quality improved growth performance, gut health and the colonization of beneficial microbial flora of their piglets.

Whole red paprika (Capsicum annuum L.) and its orange-red pigment capsanthin ameliorate obesity-induced skeletal muscle atrophy in mice

Red paprika (RP) is rich in carotenoids and capsanthin (CS) is its main carotenoid. We determined whether whole RP and CS can ameliorate obesogenic sarcopenia. C2C12 myotubes were co-treated with 500 μM of palmitic acid (PA) and 1 and 5 μg/mL RP or 0.5 and 1.0 μM CS for 24 h. Both RP and CS effectively attenuated PA-induced myotube atrophy. Male obese C57BL/6 mice (n = 40) were fed high-fat diets containing 5 and 10% RP or 0.025% CS for 10 weeks. Supplementation with RP and CS increased lean body mass, muscle performance, mtDNA content, and mitochondrial oxidative phosphorylation, while it decreased intracellular lipid accumulation in muscle tissue. Moreover, both RP and CS increased myosin heavy chains and decreased atrogens expression, which are associated with the activation of mTORC1 signaling. These results demonstrated that RP and CS protect against obesogenic sarcopenia by improving mitochondrial function.

MTORC1 Signaling and Negative Lens-Induced Axial Elongation.

The mechanism underlying axial elongation during myopia progression remains unknown. Epidermal growth factor receptor (EGFR) signaling is associated with axial elongation. We explored whether mammalian target of rapamycin complex 1 (mTORC1) signaling acts as the downstream pathway of EGFR and participates in negative lens-induced axial elongation (NLIAE). Three-week-old male pigmented guinea pigs underwent binocular NLIAE. (1) To investigate whether EGFR is the upstream regulator of mTORC1, an EGFR inhibitor (20µg erlotinib) was intravitreally injected once a week for three weeks. (2) To assess the effect of mTORC1 inhibition on NLIAE, an mTORC1 inhibitor (2µg, 10µg, and 20µg everolimus) was intravitreally injected once a week for three weeks. (3) To explore the long-term effect of mTORC1 overactivation on axial elongation, an mTORC1 agonist (4µg MHY1485) was intravitreally injected once a week for three months. Biometric measurements included axial length and choroidal thickness were performed. Compared with the guinea pigs without NLIAE, NLIAE was associated with activation of mTORC1 signaling, which was suppressed by intravitreal erlotinib injection. Intravitreally injected everolimus suppressed NLIAE-induced axial elongation, mTORC1 activation, choroidal thinning, and hypoxia-inducible factor-1α expression in the sclera. Immunofluorescence revealed that the retinal pigment epithelium was the primary location of mTORC1 activation during NLIAE. Combining NLIAE and MHY1485 intravitreal injections significantly promoted axial elongation, choroidal thinning, and peripapillary choroidal atrophy. The mTORC1 signaling is associated with increased axial elongation, as in NLIAE, raising the possibility of inhibiting mTORC1 as a novel treatment for slowing myopia progression.

Leucine and arginine enhance milk fat and milk protein synthesis via the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 pathways.

Amino acids (AAs) not only constitute milk protein but also stimulate milk synthesis through the activation of mTORC1 signaling, but which amino acids that have the greatest impact on milk fat and protein synthesis is still very limited. In this study, we aimed to identify the most critical AAs involved in the regulation of milk synthesis and clarify how these AAs regulate milk synthesis through the G-protein-coupled receptors (GPCRs) signaling pathway. In this study, a mouse mammary epithelial cell line (HC11) and porcine mammary epithelial cells (PMECs) were selected as study subjects. After treatment with different AAs, the amount of milk protein and milk fat synthesis were detected. Activation of mTORC1 and GPCRs signaling induced by AAs was also investigated. In this study, we demonstrate that essential amino acids (EAAs) are crucial to promote lactation by increasing the expression of genes and proteins related to milk synthesis, such as ACACA, FABP4, DGAT1, SREBP1, α-casein, β-casein, and WAP in HC11 cells and PMECs. In addition to activating mTORC1, EAAs uniquely regulate the expression of calcium-sensing receptor (CaSR) among all amino-acid-responsive GPCRs, which indicates a potential link between CaSR and the mTORC1 pathway in mammary gland epithelial cells. Compared with other EAAs, leucine and arginine had the greatest capacity to trigger GPCRs (p-ERK) and mTORC1 (p-S6K1) signaling in HC11 cells. In addition, CaSR and its downstream G proteins Gi, Gq, and Gβγ are involved in the regulation of leucine- and arginine-induced milk synthesis and mTORC1 activation. Taken together, our data suggest that leucine and arginine can efficiently trigger milk synthesis through the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 pathways. We found that the G-protein-coupled receptor CaSR is an important amino acid sensor in mammary epithelial cells. Leucine and arginine promote milk synthesis partially through the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 signaling systems in mammary gland epithelial cells. Although this mechanism needs further verification, it is foreseeable that this mechanism may provide new insights into the regulation of milk synthesis.

