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mTOR-inhibition: Its Role in Cellular Mechanisms

mTOR Inhibition Role in Cellular Mechanisms

Zaza, Gianluigi MD, PhD1; Granata, Simona PhD1; Caletti, Chiara MD1; Signorini, Lorenzo MD1; Stallone, Giovanni MD2; Lupo, Antonio MD1

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doi: 10.1097/TP.0000000000001806
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Abstract

MAMMALIAN TARGET OF RAPAMYCIN CELLULAR NETWORK AND ITS INHIBITION EFFECTS

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates a wide variety of eukaryotic cellular functions, mediating the signaling coming from nutrients and growth factors.1

mTOR is the constituent of 2 different complexes (mTORC1 and mTORC2) with different functions and signaling networks. Both contain mTOR, mammalian lethal with SEC13 protein 8 (mLST8), DEPTOR, and Tti1/Tel2.2-4 In addition, regulatory-associated protein of mTOR and proline-rich Akt substrate of 40 kDa are specific to mTORC1.5,6 Rapamycin-insensitive companion of mTOR (RICTOR), mSin1, and PROTOR1/2 are specific to mTORC2.7-12

mTORC1 promotes anabolic cellular metabolism stimulating synthesis of proteins, lipids, and nucleotides and, at the same time, inhibits catabolic processes, such as lysosome biogenesis and autophagy. mTORC2 controls cell survival, cytoskeleton organization, lipogenesis, and gluconeogenesis.1

RAPALOGS AND ATP-COMPETITIVE mTOR INHIBITORS

Due to their important antiproliferative and cellular effects, during the last 20 years, many rapalogs have been developed and used in the clinic as immunosuppressive medications. Among them, Sirolimus (SRL) (Rapamune; Wyeth Pharmaceuticals, New York, NY), isolated from a strain of fungus called Streptomyces hygroscopicus discovered at Rapa Nui (Easter Island), was the first mTOR inhibitor (mTOR-I) approved for use in renal transplantation. Subsequently, researchers worldwide have developed and optimized new semisynthetic rapamycin analogues with similar and more specific pharmacological properties.

Everolimus (Certican, Novartis), structurally like SRL (it has only an extra hydroxyl-ethyl chain in position 40) and characterized by better oral bioavailability, has been approved for solid organ transplant immunosuppression by the European Medicines Agency in 2003 and by the US Food and Drug Administration in 2010.

Other rapalogs, instead, have been introduced in oncology. Temsirolimus (Torisel; Wyeth Pharmaceuticals), a prodrug of SRL with better stability and solubility that render it suitable for intravenous administration, has been approved by Food and Drug Administration in 2007 for the treatment of renal cell carcinoma and by European Medicines Agency in 2009 for relapsed and refractory mantle cell lymphoma.13,14

Rapamycin and its analogues (SRL, everolimus, and temsirolimus)15 inhibit mTOR, preferentially in mTORC1, by directly blocking substrate recruitment and by further restricting active-site access.16 Interestingly, prolonged treatment with these drugs can inhibit also mTORC2, interfering with the binding of the mTORC2-specific components mSin1 and RICTOR.17

Deforolimus or Ridaforolimus, also known as AP23573, containing substituted phosphonate in C-43 secondary alcohol moiety of the cyclohexyl group of rapamycin, has been proposed for the treatment of estrogen-receptor-positive breast tumors18 and other solid cancers.19-21 However, the incomplete inhibition of the 4E binding proteins (4EBP), as well as the disruption of the negative feedback loops induced by S6K on PI3K and AKT, makes all the medications inefficient in monotherapy,22 and they require the coadministration of other compounds.23

A new class of mTOR-Is, known as TORKinibs (Torin-1, Torin-2, PP242, PP30, KU-0063794, and AZD8055), then, through a more specific ATP-competitive mechanism and the consequent inhibition of both mTOR-containing complexes and phosphorylation of Akt at S473 seem to possess better antiproliferative properties.24-29 However, now, these compounds have been only tested in animal models of cancer and in a mouse model of heart transplantation.30 AZD8055 prolonged allograft survival, promoted regulatory T (Treg) cell infiltration of the graft and inhibited T cell proinflammatory cytokine (IFNγ) production.

mTOR INHIBITION IN ORGAN TRANSPLANTATION

SRL and Everolimus have been used as immunosuppressive agents in solid organ transplantation as valid alternative to calcineurin inhibitors (CNI) mainly in patients with chronic renal dysfunction31,32 or at high risk of malignancies.

In renal transplantation, several clinical trials (eg, CONVERT, CONCEPT) have reported the improvement of renal function after CNI discontinuation and conversion to SRL.33-35 Weir et al36 demonstrated that patients undergoing an early conversion from CNI to SRL in combination with mycophenolate mofetil (MMF) presented higher mean percentage change from baseline of iothalamate GFR at 1 year compared with those treated with CNI + MMF. After 2 years, the change was indistinguishable, but calculated creatinine clearance and GFR were significantly greater in the MMF/SRL group.

