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Background: Bronchopulmonary dysplasia remains one of the most common complications of prematurity, despite significant improvements in perinatal care. Functional modeling of human lung development and disease, like BPD, is limited by our ability to access the lung and to maintain relevant progenitor cell populations in culture., Methods: We supplemented Rho/SMAD signaling inhibition with mTOR inhibition to generate epithelial basal cell-like cell lines from tracheal aspirates of neonates., Results: Single-cell RNA-sequencing confirmed the presence of epithelial cells in tracheal aspirates obtained from intubated neonates. Using Rho/SMAD/mTOR triple signaling inhibition, neonatal tracheal aspirate-derived (nTAD) basal cell-like cells can be expanded long term and retain the ability to differentiate into pseudostratified airway epithelium., Conclusions: Our data demonstrate that neonatal tracheal aspirate-derived epithelial cells can provide a novel ex vivo human cellular model to study neonatal lung development and disease., Impact: Airway epithelial basal cell-like cell lines were derived from human neonatal tracheal aspirates. mTOR inhibition significantly extends in vitro proliferation of neonatal tracheal aspirate-derived basal cell-like cells (nTAD BCCs). nTAD BCCs can be differentiated into functional airway epithelium. nTAD BCCs provide a novel model to investigate perinatal lung development and diseases.

Leukemias are routinely sub-typed for risk/outcome prediction and therapy choice using acquired mutations and chromosomal rearrangements. Down syndrome acute lymphoblastic leukemia (DS-ALL) is characterized by high frequency of CRLF2-rearrangements, JAK2-mutations, or RAS-pathway mutations. Intriguingly, JAK2 and RAS-mutations are mutually exclusive in leukemic sub-clones, causing dichotomy in therapeutic target choices. We prove in a cell model that elevated CRLF2 in combination with constitutionally active JAK2 is sufficient to activate wtRAS. On primary clinical DS-ALL samples, we show that wtRAS-activation is an obligatory consequence of mutated/hyperphosphorylated JAK2. We further prove that CRLF2-ligand TSLP boosts the direct binding of active PTPN11 to wtRAS, providing the molecular mechanism for the wtRAS activation. Pre-inhibition of RAS or PTPN11, but not of PI3K or JAK-signaling, prevented TSLP-induced RAS-GTP boost. Cytotoxicity assays on primary clinical DS-ALL samples demonstrated that, regardless of mutation status, high-risk leukemic cells could only be killed using RAS-inhibitor or PTPN11-inhibitor, but not PI3K/JAK-inhibitors, suggesting a unified treatment target for up to 80% of DS-ALL. Importantly, protein activities-based principal-component-analysis multivariate clusters analyzed for independent outcome prediction using Cox proportional-hazards model showed that protein-activity (but not mutation-status) was independently predictive of outcome, demanding a paradigm-shift in patient-stratification strategy for precision therapy in high-risk ALL.

Preeclampsia (PE) is characterized by poor placentation, consequent on aberrant extravillous trophoblast (EVT) cell function during placental development. The SRC family of proteins is important during pregnancy, especially SRC-3, which regulates placental morphogenesis and embryo survival. Although SRC-3 expression in mouse trophoblast giant cells has been documented, its role in the functional regulation of extravillous trophoblasts and the development of PE remains unknown. This study found that SRC-3 expression was significantly lower in placentas from PE pregnancies as compared to uncomplicated pregnancies. Additionally, both CoCl 2 -mimicked hypoxia and suppression of endogenous SRC-3 expression by lentivirus short hairpin RNA attenuated the migration and invasion abilities of HTR-8/SVneo cells. Moreover, we demonstrated that SRC-3 physically interacts with AKT to regulate the migration and invasion of HTR-8 cells, via the AKT/mTOR pathway. We also found that the inhibition of HTR-8 cell migration and invasion by CoCl 2 -mimicked hypoxia was through the SRC-3/AKT/mTOR axis. Our findings indicate that, in early gestation, accumulation of HIF-1α inhibits the expression of SRC-3, which impairs extravillous trophoblastic invasion and migration by directly interacting with AKT. This potentially leads to insufficient uterine spiral artery remodeling and placental hypoperfusion, and thus the development of PE.

