mTORC1 Activity Research Articles

Nup358 restricts ER-mitochondria connectivity by modulating mTORC2/Akt/GSK3β signalling

ER–mitochondria contact sites (ERMCSs) regulate processes, including calcium homoeostasis, energy metabolism and autophagy. Previously, it was shown that during growth factor signalling, mTORC2/Akt gets recruited to and stabilizes ERMCSs. Independent studies showed that GSK3β, a well-known Akt substrate, reduces ER–mitochondria connectivity by disrupting the VAPB–PTPIP51 tethering complex. However, the mechanisms that regulate ERMCSs are incompletely understood. Here we find that annulate lamellae (AL), relatively unexplored subdomains of ER enriched with a subset of nucleoporins, are present at ERMCSs. Depletion of Nup358, an AL-resident nucleoporin, results in enhanced mTORC2/Akt activation, GSK3β inhibition and increased ERMCSs. Depletion of Rictor, a mTORC2-specific subunit, or exogenous expression of GSK3β, was sufficient to reverse the ERMCS-phenotype in Nup358-deficient cells. We show that growth factor-mediated activation of mTORC2 requires the VAPB–PTPIP51 complex, whereas, Nup358’s association with this tether restricts mTORC2/Akt signalling and ER–mitochondria connectivity. Expression of a Nup358 fragment that is sufficient for interaction with the VAPB–PTPIP51 complex suppresses mTORC2/Akt activation and disrupts ERMCSs. Collectively, our study uncovers a novel role for Nup358 in controlling ERMCSs by modulating the mTORC2/Akt/GSK3β axis.

Cytoplasmic vacuolization and ectopic formation of perineuronal nets are characteristic pathologies of cytomegalic neurons in tuberous sclerosis.

Cytomegalic neurons, characterized by increased size and a hyperactive mechanistic target of rapamycin complex 1 (mTORC1), are pathognomonic for tuberous sclerosis complex (TSC). To model these neurons, we recently generated a murine Tsc1 conditional knockout model in which Tsc1 deletion in late embryonic radial glia results in neuronal hypertrophy of a subset of isocortical pyramidal neurons. In the current study, we compared the cellular pathology of these cytomegalic neurons to those of the enlarged neurons in human cortical tubers. Neurons from the mice showed unique features, such as cytoplasmic vacuoles associated with Golgi complexes and the ectopic formation of perineuronal nets (PNNs), a feature of inhibitory neurons, rarely present in excitatory cortical neurons. The membranes of these vacuoles were enriched for the plasma membrane proteins CD44, KCC2, and Na+/K+ ATPase, suggesting deficits in Golgi membrane trafficking. These aberrant features in the mouse appeared only after the onset of seizures, probably due to the prolonged seizure activity in the context of constitutive mTORC1 activation. Similar PNNs and cytoplasmic vacuoles were present in the cytomegalic neurons of human cortical tubers. Our findings reveal novel pathological features of Golgi complexes and PNNs in the cytomegalic neurons in TSC.

Metformin induces ZFP36 by mTORC1 inhibition in cervical cancer-derived cell lines

BackgroundMetformin, a widely prescribed antidiabetic drug, has shown several promising effects for cancer treatment. These effects have been shown to be mediated by dual modulation of the AMPK-mTORC1 axis, where AMPK acts upstream of mTORC1 to decrease its activity. Nevertheless, alternative pathways have been recently discovered suggesting that metformin can act through of different targets regulation.MethodsWe performed a transcriptome screening analysis using HeLa xenograft tumors generated in NOD-SCID mice treated with or without metformin to examine genes regulated by metformin. Western Blot analysis, Immunohistochemical staining, and RT-qPCR were used to confirm alterations in gene expression. The TNMplot and GEPIA2 platform were used for in silico analysis of genes found up-regulated by metformin, in cervical cancer patients. We performed an AMPK knock-down using AMPK-targeted siRNAs and mTOR inhibition with rapamycin to investigate the molecular mechanisms underlying the effect of metformin in cervical cancer cell lines.ResultsWe shown that metformin decreases tumor growth and increased the expression of a group of antitumoral genes involved in DNA-binding transcription activator activity, hormonal response, and Dcp1-Dcp2 mRNA-decapping complex. We demonstrated that ZFP36 could act as a new molecular target increased by metformin. mTORC1 inhibition using rapamycin induces ZFP36 expression, which could suggest that metformin increases ZFP36 expression and requires mTORC1 inhibition for such effect. Surprisingly, in HeLa cells AMPK inhibition did not affect ZFP36 expression, suggesting that additional signal transducers related to suppressing mTORC1 activity, could be involved.ConclusionsThese results highlight the importance of ZFP36 activation in response to metformin treatment involving mTORC1 inhibition.Graphical

