Role Of mTORC1 Research Articles

Role of mTORC1 in intestinal epithelial repair and tumorigenesis.

mTORC1 signaling is the prototypical pathway regulating protein synthesis and cell proliferation. mTORC1 is active in stem cells located at the base of intestinal crypts but silenced as transit-amplifying cells differentiate into enterocytes or secretory cells along the epithelium. After an insult or injury, self-limiting and controlled activation of mTORC1 is critical for the renewal and repair of intestinal epithelium. mTORC1 promotes epithelial cell renewal by driving cryptic stem cell division, and epithelial cell repair by supporting the dedifferentiation and proliferation of enterocytes or secretory cells. Under repeated insult or injury, mTORC1 becomes constitutively active, triggering an irreversible return to stemness, cell division, proliferation, and inflammation among dedifferentiated epithelial cells. Epithelium-derived cytokines promulgate inflammation within the lamina propria, which in turn releases inflammatory factors that act back on the epithelium where undamaged intestinal epithelial cells participate in the pervading state of inflammation and become susceptible to tumorigenesis.

The "other" mTOR complex: New insights into mTORC2 immunobiology and their implications.

A central role of the mechanistic target of rapamycin (mTOR) in regulation of fundamental cell processes is well recognized. mTOR functions in two distinct complexes: rapamycin-sensitive mTOR complex (C) 1 and rapamycin-insensitive mTORC2. While the role of mTORC1 in shaping immune responses, including transplant rejection, and the influence of its antagonism in promoting allograft tolerance have been studied extensively using rapamycin, lack of selective small molecule inhibitors has limited understanding of mTORC2 biology. Within the past few years, however, intracellular localization of mTORC2, its contribution to mitochondrial fitness, cell metabolism, cytoskeletal modeling and cell migration, and its role in differentiation and function of immune cells have been described. Studies in mTORC2 knockdown/knockout mouse models and a new class of dual mTORC1/2 inhibitors, have shed light on the immune regulatory functions of mTORC2. These include regulation of antigen-presenting cell, NK cell, T cell subset, and B cell differentiation and function. mTORC2 has been implicated in regulation of ischemia/reperfusion injury and graft rejection. Potential therapeutic benefits of antagonizing mTORC2 to inhibit chronic rejection have also been described, while selective in vivo targeting strategies using nanotechnology have been developed. We briefly review and discuss these developments and their implications.

Role of mTORC1 in mechanically induced increases in translation and skeletal muscle mass.

Skeletal muscle mass is, in part, regulated by the rate of mRNA translation (i.e., protein synthesis). The conserved serine/threonine kinase, mTOR (the mammalian/mechanistic target of rapamycin), found in the multiprotein complex, mTOR complex 1 (mTORC1), is a major positive regulator of protein synthesis. The purpose of this review is to describe some of the critical steps in translation initiation, mTORC1 and its potential direct and indirect roles in regulating translation, and evidence that mTORC1 regulates protein synthesis and muscle mass, with a particular focus on basal conditions and the response to mechanical stimuli. Current evidence suggests that for acute contraction models of mechanical stimuli, there is an emerging pattern suggesting that there is an early increase in protein synthesis governed by a rapamycin-sensitive mTORC1-dependent mechanism, while at later poststimulation time points, the mechanism may change to a rapamycin-insensitive mTORC1-dependent or even an mTORC1-independent mechanism. Furthermore, evidence suggests that mTORC1 appears to be absolutely necessary for muscle fiber hypertrophy induced by chronic mechanical loading but may only play a partial role in the hypertrophy induced by more intermittent types of acute resistance exercise, with the possibility of mTORC1-independent mechanisms also playing a role. Despite the progress that has been made, many questions about the activation of mTORC1, and its downstream targets, remain to be answered. Further research will hopefully provide novel insights into the regulation of skeletal muscle mTORC1 that may eventually be translated into novel exercise programing and/or targeted pharmacological therapies aimed at preventing muscle wasting and/or increasing muscle mass.

Role of mTORC1 and mTORC2 in Breast Cancer: Therapeutic Targeting of mTOR and Its Partners to Overcome Metastasis and Drug Resistance.