Breast cancer is the most common cancer in women worldwide. Hormone receptor breast cancers are the most common ones and, about 2 out of every 3 cases of breast cancer are estrogen receptor (ER) positive. Selective ER modulators, such as tamoxifen, are the first line of endocrine treatment of breast cancer. Despite the expression of hormone receptors some patients develop tamoxifen resistance and 50% present de novo tamoxifen resistance. Recently, we have demonstrated that activated mammalian target of rapamycin (mTOR) is positively associated with overall survival and recurrence free survival in ER positive breast cancer patients who were later treated with tamoxifen. Since altered expression of protein kinase B (PKB)/Akt in breast cancer cells affect N-myristoyltransferase 1 (NMT1) expression and activity, we investigated whether mTOR, a downstream target of PKB/Akt, regulates NMT1 in ER positive breast cancer cells (MCF7 cells). We inhibited mTOR by treating MCF7 cells with rapamycin and observed that the expression of NMT1 increased with rapamycin treatment over the period of time with a concomitant decrease in mTOR phosphorylation. We further employed mathematical modelling to investigate hitherto not known relationship of mTOR with NMT1. We report here for the first time a collection of models and data validating regulation of NMT1 by mTOR.

Adrenocortical carcinoma (ACC) is a rare cancer with poor prognosis. Pan-genomic analyses identified p53/Rb and WNT/β-catenin signaling pathways as main contributors to the disease. However, isolated β-catenin constitutive activation failed to induce malignant progression in mouse adrenocortical tumors. Therefore, there still was a need for a relevant animal model to study ACC pathogenesis and to test new therapeutic approaches. Here, we have developed a transgenic mice model with adrenocortical specific expression of SV40 large T-antigen (AdTAg mice), to test the oncogenic potential of p53/Rb inhibition in the adrenal gland. All AdTAg mice develop large adrenal carcinomas that eventually metastasize to the liver and lungs, resulting in decreased overall survival. Consistent with ACC in patients, adrenal tumors in AdTAg mice autonomously produce large amounts of glucocorticoids and spontaneously activate WNT/β-catenin signaling pathway during malignant progression. We show that this activation is associated with downregulation of secreted frizzled related proteins (Sfrp) and Znrf3 that act as inhibitors of the WNT signaling. We also show that mTORC1 pathway activation is an early event during neoplasia expansion and further demonstrate that mTORC1 pathway is activated in ACC patients. Preclinical inhibition of mTORC1 activity induces a marked reduction in tumor size, associated with induction of apoptosis and inhibition of proliferation that results in normalization of corticosterone plasma levels in AdTAg mice. Altogether, these data establish AdTAg mice as the first preclinical model for metastatic ACC.

The therapeutic potential of targeting CD4+Foxp3+ regulatory T cells (Tregs) remains controversial under the condition of neuroinflammation. This study aims to explore the neuroprotective role of Tregs in optic nerve ischemia (ONI) and evaluate the therapeutic strategy of Tregs transfer with a focus on targeting the mammalian target of rapamycin (mTOR) pathway. Intraocular pressure was transiently increased in adult C57BL/6 mice to induce ONI. Mucosal tolerance of myelin basic protein (MBP) markedly increased retinal ganglion cell (RGC) survival after ONI through enhanced Tregs suppression. mTOR inhibition significantly promoted the frequency of MBP-immunized Tregs in vitro with increased production of anti-inflammatory cytokines. Transient rapamycin treatment highly promoted the immunosuppressive capacity of Tregs and inhibited retinal inflammation in ONI animals. Intravenous infusion of MBP-immunized Tregs, instead of regular Tregs, beneficially modulated immune activities of host retinal CD11b+ cells and CD4+ effector T cells, leading to significant improvement of RGC survival. Importantly, rapamycin treatment further enhanced the neuroprotective effect of Tregs transfer. Taken together, these findings reveal a fine regulation of mTOR signaling on immunized Tregs after acute retinal injury. Adoptive transfer with targeting-mTOR strategy markedly improves neuronal recovery after ONI, supporting the therapeutic potentials of Tregs in acute and chronic neurological disorder.

