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Mutation Research 659 (2008) 284292

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Mutation Research/Reviews in Mutation Research


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Review

The mTOR pathway and its role in human genetic diseases


Margit Rosner, Michaela Hanneder, Nicol Siegel, Alessandro Valli, Christiane Fuchs, Markus Hengstschlager *
hringer Gu rtel 18-20, 1090 Vienna, Austria Medical Genetics, Obstetrics and Gynecology, Medical University of Vienna, Wa

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 May 2008 Received in revised form 29 May 2008 Accepted 3 June 2008 Available online 11 June 2008 Keywords: PI3K Akt TSC mTOR Ras Human genetic disease Cancer

The signalling components upstream and downstream of the protein kinase mammalian target of rapamycin (mTOR) are frequently altered in a wide variety of human diseases. Upstream of mTOR key signalling molecules are the small GTPase Ras, the lipid kinase PI3K, the Akt kinase, and the GTPase Rheb, which are known to be deregulated in many human cancers. Mutations in the mTOR pathway component genes TSC1, TSC2, LKB1, PTEN, VHL, NF1 and PKD1 trigger the development of the syndromes tuberous sclerosis, Peutz-Jeghers syndrome, Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome, LhermitteDuclos disease, Proteus syndrome, von Hippel-Lindau disease, Neurobromatosis type 1, and Polycystic kidney disease, respectively. In addition, the tuberous sclerosis proteins have been implicated in the development of several sporadic tumors and in the control of the cyclin-dependent kinase inhibitor p27, known to be of relevance for several cancers. Recently, it has been recognized that mTOR is regulated by TNF-a and Wnt, both of which have been shown to play critical roles in the development of many human neoplasias. In addition to all these human diseases, the role of mTOR in Alzheimers disease, cardiac hypertrophy, obesity and type 2 diabetes is discussed. 2008 Elsevier B.V. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. The mTOR pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Localization of mTOR pathway components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The mTOR pathway and specic human genetic diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Tuberous sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Peutz-Jeghers syndrome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome, Proteus syndrome, Lhermitte-Duclos 2.4. von Hippel-Lindau disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Neurobromatosis type 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Polycystic kidney disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The mTOR pathway in Alzheimers syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... ....... ....... ....... ....... ....... disease . ....... ....... ....... ....... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 285 286 286 286 287 287 287 288 288 288

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* Corresponding author. Tel.: +43 1 40400 7847; fax: +43 1 4040 7848. E-mail address: markus.hengstschlaeger@meduniwien.ac.at (M. Hengstschlager). Abbreviations: 4EBP1, eukaryotic initiation factor 4E binding protein-1; ADPKD, autosomal dominant polycystic kidney disease; AMPK, 50 AMP-activated protein kinase; Cdk, cyclin-dependent kinase; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FGFR, broblasts growth factor receptor; FKBP, FK506-binding protein; GAP, GTPase activating protein; GSK3b, glycogen synthase kinase 3b; HIF, hypoxia-inducible transcription factor; IGF-1R, insulin-like growth factor 1 receptor; IGF1, insulin-like growth factor 1; IKK, inhibitor of kB kinase; IRS1, insulin receptor substrate 1; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; NF1, neurobromatosis type 1; p70S6K, ribosomal p70S6 kinase; PC1, polycystin-1; PDGFb, plateled-derived growth factor b; PDGFR, platelet-derived growth factor receptor; PDK1, phosphoinositide-dependent kinase-1; PI3K, phosphatidylinositol 3-kinase; PIP2, lipid phosphatidylinositol-4,5-biphosphate; PIP3, phosphatidylinositol-3,4,5-triphosphate; PML, promyelocytic leukaemia; PRAS40, proline-rich AKT substrate 40 kDa; protor, protein observed with rictor; PTEN, phosphatase and tensin homolog; raptor, regulatory associated protein of mTOR; Rheb, Ras homolog enriched in brain; rictor, rapamycin-insensitive companion of mTOR; RTK, receptor tyrosine kinase; siRNA, small interfering RNA; sin1, stress-activated protein kinase-interacting protein; TGF-a, and transforming growth factor a; TNF-a, tumor necrosis factor-a; TSC, tuberous sclerosis complex; TSC1, tuberous sclerosis complex gene 1; TSC2, tuberous sclerosis complex gene 2; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; VHL, von Hippel-Lindau disease. 1383-5742/$ see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mrrev.2008.06.001