Based on the insights gleaned from decades of research, it seems clear that mechanistic target of rapamycin (mTOR) is an essential signaling node that integrates environmental clues for regulation of cell survival, metabolism and proliferation of the cells. However, overwhelmingly increasing scientific evidence has added a new layer of intricacy to already complicated and versatile signaling pathway of mTOR. Deregulation of spatio-temporally controlled mTOR-driven pathway played contributory role in breast cancer development and progression. Pharmacologists and molecular biologists have specifically emphasized on the identification and development of mTOR-pathway inhibitors. In this chapter we have attempted to provide an overview of the most recent findings related to therapeutic targeting of mTOR-associated mTORC1 and mTORC2 in breast cancer. We have also comprehensively summarized regulation of mTOR and its partners by microRNAs in breast cancer.

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, TGFβ), 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.

Targeting mTOR in Glioblastoma: Rationale and Preclinical/Clinical Evidence.

The mechanistic target of rapamycin (mTOR) drives several physiologic and pathologic cellular processes and is frequently deregulated in different types of tumors, including glioblastoma (GBM). Despite recent advancements in understanding the molecular mechanisms involved in GBM biology, the survival rates of this tumor are still disappointing, primarily due to the lack of efficacious treatments. The phosphatase and tensin homolog (PTEN)/phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mTOR pathway has emerged as a crucial player in GBM development and progression. However, to date, all the attempts to target this pathway with PI3K, AKT, or mTORC1 inhibitors failed to improve the outcome of patients with GBM. Despite these discouraging results, recent evidence pointed out that the blockade of mTORC2 might provide a useful therapeutic strategy for GBM, with the potential to overcome the limitations that mTORC1 inhibitors have shown so far. In this review, we analyzed the rationale of targeting mTOR in GBM and the available preclinical and clinical evidence supporting the choice of this therapeutic approach, highlighting the different roles of mTORC1 and mTORC2 in GBM biology.

Macrophage Raptor Deficiency-Induced Lysosome Dysfunction Exacerbates Nonalcoholic Steatohepatitis.

Background & AimsNonalcoholic steatohepatitis (NASH) is an increasingly prevalent nonalcoholic fatty liver disease, characterized by inflammatory cell infiltration and hepatocellular damage. Mammalian target of rapamycin complex 1 (mTORC1) has been investigated extensively in the context of cancer, including hepatocellular carcinoma. However, the role of mTORC1 in NASH remains largely unknown.MethodsmTORC1 activity in macrophages in human mild and severe NASH liver was compared. Mice with macrophage-specific deletion of the regulatory-associated protein of mTOR (Raptor) subunit and littermate controls were fed a high-fructose, palmitate, and cholesterol diet for 24 weeks or a methionine- and choline-deficient diet for 4 weeks to develop NASH.ResultsWe report that in human beings bearing NASH, macrophage mTORC1 activity was lower in livers experiencing severe vs mild NASH liver. Moreover, macrophage mTORC1 disruption exacerbated the inflammatory response in 2 diet-induced NASH mouse models. Mechanistically, in response to apoptotic hepatocytes (AHs), macrophage polarization toward a M2 anti-inflammatory phenotype was inhibited in Raptor-deficient macrophages. During the digestion of AHs, macrophage mTORC1 was activated and coupled with dynamin-related protein 1 to facilitate the latter’s phosphorylation, leading to mitochondrial fission-mediated calcium release. Ionomycin or A23187, calcium ionophores, prevented Raptor deficiency–mediated failure of lysosome acidification and subsequent lipolysis. Blocking dynamin-related protein 1–dependent mitochondria fission impaired lysosome function, resulting in reduced production of anti-inflammatory factors such as interleukins 10 and 13.ConclusionsPersistent mTORC1 deficiency in macrophages contributes to the progression of NASH by causing lysosome dysfunction and subsequently attenuating anti-inflammatory M2-like response in macrophages during clearance of AHs.

MTORC2 deficiency in cutaneous dendritic cells potentiates CD8+ effector T cell responses and accelerates skin graft rejection.