Mutations to fibroblast growth factor receptor 3 (FGFR3) and phosphatase and tensin homologue (PTEN) signalling pathway components (for example, PTEN loss, PIK3CA, AKT1, TSC1/2) are common in bladder cancer, yet small-molecule inhibitors of these nodes (FGFR/PTENi) show only modest activity in preclinical models. As activation of autophagy is proposed to promote survival under FGFR/PTENi, we have investigated this relationship in a panel of 18 genetically diverse bladder cell lines. We found that autophagy inhibition does not sensitise bladder cell lines to FGFR/PTENi, but newly identify an autophagy-independent cell death synergy in FGFR3-mutant cell lines between mTOR (mammalian target of rapamycin) pathway inhibitors and chloroquine (CQ)-an anti-malarial drug used as a cancer therapy adjuvant in over 30 clinical trials. The mechanism of synergy is consistent with lysosomal cell death (LCD), including cathepsin-driven caspase activation, and correlates with suppression of cSREBP1 and cholesterol biosynthesis in sensitive cell lines. Remarkably, loss of viability can be rescued by saturating cellular membranes with cholesterol or recapitulated by statin-mediated inhibition, or small interfering RNA knockdown, of enzymes regulating cholesterol metabolism. Modulation of CQ-induced cell death by atorvastatin and cholesterol is reproduced across numerous cell lines, confirming a novel and fundamental role for cholesterol biosynthesis in regulating LCD. Thus, we have catalogued the molecular events underlying cell death induced by CQ in combination with an anticancer therapeutic. Moreover, by revealing a hitherto unknown aspect of lysosomal biology under stress, we propose that suppression of cholesterol metabolism in cancer cells should elicit synergy with CQ and define a novel approach to future cancer treatments., Competing Interests: MAK and VF are full-time employees of AstraZeneca. IGG declares no conflict of interest.

Photosensitizers (PS) are ideally devoid of any activity in the absence of photoactivation, and rely on molecular oxygen for the formation of singlet oxygen ((1)O2) to produce cellular damage. Off-targets and tumor hypoxia therefore represent obstacles for the use of PS for cancer photodynamic therapy. Herein, we describe the characterization of OR141, a benzophenazine compound identified through a phenotypic screening for its capacity to be strictly activated by light and to kill a large variety of tumor cells under both normoxia and hypoxia. This new class of PS unraveled an unsuspected common mechanism of action for PS that involves the combined inhibition of the mammalian target of rapamycin (mTOR) signaling pathway and proteasomal deubiquitinases (DUBs) USP14 and UCH37. Oxidation of mTOR and other endoplasmic reticulum (ER)-associated proteins drives the early formation of high molecular weight (MW) complexes of multimeric proteins, the concomitant blockade of DUBs preventing their degradation and precipitating cell death. Furthermore, we validated the antitumor effects of OR141 in vivo and documented its highly selective accumulation in the ER, further increasing the ER stress resulting from (1)O2 generation upon light activation.

Bacterial pathogens possess complex type III effector (T3E) repertoires that are translocated inside the host cells to cause disease. However, only a minor proportion of these effectors have been assigned a function. Here, we show that the T3E AWR5 from the phytopathogen Ralstonia solanacearum is an inhibitor of TOR, a central regulator in eukaryotes that controls the switch between cell growth and stress responses in response to nutrient availability. Heterologous expression of AWR5 in yeast caused growth inhibition and autophagy induction coupled to massive transcriptomic changes, unmistakably reminiscent of TOR inhibition by rapamycin or nitrogen starvation. Detailed genetic analysis of these phenotypes in yeast, including suppression of AWR5-induced toxicity by mutation of CDC55 and TPD3, encoding regulatory subunits of the PP2A phosphatase, indicated that AWR5 might exert its function by directly or indirectly inhibiting the TOR pathway upstream PP2A. We present evidence in planta that this T3E caused a decrease in TOR-regulated plant nitrate reductase activity and also that normal levels of TOR and the Cdc55 homologues in plants are required for R. solanacearum virulence. Our results suggest that the TOR pathway is a bona fide T3E target and further prove that yeast is a useful platform for T3E function characterisation.

