S6K1 Activation Research Articles

Alcohol induces p53-mediated apoptosis in neural crest by stimulating an AMPK-mediated suppression of TORC1, S6K, and ribosomal biogenesis.

Prenatal alcohol exposure is a leading cause of permanent neurodevelopmental disability and can feature distinctive craniofacial deficits that partly originate from the apoptotic deletion of craniofacial progenitors, a stem cell lineage called the neural crest (NC). We recently demonstrated that alcohol causes nucleolar stress in NC through its suppression of ribosome biogenesis (RBG) and this suppression is causative in their p53/MDM2-mediated apoptosis. Here, we show that this nucleolar stress originates from alcohol's activation of AMPK, which suppresses TORC1 and the p70/S6K-mediated stimulation of RBG. Alcohol-exposed cells of the pluripotent, primary cranial NC line O9-1 were evaluated with respect to their S6K, TORC1, and AMPK activity. The functional impact of these signals with respect to RBG, p53, and apoptosis were assessed using gain-of-function constructs and small molecule mediators. Alcohol rapidly (<2hr) increased pAMPK, pTSC2, and pRaptor, and reduced both total and pS6K in NC cells. These changes persisted for at least 12hr to 18hr following alcohol exposure. Attenuation of these signals via gain- or loss-of-function approaches that targeted AMPK, S6K, or TORC1 prevented alcohol's suppression of rRNA synthesis and the induction of p53-stimulated apoptosis. We conclude that alcohol induces ribosome dysbiogenesis and activates their p53/MDM2-mediated apoptosis via its activation of pAMPK, which in turn activates TSC2 and Raptor to suppress the TORC1/S6K-mediated promotion of ribosome biogenesis. This represents a novel mechanism underlying alcohol's neurotoxicity and is consistent with findings that TORC1/S6K networks are critical for cranial NC survival.

CLONING OF CDNA-GENE OF ARABIDOPSIS THALIANA RIBOSOMAL PROTEIN S6, ITS EXPRESSION IN ESCHERICHIA COLI AND PURIFICATION OF ATRPS6B1 RECOMBINANT PROTEIN

Living organisms must adapt their growth and development in response to external factors like stress and nutrient availability throughout their lifespan. Consequently, they have evolved various regulatory pathways to enhance environmental perception and facilitate necessary metabolic adjustments. These pathways involve key proteins that, under stress and nutrient limitation, initiate anabolic and catabolic cellular processes. One critical pathway present in all eukaryotes involves the protein kinase TOR (Target of rapamycin), a large kinase that governs numerous biological processes, including the activation of S6K kinase, which phosphorylates the S6 protein (RPS6). Crystal structures have revealed the precise location of RPS6 within the 40S ribosomal subunit, highlighting structural features such as the C-terminal helix containing multiple phosphorylation sites. RPS6 primarily regulates mechanisms controlling cell growth and division. In plants, eS6 is encoded by two conserved genes, and its activation and phosphorylation are closely linked to S6Ks. Recent studies emphasize the significance of S6 protein phosphorylation as a key event in signaling pathways related to cell growth and survival factors, crucially regulating translation initiation and protein synthesis. S6 protein can be phosphorylated on various serine and threonine residues by S6K kinase, and further research shows its interaction with other molecules and proteins, expanding its functional roles in cell signaling. Further exploration of ribosomal protein S6 could unveil new insights into its molecular interactions, roles in cellular physiology and pathophysiology, and potential applications in enhancing plant biomass and yield. This study involved cloning and site-directed mutagenesis of cDNA for the second isoform of AtRPS6 protein (AtRPS6B), followed by expression of natural and phosphomimetic forms of the protein in E. coli ArcticExpress (DE3) cells. The proteins were then purified using metal-chelate affinity chromatography (IMAC), and their presence and degree of purification were confirmed via Western blotting. These findings contribute to the broader research context surrounding ribosomal protein S6. RPS6, a key effector in the TOR signaling pathway, plays a crucial role in regulating ribosome biosynthesis by controlling transcription and translation processes. Phosphorylation of RPS6, indicative of S6K activity, is associated with actively dividing cells. However, the precise role of RPS6 in ribosome biosynthesis regulation remains a subject of ongoing investigation. The data obtained from the study on AtRPS6B expand our understanding of the molecular mechanisms underlying ribosomal biosynthesis regulation. These insights may significantly contribute to our knowledge of the interplay between signaling pathways, ribosomal proteins, cellular metabolism, and offer new perspectives for therapeutic interventions targeting cellular processes associated with ribosomes.

Leptin promotes proliferation of human undifferentiated spermatogonia by activating the PI3K/AKT/mTOR pathway.

