mTOR and HIF-1?-mediated tumor metabolism in an
LKB1 mouse model of Peutz-Jeghers syndrome
David B. Shackelforda,b, Debbie S. Vasqueza,b, Jacqueline Corbeilc, Shulin Wud,e, Mathias Leblanca, Chin-Lee Wud,e,
David R. Verac, and Reuben J. Shawa,b,1
aDulbecco Center for Cancer Research,bMolecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, 92037;cDepartment of
Radiology, University of California San Diego Molecular Imaging Program, Moores Cancer Center, University of California San Diego, La Jolla, CA, 92093;
dDepartment of Urology, Massachusetts General Hospital, Boston, MA 02114; andeDepartment of Pathology, Harvard Medical School, Boston, MA 02115
Edited by Inder M. Verma, The Salk Institute for Biological Studies, La Jolla, CA, and approved May 14, 2009 (received for review January 15, 2009)
Peutz-Jeghers syndrome (PJS) is a familial cancer disorder due to
inherited loss of function mutations in the LKB1/ STK11 serine/
threonine kinase. PJS patients develop gastrointestinal hamarto-
mas with 100% penetrance often in the second decade of life, and
demonstrate an increased predisposition toward the development
of a number of additional malignancies. Among mitogenic signal-
ing pathways, the mammalian-target of rapamycin complex 1
(mTORC1) pathway is hyperactivated in tissues and tumors derived
from LKB1-deficient mice. Consistent with a central role for
mTORC1 in these tumors, rapamycin as a single agent results in a
dramatic suppression of preexisting GI polyps in LKB1?/? mice.
However, the key targets of mTORC1 in LKB1-deficient tumors
remain unknown. We demonstrate here that these polyps, and
LKB1- and AMPK-deficient mouse embryonic fibroblasts, show
dramatic up-regulation of the HIF-1? transcription factor and its
downstream transcriptional targets in an rapamycin-suppressible
manner. The HIF-1? targets hexokinase II and Glut1 are up-regu-
lated in these polyps, and using FDG-PET, we demonstrate that
LKB1?/? mice show increased glucose utilization in focal regions
of their GI tract corresponding to these gastrointestinal hamarto-
mas. Importantly, we demonstrate that polyps from human Peutz-
Jeghers patients similarly exhibit up-regulated mTORC1 signaling,
HIF-1?, and GLUT1 levels. Furthermore, like HIF-1? and its target
genes, the FDG-PET signal in the GI tract of these mice is abolished
by rapamycin treatment. These findings suggest a number of
therapeutic modalities for the treatment and detection of hamar-
tomas in PJS patients, and potential for the screening and treat-
ment of the 30% of sporadic human lung cancers bearing LKB1
AMPK ? FDG-PET ? glycolysis ? hamartoma ? polyposis
tomatous polyps in the gastrointestinal tract and are predisposed
to developing cancer (1). Inactivating mutations in the LKB1/
STK11 tumor suppressor gene underlie PJS and have also been
associated with sporadic lung adeno- and squamous carcinomas
(2–8). Homozygous deletion of Lkb1 is embryonic lethal to mice
while heterozygous deletion of Lkb1 results in late onset gas-
trointestinal polyposis between 6–13 months of age that closely
models human PJS (9–13). Gastrointestinal hamartomas are
benign tumors that consist of hyperplastic glandular epithelial
cells, disorganized tissue architecture, and a characteristic ar-
borizing smooth muscle stalk. Several hamartomatous syn-
dromes involve inactivating mutations in genes that negatively
regulate the mTORC1 pathway, which promotes cell growth and
proliferation. In addition to PJS, these diseases include Cow-
type I, due to inactivating mutation in the PTEN, TSC1, TSC2,
or NF1 genes, respectively (14).
The mammalian target of rapamycin (mTOR) is a central
regulator of cell growth in all eukaryotes that is found in 2
functionally distinct multiprotein complexes (15). The mTOR
eutz-Jeghers syndrome (PJS) is an inherited autosomal
dominant disorder in that patients develop benign hamar-
complex 1 (mTORC1) is composed of mTOR and its scaffolding
protein raptor. Signaling from mTORC1 is nutrient-sensitive,
acutely inhibited by the bacterial macrolide rapamycin, and
controls protein translation, cell growth, angiogenesis, and me-
tabolism. Activation of mTORC1 results in phosphorylation of
a number of downstream targets involved in promoting cell
growth and proliferation. These substrates include proteins
involved in the regulation of protein translation such as the p70
S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding
protein 1 (4EBP1) (16). Among the mRNAs known to be
translationally up-regulated by mTORC1 are a number of key
progrowth proteins including cyclin D1, cyclin D3, Mcl-1, c-myc,
and the hypoxia inducible factor 1 alpha (HIF-1?) (17–21).