Nevertheless, in the ZEUS trial, the conversion from CNI to Everolimus increased the risk for de novo donor-specific antibodies production.37 This condition could reflect underimmunosuppression rather than a drug effect.38 Undoubtedly, more studies are necessary to better address this point.

Moreover, the Symphony study reported a higher rejection rate and drop-out frequency in renal transplant recipients under mTOR-I–based regimen without CNI compared with a standard CNI-based therapy.39

Then, as reported by Furian et al,40 induction with thymoglobuline and maintenance therapy with low-dose CNIs plus mTOR-I and steroids appears to offer the best risk/benefit profile for elderly renal transplant patients.

In liver transplantation, most published studies demonstrated that conversion to SRL or everolimus from standard CNI-based therapy improved renal function, with some evidence that benefits are sustained for several years of follow-up. Converting to mTOR-I within 3 months of transplantation was associated with better renal function than converting later, presumably because irreversible glomerular damage caused by chronic CNI use was limited.41-44

In heart transplantation, in the maintenance phase, total conversion to mTOR-Is45-48 or a protocol combining mTOR-I plus low-dose CNI49-52 preserved renal function better than standard CNI-based immunosuppression.53

Furthermore, recent studies have suggested a better outcome in renal transplant patients with cancer after conversion to mTOR-I from CNI.54-56

The Rapamune Maintenance Study revealed that patients on SRL/cyclosporine/prednisone had a 5-year incidence of nonskin cancers of approximately 10% as opposed to 4% in patients converted to SRL-based immunosuppression alone. However, SRL-treated patients were exposed to high dose of SRL (trough levels, 20-30 ng/mL), which are generally not well tolerated and unused for preventing transplant rejection in current clinical practice.57

Moreover, Knoll et al,58 in a recent systematic review and meta-analysis concluded that although SRL was associated with a reduction in the risk of malignancy and nonmelanoma skin cancer in transplant recipients with a more pronounced benefit in patients who converted from an established immunosuppressive regimen to SRL, this effect was balanced with an increased risk of patient death compared with controls.

Recent clinical studies, afterward, suggest potential chemopreventive benefits of mTOR inhibitors in liver transplant recipients with hepatocellular carcinoma.59-61

Several studies, then, have demonstrated that patients treated with mTOR-I have lower cardiovascular risk factors (eg, hypertension, left ventricular hyperplasia) compared with those under CNI treatment.62-64 Joannidès et al65 reported that a cyclosporine A-free regimen based on SRL reduces aortic stiffness, plasma endothelin-1, and oxidative stress in renal transplant recipients. Conversely, the use of mTOR-I could induce hyperlipidemia and enhance levels of low density lipoprotein, cholesterol and triglycerides66-68 that are well-known cardiovascular risk factors. However, although these contrasting cardiovascular effects, to the best of our knowledge, at the state of art, no reports (including the most important clinical trials) have strongly demonstrated that in mTOR-I-treated patients the presence of cardiovascular risk factors translate into an increased incidence of cardiovascular events or cardiovascular diseases.

Although the clinical utility of mTOR-Is, their use could be associated with renal (eg, proteinuria) and systemic side effects including pulmonary toxicity, hematological disorders, dysmetabolism, lymphedema, stomatitis, cutaneous adverse effects, and fertility/gonadic toxicity.69-71

Notably, concern about the use of mTOR-I in liver transplantation stemmed from studies that reported an apparent link between hepatic artery thrombosis and regimens including SRL plus cyclosporine or tacrolimus.44

Most of the adverse effects are dose-related, and it is extremely important for clinicians to early recognize them to reduce dosage or discontinue mTOR-I treatment avoiding the onset and development of severe clinical complications.

BIOLOGICAL EFFECTS OF mTOR INHIBITION

Due to the numerous processes regulated by mTOR, its inhibition has multiple effects on: (a) protein synthesis, (b) cell cycle, (c) lipid metabolism, (d) energy metabolism, (e) autophagy, (f) angiogenesis, (g) glucose-metabolism, (h) cytoskeleton remodeling, (i) epithelial to mesenchymal transition (EMT), and (j) immune cells development and function (Figure 1 and Table 1).

FIGURE 1
FIGURE 1:
mTOR pathway reporting the proteins mediating the cellular effects influenced by mTOR inhibitors. Adapted from: Granata S, Dalla Gassa A, Carraro A, et al. Sirolimus and everolimus pathway: reviewing candidate genes influencing their intracellular effects. Int J Mol Sci. 2016; 17(5):735, published under a CC-BY 4.0 license.
TABLE 1
TABLE 1:
Main biological effects of mTOR-I

mTOR Inhibition and Protein Synthesis

The control of protein synthesis by mTOR-Is has been largely reported in literature, but the entire underlying biological machinery is still not completely defined.

