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Vol. 16, No. 20, pp. 2627-2632, October 15, 2002
1 Friedrich Miescher Institute for Biomedical Research, CH-4058, Basel, Switzerland; 2 Novartis Pharma AG, CH-4056 Basel, Switzerland
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ABSTRACT |
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 Top
 Abstract
 Introduction
Results and Discussion
Materials and methods
References
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Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in one of two tumor suppressor genes, TSC1 and TSC2. Here, we show that absence of Drosophila Tsc1/2 leads to constitutive dS6K activation and inhibition of dPKB, the latter effect being relieved by loss of dS6K. In contrast, the dPTEN tumor suppressor, a negative effector of PI3K, has little effect on dS6K, but negatively regulates dPKB. More importantly, we demonstrate that reducing dS6K signaling rescues early larval lethality associated with loss of dTsc1/2 function, arguing that the S6K pathway is a promising target for the treatment of TSC.
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Introduction |
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Abstract
Introduction
Results and Discussion
Materials and methods
References
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Hereditary cancers have revealed the existence of a
number of tumor suppressor genes that function to
control cell proliferation and maintain normal tissue homeostasis
(Macleod 2000
). Two tumor suppressors have been implicated in the
PI3K-signaling pathway, PTEN (Cantley and Neel 1999) and more recently,
a complex composed of two proteins, hamartin (TSC1) and tuberin (TSC2;
Montagne et al. 2001). The lipid phosphatase PTEN, which constrains
PI3K signaling by dephosphorylating its product phosphatidylinositol
3,4,5-trisphosphate (PIP3), is found mutated in a number of cancers
(Cantley and Neel 1999). Mutations in either TSC1 or TSC2 are
associated with widespread medically distinct tumors of the brain,
eyes, skin, heart, lungs, and kidneys (Young and Povey 1998). TSC1 and
TSC2 contain putative coiled-coil domains, with TSC1 having a predicted
transmembrane domain and TSC2 a region similar to that of
small-GTPase-activating protein (GAP) domains (Montagne et al. 2001).
Although little is known concerning the growth regulatory targets of
TSC1 and TSC2, genetic studies in Drosophila (Potter et al.
2001; Tapon et al. 2001) have led recently to the hypothesis that
dTsc1/2 acts as a negative effector of dS6K or of a dS6K target (Potter et al. 2001). In these models, dS6K was placed as a downstream effector
of dPI3K, via dPKB (Potter et al. 2001). However, dS6K and dPI3K/dPKB
appear to reside on parallel growth-promoting pathways rather than
functioning in a linear-signaling cascade (Radimerski et al. 2002).
Seemingly consistent with these findings, in Drosophila devoid
of dPTEN, dPKB appears to be the sole critical target
activated by elevated PIP3 levels (Stocker et al. 2002).
Here, we use double-stranded RNA mediated interference (dsRNAi) in Drosophila Kc167 cultured cells to demonstrate that dTsc1/2 acts to suppress dS6K activation, whereas dPTEN negatively regulates dPKB activation but has little effect on dS6K activity. Similar findings are obtained in second instar larvae deficient for either dTsc or dPTEN function, whereas overexpression of either dTsc1/2 or dPTEN in second instar larvae selectively inhibits dS6K or dPKB activity, respectively. In addition, loss of dTsc1/2 function in Kc167 cells or in larvae also leads to suppression of dPKB activity, an effect that is relieved by loss of dS6K. More strikingly, we demonstrate that a relative subtle pharmacological or genetic reduction in dS6K signaling is sufficient to rescue larval lethality associated with loss of dTsc function. These latter findings strongly suggest that the S6K pathway is a promising target for pharmaceutical intervention in tuberous sclerosis treatment.
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Results and Discussion |
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Top
Abstract
Introduction
Results and Discussion
Materials and methods
References
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To determine whether loss of dTsc1/2 or dPTEN directly affected
dS6K activity, each was depleted in Drosophila Kc167 cells by
dsRNAi (Clemens et al. 2000). Quantitative Real Time PCR showed that
such treatment strongly reduced levels of both transcripts (Fig.