through the PI3K/Akt and Erk signaling pathways (15). In
contrast, under conditions of low intracellular ATP such as after
nutrient deprivation or other stresses, the LKB1 tumor suppres-
sor activates the AMP-activated protein kinase (AMPK), which
raptor (22) and the TSC2 tumor suppressor (23, 24). Hence,
treatment with AMPK activating drugs, or overexpression of
LKB1 or AMPK, results in suppression of mTORC1, whereas
targeted deletion of LKB1 in mice leads to increased mTORC1
activity in murine fibroblasts, liver, and in polyps of LKB1-
heterozygous mice (24–26).
Despite the common feature of elevated mTORC1 signaling
in these hamartoma syndromes, the important targets of
mTORC1 in LKB1-deficient tumors remain to be defined. We
show here that HIF-1? and its transcriptional targets in glucose
metabolism are up-regulated in LKB1-deficient tumors in mice
and human Peutz-Jeghers patients. Increased Glut1 and Hex-
okinase II expression in these polyps of Lkb1?/? mice allows
them to be visualized by FDG-PET. Because rapamycin strongly
suppresses polyposis in the Lkb1?/? mice, mTORC1 inhibitors
and FDG-PET imaging may be useful clinically in the treatment
of PJS patients.
Rapamycin Reduces Tumor Burden and Proliferation in Lkb1?/? Mice.
We investigated the effect of rapamycin on preexisting PJS-like
polyps, by treating 9-month-old Lkb1?/? or Lkb1?/? mice for
a period of 2 months with rapamycin or vehicle (Fig. S1A). Our
preliminary studies revealed that at 9 months of age, 100% of the
Lkb1?/? mice have developed multiple gastrointestinal hamar-
tomas, consistent with previous reports (10–13). Both Lkb1?/?
Author contributions: D.B.S. and R.J.S. designed research; D.B.S., D.S.V., J.C., and S.W.
performed research; D.B.S., M.L., C.-L.W., D.R.V., and R.J.S. analyzed data; and D.B.S. and
R.J.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
July 7, 2009 ?
vol. 106 ?
no. 27 ?
and Lkb1?/? mice tolerated rapamycin treatment with no
obvious cytotoxicity or immunosuppression at the doses used.
After 2 months of treatment, polyp size and number in each
mouse were quantitated. The wild-type Lkb1?/? mice were free
of polyps, while all of the Lkb1?/? mice treated with vehicle
presented severe polyp burden at or before 11–12 months of age,
consistent with previous studies of these mice (10–13, 27). These
mice had multiple large polyps in the stomach and pylorus and
suffered from severe distention of the stomach and anemia (Fig.
1A i and iv). Histological analysis of H&E-stained polyps from
untreated mice were classified as pedunculated, hyperplastic
lesions consisting of differentiated glandular epithelium, stroma,
and a smooth muscle stalk. In contrast, Lkb1?/? mice treated
with rapamycin had a dramatic reduction in polyp burden. These
mice uniformly had reduced polyp size (Fig. 1E) and lower
frequency of polyps (Fig. 1D), no distention of the stomach (Fig.
1A ii and v) and appeared active and vigorous at 2 months of
treatment. Comparison of the polyp burden between Lkb1?/?
mice treated or untreated with rapamycin showed an 80%
reduction in the overall mass of polyps (Fig. 1C). Polyps isolated
from treated mice were greatly reduced in size, although still
retained some disruption of the normal tissue architecture as
shown by H&E staining (Fig. 1B ii).
We analyzed mTORC1 signaling in the polyps of Lkb1?/?
mice to determine whether rapamycin was effectively inhibiting
the pathway. Immunohistochemical staining of polyps for phos-
pho-S6 (P-S6) revealed that untreated polyps displayed high
levels of P-S6 staining indicative of hyperactive mTOR signaling,
while the polyps from rapamycin treated mice were greatly
reduced for P-S6 staining indicating successful inhibition of
corroborated by western blot analysis of polyp lysates (Fig. S1B).
Rapamycin has been shown to suppress tumor growth and
induce apoptosis, resulting in cytostatic or cytotoxic responses in
arising tumors (15). We sought to examine the mechanism(s) by
which rapamycin reduced polyp burden in the Lkb1?/? mice.