The Spectrum of Renal "TFEopathies": Flipping the mTOR Switch in Renal Tumorigenesis.

The mammalian target of Rapamycin complex 1 (mTORC1) is a serine/threonine kinase that couples nutrient and growth factor signaling to the cellular control of metabolism and plays a fundamental role in aberrant proliferation in cancer. mTORC1 has previously been considered an "on/off" switch, capable of phosphorylating the entire pool of its substrates when activated. However, recent studies have indicated that mTORC1 may be active toward its canonical substrates, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and S6 kinase (S6K), involved in mRNA translation and protein synthesis, and inactive toward TFEB and TFE3, transcription factors involved in the regulation of lysosome biogenesis, in several pathological contexts. Among these conditions are Birt-Hogg-Dubé syndrome (BHD) and, recently, tuberous sclerosis complex (TSC). Furthermore, increased TFEB and TFE3 nuclear localization in these syndromes, and in translocation renal cell carcinomas (tRCC), drives mTORC1 activity toward the canonical substrates, through the transcriptional activation of the Rag GTPases, thereby positioning TFEB and TFE3 upstream of mTORC1 activity toward 4EBP1 and S6K. The expanding importance of TFEB and TFE3 in the pathogenesis of these renal diseases warrants a novel clinical grouping that we term "TFEopathies." Currently, there are no therapeutic options directly targeting TFEB and TFE3, which represents a challenging and critically required avenue for cancer research.

Loss of Tuberous Sclerosis Complex 2 confers inflammation via dysregulation of Nuclear factor kappa-light-chain-enhancer of activated B cells.

Aberrant activation of mTORC1 is clearly defined in TSC, causing uncontrolled cell growth. While mTORC1 inhibitors show efficacy to stabilise tumour growth in TSC, they are not fully curative. Disease facets of TSC that are not restored with mTOR inhibitors might involve NF-κB. The study aimed to characterise NF-κB in the context of TSC. Enrichment of NF-κB-regulated genes was observed in TSC patient tumours, SEN/SEGAs, cortical tubers and a TSC tumour-derived cell line (621 - 101). Highlighting an inflammatory component of TSC, TSC cell models showed an elevated level of NF-κB and STAT3 activation. Herein, we report a dysregulated inflammatory phenotype of TSC2-deficient cells where NF-κB promotes autocrine signalling involving IL-6. Of importance, mTORC1 inhibition does not block this inflammatory signal to promote STAT3, while NF-κB inhibition was much more effective. Combined mTORC1 and NF-κB inhibition was potent at preventing anchorage-independent growth of TSC2-deficient cells, and unlike mTORC1 inhibition alone was sufficient to prevent colony regrowth after cessation of treatment. This study reveals autocrine signalling crosstalk between NF-κB and STAT3 in TSC cell models. Furthermore, the data presented indicate that NF-κB pathway inhibitors could be a viable adjunct therapy with the current mTOR inhibitors to treat TSC.

