G Model

DESC-2829; No. of Pages 8 Journal of Dermatological Science xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Journal of Dermatological Science journal homepage: www.jdsjournal.com

Invited review article

Mammalian target of rapamycin and tuberous sclerosis complex Mari Wataya-Kaneda * Department of Dermatology, Course of Integrated Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 8 April 2015 Accepted 16 April 2015

Mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase that is a member of the phosphoinositide 3-kinase (PI3K)-related kinase (PIKK) family. mTOR forms two distinct complexes, mTORC1 and mTORC2. mTORC1 has emerged as a central regulator of cellular metabolism, cell proliferation, cellular differentiation, autophagy and immune response regulation. In contrast to mTORC1, mTORC2, which is not well understood, participates in cell survival and the regulation of actin and cytokeratin organization. In addition, mTORC1 has been implicated in many diseases, including cancer, metabolic diseases, neurological disease, genetic diseases and longevity/aging. One of the diseases resulting from dysfunction of mTORC1 is tuberous sclerosis complex (TSC), which reflects all the symptoms that arise in response to mTORC1 dysfunction. TSC is a multiple hamartomas syndrome with epilepsy, autism, mental retardation and hypopigmented macules that are caused by the constitutive activation of mTORC1 resulting from genetic mutation of TSC1 or TSC2. Inhibitors of mTORC1, such as rapamycin, effectively suppress the symptoms of TSC. This article summarizes the current knowledge on mTOR and the efficacy of mTORC1 inhibitors in the treatment of TSC. ß 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Keywords: Mammalian target of rapamycin (mTOR) Tuberous sclerosis complex (TSC) Rapamycin Autophagy Energy metabolism PI3K-Akt-mTOR pathway

Contents 1. 2. 3. 4. 5.

6. 7. 8. 9.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . History of mammalian target of rapamycin (mTOR) . Structures of mTORC1 and mTORC2 . . . . . . . . . . . . . . mTORC1 and mTORC2 signal transduction pathways Downstream mTOR cascade . . . . . . . . . . . . . . . . . . . . Growth and proliferation . . . . . . . . . . . . . . . . . 5.1. Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. mTOR signaling pathway and disease. . . . . . . . . . . . . Tuberous sclerosis complex. . . . . . . . . . . . . . . . . . . . . Rapamycin, an mTOR inhibitor . . . . . . . . . . . . . . . . . . Treatment of TSC using mTORC1 inhibitors . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

000 000 000 000 000 000 000 000 000 000 000 000 000 000

1. Introduction

* Correspondence to: Department of Dermatology, Course of Integrated Medicine, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: +81 668 79 3031; fax: +81 668 79 3039. E-mail address: [email protected]

Mammalian target of rapamycin (mTOR) is an evolutionally conserved protein kinase and is an essential regulator of a wide range of functions. The pathway is involved in cell viability, growth, autophagy, cellular senescence and immune reactions. Therefore, deregulation of the mTOR pathways has been

http://dx.doi.org/10.1016/j.jdermsci.2015.04.005 0923-1811/ß 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Wataya-Kaneda M. Mammalian target of rapamycin and tuberous sclerosis complex. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.04.005

G Model

DESC-2829; No. of Pages 8 M. Wataya-Kaneda / Journal of Dermatological Science xxx (2015) xxx–xxx

2

3. Structures of mTORC1 and mTORC2

implicated in many diseases or disorders, such as cancers, neurodegenerative diseases, metabolic diseases, genetic disorders, and immune diseases. Over the past two decades, significant progress has been made in elucidating the regulatory mechanisms and functions of mTOR. This review article summarizes the current knowledge on mTOR and human diseases affected by the mTOR pathway, and refers to the topics of tuberous sclerosis complex (TSC), a representative genetic disease that results from mTORC1 hyperactivity.

The two mTORCs, mTORC1 and mTORC2, are multiprotein complexes. Mammalian target of rapamycin complex 1 (mTORC1) is composed of mTOR, regulatory-associated protein of mTOR (Raptor), the DEP domain-containing mTOR-interacting protein (Deptor), mammalian lethal with SEC13 protein 8/G-protein b-subunit-like protein (mLST8/GbL), and protein-rich Akt substrate of 40-kDa (PRAS40) [2,3]. Mammalian target of rapamycin complex 2 (mTORC2) is composed of mTOR, rapamycin-insensitive companion of mTOR (Rictor), mLST8 and mSin1 (mammalian stress-activated protein kinase-interacting protein 1). Recently, two new mTORC related proteins, Tel2-interacting protein 1 (Tti1) and telomere maintenance 2 (Tel2), were identified that anchor mLST8 and Raptor/Rictor to mTOR in mTORC1 and mTORC2, respectively [4] (Fig. 1). mTOR is a large multi-domain protein. The N-terminal portion of mTOR contains huntingtin, elongation factor 3, A subunit of PP2A, TOR1 (HEAT) repeats. These HEAT repeats form a large helical secondary structure that provides a protein interaction surface for Raptor and Rictor. The C-terminal portion of mTOR contains several important domains. FRAP-ATM-TRRAP (FAT) domains and FAT-C-terminal (FATC) domains are conserved domains among PIKK family members and are necessary for mTOR catalytic function. The FKBP12–rapamycin complex binds to the FKBP12–rapamycin complex binding (FRB) domain, which is adjacent to the FAT domain. The FRB domain is also involved in the

2. History of mammalian target of rapamycin (mTOR) Target of rapamycin (TOR) is a large (300 kDa) conserved serine/threonine kinase that is part of the PI3K-related kinase family. Over the past two decades, significant progress has been made in elucidating the regulation and function of TOR. TOR was discovered in yeast by genetic selection for mutants that confer rapamycin resistance [1]. In 1994, TOR was shown to be conserved in mammalian cells. Therefore, the mammalian homolog of TOR is called mammalian target of rapamycin (mTOR). In 2002, TOR was discovered to form two distinct kinases complexes, target of rapamycin complex 1 (TORC1) and target of rapamycin complex 2 (TORC2). TORC1 and TORC2 are structurally and functionally distinct kinases, each of which phosphorylates its own substrates to control different cellular processes. Similar to TOR, the two TOR complexes (TORC1 and TORC2) are conserved from yeast to human.

