Role Of mTORC1 Research Articles

Abstract 670: Targeting PI3K/mTOR leads to MEK/ERK over-activation in pancreatic cancer through suppression of mTORC2

Abstract Significant attention has been given to molecular target therapies in pancreatic cancer due to the limited survival benefits provided by standard chemotherapy. The phosphatidylinositol 3-kinase (PI3K)/Akt/mTORC1/S6K pathway which is aberrantly stimulated in pancreatic ductal adenocarcinoma (PDAC) has emerged as a target for therapy. Inhibitors of PI3K and/or mTOR (PI3K/TOR-KIs) are been developed to target this pathway. However, mTORC1/S6K also mediates negative feedback loops that attenuate upstream signaling. Suppression of these feedback loops opposes the growth-suppressive effects of mTOR inhibitors and leads to drug resistance. Here, we demonstrate that treatment of a panel of pancreatic ductal adenocarcinoma (PDAC) cells including PANC-1, MiaPaCa-2, AsPC-1 and BxPC-3 with the dual PI3K/mTOR kinase inhibitor (PI3K/TOR-KI) NPV-BEZ23 induces a novel negative feedback loop leading to increased activity of MEK/ERK pathway in PDAC cells. Exposure of the above mentioned PDAC cells to NPV-BEZ235 potently blocked mTORC1 activation mTORC1/S6K activation (scored by S6 phosphorylation at Ser240/244), mTORC1/4E-BP1 (assayed by 4E-BP1 phosphorylation at Thr37/46) and mTORC2-mediated Akt phosphorylation at Ser473, in a concentration-dependent manner. Strikingly, NPV-BEZ235 markedly enhanced the MEK/ERK pathway in a dose-dependent manner. Maximal ERK over-activation coincided with complete inhibition of phosphorylation of Akt and 4E-BP1. Other PI3K/TOR-KIs, including PKI-587 and GDC-0980 also induced ERK over-activation. The MEK inhibitors PD0325901 or Trametinib prevented ERK over-activation induced by PI3K/TOR-KIs at least acutely. The combination of NPV-BEZ235 and PD0325901 caused a more pronounced inhibition of cell growth than that produced by each inhibitor individually. Mechanistic studies assessing PI3K activity in single PDAC cells using a PIP3 fluorescent reporter indicated that NPV-BEZ235, PKI-587 and GDC-0980 act through a PI3K-independent pathway. Doses of PI3K/TOR-KIs that enhanced MEK/ERK activation coincided with those that inhibited mTORC2-mediated Akt phosphorylation on Ser473, suggesting a role of mTORC2. Knockdown of Rictor markedly increased baseline levels of ERK phosphorylation and treatment with NVP-BEZ235 did not produce further enhancement of ERK activation. These results imply that Rictor or mTORC2 exerts feedback inhibition of the MEK/ERK pathway in pancreatic cancer cells. We propose that dual PI3K/mTOR inhibitors suppress a novel negative feedback loop mediated by mTORC2 thereby leading to enhanced MEK/ERK pathway activity in pancreatic cancer cells. The therapeutic effectiveness of PI3K/TOR-KIs inhibitors in PDAC and possibly other malignancies can be diminished by activation of MEK/ERK that opposes their anti-proliferative effects and leads to drug resistance. Note: This abstract was not presented at the meeting. Citation Format: Heloisa P. Soares, Ming Ming, Michelle Mellon, Steven H. Young, Liang Han, James Sinnet-Smith, Enrique Rozengurt. Targeting PI3K/mTOR leads to MEK/ERK over-activation in pancreatic cancer through suppression of mTORC2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 670. doi:10.1158/1538-7445.AM2015-670