Skeletal muscle weight loss is accompanied by small fiber size and low protein content. Alpha-ketoglutarate (AKG) participates in protein and nitrogen metabolism. The effect of AKG on skeletal muscle hypertrophy has not yet been tested, and its underlying mechanism is yet to be determined. In this study, we demonstrated that AKG (2%) increased the gastrocnemius muscle weight and fiber diameter in mice. Our in vitro study also confirmed that AKG dose increased protein synthesis in C2C12 myotubes, which could be effectively blocked by the antagonists of Akt and mTOR. The effects of AKG on skeletal muscle protein synthesis were independent of glutamate, its metabolite. We tested the expression of GPR91 and GPR99. The result demonstrated that C2C12 cells expressed GPR91, which could be upregulated by AKG. GPR91 knockdown abolished the effect of AKG on protein synthesis but failed to inhibit protein degradation. These findings demonstrated that AKG promoted skeletal muscle hypertrophy via Akt/mTOR signaling pathway. In addition, GPR91 might be partially attributed to AKG-induced skeletal muscle protein synthesis.

Autophagy is activated in hypertension-induced cardiac hypertrophy. However, the mechanisms and significance of an activated autophagy are not clear. This study was designed to determine the role of atorvastatin (ATO) in cardiac autophagy and associated benefits on cardiac remodeling and left ventricular function in spontaneously hypertensive rats (SHRs). Twenty-eight male SHRs at 8 weeks of age were randomized to treatment with vehicle (saline solution; SHR+V) or ATO (SHR+ATO; 50 mg kg(-1) per day) for 6 or 12 months. Age-matched male Wistar-Kyoto (WKY) rats were used as normotensive controls. Cardiac magnetic resonance was used to evaluate cardiac function and structure. Compared with WKY rats, SHRs showed significant left ventricle (LV) dysfunction, remodeling and increases in cardiomyocyte size, which were all attenuated by 6 and 12 months of ATO treatment. Compared with WKY rats, autophagy was activated in the hearts of SHRs and this effect was amplified by chronic ATO treatment, particularly following 12 months of treatment. Protein expression levels of microtubule-associated protein-1 light chain 3-II and beclin-1, the biomarkers of an activated cardiac autophagy, were significantly elevated in ATO-treated versus vehicle-treated SHRs and control WKY rats. Cardiac Akt and phosphorylated mammalian target of rapamycin (mTOR) expression were also increased in the hearts of SHR versus WKY rats, and this effect was attenuated by ATO treatment. These findings suggest that ATO-mediated improvements in LV function and structure in SHRs may be, in part, through its regulation of cardiac autophagy via the Akt/mTOR pathway.

RNA-binding proteins (RBPs) bind to and post-transcriptionally regulate the stability of mRNAs. La-related protein 1 (LARP1) is a conserved RBP that interacts with poly-A-binding protein and is known to regulate 5'-terminal oligopyrimidine tract (TOP) mRNA translation. Here, we show that LARP1 is complexed to 3000 mRNAs enriched for cancer pathways. A prominent member of the LARP1 interactome is mTOR whose mRNA transcript is stabilized by LARP1. At a functional level, we show that LARP1 promotes cell migration, invasion, anchorage-independent growth and in vivo tumorigenesis. Furthermore, we show that LARP1 expression is elevated in epithelial cancers such as cervical and non-small cell lung cancers, where its expression correlates with disease progression and adverse prognosis, respectively. We therefore conclude that, through the post-transcriptional regulation of genes such as mTOR within cancer pathways, LARP1 contributes to cancer progression.

