Single-cell transcriptome reveals the novel role of T-bet in suppressing the immature NK gene signature.

The transcriptional activation and repression during NK cell ontology are poorly understood. Here, using single-cell RNA-sequencing, we reveal a novel role for T-bet in suppressing the immature gene signature during murine NK cell development. Based on transcriptome, we identified five distinct NK cell clusters and define their relative developmental maturity in the bone marrow. Transcriptome-based machine-learning classifiers revealed that half of the mTORC2-deficient NK cells belongs to the least mature NK cluster. Mechanistically, loss of mTORC2 results in an increased expression of signature genes representing immature NK cells. Since mTORC2 regulates the expression of T-bet through AktS473-FoxO1 axis, we further characterized the T-bet-deficient NK cells and found an augmented immature transcriptomic signature. Moreover, deletion of Foxo1 restores the expression of T-bet and corrects the abnormal expression of immature NK genes. Collectively, our study reveals a novel role for mTORC2-AktS473-FoxO1-T-bet axis in suppressing the transcriptional signature of immature NK cells.

Differential roles and activation of mammalian target of rapamycin complexes 1 and 2 during cell migration in prostate cancer cells.

BACKGROUND: Mammalian target of rapamycin (mTOR) is a downstream substrate activated by PI3K/AKT pathway and it is essential for cell migration. It exists as two complexes: mTORC1 and mTORC2. mTORC1 is known to be regulated by active AKT, but the activation of mTORC2 is poorly understood. In this study, we investigated the roles and differential activation of the two mTOR complexes during cell migration in prostate cancer cells. METHODS: We used small interfering RNA to silence the expression of Rac1 and the main components of mTOR complexes (regulatory associated protein of mTOR [RAPTOR] and rapamycin-insensitive companion of mTOR [RICTOR]) in LNCaP, DU145, and PC3 prostate cancer cell lines. We performed transwell migration assay to evaluate the migratory capability of the cells, and Western blot analysis to study the activation levels of mTOR complexes. RESULTS: Specific knockdown of RAPTOR and RICTOR caused a decrease of cell migration, suggesting their essential role in prostate cancer cell movement. Furthermore, epidermal growth factor (EGF) treatments induced the activation of both the mTOR complexes. Lack of Rac1 activity in prostate cancer cells blocked EGF-induced activation of mTORC2, but had no effect on mTORC1 activation. Furthermore, the overexpression of constitutively active Rac1 resulted in significant increase in cell migration and activation of mTORC2 in PC3 cells, but had no effect on mTORC1 activation. Active Rac1 was localized in the plasma membrane and was found to be in a protein complex, with RICTOR, but not RAPTOR. CONCLUSION: We suggest that EGF-induced activation of Rac1 causes the activation of mTORC2 via RICTOR. This mechanism plays a critical role in prostate cancer cell migration.

Rictor ablation in BMSCs inhibits bone metastasis of TM40D cells by attenuating osteolytic destruction and CAF formation.

The mTOR complex 2 (mTORC2) is recognized as a promising target for breast cancer treatment. As mTORC2-specific inhibitors do not yet exist, studies into the role of mTORC2 in cancer are performed by deleting Rictor or by RNAi-mediated Rictor silencing. The purpose of this study was to explore the effects of Rictor ablation in bone mesenchymal stromal cells (BMSCs) on bone metastasis of breast cancer. First, female mice with the genotype of Prx1-Cre;Rictorf/f (hereafter RiCKO) or Rictorf/f (as control) were injected intratibially with cells of the breast cancer cell line (TM40D) at 4 months of age. Three weeks later, osteolytic bone destruction was detected in metastatic legs by X-ray and micro-CT. We found that Rictor ablation in BMSCs inhibited TM40D-induced osteolytic bone destruction and resulted in greater bone volume maintenance in vivo. Lower CTX-I serum level, a decreased number of TRAP+ osteoclasts and lower Cathepsin-K expression observed at the tumor-bone interface indicated that osteoclastogenesis was inhibited in RiCKO mice. Additionally, co-culture experiments confirmed that Rictor deletion in BMSCs diminished osteoclast differentiation partly via down regulation of RANKL expression. Furthermore, Rictor deficiency was found to reduce the transition of BMSCs to CAFs coupled with decreased secretion of cytokines (IL-6, RANKL, TGFß), which resulted in lower chemotaxis and less proliferation in TM40D cells. These results suggest that Rictor ablation in BMSCs plays dual roles in breast cancer bone metastasis: (1) repression of osteolytic bone destruction; (2) inhibition of tumor growth.

Crosstalks between mTORC1 and mTORC2 variagate cytokine signaling to control NK maturation and effector function.