Abstract IA07: The TSC complex links PI3K to mTOR and cancer metabolism

Abstract Metabolic processes within cells must be managed by integrated control mechanisms that sense the nutrient status of both the cell and organism. The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) is a key signaling node, universal to eukaryotic cells, which links the sensing of nutrients to the coordinated regulation of cellular metabolism. mTORC1 has the ability to sense and integrate signals from a variety of sources, including intracellular nutrients and secreted growth factors. mTORC1 senses many of these signals through a small G protein switch involving the TSC1-TSC2-TBC1D7 complex (the TSC complex) and the Ras-related small G protein Rheb. The direct phosphorylation and regulation of the TSC complex by Akt provides the major mechanistic link between PI3K signaling and the control of mTORC1. Recent studies from our lab have revealed the molecular nature of this regulation and how it serves to spatially integrate the major signals upstream of mTORC1. Our studies reveal that the TSC complex blocks mTORC1 activation by inhibiting Rheb on the lysosomal surface, where mTORC1 is recruited in response to amino acids. PI3K-Akt signaling results in acute, phosphorylation-dependent dissociation of the TSC complex from lysosomal Rheb, allowing Rheb to become GTP loaded and activate mTORC1. Importantly, oncogenic PI3K signaling activates mTORC1 in cancer cells by promoting chronic dissociation of the TSC complex from Rheb, resulting in uncontrolled mTORC1 signaling. The physiological and pathological activation of mTORC1 results in downstream changes in cellular metabolism, with a shift from catabolic processes to anabolic biosynthetic processes required for cell growth and proliferation. Through unbiased genomic and metabolomic approaches, we have found that, in addition to its established role in promoting protein synthesis, mTORC1 stimulates changes in specific metabolic pathways through transcriptional and post-translational effects on metabolic enzymes. In this manner, mTORC1 serves to link growth signals to metabolic processes that promote growth, including the de novo synthesis of proteins, lipids, and nucleic acids. In addition, we have recently uncovered a novel and surprising role for mTORC1 in controlling cellular protein and amino acid homeostasis through the coordinated induction of both protein synthesis and proteasome-dependent protein degradation. This seemingly paradoxical function of mTORC1 serves as both a quality control mechanism to handle the increase in misfolded proteins that accompanies elevated rates of protein synthesis and as a means of maintaining adequate pools of intracellular amino acids to sustain new protein synthesis. The potential implications of these novel downstream functions of mTORC1 under physiological and pathological states will be discussed. Citation Format: Brendan D. Manning. The TSC complex links PI3K to mTOR and cancer metabolism. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA07.

Abstract B28: Rictor/mTORC2 drives formation, progression, and therapeutic resistance of HER2-amplified breast cancers

Abstract Although HER2-targeted therapies substantially improve the outcome for patients with HER2-amplified breast cancer, resistance to HER2 targeting is frequently observed. HER2-amplified tumors depend on the HER2-mediated activation of the phosphatidylinositol-3kinase (PI3K)/Akt pathway, which becomes reactivated in many therapeutically resistant HER2-amplified tumors. We investigated the mTOR complexes mTORC1 and mTORC2 as potential therapeutic targets in HER2-amplified breast cancers, based on their roles as activators (mTORC2) or effectors (mTORC1) of Akt. We found that genomic gains in Rictor, encoding a necessary mTORC2 cofactor, correlated with poor survival in breast cancer patients, while genomic gains in Raptor, an mTORC1 cofactor, did not. Rictor protein levels were increased in invasive breast cancers as compared to ductal carcinoma in situ (DCIS). Conditional Rictor gene ablation decreased growth, cell survival and invasion of human HER2-amplified breast cancer cells by blocking Akt signaling. Treatment of HER2 positive cells with the HER2/EGFR inhibitor Lapatinib caused Rictor upregulation. However, Rictor knockdown improved cell killing in Lapatinib-treated cells and impaired Akt signaling and cell survival in Lapatinib-resistant HER2 positive breast cancer cells. Together, these results demonstrate the pivotal role of mTORC2 in HER2-amplified breast cancers and support efforts to develop mTORC2-targeted therapies. Citation Format: Meghan M. Morrison, Bayley A. Jones, Donna J. Hicks, Violeta Sanchez, Valeria M. Estrada, Michelle M. Williams, Dana Brantley-Sieders, Rebecca S. Cook. Rictor/mTORC2 drives formation, progression, and therapeutic resistance of HER2-amplified breast cancers. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B28.

Abstract IA11: Targeting PI3K-mTOR signaling in glioblastoma: A central role for mTORC2 in drug resistance and metabolic reprogramming

Abstract Metabolic reprogramming is a central hallmark of cancer, enabling tumor cells to obtain the macromolecular precursors and energy needed for rapid tumor growth. mTOR plays a central role in this process. Understanding how mTOR coordinates metabolic reprogramming may yield new insights into tumor pathogenesis, and provide a landscape of promising drug targets, while yielding important clues into mechanisms of resistance to the signal transduction inhibitors currently in use. This talk will focus on the emerging central regulatory role for mTOR complex 2 (mTORC2) in cancer metabolic reprogramming and targeted therapy resistance highlighting a recently identified, novel, Akt-independent, c-Myc-dependent pathway. Citation Format: Paul Mischel. Targeting PI3K-mTOR signaling in glioblastoma: A central role for mTORC2 in drug resistance and metabolic reprogramming. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA11.