Growth factors IRS1

S6K

PI3K RAS RAf

PDK1

Translaon Transcripon

GSK3

Akt TNFα

MEK

IKK- b ERK hamarn

GATOR1

V-APTase

Amino acid

Ragulators

RSK tuberin

AMPK

RagA/B

Wnt fizzled

RagC/D hypoxia tuberin

hamarn Akt

14-3-3 protein

PRAS40

hamarn

mLST8 mTOR

TTL1/ TEL2

tuberin

mTORC1

Protor

AMPK raptor

RSK

LKB1

GSK3

REDD1

mLST8 TTL1/ TEL2

mTOR

mTORC2

mSIN1

PRA540

HiF-1a

4E-BP1

S6K

SREBP1

ULK1

NF- κB

PGC1a

S6K/Akt SGK1

ATG13

Rho, Rac

PKC

GATA3

SGK1

PKC α

FIP200 elF 4E

Glycolysis Angiogenesis

S6

elf4B

PPAR γ

Translaonal regulaon Protein synthesis Lipid synthesis Cell growth Tumorigenesis

YY1

Autophagy

FOXO1/3A

Inflammaon

Mitochondrial biogenesis

Cell survival

Differenaon and funcon of Acn cytoskeleton immune cells Sodium organizaon transport Cell to cell contact

Fig. 1. Overview of mTOR signaling pathway. Critical inputs regulating mTORC1 and mTORC2 and the key outputs of the mTOR1 and mTORC2 pathways are summarized. mTORC1 controls large amount of biological processes and regulates protein synthesis, lipogenesis energy metabolism/mitochondrial biogenesis, autophagy and immunity. mTORC2 regulates survival/metabolism and cytoskeleton organization.

Please cite this article in press as: Wataya-Kaneda M. Mammalian target of rapamycin and tuberous sclerosis complex. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.04.005

G Model

DESC-2829; No. of Pages 8 M. Wataya-Kaneda / Journal of Dermatological Science xxx (2015) xxx–xxx

interaction between mTOR and other mTORC members, including Raptor and ras homolog enriched in the brain (Rheb). The catalytic or kinase (KIN) domain is flanked by the FAT domains and encodes the serine/threonine kinase activity of mTOR. Within the KIN domain, there is a region known as the negative regulatory domain (NRD). The NRD domain contains serine and threonine residues that are phosphorylated and are involved in the regulation of mTOR activity. For example, threonine 2246 is targeted by AMPK and S6K, serine 2448 is a target of Akt and S6K, and serine 2481 is an autocatalytic target of mTOR (Fig. 2).

4. mTORC1 and mTORC2 signal transduction pathways mTOR governs and integrates input from multiple signals, including insulin, growth factors, energy, stress, mitogens, and amino acids. Insulin-like growth factors can activate mTORC1 through the receptor tyrosine kinase (RTK)-Akt/protein kinase B (PKB)-TSC signaling pathway. Growth factors, cytokines, and other factors stimulate mTORC1 signaling via engagement of the PI3K pathway, which induces phosphorylation of Akt at threonine 308. Akt phosphorylates TSC2 at serine 939, serine 981, and threonine 1462. Phosphorylation of these sites facilitates the binding of TSC2 to the cytosolic anchoring 14-3-3 protein, thereby disrupting the TSC1–TSC2 complex and partitioning TSC2 from Rheb on the membrane. TSC1 physically prevents the ubiquitin-mediated proteolytic degradation of TSC2. Additionally, TSC2 loses its GAP activity when it is not associated with TSC1; TSC2 can no longer inhibit the conversion of GDP-bound Rheb to its GTP-bound state [5], and GTP-bound Rheb directly interacts with and activates mTORC1. Thereby, converting Rheb to the GDP-bound state inhibits mTORC1 activity. Activated Akt also phosphorylates PRAS40, causing it to release from Raptor in mTORC1 [3]. The MAPK/ERK pathway activates mTORC1 by inhibiting the TSC1–TSC2 complex [7]. Erk (MAPK) phosphorylates serine 644 on TSC2, whereas ribosomal S6 kinase (RSK) phosphorylates serine 1798 on TSC2 [6]. These phosphorylation events cause the TSC1– TSC2 complex to dissociate and become inactivated. Dissociation of the TSC1–TSC2 complex prevents the inactivation of Rheb, which keeps mTORC1 active. RSK also phosphorylates Raptor and prevents the inhibitory functions of PRAS40 [7]. Inactivation of the Wnt pathway inhibits glycogen synthase kinase 3 (GSK3) beta, which phosphorylates TSC2 on two serines, 1341 and 1337, in conjunction with the phosphorylation of serine 1345 by 50 -adenosine-monophosphate-activated protein kinase (AMPK). These TSC2 phosphorylation events inactivate the TSC1– TSC2 complex and activate mTORC1.

