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Possible mechanisms of disease development in tuberous sclerosis

Jaroslaw Jozwiak, Sergiusz Jozwiak, Pawel Wlodarski

The two-hit hypothesis presented by Knudson in 1971 explains the development of tumours decient in antioncogenes. Hamartomas in patients with tuberous sclerosis usually t into this model, the rst hit is a congenital lesion of either of the tuberous sclerosis genes (TSC1 or TSC2), and the second hit is loss of heterozygosity of this gene. Although this mechanism is true for most tumours associated with tuberous sclerosis, only 3060% of brain and cardiac tumours show loss of heterozygositythe remaining tumours develop despite the presence of an intact allele. Tumours in which loss of heterozygosity is rare, such as subependymal giant-cell astrocytoma, might all share a common feature that mimics loss of heterozygosity either by inactivation of the TSC complex or by direct activation of mammalian target of rapamycin (mTOR) or its downstream targets. Because phosphorylation of the TSC complex can inactivate it, expression and activation patterns of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK), two potent protein kinases that are activators of the mTOR pathway, have been implicated. AKT activation is detected only in few samples, whereas ERK is hyperactive in all subependymal giant-cell astrocytomas. We postulate that ERK activation consistently detected in dierent tuberous-sclerosis-associated tumours is a molecular trigger for the development of these neoplasms.

Lancet Oncol 2008; 9: 7379 Department of Histology and Embryology, Center for Biostructure Research, Medical University of Warsaw, Warsaw, Poland (J Jozwiak PhD, P Wlodarski MD); and Department of Pediatric Neurology, Childrens Memorial Health Institute, Warsaw, Poland (Prof S Jozwiak MD) Correspondence to: Dr Jaroslaw Jozwiak, Department of Histology and Embryology, Center for Biostructure Research, Medical University of Warsaw, 02-004 Warsaw, Poland jjozwiak@atdv.com.pl

Introduction
Tuberous sclerosis (gure 1) is an autosomal dominant disease that aects about one in 6000 people1. Patients with tuberous sclerosis present with hamartomas of the brain, heart, kidneys, skin, or lungs (gure 2). The disease is caused by the mutation of one of two tumour-suppressor genes, TSC1 and TSC2, which encode hamartin and tuberin, respectively.1,2 Both proteins form a ubiquitous intracellular complex (called the TSC complex), a universal sensor that gathers several intracellular and extracellular signals: growth factor stimulation (acting via protein kinases); hypoxia via DNA-damage-inducible transcript 4 protein (REDD-1); and energy levels through 5-AMPactivated protein kinase. Additionally, mammalian target of rapamycin (mTOR) is a sensor of intracellular aminoacid availability via a mechanism that uses phosphatidylinositol 3-kinase (PI3K). The TSC complex has GTPase-activating properties for a small G protein, Ras homologue enriched in brain (Rheb), which directly activates mTOR.3 When one of the genes is mutated, the TSC complex does not function properly, which leads to upregulation of mTOR. This kinase in turn phosphorylates eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1; also known as phosphorylated heat and acid-stable protein regulated by insulin 1), ribosomal protein S6 kinase beta-1 (S6K1), and eukaryotic translation initiation factor 2, proteins involved in ribosome biogenesis and recruitment as well as translation initiation (gure 3).4 Cells trigger the translation of proteins, including those responsible for cell-cycle progression (eg, cyclin D1 or ornithine decarboxylase). An alternative mechanism of cell-cycle regulation by the TSC complex is connected with tuberins ability to bind cyclin-dependent kinase inhibitor 1B (p27Kip1) and protect it from degradation.5 Loss of the TSC complex leads to the formation of hamartomas (benign tumours composed of an abnormal
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mixture of tissue elements) or more aggressive neoplasms. Despite the eort of numerous research groups, there are still many unexplained features of tuberous sclerosis. Subependymal giant-cell astrocytomas grow slowly whereas renal angiomyolipomas progress rapidly and are the main cause of death in patients with the disease.6 Cardiac rhabdomyomas appear during fetal development and regress during the rst years of life,7 but angiomyolipomas usually appear during adolescence. Loss of heterozygosity in genes encoding tuberin or hamartin, which is believed to be required for neoplastic transformation, is found in all types of tumours associated with tuberous sclerosis except subependymal giant-cell astrocytomas810 and rhabdomyomas,1113 in which it occurs sporadically. Finally, 20% of patients with the characteristic tuberous sclerosis phenotype do not have mutations in TSC1 or TSC2.14 The absence of mutations in TSC1 and TSC2 seems to be directly related to the absence of loss of heterozygosity