MTORC2 Deficiency in Myeloid Dendritic Cells Enhances Their Allogeneic Th1 and Th17 Stimulatory Ability after TLR4 Ligation In Vitro and In Vivo.

The mammalian/mechanistic target of rapamycin (mTOR) is a key integrative kinase that functions in two independent complexes, mTOR complex (mTORC) 1 and mTORC2. In contrast to the well-defined role of mTORC1 in dendritic cells (DC), little is known about the function of mTORC2. In this study, to our knowledge, we demonstrate for the first time an enhanced ability of mTORC2-deficient myeloid DC to stimulate and polarize allogeneic T cells. We show that activated bone marrow-derived DC from conditional Rictor(-/-) mice exhibit lower coinhibitory B7-H1 molecule expression independently of the stimulus and enhanced IL-6, TNF-α, IL-12p70, and IL-23 production following TLR4 ligation. Accordingly, TLR4-activated Rictor(-/-) DC display augmented allogeneic T cell stimulatory ability, expanding IFN-γ(+) and IL-17(+), but not IL-10(+) or CD4(+)Foxp3(+) regulatory T cells in vitro. A similar DC profile was obtained by stimulating Dectin-1 (C-type lectin family member) on Rictor(-/-) DC. Using novel CD11c-specific Rictor(-/-) mice, we confirm the alloreactive Th1 and Th17 cell-polarizing ability of endogenous mTORC2-deficient DC after TLR4 ligation in vivo. Furthermore, we demonstrate that proinflammatory cytokines produced by Rictor(-/-) DC after LPS stimulation are key in promoting Th1/Th17 responses. These data establish that mTORC2 activity restrains conventional DC proinflammatory capacity and their ability to polarize T cells following TLR and non-TLR stimulation. Our findings provide new insight into the role of mTORC2 in regulating DC function and may have implications for emerging therapeutic strategies that target mTOR in cancer, infectious diseases, and transplantation.

Inhibition of mTOR complex 2 induces GSK3/FBXW7-dependent degradation of sterol regulatory element-binding protein 1 (SREBP1) and suppresses lipogenesis in cancer cells.

Cancer cells feature increased de novo lipogenesis. Sterol regulatory element-binding protein 1 (SREBP1), when presented in its mature form (mSREBP1), enhances lipogenesis through increasing transcription of several of its target genes. Mammalian target of rapamycin (mTOR) complexes, mTORC1 and mTORC2, are master regulators of cellular survival, growth and metabolism. A role for mTORC1 in the regulation of SREBP1 activity has been suggested; however the connection between mTORC2 and SREBP1 has not been clearly established and hence is the focus of this study. mTOR kinase inhibitors (e.g., INK128), which inhibit both mTORC1 and mTORC2, decreased mSREBP1 levels in various cancer cell lines. Knockdown of rictor, but not raptor, also decreased mSREBP1. Consistently, reduced mSREBP1 levels were detected in cells deficient in rictor or Sin1 compared to parent or rictor-deficient cells with re-expression of ectopic rictor. Hence it is mTORC2 inhibition that causes mSREBP1 reduction. As a result, expression of the mSREBP1 target genes acetyl-CoA carboxylase and fatty acid synthase was suppressed, accompanied with suppressed lipogenesis in cells exposed to INK128. Moreover, mSREBP1 stability was reduced in cells treated with INK128 or rictor knockdown. Inhibition of proteasome, GSK3 or the E3 ubiquitin ligase, FBXW7, prevented mSREBP1 reduction induced by mTORC2 inhibition. Thus mTORC2 inhibition clearly facilitates GSK3-dependent, FBXW7-mediated mSREBP1 degradation, leading to mSREBP1 reduction. Accordingly, we conclude that mTORC2 positively regulates mSREBP1 stability and lipogenesis. Our findings reveal a novel biological function of mTORC2 in the regulation of lipogenesis and warrant further study in this direction.

Mammalian target of rapamycin complex 2 (mTORC2) regulates LPS-induced expression of IL-12 and IL-23 in human dendritic cells.