We first analyzed expression of the proliferation marker Ki67 in
rapamycin or vehicle-treated polyps. The highest expression of
Ki67 was found in proliferating epithelial cells at the base of the
crypts. While staining was extensive in the vehicle-treated pol-
yps, rapamycin-treated polyps showed a clear reduction in Ki67
staining (Fig. 1 B v and vi and F). However, polyps from treated
mice did not show evidence of apoptosis as detected by cleaved
caspase-3 protein using immunohistochemistry or immunoblot-
ting. These results suggest that in this tumor model rapamycin
may be having a cytostatic effect rather than a cytotoxic effect,
consistent with observations in other tumor types (15).
Rapamycin Down-Regulates Expression of HIF-1? and HIF-1? Targets.
Activation of mTORC1 results in increased translation of a
number of key downstream targets, including cyclin D1 and the
hypoxia inducible factor 1 alpha gene (HIF-1?) (17–20). We
examined the protein levels of cyclin D1 and HIF-1? in the
stomachs and polyps of Lkb1?/? and Lkb1?/? mice by western
blot. HIF-1? but not cyclin D1 levels were elevated in polyps of
vehicle-treated Lkb1?/? mice. In contrast, the polyps of mice
treated with rapamycin showed reduced HIF-1? levels similar to
the basal levels seen in Lkb1?/? mice (Fig. 2A). Next, we
% Ki67+ Cells
Ave Polyp Size (mm3)
Ave Polyp Number
Polyp Burden (mm3)
H & EP-S6Ki67
40X 40X 4X4X40X
mTORC1 signaling, and proliferation in
Lkb1?/? polyps. (A) Top are images of whole
stomach and duodenum and Bottom are im-
ages of the open stomachs (S) showing the
with either vehicle (VEH) (i, iv) or rapamycin
(RAPA) (ii, v) and Lkb1?/? mice treated with
vehicle (VEH) (iii, vi). (B) Immunohistochemical
analysis of polyps from VEH- or RAPA-treated
(iii, iv), and Ki67 staining (v, vi). Results are
representative of polyps from 5 mice of each
burden in Lkb1?/? mice treated with either
was significantly reduced (*, P ? 0.00026; Stu-
dent t test, 2 tail) compared with those mice
or RAPA-treated mice (gray bar, n ? 10). Only
visible polyps between 1 and ?5 mm were
scored in both VEH- and RAPA-treated mice.
The mean polyp number for RAPA-treated
mice (2.8 ? 1.4) was significantly reduced (*,
P ? 0.00022; Student t test, 2 tail) compared
with VEH-treated mice (5.3 ? 1.8). (E) Average
polyp size in VEH-treated mice (black bar, n ?
mean polyp size in RAPA mice (1.2 ? 0.9) was
significantly reduced (*, P ? 0.0001; Student t
test, 2 tail) compared with VEH-treated mice
(4.4 ? 0.8). (F) Average percentage of Ki67-
The mean percentage of Ki67-positive cells in
(*, P ? 0.0002; Student t test, 2 tail) compared
Rapamycin reduces polyposis,
www.pnas.org?cgi?doi?10.1073?pnas.0900465106Shackelford et al.
including Glut1, hexokinase II, and bNIP3, and observed that
their expression was up in the polyps proportional to HIF-1?
up-regulation and similarly, that the expression of these HIF-1?
targets was suppressed by rapamycin (Fig. 2A). To confirm that
HIF-1? and its targets were up-regulated within the epithelial
cell population that are P-S6 and Ki67 positive, as opposed to
originating from any stromal or infiltrating cells, we performed
immunohistochemistry with anti- HIF-1? and Glut1 antibodies
Glut1 protein expression levels were much higher in epithelial
cells in the untreated polyps and were diminished with rapamy-
cin treatment (Fig. 2B).
Lkb1 gene and not simply a consequence of hypoxia within the
polyp microenvironment or a secondary mutation that arose
during polyp formation, we first examined the functional levels
of hypoxia present in the polyps and surrounding epithelium
using hypoxyprobe-1 (28). No significant levels of hypoxia were
observed in the polyps, in contrast to widespread HIF-1?
elevation throughout the epithelial cells of the polyps (Fig. S2).
To further extend this analysis in a controlled normoxic envi-
ronment, and to rule out the potential impact of any secondary
mutations that may have arisen in the polyps that might con-
tribute to up-regulation of HIF-1?, we examined HIF-1? levels
in primary non-immortalized wild-type and Lkb1-deficient
MEFs grown in normoxic conditions. Expression of HIF-1?
protein, and the HIF-1? targets hexokinase II and bNIP3, were
by rapamycin treatment (Fig. 2C). Because we have shown that
AMPK is a key target of LKB1 that controls mTORC1 activity
via its phosphorylation of TSC2 and raptor, we next examined
whether HIF-1? and its targets were similarly up-regulated in
immortalized MEFs lacking both catalytic isoforms of AMPK.