Mechanistic target of rapamycin (mTOR) complex (mTORC)1 and mTORC2 regulate the differentiation and function of immune cells. While inhibition of mTORC1 antagonizes dendritic cell (DC) differentiation and suppresses graft rejection, the role of mTORC2 in DCs in determining host responses to transplanted tissue remains undefined. Using a mouse model in which mTORC2 was deleted specifically in CD11c+ DCs (TORC2DC-/- ), we show that the transplant of minor histocompatibility Ag (HY)-mismatched skin grafts from TORC2DC-/- donors into wild-type recipients results in accelerated rejection characterized by enhanced CD8+ T cell responses in the graft and regional lymphoid tissue [Correction added on January 9, 2019, after first online publication: in the previous sentence, major was changed to minor]. Similar enhancement of CD8+ effector T cell responses was observed in MHC-mismatched recipients of TORC2DC-/- grafts. Augmented CD8+ T cell responses were also observed in a delayed-type hypersensitivity model in which mTORC2 was absent in cutaneous DCs. These elevated responses could be ascribed to an increased T cell stimulatory phenotype of TORC2DC-/- and not to enhanced lymph node homing of the cells. In contrast, rejection of ovalbumin transgenic skin grafts in TORC2DC-/- recipients was unaffected. These findings suggest that mTORC2 in skin DCs restrains effector CD8+ T cell responses and have implications for understanding of the influence of mTOR inhibitors that target mTORC2 in transplant.

Coordination of Necessary and Permissive Signals by PTEN Inhibition for CNS Axon Regeneration.

In the nearly 10 years since PTEN was identified as a prominent intrinsic inhibitor of CNS axon regeneration, the PTEN negatively regulated PI3K-AKT-mTOR pathway has been intensively explored in diverse models of axon injury and diseases and its mechanism for axon regeneration is becoming clearer. It is therefore timely to summarize current knowledge and discuss future directions of translational regenerative research for neural injury and neurodegenerative diseases. Using mouse optic nerve crush as an in vivo retinal ganglion cell axon injury model, we have conducted an extensive molecular dissection of the PI3K-AKT pathway to illuminate the cross-regulating mechanisms in axon regeneration. AKT is the nodal point that coordinates both positive and negative signals to regulate adult CNS axon regeneration through two parallel pathways, activating mTORC1 and inhibiting GSK3ββ. Activation of mTORC1 or its effector S6K1 alone can only slightly promote axon regeneration, whereas blocking mTORC1 significantly prevent axon regeneration, suggesting the necessary role of mTORC1 in axon regeneration. However, mTORC1/S6K1-mediated feedback inhibition prevents potent AKT activation, which suggests a key permissive signal from an unidentified AKT-independent pathway is required for stimulating the neuron-intrinsic growth machinery. Future studies into this complex neuron-intrinsic balancing mechanism involving necessary and permissive signals for axon regeneration is likely to lead eventually to safe and effective regenerative strategies for CNS repair.

Retraction: Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines.

Retraction: Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines.

Active mTORC2 Signaling in Naive T Cells Suppresses Bone Marrow Homing by Inhibiting CXCR4 Expression.

Recirculation of naive T cells between secondary lymphoid organs to receive survival cues and scan for signs of infection or other pathologic conditions is important for immune homeostasis and effective immune responses. Although the mechanisms that specifically guide the entry of naive T cells into secondary lymphoid organs are well studied, the mechanisms that keep them from fluxing into inappropriate or undesirable compartments, such as healthy tissues or bone marrow, are less well understood. In this study, we report an unexpected finding that under steady state, bone marrow homing of naive T cells is actively suppressed by mTORC2 signaling. We found that in mice, T cell-specific deletion of an essential mTORC2 component Sin1 results in increased accumulation of naive T cells in the bone marrow. Mechanistically, we show that loss of mTORC2 signaling in naive T cells results in enhanced FOXO1 activity, which leads to increased CXCR4 expression and chemotactic response to CXCL12, a key chemokine that promotes bone marrow homing and retention of T cells. Together, the results of our study reveal a novel role of mTORC2 in T cell homeostasis via active suppression of naive T cell bone marrow homing by the mTORC2-FOXO1-CXCR4 axis.

Glycolytic Bias in mTORC2 Deficient Dendritic Cells Potentiates Antigen-Specific Immunity and Accelerates Graft Rejection