IL-12 p40, a common subunit for both IL-12 p70 and IL-23, plays a critical role in the development of Th1 and Th17 cells and autoimmune diseases. Regulation of IL-12 p40 expression is thus considered to be a strategy for developing therapies for Th1- and Th17-mediated autoimmune diseases. The mTOR protein is a subunit mTORC1 and mTORC2. Although mTORC1 has been shown to mediate IL-12 p40 expression in DCs and relevant signaling, the role of mTORC2 in IL-12 p40 expression remains largely unclear. In the present study, we demonstrate that blocking mTORC2 activity using the phytochemical cytopiloyne can specifically inhibit LPS-induced expression of IL-12 p70, IL-23, and IL-12 p40 in human DCs. This regulation by mTORC2 involving Akt activation and the persistent phase of NF-κB activation is further confirmed by siRNA knockdown of Rictor and Sin1 gene expression and the use of alternative inhibition approaches. In terms of IL-12 p40 expression, our findings reveal a new role for the mTORC2 pathway that is antagonistic to that of mTORC1. Our study provides new insight into mTOR regulation of IL-12 p40-mediated Th1 (IFN-γ) and Th17 (IL-17) responses and suggests that the phytochemical cytopiloyne might have useful applications in therapies for Th1 and Th17 cell-mediated inflammatory diseases.

Roles of mTORC1 in acute myeloid leukemia

Constitutive activation of mTOR is associated with acceleration of leukemia development. However, mTORC1 functions in established leukemia are unclear. We evaluated the role of mTORC1 in mouse acute myeloid leukemia (AML) cells using a murine model of conditional deletion of Raptor, an essential component of mTORC1, and an MLL-AF9-driven leukemia model. mTORC1 is essential for leukemia initiation, but a subset of AML cells with undifferentiated phenotypes survived long-term in the absence of mTORC1 activity. Our study demonstrated AML cells lacking mTORC1 activity to be capable of self-renewal as AML stem cells. This review will summarize our study on the roles of mTORC1 in AML, and discuss potential benefits and risks of mTORC2 inhibition and the potential use of mTOR inhibitors as AML therapy.

Tu1973 The Novel Observation of Irreversible Electroporation Phenomenon on Gastrointestinal Cancer Cells in Physiological Conditions, With Sub-Micrometer Resolution by Scanning Ion Conductance Microscopy

G A A b st ra ct s enhanced MEK/ERK activation coincided with those that inhibited mTORC2-mediated Akt phosphorylation on Ser473, suggesting a role of mTORC2. Knockdown of Rictor markedly increased baseline levels of ERK phosphorylation and treatment with NVP-BEZ235 did not produce further enhancement of ERK activation. These results imply that Rictor or mTORC2 exerts feedback inhibition of the MEK/ERK pathway in pancreatic cancer cells. Conclusion: We propose that dual PI3K/mTOR inhibitors suppress a novel negative feedback loop mediated by mTORC2 thereby leading to enhanced MEK/ERK pathway activity in pancreatic cancer cells.

Tu1975 H2S Inhibition of Cystathionine-β-Synthase (CBS) Using Novel Prodrug Decreases Colorectal Cancer Xenograft Growth With Less Toxicity Than Aminooxyacetic Acid (AOAA)

G A A b st ra ct s enhanced MEK/ERK activation coincided with those that inhibited mTORC2-mediated Akt phosphorylation on Ser473, suggesting a role of mTORC2. Knockdown of Rictor markedly increased baseline levels of ERK phosphorylation and treatment with NVP-BEZ235 did not produce further enhancement of ERK activation. These results imply that Rictor or mTORC2 exerts feedback inhibition of the MEK/ERK pathway in pancreatic cancer cells. Conclusion: We propose that dual PI3K/mTOR inhibitors suppress a novel negative feedback loop mediated by mTORC2 thereby leading to enhanced MEK/ERK pathway activity in pancreatic cancer cells.

The necessary role of mTORC1 in central nervous system axon regeneration.