3

AMPK can phosphorylate TSC2 on serine 1387, which activates the GAP activity of the TSC complex, causing the hydrolysis of Rheb-GTP into Rheb-GDP and the inactivation of mTORC1 [8]. AMPK can also phosphorylate Raptor. Phosphorylated Raptor recruits the binding of 14-3-3 protein and prevents Raptor from inclusion in mTORC1 [9]. Cytokines such as tumor necrosis factor alpha (TNF-a) can induce mTOR activity through ikappa kinase beta (IKK-b), which causes the dissociation of the TSC complex by phosphorylating TSC1 at serines 487 and 511 [10]. Under hypoxic stress, hypoxia-inducible factor one alpha (HIF1a) activates the transcription of regulated in development and DNA damage response 1 (REDD1). REDD1 binds to TSC2 and prevents 14-3-3 protein from disrupting the TSC complex, which keeps mTORC1 inactive [11]. As described above, mTORC1 regulates multiple signals and signaling pathways. mTORC1 resides in the cytoplasm when amino acid levels are low. Amino acids, especially leucines, activate Rag guanosine triphosphatases (Rag GTPases). Activated Rag A/B heterodimers interact with small protein complexes collectively known as Ragulators [12], which facilitate the docking of Rag to the surface of late endosomes or lysosomes. This association promotes the translocation of mTORC1 to the surface of lysosomes, where Rheb-GTP is located [13]. Activated Rag heterodimers interact with Raptor in mTORC1, which localizes near the Ragulators on the surface of late endosomes and lysosomes. Growth factors stimulate PI3K and produce PIP3 at the plasma membrane, which in turn activates PDK1 and Akt. Activated Akt phosphorylates and thereby inhibits the TSC complex, possibly at lysosomes and peroxisomes. Reduced TSC complex GAP activity leads to an increase in GTPbound Rheb. Rheb-GTP at the lysosomal surface directly binds to and activates mTORC1. Then, mTORC1 is activated by Rheb-GTP. Amino acid levels may also control mTORC1 via the Ca2+/CaMdependent activation of the lysosomal membrane protein Vps34, a class III phosphatidylinositol 3-kinase (PI3K) [14]. By contrast, the regulation and function of mTORC2 have not been well elucidated. mTORC2 interacts with ribosomes in a PI3Kdependent manner. Upon growth factor stimulation, mTORC2 is recruited to mitochondria-associated endoplasmic reticulum membranes (MAM), presumably from the cytoplasm, and regulates mitochondrial physiology [15]. The role of mTORC2 in protein synthesis is not clear. It has been shown that upon growth factor stimulation, mTORC2 is activated through association with ribosomes [16]. Activated mTORC2 phosphorylates phosphokinases A, C, and G as well as glucocorticoid-regulated kinase 1 (SGK1) [17], protein kinase C (PKC), and Akt. Phosphorylation of Akt at Ser473 by mTORC2 promotes the further phosphorylation by 3-phosphoinositide-dependent protein kinase 1 (PDK1) at S6K AMPK

HEAT repeats

FAT

FRB

KIN

Akt

P P P

FATC

PRD

rapamycin

2549AA

Rheb

FKBP12 Torin1 Fig. 2. Schematic view of the structure of mTOR protein. mTOR has the characteristic five domains. These are the HEAT repeat domain, the FRAP-ATM-TRRAP (FAT) domain, the kinase domain (KD), the PIKK-regulatory domain (PRD) and the FAT-C-terminal (FATC) domain (from N-terminus to C-terminus). FATC and FAT domains are essential for mTOR catalytic function. FKBP12–rapamycin complex binds to the FRB domain.

Please cite this article in press as: Wataya-Kaneda M. Mammalian target of rapamycin and tuberous sclerosis complex. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.04.005

G Model

DESC-2829; No. of Pages 8 4

M. Wataya-Kaneda / Journal of Dermatological Science xxx (2015) xxx–xxx

Thr308, which in turn can induce mTORC1 signaling in the presence of several stimuli. Once activated by mTORC2, Akt inhibits the function of the transcription factors forkhead box protein O1 (FOXO1) and FOXO3. Similarly, SGK1 also inhibits the transcriptional activity of FoxO family members [18]. mTORC2 inhibits FOXO activity via acetylation, which bypasses PI3K/Akt inhibition, leading to the up-regulation of c-Myc, a key downstream effector of cell proliferation and tumor metabolism. 5. Downstream mTOR cascade 5.1. Growth and proliferation The most elucidated mechanisms of action of mTORC1 are related to cell growth and proliferation (Fig. 1). mTORC1 positively regulates anabolic processes, such as protein synthesis, ribosome biogenesis, transcription, lipid synthesis, nucleotide biosynthesis, and nutrient uptake [19]. Wellcharacterized downstream targets of mTORC1 include ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E (eIF4E) binding proteins (4E-BPs) [20]. S6K and 4E-BPs are the main regulators of protein synthesis downstream of mTORC1 [20]. Activated mTORC1 initiates mRNA translation by activating S6 kinase 1 (S6K1) activity and inhibiting 4E-binding protein 1 (4EBP1) activity, which promotes the initiation of cap-dependent translation. S6K1 phosphorylates both S6 ribosomal protein, which enhances translation efficiency, and eukaryotic initiation factor 4B (eIF4B), which associates with the eIF3 complex to form the translation–initiation complex. As a part of this complex, eIF4E dissociates from 4E-BP1, binds to eIF4G at the 50 -end of mRNAs, and triggers the initiation of protein synthesis. Then, S6K indirectly affects protein synthesis by phosphorylating S6, leading to the expression of mRNAs involved in ribosome biogenesis [21]. The above findings suggest that activation of mTORC1 signaling specifically promotes tumorigenesis, such as the development of generalized hamartomas in tuberous sclerosis complex (TSC), and the development of many other types of cancer. 5.2. Autophagy Another characteristic downstream target of mTORC1 is autophagy. Autophagy is a self-degrading process that is conserved from yeast to man. It is a well-established survival mechanism that maintains cellular homeostasis under both normal and stress conditions [22]. Therefore, cells have developed control mechanisms that tightly regulate autophagic activity. Autophagy is initiated by the formation of a membrane structure, the phagophore, which engulfs part of the cytoplasm and forms a double membrane vesicle termed the autophagosome [23]. The outer autophagosomal membrane fuses with the lysosome and degrades the inner membrane contents. To control autophagy, more than 30 specific proteins (Atgs) regulate the progression of autophagy in a hierarchical manner upon starvation. The initiator of this cascade in mammals is the Ulk1 complex (Ulk1/2, Atg13, FIP200 and Atg101). This complex is directly regulated by mTORC1 in a nutrient-dependent manner. mTORC1 associates with the Ulk1 complex via a direct interaction between Raptor and Ulk1/2. Raptor undergoes multi-site Ulk1-dependent phosphorylation, which either results in direct inhibition of mTORC1 activity [24] or interferes with Raptor–substrate interaction [25], ultimately leading to reduced phosphorylation of mTORC1 downstream targets. In mammals, Ulk1 and Ulk2 have been linked to starvationinduced autophagy. In response to starvation, the mTORC1dependent phosphorylation sites in Ulk1/2 are rapidly dephosphorylated. Then, Ulk1/2 auto-phosphorylates itself and phosphorylates Atg13 and FIP200. Alternatively, Ulk1/2 is