They affect the first step of protein synthesis, the translation initiation, during which the small ribosome subunit is recruited to the 5′ end of mRNA and scans toward the start codon, where the complete ribosome is subsequently assembled to begin polypeptide synthesis.72 This process needs the assemblage of the eukaryotic translation initiation factor 4F (eIF4F) including 3 factors: eIF4E, eIF4G, and eIF4A. mTORC1, when activated, phosphorylates the inhibitory 4EBP1 leading to its dissociation from eIF4E and the assembly of eukaryotic translation initiation factor 4F complex.73

After mTOR inhibition, 4EBP1 is hypophosphorylated, and sequesters the cap-binding protein eIF4E interfering with its interaction with eIF4A and eIF4G and their subsequent binding to mRNA74 (Figure 2A).

FIGURE 2
FIGURE 2:
Schematic representation of mTOR mechanism of action on (A) mRNA translation, (B) cell cycle, and (C) autophagy.

Nonetheless, it has been recently proposed that the suppression of some mRNAs is due not only to the binding of 4EBP to eIF4E but also to the influence of mTOR inhibition on other factors, such as eIF4A.75 The authors supposed that some mRNAs (sensible to mTOR inhibition) are highly sensitive to suppression of the eIF4E activity or to inhibition of eIF4A activity, whereas others depend on both eIF4E and eIF4A activities.75

However, prolonged rapamycin treatment confers to mTORC1 the capacity to phosphorylate 4EBP in a rapamycin-resistant fashion. This could be due to the existence of phosphorylation sites on 4EBP1, insensitive to rapamycin.24 Alternatively, prolonged rapamycin treatment, interrupting the inhibitory feedback loop through S6K, PI3K, and AKT, could overcome the partial inhibition caused by rapamycin.24,76 Therefore, it is indisputable that pharmacological substances able to inhibit the protein kinase activity of mTOR are much more efficient to inhibit protein synthesis compared to rapamycin.77

The ribosomal proteins S6K1 and S6K2 are serine/threonine kinases, targets of mTORC1 involved in protein synthesis. mTORC1 activates S6Ks78 that in turn phosphorylate several proteins involved in mRNA translation (eg, ribosomal protein S6, eukaryotic initiation factor 4B,79 eukaryotic elongation factor 2 kinase, programmed cell death 4).

S6K1 phosphorylates the cap binding complex component eIF4B at S422 promoting its association with eIF4A at the translation initiation complex where it functions as a cofactor of eIF4A increasing its helicase activity.80 It also controls initiation of translation by phosphorylating programmed cell death 4 a tumor suppressor that inhibits eIF4A,81 and mediates its degradation by the ubiquitin ligase, β-TrCP.82

S6K1 inactivates eukaryotic elongation factor-2 kinase which is a negative regulator of eukaryotic elongation factor 2, by phosphorylating it at S366, and thus regulates the elongation step of translation.83

Another target of S6K1 is S6K1 Aly/REF-like target, a scaffold protein involved in mRNA splicing.84

Effects of the mTOR Inhibition on Cell Cycle

It is largely described that mTOR-Is inhibit cell proliferation and induce cell cycle arrest with accumulation of cells in G1 phase.85,86

Inhibition of mTOR may regulate cell proliferation through the down-regulation of cyclin D1 and upregulation of cyclin-dependent kinase inhibitors (CDKI).86 mTORC2 activates AKT and glucocorticoid-inducible kinase 187 that phosphorylate Fork head box O 3a (FOXO3a) at Thr32/Ser253 and Ser314, respectively.88 These modifications result in nuclear export of the FoxO3a with the consequent inhibition of CDKI and ultimately in cell proliferation. Based on this model mTORC2 must be constantly active to promote cell proliferation.

By contrast, inhibition of mTORC1 leads to mTORC2 and AKT hyperactivation with consequent RICTOR-mediated ubiquitination and degradation of glucocorticoid-inducible kinase 1.87 Therefore, phosphorylation of FOXO3a at Ser314 is reduced and, retained in the nucleus, it can activate the expression of CDKIs, blocking cell growth89 (Figure 2B).

Biological Consequences of the mTOR Inhibition on Lipid Metabolism

It is almost completely accepted that mTOR inhibition has a major impact on lipid metabolism with several mTORC1-mediated mechanisms having a central role.

mTORC1 positively regulates the activation of sterol regulatory element-binding proteins (SREBPs) transcription factors that regulate the synthesis of lipogenic genes and cholesterol synthesis.90,91 The exact mechanism underlying this interaction is still unidentified and several mechanisms have been proposed.