Indeed, HIF-1? and its targets were up-regulated in Ampk?1/
?2?/? fibroblasts compared with wild-type cells and treatment
of 4ebp1 and S6K phosphorylation (Fig. 2C).
Lkb1?/? Polyps Exhibit Dramatic Increases in Glucose Metabolism.
One of the earliest defined biochemical hallmarks of tumor cells
is the propensity to rely on glycolysis for ATP production, even
when oxygen is not limiting, unlike their normal counterparts.
This conversion from oxidative phosphorylation to glycolysis
that accompanies tumorigenesis was termed the Warburg Effect
after its discoverer Otto Warburg (for review, see ref. 29). In the
past decade, interest in the Warburg effect has been renewed in
part due to the increased use of 18F-fluoro-deoxyglucose
(FDG)-positron emission tomography (PET) in human cancer
patients to detect tumors due to their higher rates of glucose
utilization (30). The molecular underpinning for increased
FDG-PET has been hypothesized to involve increased levels of
cell surface glucose transporters including GLUT1, as well the
enzyme for the first committed step of glycolysis, hexokinase II
(31). Because immunoblotting had revealed increased expres-
sion of both GLUT1 and hexokinase II in the polyps of
LKB1?/? mice, we were interested in whether these tumors
could be visualized by FDG-PET.
We analyzed 11-month-old Lkb1?/? and Lkb1?/? mice by
FDG PET to scan for the presence of GI polyps. In addition to
the excretion to the bladder, we observed the expected uptake
of FDG in the heart and kidney of all mice regardless of
focal masses located in the Lkb1?/? mice in their midline below
the heart where the stomach and pylorus are located, whereas
the Lkb1?/? were negative (P ? 0.06) for FDG signal in this
area (Fig. 3A). Several of the Lkb1?/? mice were killed after
imaging, and it was confirmed that these animals had large
polyps in the pylorus and stomach corresponding exactly to the
regions of greatest FDG uptake. Treatment of animals with
rapamycin for 4 weeks abolished the FDG-PET signal. Imme-
diate autopsy of the animals imaged by FDG-PET revealed that
the rapamycin-treated mice had minimal detectable GI polyps
while the vehicle-treated mice all exhibited the presence of large
GI polyps (Fig. 3B). These results demonstrate that FDG-PET
analysis is a viable method by which to detect polyps in Lkb1?/?
animals and confirms that rapamycin reverses polyp growth in
Lkb1?/? mice. All experimental procedures in mice were
approved by the Salk Institute and University of California at
San Diego Institutional Animal Care and Use Committees.
mTORC1 and HIF-1? Signaling Increased in Human PJS Polyps.Finally,
we examined whether the increased mTORC1 and HIF-1?
dependent signaling we observed in the LKB1?/? murine
model are relevant to human Peutz-Jeghers patients. mTORC1
signaling, HIF-1? protein, and GLUT1 protein expression were
analyzed by immunohistochemistry in small bowel and colon
+/+ -/- +/+ -/- +/+ -/-
+/+ -/- +/+ -/- +/+ -/-
Rapa CoCl2 NT
Ampk 1/ 2 MEFs
tissue or polyps from Lkb1?/? and Lkb1?/? mice treated VEH or RAPA. Immunoblots were probed against the indicated antibodies. (B) Immunohistochemical
analysis of polyps from VEH- or RAPA-treated Lkb1?/? mice probed with antibodies against Glut1 or Hif-1?. Results are representative of polyps from 3 mice
of each treatment group. (C) Immunoblots of lysates from Lkb1?/? or Lkb1?/? MEFs (Left) or Ampk?/? or Ampk?/? MEFs (Right) probed with antibodies
against the indicated proteins. MEFs were either untreated (NT) or treated with RAPA or cobalt chloride (CoCl2).
Up-regulated HIF-1? and HIF-1? targets in LKB1-deficient polyps and fibroblasts are reduced by rapamycin. (A) Immunoblots of lysates made from GI
Shackelford et al.PNAS ?
July 7, 2009 ?
vol. 106 ?
no. 27 ?
samples from PJS patients and compared with samples of small
bowel and colonic mucosa from normal patient controls. Ex-
pression of the mTOR target P-S6 was increased in the epithe-
tissue (Fig. 4 A and B). Likewise, strong immunostaining of
HIF-1? and GLUT1 was observed in glandular epithelial cells in
7 of 8 PJP colonic polyp specimens (Fig. 4 D and F). In the
normal colonic mucosa specimens, weak immunohistochemical
staining of HIF-1? and Glut1 was observed relative to the highly
elevated levels observed in the PJS samples (Fig. 4 C and E). These
results suggest that loss of the LKB1 gene leads to both
mTORC1 hyperactivation and increased HIF-1? and GLUT1
expression in PJS patients in a manner that closely follows the
murine model of PJS.