Figure 1: Facial angiobromas in tuberous sclerosis

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Figure 2: More symptoms of tuberous sclerosis (A) Periungual bromas. (B) Subependymal giant-cell astrocytoma in the lateral ventricle. (C) Hypomelanotic macule. (D) Shagreen patch. (E) Renal angiomyolipomas.

in tumours associated with tuberous sclerosis and suggests that there is still another, more universal, mechanism. For the past few years, researchers have sought a mechanism that inhibits the activity of the TSC complex, leading to tumour development, even when hamartin and tuberin are intact. In 2002, Dan and colleagues15 reported that protein kinase B (AKT) phosphorylates and inhibits tuberins GTPase activity towards Rheb, and in 2004 Roux and co-workers16 showed that a similar eect is also achieved by tuberin phosphorylation (on dierent moieties, though) by ribosomal protein S6 kinase alpha-1, which is activated by extracellular signal-regulated kinase (ERK; also known as mitogen-activated protein kinase). AKT, a downstream eector of PI3K, is a typical activator of mTOR-dependent translation, and numerous researchers have called the pathway the PI3K/mTOR pathway or the PI3K/AKT/ S6K1 pathway.17 Because of its strong anti-apoptotic and proproliferative functions, AKT is implicated in the pathogenesis of various human malignancies. Since AKT can suppress the activity of tuberin, this kinase can also abrogate TSC-complex activity in the presence of wildtype hamartin and tuberin. Until very recently, the role of ERK in these pathologies was poorly understood.
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AKT and tumour progression

Most research on tuberous sclerosis is done with either Tsc2/+ mice and rats (Tsc2/ mutation is lethal) or cells overexpressing altered tuberin. These are not optimum models, because of the role of AKT in tuberin inactivation. Phosphomimicking of mutations in Tsc2 induces mTOR activity, whereas a phosphorylation-decient mutation in the same transgene inhibits mTOR activity and the growth of developing Drosophila.18 Dong and Pan19 introduced the nonphosphorylatable, phosphomimicking, and wild-type Tsc2 into a Tsc2-null mutant Drosophila. This setting had the advantage of providing intracellular concentrations of Tsc2 similar to those of the normal protein, in the absence of the endogenous gene. Neither of the mutated transgenes was lethal to Drosophila larvae, and both were nearly as ecient as the wild-type gene in fullling their physiological role. All the adult ies had similar bodyweight, wing size, and cell size. As far as we are aware, this was the rst suggestion of a possible overestimation of AKT importance in the mTOR pathway. More doubts on the role of AKT in tuberous sclerosis pathogenesis came from studies on the activation status of this kinase in TSC-complex-decient cells. First, Kwiatkowski and colleagues17 and Jaeschke and
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co-workers20 found that TSC1 and TSC2 decient cells are unable to activate AKT in response to insulin stimulation. Harrington and co-workers21 explored the inability of insulin and insulin-like growth factor-1 to activate AKT in Tsc2/ broblasts. In such cells, insulinlike growth factors do not activate PI3K due to inactivation of adaptor proteins: insulin receptor substrates 1 and 2. Activation of S6K1 in Tsc2/ cells suppresses insulin receptor substrate 1 (IRS-1), both at the level of gene transcription and by coupling activated receptors to PI3K. However, the eect of TSC-complex loss on PI3K activity is limited to signals received via insulin or IGF receptors, whereas signalling via epidermal growth factor and platelet-derived growth factor is unaected. Another hint pertaining to lower AKT signicance came quite recently. As mentioned above, tuberin is a negative regulator of cell-cycle progression, as it protects p27 from degradation and increases its anity to cyclindependent kinase 2. This mechanism is thought to contribute substantially to hamartoma formation if tuberin cannot full its physiological role. AKT phosphorylation of tuberin does not abrogate this function.5 Thus, it seems that AKT cannot participate in cell-cycle regulation via p27. Last, but not least, we assessed the concentration of active AKT in tumours from patients with tuberous sclerosis. The concentration of active AKT was not high in subependymal giant-cell astrocytomas, angiomyolipomas, rhabdomyomas, or skin tumours, but was at the same or even lower concentrations as noted in healthy tissues. Additionally, in a cell line derived from a subependymal giant-cell astrocytoma, active AKT concentration was not high, nor did AKT promote growth.22 These observations agree with the reports presented earlier in this Personal View that TSC-complexdecient or mTOR-activated cells have attenuated AKT pathways.