Activation of nemo-like kinase in diamond blackfan anemia suppresses early erythropoiesis by preventing mitochondrial biogenesis

Diamond Blackfan Anemia (DBA) is a rare macrocytic red blood cell aplasia that usually presents within the first year of life. The vast majority of patients carry a mutation in one of approximately 20 genes that results in ribosomal insufficiency with the most significant clinical manifestations being anemia and a predisposition to cancers. Nemo-like Kinase (NLK) is hyperactivated in the erythroid progenitors of DBA patients and inhibition of this kinase improves erythropoiesis, but how NLK contributes to the pathogenesis of the disease is unknown. Here we report that activated NLK suppresses the critical upregulation of mitochondrial biogenesis required in early erythropoiesis. During normal erythropoiesis, mTORC1 facilitates the translational upregulation of Transcription factor A, mitochondrial (TFAM) and Prohibin 2 (PHB2) to increase mitochondrial biogenesis. In our models of DBA, active NLK phosphorylates the regulatory component of mTORC1, thereby suppressing mTORC1 activity and preventing mTORC1-mediated TFAM and PHB2 upregulation and subsequent mitochondrial biogenesis. Improvement of erythropoiesis that accompanies NLK inhibition is negated when TFAM and PHB2 upregulation is prevented. These data demonstrate that a significant contribution of NLK on the pathogenesis of DBA is through loss of mitochondrial biogenesis.

Erdafitinib promotes ferroptosis in human uveal melanoma by inducing ferritinophagy and lysosome biogenesis via modulating the FGFR1/mTORC1/TFEB signaling axis

Uveal melanoma (UM) is a rare yet lethal primary intraocular malignancy affecting adults. Analysis of data from The Cancer Genome Atlas (TCGA) database revealed that FGFR1 expression was increased in UM tumor tissues and was linked to aggressive behavior and a poor prognosis. This study assessed the anti-tumor effects of Erdafitinib, a selective pan-FGFR inhibitor, in both in vitro and in vivo UM models. Erdafitinib exhibited a robust anti-cancer activity in UM through inducing ferroptosis in the FGFR1-dependent manner. Transcriptomic data revealed that Erdafitinib mediated its anti-cancer effects via modulating the ferritinophagy/lysosome biogenesis. Subsequent research revealed that Erdafitinib exerted its effects by reducing the expression of FGFR1 and inhibiting the activity of mTORC1 in UM cells. Concurrently, it enhanced the dephosphorylation, nuclear translocation, and transcriptional activity of TFEB. The aggregation of TFEB in nucleus triggered FTH1-dependent ferritinophagy, leading to lysosomal activation and iron overload. Conversely, the overexpression of FGFR1 served to mitigate the effects of Erdafitinib on ferritinophagy, lysosome biogenesis, and the activation of the mTORC1/TFEB signaling pathway. In vivo experiments have convincingly shown that Erdafitinib markedly curtails tumor growth in an UM xenograft mouse model, an effect that is closely correlated with a decrease in FGFR1 expression levels. The present study is the first to demonstrate that Erdafitinib powerfully induces ferroptosis in UM by orchestrating the ferritinophagy and lysosome biogenesis via modulating the FGFR1/mTORC1/TFEB signaling. Consequently, Erdafitinib emerges as a strong candidate for clinical trial investigation, and FGFR1 emerges as a novel and promising therapeutic target in the treatment of UM.

TFEB safeguards trophoblast syncytialization in humans and mice

Nutrient sensing and adaptation in the placenta are essential for pregnancy viability and proper fetal growth. Our recent study demonstrated that the placenta adapts to nutrient insufficiency through mechanistic target of rapamycin (mTOR) inhibition-mediated trophoblast differentiation toward syncytiotrophoblasts (STBs), a highly specialized multinucleated trophoblast subtype mediating extensive maternal-fetal interactions. However, the underlying mechanism remains elusive. Here, we unravel the indispensable role of the mTORC1 downstream transcriptional factor TFEB in STB formation both in vitro and in vivo. TFEB deficiency significantly impaired STB differentiation in human trophoblasts and placenta organoids. Consistently, systemic or trophoblast-specific deletion of Tfeb compromised STB formation and placental vascular construction, leading to severe embryonic lethality. Mechanistically, TFEB conferred direct transcriptional activation of the fusogen ERVFRD-1 in human trophoblasts and thereby promoted STB formation, independent of its canonical function as a master regulator of the autophagy-lysosomal pathway. Moreover, we demonstrated that TFEB directed the trophoblast syncytialization response driven by mTOR complex 1 (mTORC1) signaling. TFEB expression positively correlated with the reinforced trophoblast syncytialization in human fetal growth-restricted placentas exhibiting suppressed mTORC1 activity. Our findings substantiate that the TFEB-fusogen axis ensures proper STB formation during placenta development and under nutrient stress, shedding light on TFEB as a mechanistic link between nutrient-sensing machinery and trophoblast differentiation.

