mTORC2 Formation Research Articles

Liraglutide Improves Endothelial Function via the mTOR Signaling Pathway.

Background Mammalian target of rapamycin (mTOR) is crucial for endothelial function. This study is aimed at assessing whether the glucagon-like peptide-1 (GLP-1) analogue liraglutide has a protective effect on endothelial function via the mTOR signaling pathway. Methods Human umbilical vein endothelial cells (HUVECs) were administered liraglutide (100 nM) for 0, 10, 30, 60, 720, and 1440 minutes, respectively. Then, the expression and phosphorylation levels of mTOR, mTOR-Raptor complex (mTORC1), and mTOR-Rictor complex (mTORC2) were determined by Western blot and immunoprecipitation, while mTORC1 and mTORC2 expression was blocked by siRNA-Raptor and siRNA-Rictor, respectively. Akt phosphorylation was detected by Western blot. HUVECs were then incubated with liraglutide in the absence or presence of Akt inhibitor IV. Nitric oxide (NO) release was assessed by the nitrate reductase method. Phosphorylated endothelial nitric oxide synthase (eNOS), human telomerase reverse transcriptase (hTERT), and apoptosis-related effectors were assessed for protein levels by Western blot. Telomerase activity was evaluated by ELISA. Results Sustained mTOR phosphorylation, mTORC2 formation, and mTORC2-dependent Akt phosphorylation were induced by liraglutide. In addition, eNOS phosphorylation, NO production, nuclear hTERT accumulation, and nuclear telomerase activity were enhanced by mTORC2-mediated Akt activation. Liraglutide also showed an antiapoptotic effect by upregulating antiapoptotic proteins and downregulating proapoptotic proteins in an mTORC2-Akt activation-dependent manner. Conclusion Liraglutide significantly improves endothelial function, at least partially via the mTORC2/Akt signaling pathway.

RICTOR Amplification Promotes NSCLC Cell Proliferation through Formation and Activation of mTORC2 at the Expense of mTORC1.

Non-small cell lung cancer (NSCLC) is characterized by genomic alterations, yet a targetable mutation has not been discovered in nearly half of all patients. Recent studies have identified amplification of RICTOR, an mTORC2-specific cofactor, as a novel actionable target in NSCLC. mTORC2 is one of two distinct mTOR complexes to sense environmental cues and regulate a variety of cellular processes, including cell growth, proliferation, and metabolism, all of which promote tumorigenesis when aberrantly regulated. Interestingly, other components of mTORC2 are not coamplified with RICTOR in human lung cancer, raising the question as to whether RICTOR amplification-induced changes are dependent on mTORC2 function. To model RICTOR amplification, we overexpressed Rictor using the Cas9 Synergistic Activation Mediator system. Overexpression of Rictor increased mTORC2 integrity and signaling, but at the expense of mTORC1, suggesting that overexpressed Rictor recruits common components away from mTORC1. Additionally, Rictor overexpression increases the proliferation and growth of NSCLC 3D cultures and tumors in vivo. Conversely, knockout of RICTOR leads to decreased mTORC2 formation and activity, but increased mTORC1 function. Because Rictor has mTOR-dependent and -independent functions, we also knocked out mLST8, a shared mTOR cofactor but is specifically required for mTORC2 function. Inducible loss of mLST8 in RICTOR-amplified NSCLC cells inhibited mTORC2 integrity and signaling, tumor cell proliferation, and tumor growth. Collectively, these data identify a mechanism for Rictor-driven tumor progression and provide further rationale for the development of an mTORC2-specific inhibitor. IMPLICATIONS: RICTOR amplification drives NSCLC proliferation through formation of mTORC2, suggesting mTORC2-specific inhibition could be a beneficial therapeutic option.

Desialylation of Sonic-Hedgehog by Neu2 Inhibits Its Association with Patched1 Reducing Stemness-Like Properties in Pancreatic Cancer Sphere-forming Cells.