phosphorylated by AMPK and thereby activated [26]. In addition, AMPK indirectly leads to the induction of autophagy by inhibiting mTORC1 through phosphorylating Raptor [9]. The G proteincoupled receptors (GPCRs) T1R1 and T1R3 have been implicated in the extracellular sensing of amino acid availability. Leucyl-tRNA synthetase (LRS) also senses the intracellular leucine level. Leucine regulates mTOR activity through glutamate dehydrogenase (GLUD). Amino acid and glucose starvation leads to the induction of autophagy mediated by a decrease in mTORC1 activity. In contrast, when amino acids are in excess, mTORC1 is targeted to the lysosomal membrane, where it is activated by Rheb and inhibits autophagy. Autophagy is also down-regulated through insulin receptor signaling. In low glucose conditions, hexokinase II, the initial enzyme in the glycolysis pathway, is postulated to directly bind to and thereby inhibit mTORC1. An important link exists between mTORC1, the Rag GTPase complexes and the scaffold protein p62, which is an autophagic cargo receptor. p62 recruits TNF receptor associated factor 6 (TRAF6), an E3 ubiquitin ligase that is essential for the activation of mTORC1. Toll-like receptor 4 (TLR4) that has been activated by lipopolysaccharide (LPS) binding also recruits TRAF6. The recruitment of TRAF6 results in the Lys63-linked ubiquitination of Beclin1, allowing it to bind to PI3KC3 and induce autophagy. Activation of epidermal growth factor receptor (EGFR) inhibits autophagy directly by inhibiting (via phosphorylation) Beclin1 or indirectly by activating mTORC1 via growth factor receptor-bound protein 2 (GRB2) and GRB2associated binding protein 2 (GAB2) and via the phosphorylation of STAT3, which induces the expression of autophagy-related proteins (Fig. 3). As described above, mTORC1 and AMPK are known to regulate autophagy in an opposing manner. However, the link between ULK1 and autophagy is not well known. Two alternative mechanisms have been proposed recently. One report suggests that Ulk1 directly phosphorylates activating molecule in Beclin1regulated autophagy (AMBRA1), a Beclin1-interacting protein and regulatory component of the PI3K class III complex. Under normal growth conditions, the PI3K complex associates with the dynein motor complex via direct interaction between AMBRA1 and dynein light chain 1 (DLC1). Upon starvation, activated Ulk1 phosphorylates AMBRA1; thereupon, the PI3K complex is released and translocates to the ER, where autophagosome formation is initiated [27]. Another group reported that Ulk1 can regulate the actin motor protein myosin II [28]. In mammalian systems, zipper-interacting protein kinase (ZIPK/DAPK3) is phosphorylated by Ulk1, which attenuates myosin II activation and initiates starvation-induced autophagy. Long term starvation leads to the reactivation of mTORC1 by the enhanced autophagic generation of nutrients, resulting in the inhibition of autophagy. However, prolonged inhibition of autophagy results in the accumulation of protein aggregates and damaged organelles, causing pathological disorders, such as neurodegenerative and myodegenerative diseases. Thus, autophagy affects various diseases, including cancer, neurodegenerative disease, autism and epilepsy, and is linked to the mTOR pathway [29]. 5.3. Immunity mTORC1 and mTORC2 are essential for the regulation of immune systems. In the innate immune system, mTOR senses the immune microenvironment and acts as an integrator of nutrient sensing pathways according to the requirements to proliferate or differentiate. mTOR drives the differentiation and function of antigen presenting cells, such as dendritic cells. In the acquired immune response, mTOR also plays a significant role. mTOR regulates B cell maturation and differentiation. Recently, mTOR

Please cite this article in press as: Wataya-Kaneda M. Mammalian target of rapamycin and tuberous sclerosis complex. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.04.005

G Model

DESC-2829; No. of Pages 8 M. Wataya-Kaneda / Journal of Dermatological Science xxx (2015) xxx–xxx

5

Fig. 3. Network of the regulation of autophagy. Signal cascades of autophagy regulation are summarized.