Aberrant activation of the mTORC1 pathway has been observed
in spontaneously arising tumors in mice genetically engineered
for loss of the tumor suppressors Pten, Nf1, Tsc2, or Lkb1
(32–38). Mutations in these genes are responsible for the inher-
I, tuberous sclerosis complex, and Peutz-Jeghers syndrome;
collectively referred to as Phakomatoses, and all sharing over-
lapping clinical features including the development of hamarto-
mas. Biochemical and cell biological studies from the past
decade have revealed that these tumor suppressors all are direct
components of the mTOR signaling pathway that serve to inhibit
mTORC1 activity (15).
The underlying hypothesis is that mutational inactivation of
these tumor suppressors in individual cells leads to cell-
autonomous hyperactivation of mTORC1, promoting cell
growth and ultimately resulting in tumors that are subsequently
reliant on mTORC1 signaling for tumor maintenance. Consis-
tent with this possibility, rapamycin analogs have been examined
for their therapeutic efficacy in the suppression of tumors that
arise in a number of the aforementioned mouse models. The
Pten?/?, Nf1?/?, Tsc?/?, Lkb1?/?, and activated Akt trans-
genic mouse models have also proven to be responsive to the
mTOR inhibitors rapamycin or rapamycin analogs RAD001
(Novartis), CCI0779 (Wyeth) and AP23573 (Ariad) (34, 39–43).
These drugs have been proven to effectively inhibit mTORC1 in
vivo and reduce tumor burden through mTORC1 dependent
and its targets (35, 39–41).
In recent clinical trials, rapamycin and its analog temsirolimus
were shown to have palliative success in clinical trials on patients
with PTEN-deficient glioblastomas and metastatic renal cell
carcinoma (44, 45). Furthermore, in a pair of phase II clinical
trials involving tuberous sclerosis (TSC) and lymphangio-
leiomyomatosis (LAM) patients, partial responses to the rapa-
mycin analog Sirolimus were observed, including regression of
angiomyoliomas with continuous therapy (46, 47), consistent
with previous clinical observations in TSC patients given rapa-
mycin (48, 49). Combined with data from mouse models, these
clinical data suggest that hamartoma syndromes with hyperac-
tivation of mTORC1 may be particularly responsive to rapamy-
cin analogs as a single agent. To date there are no therapies to
treat PJS and the only course of treatment is resection of arising
gastrointestinal hamartomatous polyps. Consistent with a pre-
vious report (34), we found here that rapamycin greatly reduced
the polyp burden in the Lkb1?/? mouse model of PJS. This
suppression was correlated with inhibition of mTORC1 and
downregulation of HIF-1? and its transcriptional targets. While
these results are encouraging for the use of rapamycin analogs
as therapeutics for PJS, like the recent phase II clinical trial
findings with TSC patients, removal of the drug may result in
rapid return of the initial tumor due to the largely cytostatic
nature of the response (46). Perhaps new, targeted inhibitors
directed at the kinase domain of mTOR will produce greater
therapeutic response with targeted cytotoxicity, or perhaps
PI3K provides a survival signal in most epithelial cell types. As
observed in most cancers studied to date, combinations of
targeted therapeutics, or of targeted and traditional chemo-
therapeutics may find the ultimate utility in the treatment of this
disease. Importantly, it is worth noting here that rapamycin
treatment may not only be therapeutically useful for the hamar-
tomas that arise in Peutz-Jeghers patients, but also in preventing
and reducing any secondary malignancies that arise in these
patients at additional sites (breast, pancreas, and ovary).
This study also finds the transcription factor HIF-1? as a
relevant target of mTORC1 in LKB1-dependent hamartomas,
and the up-regulation of HIF-1? targets Glut1 and hexokinase
II may be responsible for the ability of these tumors to be
visualized by FDG-PET, because both Glut1 and hexokinase II
have been reported as rate-limiting steps for FDG uptake and
imaging (31). HIF-1? has been shown to be an excellent corre-
late of rapamycin response in a transgenic model of prostate
neoplasia dependent on activated Akt, and in VHL-deficient
renal cell carcinoma xenografts (20, 39). Consistent with the
findings here in spontaneously arising hamartomas in LKB1?/?
mice, recent studies using human glioblastoma xenografts and
Lkb1+/- #1 Lkb1+/- #2Lkb1+/+ #1
shows FDG PET images of axial, sagital and coronal views of untreated
12-month-old Lkb1?/? mice. Right shows the same views of untreated
Lkb1?/? mice. The FDG PET images of the mice are labeled accordingly: K,
kidney; S, stomach; H, heart; B, bladder; and P, polyp. (B) Left shows FDG PET
imaging of axial, sagittal, and coronal views of Lkb1?/? and Lkb1?/? mice
images of the mice are labeled accordingly: K, kidney; S, stomach; H, heart; B,
bladder; and P, polyp.