1A). Compared with control cells, depletion
of dTsc1 increased dS6K activity and T398 phosphorylation, consistent
with the reduced electrophoretic mobility of dS6K (Fig. 1B). These
results are in agreement with recent findings in TSC1 null mammalian
cells (Kwiatkowski et al. 2002). Insulin treatment of either control cells or dTsc1-depleted cells did not significantly increase these responses beyond that of dTsc1 depletion alone (Fig. 1B), indicating that loss of dTsc function leads to full dS6K activation. RAD001, a
rapamycin derivative (Radimerski et al. 2002), blocked dS6K activity in
both control and dTsc1-depleted cells treated with insulin (Fig. 1B).
However, it was consistently noted that the RAD001 block of
insulin-induced dS6K activation was not as strong in dTsc1-depleted
cells (Fig. 1B), suggesting that not all the effects of dTsc on dS6K
are dependent on the Drosophila target of rapamycin, dTOR.
Similar results to those described here were obtained by dTsc2
depletion (Supplementary Fig. 1). In addition, the effects appeared
specific, as dTsc1 depletion had no effect on the basal activity of
other AGC-kinase family members, such as dPKB or Drosophila
atypical PKC (Fig. 1C,D). However, insulin-induced dPKB activation and
S505 phosphorylation were repressed in dTsc1-depleted cells as compared
with control cells (Fig. 1C), consistent with dS6K acting in a negative
feedback loop to dampen dPKB signaling (Haruta et al. 2000). In
contrast to loss of dTsc1, depletion of dPTEN had little effect on
dS6K activity and T398 phosphorylation, whereas it led to elevated
levels of both basal and insulin-stimulated dPKB activity and S505
phosphorylation (Fig. 1, cf. E and F). Thus, loss of dTsc1/2, but not
dPTEN, leads to constitutive dS6K activation.
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To determine whether the findings above could be corroborated in the
animal, dS6K activity was measured in extracts of dTsc1, dPTEN, and dS6K null larvae. The results show that
dS6K activity in extracts derived from dTsc1 null larvae was
strongly increased over that of wild-type larvae, whereas it was
slightly increased in larvae lacking dPTEN (Fig.
2A). The opposite was found for dPKB
activity, which was strongly repressed in dTsc1 null larvae, and up-regulated in dPTEN-deficient larvae (Fig. 2B). Hence,
it cannot be excluded that reduced dPKB activity contributes to larval lethality of dTsc mutants. Given that loss of dTsc function
leads to increased dS6K activity, it was reasoned that ectopic
expression of dTsc1/2, but not dPTEN, would inhibit dS6K activity. To
test this hypothesis, both tumor suppressors were expressed
ubiquitously in larvae using the GAL4/UAS system, such that the GAL4
promoter chosen in each case led to developmental arrest at late larval second instar. Extracts from larvae overexpressing dTsc1/2 display strongly reduced dS6K activity, whereas those from dPTEN overexpressing larvae have normal levels of dS6K activity (Fig. 2C). In contrast, dPKB
activity is strongly suppressed in dPTEN overexpressing larvae and
little affected in extracts from larvae overexpressing dTsc1/2 (Fig.
2D). These data corroborate previous findings that dS6K and dPKB act in
parallel signal transduction pathways (Radimerski et al. 2002), and
provide compelling evidence that they are negatively controlled by
distinct tumor suppressor genes.
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Despite the fact that dS6K and dPKB act in parallel signaling pathways,
loss of dTsc1/2 function leads to inhibition of dPKB activity (Figs.
1C, 2B; Supplementary Fig. 1B), suggesting cross-talk between the two
pathways. Compatible with such a model, recent studies have shown that
rapamycin treatment of adipocytes inhibits a negative feedback loop,
which normally functions to dampen insulin-induced PKB activation
(Haruta et al. 2000). As RAD001 inhibits dS6K activity (Fig. 1B;
Radimerski et al. 2002) and increases dPKB activity (Radimerski et al.
2002), it raised the possibility that the effects of dTsc
mutants on dPKB are mediated through dS6K. Consistent with this
hypothesis, inhibition of dPKB activity due to loss of dTsc function
was relieved in the absence of dS6K (Fig. 2E). Similar results
were obtained by using dsRNAi in cell culture (see Supplementary Fig.