signaling was revealed to be a critical regulator of T cell biology [30]. However, it is still unknown how mTORC1 and mTORC2 are involved in regulating the immune systems. Antigen-bound T cell receptor (TCR) and CD28 co-stimulation, programmed death 1 (PD-1) and interleukin-2 receptor (IL-2R) signal to the PI3K-Akt pathway to activate mTORC1 under nutrient-sensing conditions, which suggests that effector CD8T cell proliferation occurs in a PI3K-dependent, Akt-independent manner [31] (Fig. 4A). Recently, Rosborough et al. reported that mTORC1 promotes effector T cell expansion, while mTORC2 restrains the induction of regulatory T (Treg) cells [32]. The depletion of essential amino acids leads to the inhibition of mTORC1 signaling and the activation of Forkhead box P3 (FOXP3) expression, which converts more naive T cells into Treg cells [33,34]. In contrast, mTORC2 is a negative regulator of the transcription factors FOXO1 and FOXO3a, which promote FOXP3 transcription [35]. Thus, mTORC2 inhibition promotes FOXP3 expression and leads to Treg cell expansion. Taken together, both the mTORC1 and mTORC2 complexes have been shown to serve as negative regulators of Treg lineage commitment. However, mTOR activation antagonizes Treg cells and enhances the development of helper T cells. Although the roles of mTORC1 and mTORC2 for Th1 and Th2 cell development remain controversial, mTORC1 and mTORC2 play a fundamental role in integrating signals that facilitate the differentiation of helper T (Th) cells into Th1 and Th17 cells [36], and inhibit the differentiation into Treg cells. mTORC2 also has been shown to promote Th2 differentiation. As described above, mTORC1 and mTORC2 regulate T cell differentiation into Th1, Th2, and Th17 cells as well as T helper functions in different manners.

It is currently thought that mTOR plays an important role in reversing the quiescent state of CD8+ T cells by inhibiting the transcription factor ELF4 that functions to induce quiescence in naive CD8+ T cells. mTORC1 has been shown to regulate the transcriptional programs that determine the fate of CD8+ cytotoxic T cells (CTL). In T cells, mTORC1 increases effector CD8+ activity. Several investigators have implicated mTOR in the regulation of the effector-to-memory ratio and the transition between both states [37] (Fig. 4B). 6. mTOR signaling pathway and disease To summarize the mechanisms of action and the functions of mTOR complexes, growth factors stimulate mTORC1 through the activation of the Ras-MAPK and PI3K-Akt signaling pathways, which are triggered by Akt. The Akt, ERK and RSK kinases directly phosphorylate and inhibit the TSC2 subunit of the TSC1–TSC2 complex that functions as a GAP toward Rheb and prevents its ability to stimulate mTORC1. Once activated, mTORC1 promotes cell growth and metabolism by regulating several anabolic processes, including protein, lipid and nucleotide synthesis. In contrast, mTORC2 regulates actin cytoskeleton assembly through protein kinase Ca (PKCa), Rho GTPases and Ras proteins. mTORC2 also inhibits FOXO3a through S6K1 and Akt, which can lead to increased longevity. Meanwhile, inhibition of FOXO3a in T cells induces inflammation. As mTORC1 and mTORC2 are essential regulators of a wide range of functions, deregulation of the mTORC1 and/or mTORC2 pathways has been implicated in many diseases or disorders, such

Please cite this article in press as: Wataya-Kaneda M. Mammalian target of rapamycin and tuberous sclerosis complex. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.04.005

G Model

DESC-2829; No. of Pages 8 M. Wataya-Kaneda / Journal of Dermatological Science xxx (2015) xxx–xxx

6

Fig. 4. mTORC1 and mTORC2 are critical regulators of immune systems. (A) Centric view of induction of mTORC1 and FOXP3. (B) mTOR signaling controls peripheral T cell fate.

as tumorigenesis, epilepsy, cognitive impairment, neurodegenerative diseases, metabolic impairments, genetic disorders, immunity, inflammation and aging. The human diseases that involve the mTOR pathway are indicated in Fig. 5. All the diseases have similar symptoms, such as tumors, pigment disorders and neuronalpsychiatric abnormalities. 7. Tuberous sclerosis complex TSC is a representative disease related to mTOR signaling. TSC is a multi-systemic disorder characterized by generalized hamartomas. Epilepsy, cognitive impairment, autism and hypopigmented macules are also involved. TSC is caused by genetic mutation in either TSC1 [38] or TSC2 [39], which encode hamartin and tuberin, respectively. The TSC1 (hamartin)–TSC2 (tuberin) complex down-regulates mTORC1 [40,41]. Therefore, the symptoms of TSC reflect all the functions of mTORC1 and mTORC2. The constitutive activation of mTORC1 that results from the genetic mutation of TSC1 or TSC2 is associated with abnormal cellular proliferation via the promotion of protein synthesis in response to the activation of the translation initiation promoter S6K and the inhibition of the inhibitory mRNA cap binding protein 4EBP1, which causes TSC-related hamartomas. Constitutive activation of mTORC1 also induces cognitive impairment or autism via the suppression of autophagy, disruption of GABAergic

interneuron development, and the development of an abnormal number and shape of synapses. Epilepsy also may be explained by reduced autophagy and altered ion channels and neurotransmitter receptors due to mTORC1 activation [42]. Another representative symptom of TSC is hypopigmented macules. Ho et al. reported the role of autophagy in melanogenesis [43]. Recently, the involvement of autophagy in determining skin color via the regulation of melanosome degradation in keratinocytes was demonstrated [44]. mTORC1 negatively regulates autophagy through multiple inputs, including inhibitory phosphorylation of ULK1, which prevents the formation of the ULK1–ATG13–FIP200 complex (which is required for the initiation of autophagy). Hypopigmented macules in TSC may depend on disordered autophagy during melanogenesis caused by constitutively activated mTORC1. The components of the PTEN-Akt-mTORC-S6 signaling pathway have a complex influence on one another. mTORC1 and mTORC2 also have a complicated relationship. Unlike tumors in which mTORC1 activity is elevated, TSC is characterized predominantly by the development of benign tumors, and malignancies are quite rare. This feature of TSC might be due to the loss of Akt signaling in TSC tumors [45], which results from the combination of mTORC1-dependent feedback mechanisms and the loss of mTORC2 activity derived from loss of function of the TSC1–TSC2 complex.

