Phosphorylation Of S6K1 Research Articles

Alcohol Acutely Antagonizes Refeeding-Induced Alterations in the Rag GTPase-Ragulator Complex in Skeletal Muscle.

The Ragulator protein complex is critical for directing the Rag GTPase proteins and mTORC1 to the lysosome membrane mediating amino acid-stimulated protein synthesis. As there is a lack of evidence on alcohol’s effect on the Rag-Ragulator complex as a possible mechanism for the development of alcoholic skeletal muscle wasting, the aim of our study was to examine alterations in various protein–protein complexes in the Rag-Ragulator pathway produced acutely by feeding and how these are altered by alcohol under in vivo conditions. Mice (C57Bl/6; adult males) were fasted, and then provided rodent chow for 30 min (“refed”) or remained food-deprived (“fasted”). Mice subsequently received ethanol (3 g/kg ethanol) or saline intraperitoneally, and hindlimb muscles were collected 1 h thereafter for analysis. Refeeding-induced increases in myofibrillar and sarcoplasmic protein synthesis, and mTOR and S6K1 phosphorylation, were prevented by alcohol. This inhibition was not associated with a differential rise in the intracellular leucine concentration or plasma leucine or insulin levels. Alcohol increased the amount of the Sestrin1•GATOR2 complex in the fasted state and prevented the refeeding-induced decrease in Sestrin1•GATOR2 seen in control mice. Alcohol antagonized the increase in the RagA/C•Raptor complex formation seen in the refed state. Alcohol antagonized the increase in Raptor with immunoprecipitated LAMPTOR1 (part of the Ragulator complex) after refeeding and decreased the association of RagC with LAMPTOR1. Finally, alcohol increased the association of the V1 domain of v-ATPase with LAMPTOR1 and prevented the refeeding-induced decrease in v-ATPase V1 with LAMPTOR1. Overall, these data demonstrate that acute alcohol intake disrupts multiple protein–protein complexes within the Rag-Ragulator complex, which are associated with and consistent with the concomitant decline in nutrient-stimulated muscle protein synthesis under in vivo conditions.

Protocatechuic Acid Suppresses Microglia Activation and Facilitates M1 to M2 Phenotype Switching in Intracerebral Hemorrhage Mice

ObjectivesMicroglia activation, a key process in secondary injury following intracerebral hemorrhage (ICH), is divided to M1 and M2 phenotype. Protocatechuic acid (PCA) is a phenolic acid been proved neuroprotection in ICH without understanding of details. Thus, this study aimed to observe the influence of PCA on microglia activation and explore underlying mechanisms. Materials and methodsTo assess PCA affected microglia activation in vivo, an experimental ICH mice model was established and then treated with PCA intraperitoneal injection. Immunofluorescence staining was performed in brain slices at day 3 post ICH. BV2 cells were stimulated with hemin for activation, then M1 and M2 biomarkers were analyzed using Western Blot and qPCR. At last, we detected the expression of mTOR and its downstream molecules to discuss possible mechanisms. ResultsAt day 3 post ICH, less activated microglia gathering around hematoma after PCA treatment. Furtherly, in hemin treated BV2 cells, PCA downregulated M1 and promoted M2 biomarkers expression in both mRNA and protein level. PCA inhibited the phosphorylation of mTOR, S6K1 and 4E-BP1, while the inhibition was disappeared after supplemented with mTOR activator. ConclusionsPCA impacted microglia activation by suppressing the mTOR signaling pathway, thereby improving M1/M2 switch and attenuated neuroinflammation.

Chaga mushroom extract induces autophagy via the AMPK-mTOR signaling pathway in breast cancer cells

Ethnopharmacological relevanceChaga mushrooms (Inonotus obliquus) are commonly used in traditional treatments in Eastern Europe and Asia due to their diverse pharmacological effects, including anti-tumor and immunologic effects. Thus, many cancer patients take Chaga mushrooms as a complementary medicine, even during chemotherapy or radiotherapy. However, few studies have investigated the effects or molecular targets of Chaga mushrooms in breast cancer. Aim of the studyHerein, we examined the anticancer effects of Chaga mushrooms in different types of breast cancer cell lines, and explored the underlying molecular mechanism to better understand their effects and benefits. Materials and methodsChaga mushroom extract (CME) was prepared by extracting Chaga mushrooms with 70% ethanol. The cytotoxic effects of CME were assessed by MTT assay and protein expressions were evaluated by western blotting. To evaluate in vivo anti-tumor effects of CME, CME (2 g/kg) was orally administered to 4T1 tumor-bearing BALB/c mice every other day over 30 days (15 administrations), and tumor sizes were measured. Silica gel column chromatography was used to fractionate CME, and major constituents responsible for cytotoxic effects of CME were identified by 1H/13C-NMR and LC-MS. ResultsCME inhibited the proliferation of 4T1 mouse breast cancer cells in a dose and time-dependent manner. The expression of LC3 and phosphorylation of AMPK were increased by CME, while the phosphorylation of mTOR, S6, and S6K1 were suppressed, suggesting that CME induced autophagy by activating AMPK and inhibiting mTOR signaling pathways. Consistent with its observed cytotoxic effect in vitro, CME effectively suppressed tumor growth in 4T1 tumor-bearing BALB/c mice. In addition, inotodiol and trametenolic acid were identified as the major constituents responsible for the cytotoxic effects of CME on breast cancer cells. Moreover, inotodiol and trametenolic acid-enriched fractions both exhibited cytotoxic effects regardless of breast cancer cell subtypes and did not interfere with the cytotoxic effects of conventional drugs. ConclusionsTaken together, Chaga mushroom extract induced autophagy by activating AMPK and inhibiting the mTOR signaling pathway. Our data suggest Chaga mushrooms may be a beneficial complementary medicine for breast cancer patients.