There is controversy over the role of glycogen synthase kinase-3 (GSK-3) in cancer progression. Recent work has implicated GSK-3 in the regulation of mammalian target of rapamycin (mTOR), a known player in malignant transformation. Autophagy, a self-degradation pathway, is inhibited by mTOR and is tightly associated with cell survival and tumor growth. Here we show that GSK-3 suppresses autophagy via mTOR complex-1 (mTORC1) and lysosomal regulation. We show that overexpression of GSK-3 isoforms (GSK-3α and GSK-3β) activated mTORC1 and suppressed autophagy in MCF-7 human breast cancer cells as indicated by reduced beclin-1 levels and upregulation of sequestosome 1 (p62/SQSTM1). Further, overexpression of GSK-3 increased the number of autophagosomes and inhibited autophagic flux. This activity was directly related to reduced lysosomal acidification triggered by GSK-3 (in which GSK-3β has a stronger impact). We found that lysosomal acidification is reduced in MCF-7 cells that also exhibit increased levels of autophagosomes and p62/SQSTM1 and increased activity of mTORC1. Subsequently, treating cells with GSK-3 inhibitors restored lysosomal acidification, enhanced autophagic flux and inhibited mTORC1. Furthermore, GSK-3 inhibitors inhibited cell proliferation. We provide evidence that GSK3-mediated mTORC1 activity and GSK-3-mediated lysosomal acidification occur via distinct pathways, yet both mTORC1 and lysosomes control cell growth. Finally, we show that GSK-3-reduced lysosomal acidification inhibits endocytic clearance as demonstrated by reduced endocytic degradation of the epidermal growth factor receptor. Taken together, our study places GSK-3 as a key regulator coordinating cellular homeostasis. GSK-3 inhibitors may be useful in targeting mTORC1 and lysosomal acidification for cancer therapy.

The metastatic cascade is a complex and multistep process with many potential barriers. Recent evidence has shown that microRNAs (miRNAs) are involved in carcinogenesis and tumor progression in non-small-cell lung cancer (NSCLC). In this study, by comparing the miRNA expression profiles of SPC-A-1sci (high metastatic) and SPC-A-1 (weakly metastatic) cells, we demonstrated that the downregulation and function of miR-193a-3p and miR-193a-5p in NSCLC metastasis and the expression of these miRNAs was suppressed in NSCLC compared with corresponding non-tumorous tissues. Decreased miR-193a-3p/5p expression was significantly associated with tumor node metastasis (TNM) and lymph node metastasis. Furthermore, functional assays showed that the overexpression of miR-193a-3p/5p inhibited NSCLC cell migration, invasion and epithelial-mesenchymal transition (EMT) in vitro and lung metastasis formation in vivo. In addition, we discovered that ERBB4 and S6K2 were the direct targets of miR-193a-3p and that PIK3R3 and mTOR were the direct targets of miR-193a-5p in NSCLC. We also observed that miR-193a-3p/5p could inactivate the AKT/mTOR signaling pathway. Thus, miR-193a-3p/5p functions as a tumor suppressor and has an important role in NSCLC metastasis through ERBB signaling pathway.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer. Despite response to chemotherapy, relapses are frequent and resistance to available treatments is often seen in the metastatic setting. Therefore, identification of new therapeutic targets is required. With this aim, we have profiled the activation status of 44 receptor tyrosine kinases (RTKs) and their major signaling pathways in patient-derived TNBC tumors. Frequent co-activation of several RTKs as well as the extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) and mammalian target of rapamycin (mTOR) routes was found. Pharmacologic targeting of the activated kinases indicated that agents that attack the mTOR route are more potent and efficient antitumoral treatments than agents targeting RTKs. mTOR signals through two multiprotein complexes, mTORC1 and mTORC2. We used a genetic approach to explore the contribution of each of the two mTOR branches to the regulation of cell number of TNBC cells. RNA interference experiments indicated that mTORC1 predominated over mTORC2 in the control of TNBC cell proliferation. Moreover, RNA interference of mTOR had a superior antiproliferative action than separately acting on mTORC1 or mTORC2. To analyze the relevance of mTOR targeting in vivo, we used mice with TNBC. Treatment of these mice with BEZ235, a drug that targets mTOR, slowed tumor growth. Mechanistically, BEZ235 delayed cell cycle progression without affecting cell viability. Our results show that TNBCs are particularly sensitive to inhibition of the mTOR pathway, and indicate that mTOR targeting may be a more efficient anti-TNBC therapy than exclusively acting on the mTORC1 branch of the pathway. This is relevant as most mTOR inhibitors used in the clinic act on mTORC1. Collectively with the fact that BEZ235 synergized with drugs commonly used in the treatment of TNBC, our data support the clinical development of agents that act on mTOR as a therapy for this disease.