Cancer stem cells (CSCs) are crucial regulators of tumor recurrence/progression. The maintenance of CSCs is dependent on aberrant activation of various pathways, including Hedgehog. Prevalent sialylations contribute to aggressiveness in CSCs. Here, we have addressed the role of sialylation in regulating stemness-like properties of pancreatic cancer sphere-forming cells (PCS) through modulation of the Hedgehog (Hh) pathway. The status of CD133/CD44/surface-sialylation was checked by flow cytometry and effects of Neu2 overexpression in PCS were compared using qPCR, immunoblotting, co-immunoprecipitation and also by colony-formation assays. The work was also validated in a xenograft model after Neu2 overexpression. Neu2 and Shh status in patient tissues were examined by immunohistochemistry. PCS showed higher Hh-pathway activity and sialylation with reduced cytosolic-sialidase (Neu2). Neu2 overexpression caused desialylation of Shh, thereby reducing Shh-Patched1 binding thus causing decreased Hh-pathway activity with lower expression of Snail/Slug/CyclinD1 leading to reduction of stemness-like properties. Neu2-overexpression also induced apoptosis in PCS. Additionally, Neu2-overexpressed PCS demonstrated lower mTORC2 formation and inhibitory-phosphorylation of Gsk3β, reflecting a close relationship with reduced Hh pathway. Moreover, both Neu2 and Rictor (a major component of mTORC2) co-transfection reduced stem cell markers and Hh-pathway activity in PCS. Neu2-overexpressed tumors showed reduction in tumor mass with downregulation of stem cell markers/Shh/mTOR and upregulation of Bax/Caspase8/Caspase3. Thus, we established that reduced sialylation by Neu2 overexpression leads to decreased stemness-like properties by desialylation of Shh, which impaired its association with Patched1 thereby inhibiting the Hh pathway. All these may be responsible for enhanced apoptosis in Neu2-overexpressed PCS.

Dynamic modeling of signal transduction by mTOR complexes in cancer

Signal integration has a crucial role in the cell fate decision and dysregulation of the cellular signaling pathways is a primary characteristic of cancer. As a signal integrator, mTOR shows a complex dynamical behavior which determines the cell fate at different cellular processes levels, including cell cycle progression, cell survival, cell death, metabolic reprogramming, and aging. The dynamics of the complex responses to rapamycin in cancer cells have been attributed to its differential time-dependent inhibitory effects on mTORC1 and mTORC2, the two main complexes of mTOR. Two explanations were previously provided for this phenomenon: 1-Rapamycin does not inhibit mTORC2 directly, whereas it prevents mTORC2 formation by sequestering free mTOR protein (Le Chatelier’s principle). 2-Components like Phosphatidic Acid (PA) further stabilize mTORC2 compared with mTORC1. To understand the mechanism by which rapamycin differentially inhibits the mTOR complexes in the cancer cells, we present a mathematical model of rapamycin mode of action based on the first explanation, i.e., Le Chatelier’s principle. Translating the interactions among components of mTORC1 and mTORC2 into a mathematical model revealed the dynamics of rapamycin action in different doses and time-intervals of rapamycin treatment. This model shows that rapamycin has stronger effects on mTORC1 compared with mTORC2, simply due to its direct interaction with free mTOR and mTORC1, but not mTORC2, without the need to consider other components that might further stabilize mTORC2. Based on our results, even when mTORC2 is less stable compared with mTORC1, it can be less inhibited by rapamycin.

DNA polymerase gamma (Pol\u03b3) deficiency triggers a selective mTORC2 prosurvival autophagy response via mitochondria-mediated ROS signaling

Autophagy is a highly regulated evolutionarily conserved metabolic process induced by stress and energy deprivation. Here, we show that DNA polymerase gamma (Polγ) deficiency activates a selective prosurvival autophagic response via mitochondria-mediated reactive oxygen species (ROS) signaling and the mammalian target of rapamycin complex 2 (mTORC2) activities. In keratinocytes, Polγ deficiency causes metabolic adaptation that triggers cytosolic sensing of energy demand for survival. Knockdown of Polγ causes mitochondrial stress, decreases mitochondrial energy production, increases glycolysis, increases the expression of autophagy-associated genes, and enhances AKT phosphorylation and cell proliferation. Deficiency of Polγ preferentially activates mTORC2 formation to increase autophagy and cell proliferation, and knocking down Rictor abrogates these responses. Overexpression of Rictor, but not Raptor, reactivates autophagy in Polγ-deficient cells. Importantly, inhibition of ROS by a mitochondria-selective ROS scavenger abolishes autophagy and cell proliferation. These results identify Rictor as a critical link between mitochondrial stress, ROS, and autophagy. They represent a major shift in our understanding of the prosurvival role of the mTOR complexes and highlight mitochondria-mediated ROS as a prosurvival autophagy regulator during cancer development.