AKT

REDD-1

pO

RSK-1 tuberin hamartin Pi Rheb GTP Rheb

AMPK

LKB1

ERK

AMP

GDP

mTOR

PI3K

Aminoacid

S6K1

4E-BP1

eEF-2K

Figure 3: Regulation of TSC complex Diagram showing regulation of TSC complex controlling mammalian target of rapamycin (mTOR) activity. AKT=Protein kinase B. REDD-1=DNA-damageinducible transcript 4 protein. RSK-1=ribosomal protein S6 kinase alpha-1. LKB1=Serine/threonine-protein kinase 11. ERK=extracellular signal-related kinase. Rheb=Ras homologue enriched in brain. Pi=phosphate group. PI3K=phosphatidylinositol 3-kinase. S6K1=ribosomal protein S6 kinase beta-1. 4E-BP1=eukaryotic translation initiation factor 4E-binding protein 1. eEF-2K=elongation factor 2 kinase.

AKT and pathogenesis of tuberous sclerosis

AKT is constitutively active in many types of human malignancies. Such activation can occur as the result of amplication of the AKT gene or as a consequence of activating mutations in components of the PI3K signalling pathway. As AKT promotes increased cell survival and cell proliferation, it contributes to cancer progression. When Dan and colleagues15 identied AKT as an inactivating kinase for tuberin, it seemed that the mystery of loss of TSC-complex function despite the presence of wild-type hamartin and tuberin was solved. Soon after, researchers provided substantial evidence for this hypothesis. There are, however, at least two other explanations as to why AKT is deemed to underlie tumour progression in tuberous sclerosis. The rst is connected with how inhibitors of PI3K and AKT are used. LY294002, a commonly used PI3K inhibitor, interacts directly with mTOR and inhibits its catalytic activity,23 which makes this compound a awed choice for studies on relations
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between AKT and mTOR pathways. Use of this compound, which inhibits mTOR at equimolar concentrations,24 is commonly the reason why the contribution of the PI3K/AKT pathway in mTOR activation tends to be overestimated. Another inhibitor, wortmannin, in higher but commonly used concentrations, also has dual inhibitory specicity. Second, phosphorylation of glycogen synthase kinase-3 beta, a substrate of AKT commonly used as a marker of AKT activation, is regulated directly by S6K1.25 Hence, loss of TSC-complex function can lead to constitutive phosphorylation (at the same residue as AKT) and inhibition of glycogen synthase kinase-3 beta, which is PI3K/AKT-pathway-independent and can be observed even during transient loss of hamartin or tuberin. Thus, not only is AKT phosphorylation probably not increased in tuberous-sclerosis-associated tumours, but also the eect of phosphorylation of glycogen synthase kinase-3 beta and the resulting pro-proliferative stimulus attributed to AKT might be secondary to the suppressed activity of the TSC complex. The role of active AKT, however, might be more pronounced in rapidlyprogressing malignancies.26

Epigenetic silencing, TSC3, mosaicism, or phosphorylation of tuberin?