Please cite this article in press as: Wataya-Kaneda M. Mammalian target of rapamycin and tuberous sclerosis complex. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.04.005

G Model

DESC-2829; No. of Pages 8 M. Wataya-Kaneda / Journal of Dermatological Science xxx (2015) xxx–xxx

7

IRS1

SHC

Noonan synd LEOPARD synd

Gab2

SHP2

mTORC2

PI3K PTEN

CRB2

CRB2 PDK1

Sos

Cowden synd Lhermite-Duclops synd Bannayan-Riley Ruvalcaba synd Proteus synd Proteus-like synd

NF1 HRas GTD NF1

KRas

Akt

GEF

GAP

Sos1/2 ATM

TSC2

KRas

HRas GTP

Tuberous Sclerosis Complex

Legius synd

FLCN

BRAF

LKB1

p18 P14 MP1

Noonan synd LEOPARD synd

ERK1/2

CFCsynd

Peutz-Jeghers synd

Rheb

mTORC2 AMPK

Rag C/D MEK

Ataxia

TSC1

GTP

Costello synd SPRED1

Inconnena Pigmen

NFkB

GDP

Rag A/B

P14 associated growth defects immunodeficiency hypopigmentaon VHL

FKBP38 FKBP38 FLCN

mTORC1 Birt-Hogg-Dube synd

Hif-1a Rac1

Von-Hippel-Lindau 4EBP1

S6K1 STAT3

eIF4E

S6

eIF4B

Fig. 5. Involvement of mTOR pathway in human disease. P14-associated growth defects-immunodeficiency-hypopigmentation is a rare disease with an almost complete loss of function of the ragulator component LAMTOR2/p14. This is the only disease that results from mTORC1 inactivity.

8. Rapamycin, an mTOR inhibitor In 1975, rapamycin was identified as an antifungal antibiotic produced by Streptomycin hygroscopicus, which was isolated from a soil sample taken from Rapa Nui (Easter Island). Like other naturally occurring antibiotics, rapamycin is classified as a macrolide. A large part of the rapamycin molecule is identical to the macrolide FK506 (tacrolimus), and both compounds have immunosuppressive functions. Both rapamycin and rapamycin analogs bind to the 12 kDa immunophilin FK506 binding protein12 (FKBP-12). The rapamycin–FKBP12 complex binds to mammalian target of rapamycin (mTOR) and inhibits mTORC1 kinase activity by disrupting the interaction between mTOR and Raptor. As described in the previous section, mTOR forms two distinct complexes, mTORC1 and mTORC2, and only mTORC1 is sensitive to inhibition by rapamycin. Although mTORC2 was originally thought to be rapamycin insensitive [46], long-term exposure to rapamycin inhibits mTORC2 in some cell types by sequestering newly synthesized mTOR molecules [47]. 9. Treatment of TSC using mTORC1 inhibitors Activation of mTORC1 is observed in numerous human cancers. In 2007 and 2009, two rapamycin analogs, temsirolimus and everolimus, were independently approved by the Food and Drug Administration (FAD) for the treatment of advanced renal cell carcinoma. In 2011, everolimus was also approved for the treatment of neuroendocrine tumors of the pancreas. Rapamycin may be effective in the many diseases in which the PI3K-Akt-TSCmTOR pathway has been implicated as well as in those with gain of function of oncogenes such as PI3K or Akt or loss of function of tumor suppressors such as PTEN, TSC1/2, LKB1 or Folliculin (FLCN). Rapamycin may be effective for the lesions in TSC. Last year, rapamycin was approved as a therapeutic drug for Lymphangioleiomyomatosis, the typical pulmonary legions in TSC. Everolimus,

an analog of rapamycin, is also used for the treatment of renal angiomyolipomas and subependymal giant cell astrocytoma (SEGA). Both mTORC1 inhibitors are effective not only for the tumorous lesions, such as the renal and pulmonary lesions, but also for the epilepsy and autism. A report demonstrated that rapamycin reduced the seizure frequency in a 10-year-old girl with TSC [48]. Recently, the efficacy of rapamycin in the improvement of impaired social interaction in a TSC model mouse was reported [49]. Although rapamycin seems useful for the treatment of lesions due to TSC, discontinuation of rapamycin treatment causes the regrowth of the tumors. Consequently, long-term administration is required. To prevent the adverse reactions arising from long-term administration of rapamycin, a topical formulation of rapamycin has been developed. Skin lesions, such as facial angiofibromas and hypomelanotic macules, are improved by the topical formulation of rapamycin [50]. Rapamycin and its analogs (rapalogs) have been used in clinical trials to treat many diseases, such as advanced mantle cell lymphoma and advanced cancers. However, rapalogs have achieved modest effects in many clinical trials. Although the reasons for the limited clinical success of rapalogs have not been established, the large number of mTORC1-regulated negative feedback loops, such as activation of receptor tyrosine kinases (RTKs), PI3K-Akt signaling and the Ras-MAPK pathway, might be involved. To improve this problem, new drugs that inhibit both mTORC1 and mTORC2 are under development. To remove the numerous feedback mechanisms, combination therapies with rapamycin and several chemotherapeutic agents are in development. Acknowledgements This study was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan

Please cite this article in press as: Wataya-Kaneda M. Mammalian target of rapamycin and tuberous sclerosis complex. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.04.005

G Model

DESC-2829; No. of Pages 8 M. Wataya-Kaneda / Journal of Dermatological Science xxx (2015) xxx–xxx

8

(Grant-in Aid for Scientific Research No. 25461690), and by a grant from the Ministry of Health, Labor and Welfare of Japan (No. H24Nanchi-ippan-008).