MTORC2 facilitates endothelial cell senescence by suppressing Nrf2 expression via the Akt/GSK-3β/C/EBPα signaling pathway.

Vascular endothelial cell senescence is a leading cause of age-associated and vascular diseases. Mammalian target of rapamycin complex 2 (mTORC2) is a conserved serine/threonine (Ser/Thr) protein kinase that plays an important regulatory role in various cellular processes. However, its impact on endothelial senescence remains controversial. In this study we investigated the role and molecular mechanisms of mTORC2 in endothelial senescence. A replicative senescence model and H2O2-induced premature senescence model were established in primary cultured human umbilical vein endothelial cells (HUVECs). In these senescence models, the formation and activation of mTORC2 were significantly increased, evidenced by the increases in binding of Rictor (the essential component of mTORC2) to mTOR, phosphorylation of mTOR at Ser2481 and phosphorylation of Akt (the effector of mTORC2) at Ser473. Knockdown of Rictor or treatment with the Akt inhibitor MK-2206 attenuated senescence-associated β-galactosidase (β-gal) staining and expression of p53 and p21 proteins in the senescent endothelial cells, suggesting that mTORC2/Akt facilitates endothelial senescence. The effect of mTORC2/Akt on endothelial senescence was due to suppression of nuclear factor erythroid 2-related factor 2 (Nrf2) at the transcriptional level, since knockdown of Rictor reversed the reduction of Nrf2 mRNA expression in endothelial senescence. Furthermore, mTORC2 suppressed the expression of Nrf2 via the Akt/GSK-3β/C/EBPα signaling pathway. These results suggest that the mTORC2/Akt/GSK-3β/C/EBPα/Nrf2 signaling pathway is involved in both replicative and inducible endothelial senescence. The deleterious role of mTORC2 in endothelial cell senescence suggests therapeutic strategies (targeting mTORC2) for aging-associated diseases and vascular diseases.

RETRACTED ARTICLE: mTORC2 regulates hedgehog pathway activity by promoting stability to Gli2 protein and its nuclear translocation

mTORC2 is aberrantly activated in cancer and therefore is considered to be an important therapeutic target. The hedgehog pathway, which is also often hyperactivated, regulates transcription of several genes associated with angiogenesis, metastasis, cellular proliferation and cancer stem cell (CSC) regeneration. However, the contribution of mTORC2 toward hedgehog pathway activity has not been explored yet. Here we have addressed the molecular cross talk between mTORC2 and hedgehog pathway activities in the context of glioblastoma multiforme, a malignant brain tumor using as a model system. We observed that higher mTORC2 activity enhanced the expression of a few hedgehog pathway molecules (Gli1, Gli2 and Ptch1) and amplified its target genes (Cyclin D1, Cyclin D2, Cyclin E, Snail, Slug and VEGF) both in mRNA and protein levels as corroborated by increased metastasis, angiogenesis, cellular proliferation and stem cell regeneration. Inhibition of mTORC2 formation decreased hedgehog pathway activity and attenuated all these above-mentioned events, suggesting their cross talk with each other. Further investigations revealed that mTORC2 inhibited ubiquitination of Gli2 by inactivating GSK3β, and thus it promotes stability to Gli2 and its nuclear translocation. Moreover, enhanced mTORC2 activity led to the increased clonogenic properties and CD133+ cells, indicating its role in CSC regeneration. mTORC2 inhibitor directed the reduction of hedgehog pathway proteins and also reduced CSCs. Thus, our observations support a role for elevated mTORC2 activity in regulating angiogenesis, metastasis, cellular proliferation and CSC regeneration via hedgehog pathway activity. Taken together, it provides a rationale for including the mTOR2 inhibitor as part of the therapeutic regimen for CSCs.