2). Thus, the suppression of dPKB by loss of dTsc function requires dS6K.
To genetically test the specificity of dTsc1/2 and
dPTEN tumor suppressor function, either dTsc1
or dPTEN were removed in cells giving rise to the adult
eye structure, by inducing mitotic recombination with the
FLP/FRT system under the control of the eyeless
promoter (Newsome et al. 2000). In a wild-type genetic background, loss
of either dTsc1 or dPTEN within the developing eye
causes strong overgrowth of the head (Fig. 3, cf. A-C; Gao et al.
2000; Gao and Pan 2001). Eye overgrowth by removal of dTsc1 is
strongly suppressed in a genetic background null for dS6K, as
is ommatidia size (Fig. 3, cf. D and E), in agreement with a previous
report analyzing double mutant clones of dTsc2 and dS6K in the eye (Potter et al. 2001). In contrast, removal of dPTEN in the eyes of dS6K null flies still induces
overgrowth of clones with enlarged ommatidia (Fig. 3, cf. D and F).
These findings are supported by results showing that eye overgrowth by
removal of dTsc1 is still observed in clones devoid of dPKB function (Potter et al. 2001) and overgrowth by removal of
dPTEN is suppressed in a viable dPKB mutant genetic
background (Stocker et al. 2002). Thus, dTsc1/2 appears to be specific
for the dS6K-signaling pathway, whereas dPTEN antagonizes PI3K
signaling to counteract dPKB activation by decreasing PIP3 levels
(Stocker et al. 2002).
As dTsc1/2 loss-of-function overgrowth in clones is suppressed by
removing dS6K (Fig. 3E), it was
reasoned that reducing increased dS6K activity in dTsc1
loss-of-function larvae (Fig. 2A) might rescue second larval instar
lethality. Consistent with this, feeding dTsc1 null larvae low
doses of RAD001, which induces a developmental delay of 3 d in
wild-type larvae, allowed them to reach late wandering third larval
instar (Fig. 4A). The dTsc1 null
larvae died shortly after pupation, presumably because wandering third
instar larvae stop feeding and thus fail to receive the drug during
pupal stages. To circumvent the problem of feeding, we attempted to
reduce dS6K signaling by reducing the dosage of the gene. Compared with
wild-type pupae (Fig. 4B, pupae 1), dS6K null larvae are
significantly reduced in size (Fig. 4B, pupae 2), and lack of one
allele of dTsc1 in this background had no significant effect
on the dS6K null phenotype (Fig. 4B, pupae 3). Strikingly, the
second instar lethality caused by lack of both dTsc1 alleles
was rescued to early pupal stages in the dS6K null background
(Fig. 4B, pupae 4); however, these larvae were still small and severely
delayed in development. In contrast, larvae in which one allele of
dS6K had been removed in a dTsc1 null background
developed to early pupal stages with little developmental delay,
although they were now significantly larger than wild type (Fig. 4B,
pupae 5). On the basis of these latter findings, it was reasoned that
further reduction of dS6K signaling, but not its abolishment, may allow
dTsc1 null animals to develop beyond early pupal stages. To
test this in the dTsc1 null background, we used either one
allele of dTOR bearing a mutation in the kinase domain alone
or in combination with one null allele of dS6K. dTsc1 null
larvae bearing one kinase mutant dTOR allele survived with
higher frequency to pupae than animals with one null allele of
dS6K, with a few emerging as adults (Table
1). However, genetically lowering dS6K
signaling further by combining the dTOR and dS6K
loss-of function alleles, resulted in more than 60% of animals
surviving to the adult stage (Table 1). The rescued females and males
were slightly larger than wild-type flies, with overall patterning
appearing normal (Fig. 4C). Furthermore, the rescued females were
semifertile when crossed to wild-type males, whereas the rescued males
were fully fertile when crossed to wild-type females (data not shown).
