Astrinidis A, Henske EP.. Tuberous sclerosis complex: linking growth and energy signaling pathways with human disease. Oncogene 24: 7475-7481

ArticleinOncogene 24(50):7475-81 · December 2005with29 Reads
Impact Factor: 8.46 · DOI: 10.1038/sj.onc.1209090 · Source: PubMed

The most exciting advances in the tuberous sclerosis complex (TSC) field occurred in 1993 and 1997 with the cloning of the TSC2 and TSC1 genes, respectively, and in 2003 with the identification of Rheb as the target of tuberin's (TSC2) GTPase activating protein (GAP) domain. Rheb has a dual role: it activates mTOR and inactivates B-Raf. Activation of mTOR leads to increased protein synthesis through phosphorylation of p70S6K and 4E-BP1. Upon insulin or growth factor stimulation, tuberin is phosphorylated by several kinases, including AKT/PKB, thereby suppressing its GAP activity and activating mTOR. Phosphorylation of hamartin (TSC1) by CDK1 also negatively regulates the activity of the hamartin/tuberin complex. Despite these biochemical advances, exactly how mutations in TSC1 or TSC2 lead to the clinical manifestations of TSC is far from being understood. Two of the most unusual phenotypes in TSC are the apparent metastasis of benign cells carrying TSC1 and TSC2 mutations, resulting in pulmonary lymphangiomyomatosis, and the ability of cells with TSC1 or TSC2 mutations to differentiate into the separate components of renal angiomyolipomas (vessels, smooth muscle and fat). We will discuss how the TSC signaling pathways are affected by mutations in TSC1 or TSC2, focusing on how these mutations may lead to the renal and pulmonary manifestations of TSC.

Tuberous sclerosis complex: linking growth and energy signaling pathways
with human disease
Aristotelis Astrinidis
and Elizabeth P Henske*
Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA
The most exciting advances in the tuberous sclerosis
complex (TSC) field occurred in 1993 and 1997 with the
cloning of the TSC2 and TSC1 genes, respectively, and in
2003 with the identification of Rheb as the target of
tuberin’s (TSC2) GTPase activating protein (GAP)
domain. Rheb has a dual role: it activates mTOR and
inactivates B-Raf. Activation of mTOR leads to increased
protein synthesis through phosphorylation of p70S6K and
4E-BP1. Upon insulin or growth factor stimulation,
tuberin is phosphorylated by several kinases, including
AKT/PKB, thereby suppressing its GAP activity and
activating mTOR. Phosphorylation of hamartin (TSC1)
by CDK1 also negatively regulates the activity of the
hamartin/tuberin complex. Despite these biochemical
advances, exactly how mutations in TSC1 or TSC2 lead
to the clinical manifestations of TSC is far from being
understood. Two of the most unusual phenotypes in TSC
are the apparent metastasis of benign cells carrying TSC1
and TSC2 mutations, resulting in pulmonary lymphangio-
myomatosis, and the ability of cells with TSC1 or TSC2
mutations to differentiate into the separate components of
renal angiomyolipomas (vessels, smooth muscle and fat).
We will discuss how the TSC signaling pathways are
affected by mutations in TSC1 or TSC2, focusing on how
these mutations may lead to the renal and pulmonary
manifestations of TSC.
Oncogene (2005) 24, 7475–7481. doi:10.1038/sj.onc.1209090
Keywords: tuberous sclerosis complex; lymphangio-
myomatosis; mammalian target of Rapamycin; Rheb;
Tuberous sclerosis complex (TSC) is a tumor suppressor
gene syndrome with a wide spectrum of clinical
manifestations, many of which are age-related. It often
manifests in early age with infantile seizures and benign
cerebral cortical tubers, and patients may have mental
retardation and autism. Other brain lesions include
subependymal nodules, subependymal giant cell astro-
cytomas (SEGAs) and retinal hamartomas. Patients
often develop skin lesions, including facial angiofibro-
mas, hypomelanotic macules, ungual fibromas and
shagreen patches. In the kidney, benign angiomyolipo-
mas and cysts occur frequently, and malignant angio-
myolipomas and renal cell carcinomas are less common.