S6K1 regulates the expression of early adipogenic transcription factors including CCAAT-enhancer binding protein delta (C/EBPδ) and C/EBPβ and affects the terminal differentiation of preadipocytes.92

Another hypothesis is that mTORC1 could endorse SREBP1 processing through the induction of lipin-1, a phosphatidic acid phosphatase required for glycerolipid biosynthesis. Lipin-1 also acts as a transcriptional co-activator for many transcription factors, including peroxisome proliferator-activated receptor gamma (PPARγ) playing key roles in adipogenesis.93 When active, mTORC1 phosphorylates lipin-1, that is excluded from the nucleus. Conversely, inhibition of mTOR causes lipin-1 accumulation in the nucleus, which represses SREBP-dependent gene transcription by reducing the nuclear SREBP protein levels.94,95

Recent reports have suggested also the involvement of mTORC2 in the control of lipid biosynthesis through AKT.96

Despite these effects, the use of mTOR-I in transplantation is associated with a dose dependent dyslipidemia and consequent increased risk of cardiovascular disease. The pathogenesis of dyslipidemia seems to be due to reduced catabolism of apoB100-containing lipoproteins97 and up-regulation of apolipoprotein CIII followed by inhibition of lipoprotein lipase activity.98

Effects of mTOR-I on Energetic Metabolism

Biogenesis and activity of mitochondria, organelles centrally involved in the energetic cellular network and ATP synthesis, are regulated by several transcription factors such as nuclear respiratory factors, estrogen-related receptor and ying-yang 1 (YY1).99 All these elements interact with transcriptional coactivators peroxisome proliferator-activated receptor gamma coactivator (PGC)1-α, PGC1-β and PGC-1-related coactivator (PRC)100 with a process stimulated by mTORC1. Contrarily, mTOR inhibition, preventing the interaction between PGC1α and YY1, reduced expression of mitochondrial genes.101

Likewise, rapamycin, through the inhibition of 4EBPs, suppresses the translation of a subset of mitochondria related mRNAs: mitochondrial ribosomal proteins, subunits of complex I and V, mitochondrial transcription factor A together with a reduction in mitochondrial respiration and activity, mitochondrial DNA content and mitochondrial mass.102

Additionally, there are reports that mTOR colocalizes with mitochondria, integrating a variety of stress signals that affect mitochondrial function.103

Finally, it has been demonstrated that mTOR could mediate the activity of mitochondria through the phosphorylation and consequent activation of the antiapoptotic protein Bcl-xl.104

mTOR Inhibition and Autophagy

Autophagy is a cellular digestion process that removes damaged macromolecules and organelles. Thus, macromolecules are recycled and molecular building blocks are provided in response to nutrient deprivation and environmental stress.

Under physiological condition, mTOR inhibits autophagy to promote protein synthesis and cellular growth. mTOR inhibition caused by nutrient starvation or pharmacological treatments may induce autophagy to obtain essential molecules for cellular survival.105

The regulation of autophagy by mTOR is mainly performed on autophagy-initiating UNC-5 like autophagy activating kinase (ULK) complex, constituted by ULK1, mammalian autophagy-related gene 13 (ATG13), focal adhesion kinase family-interacting protein of 200 kDa (FIP200) and ATG101.106-108 mTORC1 inhibition increases kinase activity of ULK1 leading to downstream signal of phosphorylation and activation of ATG13 that is essential for the subsequent interaction of ULK1 with ATG14 in the PIK3C3 complex formed by AMBRA1, Beclin1, ATG14, PIK3C3 and PIK3R4. ULK1 phosphorylates also AMBRA1 and Beclin1 to initiate autophagosome formation and activation of PIK3C3 which produces phosphatidylinositol 3-phosphate (PI3P), essential for autophagy.109,110

Moreover, mTORC1 regulates the expression of lysosomal and autophagy genes through the transcription factor EB (TFEB). mTOR inhibition activates TFEB by promoting its nuclear translocation with consequent transcription of lysosomal and autophagic genes111,112 (Figure 2C).

More interestingly, Kapuy et al113 placed mTOR at a crosstalk between pathways in the control of cell survival-cell death decision. Under endoplasmic reticulum stress, inhibition of mTOR by rapamycin enhance cell viability through activation of autophagy but if endoplasmic reticulum stress is prolonged autophagy is inhibited and apoptosis is initiated.