Male infertility is a common disease affecting male reproductive health. Leptin is an important hormone that regulates various physiological processes, including reproductive function. However, few experimental studies have been carried out to elucidate the mechanism of leptin's effects on male reproductive function. The purpose of this study was to investigate the effects of leptin on testicular spermatogenesis and its mechanism, so as to provide potential targets for the treatment of patients with spermatogenic dysfunction. Testicular tissues were collected from eight prostate cancer patients undergoing surgical castration. GPR125-positive spermatogonia were isolated by two consecutive magnetic activated cell sorting (MACS), followed by incubation with conditioned medium. To identify the signaling pathway(s) involved in the effects of leptin, undifferentiated spermatogonia were treated with different concentrations of leptin and antagonists of leptin-related pathways. The proliferative effect of leptin was evaluated by cell counting using a hemocytometer. Expressions of p-AKT, p-ERK, p-STAT, and p-S6K were determined by western blotting analysis. Leptin promoted the growth of human GPR125-positive spermatogonia in a concentration-dependent manner. The most significant proliferative effect was observed using 100ng/mL leptin after 6 days of culture. Leptin significantly increased the phosphorylation of STAT3, AKT, and ERK in undifferentiated spermatogonia. Phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 inhibited the leptin-induced activation of AKT, ERK, and downstream S6K. Treatment with the mammalian target of rapamycin (mTOR) inhibitor rapamycin also inhibited S6K phosphorylation. Moreover, both LY294002 and rapamycin were found to inhibit the leptin-induced proliferation of undifferentiated spermatogonia. These results suggested that the leptin-induced proliferation of GPR125-positive spermatogonia was dependent on the PI3K/AKT/mTOR pathway. Further exploration of proliferation and apoptotic markers suggested that leptin may alleviate cell apoptosis by regulating the expression of Bax and FasL. A certain concentration of leptin (25∼100ng/mL) could promote proliferation of undifferentiated spermatogonia, which was mediated by PI3K/AKT/mTOR pathway.

Endothelial cell-specific reduction in mTOR ameliorates age-related arterial and metabolic dysfunction.

Systemic inhibition of the mammalian target of rapamycin (mTOR) delays aging and many age-related conditions including arterial and metabolic dysfunction. However, the mechanisms and tissues involved in these beneficial effects remain largely unknown. Here, we demonstrate that activation of S6K, a downstream target of mTOR, is increased in arteries with advancing age, and that this occurs preferentially in the endothelium compared with the vascular smooth muscle. Induced endothelial cell-specific deletion of mTOR reduced protein expression by 60-70%. Although this did not significantly alter arterial and metabolic function in young mice, endothelial mTOR reduction reversed arterial stiffening and improved endothelium-dependent dilation (EDD) in old mice, indicating an improvement in age-related arterial dysfunction. Improvement in arterial function in old mice was concomitant with reductions in arterial cellular senescence, inflammation, and oxidative stress. The reduction in endothelial mTOR also improved glucose tolerance in old mice, and this was associated with attenuated hepatic gluconeogenesis and improved lipid tolerance, but was independent of alterations in peripheral insulin sensitivity, pancreatic beta cell function, or fasted plasma lipids in old mice. Lastly, we found that endothelial mTOR reduction suppressed gene expression of senescence and inflammatory markers in endothelial-rich (i.e., lung) and metabolically active organs (i.e., liver and adipose tissue), which may have contributed to the improvement in metabolic function in old mice. This is the first evidence demonstrating that reducing endothelial mTOR in old age improves arterial and metabolic function. These findings have implications for future drug development.

Neuroligin-3 activates Akt-dependent Nrf2 cascade to protect osteoblasts from oxidative stress

Excessive oxidative stress will cause significant injury to osteoblasts, serving as one major pathological mechanism of osteoporosis. Neuroligin-3 (NLGN3) is a postsynaptic cell adhesion protein and is expressed in the bone. We here explored its potential activity against hydrogen peroxide (H2O2)-induced oxidative injury in cultured osteoblasts. In primary murine and human osteoblasts, NLGN3 stimulation dose-dependently induced Akt, Erk1/2 and S6K activation. NLGN3 pretreatment ameliorated H2O2-induced cytotoxicity and death in osteoblasts. Moreover, H2O2-induced reactive oxygen species (ROS) production and oxidative injury were alleviated with NLGN3 pretreatment in cultured osteoblasts. Further studies showed that NLGN3 activated Nrf2 signaling cascade and induced Nrf2 protein Serine-40 phosphorylation, Keap1-Nrf2 dissociation, Nrf2 protein stabilization and nuclear translocation in osteoblasts. NLGN3 also increased antioxidant response element (ARE) activity and induced expression of Nrf2-ARE-dependent genes (HO1, GCLC and NQO1) in osteoblasts. Moreover NLGN3 mitigated osteoblast oxidative injury by dexamethasone or sodium fluoride (NaF). Nrf2 cascade activation is essential for NLGN3-induced cytoprotective activity in osteoblasts. Nrf2 shRNA or knockout (KO) abolished NLGN3-induced osteoblast cytoprotection against H2O2. Contrarily forced Nrf2 cascade activation by Keap1 KO mimicked NLGN3-induced anti-oxidative activity in murine osteoblasts. Importantly, NLGN3-induced Serine-40 phosphorylation and Nrf2 cascade activation were blocked by an Akt inhibitor MK-2206 or by Akt1 shRNA. Importantly, Akt inhibition, Akt1 silencing or Nrf2 S40T mutation largely inhibited NLGN3-induced osteoblast cytoprotection against H2O2. At last, we showed that NLGN3 mRNA and protein expression was significantly downregulated in necrotic bone tissues of dexamethasone-taken patients. Taken together, NLGN3 activated Akt-dependent Nrf2 cascade to protect osteoblasts from oxidative stress.