Role of vaccinia virus growth factor in stimulating the mTORC1-CAD axis of the de novo pyrimidine pathway under different nutritional cues.

Vaccinia virus (VACV), the prototype poxvirus, actively reprograms host cell metabolism upon infection. However, the nature and molecular mechanisms remain largely elusive. Given the diverse nutritional exposures of cells in different physiological contexts, it is essential to understand how VACV may alter various metabolic pathways in different nutritional conditions. In this study, we established the importance of de novo pyrimidine biosynthesis in VACV infection. We elucidated the significance of vaccinia growth factor (VGF), a viral early protein and a homolog of cellular epidermal growth factor, in enabling VACV to phosphorylate the key enzyme CAD of the de novo pyrimidine pathway at serine 1859, a site known to positively regulate CAD activity. While nutrient-poor conditions typically inhibit mTORC1 activation, VACV activates CAD via mTORC1-S6K1 signaling axis, in conditions where glutamine and asparagine are absent. However, unlike its cellular homolog, epidermal growth factor (EGF), VGF peptide alone in the absence of VACV infection has minimal ability to activate CAD, suggestive of the involvement of other viral factor(s) and differential functions to EGF acquired during poxvirus evolution. Our research provides a foundation for understanding the regulation of a significant metabolic pathway, namely, de novo pyrimidine synthesis during VACV infection, shedding new light on viral regulation under distinct nutritional environments. This study not only has the potential to contribute to the advancement of antiviral treatments but also improve the development of VACV as an oncolytic agent and vaccine vector.

MTORC1-Driven Protein Translation Correlates with Clinical Benefit of Capivasertib within a Genetically Preselected Cohort of PIK3CA-Altered Tumors.

Capivasertib's first-in-class FDA approval demonstrates its promise, yet there remains an opportunity to optimize its use. Our results provide new evidence that proteomics can stratify genetically preselected patients on clinical benefit. Characterization of the same profile in cell lines furnishes additional validation. Among PIK3CA-altered tumors, increased mTORC1-driven translation appears to confer intrinsic resistance. Assessing mTORC1 activation could therefore prove a useful complement to the existing genetic selection strategy for capivasertib.

Resolvin E1 as a potential lead for the treatment of depression

Typical monoamine-based antidepressants have significant limitations, including a time lag for therapeutic response and low efficacy (more than one-third of depressed patients fail to respond to multiple antidepressant medications and are considered treatment-resistant). Conversely, ketamine, an N-methyl-D-aspartate receptor antagonist, exhibits rapid and sustained antidepressant actions in patients with treatment-resistant depression. However, clinical use of ketamine is limited due to its serious side effects. Thus, there is a significant need to develop novel ketamine-like antidepressants with fewer side effects. We previously demonstrated that intracerebroventricular infusion of resolvins (RvD1, RvD2, RvE1, RvE2, and RvE3), specialized pro-resolving lipid mediators derived from docosahexaenoic and eicosapentaenoic acids, produce antidepressant-like effects in mouse models of depression. Among resolvins, RvE1 produces the most potent antidepressant-like effects likely via ChemR23 in several mouse models of depression. Local infusion of RvE1 into the medial prefrontal cortex (mPFC) or dorsal hippocampal dentate gyrus (DG) also produces antidepressant-like effects, suggesting that these brain regions are sites of action of RvE1. Additionally, intranasal (i.n.) administration of RvE1 produces antidepressant-like effects through mechanisms similar to ketamine: activity-dependent release of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF), and subsequent mechanistic target of rapamycin complex 1 (mTORC1) activation in the mPFC play a crucial role in the rapid and sustained antidepressant-like actions of i.n. RvE1. Moreover, the antidepressant-like effects of i.n. RvE1 require BDNF and VEGF release, but not mTORC1 activation, in the dorsal DG. These findings suggest that RvE1 can be a promising lead for a novel rapid-acting antidepressant.