The adaptation of the cells to nutrient starvation or stress is finalized to slow growth and to obtain essential amino acids for the synthesis of the proteins necessary for cell survival. This is obtained not only by autophagy but also through the proteolysis mediated by the ubiquitin proteasome system, a highly selective process mediated by the attachment of ubiquitin chain to the substrate through several sequential enzymes.114

It has been recently reported that mTOR inhibition causes the increment of overall protein degradation by the ubiquitin proteasome system enhancing the ubiquitination of proteins that are then degraded by the 26S proteasome.115 Opposite findings were previously reported by Zhang et al,116 who concluded that rapamycin treatment for 16 hours or longer reduces overall proteolysis by decreasing proteasome expression through decreases in the expression of the transcription factor Nrf1.

mTOR Inhibition and Angiogenesis

The antitumor properties of mTOR inhibition are related also to its role in angiogenesis. In several in vivo mouse models, rapamycin and mTOR kinase inhibitors display strong inhibitory effects on tumor growth and angiogenesis, through reduced production of vascular endothelial growth factor (VEGF).117-120 In addition, rapamycin interferes with HIF1α expression and activity reducing the expression of several genes under HIF1α control, such as VEGF.121,122

HIF-1 is a dimeric protein complex consisting of HIF-1α and HIF-1β subunits, both of which are members of the basic helix-loop-helix family of transcription factors. It regulates the expression of numerous genes for glycolytic flux and genes involved in maintaining homeostasis during hypoxia.123 In fact, the expression of the HIF-1α subunit is tightly regulated by the oxygen tension, whereas HIF-1β is a constitutively expressed nuclear protein.

The oxygen-dependent turnover of HIF-1α is controlled by many physiological conditions/regulators including mTOR.121

mTOR-I, reducing the expression of VEGF120,124 through HIF-1α, could be effective inhibitors of hypoxic adaptation and have dramatic effects on tumor growth, invasiveness, and metastatic potential in human cancer patients. Rapamycin reduces HIF1α expression and function, although with a mechanism not completely clarified. Some authors suggested that rapamycin reduces the HIF1α stability without interfere with its transcription or translation. Others have proposed the reduction of EBP1 and S6K activity mediated by mTOR inhibition91,121,125,126 but inhibition of S6K1 has no effect on VEGF levels, suggesting that VEGF expression is mediated via both HIF-1α–dependent and –independent mechanisms.127-129

Moreover, mTOR-Is induce endothelial cell elongation, an indispensable cellular event that drives angiogenesis in presence of high VEGF levels.130

Recent Evidences on Glucose Metabolism of mTOR-Is

Under physiological condition, the signal mediated by insulin leads to activation of mTORC2 that activates glycolysis by activating glucokinase and inhibits gluconeogenesis by inhibiting FoxO1 nuclear accumulation by phosphorylating AKT at Ser473.131

Chronic administration of rapamycin causes glucose intolerance, hyperglycemia, hyperinsulinemia as well as insulin resistance.132-135

Glucose intolerance and hyperglycemia are due to increased hepatic gluconeogenesis135 and impaired insulin-stimulated glucose uptake in skeletal muscle.134 Rapamycin upregulates gluconeogenesis by several mechanisms: increasing the expression of gluconeogenic enzymes (phosphoenolpyruvate carboxykinase, glucose 6-phosphatase) via increment of FOXO1 nuclear content,135 and PGC-1α mRNA expression.136 Moreover, rapamycin treatment increases nuclear recruitment of both cAMP response element–binding protein (CREB) and CREB-regulated transcription coactivator 2 (CRTC2) that stimulate the gluconeogenic program.137

It has been recently proposed that insulin resistance could be caused by inhibition of mTORC2 and reduced AKT phosphorylation.138,139

Because rapamycin treatment reduces the content and activity of PPARγ, a ligand-dependent transcription factor that enhances the expression of genes regulating glucose and lipid metabolism, it is plausible that pharmacological PPARγ activation could ameliorate rapamycin-mediated glucose intolerance.140

mTOR Inhibition Consequences on Cytoskeletal Remodeling

mTOR has a pivotal role in tumor cell migration and cancer metastasis, and rapamycin suppresses IGF-1–stimulated F-actin reorganization and migration in various tumor cell lines by inhibiting mTORC1-mediated 4EBP1 and S6K1 pathway.141

Rapamycin inhibits F-actin reorganization and cell motility through the reduced protein synthesis of the small GTPases RhoA, Rac1, and Cdc42, critical molecules for cytoskeleton organization and cell migration.142

Moreover, Letavernier et al143 reported podocyte damage after SRL treatment in renal transplant recipients. Podocytes are complex cells that develop primary and secondary foot processes forming the slit diaphragm, a unique cell-cell contact that serves as a final filtration barrier.144,145 It has been demonstrated that prolonged rapamycin treatment decreases the expression level of some proteins belonging to slit diaphragm: nephrin, transient receptor potential cation channel (TRPC6), and the cytoskeletal adaptor Nck. Similar results were obtained by another group that compared the effect of everolimus and SRL on podocyte.146 Because injury of the slit diaphragm or the actin cytoskeleton causes foot process effacement and proteinuria,144 it is plausible that rapamycin-dependent reduction of actin cytoskeleton adapters and/or core components of the slit diaphragm, like nephrin, might be responsible for mTOR inhibitors-associated proteinuria.147,148

Then, an interesting recent study has described a close link between autophagy impairment and proteinuria.149 Mice carrying a podocyte-selective knock-out of the mTOR gene exhibited an aberrant accumulation of autophagolysosomal vesicles and damaged mitochondria with suppression of the reformation of lysosomes and autophagosomes. This severe cellular deregulation has been reported in proteinuric diseases150 and in vitro model of rapamycin-treated human podocytes.