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The mTOR pathway and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Upstream and downstream of mTOR in tumor development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Ras, mTOR and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. TNF-a activates mTOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Wnt regulates mTOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. mTOR and renal cell carcinoma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. The TSC proteins in sporadic cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7. The TSC proteins and p27 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The mTOR pathway and cardiac hypertrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The mTOR pathway in obesity and type 2 diabetes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction 1.1. The mTOR pathway The mammalian target of rapamycin (mTOR) is a member of the phosphoinositide-3-kinase-related kinase family, which is centrally involved in growth regulation, proliferation control and cancer cell metabolism. In mammalian cells, two structurally and functionally distinct mTOR-containing complexes have been identied. mTORC1 contains raptor (regulatory associated protein of mTOR), mLST8 (also known as GbL) and PRAS40 (proline-rich Akt substrate 40 kDa). Whereas the function of mLST8 is not really claried, raptor regulates mTORC1 functioning as a scaffold for recruiting TORC1 substrates. PRAS40 is phosphorylated by Akt at T246 releasing its inhibitory effects on mTORC1. The major substrates of mTORC1 known so far are 4EBP1 (eukaryotic initiation factor 4E binding protein-1) and p70S6K (ribosomal p70S6 kinase), both regulators of protein translation. mTORC1 phosphorylates and activates p70S6K at T389 to activate the ribosomal protein S6 via phosphorylation at S240/244 (Fig. 1) [15]. mTORC2 also contains the mLST8 protein, but instead of raptor, mTORC2 contains rictor (rapamycin-insensitive companion of mTOR) and sin1 (stress-activated protein kinase-interacting protein) proteins. Rictor and sin1 appear to stabilize each other through binding, building the structural foundation for mTORC2. The interaction between rictor and mTOR is not blocked by the drug rapamycin nor affected by nutrient levels, which are conditions known to regulate mTORC1. mTORC2 additionally contains protor (protein observed with rictor), which lacks any obvious functional domains. The role of protor for mTORC2 activity (if any) still remains elusive. mTORC2 phosphorylates the oncogenic kinase Akt (also known as protein kinase B) at S473, what in conjunction with the PDK1 (phosphoinositide-dependent kinase-1)-mediated phosphorylation of Akt at T308 drives full activation of Akt (Fig. 1) [15]. Over 100 putative Akt substrates have been reported, although many of those have not been characterized in more detail [6]. One important substrate of Akt has been shown to function upstream of mTORC1: the TSC1/TSC2 protein complex. The TSC1 gene on chromosome 9q34 encodes hamartin, and TSC2 on chromosome 16p13.3 encodes tuberin [7,8]. Mutations in either the TSC1 or TSC2 gene cause tuberous sclerosis (TSC), a multisystem autosomal dominant disorder (see below) [9,10]. Activated receptor tyrosine kinases activate the phosphatidylinositol-3kinase (PI3K) through phosphorylation of adaptors, such as the insulin receptor substrate 1 (IRS1). Phosphorylation of the membrane lipid phosphatidylinositol-4,5-biphosphate (PIP2) by PI3K produces the second messenger phosphatidylinositol-3,4,5triphosphate (PIP3) in a reaction that can be reversed by the phosphatase PTEN (phosphatase and tensin homolog) (Figs. 1 and 2). PDK1 and Akt bind to PIP3 at the plasma membrane, and PDK1

phosphorylates Akt at T308. Akt-mediated phosphorylation on S939 and T1462 downregulates tuberins GTPase activating (GAP) potential toward Rheb (Ras homolog enriched in brain), which is a potent regulator of mTOR. It was recently shown that Rheb regulates mTOR through FKBP38, a member of the FK506-binding protein (FKBP) family. FKBP38 binds to mTOR and inhibits its activity and Rheb interacts directly with FKBP38 and prevents its association with mTOR (Fig. 1) [1,2,4,11]. p70S6K has been demonstrated to phosphorylate IRS1 on multiple inhibitory sites promoting its degradation (Fig. 1) [12]. This nding is one possible explanation for the observation that loss of TSC1/2 function results in downregulated Akt phosphorylation [13,14]. It has been suggested that tumors in TSC patient are less aggressive because of this S6K-dependent negative feedback inhibition. Furthermore, it has recently been reported that Rheb has a negative effect on mTORC2. This study provides evidence that Rheb may affect mTORC2 indirectly probably through this S6K-dependent negative feedback loop [15]. In addition, recently it was demonstrated that the TSC1TSC2

Fig. 1. mTORC1 and mTORC2 in the insuling signalling pathway. For details see the text.