Because as many as 20% of patients do not carry any mutation of TSC1 or TSC2, it is possible there might be a
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Somatic mutation Hereditary

Mutation

Somatic mutation Non-hereditary

Somatic mutation

Figure 5: Knudsons two-hit model of oncogenesis Two genetic events are required to initiate neoplastic transformation. In the hereditary form of the disease, the rst sporadic mutation leads directly to a neoplastic phenotype. In the nonhereditary form, two events have to occur, one on each copy of the gene. Figure 4: Development of mosaic embryos Sporadic mutation occurs during early divisions of the embryo, which are therefore formed from a mixture of genetically dierent cells. Depending on the distribution of mutated cells between the trophoblast and embryoblast dierent organs will be aected with the mutation with dierent severity.

third protein in the TSC complex (putative TSC3), although there is no evidence for the existence of such a molecule. Furthermore, epigenetic silencing is an unlikely explanation in such patients, as no evidence of methylation of the TSC2 promoter region has been noted.27 Patients with no mutation detected in their leucocytes are diagnosed with tuberous sclerosis solely on the basis of clinical criteria, and mosaicism is thought to explain the pathogenesis. Mosaicism is a condition in which the rst mutation occurs at an early stage of embryonal development, resulting in an individual having both normal and altered cells (gure 4). Depending on the distribution of mutated cells in the embryo, various organs can be aected, thus there is the risk of false-negative genetic diagnosis. Mosaicism cannot, however, explain another enigma. According to Knudsons hypothesis28, loss of heterozygosity, or mutation of both alleles of an oncosuppressor gene, is necessary for tumour progression (gure 5). Loss of heterozygosity is common in several types of tuberous-sclerosis-associated tumours, but not in cardiac or brain hamartomas, where the presence of both wild-type proteins is typical. The hypothesis of a putative TSC3 again seems appealing. In view of recent publications showing a lack of clinical features that dierentiate mutation-free patients with tuberous sclerosis from those decient in either hamartin or tuberin,14 a third protein mutated in tuberous sclerosis (putative TSC3) would have to be very closely associated with the TSC complex, otherwise its mutation would naturally cause symptoms that are characteristic only for this genetic change. Patients with mutations in TSC1 have a lower frequency of seizures and moderate-tosevere mental retardation, fewer subependymal nodules
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and cortical tubers, less-severe kidney involvement, no retinal hamartomas, and less-severe facial angiobroma than patients with TSC2 mutations.29 Nevertheless, clinical presentation of tuberous sclerosis in patients without loss of heterozygosity for either of these genes is similar to both phenotypes of identied mutations. Development of tumours that are heterozygous for tuberous-sclerosis genes strongly suggests an alternative mechanism that could trigger neoplastic transformation even when one allele of a tumour suppressor gene remains intact or even when both alleles are functional. Inhibition of tuberin by its phosphorylation seems to be a very good candidate for such a mechanism. As presented earlier in this Personal View, the role of AKT in this model is doubtful; hence we investigated the role of ERK in various tumours.