The Efficacy and Safety of the mTOR Signaling Pathway Activator, MHY1485, for in vitro Activation of Human Ovarian Tissue

BackgroundPremature ovarian insufficiency (POI) is characterized by abnormal ovarian function before the age of 40. POI showed that primordial follicles developed in disorder. mTOR signaling plays a vital role in the process of follicle development. It has been verified that the mTOR signaling pathway activator, MHY1485, can promote primordial follicle development in mice. We considered that MHY1485 would be a promising fertility preservation method for POI patients.MethodsThe fragmented ovarian tissues of normal woman was cultured with activator MHY1485 in vitro, and then the control and activated ovaries were transplanted into the kidney capsules of ovariectomized mice. We then used the Infinium Human Methylation EPIC BeadChip to verify the DNA methylation level of ovarian tissues, thus exploring the effectiveness of them.ResultsMHY1485 stimulated mTOR, S6K1, and rpS6 phosphorylation. Cultured with MHY1485, ovarian weights increased and endocrine function was restored. The number of growing follicles was increased. The in vitro activation process did not induce histological changes or abnormal DNA methylation occurrence.ConclusionMHY1485 for in vitro activation (IVA) is effective for ovarian rejuvenation and is a potential therapeutic treatment for POI patients.

Brusatol Inhibits Tumor Growth and Increases the Efficacy of Cabergoline against Pituitary Adenomas

Cabergoline (CAB) is the first choice for treatment of prolactinoma and the most common subtype of pituitary adenoma. However, drug resistance and lack of effectiveness in other pituitary tumor types remain clinical challenges to this treatment. Brusatol (BT) is known to inhibit cell growth and promote apoptosis in a variety of cancer cells. In our present studies, we investigate the effects of BT on pituitary tumor cell proliferation in vitro and in vivo. BT treatment resulted in an increase in Annexin V-expressing cells and promoted the expression of apoptosis-related proteins in rat and human pituitary tumor cells. Investigation of the mechanism underlying this effect revealed that BT increased the production of reactive oxygen species (ROS) and inhibited the phosphorylation of 4EBP1 and S6K1. Furthermore, treatment with a combination of BT and CAB resulted in greater antitumor effects than either treatment alone in nude mice and pituitary tumor cells. Collectively, our results suggest that the BT-induced ROS accumulation and inhibition of mTORC1 signaling pathway leads to inhibition of tumor growth. Combined use of CAB and BT may increase the clinical effectiveness of treatment for human pituitary adenomas.

B and T lymphocyte attenuator regulates autophagy in mycobacterial infection via the AKT/mTOR signal pathway

The survivability of Mycobacterium tuberculosis (M.tb) in macrophages in granuloma is a predominant cause for tuberculosis (TB) infection and recurrence. However, the mechanism of mycobacterial clearance in macrophages still needs further study. Here, we explored a novel role of B and T lymphocyte Attenuator (BTLA) in macrophage-mediated host defense against mycobacterial infection. We found that the surface expression of BTLA was increased in CD14+ monocytes from active TB patients. The mRNA levels of BTLA were induced in human and mice monocytes/macrophages during Mycobacterium bovis BCG or M.tb H37Rv infection, as well as spleen and lung of H37Rv-infected mice. Furthermore, silencing of BTLA promoted the intracellular survival of BCG and H37Rv by suppressing the autophagy in macrophages but not effecting phagocytosis, reactive oxygen species (ROS) and apoptosis. Silence of BTLA reduced bacterial-autophagosome and bacterial-lysosome colocalization. Moreover, BTLA inhibited AKT and mTOR signaling substrates S6K and 4EBP1 phosphorylation in BCG and H37Rv infected macrophages, and BTLA-mediated AKT-mTOR signaling and intracellular BCG survival were reversed by PI3K inhibitors in macrophages. Finally, treatment with BTLA agonist ameliorated lung pathology and promoted autophagy and mycobacterial clearance during mycobacterial infection in vivo. These results demonstrate that BTLA promotes host defense against mycobacteria by enhancing autophagy, which may provide potential therapeutic interventions against tuberculosis.