β-catenin/CBP activation of mTORC1 signaling promotes partial epithelial-mesenchymal states in head and neck cancer

Head and neck cancers, which include oral squamous cell carcinoma (OSCC) as a major subsite, exhibit cellular plasticity that includes features of an epithelial-mesenchymal transition (EMT), referred to as partial-EMT (p-EMT). To identify molecular mechanisms contributing to OSCC plasticity, we performed a multiphase analysis of single cell RNA sequencing (scRNAseq) data from human OSCC. This included a multiresolution characterization of cancer cell subgroups to identify pathways and cell states that are heterogeneously represented, followed by casual inference analysis to elucidate activating and inhibitory relationships between these pathways and cell states. This approach revealed signaling networks associated with hierarchical cell state transitions, which notably included an association between β-catenin-driven CREB-binding protein (CBP) activity and mTORC1 signaling. This network was associated with subpopulations of cancer cells that were enriched for markers of the p-EMT state and poor patient survival. Functional analyses revealed that β-catenin/CBP induced mTORC1 activity in part through the transcriptional regulation of a raptor-interacting protein, chaperonin containing TCP1 subunit 5 (CCT5). Inhibition of β-catenin-CBP activity through the use of the orally active small molecule, E7386, reduced the expression of CCT5 and mTORC1 activity in vitro, and inhibited p-EMT-associated markers and tumor development in a murine model of OSCC. Our study highlights the use of multiresolution network analyses of scRNAseq data to identify targetable signals for therapeutic benefit, thus defining an underappreciated association between β-catenin/CBP and mTORC1 signaling in head and neck cancer plasticity.

Effects of Leucine Ingestion and Contraction on the Sestrin/GATOR2 Pathway and mTORC1 Activation in Rat Fast-Twitch muscle

BackgroundLeucine activates the mechanistic/mammalian target of rapamycin complex 1 (mTORC1) in mammalian skeletal muscle. Recent studies have shown that Sestrin, a leucine sensor, might play a role in this process. However, it remains unknown whether Sestrin dissociates from GATOR2 in a dose- and time-dependent manner and whether an acute bout of muscle contraction augments this dissociation. ObjectiveThis study aimed to examine the effects of leucine ingestion and muscle contraction on the interaction between Sestrin1/2 and GATOR2 and on mTORC1 activation. MethodsMale Wistar rats were randomly assigned to control (C), leucine 3 (L3), or leucine 10 (L10) groups. Intact gastrocnemius muscles were subjected to 30 repetitive unilateral contractions. The L3 and L10 groups were then orally administered 3 and 10 mmol/kg body weight of L-leucine 2 h after the end of the contractions, respectively. Blood and muscle samples were collected 30, 60, or 120 min after the administration. ResultsThe blood and muscle leucine concentrations increased in a dose-dependent manner. The ratio of phosphorylated ribosomal protein S6 kinase (S6K) to total S6K (which indicates mTORC1 signaling activation) was markedly increased by muscle contraction and increased in a dose-dependent manner only in rested muscle. Leucine ingestion but not muscle contraction increased Sestrin1 dissociation from GATOR2 and Sestrin2 association with GATOR2. A negative relationship was observed between the blood and muscle leucine concentrations and the Sestrin1 association with GATOR2. ConclusionsThe results suggest that Sestrin1, but not Sestrin2, regulates leucine-related mTORC1 activation via its dissociation from GATOR2 and that acute exercise-induced mTORC1 activation involves pathways other than the leucine-related Sestrin1/GATOR2 pathway.