[29] [30]

References [1] Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell 2006;124:471–84. [2] Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H, et al. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 2002;110:163–75. [3] Oshiro N, Takahashi R, Yoshino K, Tanimura K, Nakashima A, Eguchi S, et al. The proline-rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mammalian target of rapamycin complex 1. J Biol Chem 2007;282:20329–39. [4] Kaizuka T, Hara T, Oshiro N, Kikkawa U, Yonezawa K, Takehana K, et al. Tti1 and Tel2 are critical factors in mammalian target of rapamycin complex assembly. J Biol Chem 2010;285:20109–16. [5] Saucedo LJ, Gao X, Chiarelli DA, Li L, Pan D, Edgar BA. Rheb promotes cell growth as a component of the insulin/TOR signalling network. Nat Cell Biol 2003;5:566–71. [6] Ma L, Chen Z, Erdjument-Bromage H, Tempst P, Pandolfi PP. Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis. Cell 2005;121:179–93. [7] Carriere A, Cargnello M, Julien LA, Gao H, Bonneil E, Thibault P, et al. Oncogenic MAPK signaling stimulates mTORC1 activity by promoting RSK-mediated raptor phosphorylation. Curr Biol 2008;18:1269–77. [8] Mihaylova MM, Shaw RJ. The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat Cell Biol 2011;13:1016–23. [9] Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS, et al. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell 2008;30:214–26. [10] Salminen A, Hyttinen JM, Kauppinen A, Kaarniranta K. Context-dependent regulation of autophagy by IKK-NF-kappaB signaling: impact on the aging process. Int J Cell Biol 2012;2012:849541. [11] Horak P, Crawford AR, Vadysirisack DD, Nash ZM, DeYoung MP, Sgroi D, et al. Negative feedback control of HIF-1 through REDD1-regulated ROS suppresses tumorigenesis. Proc Natl Acad Sci U S A 2010;107:4675–80. [12] Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, Sabatini DM. Ragulator– Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 2010;141:290–303. [13] Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L, et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 2008;320:1496–501. [14] Gulati P, Gaspers LD, Dann SG, Joaquin M, Nobukuni T, Natt F, et al. Amino acids activate mTOR complex 1 via Ca2+/CaM signaling to hVps34. Cell Metab 2008;7:456–65. [15] Betz C, Stracka D, Prescianotto-Baschong C, Frieden M, Demaurex N, Hall MN. Feature article: mTOR complex 2-Akt signaling at mitochondria-associated endoplasmic reticulum membranes (MAM) regulates mitochondrial physiology. Proc Natl Acad Sci U S A 2013;110:12526–34. [16] Oh WJ, Wu CC, Kim SJ, Facchinetti V, Julien LA, Finlan M, et al. mTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt polypeptide. EMBO J 2010;29:3939–51. [17] Garcia-Martinez JM, Alessi DR. mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoidinduced protein kinase 1 (SGK1). Biochem J 2008;416:375–85. [18] Masui K, Tanaka K, Akhavan D, Babic I, Gini B, Matsutani T, et al. mTOR complex 2 controls glycolytic metabolism in glioblastoma through FoxO acetylation and upregulation of c-Myc. Cell Metab 2013;18:726–39. [19] Shimobayashi M, Hall MN. Making new contacts: the mTOR network in metabolism and signalling crosstalk. Nat Rev Mol Cell Biol 2014;15:155–62. [20] Ma XM, Blenis J. Molecular mechanisms of mTOR-mediated translational control. Nat Rev Mol Cell Biol 2009;10:307–18. [21] Shahbazian D, Roux PP, Mieulet V, Cohen MS, Raught B, Taunton J, et al. The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity. EMBO J 2006;25:2781–91. [22] Boya P, Reggiori F, Codogno P. Emerging regulation and functions of autophagy. Nat Cell Biol 2013;15:713–20. [23] Weidberg H, Shvets E, Elazar Z. Biogenesis and cargo selectivity of autophagosomes. Annu Rev Biochem 2011;80:125–56. [24] Jung CH, Seo M, Otto NM, Kim DH. ULK1 inhibits the kinase activity of mTORC1 and cell proliferation. Autophagy 2011;7:1212–21. [25] Dunlop EA, Hunt DK, Acosta-Jaquez HA, Fingar DC, Tee AR. ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding. Autophagy 2011;7:737–47. [26] Kim E, Goraksha-Hicks P, Li L, Neufeld TP, Guan KL. Regulation of TORC1 by Rag GTPases in nutrient response. Nat Cell Biol 2008;10:935–45. [27] Di Bartolomeo S, Corazzari M, Nazio F, Oliverio S, Lisi G, Antonioli M, et al. The dynamic interaction of AMBRA1 with the dynein motor complex regulates mammalian autophagy. J Cell Biol 2010;191:155–68. [28] Tee AR, Fingar DC, Manning BD, Kwiatkowski DJ, Cantley LC, Blenis J. Tuberous sclerosis complex-1 and -2 gene products function together to