Polyps from Lkb1?/? mice visualized by FDG PET analysis. (A) Left
www.pnas.org?cgi?doi?10.1073?pnas.0900465106Shackelford et al.
transplanted murine breast carcinomas also found rapamycin-
sensitivity of FDG-PET imaging of these tumors (50, 51). Data
from LKB1 and AMPK-deficient MEFs demonstrate that
HIF-1? and HIF-1? targets are dramatically up-regulated in
these cells under normoxic conditions, indicating that HIF-1?
up-regulation is not a secondary consequence of other tumor
mutations or hypoxia present within the hamartomas. These
findings also suggest that AMPK may be a key effector of LKB1
in the suppression of HIF-1? in the normal gastrointestinal
epithelium that when disrupted gives rise to hamartomas. Inter-
estingly, the increase in HIF-1? was observed in the deficient
fibroblasts under conditions of increased cell density when basal
depletion of the media. In cells that genetically lack the ability to
activate AMPK, HIF-1? is up-regulated under these conditions.
Consistent with these findings, hypoxia-independent up-
regulation of HIF-1? has been observed in murine embryonic
fibroblasts deficient in TSC2 or TSC1 (52, 53). Furthermore,
that mTORC1 signaling stimulates increases in HIF-1? levels
independent of VHL and hypoxia-driven stabilization, but instead
dependent on increased translation of HIF-1? due to a functional
5? TOP element in its 5? UTR (20, 39, 52, 54–58). Indeed, fusion
of the 5? UTR of HIF-1? to multiple reporter constructs confers
It will be critical for future studies to define whether the
distinct downstream targets of mTORC1 are key for tumorigen-
esis in different cell types. Preliminary evidence suggests that
different effectors may be important, because in a mouse model
of Neurofibromatosis type 1, rapamycin dramatically suppressed
tumors, although no effects were observed on HIF-1? or on
vascularization; instead, cyclin D appears to be the critical target
of mTORC1 in this setting. Notably, no change in cyclin D1 was
observed in LKB1?/? hamartomas, consistent with recent
reports on tissue variability of the reliance of cyclin D on
mTORC1 (18). Interestingly however, we did observe increases
in cyclinD1 in LKB1- and AMPK-deficient fibroblasts parallel-
ing increases in HIF-1?, suggesting that different cell types
exhibit distinct effectors downstream of mTORC1.
Although most often used in the detection of malignant
tumors, our findings suggest that FDG-PET may find clinical
utility for the identification of polyps in PJS patients. Moreover,
FDG-PET may be useful for monitoring the efficacy of treat-
ment or surgical resection of these polyps. It will also be very
interesting to determine whether secondary cancers that arise in
PJS patients at other sites (breast, pancreas, and endometrium)
can also be visualized by FDG-PET, and whether rapamycin
analogs or mTOR kinase inhibitors will demonstrate clinical
efficacy in the treatment of those tumors. Finally, given the
lung carcinomas, it will be of great interest to examine if the
presence of LKB1 mutations in NSCLC will correlate with the
propensity of lung tumors to be imaged with FDG-PET, or their
response to mTOR-targeted therapeutics.
Materials and Methods
For immunoblotting, anti-phospho-S6K1 (T389), phospho-ribosomal protein
Akt (Thr-308), and bNIP3 antibodies were obtained from Cell Signaling Tech-
nology. Antibodies against HIF-1? (C-term) polyclonal antibody (Cayman
Cyclin D1 (BD PharMingen) and tubulin (Sigma Chemicals) were also used.
the other methods appears in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Lauri Aaltonen and Sini Marttinen (Bio-
medicum Helsinki, Helsinki)for the Finnish Peutz-Jeghers patient samples,
Keith Laderoute (SRI International, Menlo Park, CA) and Benoit Viollet (Insti-
tut Cochin, Paris) for their generous donation of the isogenic SV40-
immortalized wild-type and AMPKa1/a2 double deficient MEFs, and Dr. Carl
Ho (University of California San Diego, La Jolla, CA) for his assistance in
analyzing the FDG-PET data. We thank Katja Lamia for critical reading of the
manuscript. This work was supported in part from grants from the National
Institutes of Health (P01 CA120964 to R.J.S. and C.-L.W.), National Cancer
Institute (P50 CA128346 to D.R.V.), American Cancer Society (R.J.S.), and V
T32 CA009370 to the Salk Institute Center for Cancer Research.