A motif in the 5'untranslated region of messenger RNAs regulates protein synthesis in a S6 kinase-dependent manner.

The 5' untranslated regions (UTRs) in messenger RNAs (mRNAs) play an important role in the regulation of protein synthesis. We had previously identified a group of mRNAs that includes human semaphorin 7A (SEMA7A) whose translation is upregulated by the Erk/p90S6K pathway in human eosinophils, with a potential negative impact in asthma and airway inflammation. In the current study, we aimed to find a common 5'UTR regulatory cis-element, and determine its impact on protein synthesis. We identified a common and conserved 5'UTR motif GGCTG-[(C/G)T(C/G)]n-GCC that was present in this group of mRNAs. Mutations of the first two GG bases in this motif in SEMA7A 5'UTR led to a complete loss of S6K activity dependence for maximal translation. In conclusion, the newly identified 5'UTR motif present in SEMA7A has a critical role in regulating S6K-dependent protein synthesis.

Impact of ergot alkaloid and steroidal implant on whole-body protein turnover and expression of mTOR pathway proteins in muscle of cattle.

Holstein steers (n = 32) were used to determine if the ergot analog, bromocriptine decreases muscle protein synthesis through inhibitory action on the mTOR pathway via a direct effect on signal proteins, and if these negative effects can be alleviated with anabolic agents. Steers were treated with intramuscular administration of bromocriptine (vehicle or 0.1 mg/kg BW) and a subdermal commercial steroidal implant containing trenbolone acetate (TBA) and estradiol 17β (with or without), in a 2×2 factorial design. During the 35 day experiment, intake was restricted to 1.5 times maintenance energy requirement. On days 27 through 32, steers were moved to metabolism stalls for urine collection, and whole-body protein turnover was determined using a single pulse dose of [15N] glycine into the jugular vein on day 28. On day 35, skeletal muscle samples were collected before (basal state) and 60 min after (stimulated state) an i.v. glucose challenge (0.25 g glucose/kg). Blood samples were collected at regular intervals before and after glucose infusion for determination of circulating concentrations of glucose and insulin. Bromocriptine reduced insulin and glucose clearance following the glucose challenge, indicating decreased insulin sensitivity and possible disruption of glucose uptake and metabolism in the skeletal muscle. Conversely, analysis of whole-body protein turnover demonstrated that bromocriptine does not appear to affect protein synthesis or urea excretion. Western immunoblot analysis of skeletal muscle showed that it did not affect abundance of S6K1 or 4E-BP1, so bromocriptine does not appear to inhibit activation of the mTOR pathway or protein synthesis. Estradiol/TBA implant decreased urea excretion and protein turnover but had no effect on protein synthesis, suggesting that steroidal implants promote protein accretion through unchanged rates of synthesis and decreased degradation, even in the presence of bromocriptine, resulting in improved daily gains. Implanted steers likely experienced increased IGF-1 signaling, but downstream activation of mTOR, S6K and 4E-BP1, and thus increased protein synthesis did not occur as expected. Overall, this data suggests that bromocriptine does not have a negative impact on muscle protein synthetic pathways independent of DMI.

Abstract 4879: Inhibition of amino acid transporters as a novel mechanism of action of the repurposed anti-cancer agent tranexamic acid

Abstract Tranexamic Acid (TA) is an anti-fibrinolytic agent that inhibits Plasminogen activation and is used to control bleeding. Past studies have suggested that the ability of TA to block Plasminogen activation may be useful for ablating the growth or invasion of cancer. Based on the similarity of TA to the amino acid Lysine we hypothesize that TA might exhibit novel mechanisms of action independent of blocking Plasminogen activation. Analysis of a series of signaling pathways indicated that TA inhibits phosphorylation of S6K1 and STAT3 on sites required for their activation and reduces expression of the MYC oncogene in a concentration-dependent manner. Interestingly, MYC transformed MCF10A human mammary epithelial cells exhibited enhanced sensitivity to TA-mediated reduction of cell viability, suggesting that TA may exhibit selectivity for MYC overexpressing breast cancers. Treatment of breast tumor bearing mice with TA strongly blocked tumor growth and was associated with extensive cancer cell death compared with control tumors. Analysis of extracts from TA or vehicle treated tumors showed that TA blocked S6K1 phosphorylation in vivo in a statistically significant manner. Since S6K1 phosphorylation is regulated by amino acid levels, we hypothesized that due to the structural similarity between TA and Lysine and Arginine, TA may block Lysine or Arginine uptake by blocking cationic amino acid transporters (CATs). Molecular docking simulations utilizing the structure of the bacterial CAT predicted that TA binds to CAT with affinity similar to that of Lysine. Amino acid uptake experiments performed with radiolabeled Arginine and Lysine showed that TA blocked both Arginine and Lysine absorption by cancer cells in a concentration-dependent manner. Together, these results indicate a novel mechanism by which TA inhibits uptake of Arginine and Lysine, triggering loss of S6K1 activity in parallel with tumor growth inhibition. Initial studies suggest the feasibility of designing TA analogs with significantly improved potency for amino acid transporters, and with corresponding increases in selective toxicity to breast cancer cells. Based on these novel mechanisms of TA action, we expect TA to exhibit anti-cancer activity against a broad range of human malignancies. Citation Format: Brian K. Law, Mary E. Law, Amanda F. Ghilardi, Elham Yaaghubi, Brad J. Davis, Zaafir M. Dulloo, Mengxiong Wang, Olga A. Guryanova, Coy D. Heldermon, Stephan C. Jahn, Ronald K. Castellano. Inhibition of amino acid transporters as a novel mechanism of action of the repurposed anti-cancer agent tranexamic acid. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4879.