TRAF2 and OTUD7B govern a ubiquitin-dependent switch that regulates mTORC2 signalling.

The mechanistic target of rapamycin (mTOR) has a key role in the integration of various physiological stimuli to regulate several cell growth and metabolic pathways. mTOR primarily functions as a catalytic subunit in two structurally related but functionally distinct multi-component kinase complexes, mTOR complex 1 (mTORC1) and mTORC2 (refs 1, 2). Dysregulation of mTOR signalling is associated with a variety of human diseases, including metabolic disorders and cancer. Thus, both mTORC1 and mTORC2 kinase activity is tightly controlled in cells. mTORC1 is activated by both nutrients and growth factors, whereas mTORC2 responds primarily to extracellular cues such as growth-factor-triggered activation of PI3K signalling. Although both mTOR and GβL (also known as MLST8) assemble into mTORC1 and mTORC2 (refs 11, 12, 13, 14, 15), it remains largely unclear what drives the dynamic assembly of these two functionally distinct complexes. Here we show, in humans and mice, that the K63-linked polyubiquitination status of GβL dictates the homeostasis of mTORC2 formation and activation. Mechanistically, the TRAF2 E3 ubiquitin ligase promotes K63-linked polyubiquitination of GβL, which disrupts its interaction with the unique mTORC2 component SIN1 (refs 12, 13, 14) to favour mTORC1 formation. By contrast, the OTUD7B deubiquitinase removes polyubiquitin chains from GβL to promote GβL interaction with SIN1, facilitating mTORC2 formation in response to various growth signals. Moreover, loss of critical ubiquitination residues in GβL, by either K305R/K313R mutations or a melanoma-associated GβL(ΔW297) truncation, leads to elevated mTORC2 formation, which facilitates tumorigenesis, in part by activating AKT oncogenic signalling. In support of a physiologically pivotal role for OTUD7B in the activation of mTORC2/AKT signalling, genetic deletion of Otud7b in mice suppresses Akt activation and Kras-driven lung tumorigenesis in vivo. Collectively, our study reveals a GβL-ubiquitination-dependent switch that fine-tunes the dynamic organization and activation of the mTORC2 kinase under both physiological and pathological conditions.

PTEN negatively regulates mTORC2 formation and signaling in grade IV glioma via Rictor hyperphosphorylation at Thr1135 and direct the mode of action of an mTORC1/2 inhibitor.

To investigate the role of PTEN (phosphatase and tensin homolog) in mammalian target of rapamycin complex 2 (mTORC2) signaling in glioblastoma multiforme (GBM), we found higher activation of mTORC2 in PTENmu cells, as evidenced by enhanced phosphorylation of mTOR (Ser2481), AKT (Ser473) and glycogen synthase kinase 3 beta (GSK3β) (Ser9) as compared with PTENwt cells. In addition, PTENwt cells upon PTEN depletion showed mTORC2 activation. The reduced mTORC2 signaling in PTENwt cells was related to higher Rictor phosphorylation at Thr1135 residue. Phosphorylation of Rictor at Thr1135 inhibited its association with mTORC and thus there was a reduction in mTORC2 complex formation. In addition, PTENwt cells expressing mutated Rictor in which Thr1135 was substituted with alanine, showed enhanced mTORC2 formation and signaling. This enhanced mTORC2 signaling promoted inactivation of GSK3β. Thus, we established the reciprocal activation of mTORC2 and GSK3β in GBM. To the best of our knowledge, this is the first report describing role of PTEN in mTORC2 formation by promoting Rictor phosphorylation (Thr1135) in GBM. Furthermore, the drug sensitivity of mTORC2 was evaluated. A newly identified carbazole alkaloid, mahanine, showed cytotoxicity in both PTENmu and PTENwt cells. It inhibited both mTORC1/2 and AKT completely in PTENmu cells, whereas it inhibited only mTORC1 in PTENwt cells. Cytotoxity and AKT-inhibitory activity of the mTORC1/2 inhibitor was increased either by depleting PTEN or in combination with phosphatidylinositol 3 kinase inhibitors in PTENwt cells. In contrast, depletion of Rictor decreased the cytotoxicity of the mTORC1/2 inhibitor in PTENmu cells. Thus, PTEN has an important role in mTORC2 formation and also influences the effectiveness of an mTORC1/2 inhibitor in GBM.