Background/Hypothesis The mechanistic target of rapamycin (mTOR) is known to function in two discrete complexes: mTOR complex 1 (mTORC1) and mTORC2. The function of mTORC1 in dendritic cells (DCs) has been studied extensively using rapamycin (RAPA) as an inhibitor. RAPA inhibition of mTORC1 prevents DC maturation, leading to decreased T effector cell proliferation and increased regulatory T cell (Treg) differentiation. Our group has recently demonstrated that mTORC2 deletion in DCs leads to both an enhanced pro-inflammatory DC phenotype and Th1/Th17 allogeneic T cell polarization and proliferation. However, the underlying mechanism has not been resolved. In addition, the role of mTORC2 in DCs in the context of transplantation has not been defined. We hypothesized that ablation of mTORC2 in DCs would alter metabolic activity, resulting in augmented antigen-specific T cell responses and accelerated graft rejection. Methods To assess the role of mTORC2 in DCs in transplantation, we used models of non-MHC mismatched skin transplantation in which either the graft donor or recipient was deficient in mTORC2 specifically in conventional CD11c+ DC (TORC2DC-/-). Graft survival was monitored; Banff rejection scoring was performed by a blinded pathologist. T cell infiltration and collagen degradation in the graft were determined by immunohistochemistry. To ascertain whether skin-resident TORC2DC-/- could augment inflammatory responses, we performed a cell-mediated delayed-type hypersensitivity assay. As mTORC2 has been implicated in cytoskeletal dynamics, we measured DC migration into secondary lymphoid tissue. To elucidate the role of mTORC2 in regulating DC metabolism we analyzed glycolytic capacity and mitochondrial respiratory activity of wild-type (WT) DC and TORC2DC-/- using a Seahorse XF Bioanalyzer. Mitochondrial mass and activity were determined via flow cytometric analysis of MitoTracker Green and TMRE uptake, respectively. ATP production was assessed using a luciferase-based luminescence assay. Results/Conclusions We demonstrate for the first time, that TORC2DC-/- deficiency in either skin graft donors or recipients accelerates immune-mediated rejection. TORC2DC-/- mice also exhibit enhanced T cell and inflammatory monocytic infiltration in the course of delayed-type hypersensitivity responses. These effects are not due to differential migration of TORC2DC-/- to secondary lymphoid tissue. TORC2DC-/- utilize an altered metabolic program, wherein glycolytic function is enhanced as compared to WT DCs. This metabolic phenotype corresponds with increased viability of TORC2DC-/- after stimulation, which may allow these TORC2DC-/- to persist in secondary lymphoid tissue longer than WT DCs. These findings reveal a novel role for mTORC2 in regulating DC immunometabolism, and may provide a basis for therapeutic targeting of DC metabolism to regulate immune responses in transplantation. NIH T32 AI74490.

Role of mTORC2 and Nitric oxide in Bladder Cancer Invasion

BackgroundDespite accounting for around 17,000 deaths and 79,000 new diagnoses annually in the United States, treatment options for bladder cancer remain limited. The key driver of bladder cancer stage progression is increasing invasion into the bladder wall, which has limited response to current chemotherapy options and hence newer therapies directed against invasion are key to improving the treatment of bladder cancer. We have recently shown that arginine, the nitric oxide (NO) precursor, is implicated in bladder cancer progression. Our laboratory has also identified mammalian target of rapamycin complex 2 (mTORC2) as a driver of bladder cancer cell invasion. We evaluated the interaction between these two pathways to promote invasion.MethodsExpression of inducible and endothelial nitric oxide synthases (iNOS and eNOS) in FFPE tissue sections of progressive disease was assessed by IHC and correlated with histopathology, progression and stage based on moderate to strong expression (2+, 3+) compared to absent to weak expression (0, 1+). We utilized gene silencing methods and NOS inhibitors and NO scavenger for effects on bladder cancer invasion and migration. We assessed mTORC2 pathway activity and NOS levels in invasive cell tip protrusions called “invadopodia” and evaluated if mTORC2 regulates NOS localization. We used a novel zebrafish model to characterize the effects of mTORC2 and NO on bladder cancer metastases.ResultWe found that eNOS and iNOS are elevated in invasive human bladder tumors and cell lines and their ablation reduces bladder cancer cell migration and invasion by reorganizing the actin cytoskeleton and reducing invadopodia. mTORC2 silencing can affect levels of iNOS and eNOS in bladder cancer cell lines. Silencing of mTORC2, eNOS or iNOS reduced metastases of bladder cancer cells within zebrafish.ConclusionmTORC2 pathway is a key driver of bladder cancer invasion and metastases by regulating the NO pathway and both mTORC2 and NO can be targets for bladder cancer therapy, which would benefit patient outcomes.Support or Funding InformationPrevent Cancer Foundation Fellowship to DSCase Western Reserve University/Cleveland Clinic CTSA Grant Number UL1 RR024989 from the National Center for Research Resources (NCRR), a KL2 career development award (RR024990) and UCSD start‐up funding to DEHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Potassium Can Regulate Its Own Excretion in Collecting Duct Cells: Role of mTORC2, SGK1 and ENaC