Phosphorylated S6K1 and 4E-BP1 play different roles in constitutively active Rheb-mediated retinal ganglion cell survival and axon regeneration after optic nerve injury.

Ras homolog enriched in brain (Rheb) is a small GTPase that activates mammalian target of rapamycin complex 1 (mTORC1). Previous studies have shown that constitutively active Rheb can enhance the regeneration of sensory axons after spinal cord injury by activating downstream effectors of mTOR. S6K1 and 4E-BP1 are important downstream effectors of mTORC1. In this study, we investigated the role of Rheb/mTOR and its downstream effectors S6K1 and 4E-BP1 in the protection of retinal ganglion cells. We transfected an optic nerve crush mouse model with adeno-associated viral 2-mediated constitutively active Rheb and observed the effects on retinal ganglion cell survival and axon regeneration. We found that overexpression of constitutively active Rheb promoted survival of retinal ganglion cells in the acute (14 days) and chronic (21 and 42 days) stages of injury. We also found that either co-expression of the dominant-negative S6K1 mutant or the constitutively active 4E-BP1 mutant together with constitutively active Rheb markedly inhibited axon regeneration of retinal ganglion cells. This suggests that mTORC1-mediated S6K1 activation and 4E-BP1 inhibition were necessary components for constitutively active Rheb-induced axon regeneration. However, only S6K1 activation, but not 4E-BP1 knockdown, induced axon regeneration when applied alone. Furthermore, S6K1 activation promoted the survival of retinal ganglion cells at 14 days post-injury, whereas 4E-BP1 knockdown unexpectedly slightly decreased the survival of retinal ganglion cells at 14 days post-injury. Overexpression of constitutively active 4E-BP1 increased the survival of retinal ganglion cells at 14 days post-injury. Likewise, co-expressing constitutively active Rheb and constitutively active 4E-BP1 markedly increased the survival of retinal ganglion cells compared with overexpression of constitutively active Rheb alone at 14 days post-injury. These findings indicate that functional 4E-BP1 and S6K1 are neuroprotective and that 4E-BP1 may exert protective effects through a pathway at least partially independent of Rheb/mTOR. Together, our results show that constitutively active Rheb promotes the survival of retinal ganglion cells and axon regeneration through modulating S6K1 and 4E-BP1 activity. Phosphorylated S6K1 and 4E-BP1 promote axon regeneration but play an antagonistic role in the survival of retinal ganglion cells.

Oligomeric β-Amyloid Suppresses Hippocampal γ-Oscillations through Activation of the mTOR/S6K1 Pathway.

Neuronal synchronization at gamma frequency (30-100 Hz: γ) is impaired in early-stage Alzheimer's disease (AD) patients and AD models. Oligomeric Aβ1-42 caused a concentration-dependent reduction of γ-oscillation strength and regularity while increasing its frequency. The mTOR1 inhibitor rapamycin prevented the Aβ1-42-induced suppression of γ-oscillations, whereas the mTOR activator leucine mimicked the Aβ1-42-induced suppression. Activation of the downstream kinase S6K1, but not inhibition of eIF4E, was required for the Aβ1-42-induced suppression. The involvement of the mTOR/S6K1 signaling in the Aβ1-42-induced suppression was confirmed in Aβ-overexpressing APP/PS1 mice, where inhibiting mTOR or S6K1 restored degraded γ-oscillations. To assess the network changes that may underlie the mTOR/S6K1 mediated γ-oscillation impairment in AD, we tested the effect of Aβ1-42 on IPSCs and EPSCs recorded in pyramidal neurons. Aβ1-42 reduced EPSC amplitude and frequency and IPSC frequency, which could be prevented by inhibiting mTOR or S6K1. These experiments indicate that in early AD, oligomer Aβ1-42 impairs γ-oscillations by reducing inhibitory interneuron activity by activating the mTOR/S6K1 signaling pathway, which may contribute to early cognitive decline and provides new therapeutic targets.

Sublinear scaling of the cellular proteome with ploidy

Ploidy changes are frequent in nature and contribute to evolution, functional specialization and tumorigenesis. Analysis of model organisms of different ploidies revealed that increased ploidy leads to an increase in cell and nuclear volume, reduced proliferation, metabolic changes, lower fitness, and increased genomic instability, but the underlying mechanisms remain poorly understood. To investigate how gene expression changes with cellular ploidy, we analyzed isogenic series of budding yeasts from 1N to 4N. We show that mRNA and protein abundance scales allometrically with ploidy, with tetraploid cells showing only threefold increase in protein abundance compared to haploids. This ploidy-dependent sublinear scaling occurs via decreased rRNA and ribosomal protein abundance and reduced translation. We demonstrate that the activity of Tor1 is reduced with increasing ploidy, which leads to diminished rRNA gene repression via a Tor1-Sch9-Tup1 signaling pathway. mTORC1 and S6K activity are also reduced in human tetraploid cells and the concomitant increase of the Tup1 homolog Tle1 downregulates the rDNA transcription. Our results suggest that the mTORC1-Sch9/S6K-Tup1/TLE1 pathway ensures proteome remodeling in response to increased ploidy.