The brain, skin, and renal manifestations are hallmarks
of TSC and can be used as the basis for differential
diagnosis. Brain and renal lesions cause the highest mor-
bidity and mortality among TSC patients. Heart rhabdo-
myomas, often observed in infancy, spontaneously
regress. A particularly striking manifestation of TSC
is pulmonary lymphangiomyomatosis (LAM). It is
observed exclusively in female TSC patients. LAM can
also occur in women who do not have TSC (sporadic
LAM). LAM is characterized by benign smooth muscle
cell proliferation in the lungs, which thicken the alveolar
wall and reduce gas exchange, and by cystic degenera-
tion of the lungs, resulting in lung collapse. LAM
patients often become oxygen-dependent.
The genetic basis of TSC
TSC is inherited as an autosomal dominant trait with
very high penetrance, but only approximately 20% of
the patients have positive family history of TSC. The
majority of the remaining 80% represents de novo
mutations in either TSC1 or TSC2. TSC2, which was
cloned in 1993, has 41 exons and a 5.5 kb mRNA
transcript (The European Chromosome 16 Tuberous
Sclerosis Consortium, 1993). TSC1, which was cloned in
1997, consists of 21 coding exons (van Slegtenhorst
et al., 1997). The 8.6 kb mRNA transcript contains a
small 5
and a large 4 kb 3
un-translated region.
More than three hundred germline mutations have
been reported for TSC2, including missense, nonsense,
frameshift deletions/insertions and splice junction muta-
tions (Au et al., 1998; Jones et al., 1999; Dabora et al.,
2001; Sancak et al., 2005). The most frequent TSC2
mutations are missense changes in codons 611 and 1675,
and an 18-bp in-frame deletion in exon 40. However
together they represent less than 5% of the known TSC2
mutations. Of the TSC2 mutations, 20% are missense or
nonsense. In contrast, nearly all the reported TSC1
mutations are either nonsense or frameshift, causing
premature protein truncation (Kwiatkowska et al., 1998;
Jones et al., 1999; van Slegtenhorst et al., 1999; Sancak
et al., 2005). Although no significant genotype–pheno-
type correlations have been established, patients with
TSC1 mutations are less severely affected than those
with TSC2 mutations (Jones et al., 1999; Dabora et al.,
In agreement with Knudson’s two-hit tumor suppres-
sor gene model (Knudson, 1971), inactivation of both
*Correspondence: EP Henske; E-mail:
Oncogene (2005) 24, 7475–7481
2005 Nature Publishing Group
All rights reserved 0950-9232/05 $30.00
Page 1
alleles of either TSC1 or TSC2 is necessary at least for
some of the clinical manifestations of TSC. Most
‘second hits’ are large deletions involving loss of
surrounding loci. These mutations are referred to as
loss-of-heterozygosity (LOH) since they affect neighbor-
ing heterozygous polymorphic markers. LOH in TSC1
or TSC2 has been observed in angiomyolipomas,
rhabdomyomas and LAM cells (Carbonara et al.,
1994; Green et al., 1994; Henske et al., 1995, 1996;
Smolarek et al., 1998). LOH has not been consistently
observed in cerebral cortical tubers (Henske et al., 1996;
Niida et al., 2001).
The protein products of TSC1 and TSC2 are
hamartin and tuberin, respectively (Figure 1). Hamartin
and tuberin physically interact to form heterodimers
(Plank et al., 1998; van Slegtenhorst et al., 1998).
Hamartin and tuberin are expressed by the same cell
types within multiple organs including the kidney, brain,
lung and pancreas (Plank et al., 1999). A definitive
subcellular localization has not been established yet for
either hamartin or tuberin. In most studies, hamartin
and tuberin are diffusely cytoplasmic, although tuberin
has been reported in the Golgi apparatus (Wienecke
et al., 1996) and the nucleus (Lou et al., 2001). It is not
clear whether these differences reflect cell type or cell
cycle-specific differential localization, or antibody
Signaling pathways involving the TSC proteins
Hamartin and tuberin participate in the insulin/AKT/
mTOR signaling pathway
The first critical clues for the function of hamartin
and tuberin came from genetic epistasis studies in
Drosophila. Mutant cells for dTsc1 and dTsc2 (the
Drosophila TSC1 and TSC2 homologues, respectively)
exhibit increase in cell and organ size, phenotypes that
are not rescued by insulin receptor (dinr)ordAkt
mutants. However, dS6k mutants reverted the dTsc1
phenotype, suggesting that dTsc1/dTsc2 are epistatic to
dinr and dAkt, and dS6k is epistatic to dTsc1 /dTsc2
(Figure 2a) (Gao and Pan, 2001; Potter et al., 2001).