Time-dependent effects of endogenous hyperglucagonemia on glucose homeostasis and hepatic glucagon action.

Elevation of glucagon levels and increase in α cell proliferation is associated with states of hyperglycemia in diabetes. A better understanding of the molecular mechanisms governing glucagon secretion could have major implications for understanding abnormal responses to hypoglycemia in patients with diabetes and provide novel avenues for diabetes management. Using mice with inducible induction of Rheb1 in α cells (αRhebTg mice), we showed that short-term activation of mTORC1 signaling is sufficient to induce hyperglucagonemia through increased glucagon secretion. Hyperglucagonemia in αRhebTg mice was also associated with an increase in α cell size and mass expansion. This model allowed us to identify the effects of chronic and short-term hyperglucagonemia on glucose homeostasis by regulating glucagon signaling in the liver. Short-term hyperglucagonemia impaired glucose tolerance, which was reversible over time. Liver glucagon resistance in αRhebTg mice was associated with reduced expression of the glucagon receptor and genes involved in gluconeogenesis, amino acid metabolism, and urea production. However, only genes regulating gluconeogenesis returned to baseline upon improvement of glycemia. Overall, these studies demonstrate that hyperglucagonemia exerts a biphasic response on glucose metabolism: Short-term hyperglucagonemia lead to glucose intolerance, whereas chronic exposure to glucagon reduced hepatic glucagon action and improved glucose tolerance.

The duration of glucocorticoid treatment alters the anabolic response to high-force muscle contractions.

Glucocorticoids induce a myopathy that includes loss of muscle mass and strength. Resistance exercise may reverse the muscle loss because it induces an anabolic response characterized by increases in muscle protein synthesis and potentially suppressing protein breakdown. Whether resistance exercise induces an anabolic response in glucocorticoid myopathic muscle is unknown, which is a problem because long-term glucocorticoid exposure alters the expression of genes that may prevent an anabolic response by limiting activation of pathways such as the mechanistic target of rapamycin in complex 1 (mTORC1). The purpose of this study was to assess whether high-force contractions initiate an anabolic response in glucocorticoid myopathic muscle. The anabolic response was analyzed by treating female mice with dexamethasone (DEX) for 7 days or 15 days. After treatment, the left tibialis anterior muscle of all mice was contracted via electrical stimulation of the sciatic nerve. Muscles were harvested 4 h after contractions. Rates of muscle protein synthesis were estimated using the SUnSET method. After 7 days of treatment, high-force contractions increased protein synthesis and mTORC1 signaling in both groups. After 15 days of treatment, high-force contractions activated mTORC1 signaling equally in both groups, but protein synthesis was only increased in control mice. The failure to increase protein synthesis may be because baseline synthetic rates were elevated in DEX-treated mice. The LC3 II/I ratio marker of autophagy was decreased by contractions regardless of treatment duration. These data show duration of glucocorticoid treatment alters the anabolic response to high-force contractions.NEW & NOTEWORTHY Glucocorticoid myopathy is the most common, toxic, noninflammatory myopathy. Our work shows that high-force contractions increase protein synthesis in skeletal muscle following short-term glucocorticoid treatment. However, longer duration glucocorticoid treatment results in anabolic resistance to high-force contractions despite activation of the mechanistic target of rapamycin in complex 1 (mTORC1) signaling pathway. This work defines potential limits for high-force contractions to activate the processes that would restore lost muscle mass in glucocorticoid myopathic patients.

Yeast UPS1 deficiency leads to UVC radiation sensitivity and shortened lifespan.

UPS1/YLR193C of Saccharomyces cerevisiae (S. cerevisiae) encodes a mitochondrial intermembrane space protein. A previous study found that Ups1p is needed for normal mitochondrial morphology and that UPS1 deficiency disrupts the intramitochondrial transport of phosphatidic acid in yeast cells and leads to an altered unfolded protein response and mTORC1 signaling activation. In this paper, we first provide evidence showing that the UPS1 gene is involved in the UVC-induced DNA damage response and aging. We show that UPS1 deficiency leads to sensitivity to ultraviolet C (UVC) radiation and that this effect is accompanied by elevated DNA damage, increased intracellular ROS levels, abnormal mitochondrial respiratoryfunction, an increased early apoptosis rate, and shortened replicative lifespan and chronological lifespan. Moreover, we show that overexpression of the DNA damage-induced checkpoint gene RAD9 effectivelyeliminates the senescence-related defects observed in the UPS1-deficient strain. Collectively, these results suggest a novel role for UPS1 in the UVC-induced DNA damage response and aging.