[31]

[32]

[33]

[34] [35]

[36]

[37]

[38]

[39]

[40] [41] [42] [43] [44]

[45]

[46]

[47] [48] [49]

[50]

inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling. Proc Natl Acad Sci U S A 2002;99:13571–76. Choi AM, Ryter SW, Levine B. Autophagy in human health and disease. N Engl J Med 2013;368:651–62. Chi H. Regulation and function of mTOR signalling in T cell fate decisions. Nat Rev Immunol 2012;12:325–38. Macintyre AN, Finlay D, Preston G, Sinclair LV, Waugh CM, Tamas P, et al. Protein kinase B controls transcriptional programs that direct cytotoxic T cell fate but is dispensable for T cell metabolism. Immunity 2011;34:224–36. Rosborough BR, Raich-Regue D, Matta BM, Lee K, Gan B, DePinho RA, et al. Murine dendritic cell rapamycin-resistant and rictor-independent mTOR controls IL-10, B7-H1, and regulatory T-cell induction. Blood 2013;121:3619–30. Cobbold SP, Adams E, Farquhar CA, Nolan KF, Howie D, Lui KO, et al. Infectious tolerance via the consumption of essential amino acids and mTOR signaling. Proc Natl Acad Sci U S A 2009;106:12055–60. Peter C, Waldmann H, Cobbold SP. mTOR signalling and metabolic regulation of T cell differentiation. Curr Opin Immunol 2010;22:655–61. Sauer S, Bruno L, Hertweck A, Finlay D, Leleu M, Spivakov M, et al. T cell receptor signaling controls Foxp3 expression via PI3K, Akt, and mTOR. Proc Natl Acad Sci U S A 2008;105:7797–802. Delgoffe GM, Pollizzi KN, Waickman AT, Heikamp E, Meyers DJ, Horton MR, et al. The kinase mTOR regulates the differentiation of helper T cells through the selective activation of signaling by mTORC1 and mTORC2. Nat Immunol 2011;12:295–303. Rao RR, Li Q, Odunsi K, Shrikant PA. The mTOR kinase determines effector versus memory CD8+ T cell fate by regulating the expression of transcription factors T-bet and eomesodermin. Immunity 2010;32:67–78. van Slegtenhorst M, de Hoogt R, Hermans C, Nellist M, Janssen B, Verhoef S, et al. Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science 1997;277:805–8. European Chromosome 16 Tuberous Sclerosis C. Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell 1993;75: 1305–15. Inoki K, Li Y, Zhu T, Wu J, Guan KL. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol 2002;4:648–57. Inoki K, Li Y, Xu T, Guan KL. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev 2003;17:1829–34. Curatolo P. Mechanistic target of rapamycin (mTOR) in tuberous sclerosis complex-associated epilepsy. Pediatr Neurol 2015;52:281–9. Ho H, Ganesan AK. The pleiotropic roles of autophagy regulators in melanogenesis. Pigment Cell Melanoma Res 2011;24:595–604. Murase D, Hachiya A, Takano K, Hicks R, Visscher MO, Kitahara T, et al. Autophagy has a significant role in determining skin color by regulating melanosome degradation in keratinocytes. J Investig Dermatol 2013;133: 2416–24. Manning BD, Logsdon MN, Lipovsky AI, Abbott D, Kwiatkowski DJ, Cantley LC. Feedback inhibition of Akt signaling limits the growth of tumors lacking Tsc2. Genes Dev 2005;19:1773–8. Jacinto E, Loewith R, Schmidt A, Lin S, Ruegg MA, Hall A, et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol 2004;6:1122–8. Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell 2012;149:274–93. Muncy J, Butler IJ, Koenig MK. Rapamycin reduces seizure frequency in tuberous sclerosis complex. J Child Neurol 2009;24:477. Sato A, Kasai S, Kobayashi T, Takamatsu Y, Hino O, Ikeda K, et al. Rapamycin reverses impaired social interaction in mouse models of tuberous sclerosis complex. Nat Commun 2012;3:1292. Wataya-Kaneda M, Tanaka M, Nakamura A, Matsumoto S, Katayama I. A novel application of topical rapamycin formulation, an inhibitor of mTOR, for patients with hypomelanotic macules in tuberous sclerosis complex. Arch Dermatol 2012;148:138–9.

Dr. Mari Wataya-Kaneda is an associate professor of Department of Dermatology, Graduate School of Medicine, Osaka University and also a professor of Osaka University Hospital. She graduated from Ehime University School of Medicine and obtained Ph.D. degree at Graduate School of Medicine, Osaka University, in 1985. She completed dermatology residency at Osaka University, and became a medical stuff at Mino-city hospital. She was also a post-doctoral fellow in the Department of Dermatology at the University of California, San Francisco, from 1988 to 1990. Since 1996, she has treated many genetic skin diseases including tuberous sclerosis complex (TSC) and von Recklinghausen diseases in Osaka University Hospital. She has treated approximately 300 TSC patients in Japan. TSC results from the constitutive activation of mTOR, which is related to many symptoms including convulsion, autism and white macules besides tumorigenesis. She has been focusing on the function of mTOR involved in the symptoms. Recently, she has started the treatment of TSC skin lesions using a novel topical rapamycin formulation.

Please cite this article in press as: Wataya-Kaneda M. Mammalian target of rapamycin and tuberous sclerosis complex. J Dermatol Sci (2015), http://dx.doi.org/10.1016/j.jdermsci.2015.04.005