Contrarily, Jeruschke et al,151 in an in vitro model of puromycin aminonucleoside (PAN) proteinuria (a reliable model for glomerular diseases), reported that everolimus recovered aberrant podocyte behavior by reestablishing a stationary phenotype with decreased migration efficiency, enhanced cell adhesion and recovery of actin stress fibers.152 More recently, the same authors suggested that the protective role of everolimus could be mediated also by the upregulation of tubulin beta 2B class IIb (TUBB2B) and double cortin domain containing 2, 2 proteins involved in microtubules formation.

Additionally, the dual opposing effects of mTOR-I could be probably influenced by the different structure and function of podocytes. As reported by Torras et al,153 rapamycin increased proteinuria, together with a significant fall in podocyn expression and damage of foot processes in rats treated with PAN, whereas in animal undergoing 5/6 nephrectomy (chronic damage) this drug caused lower levels of proteinuria, amelioration of inflammatory and reduction of profibrotic injury. Additionally, contrarily to the puromycin model, higher glomerular podocin and nephrin expression and amelioration of glomerular ultrastructural damage were found.

m-TOR-Is, then, may have important tubular effects (including the reduction of proliferation rate of the renal epithelial tubular cells)154 that may explain the slow recovery of renal function (delayed graft function, DGF) in patients treated with these agents.155,156

EMT Induced by mTOR-I

EMT is the conversion of epithelial cells into mesenchymal phenotype through multiple biochemical changes such as enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, and greatly increased production of extracellular matrix components.157 This process is fundamental in morphogenesis and wound healing but when persistent it can lead to tissue fibrosis.

Inhibition of mTOR has been associated with a protective role in fibrosis in different tissue (kidney, skin, and liver).158-161

In an unilateral ureteral obstruction rat model mTOR is activated in myofibroblast and interstitial infiltrating macrophages but not in tubular epithelial cells during the progression of kidney interstitial fibrosis. Therefore, rapamycin ameliorates renal fibrosis by inhibiting mTOR signaling.162 This effect could be also responsible of the impaired wound healing observed in several renal transplant recipients undergoing mTOR-I treatment.162,163

It has been recently reported that rapamycin is effective in suppressing the EMT of peritoneal mesothelial cells and preventing peritoneal fibrosis.164-166 Xiang et al, infused in a rat peritoneal dialysis model, high-glucose (HG) dialysis solution for 6 weeks to induce peritoneal fibrosis. Rapamycin administered 1 or 2 mg/kg, inhibited the HG-induced EMT process by inhibiting the activation of Rho GTPases (RhoA, Rac1, and Cdc42),167 a subset of the Ras superfamily of GTPases, which can induce coordinated changes in the actin cytoskeleton dynamics and in gene transcription to drive several biological responses including cell polarity, adhesion, migration, morphogenesis, proliferation.168

In this context, we have previously demonstrated that Everolimus could have antifibrotic or profibrotic effect depending on the dosage used. HK2 cells treated with high concentrations of everolimus (higher than 100 nM) showed a significant upregulation of EMT markers (α-smooth muscle actin, vimentin, fibronectin, and matrix metalloproteinase-9) together with increment of cells motility.169 Similar results were obtained in hepatic stellate cell, human liver cancer cells (HepG2) and in bronchial/pulmonary cells.170,171 Our hypothesis is that a substantial mTORC1 inhibition may lead to a downregulation of S6K and a subsequent hyperactivation of mTORC2 that, sustaining the phosphorylation of AKT at S473, could induce a feedback loop that stimulates PI3K-AKT signaling activating the cellular/molecular machinery leading to fibrosis. AKT, once activated, could induce, through the inhibition of GSK3, the nuclear translocation of β-catenin which stimulates the expression of EMT-associated genes. All together, our results suggested that mTOR-I should be administered at the lowest dose able to maximize their important and specific therapeutic properties minimizing or avoiding fibrosis-related adverse effects.69-71