Biochemical studies showed that mammalian tuberin is
directly phosphorylated by AKT/protein kinase B
(PKB) at two residues, S939 and T1462 (Dan et al.,
2002; Manning et al., 2002; Potter et al., 2002). The
hamartin/tuberin complex acts upstream of the target of
Rapamycin (TOR) in yeast, insects and mammals (Gao
et al., 2002; Inoki et al., 2002; van Slegtenhorst et al.,
2004). Mammalian TOR (mTOR) is a serine–threonine
kinase, which increases cell growth and proliferation
through phosphorylation of two effector molecules:
p70S6K and 4E-BP1. p70S6K is responsible for
increased ribosome biogenesis through phosphorylation
of the ribosomal protein S6. 4E-BP1 binds to eIF4E and
inhibits the 5
cap-dependent mRNA translation
(reviewed by Fingar and Blenis, 2004). Loss of hamartin
or tuberin results in TOR-dependent increased phos-
phorylation of the ribosomal protein S6, p70S6K and
4E-BP1 (Gao et al., 2002; Goncharova et al., 2002; Tee
et al., 2002).
Although a highly conserved GTPase activating
protein (GAP)-related domain was identified in tuberin
in 1993, it was not until 2003 that Rheb, a member of
the Ras super-family, was identified as the specific
GTPase downstream of tuberin (Garami et al., 2003;
Inoki et al., 2003a). Overexpression of Rheb leads to
increased mTOR and S6 phosphorylation, which is
blocked by the immunosuppressant drug Rapamycin
(Castro et al., 2003; Garami et al., 2003; Stocker et al.,
2003; Tee et al., 2003). Several patient-derived TSC2
mutations were identified within the GAP domain
(Maheshwar et al., 1997; Jones et al., 1999). These
mutants have low GAP activity toward Rheb (Li et al.,
Figure 1 (a) Hamartin (encoded by TSC1) is 1164 amino acids long with molecular mass 130 kDa. It interacts with tuberin through
amino acids 302–430 (Hodges et al., 2001) and with ezrin–radixin–moesin through amino acids 881–1084 (Lamb et al., 2000). It is
phosphorylated by CDK1/cyclin B1 at residues T417, S584 and T1047, and by GSK3b at T357 and T390. (b) Tuberin (encoded by
TSC2) is 1807 amino acids long with molecular mass 200 kDa. It interacts with hamartin through amino acids 1–418 (Hodges et al.,
2001). It has a GTPase activating protein (GAP) domain with specificity for Rheb. It is phosphorylated by AKT/PKB (residues S939
and T1462), MK2 (S1210), RSK1 (S1798), AMPK (T1227 and S1345) and ERK2 (S664)
Regulation of cell growth and differentiation by the TSC genes
A Astrinidis and EP Henske
Page 2
2004) confirming that the GAP activity of tuberin is
essential for its physiological function. Moreover,
patient-derived TSC2 mutations that disrupt the asso-
ciation of tuberin with hamartin also have decreased
GAP activity (Nellist et al., 2005b), indicating that
interaction with hamartin is important for tuberin’s
function as a GAP. This is consistent with the similar
clinical phenotype of TSC1 and TSC2-associated
Rheb inhibits B-Raf kinase a Rapamycin-independent
Two groups have demonstrated that Rheb directly binds
and inhibits the kinase B-Raf (Im et al., 2002;
Karbowniczek et al., 2004). Stable or transient expres-
sion of tuberin increases the phosphorylation of p42/44
mitogen-activated protein kinase (MAPK) (Karbow-
niczek et al ., 2004). Tuberin inhibition by siRNA or
expression of Rheb negatively regulates the B-Raf-
induced phosphorylation of p42/44 MAPK in a
Rapamycin-independent manner, indicating that the
Rapamycin-sensitive mTOR complex is not involved
in this process.