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Fig. 2. Regulatory components upstream of the TSC1/TSC2 complex The phosphorylation sites on tuberin or hamartin are shown under the corresponding enzymes. For details see the text.

complex can physically associate with mTORC2 but not mTORC1, and that the TSC protein complex positively regulates mTORC2 in a manner independent of its GTPase-activating protein activity toward Rheb (Fig. 1) [16]. 1.2. Localization of mTOR pathway components IRS1, PI3K, PDK1 and Akt function at the plasma membrane. In agreement with its role in the regulation of translation, all the other components of the described pathway including TSC1/2, Rheb, mTOR and p70S6K have also been localized to the cytoplasm. Besides regulation of translation, mTOR has also been implicated in the regulation of ribosome biogenesis, macro-autophagy or transcription [17]. Accordingly, it is not surprising that the proteins involved in the PI3K signalling pathway are also localized within the nucleus. PI3K has been shown to be nuclear [18] PDK1 is a nucleo-cytoplasmic shuttling protein [19]. Akt translocates also to the nucleus [2023] and it was reported that IGF-1 (insulin-like growth factor 1) stimulates phosphorylation of Akt T308 and of Akt S473 in the cytoplasm and in the nucleus [24]. It has even recently been shown that the PML (promyelocytic leukaemia) tumor suppressor prevents cancer by inactivating Akt in the nucleus [25]. Several recent reports have brought conclusive evidence that the tumor suppressor PTEN, once considered as a strictly cytoplasmic protein, also shuttles to the nuclear compartment, where it joins the components PI3K, PDK1 and Akt [26,27]. Tuberin has been reported to be cytoplasmic and nuclear [2831]. Furthermore, mTOR and its substrate p70S6K have been found to be localized to both the cytoplasm and the nucleus, and cytoplasmic nuclear shuttling of mTOR has been shown to be involved in rapamycinsensitive signalling and translation initiation [3236]. The p70S6K target S6 is dispersed throughout the cytoplasm. Within the nucleus S6 protein is concentrated to the nucleoli and almost absent from the nucleoplasm, what is a consequence of the fact that eukaryotic ribosomes are assembled in the nucleolus before export to the cytoplasm [37,38]. 2. The mTOR pathway and specic human genetic diseases 2.1. Tuberous sclerosis Hamartin and tuberin are believed to function in the same complex, because they associate physically in vivo with high

afnity to form heterodimers [39,40]. Until now, over 50 proteins have been demonstrated to interact with hamartin and/or tuberin and a wide variety of functions have been described for this protein complex [41]. However, in addition, evidence for functional differences of these two proteins comes from knockout studies and from microarray and proteomic analyses [4246]. As described above (Fig. 1), a major function of the hamartin/tuberin (TSC1/ TSC2) protein complex is to antagonize the mTOR pathway. Aktmediated phosphorylation on S939 and T1462 downregulates tuberins GTPase activating (GAP) potential toward Rheb (Figs. 1 and 2). In summary, Akt stimulates mTOR signalling by inhibiting the function of tuberin [4752]. Via this pathway mTOR controls the translation rate of RNAs specically regulated via the translation initiation factor eIF4E. Cap-dependent translation is facilitated by mTOR phosphorylation and inactivation of 4E-BPs, which are suppressors of eIF4E. In addition, mTOR regulates the translation rates of specic messages (mainly from genes involved in ribosome biogenesis) via its effects on the phosphorylation of the 40S ribosomal protein S6 by S6K. S6K is considered to be involved in translation control of a small subset of mRNAs that contain a 50 -terminal oligopyrimidine tract [1,2,53]. The majority of TSC disease-causing mutations occur de novo in either TSC1 or TSC2. Linkage analysis suggests that about half of large families are linked to TSC1 and half to TSC2, whereas in the sporadic TSC population mutations in TSC2 are about ve times more common than mutations in TSC1. The mutation spectra of the TSC genes are very heterogenous and no hotspots of mutations have been found. No signicant genotypephenotype correlations have been established. Patients with TSC2 mutations seem to be more severely affected than patients with mutations in the TSC1 gene, which appears to be due to a higher rate of second hit events. TSC patients carry a mutant TSC1 or TSC2 gene in each of their somatic cells and loss of heterozygosity has been documented in a wide variety of TSC tumors. Tumor development is assumed to be the result of somatic second hit mutations according to Knudsons tumor suppressor model. Accordingly, although TSC is a disease, which is transmitted in an autosomal dominant fashion, mutations in the TSC genes are believed to be recessive at the level of the affected cell [710,53]. TSC affects about 1 in 6000 live births and is characterized by the development of tumor-like growths, named hamartomas, in the kidneys, heart, skin and brain. Primary diagnostic criteria for TSC include facial angiobromas, peringual bromas, calcied retinal hamartomas, cortical tubers or

