ß-Estradiol Enhanced Secretion of Lipoprotein Lipase from Mouse Mammary Tumor FM3A Cells.

The role of ß-estradiol (E2) in lipoprotein metabolism in mammary tumors is unclear, therefore, we investigated the effect of E2 on the secretion of lipoprotein lipase (LPL) from mouse mammary tumor FM3A cells. E2-treated cells increased the secretion of active LPL from FM3A cells in a time- and dose-dependent manner. Activity of mitogen-activated protein kinase (MAPK) was increased in the tumor cells treated with E2, and enhanced secretion of LPL was suppressed by MAPK kinase 1/2 inhibitor, PD98059, extracellular signal-regulated kinase (ERK) 1/2 inhibitor, FR180204, p38 MAPK inhibitor, SB202190, and phosphatidyl inositol 3-kinase (PI3K) inhibitor, LY294002. In addition, the effect of E2 on LPL secretion was markedly suppressed by an inhibitor of mammalian target of rapamycin complex (mTORC) 1 and 2, KU0063794, but were not by a mTORC1 inhibitor, rapamycin. Furthermore, a small interfering RNA (siRNA)-mediated decrease in the expression of rapamycin-insensitive companion of mTOR (Rictor), a pivotal component of mTORC2, suppressed secretion of LPL by E2. These results suggest that the stimulatory secretion of LPL by E2 from the tumor cells is closely associated with an activation of mTORC2 rather than mTORC1 possibly via the MAPK cascade.

The role of RICTOR amplification in targeted therapy and drug resistance.

The emergence of tyrosine kinase inhibitors (TKIs) has changed the current treatment paradigm and achieved good results in recent decades. However, an increasing number of studies have indicated that the complex network of receptor tyrosine kinase (RTK) co-activation could influence the characteristic phenotypes of cancer and the tumor response to targeted treatments. One of strategies to blocking RTK co-activation is targeting the downstream factors of RTK, such as PI3K-AKT-mTOR pathway. RICTOR, a core component of mTORC2, acts as a key effector molecule of the PI3K-AKT pathway; its amplification is often associated with poor clinical outcomes and resistance to TKIs. Here, we discuss the biology of RICTOR in tumor and the prospects of targeting RICTOR as a complementary therapy to inhibit RTK co-activation.

Upregulation of 6-phosphofructo-2-kinase (PFKFB3) by hyperactivated mammalian target of rapamycin complex 1 is critical for tumor growth in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by the benign tumor formation in multiple organs. The main etiology of TSC is the loss-of-function mutation of TSC1 or TSC2 gene, which leads to aberrant activation of mammalian target of rapamycin complex 1 (mTORC1). In this research, we found a significant increase of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) expression in Tsc1-/- and Tsc2-/- mouse embryonic fibroblasts (MEFs) compared with the control cells. Inhibition of mTORC1 led to a dramatic decrease of PFKFB3 expression, indicating PFKFB3 regulation by mTORC1. Moreover, suppression of mTORC1 inhibited the expression of PFKFB3 in rat uterine leiomyoma-derived Tsc2-null ELT3 cells and human tumor cells. Furthermore, we identified hypoxia-inducible factor 1α (HIF-1α) as a mediator transmitting the signal from mTORC1 to PFKFB3. Depletion of PFKFB3 inhibited proliferation and tumorigenicity of Tsc1- or Tsc2-deficient cells. In addition, combination of rapamycin with PFK15, a PFKFB3 inhibitor, exerts a stronger inhibitory effect on cell proliferation of Tsc1- or Tsc2-null MEFs than treatment with single drug. We conclude that loss of TSC1 or TSC2 led to upregulated expression of PFKFB3 through activation of mTORC1/HIF-1α signaling pathway and co-administration of rapamycin and PFK15 may be a promising strategy for the treatment of TSC tumors as well as other hyperactivated mTORC1-related tumors.

Therapeutic inhibition of mTORC2 rescues the behavioral and neurophysiological abnormalities associated with Pten-deficiency.