E-cadherin, a cell-cell adhesion glycoprotein, is frequently downregulated with tumorigenic progression. The extracellular domain of E-cadherin is cleaved by proteases to generate a soluble ectodomain fragment, termed sEcad, which is elevated in the urine or serum of cancer patients. In this study, we explored the functional role of sEcad in the progression of skin squamous cell carcinomas (SCCs). We found that full-length E-cadherin expression was decreased and sEcad increased in human clinical tumor samples as well as in ultraviolet (UV)-induced SCCs in mice. Interestingly, sEcad associated with members of the human epidermal growth factor receptor (HER) and insulin-like growth factor-1 (IGF-1R) family of receptors in human and UV-induced mouse tumors. Moreover, in both E-cadherin-positive (E-cadherin(+)) and -negative (E-cadherin(-)) cells in vitro, sEcad activated downstream mitogen-activated protein (MAP) kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling and enhanced tumor growth, motility and invasion, the latter via activation of matrix metalloproteinase-2 (MMP-2) and MMP-9. To this end, HER, PI3K or MEK inhibitors suppressed sEcad's tumorigenic effects, including proliferation, migration and invasion. Taken together, our data suggest that sEcad contributes to skin carcinogenesis via association with the HER/IGF-1R-family of receptors and subsequent activation of the MAPK and PI3K/Akt/mTOR pathways, thereby implicating sEcad as a putative therapeutic target in cutaneous SCCs.

The protein complex of tuberous sclerosis complex (TSC)1 and TSC2 tumor suppressors is a key negative regulator of mammalian target of rapamycin (mTOR). Hyperactive mTOR signaling due to the loss-of-function of mutations in either TSC1 or TSC2 gene causes TSC, an autosomal dominant disorder featured with benign tumors in multiple organs. As the ubiquitous second messenger calcium (Ca(2+)) regulates various cellular processes involved in tumorigenesis, we explored the potential role of mTOR in modulation of cellular Ca(2+) homeostasis, and in turn the effect of Ca(2+) signaling in TSC-related tumor development. We found that loss of Tsc2 potentiated store-operated Ca(2+) entry (SOCE) in an mTOR complex 1 (mTORC1)-dependent way. The endoplasmic reticulum Ca(2+) sensor, stromal interaction molecule 1 (STIM1), was upregulated in Tsc2-deficient cells, and was suppressed by mTORC1 inhibitor rapamycin. In addition, SOCE repressed AKT1 phosphorylation. Blocking SOCE either by depleting STIM1 or ectopically expressing dominant-negative Orai1 accelerated TSC-related tumor development, likely because of restored AKT1 activity and enhanced tumor angiogenesis. Our data, therefore, suggest that mTORC1 enhancement of store-operated Ca(2+) signaling hinders TSC-related tumor growth through suppression of AKT1 signaling. The augmented SOCE by hyperactive mTORC1-STIM1 cascade may contribute to the benign nature of TSC-related tumors. Application of SOCE agonists could thus be a contraindication for TSC patients. In contrast, SOCE agonists should attenuate mTOR inhibitors-mediated AKT reactivation and consequently potentiate their efficacy in the treatment of the patients with TSC.