Reprogramming of Energy Metabolism in Human PKD1 Polycystic Kidney Disease: A Systems Biology Analysis.

Multiple alterations of cellular metabolism have been documented in experimental studies of autosomal dominant polycystic kidney disease (ADPKD) and are thought to contribute to its pathogenesis. To elucidate the molecular pathways and transcriptional regulators associated with the metabolic changes of renal cysts in ADPKD, we compared global gene expression data from human PKD1 renal cysts, minimally cystic tissues (MCT) from the same patients, and healthy human kidney cortical tissue samples. We found gene expression profiles of PKD1 renal cysts were consistent with the Warburg effect with gene pathway changes favoring increased cellular glucose uptake and lactate production, instead of pyruvate oxidation. Additionally, mitochondrial energy metabolism was globally depressed, associated with downregulation of gene pathways related to fatty acid oxidation (FAO), branched-chain amino acid (BCAA) degradation, the Krebs cycle, and oxidative phosphorylation (OXPHOS) in renal cysts. Activation of mTORC1 and its two target proto-oncogenes, HIF-1α and MYC, was predicted to drive the expression of multiple genes involved in the observed metabolic reprogramming (e.g., GLUT3, HK1/HK2, ALDOA, ENO2, PKM, LDHA/LDHB, MCT4, PDHA1, PDK1/3, MPC1/2, CPT2, BCAT1, NAMPT); indeed, their predicted expression patterns were confirmed by our data. Conversely, we found AMPK inhibition was predicted in renal cysts. AMPK inhibition was associated with decreased expression of PGC-1α, a transcriptional coactivator for transcription factors PPARα, ERRα, and ERRγ, all of which play a critical role in regulating oxidative metabolism and mitochondrial biogenesis. These data provide a comprehensive map of metabolic pathway reprogramming in ADPKD and highlight nodes of regulation that may serve as targets for therapeutic intervention.

IL-37d suppresses Rheb-mTORC1 axis independently of TCS2 to alleviate alcoholic liver disease

Tuberous sclerosis complex 2 (TSC2) crucially suppresses Rheb activity to prevent mTORC1 activation. However, mutations in TSC genes lead to mTORC1 overactivation, thereby causing various developmental disorders and cancer. Therefore, the discovery of novel Rheb inhibitors is vital to prevent mTOR overactivation. Here, we reveals that the anti-inflammatory cytokine IL-37d can bind to lysosomal Rheb and suppress its activity independent of TSC2, thereby preventing mTORC1 activation. The binding of IL-37d to Rheb switch-II subregion destabilizes the Rheb-mTOR and mTOR-S6K interactions, further halting mTORC1 signaling. Unlike TSC2, IL-37d is reduced under ethanol stimulation, which results in mitigating the suppression of lysosomal Rheb-mTORC1 activity. Consequently, the recombinant human IL-37d protein (rh-IL-37d) with a TAT peptide greatly improves alcohol-induced liver disorders by hindering Rheb-mTORC1 axis overactivation in a TSC2- independent manner. Together, IL-37d emerges as a novel Rheb suppressor independent of TSC2 to terminate mTORC1 activation and improve abnormal lipid metabolism in the liver.

Protein restriction slows the development and progression of pathology in a mouse model of Alzheimer’s disease

Dietary protein is a critical regulator of metabolic health and aging. Low protein diets are associated with healthy aging in humans, and dietary protein restriction extends the lifespan and healthspan of mice. In this study, we examined the effect of protein restriction (PR) on metabolic health and the development and progression of Alzheimer’s disease (AD) in the 3xTg mouse model of AD. Here, we show that PR promotes leanness and glycemic control in 3xTg mice, specifically rescuing the glucose intolerance of 3xTg females. PR induces sex-specific alterations in circulating and brain metabolites, downregulating sphingolipid subclasses in 3xTg females. PR also reduces AD pathology and mTORC1 activity, increases autophagy, and improves the cognition of 3xTg mice. Finally, PR improves the survival of 3xTg mice. Our results suggest that PR or pharmaceutical interventions that mimic the effects of this diet may hold promise as a treatment for AD.