Dysregulation of the mammalian target of rapamycin (mTOR) signaling, which is mediated by two structurally and functionally distinct complexes, mTORC1 and mTORC2, has been implicated in several neurological disorders1-3. Individuals carrying loss-of-function mutations in the phosphatase and tensin homolog (PTEN) gene, a negative regulator of mTOR signaling, are prone to developing macrocephaly, autism spectrum disorder (ASD), seizures and intellectual disability2,4,5. It is generally believed that the neurological symptoms associated with loss of PTEN and other mTORopathies (for example, mutations in the tuberous sclerosis genes TSC1 or TSC2) are due to hyperactivation of mTORC1-mediated protein synthesis1,2,4,6,7. Using molecular genetics, we unexpectedly found that genetic deletion of mTORC2 (but not mTORC1) activity prolonged lifespan, suppressed seizures, rescued ASD-like behaviors and long-term memory, and normalized metabolic changes in the brain of mice lacking Pten. In a more therapeutically oriented approach, we found that administration of an antisense oligonucleotide (ASO) targeting mTORC2's defining component Rictor specifically inhibits mTORC2 activity and reverses the behavioral and neurophysiological abnormalities in adolescent Pten-deficient mice. Collectively, our findings indicate that mTORC2 is the major driver underlying the neuropathophysiology associated with Pten-deficiency, and its therapeutic reduction could represent a promising and broadly effective translational therapy for neurological disorders where mTOR signaling is dysregulated.

mTORC2 Regulates Lipogenic Gene Expression through PPARγ to Control Lipid Synthesis in Bovine Mammary Epithelial Cells.

The mechanistic target of rapamycin complex 2 (mTORC2) primarily functions as an effector of insulin/PI3K signaling to regulate cell proliferation and is associated with cell metabolism. However, the function of mTORC2 in lipid metabolism is not well understood. In the present study, mTORC2 was inactivated by the ATP-competitive mTOR inhibitor AZD8055 or shRNA targeting RICTOR in primary bovine mammary epithelial cells (pBMECs). MTT assay was performed to examine the effect of AZD8055 on cell proliferation. ELISA assay and GC-MS analysis were used to determine the content of lipid. The mRNA and protein expression levels were investigated by RT/real-time PCR and western blot analysis, respectively. We found that cell proliferation, mTORC2 activation, and lipid secretion were inhibited by AZD8055. RICTOR was knocked down and mTORC2 activation was specifically attenuated by the shRNA. Compared to control cells, the expression of the transcription factor gene PPARG and the lipogenic genes LPIN1, DGAT1, ACACA, and FASN was downregulated in RICTOR silencing cells. As a result, the content of intracellular triacylglycerol (TAG), palmitic acid (PA), docosahexaenoic acid (DHA), and other 16 types of fatty acid was decreased in the treated cells; the accumulation of TAG, PA, and DHA in cell culture medium was also reduced. Overall, mTORC2 plays a critical role in regulating lipogenic gene expression, lipid synthesis, and secretion in pBMECs, and this process probably is through PPARγ. This finding provides a model by which lipogenesis is regulated in pBMECs.

MITF-MIR211 axis is a novel autophagy amplifier system during cellular stress.