M. Rosner et al. / Mutation Research 659 (2008) 284292 Table 1 mTOR pathway components in specic human genetic diseases Gene TSC1 Protein: function Hamartin: binding partner for tuberin Disease: clinical characteristics Tuberous sclerosis: hamartomas, epilepsy, mental retardation Lymphangiomyomatosis: lung cysts, dyspnea, pneumothorax Tuberous sclerosis: hamartomas, epilepsy, mental retardation Lymphangiomyomatosis: lung cysts, dyspnea, pneumothorax Peutz-Jeghers syndrome: hamartomas in the intestine Cowden syndrome: hamartomas in multiple organs Bannayan-Riley-Ruvalcaba syndrome: hamartomas in multiple organs Lhermitte-Duclos disease: hamartomas in brain Proteus syndrome: hamartomas in multiple organs

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TSC2

Tuberin: Rheb GTPase activation, regulation of mTORC2, p27, etc.

LKB1 PTEN

LKB1: ser/thr kinase PTEN: phosphatase

VHL NF1 PKD1

VHL: ubiquitination of HIF Neurobromin: Ras-GTPase activating Polycystin-1: interaction, block of mTOR

von Hippel-Lindau disease: angiomas, hemangioblastomas, renal carcinomas Neurobromatosis type 1: neurobromas, hamartomas Polycystic kidney disease: cysts in both kidneys

renal angiomyolipomas. In addition, nearly all patients exhibit skin signs, such as, e.g. hypomelanotic macules, forehead brous plaques, facial angiobromas or shagreen patches. In summary, the major features of TSC include dermatologic manifestations, renal angiomyolipomas, and neurologic manifestations, such as epilepsy, mental retardation, and autism. The severity of TSC and its impact on the quality of life is extremely variable. Many patients have minimal signs and symptoms with no neurologic disability. The greatest source of morbidity is the brain tumors, named cortical tubers, causing seizures in 8090% of affected individuals, and behavioural abnormalities (mostly autism) in over half of affected individuals [9,10,53]. Lymphangiomyomatosis (also known as lymphangioleiomyomatosis) is characterized by widespread pulmonary proliferation of abnormal smooth-muscle cells and cystic changes within the lung parenchyma manifested by dyspnea or pneumothorax. It affects women almost exclusively. Among women with TSC the incidence for lymphangiomyomatosis is 2639% with many of these women being asymptomatic [9,53] (Table 1). The mTORC1 inhibitor sirolimus has already been studied in clinical trials analysing its effects in TSC and lymphangiomyomatosis therapy justifying the need for larger, well-powered trials to test whether sirolimus therapy should become the standard of care for such patients [54,55]. 2.2. Peutz-Jeghers syndrome In the recent past, in addition to Akt other enzymes have been identied to regulate tuberins functions. The LKB1 tumor suppressor gene is responsible for the hamartomatous PeutzJeghers syndrome and encodes a serine/threonine kinase, which phosphorylates and activates AMPK (50 AMP-activated protein kinase). Under energy starvation conditions phosphorylation by AMPK activates tuberin via phosphorylation at T1227 and S1345, is required for the regulation of cell size control, and protects cells from energy deprivation-induced apoptosis [56,57]. Recently, it was demonstrated that Wnt inhibits the GSK3b (glycogen synthase kinase 3b)-mediated phosphorylation of tuberin. GSK3 inhibits the mTOR pathway via phosphorylating tuberin at S1337 and at S1341. This phosphorylation depends on AMPK-priming phosphorylation of tuberin at S1345 and triggers activation of tuberins potential to inhibit mTOR. Accordingly, Wnt inhibits the GSK3-mediated activation of tuberin to block mTOR [58] (Fig. 2). The Peutz-Jeghers syndrome, caused by mutation in the LKB1 tumor suppressor gene, is characterized by hamartomas primarily in the intestine, where they grow as polyps, by abnormal