Correction to: Downregulation of LAPTM4B Contributes to the Impairment of the Autophagic Flux via Unopposed Activation of mTORC1 Signaling During Myocardial Ischemia/Reperfusion Injury.

HomeCirculation ResearchVol. 132, No. 5Correction to: Downregulation of LAPTM4B Contributes to the Impairment of the Autophagic Flux via Unopposed Activation of mTORC1 Signaling During Myocardial Ischemia/Reperfusion Injury Free AccessCorrectionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessCorrectionPDF/EPUBCorrection to: Downregulation of LAPTM4B Contributes to the Impairment of the Autophagic Flux via Unopposed Activation of mTORC1 Signaling During Myocardial Ischemia/Reperfusion Injury Originally published2 Mar 2023https://doi.org/10.1161/RES.0000000000000597Circulation Research. 2023;132:e94This article corrects the followingDownregulation of LAPTM4B Contributes to the Impairment of the Autophagic Flux via Unopposed Activation of mTORC1 Signaling During Myocardial Ischemia/Reperfusion InjuryIn the article by Gu et al. entitled “Downregulation of LAPTM4B Contributes to the Impairment of the Autophagic Flux via Unopposed Activation of mTORC1 Signaling During Myocardial Ischemia/Reperfusion Injury,” which was published in the September 11, 2020 issue of the journal (Circ Res. 2020;127:e148–e165. doi: 10.1161/CIRCRESAHA.119.316388), corrections were needed.After concerns were raised in a PubPeer post about potential included duplicated images in image figures and blots, the authors thoroughly checked the data and provided an explanation. For Supplemental Figure IV-I, the WGA image in WT was accidentally presented as a ‘LAPTM4B-/-’ image during figure preparation. For Figure 4C, the representative blot image of GAPDH for Figure 7B was inadvertently duplicated to Figure 4C during figure processing. The authors identified a similar accidental mistake of P62 blot image in Figure 7B.The authors have provided original images and uncropped original gel scan to the journal for correction of these mistakes and updated the Figure 4C, Figure 7B and Supplemental Figure IV-I.The authors apologize for these errors and state that the corrections do not affect the original conclusions.The corrected version of this article is available at http://www.ahajournals.org/doi/10.1161/CIRCRESAHA.119.316388. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesDownregulation of LAPTM4B Contributes to the Impairment of the Autophagic Flux via Unopposed Activation of mTORC1 Signaling During Myocardial Ischemia/Reperfusion InjuryShanshan Gu, et al. Circulation Research. 2020;127:e148-e165 March 3, 2023Vol 132, Issue 5 Advertisement Article InformationMetrics © 2023 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000597PMID: 36862816 Originally publishedMarch 2, 2023 PDF download Advertisement

Abstract P1-13-19: ESAM reduces anti-HER2 therapy sensitivity by activating mTOR pathway in HER2 positive breast cancer