Macroautophagy (autophagy) is an evolutionarily conserved recycling and stress response mechanism. Active at basal levels in eukaryotes, autophagy is upregulated under stress providing cells with building blocks such as amino acids. A lysosome-integrated sensor system composed of RRAG GTPases and MTOR complex 1 (MTORC1) regulates lysosome biogenesis and autophagy in response to amino acid availability. Stress-mediated inhibition of MTORC1 results in the dephosphorylation and nuclear translocation of the TFE/MITF family of transcriptional factors, and triggers an autophagy- and lysosomal-related gene transcription program. The role of family members TFEB and TFE3 have been studied in detail, but the importance of MITF proteins in autophagy regulation is not clear so far. Here we introduce for the first time a specific role for MITF in autophagy control that involves upregulation of MIR211. We show that, under stress conditions including starvation and MTOR inhibition, a MITF-MIR211 axis constitutes a novel feed-forward loop that controls autophagic activity in cells. Direct targeting of the MTORC2 component RICTOR by MIR211 led to the inhibition of the MTORC1 pathway, further stimulating MITF translocation to the nucleus and completing an autophagy amplification loop. In line with a ubiquitous function, MITF and MIR211 were co-expressed in all tested cell lines and human tissues, and the effects on autophagy were observed in a cell-type independent manner. Thus, our study provides direct evidence that MITF has rate-limiting and specific functions in autophagy regulation. Collectively, the MITF-MIR211 axis constitutes a novel and universal autophagy amplification system that sustains autophagic activity under stress conditions. Abbreviations: ACTB: actin beta; AKT: AKT serine/threonine kinase; AKT1S1/PRAS40: AKT1 substrate 1; AMPK: AMP-activated protein kinase; ATG: autophagy-related; BECN1: beclin 1; DEPTOR: DEP domain containing MTOR interacting protein; GABARAP: GABA type A receptor-associated protein; HIF1A: hypoxia inducible factor 1 subunit alpha; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPKAP1/SIN1: mitogen-activated protein kinase associated protein 1; MITF: melanogenesis associated transcription factor; MLST8: MTOR associated protein, LST8 homolog; MRE: miRNA response element; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; MTORC2: MTOR complex 2; PRR5/Protor 1: proline rich 5; PRR5L/Protor 2: proline rich 5 like; RACK1: receptor for activated C kinase 1; RPTOR: regulatory associated protein of MTOR complex 1; RICTOR: RPTOR independent companion of MTOR complex 2; RPS6KB/p70S6K: ribosomal protein S6 kinase; RT-qPCR: quantitative reverse transcription-polymerase chain reaction; SQSTM1: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TSC1/2: TSC complex subunit 1/2; ULK1: unc-51 like autophagy activating kinase 1; UVRAG: UV radiation resistance associated; VIM: vimentin; VPS11: VPS11, CORVET/HOPS core subunit; VPS18: VPS18, CORVET/HOPS core subunit; WIPI1: WD repeat domain, phosphoinositide interacting 1.

The role of AKT and FOXO3 in preventing ovarian toxicity induced by cyclophosphamide.

Cyclophosphamide (CTX) has immunosuppressive effects and has been wildly used as one anti-cancer drug in clinical. Significant toxicity has been noticed particularly in the reproductive system. CTX promotes the maturation of ovarian follicles, decreases follicular reserve, and ultimately lead to ovarian failure or even premature ovarian failure (POF). The placental extract (HPE) has been shown to have some beneficial impact on reproductive system; however, little is known regarding to the effect of HPE on protecting CTX-induced ovarian injury and the mechanism involved. Whether human placental extracts (HPE) has a protective effect on CTX-induced toxicity on ovarian was studied by using a CTX-induced ovarian injury animal model. The effects of HEP on histopathology, the number of atretic follicles, the weight of the ovary, serum hormone levels, and apoptosis in granulosa cells were studied in mice with CTX or control vehicle. Our results have demonstrated that HPE inhibited p-Rictor, reduced the expression of Bad, Bax and PPAR, and activated Akt and Foxo3a (increased their phosphorylation). Mice treated with HPE showed higher ovarian weight, lower number of atretic follicles, higher serum levels of the hormones E2 and progesterone, and lower apoptosis and serum levels of LH and FSH in granulosa cells, than that in the control animal group. Our data show that ovarian injury can be attenuated by HPE. HPE likely protects follicular granulosa cells from undergoing significant apoptosis and reduce atresia follicle formation, therefore, alleviates CTX-induced ovarian injury.

Unmasking the impact of Rictor in cancer: novel insights of mTORC2 complex.