BackgroundThe control of potassium (K+) excretion is central to the homeostatic response to variation in environmental K+ in all multicellular organisms. In mammals, the serine‐threonine kinase SGK1 plays a central role in regulating K+ excretion by stimulating electrogenic Na+ transport in the kidney tubules through the epithelial Na+ channel, ENaC. The resulting increase in lumen negative electrical potential enhances the driving force for K+ secretion through apical membrane K+ channels. It is well established that SGK1 phosphorylation state and activity are controlled by the kinase mTORC2, however, the central physiological inputs that control this phosphorylation are poorly characterized. We postulated that K+ itself might act directly in kidney tubule cells to regulate SGK1 through effects on mTORC2 activity.MethodsmpkCCD cortical collecting duct (CCD) cells were grown on Transwell filters, and treated for 4 h with aldosterone to increase SGK1 expression and adapted to either 1 mM or 5 mM [K+]. At t = 0, basolateral [K+] was changed from 1 to 5 mM or from 5 mM to 1 mM, followed by a return to 5 mM in the presence or absence of either an SGK1 (GSK650394) or mTOR inhibitor. Additional experiments were performed using patch clamp to detect ENaC currents in CCD isolated from mice and subjected to acute change in bath [K+] from 5 mM to 1 mM. SGK1 phosphorylation state in response to changes in [K+] was also assessed in wild type and WNK1 deficient HEK‐293 cells.ResultsIn mpkCCD cells, raising basolateral [K+] from 1 to 5 mM increased (approximately 3‐fold), while lowering [K+] from 5 mM to 1 mM decreased (approximately 4.5‐fold) SGK1 phosphorylation (p < 0.01 for both manipulations). Changing apical [K+] had no significant effect. Further, shifting basolateral [K+] from 5 mM to 1 mM markedly reduced ENaC‐dependent Na+ current, and returning [K+] to 5 mM induced a rapid increase in Na+ current. The stimulatory effects on both SGK1 phosphorylation and Na+ current were dependent on mTOR and SGK1 in that they were inhibited by the mTOR inhibitor, AZD 8055 and the SGK1 inhibitor, GSK650394. SGK1 phosphorylation was also strongly stimulated in a graded fashion by raising medium [K+] in HEK‐293 cells. Interestingly, the K+ stimulatory effect on SGK1 was lost in WNK1‐deficient HEK‐293 cells. Finally, in patch clamp performed on cortical collecting duct isolated from mice, ENaC currents were significantly greater in the presence of 5 mM (330 +/−25 pA, N=5) than in the presence of 1 mM (250 +/− 20 pA, N=5) [K+] (p < 0.05).ConclusionThese data strongly support the idea that changes in extracellular [K+] act directly in CCD cells to modulate mTORC2‐dependent SGK1 phosphorylation resulting in altered ENaC‐mediated Na+ transport, and thereby stimulating K+ secretion. This effect appears to be highly dependent on WNK1. In light of recent evidence that Na‐Cl cotransporter (NCC) is directly regulated in distal convoluted tubule cells by extracellular fluid [K+], these data support a new model of coordinated regulation of Na+ transport between distal convoluted tubule and CCD, which is directly modulated by local renal [K+].Support or Funding InformationAm Heart Assoc. 17GRNT33410667;NIH/NIDDK, R01‐DK56695This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hydrogen Peroxide Increases Intracellular Sodium Concentration in the Medullary Thick Ascending Limb Isolated from Sprague‐Dawley Rats