Abstract Background: Trastuzumab combined with pertuzumab is the main therapy for HER2 positive breast cancer, but poor patient response due to drug resistance remains a clinical challenge. Organoids derived from tumor patients can highly maintain the heterogeneity of the original tumor, and have been used in drug sensitivity testing and new drug development. To screen for genes that associated with trastuzumab or pertuzumab drug resistance, this study used HER2-positive breast cancer organoids to establish a model of trastuzumab combined with pertuzumab and conducted high-throughput sequencing to screen the differential genes. We carried out further research to explore the effect of ESAM on drug sensitivity of HER2-targeted therapy and clarify the specific regulatory mechanisms and potential therapeutic targets of ESAM. Method: 8 HER2 positive breast cancer organoids were cultured. Immunohistochemistry, hematoxylin-eosin staining and Immunofluorescence were performed to identify the consistency between organoids and primitive tumors. According to drug sensitivity results of organoids to trastuzumab combined with pertuzumab, organoids were divided into a relatively sensitive group and a relatively insensitive group. Trastuzumab combined with pertuzumab was regularly used to stimulate the two groups. After 3 cycles, high-throughput transcriptome sequencing was used to evaluate their gene expression to find a gene responsible for trastuzumab and pertuzumab resistance. METABRIC and our center database were used to determine the prognostic value of ESAM. ESAM overexpression and knockdown stably transfected cell lines were constructed in SK-BR-3 and BT474 breast cancer cell lines by lentivirus. Colony formation assay, CCK-8 assay, organoid model and mouse xenograft model were conducted to examine the influence of ESAM on proliferation and trastuzumab or pertuzumab sensitization in vitro and in vivo. RNA-seq and GSEA analysis were performed to investigate the downstream pathways of ESAM. Western blot was used to confirm the relationship between ESAM and mTOR pathway. Small interfering RNA and mTOR/PI3K pathway inhibitors were used to confirm the function of mTOR in ESAM-induced drug resistance of trastuzumab. Results: HER2 positive breast cancer organoids can maintain the pathological characteristics of primitive tumors. The results of high-throughput transcriptome show that ESAM is significantly up-regulated in the relatively insensitive group compared to the relatively sensitive group. METABRIC and our center database suggest that high ESAM mRNA expression was associated with poor OS, RFS, DFS and PFS of HER2-positive breast cancer patients. CCK-8 and cell colony formation assay confirm that ESAM can promote the proliferation of HER2-positive breast cancer cells and inhibit the drug sensitivity of HER2-positive breast cancer cells to trastuzumab and pertuzumab. GSEA analysis shows high ESAM can activate mTORC1 signaling pathway. Western blotting analysis proves the expression of ESAM is positively correlated with mTOR pathway. Inhibition of mTOR pathway in ESAM overexpression cells can suppress ESAM-mediated proliferation and reverse the drug sensitivity of cells to trastuzumab and pertuzumab. In vivo, overexpression of ESAM can promote the proliferation of mouse mammary tumors and reduce the sensitivity of mouse mammary tumors to trastuzumab, combined use of PI3K inhibitor could reverse drug sensitivity of mouse mammary tumors to trastuzumab. Conclusion: High ESAM expression is associated with poor prognosis in patients with HER2 positive breast cancer. ESAM can activate mTOR pathway, promote cell proliferation and reduce the sensitivity of HER2 positive breast cancer cells to trastuzumab and pertuzumab, which can be inhibited by the application of PI3K/mTOR inhibitors. Keywords: ESAM; HER2 positive breast cancer; HER2 targeted therapy; mTOR; drug sensitivity. Citation Format: Jiong Wu, Xujie Zhou, Yayun Chi, Jingyan Xue. ESAM reduces anti-HER2 therapy sensitivity by activating mTOR pathway in HER2 positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-19.

Maternal intermittent fasting deteriorates offspring metabolism via suppression of hepatic mTORC1 signaling.

The metabolic benefits of intermittent fasting (IF) have been well recognized. However, limited studies have examined the relationship between long-term maternal IF before pregnancy and offspring health. In this study, a C57BL/6J mouse model of long-term IF before pregnancy was established: 4-week-old female mice were subjected to alternate-day fasting for 12 weeks and resumed normal diet after mating. Female mice in the control group were fed ad libitum. Offspring mice were weaned at 6 weeks of age and fed a normal chow diet or a 60% high-fat diet. The effects of long-term pre-pregnancy IF on offspring metabolism and its underlying mechanism were examined. We found that neonatal IF offspring weighted significantly less relevant to control mice. This difference gradually disappeared as a result of catch-up growth. In the IF offspring, adipose tissue mass was significantly increased. This alteration was associated with a considerable deterioration in glucose tolerance. No significant difference in food intake was observed. Further, lipid deposition as well as triglyceride contents in the liver were greatly increased. Maternal IF significantly decreased levels of DNA methyltransferase in the liver of offspring. DNA methylation modifications of molecules associated with the mTORC1 signaling pathway were significantly altered, leading to the significant inhibition of mTORC1 signaling. Overexpression of S6K1 activated hepatic mTORC1 signaling and reversed the metabolic dysfunction in IF offspring. In conclusion, long-term pre-pregnancy IF increases hepatic steatosis and adiposity, as well as impairs glucose metabolism in adult offspring. This occurs through DNA methylation-dependent suppression of hepatic mTORC1 signaling activity.