show increased P-S6, GLUT1, and HIF-1? expression. A
and B represent immunohistochemistry performed on
human small bowel samples from normal patients
(Left) or Peutz Jeghers patients (Right) that were
probed with antibodies against the mTORC1 marker
P-S6. C–F represent immunohistochemistry performed
on normal colonic mucosa (Left) and colonic Peutz-
Jeghers polyps (Right) probed with antibodies against
the GLUT1 protein (C and D) or the HIF-1? protein (E
Polyps from human Peutz-Jeghers patients
Shackelford et al. PNAS ?
July 7, 2009 ?
vol. 106 ?
no. 27 ?
1. HemminkiA(1999)ThemolecularbasisandclinicalaspectsofPeutz-Jegherssyndrome. Download full-text
Cell Mol Life Sci 55:735–750.
2. Sanchez-Cespedes M, et al. (2002) Inactivation of LKB1/STK11 is a common event in
adenocarcinomas of the lung. Cancer Res 62:3659–3662.
3. Carretero J, Medina PP, Pio R, Montuenga LM, Sanchez-Cespedes M (2004) Novel and
natural knockout lung cancer cell lines for the LKB1/STK11 tumor suppressor gene.
4. Ji H, et al. (2007) LKB1 modulates lung cancer differentiation and metastasis. Nature
5. Hemminki A, et al. (1998) A serine/threonine kinase gene defective in Peutz-Jeghers
syndrome. Nature 391:184–187.
threonine kinase. Nat Genet 18:38–43.
7. Aretz S, et al. (2005) High proportion of large genomic STK11 deletions in Peutz-
Jeghers syndrome. Hum Mutat 26:513–519.
8. Hearle NC, et al. (2006) Exonic STK11 deletions are not a rare cause of Peutz-Jeghers
syndrome. J Med Genet 43:e15.
9. Ylikorkala A, et al. (2001) Vascular abnormalities and deregulation of VEGF in Lkb1-
deficient mice. Science 293:1323–1326.
10. Bardeesy N, et al. (2002) Loss of the Lkb1 tumour suppressor provokes intestinal
polyposis but resistance to transformation. Nature 419:162–167.
11. Miyoshi H, et al. (2002) Gastrointestinal hamartomatous polyposis in Lkb1 heterozy-
gous knockout mice. Cancer Res 62:2261–2266.
12. Jishage K, et al. (2002) Role of Lkb1, the causative gene of Peutz-Jegher’s syndrome, in
embryogenesis and polyposis. Proc Natl Acad Sci USA 99:8903–8908.
polyposis. Proc Natl Acad Sci USA 99:12327–12332.
14. Shaw RJ, Cantley LC (2006) Ras, PI(3)K and mTOR signalling controls tumour cell
growth. Nature 441:424–430.
15. Guertin DA, Sabatini DM (2007) Defining the role of mTOR in cancer. Cancer Cell
16. Fingar DC, et al. (2004) mTOR controls cell cycle progression through its cell growth
effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E. Mol Cell Biol
17. Laughner E, Taghavi P, Chiles K, Mahon PC, Semenza GL (2001) HER2 (neu) signaling
increases the rate of hypoxia-inducible factor 1alpha (HIF-1alpha) synthesis: Novel
signaling requires eukaryotic initiation factor 4E-binding protein 1. Oncogene
mRNA. J Cell Physiol 200:82–88.
20. Thomas GV, et al. (2006) Hypoxia-inducible factor determines sensitivity to inhibitors
of mTOR in kidney cancer. Nat Med 12:122–127.
Proc Natl Acad Sci USA 105:10853–10858.
22. Gwinn DM, et al. (2008) AMPK phosphorylation of raptor mediates a metabolic
checkpoint. Mol Cell 30:214–226.
23. Inoki K, Zhu T, Guan KL (2003) TSC2 mediates cellular energy response to control cell
growth and survival. Cell 115:577–590.
Cancer Cell 6:91–99.
Peutz-Jeghers syndrome. Genes Dev 18:1533–1538.
26. Shaw RJ, et al. (2005) The kinase LKB1 mediates glucose homeostasis in liver and
therapeutic effects of metformin. Science 310:1642–1646.
27. Katajisto P, et al. (2008) LKB1 signaling in mesenchymal cells required for suppression
of gastrointestinal polyposis. Nat Genet 40:455–459.