mucocutaneous pigmentation and an increased risk of malignant tumors in the intestine and elsewhere [59,60]. 2.3. Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome, Proteus syndrome, Lhermitte-Duclos disease The tumor suppressor gene PTEN encodes a phosphatase that catalyzes the conversion of PIP3 to PIP2. Loss of PTEN increases Akt activity, which downregulates tuberins function (Fig. 2). Germline PTEN mutations trigger a wide variety of different clinical syndromes, such as Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome, Proteus syndrome, and Lhermitte-Duclos disease. Since these four diseases are all autosomal dominant hamartoma syndromes it has been suggested that these syndromes should be grouped together as PTEN-hamartoma tumor syndromes [59,60]. As described above, LKB1, associated with the hamartomatous Peutz-Jeghers syndrome, activates tuberin via AMPK. Since PTEN, LKB1 and tuberin have been demonstrated to be involved in the regulation of HIF and VEGF, it has been speculated that increased HIF and VEGF levels may be a common feature of familial hamartoma syndromes. It was reported that tuberin regulates the expression of the vascular endothelial growth factor (VEGF) through mTOR-dependent and -independent mechanisms. Loss of functional tuberin triggers the accumulation of the hypoxiainducible transcription factor (HIF) and upregulation of the expression of HIF-responsive genes including VEGF. TSC2-negative cells, in contrast to normal cells, fail to downregulate HIF in response to growth factor deprivation. Expression of a diseasecausing mutation of TSC2 fails to normalize HIF in TSC2-decient cells, suggesting that HIF regulation plays a role in TSC2 tumor suppressor function (Table 1; Figs. 2 and 3) [5962]. 2.4. von Hippel-Lindau disease Mutations in the VHL tumor suppressor gene cause von HippelLindau disease (VHL). This genetic multisystem disorder is characterized by the abnormal growth of tumors (angiomas) in certain parts of the body. Hemangioblastomas (tumors of the central nervous system) can develop in the brain, the retina of the eyes, and other areas of the nervous system. Other types of tumors develop in the adrenal glands, the kidneys, or the pancreas. Patients can have headaches, problems with balance and walking, dizziness, weakness of the limbs, vision problems, and high blood pressure. Individuals with VHL also harbor increased risks for certain types of cancer, especially renal carcinomas. VHL encodes a protein, which is part of a multiprotein complex involved in

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another protein known to be involved in the etiology of polycystic kidney disease was found to interact with tuberin. Mutations in NEK1 cause the formation of kidney cysts in mice. Performing a yeast two-hybrid screen NEK1 has been identied as interacting partner of tuberin. It will be of great interest for the future to further investigate the role of this interaction for the formation of renal cysts (Table 1) [69]. Recent results demonstrate that PC1 regulates mTOR and is involved in mechanotransduction by primary cilia measuring the degree of luminal uid ow. It is currently under discussion whether a critical function of PC1 and primary cilia in the kidney may be to sense renal injury by detecting changes in luminal ow [70]. 3. The mTOR pathway in Alzheimers syndrome Hamartin has been demonstrated to interact with neurolament-light chain and hamartin and tuberin co-localize with neurolament-light chain preferentially in the proximal to central growth cone region of cultured cortical neurons suggesting that hamartin could function as an integrator of the neuronal intermediate lament and the actin cytoskeletal network [71]. In TSC the central nervous system lesions, such as cortical tubers, result in a variety of neurological manifestations, including mental retardation and seizures. Furthermore, tuberin has been reported to be involved in the regulation of neuronal differentiation [72]. Alzheimers disease, the most common form of dementia in the elderly, is a progressive neurodegenerative disorder characterized by global cognitive decline involving memory, orientation, judgment, and reasoning. Upon autopsy abundant amounts of the typical lesions, extracellular deposits of b-amyloid and intracellular deposits of neurobrillary tangles, are necessary for a conrmed diagnosis of Alzheimers disease. There is a very high incidence of Alzheimer-type neuropathology that is observed in the brains of middle-aged patients with Down syndrome (Trisomy 21). A genetic link between these two diseases has been suggested since it was shown that the b-amyloid precursor protein gene mapped to chromosome 21. Beside the b-amyloid precursor protein gene recent genetic studies have identied also other genes associated with inherited risk for Alzheimers disease, such as presenilin 1, presenilin 2, and apolipoprotein E. However, Alzheimers disease is a multifactorial illness with both genetic and non-genetic causes [73]. Tuberin levels have been found to be decreased in samples of brains positive for b-amyloid plaques and neurobrillary tangles of patients with Alzheimers disease or with Down syndrome [74]. Deregulation of PTEN and Akt [75,76], alteration of mTOR activity [77,78] and deregulated p70S6K activity [79] has also already been found to be associated with Alzheimers disease pathology. 4. The mTOR pathway and cancer 4.1. Upstream and downstream of mTOR in tumor development The signalling components upstream and downstream of mTOR are frequently altered in a wide variety of human tumors. As described above, mutations in the tumor suppressor genes TSC1, TSC2, LKB1, PTEN, VHL, NF1 and PKD1 trigger the development of the hamartoma syndromes Tuberous sclerosis, Peutz-Jeghers syndrome, Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome, Lhermitte-Duclos disease, Proteus syndrome, von Hippel-Lindau disease, Neurobromatosis type 1, and Polycystic kidney disease, respectively (Table 1). PI3K itself can function as protooncogene, since increased PI3K activity has been shown to induce cell transformation and progression and has been found in many human cancers, such as, e.g. ovarian and gastrointestinal cancer.