AD80 INHIBITS FLT3-MEDIATED SIGNALING AND HAS ANTINEOPLASTIC EFFECTS ON ACUTE MYELOID LEUKEMIA CELLS

Acute myeloid leukemia (AML) is one of the most common types of hematological malignancy in adults. Activating mutations in different tyrosine kinases have been identified as important factors in the development and progression of hematological neoplasms. For AML, FLT3-ITD has been described as a key mutation, activating signaling pathways such as PI3K, RAS, and STAT5. AD80 is a multikinase inhibitor able to inhibit the activity of kinases RET, RAF, S6K, and ERK. It is a new compound and, therefore, few studies have been carried out to date verifying its effects on tumor cells. In the present study, the cellular and molecular effects of AD80 in AML cell lines were analyzed. For the study, 6 human (FLT3-ITD: MV4-11 and MOLM13, BCR: ABL1: K562 and KU812, JAK2V617F: SET2 and HEL) and 5 murine (Ba/F3 with activating mutations: BCR::ABL1, BCR:ABL1T315I, JAK2V617F, MPLW515L, CSF3RT618I) cell lines representing different hematological neoplasms were utilized. Cell viability was assessed using MTT assay. Apoptosis, autophagy, and mitochondrial membrane potential were determined by flow cytometry. Colony formation assays were performed to analyze clonogenic ability. Protein expression/activation was assessed by western blot. In all cellular models, AD80 reduced cell viability in a time- and dose-dependent manner (all p < 0.05). MOLM13 and MV4-11 cells were the most sensitive, with IC50 ranging from 0.4 to 1.1 nM. In FLT3-ITD cell lines, AD80 induced apoptosis and autophagy, disrupted mitochondrial membrane potential, as well as decreased clonogenic ability in a dose-dependent manner (all p < 0.05). AD80 markedly decreased the activation of AKT, STAT5, S6RP, and induced PARP1 cleavage and γH2AX. AD80 was more potent in reducing the viability of FLT3-ITD AML cell lines. Indeed, it has been shown that AD80 strongly inhibits wild-type and mutated FLT3 in an in vitro assay (>90% of inhibition; Dar et al. Nature, 486:80–84, 2012). Similarly, AD80 also inhibits, in less extension, wild-type and mutated ABL1 and JAK2, which corroborate our cell viability findings. Previous studies from our group showed that AD80 has effects on AML cell lines without the tyrosine kinase-related mutations, with IC50 values ranging from 1.6 to 27.2 μM. In contrast, AD80 concentrations at 0.64 nM were able to induce apoptosis and disrupt mitochondrial membrane potential at 72 h, and induced autophagy at 24 h of treatment in FLT3-ITD AML cell lines. In the molecular scenario, this compound decreases the expression/activating of proteins related to cell growth and proliferation and increases molecular markers of autophagy and apoptosis. AD80 exhibits antineoplastic effects against FLT3-ITD mutated cell lines at nanomolar concentrations, highlighting a noticeably on-targeted effect, and opening up research possibilities that could potentially evolve to further investigations. FAPESP, CNPq, and CAPES.

Alpha-Ketoglutarate Alleviates Neuronal Apoptosis Induced by Central Insulin Resistance through Inhibiting S6K1 Phosphorylation after Subarachnoid Hemorrhage

Neuronal apoptosis after subarachnoid hemorrhage (SAH) is believed to play an important role in early brain injury after SAH. The energy metabolism of neuron is closely related to its survival. The transient hyperglycemia caused by insulin resistance (IR) after SAH seriously affects the prognosis of patients. However, the specific mechanisms of IR after SAH are still not clear. Studies have shown that α-KG takes part in the regulation of IR and cell apoptosis. In this study, we aim to investigate whether α-KG can reduce IR after SAH, improve the disorder of neuronal glucose metabolism, alleviate neuronal apoptosis, and ultimately play a neuroprotective role in SAH-induced EBI. We first measured α-KG levels in the cerebrospinal fluid (CSF) of patients with SAH. Then, we established a SAH model through hemoglobin (Hb) stimulation with HT22 cells for further mechanism research. Furthermore, an in vivo SAH model in mice was established by endovascular perforation. Our results showed that α-KG levels in CSF significantly increased in SAH patients and could be used as a potential prognostic biomarker. In in vitro model of SAH, we found that α-KG not only inhibited IR-induced reduction of glucose uptake in neurons after SAH but also alleviated SAH-induced neuronal apoptosis. Mechanistically, we found that α-KG inhibits neuronal IR by inhibiting S6K1 activation after SAH. Moreover, neuronal apoptosis significantly increased when glucose uptake was reduced. Furthermore, our results demonstrated that α-KG could also alleviate neuronal apoptosis in vivo SAH model. In conclusion, our study suggests that α-KG alleviates apoptosis by inhibiting IR induced by S6K1 activation after SAH.