ERK and tuberous sclerosis

Several researchers have noted hyperphosphorylation of ERK in cell lines developed from tuberous-sclerosisrelated neoplasms, such as human angiomyolipoma,30 subependymal giant-cell astrocytoma,22 lymphangioleiomyomatosis,31 and murine sarcoma,32 and in tissue samples from cortical tubers,33 subependymal giant-cell astrocytoma,32 angiomyolipoma,22 and periungual broma.32 Activated ERK might play an important part in tumour progression. Ma and colleagues34 assessed the tumorigenic potential of the Tsc2ang1 cell line derived from a cutaneous sarcoma in Tsc2+/ mice by transfecting the cells with wild-type TSC2, TSC2-mutant nonphosphorylatable by ERK, or the TSC2-phosphorylationmimicking mutant. Cells were cultured in soft agar or injected into athymic mice. In both assays, only the ERKnon-phosphorylatable mutant decreased the tumorigenic potential of the cell line. Similarly, Govindarajan32 found that introduction of dominant-negative ERK inhibits tuberous-sclerosis-related tumour growth in vivo.
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We investigated the role of AKT and ERK in clinical biopsy material and in a cell line that we derived from a subependymal giant-cell astrocytoma from a patient with tuberous sclerosis. Although the concentration of activated AKT in subependymal giant-cell astrocytoma and angiomyolipoma was generally the same as that in control tissues, ERK activity was much higher in tumours than in control tissues of the brain (gure 6) and kidney. To test the inuence of ERK on cell proliferation and tumour progression, we analysed the proliferation rate of a cell line derived from subependymal giant-cell astrocytoma.22 Inhibition of mitogen-activated protein kinase kinase (MAP2K), a canonical ERK kinase, with two inhibitors, U0126 and PD98059, only slightly decreased ERK phosphorylation, whereas this eect was much stronger in two control cell lines. Still, ERK inhibition evoked a decrease in the proliferation of cells derived from the subependymal giant-cell astrocytoma. We concluded that ERK inhibition in subependymal giant-cell astrocytoma is MAP2K-independent, and MAP2K inhibitors are only able to abolish a constitutive low ERK activation, without inuencing stimulus-driving ERK activation. Govindarajan and colleagues32 noted that tumours with ERK activation are likely to have inactivation of cyclindependent kinase inhibitor 2A, isoforms 1/2/3 (p16INK4A). Indeed, the growth of Tsc2ang1 cells derived from a murine model of tuberous-sclerosis-associated sarcoma is ERK-dependent, and the cell line shows inactivation of p16INK4A. Even more importantly, most renal tumours arising in Eker rats (Tsc2+/ model of tuberous sclerosis), in which loss of heterozygosity is well-documented from the early stages of tumour development, show inactivation of p16INK4A, which supports our hypothesis of a more universal signicance of ERK (than just when loss of heterozygosity is not present).

SEGA 1 2 3 4 5 6

Human brain pAKT s473 pPTEN S380 pERK T202/Y204 pRSK-1 S380 pS6rp S236/236

Figure 6: Molecular pathways implicated in tumour progression Western blot of subependymal giant-cell astrocytoma (SEGA) shows consistent activation of ERK and its substrate, ribosomal protein S6 kinase alpha-1 (RSK-1), whereas the AKT pathway is rarely activated. Protein lysates of healthy human and murine brain tissue were used as controls. PTEN=phosphatase and tensin homologue. S6rp=S6 ribosomal protein.