The liver kinase B1 (LKB1) tumor suppressor inhibits cell growth through its regulation of cellular metabolism and apical-basal polarity. The best understood mechanism whereby LKB1 limits cell growth is through activation of the AMP-activated-protein-kinase/mammalian-target-of-rapamycin (AMPK/mTOR) pathway to control metabolism. As LKB1 is also required for polarized epithelial cells to resist hyperplasia, it is anticipated to function through additional mechanisms. Recently, Yes-associated protein (Yap) has emerged as a transcriptional co-activator that modulates tissue homeostasis in response to cell-cell contact. Thus this study examined a possible connection between Yap and LKB1. Restoration of LKB1 expression in HeLa cells, which lack this tumor suppressor, or short-hairpin RNA knockdown of LKB1 in NTERT immortalized keratinocytes, demonstrated that LKB1 promotes Yap phosphorylation, nuclear exclusion and proteasomal degradation. The ability of phosphorylation-defective Yap mutants to rescue LKB1 phenotypes, such as reduced cell proliferation and cell size, suggest that Yap inhibition contributes to LKB1 tumor suppressor function(s). However, failure of Lats1/2 knockdown to suppress LKB1-mediated Yap regulation suggested that LKB1 signals to Yap via a non-canonical pathway. Additionally, LKB1 inhibited Yap independently of either AMPK or mTOR activation. These findings reveal a novel mechanism whereby LKB1 may restrict cancer cell growth via the inhibition of Yap.

Relapse to alcohol abuse is an important clinical issue that is frequently caused by cue-induced drug craving. Therefore, disruption of the memory for the cue-alcohol association is expected to prevent relapse. It is increasingly accepted that memories become labile and erasable soon after their reactivation through retrieval during a memory reconsolidation process that depends on protein synthesis. Here we show that reconsolidation of alcohol-related memories triggered by the sensory properties of alcohol itself (odor and taste) activates mammalian target of rapamycin complex 1 (mTORC1) in select amygdalar and cortical regions in rats, resulting in increased levels of several synaptic proteins. Furthermore, systemic or central amygdalar inhibition of mTORC1 during reconsolidation disrupts alcohol-associated memories, leading to a long-lasting suppression of relapse. Our findings provide evidence that the mTORC1 pathway and its downstream substrates are crucial in alcohol-related memory reconsolidation and highlight this pathway as a therapeutic target to prevent relapse.

The potential therapeutic benefits of cannabinoid compounds have raised interest in understanding the molecular mechanisms that underlie cannabinoid-mediated effects. We previously showed that the acute amnesic-like effects of delta9-tetrahydrocannabinol (THC) were prevented by the subchronic inhibition of the mammalian target of rapamycin (mTOR) pathway. In the present study, we assess the relevance of the mTOR pathway in other acute and chronic pharmacological effects of THC. The rapamycin derivative temsirolimus, an inhibitor of the mTOR pathway approved by the Food and Drug Administration, prevents both the anxiogenic- and the amnesic-like effects produced by acute THC. In contrast, THC-induced anxiolysis, hypothermia, hypolocomotion, and antinociception are not sensitive to the mTOR inhibition. In addition, a clear tolerance to THC-induced anxiolysis, hypothermia, hypolocomotion, and antinociception was observed after chronic treatment, but not to its anxiogenic- and amnesic-like effects. Temsirolimus pre-treatment prevented the amnesic-like effects of chronic THC without affecting the downregulation of CB1 receptors (CB1R) induced by this chronic treatment. Instead, temsirolimus blockade after chronic THC cessation did not prevent the residual cognitive deficit produced by chronic THC. Using conditional knockout mice lacking CB1R in GABAergic or glutamatergic neurons, we found that GABAergic CB1Rs are mainly downregulated under chronic THC treatment conditions, and CB1-GABA-KO mice did not develop cognitive deficits after chronic THC exposure. Therefore, mTOR inhibition by temsirolimus allows the segregation of the potentially beneficial effects of cannabinoid agonists, such as the anxiolytic and antinociceptive effects, from the negative effects, such as anxiogenic- and amnesic-like responses. Altogether, these results provide new insights for targeting the endocannabinoid system in order to prevent possible side effects.