MEN1 silencing triggers the dysregulation of mTORC1 and MYC pathways in ER+ breast cancer cells.

Menin, encoded by the MEN1 gene, has been identified as a critical factor regulating ESR1 transcription, playing an oncogenic role in ER+ breast cancer (BC) cells. Here, we further dissected the consequences of menin inactivation in ER+ BC cells by focusing on factors within two major pathways involved in BC, mTOR and MYC. MEN1 silencing in MCF7 and T-47D resulted in an increase in phosphor-p70S6K1, phosphor-p85S6K1 and phosphor-4EBP1 expression. The use of an AKT inhibitor inhibited the activation of S6K1 and S6RP triggered by MEN1 knockdown (KD). Moreover, MEN1 silencing in ER+ BC cells led to increased formation of the eIF4E and 4G complex. Clinical studies showed that patients with menin-low breast cancer receiving tamoxifen plus everolimus displayed a trend toward better overall survival. Importantly, MEN1 KD in MCF7 and T-47D cells led to reduced MYC expression. ChIP analysis demonstrated that menin bound not only to the MYC promoter but also to its 5' enhancer. Furthermore, E2-treated MEN1 KD MCF7 cells displayed a decrease in MYC activation, suggesting its role in estrogen-mediated MYC transcription. Finally, expression data mining in tumors revealed a correlation between the expression of MEN1 mRNA and that of several mTORC1 components and targets and a significant inverse correlation between MEN1 and two MYC inhibitory factors, MYCBP2 and MYCT1, in ER+ BC. The current work thus highlights altered mTORC1 and MYC pathways after menin inactivation in ER+ BC cells, providing insight into the crosstalk between menin, mTORC1 and MYC in ER+ BC.

Abstract 5502: In vitro characterization of tki resistant renal cell carcinoma after sequential application of cabozantinib

Abstract The VEGFR/MET/AXL receptor tyrosine kinase inhibitor, Cabozantinib (Cbz), showed clinical benefits in first- and second-line treatment for selective metastatic renal cell carcinoma (mRCC) patients. We tested the molecular effects of Cbz in treatment-naïve cells as well as after a chronic treatment with Sunitinib (Sun) and Pazopanib (Paz) in vitro. We used Sun-resistant (786-O/S and Caki-2/S), Paz-resistant cell lines (786-O/P, Caki-2/P), and the matched wild type (WT) cells (786-O/WT and Caki-2/WT). Treatment tolerance to Cbz (IC50 method) was tested by WST-1 and the drug’ effect on signaling pathways was evaluated by immunoblotting. The pre-treatment with Sun increased the IC50 by 1.3 fold in the 786-O/S cell lines (13µM), compared to the 786-O/WT cells (10µM, p-value 0.002) but no difference was observed between Caki-2/S and Caki-2/WT cell lines (13µM vs 14µM, singly). In 786-O/WT, Cbz caused a late down-regulation of the activated receptor P-MET (FC:-2.2), the coupled proteins Src and STAT3, and it impaired FAK. Unlike, P-MET was strongly decresed in Caki-2/WT cells (FC:-10.4). We saw a compensatory activation of FAK and mTOR marker S6K during the treatment. Of note, Cbz elicited a MAPK by 1.5 FC in the 786-O/WT and by 2.5 in Caki-2/WT. The Sun-treatment enhanced P-MET only in 786-O/S (FC: 2.6). Cbz caused late inhibition of P-MET in 786-O/S cells, but an early downregulation in Caki-2/S cells. In both Sun-pretreated cells, Cbz impaired MAPK, mainly in 786-O/S. Paz-pretreatment did not change either the cell viability of the 786-O/P cells (IC50 of 10.4µM vs 10.2µM of 786-O/WT), nor that of the Caki-2/P cell lines (IC50 of 16 µM vs 17 µM of the age-matched Caki-2/WT cells). In the Paz-resistant cells, P-MET was up-regulated by a FC of 6 and 2.4 in 786-O/P and Caki-2/P cells, respectively. A strong inhibition of P-MET by Cbz was observed independent on pretreatment. In both Sun-and Paz- resistant cell lines we observed a high activity of the protein complex Src-FAK, following Cbz treatment. While the protein level of MAPK was reduced by Cbz in the Sun- resistant cells, it was boosted in the 786-O/P and Caki-2/P (FC: 2). Only the Sun-chronic treatment sensitized the resistant cells to the inhibition of S6K by Cbz. To conclude, the P-MET-driven state in Sun and Paz resistant cell lines, and in Caki -2/WT cells did not sensitize the cells towards Cbz. The activation of Src-FAK protein axis, after P-MET inhibition, might suggest a synergy that could weaken Cbz’s antitumor activity in all resistant cells. Yet, a differential regulation of on mTOR or MAPK-signaling by Cbz might depend on Sun or Paz-pretreatment. Our data suggest that pretreatment with Sun could partially reduce the sensitivity towards Cbz. Still, it was cell line-dependent and thus, it might reflect the possible heterogeneity between patients. Overall, our study provides an understanding of cellular signaling of therapy sequencing using Cbz in mRCC. Citation Format: Angela Zaccagnino, Bozhena Vynnytska-Myronovska, Michael Stöckle, Kerstin Junker. In vitro characterization of tki resistant renal cell carcinoma after sequential application of cabozantinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5502.