Modulation of mTOR and epigenetic pathways as therapeutics in gallbladder cancer

Gallbladder cancer (GBC) is the most common malignancy of the biliary tract, with extremely dismal prognosis. Limited therapeutic options are available for GBC patients. We used whole-exome sequencing of human GBC to identify the ErbB and epigenetic pathways as two vulnerabilities in GBC. We screened two focused small-molecule libraries that target these two pathways using GBC cell lines and identified the mTOR inhibitor INK-128 and the histone deacetylase (HDAC) inhibitor JNJ-26481585 as compounds that inhibited proliferation at low concentrations. Both significantly suppressed tumor growth and metastases in mouse models. Both synergized with the standard of care chemotherapeutic agent gemcitabine in cell lines and in mouse models. Furthermore, the activation of the mTOR pathway, measured by immunostaining for phosphorylated mTOR and downstream effector S6K1, is correlated with poor prognosis in GBC. Phosphorylated mTOR or p-S6K1 in clinical samples is an independent indicator for overall survival in GBC patients. Taken together, our findings suggest that mTOR inhibitors and HDAC inhibitors can serve as potential therapeutics for GBC, and the phosphorylation of mTOR and S6K1 may serve as biomarkers for GBC.

Soluble RANKL exaggerates hindlimb suspension-induced osteopenia but not muscle protein balance.

We examined the hypothesis that exaggerating unloading-induced bone loss using a combination of hindlimb suspension (HLS) and exogenous injections of receptor activator of nuclear factor-κB ligand (RANKL) also exaggerates gastrocnemius and quadriceps muscle loss. Forty, male C57Bl/6J mice (16 weeks) were subjected to HLS or normal ambulation (ground control, GC) for 14 days. Mice received three intraperitoneal injections of either human recombinant soluble RANKL or phosphate-buffered saline as control (n = 10/group) at 24 h intervals starting on Day 1 of HLS. GC + RANKL and HLS mice exhibited similar decreases in trabecular bone volume and density in both proximal tibias and distal femurs. However, RANKL affected trabecular number, separation, and connectivity density, while HLS decreased trabecular thickness. The combination of RANKL and HLS exacerbated these changes. Similarly, GC + RANKL and HLS mice saw comparable decreases in cortical bone volume, thickness, and strength in femur midshafts, and combination treatment exacerbated these changes. Plasma concentrations of P1NP were increased in both groups receiving RANKL, while CTX concentrations were unchanged. HLS decreased gastrocnemius weight and was associated with a reduction in global protein synthesis, and no change in proteasome activity. This change was correlated with a decrease in S6K1 and S6 phosphorylation, but no change in 4E-BP1 phosphorylation. Injection of RANKL did not alter gastrocnemius or quadriceps muscle protein metabolism in GC or HLS mice. Our results suggest that injection of soluble RANKL exacerbates unloading-induced bone loss, but not unloading-induced gastrocnemius or quadriceps muscle loss.

100 President Oral Presentation Pick: Prematurity Blunts Protein Synthesis in Skeletal Muscle of Neonatal Pigs Independently of Birth Weight

Abstract Postnatal growth failure reduces lean mass accretion and increases adiposity. Previously we showed that preterm birth impairs the feeding-induced stimulation of mechanistic target of rapamycin (mTOR), translation initiation, and protein synthesis in skeletal muscle of neonatal pigs. Considering that pig body weight can vary widely at birth, we wished to differentiate the effect of prematurity versus BW on these responses. Pigs delivered by caesarean section at preterm (103 d) or term (112 d) gestation were fed parenterally for 4 d. On day 4, all pigs were fasted for 4 h and were either fasted one additional hour or fed an elemental meal by oral gavage, yielding four treatment groups: preterm fasted, preterm fed, term fasted, and term fed. A flooding dose of L-[4-3H]Phe was injected into pigs 30 min after feeding (30 min before euthanasia). Preterm and term pigs were stratified according to birth weight such that preterm pigs with the highest birth weight were compared to term pigs with the lowest birth weight (n = 10–13 per group). Despite similar birth weight of pigs in these two subgroups, relative body weight gain of preterm pigs was 33% lower than term pigs (22.0 vs. 32.9 ± 1.6 g·kg-1·d-1, P < 0.001). Gastrocnemius protein synthesis increased after feeding in both preterm and term pigs, but the increase was 18% lower in preterm than in term pigs pigs (Table 1). Feeding-induced AKT phosphorylation and mTOR-dependent 4EBP1 and S6K1 phosphorylation mirrored the pattern in protein synthesis. These results suggest that prematurity (rather than low birth weight, which is frequently associated with preterm birth) perturbs mTOR activation and protein synthesis after feeding. However, it is unclear whether an inadequate response to feeding also underpins poor growth in intrauterine growth restricted piglets. Research was supported by NIH and USDA.