Genomic alterations affecting components of the mechanistic target of rapamycin (mTOR) pathway are found rather frequently in cancers, suggesting that aberrant pathway activity is implicated in oncogenesis of different tumor types. mTOR functions as the core catalytic kinase of two distinct complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2), which control numerous vital cellular processes. There is growing evidence indicating that Rictor, an essential subunit of the mTORC2 complex, is inappropriately overexpressed across numerous cancer types and this is associated with poor survival. To date, the candidate mechanisms responsible for aberrant Rictor expression described in cancer are two: (i) gene amplification and (ii) epigenetic regulation, mainly by microRNAs. Moreover, different mTOR-independent Rictor-containing complexes with oncogenic role have been documented, revealing alternative routes of Rictor-driven tumorigenesis, but simultaneously, paving the way for identifying novel biomarkers and therapeutic targets. Here, we review the main preclinical and clinical data regarding the role of Rictor in carcinogenesis and metastatic behavior as well as the potentiality of its alteration as a target.

Hyperglycemia-induced Bcl-2/Bax-mediated apoptosis of Schwann cells via mTORC1/S6K1 inhibition in diabetic peripheral neuropathy.

Schwann cell apoptosis is one of the characteristics of diabetic peripheral neuropathy (DPN). The mammalian target of rapamycin (mTOR) is a multifunctional signaling pathway that regulates cell apoptosis in various types of tissues and cells. To investigate whether the mTOR pathway is involved in cell apoptosis in the Schwann cells of DPN, diabetic mice and rat Schwann cells (RSC96) were chosen to detect phospho-mTOR (Ser 2448), phospho-S6K1 (Thr 389), phospho-4EBP1 (Thr 37/46), Bcl-2, Bax and cleaved caspase-3 by diverse pathological and biological techniques. The results showed that phospho-mTOR (Ser 2448) was decreased in the sciatic nerves of diabetic mice, concomitant with decreased Bcl-2, increased Bax, cleaved caspase-3 and cell apoptosis. In addition, high glucose treatment for 72 h caused a 35.95% decrease in the phospho-mTOR (Ser 2448)/mTOR ratio, a 65.50% decrease in the phospho-S6K1 (Thr 389)/S6K1 ratio, a 3.67-fold increase in the Bax/Bcl-2 ratio and a 1.47-fold increase in the cleaved caspase-3/caspase-3 ratio. Furthermore, mTORC1 inhibition, rather than mTORC2 inhibition, resulted in mitochondrial controlled apoptosis in RSC96 cells by silencing RAPTOR or RICTOR. Again, suppression of the mTORC1 pathway by a chemical inhibitor led to mitochondrial controlled apoptosis in cultured RSC96 cells in vitro. By contrast, activation of the mTORC1 pathway with MHY1485 prevented decreased phospho-S6K1 (Thr 389) levels caused by high glucose and cell apoptosis. Additionally, constitutive activation of S6K1 avoided high glucose-induced cell apoptosis in RSC96 cells. In summary, our findings suggest that activating mTORC1/S6K1 signaling in Schwann cells may be a promising strategy for the prevention and treatment of DPN.

Rapamycin inhibits ox-LDL-induced inflammation in human endothelial cells in vitro by inhibiting the mTORC2/PKC/c-Fos pathway.

Rapamycin and its derivative possess anti-atherosclerosis activity, but its effects on adhesion molecule expression and macrophage adhesion to endothelial cells during atherosclerosis remain unclear. In this study we explored the effects of rapamycin on ox-LDL-induced adhesion molecule expression and macrophage adhesion to endothelial cells in vitro and the underlying mechanisms. Ox-LDL (6-48 µg/mL) dose-dependently increased the protein levels of two adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and E-selectin, in human umbilical vein endothelial cells (HUVECs), whereas pretreatment with rapamycin (1-10 µmol/L) dose-dependently inhibited ox-LDL-induced increase in the adhesion molecule expression and macrophage adhesion to endothelial cells. Knockdown of mTOR or rictor, rather than raptor, mimicked the effects of rapamycin. Ox-LDL (100 µg/mL) time-dependently increased PKC phosphorylation in HUVECs, which was abolished by rapamycin or rictor siRNA. Pretreatment with PKC inhibitor staurosporine significantly reduced ox-LDL-stimulated adhesion molecule expression and macrophage adhesion to endothelial cells, whereas pretreatment with PKC activator PMA/TPA attenuated the inhibitory effect of rapamycin on adhesion molecule expression. Ox-LDL (100 µg/mL) time-dependently increased c-Fos levels in HUVECs, and pretreatment with rapamycin or rictor siRNA significantly decreased expression of c-Fos. Knockdown of c-Fos antagonized ox-LDL-induced adhesion molecule expression and macrophage adhesion to endothelial cells. Our results demonstrate that rapamycin reduces ox-LDL-stimulated adhesion molecule expression and macrophage adhesion to endothelial cells by inhibiting mTORC2, but not mTORC1, and mTORC2 acts through the PKC/c-Fos signaling pathway.