Activation of the essential kinase PDK1 by phosphoinositide-driven trans-autophosphorylation

3-phosphoinositide-dependent kinase 1 (PDK1) is an essential serine/threonine protein kinase, which plays a crucial role in cell growth and proliferation. It is often referred to as a ‘master’ kinase due to its ability to activate at least 23 downstream protein kinases implicated in various signaling pathways. In this study, we have elucidated the mechanism of phosphoinositide-driven PDK1 auto-activation. We show that PDK1 trans-autophosphorylation is mediated by a PIP3-mediated face-to-face dimer. We report regulatory motifs in the kinase-PH interdomain linker that allosterically activate PDK1 autophosphorylation via a linker-swapped dimer mechanism. Finally, we show that PDK1 is autoinhibited by its PH domain and that positive cooperativity of PIP3 binding drives switch-like activation of PDK1. These results imply that the PDK1-mediated activation of effector kinases, including Akt, PKC, Sgk, S6K and RSK, many of whom are not directly regulated by phosphoinositides, is also likely to be dependent on PIP3 or PI(3,4)P2.

Protrudin regulates FAK activation, endothelial cell migration and angiogenesis

During angiogenesis, endothelial cells form protrusive sprouts and migrate towards the angiogenic stimulus. In this study, we investigate the role of the endoplasmic reticulum (ER)-anchored protein, Protrudin, in endothelial cell protrusion, migration and angiogenesis. Our results demonstrate that Protrudin regulates angiogenic tube formation in primary endothelial cells, Human umbilical vein endothelial cells (HUVECs). Analysis of RNA sequencing data and its experimental validation revealed cell migration as a prominent cellular function affected in HUVECs subjected to Protrudin knockdown. Further, our results demonstrate that knockdown of Protrudin inhibits focal adhesion kinase (FAK) activation in HUVECs and human aortic endothelial cells (HAECs). This is associated with a loss of polarized phospho-FAK distribution upon Protrudin knockdown as compared to Protrudin expressing HUVECs. Reduction of Protrudin also results in a perinuclear accumulation of mTOR and a decrease in VEGF-mediated S6K activation. However, further experiments suggest that the observed inhibition of angiogenesis in Protrudin knockdown cells is not affected by mTOR disturbance. Therefore, our findings suggest that defects in FAK activation and its abnormal subcellular distribution upon Protrudin knockdown are associated with a detrimental effect on endothelial cell migration and angiogenesis. Furthermore, mice with global Protrudin deletion demonstrate reduced retinal vascular progression. To conclude, our results provide evidence for a novel key role of Protrudin in endothelial cell migration and angiogenesis.

EIF3a regulation of mTOR signaling and translational control via HuR in cellular response to DNA damage.

eIF3a (eukaryotic translation initiation factor 3a), a subunit of the eIF3 complex, has been suggested to play a regulatory role in protein synthesis and in cellular response to DNA-damaging treatments. S6K1 is an effector and a mediator of mTOR complex1 (mTORC1) in regulating protein synthesis and integrating diverse signals into control of cell growth and response to stress. Here, we show that eIF3a regulates S6K1 activity by inhibiting mTORC1 kinase via regulating Raptor synthesis. The regulation of Raptor synthesis is via eIF3a interaction with HuR (human antigen R) and binding of the eIF3a-HuR complex to the 5’-UTR of Raptor mRNA. Furthermore, mTORC1 may mediate eIF3a function in cellular response to cisplatin by regulating synthesis of NER proteins and NER activity. Taken together, we conclude that the mTOR signaling pathway may also be regulated by translational control and mediate eIF3a regulation of cancer cell response to cisplatin by regulating NER protein synthesis.

Central Suppression of the GH/IGF Axis and Abrogation of Exercise-Related mTORC1/2 Activation in the Muscle of Phenotype-Selected Male Marathon Mice (DUhTP).