Fig. 3. The mTOR pathway downstream of the TSC1/TSC2 complex. For details see the text.

ubiquitination and degradation of the transcription factor HIF leading to the dysregulation of a variety of genes, involved in growth control. As described above, mTOR is a key upstream regulator of HIF. However, the interactive roles of mTOR and VHL for the control of HIF activity is still under investigation (Fig. 3; Table 1) [59,60,63,64]. 2.5. Neurobromatosis type 1 The familial cancer syndrome neurobromatosis type 1 (NF1) affects about 1 in 3500 individuals. It is characterized by the development of benign (neurobromas) and malignant peripheral nerve sheath tumors. Patients can exhibit cognitive decits, bone deformations and hamartomatous lesions of the iris. Neurobromatosis type 1 is often grouped clinically with TSC as a common neurocutaneous syndrome. It is caused by mutations in NF1. NF1 encodes neurobromin, which functions as a Ras-GTPase-activating protein. Accordingly, Ras is aberrantly activated in NF1decient tumors. Ras has many functions in the cell, one of which is to activate the PI3K-TSC1/TSC2-mTOR cascade. It has been demonstrated that in NF1-decient cells mTOR is constitutively activated. This activation depends on Ras, PI3K and TSC2. Furthermore, it has been shown that activation of mTOR is essential for in vivo NF1-associated tumorigenesis (Fig. 2; Table 1) [65,66]. 2.6. Polycystic kidney disease Renal cysts are the hallmark of the autosomal dominant polycystic kidney disease (ADPKD) and are also characteristic for TSC. Mutations in the PKD1 gene, which encodes polycystin-1 (PC1), account for almost 90% of ADPKD. ADPKD is considered to be one of the most common monogenic inherited diseases. The growth of thousands of cysts in both kidneys in a progressive manner during adulthood results in replacement of normal renal tissue and signicant overall growth of the organs. A synergistic role of tuberin and PC1 is suggested by the fact that patients with mutations in both, TSC2 and PKD1, have earlier-onset and more severe polycystic kidney disease than patients harboring only PKD1 mutations. It has been demonstrated that tuberin is required for the localization of PC1 to the plasma membrane [67]. Furthermore, the cytoplasmic tail of PC1 can interact, directly or indirectly, with tuberin and mTOR. This interaction would appear to normally result in inhibition of mTOR activity. Disruption of PC1 triggers high levels of mTOR activity (Fig. 3) [68]. In addition,