Permanent loss of vital functions after central nervous system (CNS) injury, e.g., blindness in traumatic optic nerve (ON) injury or paralysis in spinal cord injury, occurs in part because axons in the adult mammalian CNS do not regenerate after injury. Growth failure is due to the diminished intrinsic regenerative capacity of mature neurons and the inhibitory environment of the adult CNS. Neutralizing extracellular inhibitory molecules genetically or pharmacologically yields only limited regeneration and functional recovery, highlighting the critical importance of neuron-intrinsic factors. To explore the intrinsic regenerative signaling molecules, a relatively simple but robust in vivo model that replicates CNS traumatic injury and permits straightforward interpretation is required. Mouse retinal ganglion cell (RGC) and ON provide a valuable in vivo neural injury system to study intrinsic growth mechanisms in adult CNS neurons (Figure 1). Retina and ON are CNS structures that are essential for visual functions. RGC is the only neuronal type to relay visual information from retina to brain. The ON is formed by the projection axons sent exclusively from RGCs; it has the simplicity of a unidirectional axon pathway, which insures that any nerve fibers observed distal to a complete crush injury have regenerated and do not represent spared axons that underwent collateral sprouting or efferent axons from the brain to the retina. Its easy access allows adeno-associated viruses to be injected directly into the vitreous chamber of the eye and express transgenes specifically and efficiently in adult RGCs. This spatially and temporally controlled genetic manipulation allows us to overcome developmental issues associated with germ line manipulation and to test interventions that can potentially be translated to therapies. Exploiting the anatomical and technical advantages of the RGC/ON crush model to understand the intrinsic mechanisms of regenerative failure led us to the finding that deletion of phosphatase and tensin homolog (PTEN) promoted significant ON regeneration (Park et al., 2008). PTEN, a lipid phosphatase, is a major negative regulator of the phosphatidylinositol 3-kinase (PI3K)-mammalian target of rapamycin complex 1 (mTORC1) pathway. Similar axon regeneration phenotypes after PTEN deletion have been reported for mouse cortical motor neurons (Liu et al., 2010), drosophila sensory neurons (Song et al., 2012) and C. elegans motor neurons (Byrne et al., 2014), presumably through activating PI3K-mTORC1-controlled cell growth (Figure 2). Direct activation of mTORC1 also promotes axon regeneration in dopaminergic neurons (Kim et al., 2011) and peripheral nerves (Abe et al., 2010), further support for the critical role of mTORC1 in axon regeneration.

DNA damage-induced S and G2/M cell cycle arrest requires mTORC2-dependent regulation of Chk1.

mTOR signalling is commonly dysregulated in cancer. Concordantly, mTOR inhibitors have demonstrated efficacy in a subset of tumors and are in clinical trials as combination therapies. Although mTOR is associated with promoting cell survival after DNA damage, the exact mechanisms are not well understood. Moreover, since mTOR exists as two complexes, mTORC1 and mTORC2, the role of mTORC2 in cancer and in the DNA damage response is less well explored. Here, we report that mTOR protein levels and kinase activity are transiently increased by DNA damage in an ATM and ATR-dependent manner. We show that inactivation of mTOR with siRNA or pharmacological inhibition of mTORC1/2 kinase prevents etoposide-induced S and G2/M cell cycle arrest. Further results show that Chk1, a key regulator of the cell cycle arrest, is important for this since ablation of mTOR prevents DNA damage-induced Chk1 phosphorylation and decreases Chk1 protein production. Furthermore, mTORC2 was essential and mTORC1 dispensable, for this role. Importantly, we show that mTORC1/2 inhibition sensitizes breast cancer cells to chemotherapy. Taken together, these results suggest that breast cancer cells may rely on mTORC2-Chk1 pathway for survival and provide evidence that mTOR kinase inhibitors may overcome resistance to DNA-damage based therapies in breast cancer.

Abstract PR02: Negative regulation of myogenesis by Mtor: A pathway toward differentiation therapy in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, is composed of skeletal myoblast-like cells that have lost the capacity to terminally differentiate. This suggests that RMS cells may contain a factor that blocks normal muscle differentiation. Because cell cycle arrest is coupled to muscle differentiation, identifying putative negative regulators of differentiation could lead to novel therapeutic approaches aimed at fostering terminal differentiation. To gain insight into the events that normally trigger the initial phase of muscle differentiation, we carried out a high content cell-based screen using detection of Myogenin, an early marker of muscle differentiation, as a readout that we measured by automated fluorescence microscopy. We focused on identifying kinases that hinder this developmental transition because of the potential to develop pharmacological inhibitors of “hit” kinases. Using an siRNA library targeting over 600 kinases, we identified 56 as putative negative regulators of myogenic differentiation: 43 with and 19 without PD332991, a Cdk4/6 inhibitor included as a sensitizer. Network analysis showed that 47% of the hits identified without the sensitizer, including SRC family kinases Src and Fyn, were just one interaction node away from MyoD- or Mef2-family myogenic regulatory factors. Although some of the hit kinases were previously implicated in myogenesis, many others were not. Further, 9 (16%) of the hits are causally linked with cancer, based on KEGG and COSMIC databases. Pathway analyses highlighted certain cancer-associated pathways, like EPHA/Ephrin B signaling, the MAPK kinase pathway, de novo pyrimidine synthesis, and mTOR signaling. Among the hits relevant to RMS, Mtor was particularly interesting because this gene encodes a kinase regarded to positively regulate skeletal muscle differentiation. We confirmed our screen findings that Mtor blocks muscle differentiation by showing that its knockdown increases the transcription of a panel of muscle-specific genes in an established myoblast cell line and primary mouse myoblasts. Induction of muscle genes by Mtor knockdown correlated with G0/G1 cell cycle arrest that normally accompanies differentiation. Rapamycin mimicked the effects of Mtor knockdown on muscle gene expression and cell proliferation, implying a role for mTorc1. Finally, preliminary analysis of RMS gene expression data demonstrated that higher expression of muscle differentiation genes correlates with improved survival. These data highlight the potential for mTOR inhibitors to foster the expression of muscle specific genes, pushing the myoblast-like tumor cells to a more differentiated state. Altering their biology in this way may improve outcome. On-going efforts are exploring the mechanisms acting downstream of Mtor to impede muscle differentiation and directly evaluating pro-differentiation effects of mTOR inhibition in RMS models. This abstract is also presented as Poster A1. Citation Format: Raphael A. Wilson, Jing Liu, Lin Xu, Yanbin Zheng, Stephen X. Skapek. Negative regulation of myogenesis by Mtor: A pathway toward differentiation therapy in rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR02.