Abstract 14395: Abnormal Regulation of P53-mTOR Pathway by MRNA-binding Protein ZFP36L2 in Peri-partum Cardiomyopathy

Introduction: A loss of function mutation in the mRNA binding protein ZFP36L2 has been associated with congenital heart diseases in humans, although the mechanism remains unclear. Objectives: We sought to elucidate the role of ZFP36L2 in the heart using mice with cardiac-specific ZFP36L2 deletion. Results: Cardiac-specific Zfp36l2 knockout (cs-L2KO) female mice exhibit a dramatic phenotype in which the majority of mice die after repeated pregnancies due to heart failure. We performed RNA-seq in H9c2 cells with ZFP36L2 KD, and found the expression of multiple mTOR pathway genes were altered, including marked repression of Redd1 and Sesn2 . Consistent with the RNA-seq data, the activity of mTORC1 was increased in cs-L2KO mouse hearts and in ZFP36L2 KD H9c2 cells. Loss of TSC2 mitigated mTORC1 hyperactivity in ZFP36L2 KD cells, indicating that ZFP36L2 regulates mTORC1 activity through the TSC2 complex. Overexpression of REDD1 rescued increased mTORC1 activity with ZFP36L2 KD, suggesting that ZFP36L2 regulates mTORC1, in-part through REDD1. Under total amino acid starvation, we observed higher p-S6K T389 levels in ZFP36L2 KD cells, and SESN2 overexpression mitigated p-S6K T389 , indicating loss of ZFP36L2 also regulates mTORC1 through amino acid sensing. p53 is a transcriptional activator of both REDD1 and SESN2, and we found that ZFP36L2 was required to maintain p53 levels by directly destabilizing MDM2 mRNA. Accordingly, administration of Nutlin3, an MDM2 inhibitor, reversed the reduction in p53, REDD1 and SESN2 protein levels, and prevented the increase in S6K phosphorylation in response to ZFP36L2 KD. Next, we treated pregnant mice with rapamycin, and cs-L2KO mice treated with rapamycin displayed significantly improved cardiac function after delivery. Finally, we analyzed human heart samples from patients with peri-partum cardiomyopathy and found ZFP36L2 protein to be significantly decreased and p-S6K T389 levels increased, compared to healthy controls. Conclusions: Our studies demonstrate that ZFP36L2 regulates mTORC1 activity through modifying p53 stability, and that the disruption of this pathway induces peri-partum cardiomyopathy. Inhibition of mTORC1 hyperactivity with rapamycin can be a potential therapy for this disease.

The therapeutic value of XL388 in human glioma cells.

XL388 is a highly efficient and orally-available ATP-competitive PI3K-mTOR dual inhibitor. Its activity against glioma cells was studied here. In established and primary human glioma cells, XL388 potently inhibited cell survival and proliferation as well as cell migration, invasion and cell cycle progression. The dual inhibitor induced significant apoptosis activation in glioma cells. In A172 cells and primary human glioma cells, XL388 inhibited Akt-mTORC1/2 activation by blocking phosphorylation of Akt and S6K1. XL388-induced glioma cell death was only partially attenuated by a constitutively-active mutant Akt1. Furthermore, it was cytotoxic against Akt1-knockout A172 glioma cells. XL388 downregulated MAF bZIP transcription factor G (MAFG) and inhibited Nrf2 signaling, causing oxidative injury in glioma cells. Conversely, antioxidants, n-acetylcysteine, pyrrolidine dithiocarbamate and AGI-106, alleviated XL388-induced cytotoxicity and apoptosis in glioma cells. Oral administration of XL388 inhibited subcutaneous A172 xenograft growth in severe combined immunodeficient mice. Akt-S6K1 inhibition and MAFG downregulation were detected in XL388-treated A172 xenograft tissues. Collectively, XL388 efficiently inhibits human glioma cell growth, through Akt-mTOR-dependent and -independent mechanisms.

Insulin potentiates essential amino acids effects on mechanistic target of rapamycin complex 1 signaling in MAC-T cells