Elevations of renal medullary hydrogen peroxide (H2O2) produce a salt‐sensitive form of hypertension as shown by chronic infusion of H2O2 into the renal medulla of a single remaining kidney of normotensive Sprague‐Dawley (SD) rats (Makino et al., Hypertension, 2003) and salt‐resistant consomic SS.13BN rats. Conversely, chronic medullary infusion of catalase (H2O2 scavenger) into the renal medulla of Dahl salt‐sensitive rats significantly reduced salt induced hypertension (Taylor and Cowley, Am J Physiol Regul Integr Comp Physiol, 2005). The goal of this study was to assess the effects of H2O2 upon intracellular sodium concentration [Na+]i and the role of mTORC1 and mTORC2 signaling in the medullary thick ascending limb (mTAL) isolated from the outer medulla (OM) of SD rats.Methodsrats were anesthetized, the kidney flushed with saline, removed, and mTALs dissected from the inner stripe of the OM at 4°C under a stereomicroscope. The mTALs were attached to poly‐Lysine coated cover slips and incubated with HBSS containing 10 nM of the Na+‐sensitive fluorescence dye NaGreen and 0.05% Pluronic acid F‐127 for 30 min at room temperature. The mTALs were washed and then placed in a heated chamber (37°C) mounted on the stage of an inverted Nikon TE‐2000U microscope for recording.ResultsIt was found that 100 uM H2O2 resulted in a significant increase in [Na+]i (p<0.05). The responses to pretreatment of mTAL with 10 uM PP242 (mTORC1/2 inhibitor), 200 nM rapamycin (mTORC1 specific inhibitor), or 1 mM furosemide (Na+/K+/2Cl− inhibitor) were then studied. We found that each of these inhibitors nearly abolished H2O2 induced increases in intracellular Na+. Western blot analysis of isolated mTAL from SD rats for each of the treatments found that 100 uM H2O2 increased the activation of AKT (pAKTSer473; a surrogate for mTORC1/2 activation) and S6 (pS6Ser235/236; a surrogate for mTORC1 activation) and were inhibited by their respective inhibitors.ConclusionTogether these observations indicate that H2O2 acts through the mTOR signaling pathways to inhibit NKCC2 activity, which would in turn be expected to lead to sodium retention and hypertension.Support or Funding InformationHL 122662This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of mTORC2 and nitric oxide in bladder cancer invasion.

504 Background: In the United States, bladder cancer is the fourth most common cancer, with an estimated 79,000 new diagnoses and 17,000 deaths in 2017, and the eleventh most common cancer worldwide. The key driver of bladder cancer stage progression is tumor cell invasion which has limited response to current chemotherapy options and hence, newer therapies directed against invasion are key to improving the treatment of bladder cancer. Our laboratory has identified mammalian target of rapamycin complex 2 (mTORC2) as a driver of bladder cancer cell invasion. We have also recently shown that arginine, the nitric oxide (NO) precursor, is implicated in bladder cancer progression. We evaluated the interaction between these two pathways to promote invasion. Methods: Expression of inducible and endothelial nitric oxide synthases (iNOS and eNOS) in FFPE tissue sections of progressive disease was assessed by IHC and correlated with histopathology, progression and stage based on moderate to strong expression (2+, 3+) compared to absent to weak expression (0, 1+).We utilized gene silencing methods and NOS inhibitors and NO scavenger for effects on bladder cancer invasion and migration. We assessed mTORC2 pathway activity and NOS levels in invasive cell tip protrusions called “invadopodia” and evaluated if mTORC2 regulates NOS localization and/or activity. We used a novel zebrafish model to characterize the effects of mTORC2 and NO on bladder cancer metastases. Results: We found that eNOS and iNOS are elevated in invasive human bladder tumors and cell lines and their ablation reduces bladder cancer cell migration and invasion. mTORC2 silencing can affect levels of iNOS and eNOS in bladder cancer cell lines. Silencing of mTORC2, eNOS or iNOS reduced metastases of bladder cancer cells within zebrafish. Conclusions: mTORC2 pathway is a key driver of bladder cancer invasion and metastases by regulating the NO pathway and both mTORC2 and NO can be targets for bladder cancer therapy, which would benefit patient outcomes.

ALTERATIONS OF ECCENTRICALLY EVOKED PROTEIN SYNTHESIS IN ISOLATED RAT SOLEUS FOLLOWING GRAVITATIONAL UNLOADING: ROLE OF FAK

ALTERATIONS OF ECCENTRICALLY EVOKED PROTEIN SYNTHESIS IN ISOLATED RAT SOLEUS FOLLOWING GRAVITATIONAL UNLOADING: ROLE OF FAK

Inactivation of mTORC1 Signaling in Osterix-Expressing Cells Impairs B-cell Differentiation.