Abstract 2441: mTOR complex 2 modulates glycosylation of CD147 via the hexosamine biosynthetic pathway

Abstract Cell surface proteins transduce extracellular signals into the cell to control metabolism and growth. In turn, their expression is linked to nutrient availability and other growth signals by mechanisms that are poorly understood. The mammalian target of rapamycin (mTOR) regulates cell growth and metabolism and is part of two distinct protein complexes, mTOR complex 1 (mTORC1) and mTORC2. We found that mTORC2 is involved in the processing and maturation of cell surface receptors such as CD147. CD147 is a highly glycosylated receptor that has been linked to tumor progression via its role in activating matrix metalloproteinases and maturation of lactate transporters. In breast cancer cells, CD147 glycosylation is highly sensitive to glucose starvation and mTOR inhibition. In mTORC2-disrupted cells CD147 is misprocessed and occurs predominantly in a low glycosylated form. CD147 misprocessing can be partly rescued by addition of exogenous UDP-GlcNAc, the end product of the hexosamine biosynthetic pathway (HBP). However, UDP-GlcNAc cannot restore the abnormal growth and metabolism in mTORC2-disrupted cells due to defects in expression of other key metabolic enzymes in these cells. Our findings define a role for mTORC2 in regulating receptor glycosylation via the HBP and reveal a broader role for mTORC2 in controlling other biosynthetic pathways that become deregulated in cancer. Citation Format: Chang-Chih Wu, Thomas Lynch, Joseph Moloughney, Aixa Navia, Olufunmilola Ibironke, Po-Chien Chou, Nicole M. Vega-Cotto, Sisi Zhang, Joshua Rabinowitz, Guy Werlen, Estela Jacinto. mTOR complex 2 modulates glycosylation of CD147 via the hexosamine biosynthetic pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2441. doi:10.1158/1538-7445.AM2014-2441

Abstract 4050: A central role for mTORC1 in CXCR4-mediated directional migration and metastasis

Abstract Tumor cells can co-opt the promigratory activity of chemokines and their cognate G protein-coupled receptors to disseminate to regional lymph nodes and distant organs. Indeed, the migration to SDF-1 of tumor cells expressing its receptor, CXCR4, is implicated in lymphatic and organ-specific metastasis in multiple human malignancies. However, the critical role of CXCR4 in bone marrow stem cell retention has hampered the development of CXCR4 inhibitors for metastasis prevention. Therefore the study of CXCR4 downstream signaling circuitry may provide new opportunities for the treatment of many human malignancies that depend on CXCR4 for their metastatic spread. The PI3K/mTOR pathway represents a major player in normal and aberrant cell growth. Consistently, mTOR inhibitors targeting its complex 1 (mTORC1) alone or mTORC1/mTORC2 have already shown promising therapeutic responses in many tumor types. Our data show that mTOR is activated upon SDF-1 stimulation of CXCR4 on epithelial-derived cancer cells, and that the disruption of mTORC1 or mTORC2 abrogates CXCR4-mediated directional migration. To study the CXCR4/mTOR axis in vivo we developed a simple and robust model for CXCR4-mediated spontaneous metastasis. Surprisingly, disruption of mTORC1 alone was sufficient to decrease tumor cell proliferation, angiogenesis, lymphangiogenesis, and CXCR4-mediated metastasis, whereas mTORC2 impairment had no effect on tumor dissemination or growth in vivo. Taken together, our data suggest that mTORC1 blockade could inhibit the migration of CXCR4 expressing cancer cells to their secondary sites, while disrupting the establishment of a permissive tumor microenvironment thereby halting the spread of highly aggressive tumors that require CXCR4 to metastasize. Citation Format: Patricia Dillenburg-Pilla, Vyomesh Patel, Constantinos M. Mikelis, Carlos Rodrigo Zarate-Blades, Panomwat Amornphimoltham, Zhiyong Wang, Daniel Martin, Kantima Leelahavanichkul, Colleen L. Doçi, Robert T. Dorsam, Andrius Masedunskas, Nijiro Nohata , Roberto Weigert, Alfredo A. Molinolo, J. Silvio Gutkind . A central role for mTORC1 in CXCR4-mediated directional migration and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4050. doi:10.1158/1538-7445.AM2014-4050