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Mutations in PI3K upstream elements, such as epidermal growth factor, ErbB2 or insulin-like growth factor 1 receptor trigger increased PI3K activity in various cancer cell types [1,3,17,80]. The protooncogene Akt is amplied in different tumors, such as, e.g. breast and ovarian cancer [1,6,17]. Rheb expression is upregulated in many human cancers [81] and p70S6K often is amplied or overexpressed in breast cancers correlating with poor prognosis [82]. 4.2. Ras, mTOR and cancer Growth factors activate receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), plateletderived growth factor receptor (PDGFR), broblasts growth factor receptor (FGFR), insulin-like growth factor 1 receptor (IGF-1R), interleukin receptors, vascular endothelial growth factor receptor (VEGFR), interferon receptors, and integrin receptors, which then activate two key signalling molecules. As described above, one is the lipid kinase PI3K and the other one is the small GTPase Ras (Fig. 2). In fact, most human tumors harbor activating mutations in K-ras, H-ras, N-ras, the p110a PI3K subunit or in RTKs [17,83]. Tuberin is also phosphorylated by ERK at S664 (Fig. 2). Mitogenic stimuli or oncogenic Ras via Raf activate the mitogen-activated protein kinase (MAPK)/Erk pathway leading to phosphorylation of tuberin and inactivation of the TSC protein complex to regulate p70S6K [84]. Furthermore, the MAPK-activated kinase RSK1 interacts with tuberin and phosphorylates it at S1798 triggering inactivation of tuberin and increased mTOR signalling to p70S6K (Fig. 2) [85]. Very recent data provide evidence that the function of Ras to trigger inactivation of tuberin via the MAPK/Erk pathway plays a major role in the regulation of cell survival upon mutational activation of the oncogene Ras [86,87]. 4.3. TNF-a activates mTOR Dysregulation of the tumor necrosis factor-a (TNF-a) signalling pathway contributes to the development of many human cancers due to enhanced IKK activity and constitutive activation of the transcription factor NF-kB. Very recently, this pathway of transcription control has also been demonstrated to affect the regulation of translation. TNF-a activates mTOR through phosphorylation and inactivation of hamartin by the kinase IKKb (Fig. 2) [88]. 4.4. Wnt regulates mTOR Components of the mTOR pathway may also be therapeutic targets for diseases linked to hyperactive Wnt signalling. The Wnt protein family regulates a wide range of cellular functions including cell growth, proliferation, polarity, differentiation and development. Deregulation of the Wnt/b-catenin pathway is a common feature in many human neoplasms [89,90]. In the absence of the secreted factor Wnt, GSK3 phosphorylates b-catenin triggering ubiquitination and degradation of b-catenin. Wnt stimulation leads to activation of the protein Dishevelled (Dsh) to inhibit the ability of GSK3 to phosphorylate b-catenin. Another component of this degradation complex is the scaffold protein Axin. Both, hamartin and tuberin, have been reported to coimmunoprecipitate with GSK3 and Axin [91,92]. As already described above, Wnt inhibits the GSK3b-mediated phosphorylation of tuberin. GSK3 inhibits the mTOR pathway via phosphorylating tuberin, what depends on AMPK-priming phosphorylation of tuberin and triggers activation of tuberins potential to inhibit mTOR. Accordingly, Wnt inhibits the GSK3-mediated activation of tuberin to block mTOR (Fig. 2) [58].

4.5. mTOR and renal cell carcinoma In about 60% of renal cell carcinomas the VHL gene is inactivated [63,64]. As already described, inactivation of VHL triggers increased activity of the transcription factor HIF leading to overexpression of vascular endothelial growth factor (VEGF), plateled-derived growth factor b (PDGFb) and transforming growth factor a (TGF-a). A role of mTOR for renal cell carcinoma development has been suggested because mutations in TSC1 or TSC2 lead to activation of mTOR, confer a predisposition to renalcell carcinoma and are accompanied by upregulated HIF activity. mTOR is a key upstream regulator of HIF (Fig. 3). Recent data suggest that inhibition of mTOR results in clinical benet in renal cell carcinoma patients with poor prognostic features [5964,93]. 4.6. The TSC proteins in sporadic cancer The nding that the TSC genes are involved in regulation of the mTOR signalling network already initiated clinical trials for the treatment with rapamycin, a negative regulator of mTOR, of renal tumors and lung cysts found in tuberous sclerosis [15,17]. Approaches like that have clear implications for the devastating disease, tuberous sclerosis, but beyond that to sufferers from cancers that may also involve the TSC proteins, such as, e.g. sporadic bladder cancer, ovarian and gall bladder carcinoma, nonsmall-cell carcinoma of the lung and breast cancer [94]. Mutations in TSC1 have been found to be associated with the development of human bladder carcinoma [9597]. Evidence has been provided that reduced expression of tuberin might be involved in the progression of human pancreatic cancer [98]. It was also reported that tuberin and hamartin are aberrantly expressed and linked to clinical outcome in human breast cancer. The TSC1 gene promoter was seen to be methylated in human breast tumor tissues [99]. Human xanthoastrocytomas consistently show low TSC1 transcript levels [100]. And recently, it was demonstrated that inactivation of tuberin via loss of expression or phosphorylation occurs frequently in endometrial carcinoma [101]. 4.7. The TSC proteins and p27 A major regulator of mammalian cell cycle control is the cyclindependent kinase (Cdk) inhibitor p27. The transition from G0/G1 to S phase is regulated via Cdk4 and Cdk6 activated by D-type cyclins and via Cdk2 activated by cyclin E and A. Due to its potential to control Cdk activity p27 is a very potent regulator of this transition [102,103]. Another important cell cycle regulator is tuberin. Downregulation of endogenous tuberin expression induces quiescent broblasts to enter the cell cycle and TSC2negative cells exhibit a shortened G1 phase. Overexpression of hamartin or tuberin negatively regulates cell cycle progression accompanied by an increase of G1 cells and of p27 protein levels. Tuberin negatively regulates the activity of Cdk2 [104107]. p27 is known to be regulated on the level of protein stability and via subcellular localization. Binding of tuberin to p27 inhibits p27 degradation by sequestering p27 from the Skp2-containing E3 ubiquitin ligase [108]. Tuberin also induces nuclear p27 localization by inhibiting its 14-3-3-mediated cytoplasmic retention [109] (Fig. 3). In bladder tumors TSC gene mutations are correlated with reduced p27 expression [9597]. Cytoplasmic localization of p27 was found in breast cancer and it was demonstrated that tuberin and hamartin expression is downregulated in breast cancer. Due to this connection between tuberin and p27, one could assume that under certain circumstances p27 or Cdk2 could also be considered as targets in addition to mTOR for therapeutics of hamartoma diseases and other tumor diseases [99,110,111].