28. Raleigh JA, et al. (1998) Hypoxia and vascular endothelial growth factor expression in
30. Garber K (2006) Energy deregulation: Licensing tumors to grow. Science 312:1158–
31. Smith TA (2001) The rate-limiting step for tumor [18F]fluoro-2-deoxy-D-glucose (FDG)
incorporation. Nucl Med Biol 28:1–4.
tumor suppressor PTEN. Cell 95:29–39.
33. Johannessen CM, et al. (2005) The NF1 tumor suppressor critically regulates TSC2 and
mTOR. Proc Natl Acad Sci USA 102:8573–8578.
34. Wei C, et al. (2008) Suppression of Peutz-Jeghers polyposis by targeting mammalian
target of rapamycin signaling. Clin Cancer Res 14:1167–1171.
35. Wendel HG, et al. (2006) Determinants of sensitivity and resistance to rapamycin-
chemotherapy drug combinations in vivo. Cancer Res 66:7639–7646.
giant cells, and show activation of an mTOR pathway. Mol Cell Neurosci 21:561–574.
protein is linked to inhibition of the phosphatidylinositol 3-kinase/Akt pathway. Proc
Natl Acad Sci USA 96:2110–2115.
38. Zhang H, et al. (2003) Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling
through downregulation of PDGFR. J Clin Invest 112:1223–1233.
39. Majumder PK, et al. (2004) mTOR inhibition reverses Akt-dependent prostate intra-
epithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways.
Nat Med 10:594–601.
therapy. Nature 428:332–337.
41. JohannessenCM,etal.(2008)TORC1isessentialforNF1-associatedmalignancies. Curr
42. Podsypanina K, et al. (2001) An inhibitor of mTOR reduces neoplasia and normalizes
p70/S6 kinase activity in Pten?/? mice. Proc Natl Acad Sci USA 98:10320–10325.
43. Lee L, Sudentas P, Dabora SL (2006) Combination of a rapamycin analog (CCI-779) and
interferon-gamma is more effective than single agents in treating a mouse model of
tuberous sclerosis complex. Genes Chromosomes Cancer 45:933–944.
with recurrent PTEN-deficient glioblastoma. PLoS Med 5:e8.
45. Hudes G, et al. (2007) Temsirolimus, interferon alfa, or both for advanced renal-cell
carcinoma. N Engl J Med 356:2271–2281.
46. Bissler JJ, et al. (2008) Sirolimus for angiomyolipoma in tuberous sclerosis complex or
lymphangioleiomyomatosis. N Engl J Med 358:140–151.
leiomyomatosis. N Engl J Med 358:200–203.
on renal angiomyolipomas in a patient with tuberous sclerosis complex. Eur J Intern
49. Franz DN, et al. (2006) Rapamycin causes regression of astrocytomas in tuberous
sclerosis complex. Ann Neurol 59:490–498.
50. Wei LH, et al. (2008) Changes in tumor metabolism as readout for mammalian target
51. Namba R, et al. (2006) Rapamycin inhibits growth of premalignant and malignant
mammary lesions in a mouse model of ductal carcinoma in situ. Clin Cancer Res
52. Brugarolas JB, Vazquez F, Reddy A, Sellers WR, Kaelin WG, Jr (2003) TSC2 regulates
VEGF through mTOR-dependent and -independent pathways. Cancer Cell 4:147–158.
53. Laderoute KR, et al. (2006) 5?-AMP-activated protein kinase (AMPK) is induced by
low-oxygen and glucose deprivation conditions found in solid-tumor microenviron-
ments. Mol Cell Biol 26:5336–5347.
54. Zundel W, et al. (2000) Loss of PTEN facilitates HIF-1-mediated gene expression. Genes
epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in
human prostate cancer cells: Implications for tumor angiogenesis and therapeutics.
Cancer Res 60:1541–1545.
56. Elstrom RL, et al. (2004) Akt stimulates aerobic glycolysis in cancer cells. Cancer Res
57. Lum JJ, et al. (2007) The transcription factor HIF-1alpha plays a critical role in the
growth factor-dependent regulation of both aerobic and anaerobic glycolysis. Genes
58. Dekanty A, Lavista-Llanos S, Irisarri M, Oldham S, Wappner P (2005) The insulin-PI3K/
TOR pathway induces a HIF-dependent transcriptional response in Drosophila by
promoting nuclear localization of HIF-alpha/Sima. J Cell Sci 118:5431–5441.
59. Choo AY, Yoon SO, Kim SG, Roux PP, Blenis J (2008) Rapamycin differentially inhibits
Acad Sci USA 105:17414–17419.
www.pnas.org?cgi?doi?10.1073?pnas.0900465106Shackelford et al.