Abstract 442: Cardiomyocyte Specific mTORC1 Deletion Precipitates Interstitial Fibrosis, Heart Failure And Loss Of Female Cardio Protection

Mechanistic target of rapamycin (mTOR) kinase operates in two functionally and structurally distinct multiprotein complexes mTORC1 and mTORC2. mTORC1 including its defining raptor protein is a critical mediator of myocardial growth. We induced cardiomyocyte specific raptor-KO (cKO) deletion to investigate a sex specific role of mTORC1 during cardiac adaptation in normotensive DOCA-salt mice. Untypical for this mild cardiac stress model, raptor-cKO females had sharper decrease in EF (51 vs. 26%) than males (50 vs. 32%) already at 3 weeks after start of DOCA-salt challenge. Male raptor-cKO mice mounted eccentric hypertrophic response upon DOCA-salt indicated by heart weight/tibia length ratios (6.7 to 8.0) as compared to their WT litters (6.6 to 7.6). In contrast female raptor-cKO (5.8 to 6.4) minimally differed from their litters (5.8 to 6.6). Severe dilative phenotype was detected in both sexes regardless of DOCA-salt challenge. In addition, we found tremendous diffuse left ventricular fibrosis which was also more prominent in raptor-cKO females. Collagen depositions in the LV visualized by Sirius red staining increased 10fold in males (1.0 to 10.6%) and 15fold in females (0.9 to 15.7%), respectively. Expression of raptor protein in the heart was diminished by 80% in raptor cKO mice of both sexes what lead to similar extent of decreased activity of direct mTORC1 downstream targets phospho-S6 ribosomal protein and phospho-4E-BP1Ser65 (68% reduction in males vs. 61% in females. Over-compensatory mTORC2 activation in cKO animals via phospho-AKTSer473 (12fold increase) and induction of anti-apoptotic signaling was similar in both sexes. However, raptor-cKO females failed to up-regulate expression of mitochondrial genes such as Mtnd1 (ND1), mt-Co1 (COX1) and Atp5i (ATP5k), stressing sex differences in mTORC1 dependent mitochondrial adaptation. In addition, transmission electron microscopy (TEM) points towards structural alterations of mitochondrial morphology. Intact mTORC1 function is important for myocardial adaptation in both sexes. More severe phenotype in females further implicates mTORC1 to be essential for intrinsic female cardio protection.

Mammalian target of rapamycin complex 1 orchestrates invariant NKT cell differentiation and effector function.

Invariant NKT (iNKT) cells play critical roles in bridging innate and adaptive immunity. The Raptor containing mTOR complex 1 (mTORC1) has been well documented to control peripheral CD4 or CD8 T cell effector or memory differentiation. However, the role of mTORC1 in iNKT cell development and function remains largely unknown. By using mice with T cell-restricted deletion of Raptor, we show that mTORC1 is selectively required for iNKT but not for conventional T cell development. Indeed, Raptor-deficient iNKT cells are mostly blocked at thymic stage 1-2, resulting in a dramatic decrease of terminal differentiation into stage 3 and severe reduction of peripheral iNKT cells. Moreover, residual iNKT cells in Raptor knockout mice are impaired in their rapid cytokine production upon αGalcer challenge. Bone marrow chimera studies demonstrate that mTORC1 controls iNKT differentiation in a cell-intrinsic manner. Collectively, our data provide the genetic evidence that iNKT cell development and effector functions are under the control of mTORC1 signaling.