Low potassium activation of proximal mTOR/AKT signaling is mediated by Kir4.2

The renal epithelium is sensitive to changes in blood potassium (K+). We identify the basolateral K+ channel, Kir4.2, as a mediator of the proximal tubule response to K+ deficiency. Mice lacking Kir4.2 have a compensated baseline phenotype whereby they increase their distal transport burden to maintain homeostasis. Upon dietary K+ depletion, knockout animals decompensate as evidenced by increased urinary K+ excretion and development of a proximal renal tubular acidosis. Potassium wasting is not proximal in origin but is caused by higher ENaC activity and depends upon increased distal sodium delivery. Three-dimensional imaging reveals Kir4.2 knockouts fail to undergo proximal tubule expansion, while the distal convoluted tubule response is exaggerated. AKT signaling mediates the dietary K+ response, which is blunted in Kir4.2 knockouts. Lastly, we demonstrate in isolated tubules that AKT phosphorylation in response to low K+ depends upon mTORC2 activation by secondary changes in Cl- transport. Data support a proximal role for cell Cl- which, as it does along the distal nephron, responds to K+ changes to activate kinase signaling.

Developing a Tanshinone IIA Memetic by Targeting MIOS to Regulate mTORC1 and Autophagy in Glioblastoma.

Tanshinone IIA (T2A) is a bioactive compound that provides promise in the treatment of glioblastoma multiforme (GBM), with a range of molecular mechanisms including the inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) and the induction of autophagy. Recently, T2A has been demonstrated to function through sestrin 2 (SESN) to inhibit mTORC1 activity, but its possible impact on autophagy through this pathway has not been investigated. Here, the model system Dictyostelium discoideum and GBM cell lines were employed to investigate the cellular role of T2A in regulating SESN to inhibit mTORC1 and activate autophagy through a GATOR2 component MIOS. In D. discoideum, T2A treatment induced autophagy and inhibited mTORC1 activity, with both effects lost upon the ablation of SESN (sesn-) or MIOS (mios-). We further investigated the targeting of MIOS to reproduce this effect of T2A, where computational analysis identified 25 novel compounds predicted to strongly bind the human MIOS protein, with one compound (MIOS inhibitor 3; Mi3) reducing cell proliferation in two GBM cells. Furthermore, Mi3 specificity was demonstrated through the loss of potency in the D. discoideum mios- cells regarding cell proliferation and the induction of autophagy. In GBM cells, Mi3 treatment also reduced mTORC1 activity and induced autophagy. Thus, a potential T2A mimetic showing the inhibition of mTORC1 and induction of autophagy in GBM cells was identified.

Alternative oxidase blunts pseudohypoxia and photoreceptor degeneration due to RPE mitochondrial dysfunction

Loss of mitochondrial electron transport complex (ETC) function in the retinal pigment epithelium (RPE) in vivo results in RPE dedifferentiation and progressive photoreceptor degeneration, and has been implicated in the pathogenesis of age-related macular degeneration. Xenogenic expression of alternative oxidases in mammalian cells and tissues mitigates phenotypes arising from some mitochondrial electron transport defects, but can exacerbate others. We expressed an alternative oxidase from Ciona intestinalis (AOX) in ETC-deficient murine RPE in vivo to assess the retinal consequences of stimulating coenzyme Q oxidation and respiration without ATP generation. RPE-restricted expression of AOX in this context is surprisingly beneficial. This focused intervention mitigates RPE mTORC1 activation, dedifferentiation, hypertrophy, stress marker expression, pseudohypoxia, and aerobic glycolysis. These RPE cell autonomous changes are accompanied by increased glucose delivery to photoreceptors with attendant improvements in photoreceptor structure and function. RPE-restricted AOX expression normalizes accumulated levels of succinate and 2-hydroxyglutarate in ETC-deficient RPE, and counteracts deficiencies in numerous neural retinal metabolites. These features can be attributed to the activation of mitochondrial inner membrane flavoproteins such as succinate dehydrogenase and proline dehydrogenase, and alleviation of inhibition of 2-oxyglutarate-dependent dioxygenases such as prolyl hydroxylases and epigenetic modifiers. Our work underscores the importance to outer retinal health of coenzyme Q oxidation in the RPE and identifies a metabolic network critical for photoreceptor survival in the context of RPE mitochondrial dysfunction.