These data indicate that Rheb has two separate
growth-related functions: B-Raf inhibition and mTOR
activation. These two opposing effects may counter-
balance each other, naturally limiting Rheb’s ability to
induce tumor growth in cells expressing B-Raf, and
contributing to the fact that the vast majority of tumors
in TSC patients are benign. Since B-Raf expression is
variable between cell types (Hagemann and Rapp,
1999), it is likely that Rheb inhibits p42/44 MAPK
activity in a cell type-specific manner. Whether Rheb
inhibits other members of the Raf family (A-Raf and
C-Raf/Raf1) is not known yet.
Tuberin integrates multiple mitogenic signals
AKT/PKB was the first kinase recognized to phosphor-
ylate and inactivate tuberin upon insulin or growth
factor stimulation. During the past 2 years, other
kinases have also been found to regulate tuberin’s
function (Figure 1b). These kinases include MK2,
ribosomal S6 kinase 1 (RSK1), ERK2 and AMP kinase
(AMPK). The phosphorylation of tuberin at residue
S1210 by MAPK-activated protein kinase 2 (MK2)
(Li et al., 2003) promotes binding of tuberin with
14-3-3 proteins, which may sequester tuberin from its
physiological substrates. Tuberin interacts with and is
phosphorylated by p90 RSK1 at S1798 (Roux et al.,
2004). This phosphorylation site is important for
PMA-induced stimulation of p70S6K and its effect is
additive to the phosphorylation by AKT at S939 and
T1462. Inactivating phosphorylation of tuberin at
residue S664 by ERK2 causes the disruption of the
hamartin/tuberin complex and activation of mTOR. A
single nonphosphorylatable tuberin mutant at this site
inhibits tumorigenicity of a cell line with constitutively
activated MAPK pathway (Ma et al., 2005). All those
phosphorylation events are inactivating and positively
regulate the mTOR signaling pathway. In contrast,
under energy deprivation, tuberin undergoes activating
phosphorylation at T1227 and S1345 by AMPK, leading
to enhanced inhibition of S6 (Inoki et al., 2003b). This
phosphorylation may protect the cells from apoptosis
caused by prolonged energy deprivation.
It is likely that tuberin is the target of additional
phosphorylation events. For example, tuberin is phos-
phorylated at S1395/1397 and S1775 by an unknown
kinase (Nellist et al., 2005a). The identification of novel
phosphorylation sites and kinases will provide us with a
more comprehensive understanding of the signaling
pathways involved in the pathogenesis of the disease.
The potential of mTOR inhibition in TSC therapy
Since mTOR is directly activated upon loss of TSC1 or
TSC2, Rapamycin has been identified as a potential
therapeutic agent for TSC and LAM. Treatment of
hamartin or tuberin null cells with Rapamycin (or the
Rapamycin analogue CCI-779) results in apoptosis and
decreased proliferation, and reduction of tumor size in
the Eker rat model for TSC and in Tsc2
þ /
(Kenerson et al., 2002, 2005; Lee et al., 2005).
Rapamycin clinical trials for individuals with angio-
myolipomas or pulmonary LAM are ongoing.
Growth factors
mTOR B-Raf
growth differentiation
Figure 2 (a) The insulin/AKT/mTOR pathway. Activation of
PI3K/AKT from insulin or growth factors leads to inactivating
phosphorylation of tuberin (TSC2) and decreases the GAP activity
toward Rheb. Rheb-GTP activates mTOR, which directly phos-
phorylates p70S6K and 4E-BP1, leading to increased S6 and eIF4E
activity. The net result of mTOR activation is increased ribosome
biogenesis (through S6) and increased 5
cap-dependent mRNA
translation (through eIF4E). Rheb inhibits B-Raf activity by direct
binding to B-Raf. A negative feedback loop from p70S6K to
insulin receptor substrate (IRS1/2) is responsible for decreased
AKT phosphorylation in hamartin and tuberin-null tumors.