Rictor Undergoes Glycogen Synthase Kinase 3 (GSK3)-dependent, FBXW7-mediated Ubiquitination and Proteasomal Degradation

Rictor, an essential component of mTOR complex 2 (mTORC2), plays a pivotal role in regulating mTOR signaling and other biological functions. Posttranslational regulation of rictor (e.g. via degradation) and its underlying mechanism are largely undefined and thus are the focus of this study. Chemical inhibition of the proteasome increased rictor ubiquitination and levels. Consistently, inhibition of FBXW7 with various genetic means including knockdown, knock-out, and enforced expression of a dominant-negative mutant inhibited rictor ubiquitination and increased rictor levels, whereas enforced expression of FBXW7 decreased rictor stability and levels. Moreover, we detected an interaction between FBXW7 and rictor. Hence, rictor is degraded through an FBXW7-mediated ubiquitination/proteasome mechanism. We show that this process is dependent on glycogen synthase kinase 3 (GSK3): GSK3 was associated with rictor and directly phosphorylated the Thr-1695 site in a putative CDC4 phospho-degron motif of rictor; mutation of this site impaired the interaction between rictor and FBXW7, decreased rictor ubiquitination, and increased rictor stability. Finally, enforced activation of Akt enhanced rictor levels and increased mTORC2 activity as evidenced by increased formation of mTORC2 and elevated phosphorylation of Akt, SGK1, and PKCα. Hence we suggest that PI3K/Akt signaling may positively regulate mTORC2 signaling, likely through suppressing GSK3-dependent rictor degradation.

Achilles' heel of triple negative cancer.

Achilles' heel of triple negative cancer.

Inhibitory effect of 14,15-EET on endothelial senescence through activation of mTOR complex 2/Akt signaling pathways

Therapies to reverse the vascular endothelial aging process may play as a novel strategy for the treatment of cardiovascular diseases. 14,15-epoxyeicosatrienoic acid (14,15-EET) is a predominant cytochrome P450 epoxygenases-derived arachidonic acid metabolite and possesses multiple biological effects on the vascular system. The present study sought to investigate the roles of mammalian target of rapamycin complex 2 (mTORC2)/Akt signaling pathways in mediating the effect of 14,15-EET on endothelial senescence. By measuring the isometric tension in rat mesenteric arteries, we demonstrated that 14,15-EET improved the impaired endothelium-dependent vasodilatation in aged rats through activating mTORC2/Akt signaling pathway. Meanwhile, by promoting the formation of mTORC2 and the phosphorylation of Akt (Ser473), 14,15-EET inhibited the senescence of rat mesenteric arterial endothelial cells, which was not influenced by rapamycin but was significantly attenuated by Akt1/2 kinase inhibitor. The knockdown of Rictor gene by RNA interference abolished the inhibitory effect of 14,15-EET on endothelial senescence. Furthermore, 14,15-EET down-regulated the expression of p53 protein in aged endothelial cells. Meanwhile, the nuclear translocation of telomerase reverse transcriptase and the nuclear telomerase activity were also enhanced by treatment with 14,15-EET. Therefore, our present study suggests the crucial role of mTORC2/Akt signaling pathways in the inhibitory effects of 14,15-EET on the endothelial senescence. Our findings reveal important mechanistic clues to understanding of the effects of 14,15-EET on the endothelial functions.