Different models of lactation offer conflicting evidence as to whether insulin signaling is required for AA to stimulate mechanistic target of rapamycin complex 1 (mTORC1) activity. We hypothesized that insulin potentiates essential AA stimulation of mTORC1 activity in the MAC-T mammary epithelial cell line. Here, our objective was to assess mTORC1 signaling activity in response to insulin and individual or grouped essential AA. Insulin and essential AA concentrations in the treatment medium ranged from normo- to supraphysiological, with insulin at 0, 1, 10, or 100 nmol/L and essential AA at approximately 0, 0.01, 0.05, 0.1, 1, or 3× reference plasma levels. Effects and interaction of insulin and total essential AA were tested in a 3 × 5 factorial design (n = 3 replicates/treatment); insulin and the individual AA Leu, Met, Ile, and Arg were likewise tested in 3 × 4 factorials (n = 4). As the remaining individual AA His, Lys, Phe, Thr, Trp, and Val were expected to not affect mTORC1, these were tested only at the highest insulin level, 100 nmol/L (n = 4). For all of these, linear and quadratic effects of total and individual AA were evaluated. Essential AA were subsequently grouped by their positive (Leu, Met, Ile, Arg, and Thr; TOR-AA) or absent-to-negative effects (His, Lys, Phe, Trp, and Val; NTOR-AA), and tested for interaction in a 2 × 2 factorial design (n = 4), with each AA at its respective 1× plasma level, and insulin held at 100 nmol/L. All experiments consisted of 1 h treatment incubation, followed by Western blotting of cell lysates to measure phosphorylation and abundance of the mTORC1 pathway proteins Akt (Ser473); ribosomal protein S6 kinase p70 (S6K1, Thr389); eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1, Ser65); and ribosomal protein S6 (S6, Ser240/244). The Akt phosphorylation was overall increased by insulin, with a possible negative interaction with both total essential AA and the individual AA Leu. Total essential AA also increased S6K1 and 4E-BP1 phosphorylation in an insulin-dependent manner. The individual AA Leu, Met, Ile, and Arg increased S6K1 phosphorylation in an insulin-dependent manner. Similarly, Met and Arg increased 4E-BP1 phosphorylation in an insulin-dependent manner. Histidine, Lys, Trp, and Val did not affect S6K1 phosphorylation. However, S6K1 phosphorylation was linearly increased by Thr and quadratically decreased by Phe. Relative to the phosphorylation of S6K1 when cells were incubated with no essential AA, the NTOR-AA group had no effect, whereas the TOR-AA increased phosphorylation to the same degree observed with all 10 essential AA. Overall, we have found that insulin is required for essential AA to stimulate mTORC1 activity in MAC-T cells. In addition, the AA responsible for the bulk of mTORC1 activation in MAC-T are limited to Leu, Met, Ile, Arg, and Thr.

MTOR Inhibition Ablates Cisplatin-Resistant Salivary Gland Cancer Stem Cells

Patients with advanced salivary gland mucoepidermoid carcinoma (MEC) are treated with surgery and radiotherapy, as current systemic therapies are largely ineffective. As such, current treatment frequently leads to poor long-term survival due to locoregional recurrence or metastases. We have shown that salivary gland cancer stem cells (CSCs) are resistant to platinum-based chemotherapy and drive tumor progression. The purpose of this study was to investigate the effect of therapeutic inhibition of mTOR (mechanistic target of rapamycin) on resistance of CSCs to cisplatin, a prototypic platinum-based chemotherapeutic agent. Viability assays determined the effect of several inhibitors of PI3k/mTOR signaling (e.g., temsirolimus, BKM120, AZD8055, PF4708671) and/or cisplatin on survival of human MEC cells. The impact of mTOR inhibitors and/or cisplatin on MEC stemness was examined with salisphere assays, flow cytometry for ALDH/CD44 (CSC markers for MEC), and Western blots for Bmi-1 expression (marker of stem cell self-renewal). Salivary gland MEC patient-derived xenografts were used to examine the effect of cisplatin and/or temsirolimus on CSCs in vivo. We observed that cisplatin induced mTOR and S6K1 phosphorylation, increased the number and size of MEC salispheres, and induced Bmi-1 expression and the fraction of CSCs in MEC models in vitro. Cisplatin also increased the fraction of CSCs in vivo. In contrast, mTOR inhibition (e.g., temsirolimus) blocked cisplatin-induced Bmi-1 expression and salisphere formation in vitro. Remarkably, temsirolimus slowed down tumor growth and decreased the fraction of CSCs (P < 0.05) even in presence of cisplatin in a short-term in vivo experiment. Collectively, these results demonstrate that therapeutic inhibition of mTOR ablates cytotoxic-resistant CSCs, and they suggest that a combination of an mTOR inhibitor and platinum-based chemotherapy might be beneficial to patients with salivary gland mucoepidermoid carcinoma.

Abstract A02: SYK kinase inhibition causes autophagy pathway activation via suppression of mTORC1 in KRAS-mutant pancreatic cancer cells

Abstract The spleen tyrosine kinase (SYK) is highly expressed in a subset of KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cell lines. This prompted our interest in investigating SYK as a candidate therapeutic target in PDAC cell lines. Here, we use a selective SYK kinase inhibitor (PRT-062607) in pharmacologic and mechanistic studies of SYK kinase signaling in PDAC cell lines. SYK kinase inhibition causes dose-dependent decreases in cell proliferation of “SYK-high” PDAC cell lines YAPC and DAN-G. Furthermore, combined SYK and MEK inhibition causes additive effects on reducing cell proliferation. Mechanistically, SYK inhibition causes reduced S6K and rpS6 phosphorylation, which is independent of MEK/ERK and PI3K/AKT signaling. Therefore, SYK activates mTORC1 activity through an unknown mechanism. The mTORC1 complex is a key activator of the macroautophagy pathway (autophagy), which is implicated as a driver of PDAC progression. However, autophagy can cause tumor suppression in some contexts. We demonstrate that SYK inhibition or shRNA-mediated depletion results in increased autolysosomal biogenesis and autophagic flux that occur with latency following treatment of cells with the SYK inhibitor. These effects are partly mediated by the nuclear localization and activation of MITF, which is a key transcriptional regulator of genes that control lysosomal biogenesis and autophagy. Taken together, our results demonstrate that SYK is a positive regulator of mTORC1 activity in a subset of PDAC cell lines. SYK kinase inhibition or shRNA-mediated depletion causes a strong reduction in mTORC1 activity and subsequent MITF activation and nuclear localization. The net effect is increased autolysosomal biogenesis and autophagic flux in YAPC and DAN-G PDAC cell lines. Therefore, SYK inhibition in combination with KRAS pathway inhibitors may serve as a potential therapeutic strategy to perturb autophagy pathway homeostasis and pancreatic cancer cell survival. Citation Format: Kevin L. Hua, Michelle Pan, Minoo Rafati, Anurag Singh. SYK kinase inhibition causes autophagy pathway activation via suppression of mTORC1 in KRAS-mutant pancreatic cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A02.

