DNA-PKcs is activated under nutrient starvation and activates Akt,MST1, FoxO3a,and NDR1

BackgroundPresence of unperfused regions containing cells under hypoxia and nutrient starvation; contributes to radioresistance in solid human tumors. We have previously reported that cultured cells; under nutrient starvation show resistance to ionizing radiation compare with cells under normal; condition, and that nutrient starvation increases ATM activity, which causes cellular resistance to; ionizing radiation (Murata et al., BBRC2018). For further investigation of molecular mechanisms; underlying radioresistance of cells under nutrient starvation, effects of nutrient starvation on activity; of DNA-PKcs have been investigated because both DNA-PKcs and ATM belong to the PIKK family; and are required for DNA DSBs repair. In addition to DNA-PKcs, effects of nutrient starvation on; activities of FoxO3a and its regulators Akt, MST1 and AMPK have been investigated because FoxO3a; mediates cellular responses to stress and is activated under nutrient starvation.MethodsA human glioblastoma cell line, T98G was used to examine the effects of nutrient starvation on activities and expression of DNA-PKcs, Akt, MST1, FoxO3a, NDR1, and AMPK. To elucidate; signal transduction pathways for FoxO3a activation under nutrient starvation, we examined effects of; specific inhibitors or siRNA for DNA-PKcs or Akt on activities and expression of MST1, FoxO3, NDR1, andAMPK.ResultsUnder nutrient starvation, phosphorylations of DNA-PKcs at Ser2056, Akt at Ser473, MST at Thr183, FoxO3a at Ser413, NDR1 at Ser281 and Thr282, and AMPK at Thr172 were increased, which suggests their activation. Nutrient starvation did not affect expression of DNA-PKcs, Akt, MST1, or NDR1, with decreased expression of FoxO3a and increased expression of AMPK. Inhibition; of DNA-PK suppressed phosphorylation of Akt under nutrient starvation. Inhibition of DNA-PK or; Akt suppressed phosphorylations of MST1, FoxO3a, and NDR1 under nutrient starvation, which; suggests DNA-PKcs and Akt activate MST1, FoxO3a, and NDR1. Inhibition of DNA-PK did not; suppress phosphorylation ofAMPK under nutrient starvation.ConclusionOur data suggest that DN-PKcs is activated under nutrient starvation and activates AktMST1, FoxO3a, and NDR1.     

Concomitant inhibition of PPARγ and mTORC1 induces the differentiation of human monocytes into highly immunogenic dendritic cells

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The E3 ubiquitin ligase Peli1 regulates the metabolic actions of mTORC1 to suppress antitumor T cell responses

Metabolic fitness of T cells is crucial for immune responses against infections and tumorigenesis. Both the T cell receptor (TCR) signal and environmental cues contribute to the induction of T cell metabolic reprogramming, but the underlying mechanism is incompletely understood. Here, we identified the E3 ubiquitin ligase Peli1 as an important regulator of T cell metabolism and antitumor immunity. Peli1 ablation profoundly promotes tumor rejection, associated with increased tumor‐infiltrating CD4 and CD8 T cells. The Peli1‐deficient T cells display markedly stronger metabolic activities, particularly glycolysis, than wild‐type T cells. Peli1 controls the activation of a metabolic kinase, mTORC1, stimulated by both the TCR signal and growth factors, and this function of Peli1 is mediated through regulation of the mTORC1‐inhibitory proteins, TSC1 and TSC2. Peli1 mediates non‐degradative ubiquitination of TSC1, thereby promoting TSC1‐TSC2 dimerization and TSC2 stabilization. These results establish Peli1 as a novel regulator of mTORC1 and downstream mTORC1‐mediated actions on T cell metabolism and antitumor immunity.     

GRB10 regulates β-cell mass by inhibiting β-cell proliferation and stimulating β-cell dedifferentiation

Decreased functional β-cell mass is the hallmark of diabetes, but the cause of this metabolic defect remains elusive. Here, we show that the levels of the growth factor receptor-bound protein 10 (GRB10), a negative regulator of insulin and mTORC1 signaling, are markedly induced in islets of diabetic mice and high glucose-treated insulinoma cell line INS-1 cells. β-cell-specific knockout of Grb10 in mice increased β-cell mass and improved β-cell function. Grb10-deficient β-cells exhibit enhanced mTORC1 signaling and reduced β-cell dedifferentiation, which could be blocked by rapamycin. On the contrary, Grb10 overexpression induced β-cell dedifferentiation in MIN6 cells. Our study identifies GRB10 as a critical regulator of β-cell dedifferentiation and β-cell mass, which exerts its effect by inhibiting mTORC1 signaling.     