Similarly, animals lacking dTsc2 function (Canal et al. 1998;
Ito and Rubin 1999) were rescued to viability (data not shown) by the
same genetic approach applied above. Importantly, flies lacking one
dS6K allele and bearing one kinase mutant dTOR allele
display no obvious mutant phenotype (data not shown). Therefore, lowering but not abolishing dS6K signaling is sufficient to allow development of Drosophila lacking dTsc function.
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Taken together, our results demonstrate that the tumor suppressor
dTsc1/2 is a critical component in controlling dS6K activation. Interestingly, this effect may be dTOR independent, as insulin-induced dS6K activation is more elevated in dTsc1/2-depleted cells pretreated with RAD001 than in control cells (Fig. 1B; Supplementary Fig. 1), and
in preliminary studies, clonal overgrowth in the eye induced by loss of
dTsc1 is not suppressed in a semiviable, heterorallelic dTOR mutant background. We further show that overexpression of dTsc1/2 selectively suppresses the dS6K-signaling pathway, whereas dPTEN operates on the dPI3K-signaling pathway (Fig. 2). It was shown
recently that double mutations for dPTEN and dTsc1
are additive for clonal overgrowth (Gao and Pan 2001), compatible with
dS6K and dPKB independently mediating growth. Nevertheless, inhibition of dPKB by loss of dTsc function shows that there is negative cross-talk between the two signaling pathways (Fig. 2). Given this
negative cross-talk, the observation that in double mutant clones
growth is additive, suggests that in the absence of dPTEN, inhibition
of dPKB by loss of dTsc is circumvented. However, despite the
observation that double mutations for dPTEN and dTsc1
are additive for clonal overgrowth, overgrowth induced by absence of
dPTEN is suppressed in clones mutant for dTOR (Oldham et al. 2000; Zhang et al. 2000). As dS6K does not prevent such overgrowth (Fig. 3), it is possible that this suppression actually represents an
intermediate phenotype, or that dPTEN negatively acts on a dTOR target
distinct from dS6K. At this point, it is important to gain a deeper
knowledge of the molecular mechanisms by which dTsc1/2 acts to suppress
dS6K function and how the signaling components of these two pathways
cross-talk with one another.
Recently, a successful Phase I clinical trial was completed for a
rapamycin analog in the treatment of solid tumors. The results of the
trial demonstrated that the drug was efficacious at subtoxic doses, and
suggested that specific tumor types may be more sensitive to inhibition
by rapamycin than others (Hidalgo and Rowinsky 2000). The question that
arose from the trial is, which tumors would be susceptible to rapamycin
treatment? Here, we demonstrate for the first time in vivo that a mild
reduction in dS6K signaling, which alone has no blatant phenotype, is
sufficient to restore viability of flies devoid of dTsc function. Thus,
these findings imply that rapamycin or its derivatives might be very
promising pharmaceutical agents in the treatment of tumors arising from TSC.
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Materials and methods |
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Top
Abstract
Introduction
Results and Discussion
Materials and methods
References
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Fly strains
The following null alleles were used: dS6Kl1
(Montagne et al. 1999), dTsc1Q87X (Tapon et al.
2001), and dPTEN117 (Stocker et al. 2002).
dS6Kl1 and dTsc1Q87X alleles were
combined on the same chromosome by recombination. dTOR2L1 (Oldham et al. 2000) and
dPKB1 (Staveley et al. 1998) encode kinase mutant
proteins. UAS-dPTEN and UAS-dTsc1 UAS-dTsc2 strains
were described previously (Goberdhan et al. 1999; Tapon et al. 2001).
For ubiquitous GAL4 expression, actin5C-GAL4
(act-GAL4) and daughterless-GAL4 (da-GAL4)
lines were used. Experiments were performed in the y w genetic
background, crosses were set at 25°C, and adult phenotype analysis
was done in females. Mosaic clones within the developing eye were
generated by mitotic recombination using the ey-FLP FRT system
as described (Newsome et al. 2000). Relative size of ommatidia was
determined by measuring the area inscribed within the black outlines of
individual eyes with the Adobe Photoshop 4.0 histogram function and
dividing this value by the number of ommatidia within each outlined
area. At least 50 ommatidia were counted for each genotype.