A major goal of biomedical research is the identification of molecular and cellular mechanisms that underlie memory storage. Here we report a previously unknown signaling pathway that is necessary for the conversion from short- to long-term memory. The mammalian target of rapamycin (mTOR) complex 2 (mTORC2), which contains the regulatory protein Rictor (rapamycin-insensitive companion of mTOR), was discovered only recently and little is known about its function. We found that conditional deletion of Rictor in the postnatal murine forebrain greatly reduced mTORC2 activity and selectively impaired both long-term memory (LTM) and the late phase of hippocampal long-term potentiation (L-LTP). We also found a comparable impairment of LTM in dTORC2-deficient flies, highlighting the evolutionary conservation of this pathway. Actin polymerization was reduced in the hippocampus of mTORC2-deficient mice and its restoration rescued both L-LTP and LTM. Moreover, a compound that promoted mTORC2 activity converted early LTP into late LTP and enhanced LTM. Thus, mTORC2 could be a therapeutic target for the treatment of cognitive dysfunction.

Levels of eukaryotic initiation factor 4E (eIF4E) are frequently elevated in human cancers and in some instances have been associated with poor prognosis and outcome. Here we utilize transgenic and allograft breast cancer models to demonstrate that increased mammalian target of rapamycin (mTOR) signalling can be a significant contributor to breast cancer progression in vivo. Suppressing mTOR activity, as well as levels and activity of the downstream translation regulators, eIF4E and eIF4A, delayed breast cancer progression, onset of associated pulmonary metastasis in vivo and breast cancer cell invasion and migration in vitro. Translation of vascular endothelial growth factor (VEGF), matrix metallopeptidase 9 (MMP9) and cyclin D1 mRNAs, which encode products associated with the metastatic phenotype, is inhibited upon eIF4E suppression. Our results indicate that the mTOR/eIF4F axis is an important contributor to tumor maintenance and progression programs in breast cancer. Targeting this pathway may be of therapeutic benefit.

Tumorigenesis is often accompanied by metabolic changes that favor rapid energy production and increased biosynthetic capabilities. These metabolic adaptations promote the survival and proliferation of tumor cells, and in conjunction with the hypoxic and metabolically challenged tumor microenvironment, influence autophagic activity. Autophagy is a catabolic process that allows cellular macromolecules to be broken down and re-utilized as metabolic precursors. Stimulation of autophagy promotes the survival of tumor cells under stressful metabolic and environmental conditions, and counters the potentially deleterious effects of mitochondrial dysfunction and the ROS that these organelles generate. However, inhibition of autophagy has also been reported to fuel tumorigenesis. In spite of the advances in our understanding of the relationship between autophagy and tumorigenesis, it remains unclear whether the therapeutic approaches targeting autophagy should aim to increase or decrease autophagic flux in tumor tissues in human patients. Here, we review how metabolic reprogramming influences autophagic activity in tumors, and discuss how inhibition of autophagy might be exploited to target tumor cells that show altered metabolism.

Release from growth factor dependence and acquisition of signalling pathway addiction are critical steps in oncogenesis. To identify genes required on mammalian target of rapamycin (mTOR) addiction, we performed a genome-wide short hairpin RNA screen on a v-H-ras-transformed Pten-deficient cell line that displayed two alternative growth modes, interleukin (IL)-3-independent/mTOR-addicted proliferation (transformed growth mode) and IL-3-dependent/mTOR-non-addicted proliferation (normal growth mode). We screened for genes required only in the absence of IL-3 and thus specifically for the transformed growth mode. The top 800 hits from this conditional lethal screen were analyzed in silico and 235 hits were subsequently rescreened in two additional Pten-deficient cell lines to generate a core set of 47 genes. Hits included genes encoding mTOR and the mTOR complex 2 (mTORC2) component rictor and several genes encoding mitochondrial functions including components of the respiratory chain, adenosine triphosphate synthase, the mitochondrial ribosome and mitochondrial fission factor. Small interfering RNA knockdown against a sizeable fraction of these genes triggered apoptosis in human cancer cell lines but not in normal fibroblasts. We conclude that mTORC2-addicted cells require mitochondrial functions that may be novel drug targets in human cancer.