Additionally, tuberin is inactivated by phosphorylation from
ERK2. (b) Hamartin and tuberin integrate growth factor, energy
and cell cycle signals through multiple kinases. Tuberin inhibits
Rheb to suppress mTOR and activate B-Raf, leading to control of
cell growth and differentiation
Regulation of cell growth and differentiation by the TSC genes
A Astrinidis and EP Henske
Page 3
Several research groups made the observation that in
tuberin-null cells, AKT is not efficiently phosphorylated
after growth factor stimulation (Jaeschke et al., 2002;
Kwiatkowski et al., 2002; Zhang et al., 2003). This led to
the identification of a negative feedback loop, which is
responsible for the inactivation of AKT signaling
downstream of insulin receptor substrate proteins,
IRS1 and IRS2 (Figure 2a) (reviewed by Manning,
2004). The stability and phosphorylation of IRS1/2 is
compromised in tuberin-null cells (Jaeschke et al ., 2002;
Harrington et al., 2004) and this is caused by direct
phosphorylation of the IRS proteins by p70S6K
(Harrington et al., 2004; Shah et al., 2004). Rapamycin
treatment restores insulin signaling toward AKT. This
feedback inhibition of AKT signaling, raises the concern
that long-term Rapamycin treatment may increase the
risk of malignant tumors in TSC patient by reactivating
AKT (Manning, 2004).
What is the primary function of hamartin?
In contrast to these advances in understanding the
function of tuberin, very little is known about the
function of hamartin. The fact that patients with TSC1
or TSC2 mutations have similar clinical manifestations
indicates that hamartin is essential to the function of
tuberin. Hamartin could be responsible for the proper
subcellular localization of tuberin or it could be required
to activate tuberin’s GAP domain perhaps by releasing
an autoinhibitory domain. Hamartin is an interacting
partner with members of the ezrin–radixin–moesin
family (Lamb et al., 2000) and neurofilament-L (Had-
dad et al ., 2002), suggesting that it may act as a
scaffolding protein for proper localization of tuberin.
Further evaluation of the subcellular localization of
both hamartin and tuberin is needed.
Whether hamartin is important for the function of
tuberin as a GAP is still controversial. Tee et al. (2003).
found that tuberin’s in vitr o GAP activity is enhanced by
the presence of hamartin, and Nellist et al. (2005b)
reported that mutations which disrupt the hamartin/
tuberin interaction decrease tuberin’s GAP activity,
while Li et al. (2004) concluded that tuberin’s function
in vivo is independent of hamartin.
Recent work from our group indicates that the
activity of the hamartin/tuberin complex is regulated
by hamartin phosphorylation. Hamartin is phosphory-
lated at three residues (T417, S584 and T1047) by the
cyclin-dependent kinase 1 (CDK1) during nocodazole-
induced G
/M arrest and during progression of syn-
chronized cells through G
/M phase of the cell cycle
(Astrinidis et al., 2003). This phosphorylation is evident
at endogenous hamartin levels. Nonphosphorylatable
hamartin mutants for the CDK1 sites inhibit p70S6K
activity more potently than wild-type hamartin, suggest-
ing that hamartin phosphorylation leads to decreased
tuberin GAP activity during G
In addition to phosphorylation by CDK1, hamartin is
the substrate of other kinases. GSK3b phosphorylates
hamartin at residues T357 and T390 (Mak et al., 2005).
This phosphorylation increases the stability of the
hamartin/tuberin complex causing attenuation of
b-catenin signaling. Finally, hamartin is phosphorylated
at residue S505 (Nellist et al., 2005a), although the
responsible kinase is unknown.
Links between signaling and clinical manifestations
of TSC
Pulmonary manifestations of TSC the ‘benign
metastasis’ model
Hyperactivation of S6 and 4E-BP1 has been observed in
LAM cells and angiomyolipomas from TSC patients
(Goncharova et al ., 2002; Kenerson et al., 2002;
El-Hashemite et al., 2003b; Karbowniczek et al.,
2003b). Pulmonary LAM occurs in female TSC patients,
and also in women without TSC (sporadic LAM).
Approximately 60% of sporadic LAM patients have
renal angiomyolipomas, and LAM cells are morpholo-
gically and immunohistochemically identical to the
smooth muscle component of renal angiomyolipomas.
Our group found the same TSC2 mutations in LAM
and angiomyolipoma cells from sporadic LAM patients,
but not in the peripheral blood, normal kidney or
normal lung cells of the patients (Carsillo et al., 2000),
indicating that the LAM and angiomyolipoma cells are
genetically identical and must arise from a common
progenitor. Similar results were obtained from the study
of Japanese sporadic LAM patients (Sato et al., 2002).