Abstract A13: Mammalian EAK-7 activates alternative mTOR signaling to regulate cell proliferation and migration

Abstract Nematode EAK-7 (enhancer-of-akt-1-7) regulates dauer formation and controls life span; however, the function of the human ortholog mammalian EAK-7 (mEAK-7) is unknown. We report that mEAK-7 activates an alternative mechanistic/mammalian target of rapamycin (mTOR) signaling pathway in human cells, in which mEAK-7 interacts with mTOR at the lysosome to facilitate S6K2 activation and 4E-BP1 repression. Despite interacting with mTOR and mammalian lethal with SEC13 protein 8 (mLST8), mEAK-7 does not interact with other mTOR complex 1 (mTORC1) or mTOR complex 2 (mTORC2) components; however, it is essential for mTOR signaling at the lysosome. This phenomenon is distinguished by S6 and 4E-BP1 activity in response to nutrient stimulation. Conventional S6K1 phosphorylation is uncoupled from S6 phosphorylation in response to mEAK-7 knockdown. mEAK-7 recruits mTOR to the lysosome, a crucial compartment for mTOR activation. Loss of mEAK-7 results in a marked decrease in lysosomal localization of mTOR, whereas overexpression of mEAK-7 results in enhanced lysosomal localization of mTOR. Deletion of the carboxyl terminus of mEAK-7 significantly decreases mTOR interaction. mEAK-7 knockdown decreases cell proliferation and migration, whereas overexpression of mEAK-7 enhances these cellular effects. Constitutively activated S6K rescues mTOR signaling in mEAK-7–knocked down cells. Thus, mEAK-7 activates an alternative mTOR signaling pathway through S6K2 and 4E-BP1 to regulate cell proliferation and migration. Citation Format: Joe T. Nguyen, Connor Ray, Alexandra L Fox, Daniela B Mendonca, Jin Koo Kim, Paul H. Krebsbach. Mammalian EAK-7 activates alternative mTOR signaling to regulate cell proliferation and migration [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A13.

Extracellular ATP Increases Glucose Metabolism in Skeletal Muscle Cells in a P2 Receptor Dependent Manner but Does Not Contribute to Palmitate-Induced Insulin Resistance.

Saturated fatty acids such as palmitate contribute to the development of Type 2 Diabetes by reducing insulin sensitivity, increasing inflammation and potentially contributing to anabolic resistance. We hypothesized that palmitate-induced ATP release from skeletal muscle cells may increase inflammatory cytokine production and contribute to insulin/anabolic resistance in an autocrine/paracrine manner. In C2C12 myotubes differentiated at physiological glucose concentrations (5.5 mM), palmitate treatment (16 h) at concentrations greater than 250 μM increased release of ATP and inflammatory cytokines IL-6 and MIF, significantly blunted insulin and amino acid-induced signaling and reduced mitochondrial function. In contrast to our hypothesis, degradation of extracellular ATP using apyrase, did not alter palmitate-induced insulin resistance nor alter release of cytokines. Moreover, treatment with ATPγS (16 h), a non-hydrolysable ATP analog, in the absence of palmitate, did not diminish insulin sensitivity. Acute treatment with ATPγS produced insulin mimetic roles; increased phosphorylation of PKB (aka AKT), S6K1 and ERK and enhanced GLUT4-mediated glucose uptake in the absence of exogenous insulin. The increases in PKB and S6K1 phosphorylation were completely prevented by pre-incubation with broad spectrum purinergic receptor (P2R) blockers PPADs and suramin but not by P2 × 4 or P2 × 7 blockers 5-BDBD or A-438079, respectively. Moreover, ATPγS increased IL-6 yet decreased MIF release, similar to the cytokine profile produced by exercise. Acute and chronic treatment with ATPγS increased glycolytic rate in a manner that was differentially inhibited by PPADs and suramin, suggesting heterogeneous P2R activation in the control of cellular metabolism. In summary, our data suggest that the palmitate-induced increase in ATP does not contribute to insulin/anabolic resistance in a cell autonomous manner.