Major Effects of mTOR Inhibition on Immune Cells

As described, mTOR kinase inhibitors have higher antiproliferative potentials compared with mTOR-I. However, interestingly, in lymphocytes, this difference is not so evident172,173 because their proliferation is tightly regulated by the 4E-BP-eIF4E axis, instead of S6K. Additionally, lymphocytes, unlike other cells, express a higher amount of the 4E-BP2 isoform that increase considerably their susceptibility to the mTOR-I.174

T cells

mTOR-Is, besides the previous described antiproliferative effects, induce T-cell anergy even in presence of an adequate costimulation.175 This is due to the downregulation of the metabolic machinery implicated in the full T-cell effector function176 and the reduction of the expression of Kruppel-like factor 2, a transcription factor that, regulating the synthesis of the homing molecules (SP1, CD62L, CCR7), may induce a sequester of activated T cells in lymphoid tissues.177,178

Moreover, these drugs could orchestrate T-cell differentiation into a specific subset. Delgoffe et al179,180 found that mTORC1 inhibition (by gene silencing) in CD4+T cells impairs Th1 and Th17 cell differentiation without affecting Th2 cell generation, whereas mTORC2-deficient T cells (KO for RICTOR) fail to differentiate into Th2 cells, but retain ability to become Th1 and Th17 cells. Contrarily others authors have reported that mTORC1 is critical for Th17 but not Th1 differentiation and mTORC2 play a role for both Th1 and Th2 differentiation. This discrepancy has not been completely clarified and additional studies are necessary to better define these biological effects.

mTOR-Is may also influence the differentiation of Treg cells, a group of cells capable of inhibiting most types of immune response and having a key role in the transplantation tolerance.181-183 mTOR inhibition can both expand naturally occurring Treg cells (CD4+CD25+FoxP3+) and induce adaptive Treg cells from conventional CD4+ T cells184-190 while inhibits the proliferation of nonsuppressive, activated T cells (CD4+CD25low) and render these cells more susceptible to apoptosis.185 Upon transfer in animal model, these cells mediate graft protection.188

However, although the protolerogenic role of mTOR-I-induced Treg cells has been clearly reported in the context of basic sciences reports and experimental organ transplantation models, in human studies, only a donor specific hyporesponsiviness favored by the effect of mTOR-I on Treg cells has been described.191,192

Additionally, mTOR inhibition may have a specific effect on CD8+ T cells with a proliferation of their memory phenotype. Araki et al193 have demonstrated that rapamycin administered in mice during an acute lymphocytic choriomeningitis virus (LCMV) infection, enhanced the LCMV-specific CD8 T cells in both lymphoid and nonlymphoid tissues. Moreover, these cells showed higher levels of CD127 (IL-7 receptor α and essential for memory T-cell maintenance), CD62L (lymph node homing receptor and associated with high proliferative capacity), and the antiapoptotic Bcl2 (expressed at high levels in memory T cells), all markers associated with increased properties of self-renewing memory cells.

This mechanism could be due to the mTOR inhibition leading to a decrement of the expression level of T-bet (transcription factor regulating genes involved in Th1 differentiation) with a consequent increase in eomesodermin that, in turn, promotes the generation of memory T cells.194

Interestingly, Ferrer et al195 have recently compared the effect of rapamycin on the CD8+ T cell response to a pathogen with its effect on the CD8+ T cell response to a skin transplant. They found that rapamycin enhanced the Ag-specific T-cell response after bacterial infection but not when the same Ag was presented in the context of a transplant. These results suggested that the environment in which an Ag is presented alters the influence of rapamycin on Ag-specific T cell expansion and highlighted a difference between Ag presented by an infectious agent as compared with allograft.195

Dendritic Cells

mTOR-Is may also have effects on dendritic cells (DCs), specialized antigen presenting cells that play a critical role in adaptive immune response.196 Rapamycin inhibits phenotypic and functional maturation of DCs after exposure to IL-4, LPS or CD40 ligation197,198 and impairs their development induced by fms-like tyrosine 3 kinase ligand (Flt3L), a potent endogenous DC growth factor.199,200 In addition, mTOR-Is induce apoptosis in both human monocyte-derived (mo)-DCs and CD34+-derived DCs, but not other immune cells such as macrophages or myeloid cell lines.201

Human mo-DCs differentiated in the presence of rapamycin have a decreased expression of antigen uptake receptors (such as CD205, CD32, CD46 and CD91), antigen uptake202 together with reduced expression of co-stimulatory expression and cytokine production.199,202

Contrarily to T cells, mTOR inhibition enhances CCR7 expression on DC increasing their migration into secondary lymphoid tissue.203 This property is crucial for the tolerogenic properties ascribed to rapamycin, because it allows these cells to reach appropriate T cell areas in the lymphoid tissue.178

Furthermore, mTOR-Is regulate plasmacytoid DCs (pDC). These cells have a key role in innate immunity and in adaptive immunity. They can rapidly produce large quantities of IFN type I during viral infection and to activate NK cells through the release of IFN, IL-12, and IL-18.204 Moreover, the production of IFN and IL-12 support the effector functions of CD8+ T cells as well as the polarization of CD4+ T cells into Th1. At the same time these cells can induce the generation of CD4+ and CD8+ Treg cell.204

Cao et al205 reported that pDC stimulated with TLR9 ligand (A-D-type CpG oligodeoxynucleotide) in presence of rapamycin, showed a suppressed IFN-α/β production together with impaired ability to stimulate antigen-specific CD4+ T cell activation, probably mediated by the inhibition of the formation of the TLR9-MyD88 complex and consequent inhibition of IRF7 phosphorylation and nuclear localization.