This led us to propose the ‘benign metastasis’ model for
LAM pathogenesis, in which histologically benign cells
with mutations in TSC1 or TSC2 have the ability to
metastasize to the lungs from other organs (Carsillo
et al., 2000). Further evidence to support the benign
metastasis model was obtained from analysis of
recurrent LAM after lung transplantation, in which
the same TSC2 mutation was present in native LAM
and recurrent LAM cells after lung transplantation
(Karbowniczek et al., 2003a).
Biochemical and cellular data support the notion that
cells lacking hamartin or tuberin have the ability to
migrate aberrantly. Both hamartin and tuberin activate
members of the Rho GTPase superfamily, which
regulate the actin cytoskeleton, cell morphology and
migration, and induce focal adhesions (Lamb et al.,
2000; Astrinidis et al., 2002; Goncharova et al., 2004).
Tuberin-deficient smooth muscle cells exhibit increased
migration in vitro, compared to cells with reintroduction
of wild-type tuberin, and decreased RhoA-GTP
(Astrinidis et al., 2002). Tuberin expression increases
the in vitro attachment of cells on plastic, consistent with
decreased expression of focal adhesion kinase (FAK)
and increase in the fraction of phospho-FAK (Astrinidis
et al., 2002). These findings are consistent with the
hypothesis that tuberin-deficient cells have increased
migratory potential. In 2004, Goncharova et al. (2004)
showed that expression of full-length tuberin or the
hamartin binding domain of tuberin results in Rac1
activation and decreases the activation of RhoA. These
results contradict our finding that, upon tuberin
Regulation of cell growth and differentiation by the TSC genes
A Astrinidis and EP Henske
Page 4
expression, the activity of Rac1 does not change and
that RhoA is strongly activated (Astrinidis et al., 2002),
and may reflect differences in the experimental appro-
aches in the two papers; stable retroviral reintroduction
(Astrinidis et al., 2002) versus transient overexpression
of tuberin (Goncharova et al., 2004).
In 2004, the groups of Sabatini and Hall found that
shRNA or siRNA inhibition of mTOR or rictor, a novel
interacting partner of mTOR, induces formation of
actin stress fibers and focal adhesions (Jacinto et al.,
2004; Sarbassov et al., 2004). These results are consistent
with the finding that hamartin expression induces
formation of stress fibers and focal adhesions (Lamb
et al., 2000). Rictor forms a complex with mTOR and
GbL (Jacinto et al., 2004; Sarbassov et al., 2004), which
is distinct from the Rapamycin-sensitive mTOR/GbL/
raptor complex (Kim et al., 2003). Whether mTOR/
rictor is regulated by the hamartin/tuberin complex, and
whether Rheb mediates the hamartin/tuberin-dependent
changes in the cytoskeleton, is unknown. Also, the
relevance of patient-derived TSC2 mutations with
respect to the aforementioned changes has not been
studied yet.
The fact that pulmonary LAM occurs only in women
has led to the hypothesis that estrogen regulates TSC
signaling. Consistent with this hypothesis, pulmonary
LAM and angiomyolipoma cells from LAM patients
express estrogen and progesterone receptors (Kinoshita
et al., 1995; Logginidou et al., 2000). The carboxy-
terminus of tuberin interacts with estrogen receptor a
(Henry et al., 1998; Finlay et al., 2004) and functions
in vitro as a transcriptional corepressor of the estrogen
receptor (Noonan et al., 2002). The growth of cells with
TSC2 mutations is stimulated by estradiol (Howe et al.,
1995; Yu et al., 2004), and re-expression of tuberin in
tuberin-null cells abrogated estradiol-induced growth
in vitro (Finlay et al., 2004). York et al. (2005) recently
showed that tuberin and estrogen receptor a interact at
endogenous expression levels in multiple cell types.
Whether the tuberin/estrogen receptor a interaction is
disrupted by patient-derived TSC2 mutations, and how
estrogen signaling might lead to LAM is not known.
Renal manifestations of TSC aberrant differentiation
of angiomyolipomas
The renal manifestations in TSC patients include both
mesenchymal (angiomyolipomas) and epithelial (cysts,
oncocytomas and carcinomas) tumors. This hetero-
geneity suggests that the hamartin/tuberin complex
may regulate the epithelial–mesenchymal transition of
progenitor renal cells (Henske, 2004).