Glucose deprivation triggers DCAF1-mediated inactivation of Rheb-mTORC1 and promotes cancer cell survival

Low glucose is a common microenvironment for rapidly growing solid tumors, which has developed multiple approaches to survive under glucose deprivation. However, the specific regulatory mechanism remains largely elusive. In this study, we demonstrate that glucose deprivation, while not amino acid or serum starvation, transactivates the expression of DCAF1. This enhances the K48-linked polyubiquitination and proteasome-dependent degradation of Rheb, inhibits mTORC1 activity, induces autophagy, and facilitates cancer cell survival under glucose deprivation conditions. This study identified DCAF1 as a new cellular glucose sensor and uncovered new insights into mechanism of DCAF1-mediated inactivation of Rheb-mTORC1 pathway for promoting cancer cell survival in response to glucose deprivation.

Unconventional mechanism of action and resistance to rapalogs in renal cancer

mTORC1 is aberrantly activated in renal cell carcinoma (RCC) and is targeted by rapalogs. As for other targeted therapies, rapalogs clinical utility is limited by the development of resistance. Resistance often results from target mutation, but mTOR mutations are rarely found in RCC. As in humans, prolonged rapalog treatment of RCC tumorgrafts (TGs) led to resistance. Unexpectedly, explants from resistant tumors became sensitive both in culture and in subsequent transplants in mice. Notably, resistance developed despite persistent mTORC1 inhibition in tumor cells. In contrast, mTORC1 became reactivated in the tumor microenvironment (TME). To test the role of the TME, we engineered immunocompromised recipient mice with a resistance mTOR mutation (S2035T). Interestingly, TGs became resistant to rapalogs in mTORS2035T mice. Resistance occurred despite mTORC1 inhibition in tumor cells and could be induced by coculturing tumor cells with mutant fibroblasts. Thus, enforced mTORC1 activation in the TME is sufficient to confer resistance to rapalogs. These studies highlight the importance of mTORC1 inhibition in nontumor cells for rapalog antitumor activity and provide an explanation for the lack of mTOR resistance mutations in RCC patients.

Inhibition of CIRBP represses the proliferation and migration of vascular smooth muscle cells via inhibiting Rheb/mTORC1 axis

The excessive migration and proliferation of vascular smooth muscle cells (VSMCs) plays a vital role in vascular intimal hyperplasia. CIRBP is involved in the proliferation of various cancer cells. This study was aimed to explore the role of CIRBP in the proliferation and migration of VSMCs. Adenovirus was used to interfere with cold-inducible RNA-binding protein (CIRBP) expression, while lentivirus was used to overexpress Ras homolog enriched in brain (Rheb). Western blotting and qRT-PCR were used to evaluate the expression of CIRBP, Rheb, and mechanistic target of rapamycin complex 1 (mTORC1) activity. The cell proliferation was determined by Ki67 immunofluorescence staining and CCK-8 assay. The wound healing assay was performed to assess cell migration. Additionally, immunohistochemistry was conducted to explore the role of CIRBP in intimal hyperplasia after vascular injury. We found that silencing CIRBP inhibited the proliferation and migration of VSMCs, decreased the expression of Rheb and mTORC1 activity. Restoration of mTORC1 activity via insulin or overexpression of Rheb via lentiviral transfection both attenuated the inhibitory effects of silencing CIRBP on the proliferation and migration of VSMCs. Moreover, Rheb overexpression abolished the inhibitory effect of silencing CIRBP on mTORC1 activity in VSMCs. CIRBP was upregulated in the injured carotid artery. Silencing CIRBP ameliorated intimal hyperplasia after vascular injury. In the summary, silencing CIRBP attenuates mTORC1 activity via reducing Rheb expression, thereby supressing the proliferation and migration of VSMCs and intimal hyperplasia after vascular injury.