Rictor is required for early B cell development in bone marrow

Mammalian target of rapamycin (mTOR) receives signals such as growth factors, nutrients and stress, and regulates cell growth and proliferation. mTOR forms mTORC1 and mTORC2 complexes and have been shown to have distinct functions in hematopoietic stem cells (HSCs). The early B cells are developed from HSC through a series of well-characterized stages in bone marrow (BM) and are regulated by PI3K/Akt/mTOR signaling. However, the role of mTORC2 in the development of early B cells is still not fully understood.

MTORC1 and JNK coordinate phosphorylation of the p70S6K1 autoinhibitory domain in skeletal muscle following functional overloading.

The present project was designed to investigate phosphorylation of p70S6K1 in an animal model of skeletal muscle overload. Within 24 h of male Sprague-Dawley rats undergoing unilateral tenotomy to induce functional overloading of the plantaris muscle, phosphorylation of the Thr³⁸⁹ and Thr⁴²¹/Ser⁴²⁴ sites on p70S6K1 was significantly elevated. Since the Thr⁴²¹/Ser⁴²⁴ sites are purportedly mammalian target of rapamycin complex 1 (mTORC1) independent, we sought to identify the kinase(s) responsible for their phosphorylation. Initially, we used IGF-I treatment of serum-deprived HEK-293E cells as an in vitro model system, because IGF-I promotes phosphorylation of p70S6K1 on both the Thr³⁸⁹ and Thr⁴²¹/Ser⁴²⁴ sites in skeletal muscle and in cells in culture. We found that, whereas the mTOR inhibitor TORIN2 prevented the IGF-I-induced phosphorylation of the Thr⁴²¹/Ser⁴²⁴ sites, it surprisingly enhanced phosphorylation of these sites during serum deprivation. JNK inhibition with SP600125 attenuated phosphorylation of the Thr⁴²¹/Ser⁴²⁴ sites, and in combination with TORIN2 both the effect of IGF-I and the enhanced Thr⁴²¹/Ser⁴²⁴ phosphorylation during serum deprivation were ablated. In contrast, both JNK activation with anisomycin and knockdown of the mTORC2 subunit rictor specifically stimulated phosphorylation of the Thr⁴²¹/Ser⁴²⁴ sites, suggesting that mTORC2 represses JNK-mediated phosphorylation of these sites. The role of JNK in mediating p70S6K1 phosphorylation was confirmed in the animal model noted above, where rats treated with SP600125 exhibited attenuated Thr⁴²¹/Ser⁴²⁴ phosphorylation. Overall, the results provide evidence that the mTORC1 and JNK signaling pathways coordinate the site-specific phosphorylation of p70S6K1. They also identify a novel role for mTORC1 and mTORC2 in the inhibition of JNK.

Fatty acids are novel nutrient factors to regulate mTORC1 lysosomal localization and apoptosis in podocytes

Podocyte apoptosis is a potent mechanism of proteinuria in diabetic nephropathy. More detailed mechanistic insight into podocyte apoptosis is needed to better understand the pathogenesis of diabetic nephropathy. An elevated level of serum free fatty acid (FFA), as well as hyperglycemia, is a clinical characteristic in diabetes, although its causal role in podocyte apoptosis remains unclear. This study examined the effect of three types of FFAs, saturated, monounsaturated and polyunsaturated FFAs, on podocyte apoptosis. Palmitate, a saturated FFA, induced endoplasmic reticulum (ER) stress-dependent apoptosis in podocytes. Oleate, a monounsaturated FFA, and eicosapentaenoic acid (EPA), an ω−3 polyunsaturated FFA did not induce apoptosis; rather, they antagonized palmitate-induced apoptosis. Palmitate activated mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a nutrient-sensing kinase regulating a wide range of cell biology. Furthermore, inhibition of mTORC1 activity by rapamycin or siRNA for Raptor, a component of mTORC1, ameliorated palmitate-induced ER stress and apoptosis in podocytes. Activity of mTORC1 is regulated by upstream kinases and Rag/Ragulator-dependent recruitment of mTOR onto lysosomal membranes. Palmitate activated mTORC1 by enhancing recruitment of mTOR onto lysosomal membranes, which was inhibited by co-incubation with oleate or EPA. Inhibition of mTOR translocation onto lysosomes by transfection with dominant-negative forms of Rag ameliorated palmitate-induced apoptosis. This study suggests that saturated and unsaturated FFAs have opposite effects on podocyte apoptosis by regulating mTORC1 activity via its translocation onto lysosomal membranes, and the results provide a better understanding of the pathogenesis in diabetic nephropathy and a novel role of mTORC1 in cell apoptosis.