Polycystin-1 Downregulation Induced Vascular Smooth Muscle Cells Phenotypic Alteration and Extracellular Matrix Remodeling in Thoracic Aortic Dissection

ObjectivePolycystin-1 (PC-1) is a protein encoded by the gene of polycystic kidney disease-1 (PKD-1). This study was designed to investigate the regulatory mechanisms of PC-1 on phenotypes of aortic vascular smooth muscle cells (VSMCs) and functions of extracellular matrix (ECM) in thoracic aortic dissection (TAD).MethodsAortic tissues from patients with TAD and healthy controls were collected, primary aortic VSMCs were also isolated. Immunohistochemistry, immunofluorescence, and immunocytochemistry was used to visualize the target proteins. Western blot and RT-qPCR were used to examine the expression of mRNA and proteins. Lentivirus infection was used to downregulate or overexpress PC-1.ResultsCompared with the control group, expression of PC-1 and the contractile phenotypic markers of VSMCs were decreased in TAD group, whereas expression of the synthetic markers of VSMCs, matrix metalloproteinase (MMP)-2, collagen I and collagen III were increased. The phosphorylation of mTOR, S6K and S6 were also elevated in TAD group. PC-1 downregulation of aortic VSMCs inhibited the expression of the contractile markers, but elevated the expression of the synthetic markers, MMP-2, collagen I and collagen III compared with the control group. The phosphorylation of mTOR, S6K and S6 were also increased in PKD-1-knockdown VSMCs. PC-1 upregulation reversed all these expression characteristics in aortic VSMCs. Furthermore, rapamycin treatment to PKD-1-knockdown VSMCs inhibited the effects caused by PC-1 downregulation.ConclusionOur study revealed PC-1 downregulation induces aortic VSMCs phenotypic alteration and ECM remodeling via activation of mTOR/S6K/S6 signaling pathway. Downregulation of PC-1 might be a potential mechanism for the development and progression of TAD. Rapamycin might be a potential inhibitor to attenuate the development and progression of TAD.

Glycogen phosphorylase inhibitor, 2,3-bis[(2E)-3-(4-hydroxyphenyl)prop-2-enamido] butanedioic acid (BF142), improves baseline insulin secretion of MIN6 insulinoma cells.

Type 2 diabetes mellitus (T2DM), one of the most common metabolic diseases, is characterized by insulin resistance and inadequate insulin secretion of β cells. Glycogen phosphorylase (GP) is the key enzyme in glycogen breakdown, and contributes to hepatic glucose production during fasting or during insulin resistance. Pharmacological GP inhibitors are potential glucose lowering agents, which may be used in T2DM therapy. A natural product isolated from the cultured broth of the fungal strain No. 138354, called 2,3-bis(4-hydroxycinnamoyloxy)glutaric acid (FR258900), was discovered a decade ago. In vivo studies showed that FR258900 significantly reduced blood glucose levels in diabetic mice. We previously showed that GP inhibitors can potently enhance the function of β cells. The purpose of this study was to assess whether an analogue of FR258900 can influence β cell function. BF142 (Meso-Dimethyl 2,3‐bis[(E)‐3‐(4‐acetoxyphenyl)prop‐2‐enamido]butanedioate) treatment activated the glucose-stimulated insulin secretion pathway, as indicated by enhanced glycolysis, increased mitochondrial oxidation, significantly increased ATP production, and elevated calcium influx in MIN6 cells. Furthermore, BF142 induced mTORC1-specific phosphorylation of S6K, increased levels of PDX1 and insulin protein, and increased insulin secretion. Our data suggest that BF142 can influence β cell function and can support the insulin producing ability of β cells.

Abstract LB-113: Positioning a selective, bi-steric inhibitor of mTORC1 as a combination partner in RAS-driven cancers