Double-stranded RNA mediated interference
Double-stranded RNA (dsRNAi) was performed essentially as
described (Clemens et al. 2000) with an incubation time of 1 wk. Cells
were maintained and treated as reported (Radimerski et al. 2002). All
primers were designed starting with the T7 RNA polymerase binding site
as follows: 5'-TTAATACGACTCACTATAGGGAGA-3'. dTsc1; accession no. AF173560, sense-primer 436-453, antisense-primer 1081-1098, dTsc2; accession no. AF172995, sense primer
591-608, antisense primer 1371-1388, dPTEN; accession no.
AF144232, sense primer 205-222, antisense primer 895-912. Total RNA
was isolated with the RNaesy kit (Qiagen) according to the supplier's protocol and dTsc1, dTsc2, and dPTEN mRNA
levels were analyzed by Quantitative Real Time PCR using the following
probes: sense-primer 1640-1664; Taq 1667-1694; antisense-primer
1696-1716 of AF144232, sense-primer 1831-1850; Taq 1857-1874;
antisense-primer 1878-1896 of AF172995, and sense-primer 1120-1138;
Taq 1144-1166; antisense-primer 1179-1197 of AF173560.
In vitro kinase activity assays
Protein extracts of cultured cells or of larvae were prepared as
reported (Oldham et al. 2000). Kinase activity of dS6K and dPKB was
measured essentially as described (Radimerski et al. 2002).
Drosophila atypical PKC was immunoprecipitated with the nPKC
(C-20) antibody (Santa Cruz Biotechnology) and activity assayed
as for dS6K except for use of myelin basic protein (0.5 µg) as substrate.
RAD feeding
RAD was prepared freshly from a 20-mM stock in ethanol by diluting to 30 µM in water, and 100 µL were distributed over the surface of standard Drosophila cornmeal-molasses medium (10 mL), onto which flies had previously deposited roughly 150 eggs in short-term collections.
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Acknowledgments |
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We thank I. Hariharan, C. Wilson, S. Oldham, and E. Hafen for the gift of flies, B. Hemmings for providing the dPKB antibody, N. Tapon and N. Ito for providing cDNAs for dTsc1 and dTsc2, and E. Hafen and R. Lamb for critical reading of the manuscript. This work was supported by the Novartis Research Foundation and by grants provided by the Roche Research Foundation to T.R. and G.T. and the Swiss Cancer League to J.M. and G.T.
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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Footnotes |
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[Key Words: growth; TSC; S6K; TOR; PTEN; PKB]
Received June 20, 2002; revised version accepted August 23, 2002.
3 Corresponding author.
E-MAIL gthomas{at}fmi.ch; FAX 41-61-697-3976.
Supplemental material is available at http://www.genesdev.org.
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.239102.
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Abstract
Introduction
Results and Discussion
Materials and methods
References
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J. R. McMullen, T. Shioi, L. Zhang, O. Tarnavski, M. C. Sherwood, A. L. Dorfman, S. Longnus, M. Pende, K. A. Martin, J. Blenis, et al. Deletion of Ribosomal S6 Kinases Does Not Attenuate Pathological, Physiological, or Insulin-Like Growth Factor 1 Receptor-Phosphoinositide 3-Kinase-Induced Cardiac Hypertrophy Mol. Cell. Biol., July 15, 2004; 24(14): 6231 - 6240. [Abstract] [Full Text] [PDF]
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M. van Slegtenhorst, E. Carr, R. Stoyanova, W. D. Kruger, and E. P. Henske Tsc1+ and tsc2+ Regulate Arginine Uptake and Metabolism in Schizosaccharomyces pombe J. Biol. Chem., March 26, 2004; 279(13): 12706 - 12713. [Abstract] [Full Text] [PDF]
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A. Astrinidis, W. Senapedis, T. R. Coleman, and E. P. Henske Cell Cycle-regulated Phosphorylation of Hamartin, the Product of the Tuberous Sclerosis Complex 1 Gene, by Cyclin-dependent Kinase 1/Cyclin B J. Biol. Chem., December 19, 2003; 278(51): 51372 - 51379. [Abstract] [Full Text] [PDF]
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