Mechanism of ERK activation: the great unknown

One of the potential mechanisms that could account for increased ERK activation in the absence of loss of heterozygosity is activation of tyrosine-kinase receptors. Cells from angiomyolipomas have active platelet-derived growth-factor receptor . Growth of such cells can be decreased by imatinib, that, apart from being an inhibitor of bcr-abl tyrosine kinase, also inhibits this receptor.35 Furthermore, the dominant-negative allele of tuberin transactivates platelet-derived growth-factor receptor .36 Resistance to MEK inhibitors in our experiments was surprising, because until recently the Ras/Raf/MEK pathway was the only known kinase cascade for ERK. When we noted that ERK activation in tuberous-sclerosis lesions is MEK-independent, we looked for alternative pathways. One of the earliest hypotheses favoured the implication of PI3K and protein kinase C in ERK
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activation.37 In Tsc2+/+ cells stimulation of platelet-derived growth factor triggered phosphorylation of ERK. This eect was completely abolished by combined application of a high dose of the PI3K inhibitor wortmannin and phorbol ester (which downregulates protein kinase C). Furthermore, ERK activation can only be decreased by bisindolyl maleimide, a protein kinase C inhibitor.38 Another study strongly supports our hypothesis of an alternative mechanism for ERK activation. Finlay and co-workers39 found that Tsc2+/+ smooth-muscle cells derived from the Eker rat respond to platelet-derived growth factor stimulation by triggering canonical MAP2K-dependent ERK activation and translocation of phosphorylated ERK to the nucleus, whereas in the absence of tuberin (Tsc2/) the same cells activate ERK without engaging MAP2K. Activation of ERK in Tsc2/ cells, but not in their Tsc2+/+ counterparts, supposedly requires an intermediate of reactive oxygen species, superoxide anion (O2). Activation of ERK in angiomyolipoma or lymphangioleiomyomatosis, tumours in which there is always loss of heterozygosity, can be caused by one of the eectors of mTOR, which is hyperactive due to loss of both functional alleles of the genes encoding either hamartin or tuberin.40,41 Occasionally, this mechanism may not be engaged, as in patients with loss of heterozygosity but without ERK activation.42 ERK activation is consistently found in tuberous-sclerosis-associated tumours such as subependymal giant-cell astrocytoma or rhabdomyoma, in which loss of heterozygosity is not found, strongly suggesting its causative role as a surrogate of loss of heterozygosity in a mechanism of post-translational activation.
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Search strategy and selection criteria Data for this Review were identied by searches of PubMed (journals published in English from Jan 1991, to Aug 2007) and references from relevant articles with the search terms tuberous sclerosis, ERK, AKT, Mek-independent ERK activation.

The concept of ERK being a surrogate of loss of heterozygosity is supported by experimental work on manipulated cell lines, where this kinase clearly had a role in neoplastic transformation.43 These observations were supported by analysis of tumours, where the potency of ERK activation was exceptionally high. Results of our experiments on a unique cell line obtained from a subependymal giant-cell astrocytoma are convincing proof for the role of this activity in the growth of such gliomas. MEK-independent activation of ERK in this cell line is by an unknown mechanism. Would hyperactive ERK be sucient to trigger formation of tuberous-sclerosis-associated tumours? Because tumour formation is usually a multistep process, one can expect the same in tuberous sclerosis. Indeed, as hyperactive ERK inhibits or bypasses the TSC complex, why is this not a universal mechanism? Small-cell lung cancers have an activated ERK pathway, but this activation does not aect mTOR activity.44 Patients with tuberous sclerosis are heterozygous for one of the TSC genes. A tempting explanation of this dilemma would be that heterozygous cells are haploinsucient in TSC genes and therefore are prone to loss of function of the TSC complex by post-translational modication caused by ERK. Data from rodent models show normal concentrations of tuberin or hamartin in heterozygous and homozygous cells that contradict this hypothesis.34,45 These models are, however, imperfect in studies of malignancies such as subependymal giant-cell astrocytoma, because neither Eker rats (Tsc2+/) nor Tsc2+/ mice develop brain tumours.46 This discrepancy alone shows how pathogenesis of tuberous sclerosis cannot be easily reproduced in animal models. Development of such gliomas is obviously unique to human beings heterozygous for TSC and we postulate ERK involvement in those patients. There are still unanswered questions; however, we feel that the puzzle of tuberous sclerosis pathogenesis is close to being solved.
Conicts of interest The authors declared no conicts of interest. References 1 European Chromosome 16 Consortium. Identication and characterization of the tuberous sclerosis gene on chromosome 16. Cell 1993; 75: 130515. 2 van Slegtenhorst M, de Hoogt R, Hermans C, et al. Identication of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science 1997; 277: 80508. 3 Zhang Y, Gao X, Saucedo LJ, et al. Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins. Nat Cell Biol 2003; 5: 57881.

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