Critical role of SCD1 in autophagy regulation via lipogenesis and lipid rafts-coupled AKT-FOXO1 signaling pathway

SCD1 (stearoyl-coenzyme A desaturase 1) is an endoplasmic reticulum-bound enzyme that catalyzes the formation of the first double bond at the cis-Δ9 position of saturated fatty acids (SFA) to form monounsaturated fatty acids (MUFA). Increasing evidence indicates that autophagy plays an important role in regulating lipid metabolism, while little is known about whether key enzymes of lipogenesis like SCD1 can regulate autophagy. In this study, we examined the role of SCD1 in autophagy using the tsc2−/− mouse embryonic fibroblasts (MEFs) possessing constitutively active MTORC1 as a cellular model. We found that mRNA and protein levels of SCD1 are significantly elevated in the tsc2−/− MEFs compared with Tsc2+/+ MEFs, resulting in significant increases in levels of various lipid classes. Furthermore, inhibition of SCD1 activity by either a chemical inhibitor or genetic knockdown resulted in an increase of autophagic flux only in the tsc2−/− MEFs. Induction of autophagy was independent of MTOR as MTORC1 activity was not suppressed by SCD1 inhibition. Loss of phosphorylation on AKT Ser473 was observed upon SCD1 inhibition and such AKT inactivation was due to disruption of lipid raft formation, without affecting the formation and activity of MTORC2. Increased nuclear translocation of FOXO1 was observed following AKT inactivation, leading to increased transcription of genes involved in the autophagic process. The tsc2−/− MEFs were also more susceptible to apoptosis induced by SCD1 inhibition and blockage of autophagy sensitized the cell death response. These results revealed a novel function of SCD1 on regulation of autophagy via lipogenesis and the lipid rafts-AKT-FOXO1 pathway.

MiR-424/503-Mediated Rictor Upregulation Promotes Tumor Progression

mTOR complex 2 (mTORC2) signaling is upregulated in multiple types of human cancer, but the molecular mechanisms underlying its activation and regulation remain elusive. Here, we show that microRNA-mediated upregulation of Rictor, an mTORC2-specific component, contributes to tumor progression. Rictor is upregulated via the repression of the miR-424/503 cluster in human prostate and colon cancer cell lines that harbor c-Src upregulation and in Src-transformed cells. The tumorigenicity and invasive activity of these cells were suppressed by re-expression of miR-424/503. Rictor upregulation promotes formation of mTORC2 and induces activation of mTORC2, resulting in promotion of tumor growth and invasion. Furthermore, downregulation of miR-424/503 is associated with Rictor upregulation in colon cancer tissues. These findings suggest that the miR-424/503–Rictor pathway plays a crucial role in tumor progression.

MTOR inhibition with rapamycin causes impaired insulin signalling and glucose uptake in human subcutaneous and omental adipocytes

Rapamycin is an immunosuppressive agent used after organ transplantation, but its molecular effects on glucose metabolism needs further evaluation. We explored rapamycin effects on glucose uptake and insulin signalling proteins in adipocytes obtained via subcutaneous (n=62) and omental (n=10) fat biopsies in human donors.At therapeutic concentration (0.01μM) rapamycin reduced basal and insulin-stimulated glucose uptake by 20–30%, after short-term (15min) or long-term (20h) culture of subcutaneous (n=23 and n=10) and omental adipocytes (n=6 and n=7). Rapamycin reduced PKB Ser473 and AS160 Thr642 phosphorylation, and IRS2 protein levels in subcutaneous adipocytes. Additionally, it reduced mTOR–raptor, mTOR–rictor and mTOR–Sin1 interactions, suggesting decreased mTORC1 and mTORC2 formation. Rapamycin also reduced IR Tyr1146 and IRS1 Ser307/Ser616/Ser636 phosphorylation, whereas no effects were observed on the insulin stimulated IRS1–Tyr and TSC2 Thr1462 phosphorylation.This is the first study to show that rapamycin reduces glucose uptake in human adipocytes through impaired insulin signalling and this may contribute to the development of insulin resistance associated with rapamycin therapy.