Targeting Glutaminolysis Shows Efficacy in Both Prednisolone-Sensitive and in Metabolically Rewired Prednisolone-Resistant B-Cell Childhood Acute Lymphoblastic Leukaemia Cells

The prognosis for patients with relapsed childhood acute lymphoblastic leukaemia (cALL) remains poor. The main reason for treatment failure is drug resistance, most commonly to glucocorticoids (GCs). The molecular differences between prednisolone-sensitive and -resistant lymphoblasts are not well-studied, thereby precluding the development of novel and targeted therapies. Therefore, the aim of this work was to elucidate at least some aspects of the molecular differences between matched pairs of GC-sensitive and -resistant cell lines. To address this, we carried out an integrated transcriptomic and metabolomic analysis, which revealed that lack of response to prednisolone may be underpinned by alterations in oxidative phosphorylation, glycolysis, amino acid, pyruvate and nucleotide biosynthesis, as well as activation of mTORC1 and MYC signalling, which are also known to control cell metabolism. In an attempt to explore the potential therapeutic effect of inhibiting one of the hits from our analysis, we targeted the glutamine-glutamate-α-ketoglutarate axis by three different strategies, all of which impaired mitochondrial respiration and ATP production and induced apoptosis. Thereby, we report that prednisolone resistance may be accompanied by considerable rewiring of transcriptional and biosynthesis programs. Among other druggable targets that were identified in this study, inhibition of glutamine metabolism presents a potential therapeutic approach in GC-sensitive, but more importantly, in GC-resistant cALL cells. Lastly, these findings may be clinically relevant in the context of relapse-in publicly available datasets, we found gene expression patterns suggesting that in vivo drug resistance is characterised by similar metabolic dysregulation to what we found in our in vitro model.

AMPK-dependent phosphorylation of the GATOR2 component WDR24 suppresses glucose-mediated mTORC1 activation.

The mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth in response to amino acid and glucose levels. However, how mTORC1 senses glucose availability to regulate various downstream signalling pathways remains largely elusive. Here we report that AMP-activated protein kinase (AMPK)-mediated phosphorylation of WDR24, a core component of the GATOR2 complex, has a role in the glucose-sensing capability of mTORC1. Mechanistically, glucose deprivation activates AMPK, which directly phosphorylates WDR24 on S155, subsequently disrupting the integrity of the GATOR2 complex to suppress mTORC1 activation. Phosphomimetic Wdr24S155D knock-in mice exhibit early embryonic lethality and reduced mTORC1 activity. On the other hand, compared to wild-type littermates, phospho-deficient Wdr24S155A knock-in mice are more resistant to fasting and display elevated mTORC1 activity. Our findings reveal that AMPK-mediated phosphorylation of WDR24 modulates glucose-induced mTORC1 activation, thereby providing a rationale for targeting AMPK-WDR24 signalling to fine-tune mTORC1 activation as a potential therapeutic means to combat human diseases with aberrant activation of mTORC1 signalling including cancer.

Niacin/β-hydroxybutyrate regulates milk fat and milk protein synthesis via the GPR109A/Gi/mTORC1 pathway.

As a crucial receptor of BHBA and niacin, GPR109A is largely expressed in the mammary gland. However, the role of GPR109A in milk synthesis and its underlying mechanism is still largely unknown. In this study, we first investigated the effect of GPR109A agonists (niacin/BHBA) on milk fat and milk protein synthesis in a mouse mammary epithelial cell line (HC11) and PMECs (porcine mammary epithelial cells). The results showed that both niacin and BHBA promote milk fat and milk protein synthesis with the activation of mTORC1 signaling. Importantly, knockdown GPR109A attenuated the niacin-induced increase of milk fat and protein synthesis and the niacin-induced activation of mTORC1 signaling. Furthermore, we found that GPR109A downstream G protein-Gαi and -Gβγ participated in the regulation of milk synthesis and the activation of mTORC1 signaling. Consistent with the finding in vitro, dietary supplementation with niacin increases milk fat and protein synthesis in mice with the activation of GPR109A-mTORC1 signaling. Collectively, GPR109A agonists promote the synthesis of milk fat and milk protein through the GPR109A/Gi/mTORC1 signaling pathway.