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5. The mTOR pathway and cardiac hypertrophy Under energy starvation conditions tuberin is phosphorylated and thereby activated by AMPK [56,57]. Dependent on AMPKpriming phosphorylation of tuberin, GSK3b phosphorylates tuberin and triggers activation of its potential to inhibit mTOR [58] (Fig. 2). AMPK g2, an important regulatory subunit of AMPK, is encoded by the gene PRKAG2. Mutations in PRKAG2 are responsible for familial cardiac hypertrophy and Wolff-Parkinson-White syndrome, a distinctive ventricular electrophysiologic abnormality [112,113]. The knowledge that an important hallmark of cardiac hypertrophy (a major risk for cardiac morbidity and mortality) is hyperactivation of the PI3K-mTOR cascade, initiated a discussion whether inhibition of the mTOR pathway could be benecial for therapies of these diseases [17,60,114,115]. 6. The mTOR pathway in obesity and type 2 diabetes p70S6K has been demonstrated to phosphorylate IRS1 on multiple inhibitory sites promoting its degradation (Fig. 1) [12]. Inhibition of IRS protein function is one means by which cells become desensitized to insulin. Phosphorylation of IRS1, which is known to antagonize IRS signalling, is elevated in animal models of obesity and in muscle from type 2 diabetic patients. Recent studies have shown that insulin resistance can be regulated by mTORC1 activation of p70S6K through the negative feedback loop. The inability to respond to insulin (insulin resistance) is a hallmark of both, obesity and type 2 diabetes [4,12,17,60,114,115]. 7. Summary The mTOR signalling pathway is activated during a wide variety of cellular responses, which includes T-lymphocyte activation, neurogenesis, muscle regeneration, insulin signalling and tumor formation. This has provoked intense interest in the TSC/mTOR cascade from virtually all major therapeutic areas related to many human diseases. mTOR plays an important role in Alzheimers disease, cardiac hypertrophy, obesity and type 2 diabetes. mTOR is regulated by TNF-a and Wnt, both of which have been shown to play critical roles in many human cancers. The TSC proteins have been implicated in the development of several sporadic tumors and in the control of p27, known to be of relevance for several cancers. The mTOR upstream regulators Ras, PI3K, Akt and Rheb are known to be deregulated in a wide variety of human tumors. Tuberous sclerosis, Peutz-Jeghers syndrome, Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome, Lhermitte-Duclos disease, Proteus syndrome, von Hippel-Lindau disease, Neurobromatosis type 1, and Polycystic kidney disease are caused by mutations in the mTOR pathway component genes TSC1, TSC2, LKB1, PTEN, VHL, NF1 and PKD1, respectively. Very recent data suggest that inhibition of mTOR results in clinical benet in renal cell carcinoma patients with poor prognostic features and in the treatment of renal tumors and lung cysts found in tuberous sclerosis. It is the hope of investigators and patients alike that a more detailed understanding of the mTOR signalling cascade will lead to new therapies for many different human diseases. Acknowledgements Research in our laboratory is supported by the FWF Austrian Science Fund (P18894-B12), by the Marie Curie Research Network of the European Community (FP6 036097-2) and by the Herzfeldersche Familienstiftung. We apologize to those colleagues whose work is not cited due to space limitations or our inability to draw connections between elements of the primary literature.

Conict of interest None. References


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