Boor et al206 added new insights analyzing simultaneously the effects of rapamycin during activation of pDC via TLR-7 and TLR-9. Interestingly the addition of rapamycin during TLR-7 activation (but not TLR-9) induces an increment of the capacity of pDC to stimulate naïve and memory CD4+ T cells proliferation, although the generation of IFN-[Latin Small Letter Gamma] and IL-10 from naïve T cells is reduced together with the induction of CD4+FOXP3+ Treg cells.

Rapamycin is also able to modify the phenotypic characteristics of antigen presenting cells by inducing the upregulation of ILT3 and ILT4 together with a reduction of CD40 expression on their surface. These changes were associated with an increase in circulating CD8+CD28T cells and CD4+CD25+Foxp3+CTLA4+ Treg cells.207,208

Besides an anti-inflammatory action of DC preconditioned with mTOR-I, several studies have reported some proinflammatory effects of rapamycin on DC. LPS-stimulated peripheral blood mononuclear cells of kidney transplanted patients on a rapamycin-based immunosuppressive regimen displayed an elevated production of proinflammatory mediators, such as IL-12, IL-6, TNF-α, and IL-1β ex vivo compared with patients on CNI.209

Treatment of human monocytes or primary myeloid DCs with rapamycin or ATP-competitive mTOR-I enhances their production of IL-12p40 and IL-12p70 after stimulation with TLR ligands.210-212 This proinflammatory action of mTOR-I is a possible explanation of the high incidence of pulmonary inflammation and aphthous ulcers in patients treated with this class of immunosuppressant.213-215

Inhibition of mTOR with rapamycin or Torin1 prevented the anti-inflammatory potency of glucocorticoids both in human monocytes and myeloid DCs, that could explain the clinical observation that rapamycin-induced pulmonary pneumonitis cannot be ameliorated by high-dose glucocorticoid therapy.209,216

Rosborough et al,217 comparing the effects of rapamycin and Torin-1 on DC, showed that both agents reduce the expression of CD80, CD86, and B7-DC but Torin-1–enhanced B7-H1 expression on DC in a STAT3-dependent mechanism. As B7-H1 has a key role in regulating the differentiation of Treg cells, Torin-1 conditioned DC induced a greater number of Treg cells compared with rapamycin-conditioned DC defining a new rapamycin-insensitive mTORC1 pathway that restrains Treg cell induction.

B cells

Several in vitro studies reported that mTOR-Is can also influence B cell proliferation and maturation into antibody secreting cells218-221 and reduce class switching recombination.173 These effects have a key role in the pathogenesis of the antibody-mediated rejection due to preformed antibody before transplantation or de novo-specific alloantibodies after transplantation.222,223 mTOR-Is treatment could influence their formation but no consensus has emerged37,224-228 and additional studies are necessary. Probably, a personalized and late posttransplant conversion to mTOR-I monotherapy may reduce the risk of formation of de novo donor-specific antibodies.229

CONCLUSIONS

It is unquestionable that the inhibition of mTOR, besides to induce immunosuppression, may deeply impact on cell homeostasis/metabolism. In fact, as extensively describe, mTOR inhibition may control several cellular functions including (a) protein synthesis, (b) cell cycle, (c) lipid metabolism, (d) energy metabolism, (e) autophagy, (f) angiogenesis, (g) glucose metabolism, (h) cytoskeleton remodeling, and (i) EMT. Additionally, these drugs may have important pleiotropic effects on several immune cell types (including T, B, and DCs). Most of these effects, whether not accurately regulated, may induce biological and biochemical changes leading to severe drug-related toxicities.

Therefore, to know the intricate mTOR-I-related biological machinery could represent a valuable tool to potentiate therapeutic effects and minimize toxicities. However, additional studies, maybe using new technologies (eg, omics methodologies), are necessary to achieve this objective.

Finally, in the future, the development of new mTOR-targeting molecules could improve drug properties, reduce adverse effects, and extend their use in other diseases.

ACKNOWLEDGMENTS

The authors thank Dr. Francesco Zaza and Dr. Andrea Donadon for technical support in the preparation of the artwork.

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