Rictor regulates the vasculogenic mimicry of melanoma via the AKT-MMP-2/9 pathway.

Vasculogenic mimicry (VM)-positive melanomas are usually associated with poor prognosis. Rictor, the key component of the rapamycin-insensitive complex of mTOR (mTORC2), is up-regulated in several cancers, especially in melanomas with poor prognosis. The aim of this study was to investigate the role of Rictor in the regulation of VM and the mechanism underlying this possible regulation. VM channels were found in 35 of 81 tested melanoma samples and high Rictor expression correlated with VM structures. Moreover, Kaplan-Meier survival curves indicated that VM structures and high Rictor expression correlated with shorter survival in patients with melanoma. In vitro, Rictor knockdown by short hairpin RNA (shRNA) significantly inhibited the ability of A375 and MUM-2B melanoma cells to form VM structures, as evidenced by most tubes remaining open. Cell cycle analysis revealed that Rictor knockdown blocked cell growth and resulted in the accumulation of cells in G2/M phase, and cell migration and invasion were greatly affected after Rictor down-regulation. Western blotting assays indicated that down-regulating Rictor significantly inhibited the phosphorylation of AKT at Ser473 and Thr308 , which subsequently inhibited the expression and activity of downstream MMP-2/9, as confirmed by real-time PCR and gelatin Zymography. MK-2206, a small-molecule inhibitor of AKT, similarly inhibited the activity of AKT and secretion of MMP-2/9, further supporting that Rictor down-regulation inhibits the phosphorylation of AKT and activity of downstream MMP-2/9 to affect VM formation. In conclusion, Rictor plays an important role in melanoma VM via the Rictor-AKT-MMP-2/9 signalling pathway.

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 GSK3ß, 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.

Inhibition of mTORC2 component RICTOR impairs tumor growth in pancreatic cancer models.

Mammalian Target of Rapamycin complex 2 (mTORC2) and its regulatory component Rapamycin-insensitive companion of mTOR (RICTOR) are increasingly recognized as important players in human cancer development and progression. However, the role of RICTOR in human pancreatic ductal adenocarcinoma (PDAC) is unclear so far. Here, we sought to analyze the effects of RICTOR inhibition in human pancreatic cancer cell lines in vitro and in vivo. Furthermore, RICTOR expression was determined in human PDAC samples. Results demonstrate that depletion of RICTOR with siRNA (transient knock-down) or shRNA (stable knock-down) has an inhibitory effect on tumor growth in vitro. Moreover, RICTOR inhibition led to impaired phosphorylation/activity of AGC kinases (AKT, SGK1). Interestingly, hypoxia-induced expression of hypoxia-induced factor-1α (HIF-1α) was diminished and secretion of vascular-endothelial growth factor-A (VEGF-A) was impaired upon targeting RICTOR. Stable RICTOR knock-down led to significant inhibition of tumor growth in subcutaneous and orthotopic tumor models which was accompanied by significant reduction of tumor cell proliferation. Finally, immunohistochemical analyses of 85 human PDAC samples revealed significantly poorer survival in patients with higher RICTOR expression. In conclusion, these findings provide first evidence for mTORC2/RICTOR as an attractive novel target for treatment of human PDAC.

Rictor/mTORC2 deficiency enhances keratinocyte stress tolerance via mitohormesis.

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