Abstract The RAS and PI3K/mTOR signaling pathways function downstream of receptor tyrosine kinases (RTK), and are hyperactivated in a wide range of human cancers. Several points of cross-talk and intersecting feedback mechanisms have also been described, in which inhibition of one pathway node leads to activation of the parallel signaling pathway, thereby decreasing the effectiveness of single-pathway targeted therapies. Concomitant inhibition of both pathways is clearly effective in RAS-driven cancers as demonstrated via genetic means and by the combination of pharmacologic inhibitors of MEK and PI3 kinase in a variety of preclinical models (Engelman 2008, Collisson 2012, Eser 2013) However, the clinical translation of this combination has been largely unsuccessful, primarily due to development of dose-limiting toxicities that prohibit the achievement of optimal therapeutic drug concentrations. Recent advances in the treatment paradigm for RAS mutant non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) include several first-in-class covalent inhibitors of KRASG12C (e.g., AMG510, MRTX849) which have demonstrated a favorable safety profile along with early promising anti-tumor activity in patients with advanced cancers harboring a KRASG12C mutation. Drug-anchored CRISPR screens and combination experiments have identified mTOR signaling as a key mediator that limits therapeutic response to KRASG12C inhibition (Molina-Arcas 2019, Hallin 2019). We have developed a novel class of selective mTORC1 inhibitors, termed ‘bi-steric', that covalently links rapamycin analogs with a mTOR kinase active-site inhibitor, and interacts with both the ATP- and FKBP12/FRB-binding sites of mTORC1. These compounds exhibit potent and selective (&amp;gt;10-fold) inhibition of mTORC1 over mTORC2, durably suppress S6K and 4EBP1 phosphorylation, and induce growth suppression and apoptosis in multiple cancer cell lines. In preclinical tests, these mTORC-1 selective inhibitors do not induce hyperglycemia and are predicted to have reduced mTORC2-mediated toxicities. We tested the combinatorial activity of these mTOR inhibitors and covalent KRASG12C inhibitors in preclinical models of two KRASG12C mutant tumor types predicted to have limited sensitivity to KRASG12C inhibitor monotherapy: a) STK11 deficient NSCLC wherein activation of mTORC1 signaling is also an oncogenic driver (~20% co-occurrence in KRAS-mutant NSCLC, Scheffler 2018) and b) colorectal cancer wherein RTK activation is thought to co-activate PI3 kinase signaling (Ebi, 2011). In each case, we showed combinatorial activity manifest as decreased tumor cell viability in vitro and tumor regressions in vivo. In contrast, single agents exhibited cytostatic effects in vitro and modest effects on tumor growth in vivo. Our data demonstrate that the combination of KRASG12C and bi-steric mTORC1 selective inhibitors elicits marked anti-tumor activity at well tolerated doses in vivo in models of cancer patient populations with current unmet medical need. The mechanistic bases for these combinatorial effects are currently under investigation. Our preclinical results reinforce the importance of mTORC1 as a concurrent target in genetically defined RAS-mutant cancers, and showcase the therapeutic potential of a novel and selective bi-steric mTORC1 inhibitor, currently in IND-enabling studies. Citation Format: Yu Chi Yang, Christopher Schulze, Jingjing Jiang, Denise F. Reyes, Tiffany Choy, Tram Nguyen, Zhican Wang, Dong Lee, Robert J. Nichols, Zhengping Wang, Jacqueline A. Smith, Steve M. Kelsey, Mallika Singh. Positioning a selective, bi-steric inhibitor of mTORC1 as a combination partner in RAS-driven cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-113.

Abstract 657: Inhibition of mTORC1 by dihydroartemisinin

Abstract mTOR functions as two complexes, mTORC1 and mTORC2, and is a central controller for cell growth and metabolism. Both mTORC1 and mTORC2 are activated in numerous cancers, and have become the focus of anticancer drug development. Rapalogs and mTOR kinase inhibitors (TORKIs) have been developed. However, the anticancer efficacy of rapalogs is clinically weak or mild to most cancers, and the adverse effects of rapalogs and TORKIs are greatly concerned. So, it is imperative to discover and develop new generation of mTOR inhibitors. Dihydroartemisinin (DHA) is an anti-malarial agent showing good safety in both children and adults. Recently, we have shown that DHA, at pharmacological concentrations, inhibits proliferation and induces apoptosis in cells derived from various cancer types but not in normal or immortalized cell lines, implying that DHA is potentially a selective anticancer agent and may be repurposed for cancer therapy. Of interest, DHA potently suppresses mTORC1-mediated phosphorylation of S6K1 and 4E-BP1, but does not obviously affect mTORC2-mediated phosphorylation of Akt, which is often activated by classic mTOR inhibitors (rapalogs) via a feedback mechanism. The results suggest that DHA is a novel mTORC1 inhibitor. This study was set to determine how DHA inhibits mTORC1. Here we found that DHA did not bind to any mTORC1 components (mTOR, mLST8 or raptor) directly, as determined by in vivo [3H]-DHA radiolabeling. Besides, our molecular docking revealed that DHA did not have the ability to bind FKBP12 or mTOR kinase domain, in contrast to the positive controls rapamycin (binding FKBP12) and INK128 (binding mTOR kinase domain). These data suggest that DHA inhibits mTORC1 via indirect mechanism(s). Further research revealed that DHA altered neither the phosphorylation of IGF-1R/PI3K/PTEN and ERK, nor the expression of REDD1, but induced phosphorylation of AMPKα (T172). Interestingly, DHA-induced mTORC1 inhibition was profoundly attenuated by inhibition with Compound C (AMPK inhibitor) or ectopic expression of dominant negative AMPKα2 (AMPKα2-DN), indicating that activation of AMPK links to inhibition of mTORC1. Collectively, the results support that DHA inhibits mTORC1 by activating AMPK. (Supported by the Feist-Weiller Cancer Center, LSU Health Sciences Center, Shreveport, LA, USA.) Citation Format: Wang Liu, Jun Luo, Yoshinobu Odaka, Shile Huang. Inhibition of mTORC1 by dihydroartemisinin [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 657.