TORC-Specific Phosphorylation of Mammalian Target of Rapamycin (mTOR): Phospho-Ser2481 Is a Marker for Intact mTOR Signaling Complex 2

The mammalian target of rapamycin (mTOR) serine/threonine kinase is the catalytic component of two evolutionarily conserved signaling complexes. mTOR signaling complex 1 (mTORC1) is a key regulator of growth factor and nutrient signaling. S6 kinase is the best-characterized downstream effector of mTORC1. mTOR signaling complex 2 (mTORC2) has a role in regulating the actin cytoskeleton and activating Akt through S473 phosphorylation. Herein, we show that mTOR is phosphorylated differentially when associated with mTORC1 and mTORC2 and that intact complexes are required for these mTORC-specific mTOR phosphorylations. Specifically, we find that mTORC1 contains mTOR phosphorylated predominantly on S2448, whereas mTORC2 contains mTOR phosphorylated predominantly on S2481. Using S2481 phosphorylation as a marker for mTORC2 sensitivity to rapamycin, we find that mTORC2 formation is in fact rapamycin sensitive in several cancer cell lines in which it had been previously reported that mTORC2 assembly and function were rapamycin insensitive. Thus, phospho-S2481 on mTOR serves as a biomarker for intact mTORC2 and its sensitivity to rapamycin.

MTOR regulates expression of slit diaphragm proteins and cytoskeleton structure in podocytes

The immunosuppressive mammalian target of rapamycin (mTOR) inhibitors can cause proteinuria, especially in kidney and heart transplanted patients. Podocytes play a major role in establishing the selective permeability of the blood-urine filtration barrier. Damage of these cells leads to proteinuria, a hallmark of most glomerular diseases. Interestingly, podocyte damage and focal segmental glomerulosclerosis can occur after treatment with an mTOR inhibitor in some transplant patients. To investigate the mechanisms of mTOR inhibitor-induced podocyte damage, we analyzed the effect of rapamycin on mTOR signaling and cellular function in human podocytes. We found that prolonged rapamycin treatment reduced the expression of total mTOR, which correlates with diminished levels of mTOR phosphorylation at Ser(2448) and Ser(2481). In addition, treatment with rapamycin reduced rictor expression and mTORC2 formation, resulting in a reduced phosphorylation of protein kinase B at Ser(473). The expression level of the slit-diaphragm proteins nephrin and transient receptor potential cation channel 6 as well as the cytoskeletal adaptor protein Nck significantly decreased. Moreover, rapamycin reduced cell adhesion and cell motility, which was accompanied by an enhanced formation of dot-like actin-rich structures. Our data provide new molecular insights explaining which pathways and molecules are affected in podocytes by an imbalanced mTOR function because of rapamycin treatment.

Targeting the Hypoxic Microenvironment by mTOR Inhibitors Sensitizes ALL Cells to Chemotherapy

The main therapeutic challenge in the treatment of Acute Lymphocytic Leukemia (ALL) is the development of strategies aimed at overcoming resistance to chemotherapy. Interactions between leukemia cells and microenvironment promote leukemia cell survival and confer resistance to drugs commonly used to treat ALL. Recent reports indicate that the endosteum at the murine bone-bone marrow (BM) interface is hypoxic, and data in a rat model demonstrate that leukemic cells infiltrating bone marrow were markedly hypoxic compared to cells in bone marrow of healthy rats. Hypoxia-inducible factor 1α (HIF-1α) is a key regulator of the cellular response to hypoxia. To characterize expression and function of HIF-1α in the bone marrow from ALL patients, HIF-1α expression was analyzed by immunohistochemistry in the bone marrow specimens from 16 newly diagnosed patients with pre-B ALL. HIF-1α was found to be expressed in 10/16 samples tested (62.5%). Of the 16 patients, 5 patients subsequently relapsed, all of which have expressed HIF-1α at diagnosis. No relapses were seen in the 6 patients with negative HIF-1α levels at presentation. To examine the molecular mechanisms of survival of leukemic cells growing under hypoxic conditions of bone marrow microenvironment, we established a co-culture system of pre-B ALL cells with BM-derived mesenchymal stem cells (MSC). Culture of REH cells under hypoxia (1% O2) resulted in induction of HIF-1α protein which was further increased in leukemia/stroma co-culture. Exposure of cells to hypoxia resulted in robust activation of AKT phosphorylation in leukemic cells. We have recently demonstrated that rapamycin analogs inhibit AKT signaling in AML cells via inhibition of mTORC2 formation (Zeng et al., Blood 109:3509-12, 2007). Likewise, mTOR inhibition by RAD001 completely blocked HIF-1α and pAKT in REH cells. Importantly, REH cells co-cultured with MSC under hypoxia/high glucose environment exhibited significantly lower apoptotic rates (p=0.02) and growth inhibition (p=0.002) in response to vincristine, and these effects were reversed by mTOR blockade with RAD001. We have further demonstrated that inhibition of mTOR signaling reduced expression of the glucose transporter Glut-1 and diminished glucose flux, decreased glycolytic rate and ATP production, both in leukemic cell lines and in primary ALL blasts (n=8). This was associated with decreased mitochondrial membrane potential and inhibition of the hypoxia-induced hexokinase (HKII) in the mitochondrial fraction of ALL cells. In summary, data suggest that mTOR/AKT signaling critically controls HIF-1α expression and function in ALL cells studied under the hypoxic conditions characteristic of bone marrow microenvironment. Hence, mTOR inhibition or blockade of HIF-1α-mediated pro-survival signaling events may reverse microenvironment-mediated chemoresistance and improve clinical outcomes in ALL.