The metabolic checkpoint kinase mechanistic/mammalian target of rapamycin (mTOR) regulates natural killer (NK) cell development and function, but the exact underlying mechanisms remain unclear. Here, we show, via conditional deletion of Raptor (mTORC1) or Rictor (mTORC2), that mTORC1 and mTORC2 promote NK cell maturation in a cooperative and non-redundant manner, mainly by controlling the expression of Tbx21 and Eomes. Intriguingly, mTORC1 and mTORC2 regulate cytolytic function in an opposing way, exhibiting promoting and inhibitory effects on the anti-tumor ability and metabolism, respectively. mTORC1 sustains mTORC2 activity by maintaining CD122-mediated IL-15 signaling, whereas mTORC2 represses mTORC1-modulated NK cell effector functions by restraining STAT5-mediated SLC7A5 expression. These positive and negative crosstalks between mTORC1 and mTORC2 signaling thus variegate the magnitudes and kinetics of NK cell activation, and help define a paradigm for the modulation of NK maturation and effector functions.

Mitochondria-Endoplasmic Reticulum Contact Sites Function as Immunometabolic Hubs that Orchestrate the Rapid Recall Response of Memory CD8+ T Cells.

Glycolysis is linked to the rapid response of memory CD8+ T cells, but the molecular and subcellular structural elements enabling enhanced glucose metabolism in nascent activated memory CD8+ T cells are unknown. We found that rapid activation of protein kinase B (PKB or AKT) by mammalian target of rapamycin complex 2 (mTORC2) led to inhibition of glycogen synthase kinase 3ß (GSK3ß) at mitochondria-endoplasmic reticulum (ER) junctions. This enabled recruitment of hexokinase I (HK-I) to the voltage-dependent anion channel (VDAC) on mitochondria. Binding of HK-I to VDAC promoted respiration by facilitating metabolite flux into mitochondria. Glucose tracing pinpointed pyruvate oxidation in mitochondria, which was the metabolic requirement for rapid generation of interferon-γ (IFN-γ) in memory T cells. Subcellular organization of mTORC2-AKT-GSK3ß at mitochondria-ER contact sites, promoting HK-I recruitment to VDAC, thus underpins the metabolic reprogramming needed for memory CD8+ T cells to rapidly acquire effector function.

Rictor/mTORC2 deficiency enhances keratinocyte stress tolerance via mitohormesis.

How metabolic pathways required for epidermal tissue growth and remodeling influence the ability of keratinocytes to survive stressful conditions is still largely unknown. The mechanistic target of rapamycin complex 2 (mTORC2) regulates growth and metabolism of several tissues, but its functions in epidermal cells are poorly defined. Rictor is an adaptor protein essential for mTORC2 activity. To explore the roles of mTORC2 in the epidermis, we have conditionally deleted rictor in mice via K14-Cre-mediated homologous recombination and found that its deficiency causes moderate tissue hypoplasia, reduced keratinocyte proliferation and attenuated hyperplastic response to TPA. Noteworthy, rictor-deficient keratinocytes displayed increased lifespan, protection from senescence, and enhanced tolerance to cellular stressors such as growth factors deprivation, epirubicin and X-ray in vitro and radioresistance in vivo. Rictor-deficient keratinocytes exhibited changes in global gene expression profiles consistent with metabolic alterations and enhanced stress tolerance, a shift in cell catabolic processes from glycids and lipids to glutamine consumption and increased production of mitochondrial reactive oxygen species (ROS). Mechanistically, the resiliency of rictor-deficient epidermal cells relies on these ROS increases, indicating stress resistance via mitohormesis. Thus, our findings reveal a new link between metabolic changes and stress adaptation of keratinocytes centered on mTORC2 activity, with potential implications in skin aging and therapeutic resistance of epithelial tumors.

Rictor/mTORC2 loss in osteoblasts impairs bone mass and strength.

Mammalian target of rapamycin (mTOR) is a Ser/Thr kinase conserved through evolution that coordinates extra cellular signals associated with cell growth. Main functions of mTOR present in the form of two complexes, namely mTORC1 and mTORC2, which are distinct in their unique components, raptor and rictor. In the current study, using a Cre/loxp system, we found an anabolic effect of mTORC2 signaling on skeleton. Osteoblast differentiation was reduced, with down-regulation of mTORC2 signaling activity in primary cultures of osteoblasts that did not contain rictor. Mice with a specific deletion of rictor in mature osteoblasts showed a significant reduction in lean mass and bone mineral density by dual energy x-ray absorptiometry analysis. Micro-computed tomography, histomorphometric, and molecular biological analyses revealed a marked impairment of the cortical bone mass and microarchitecture, as well as minor changes in trabecular bone, of the Rictorob(-/-) mice. Cortical bone mass and thickness of the femoral mid-shaft were dramatically reduced, with unusual increases in porosity and marrow area in Rictorob(-/-) mice. Thinner trabeculae were found in the L4 vertebrae with relatively normal structural indices of trabecular numbers and separation. A lower rate of bone turnover was observed, as the consequence of the decreased individual osteoblast activity and bone resorption. Furthermore, these changes were associated with significantly decreased bone biomechanical properties. In conclusion, expression of rictor in osteoblasts is essential for the maintenance of normal bone remodeling and microarchitecture, especially for the maintenance of the cortical bone.