Angiomyolipomas are by far the most extensively
studied renal manifestation of TSC. Angiomyolipomas
have three distinct components: vessels, smooth muscle
and fat cells. LOH at the TSC1 or TSC2 locus (Henske
et al., 1995; Niida et al., 2001; Karbowniczek et al.,
2003b) and hyperphosphorylation of S6 (El-Hashemite
et al., 2003b; Karbowniczek et al., 2003b) have been
observed in each of the three components of angiomyo-
lipomas. This indicates that all three components arise
from a common progenitor and suggests that the
hamartin/tuberin complex regulates cellular differentia-
tion. The vascular component of angiomyolipomas
consists of five morphologically distinct types of vessels
(Karbowniczek et al., 2003b), four of which are
neoplastic, with TSC2 LOH, and one of which is non-
neoplastic. The finding that the hamartin/tuberin com-
plex is required for mTOR inhibition during hypoxic
conditions and that disruption of the complex increases
hypoxia-induced factor 1 (HIF1) (Brugarolas et al.,
2004) raises the possibility that the non-neoplastic
vasculature of angiomyolipomas is partially a result of
aberrant mTOR activity. Vascular endothelial growth
factor expression is increased in primary renal cell
carcinomas from Tsc2
þ /
Eker rats (Liu et al., 2003), is
secreted from Tsc1 and Tsc2 null mouse embryonic
fibroblasts and is increased in the serum of Tsc1
þ /
(El-Hashemite et al., 2003a).
A new model for somatic inactivation constitutive
phosphorylation of tuberin
One intriguing aspect of TSC pathophysiology is that, in
contrast to renal angiomyolipomas and pulmonary
LAM, cortical tubers have not been shown to have
LOH at either the TSC1 or TSC2 regions (Henske et al.,
1996; Niida et al., 2001; Ramesh, 2003). It is possible
that a second hit genetic event is present only in certain
cell types within a tuber. However, some groups
proposed that constitutive phosphorylation and inacti-
vation of tuberin might substitute for a genetic second
hit (Govindarajan et al., 2003; Han et al., 2004; Ma
et al., 2005). Since second hit events can be very difficult
to detect, particularly in tumors with both neoplastic
and non-neoplastic components, whether this model
actually contributes to any of the clinical manifestations
of TSC is unknown.
The hamartin/tuberin complex as a nexus for cell growth
signaling and the future of TSC research
The hamartin/tuberin complex appears to integrate
growth factor, energy, nutrient and cell cycle cues to
regulate cell growth and differentiation in an mTOR
and B-Raf-dependent manner (Figure 2b). The dysre-
gulation of hamartin or tuberin, by genetic alterations
or perhaps by inactivating phosphorylation, has devas-
tating consequences in the homeostasis of cell growth
and proliferation, as manifested by the development of
TSC and LAM. The existence of negative feedback
loops increases the complexity of these signaling path-
ways and may influence the development of targeted
therapeutic strategies. Key priorities that will undoubt-
edly be at the forefront of basic TSC research are
elucidating hamartin’s function and its role in the
localization and function of tuberin, determining
whether Rheb is the sole downstream effector of the
hamartin/tuberin complex, and determining whether the
cytoskeletal changes observed in TSC cells are mediated
by the Rapamycin-insensitive mTOR/rictor complex. In
Regulation of cell growth and differentiation by the TSC genes
A Astrinidis and EP Henske
Page 5
addition, there are many areas of uncertainty regarding
the pathogenesis of the renal and pulmonary mani-
festations of TSC. The role of estrogen in LAM
pathogenesis, the cellular basis of the ‘benign metastasis’
model of LAM cells, and the pathways through which
the TSC proteins regulate cellular differentiation in
angiomyolipomas, need to be elucidated. The short
and long-term impact of Rapamycin therapy in
TSC and LAM is unknown. Finally, the develo-
pment of animal models that recapitulate the most
clinically significant tumors and symptoms of TSC will
be critical for progress toward targeted therapeutic
This work was sponsored by grants from the National
Institutes of Health, The LAM Foundation (Cincinnati, OH,
USA), the Tuberous Sclerosis Alliance (Silver Spring, MD,
USA), and the Rothberg Institute for Childhood Disease
(Guilford, CT, USA).
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