Hsp70 associates with Rictor and is required for mTORC2 formation and activity

mTORC2 is a multiprotein kinase composed of mTOR, mLST8, PRR5, mSIN1 and Rictor. The complex is insensitive to rapamycin and has demonstrated functions controlling cell growth, motility, invasion and cytoskeletal assembly. mTORC2 is the major hydrophobic domain kinase which renders Akt fully active via phosphorylation on serine 473. We isolated Hsp70 as a putative Rictor interacting protein in a yeast two-hybrid assay and confirmed this interaction via co-immunoprecipitation and colocalization experiments. In cells expressing an antisense RNA targeting Hsp70, mTORC2 formation and activity were impaired. Moreover, in cells lacking Hsp70 expression, mTORC2 activity was inhibited following heat shock while controls demonstrated increased mTORC2 activity. These differential effects on mTORC2 activity were specific, in that mTORC1 did not demonstrate Hsp70-dependent alterations under these conditions. These data suggest that Hsp70 is a component of mTORC2 and is required for proper assembly and activity of the kinase both constitutively and following heat shock.

Rapamycin Analogs Reduce mTORC2 Signaling and Inhibit AKT Activation in AML.

The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be rapamycin-insensitive, and was recently shown to regulate the pro-survival kinase AKT by phosphorylation on Ser473 (Sarbassov Science 2005;307 and Mol Cell 2006;22). We investigated the molecular effects of mTOR inhibition by rapamycin analog CCI-779 in AML cells. Unexpectedly, CCI-779 not only inhibited the mTOR complex 1 (mTORC1) containing mTOR and raptor with decreased phosphorylation of p70S6K, 4EPB1 and reduction in Glut-1 mRNA, but also blocked AKT activation via inhibition of mTORC2 formation. This resulted in suppression of phosphorylation of the direct AKT substrate FKHR and decreased transcription of D-Cyclins in AML cell line and 5 of 8 primary AML samples in vitro. Similar observations were made in samples from patients with hematological malignancies who were treated with the rapamycin analogs temsirolimus or everolimus: the levels of Ser473 phosphorylated AKT decreased in 3/5 patient samples at 1 or 24 hour(s) of temsirolimus treatment, and in 6/8 patient samples treated with everolimus. In the 9 samples in which AKT was inhibited, ≥2-fold decrease in Cyclin D1 mRNA was observed in 5, Cyclin D2 in 3, both, Cyclin D1 and D2 in 1 sample, and Glut-1 in 4 patient samples. In 7 of the 9 patients in whom AKT was inhibited, a >50% decrease in peripheral blood absolute blast count (3 AML, 1 ALL) or absolute lymphocyte count (1 CLL) for >1 week duration was documented, and two patients with RAEB-1 had improvements in platelet counts, one fulfilling the criteria for clinical response (hematological improvement). No change in peripheral blood counts or progression of leukemia was seen in 6 patients. Of these, decrease in pAKT was observed in 2, no change in 3 and increase in 1 (Fisher exact two-tailed p= 0.021). Altogether, our study provides first evidence that rapamycin analogs inhibit AKT signaling in primary AML cells both in vitro and in vivo, and support the therapeutic potential of mTOR inhibition strategies in leukemias.