The somatotropic axis is required for a number of biological processes, including growth, metabolism, and aging. Due to its central effects on growth and metabolism and with respect to its positive effects on muscle mass, regulation of the GH/IGF-system during endurance exercise is of particular interest. In order to study the control of gene expression and adaptation related to physical performance, we used a non-inbred mouse model, phenotype-selected for high running performance (DUhTP). Gene expression of the GH/IGF-system and related signaling cascades were studied in the pituitary gland and muscle of sedentary males of marathon and unselected control mice. In addition, the effects of three weeks of endurance exercise were assessed in both genetic groups. In pituitary glands from DUhTP mice, reduced expression of Pou1f1 (p = 0.002) was accompanied by non-significant reductions of Gh mRNA (p = 0.066). In addition, mRNA expression of Ghsr and Sstr2 were significantly reduced in the pituitary glands from DUhTP mice (p ≤ 0.05). Central downregulation of Pou1f1 expression was accompanied by reduced serum concentrations of IGF1 and coordinated downregulation of multiple GH/IGF-signaling compounds in muscle (e.g., Ghr, Igf1, Igf1r, Igf2r, Irs1, Irs2, Akt3, Gskb, Pik3ca/b/a2, Pten, Rictor, Rptor, Tsc1, Mtor; p ≤ 0.05). In response to exercise, the expression of Igfbp3, Igfbp 4, and Igfbp 6 and Stc2 mRNA was increased in the muscle of DUhTP mice (p ≤ 0.05). Training-induced specific activation of AKT, S6K, and p38 MAPK was found in muscles from control mice but not in DUhTP mice (p ≤ 0.05), indicating a lack of mTORC1 and mTORC2 activation in marathon mice in response to physical exercise. While hormone-dependent mTORC1 and mTORC2 pathways in marathon mice were repressed, robust increases of Ragulator complex compounds (p ≤ 0.001) and elevated sirtuin 2 to 6 mRNA expression were observed in the DUhTP marathon mouse model (p ≤ 0.05). Activation of AMPK was not observed under the experimental conditions of the present study. Our results describe coordinated downregulation of the somatotropic pathway in long-term selected marathon mice (DUhTP), possibly via the pituitary gland and muscle interaction. Our results, for the first time, demonstrate that GH/IGF effects are repressed in a context of superior running performance in mice.

Abstract P138: nab-Sirolimus improves mTOR pathway suppression and antitumor activity versus oral mTOR inhibitors in PTEN null bladder cancer (UMUC3) and TSC2 null liver cancer (SNU398) xenografts

Abstract Background: TSC1, TSC2, and PTEN genes are tumor suppressors in the mTOR pathway and can be inactivated or deleted across many cancers (Kwiatkowski, Clin Cancer Res 2016). The mTORC1 pathway is frequently activated in cancer and causes phosphorylation of downstream targets S6K (activation) and 4EBP1 (inactivation). Nanomolar concentrations of mTOR inhibitors (mTORi) sirolimus and everolimus can effectively inhibit S6K but not 4EBP1 and may lead to therapeutic resistance (Kang, Science 2013). In a registrational phase 2 trial (AMPECT) with malignant PEComa, nab-sirolimus (ABI-009) had a response rate of 64% (9/14) in patients with TSC1 or TSC2 mutations. In an expanded access program (NCT03817515), of 8 patients with TSC1 or TSC2 mutations (2 had prior mTORi) treated with nab-sirolimus, 5 had partial responses (all mTORi naïve). This study investigated mTOR pathway inhibition, tumor drug levels, and antitumor activity of nab-sirolimus vs equal doses of oral mTORi in PTEN-null and TSC2-null xenograft models. Methods: Athymic mice bearing subcutaneous PTEN-null UMUC3 bladder cancer xenografts were treated with either saline or equal weekly doses (15 mg/kg) of nab-sirolimus (IV, 7.5 mg/kg, 2x/wk), and sirolimus or everolimus (PO, 3 mg/kg/day, 5 days/wk). Tumors were harvested and analyzed for tumor drug levels (LC-MS/MS) and pS6 inhibition by immunohistochemistry (IHC). The same treatment conditions were repeated in a subsequent experiment with TSC2-null SNU-398 hepatocellular carcinoma xenografts, which further analyzed pS6K, pS6, and p4EBP1 via western blot (WB). Results: In UMUC3 xenografts, compared with oral mTORi, IV nab-sirolimus resulted in significantly higher drug exposure (AUC 7d) in the tumor (P&amp;lt;0.0001) and greater pS6 inhibition as measured by IHC (P=0.0001 vs sirolimus, P=0.0034 vs everolimus). Correspondingly, nab-sirolimus resulted in significantly greater tumor growth inhibition (TGI) than sirolimus (69.6% vs 24.3%, P&amp;lt;0.0001) and everolimus (36.2%, P=0.0023), and prolonged animal survival vs both oral mTORi (P&amp;lt;0.05 log-rank). Based on WB in SNU-398 xenografts, IV nab-sirolimus consistently inhibited mTOR targets pS6K, pS6, and p4EBP1, whereas oral sirolimus only partially decreased pS6K and pS6 and did not appear to reduce p4EBP1 levels. Correspondingly, nab-sirolimus resulted in significantly greater TGI than sirolimus (67.8% vs 36.2%, P&amp;lt;0.05) and prolonged animal survival (P&amp;lt;0.05 log-rank). Conclusions: The relatively low tumor concentrations achieved with oral mTORi may limit their effectiveness as anticancer therapies. IV nab-sirolimus at equal dose showed significantly higher tumor accumulation and inhibition of pS6 in a PTEN-null bladder cancer xenograft and increased inhibition of mTOR targets pS6K, pS6, and p4EBP1 in a TSC2-null hepatocellular carcinoma xenograft. This was accompanied with significantly greater antitumor activity, suggesting that nab-sirolimus may have a more optimal pharmacologic profile than the oral mTORi. Clinical studies in cancers harboring these alterations are planned. Citation Format: Shihe Hou, Heng Du, Anita N. Schmid, David J. Kwiatkowski, Neil P. Desai. nab-Sirolimus improves mTOR pathway suppression and antitumor activity versus oral mTOR inhibitors in PTEN null bladder cancer (UMUC3) and TSC2 null liver cancer (SNU398) xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P138.