Discovery and optimization of 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives as mTORC1/mTORC2 dual inhibitors

New potent mTORC1/mTORC2 dual inhibitors, 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives, were obtained by optimizing functional groups on our previously reported PI3Kα inhibitor. All the target compounds were synthesized and structural optimization on the structure of the lead compound based on cytotoxic activity. The results showed that some of the target compounds exhibited moderate to high cytotoxic activity against cell line U87MG and PC-3. The activities against mTOR kinase were investigated and the compound 12q showed excellent activity with an IC₅₀ value of 54 nM in the same level of the positive control BEZ235 with IC₅₀ value of 55 nM under the same test conditions. The western blot and cell cycle results demonstrate that compound 12q is a candidate as an mTORC1/mTORC2 dual-target inhibitor. The theoretical calculations were also performed to better understanding the binding modes of the compound 12q in the mTOR active site.     

Autophagy regulation by nutrient signaling

The ability of cells to respond to changes in nutrient availability is essential for the maintenance of metabolic homeostasis and viability. One of the key cellular responses to nutrient withdrawal is the upregulation of autophagy. Recently, there has been a rapid expansion in our knowledge of the molecular mechanisms involved in the regulation of mammalian autophagy induction in response to depletion of key nutrients. Intracellular amino acids, ATP, and oxygen levels are intimately tied to the cellular balance of anabolic and catabolic processes. Signaling from key nutrient-sensitive kinases mTORC1 and AMP-activated protein kinase (AMPK) is essential for the nutrient sensing of the autophagy pathway. Recent advances have shown that the nutrient status of the cell is largely passed on to the autophagic machinery through the coordinated regulation of the ULK and VPS34 kinase complexes. Identification of extensive crosstalk and feedback loops converging on the regulation of ULK and VPS34 can be attributed to the importance of these kinases in autophagy induction and maintaining cellular homeostasis.     

Metabolic compensation activates pro-survival mTORC1 signaling upon 3-phosphoglycerate dehydrogenase inhibition in osteosarcoma

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FUS contributes to mTOR-dependent inhibition of translation

The amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)–linked RNA-binding protein called FUS (fused in sarcoma) has been implicated in several aspects of RNA regulation, including mRNA translation. The mechanism by which FUS affects the translation of polyribosomes has not been established. Here we show that FUS can associate with stalled polyribosomes and that this association is sensitive to mTOR (mammalian target of rapamycin) kinase activity. Specifically, we show that FUS association with polyribosomes is increased by Torin1 treatment or when cells are cultured in nutrient-deficient media, but not when cells are treated with rapamycin, the allosteric inhibitor of mTORC1. Moreover, we report that FUS is necessary for efficient stalling of translation because deficient cells are refractory to the inhibition of mTOR-dependent signaling by Torin1. We also show that ALS-linked FUS mutants R521G and P525L associate abundantly with polyribosomes and decrease global protein synthesis. Importantly, the inhibitory effect on translation by FUS is impaired by mutations that reduce its RNA-binding affinity. These findings demonstrate that FUS is an important RNA-binding protein that mediates translational repression through mTOR-dependent signaling and that ALS-linked FUS mutants can cause a toxic gain of function in the cytoplasm by repressing the translation of mRNA at polyribosomes.     

Potent antitumour of the mTORC1/2 dual inhibitor AZD2014 in docetaxel-sensitive and docetaxel-resistant castration-resistant prostate cancer cells

Recent studies indicate mammalian target of rapamycin (mTOR) may play an important role in PCa progression and drug resistance. Here, we investigated the effects of a novel mTORC1/C2 dual inhibitor, AZD2014, on naive and docetaxel (Doc)-pre-treated castration-resistant PCa (CRPC) cells and explored its therapeutic potential in CRPCs. In the current study, AZD2014 has a greater inhibitory effect against 4EBP1 and AKT phosphorylation than rapamycin in CRPC cells and prevented the feedback activation of AKT signalling. Importantly, AZD2014 suppressed CRPC cell growth in vitro by suppressing proliferation, apoptosis, cell cycle arrest at G1 phase and autophagy to a greater extent than rapamycin. Moreover, AZD2014 was more efficacious than rapamycin in inhibiting migration, invasion and EMT progression in Doc-sensitive and Doc-resistant CRPC cells. Overall, AZD2014 showed significant antitumour effects. Thereby, the current study highlights a reliable theoretical basis for the clinical application of AZD2014 in both Doc-sensitive and Doc-resistant CRPCs.