Osteoblasts provide a microenvironmental niche for B-cell commitment and maturation in the bone marrow (BM). Any abnormity of osteoblasts function may result in the defect of B lymphopoiesis. Signaling from mechanistic target of rapamycin complex 1 (mTORC1) has been implicated in regulating the expansion and differentiation of osteoblasts. Thus, we raise a hypothesis that mTORC1 signaling in osteoblasts plays a vital role in B-cell development. Inactivation of mTORC1 in osterix-expressing cells (mainly osteoblast lineage) through Osx-Cre-directed deletion of Raptor (an mTORC1-specific component) resulted in a reduction in the total B-cell population in the BM, which was due to a block in early B-cell development from the pro-B to pre-B cell stage. Further mechanistic studies revealed that this defect was the result of reduction of interleukin-7 (IL-7) expression in osterix-expressing immature osteoblasts, which caused the abnormality of IL-7/Stat5 signaling in early B lymphocytes, leading to an increased apoptosis of pre-B plus immature B cells. In vitro and in vivo studies demonstrated that the addition of exogenous IL-7 partially restored B lymphopoiesis in the BM of Raptor mutant mice. Furthermore, total BM cells cultured in conditioned media from Raptor null immature osteoblasts or media with anti-IL-7 neutralizing antibody failed to differentiate into pre-B and immature B cells, indicating that inactivation of mTORC1 in immature osteoblast cannot fully support normal B-cell development. Taken together, these findings demonstrate a novel role for mTORC1 in the regulation of bone marrow environments that support B-cell differentiation via regulating IL-7 expression. © 2017 American Society for Bone and Mineral Research.

Influenza virus differentially activates mTORC1 and mTORC2 signaling to maximize late stage replication.

Influenza A virus usurps host signaling factors to regulate its replication. One example is mTOR, a cellular regulator of protein synthesis, growth and motility. While the role of mTORC1 in viral infection has been studied, the mechanisms that induce mTORC1 activation and the substrates regulated by mTORC1 during influenza virus infection have not been established. In addition, the role of mTORC2 during influenza virus infection remains unknown. Here we show that mTORC2 and PDPK1 differentially phosphorylate AKT upon influenza virus infection. PDPK1-mediated phoshorylation of AKT at a distinct site is required for mTORC1 activation by influenza virus. On the other hand, the viral NS1 protein promotes phosphorylation of AKT at a different site via mTORC2, which is an activity dispensable for mTORC1 stimulation but known to regulate apoptosis. Influenza virus HA protein and down-regulation of the mTORC1 inhibitor REDD1 by the virus M2 protein promote mTORC1 activity. Systematic phosphoproteomics analysis performed in cells lacking the mTORC2 component Rictor in the absence or presence of Torin, an inhibitor of both mTORC1 and mTORC2, revealed mTORC1-dependent substrates regulated during infection. Members of pathways that regulate mTORC1 or are regulated by mTORC1 were identified, including constituents of the translation machinery that once activated can promote translation. mTORC1 activation supports viral protein expression and replication. As mTORC1 activation is optimal midway through the virus life cycle, the observed effects on viral protein expression likely support the late stages of influenza virus replication when infected cells undergo significant stress.

MTORC1 loss impairs epidermal adhesion via TGF-β/Rho kinase activation.

Despite its central position in oncogenic intracellular signaling networks, the role of mTORC1 in epithelial development has not been studied extensively in vivo. Here, we have used the epidermis as a model system to elucidate the cellular effects and signaling feedback sequelae of mTORC1 loss of function in epithelial tissue. In mice with conditional epidermal loss of the mTORC1 components Rheb or Rptor, mTORC1 loss of function unexpectedly resulted in a profound skin barrier defect with epidermal abrasions, blistering, and early postnatal lethality, due to a thinned epidermis with decreased desmosomal protein expression and incomplete biochemical differentiation. In mice with mTORC1 loss of function, we found that Rho kinase (ROCK) signaling was constitutively activated, resulting in increased cytoskeletal tension and impaired cell-cell adhesion. Inhibition or silencing of ROCK1 was sufficient to rescue keratinocyte adhesion and biochemical differentiation in these mice. mTORC1 loss of function also resulted in marked feedback upregulation of upstream TGF-β signaling, triggering ROCK activity and its downstream effects on desmosomal gene expression. These findings elucidate a role for mTORC1 in the regulation of epithelial barrier formation, cytoskeletal tension, and cell adhesion, underscoring the complexity of signaling feedback following mTORC1 inhibition.