HIF-2 deletion promotes Kras-driven lung tumor
development
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Citation
Mazumdar, J., M. M. Hickey, D. K. Pant, A. C. Durham, A.
Sweet-Cordero, A. Vachani, T. Jacks, et al. “HIF-2 deletion
promotes Kras-driven lung tumor development.” Proceedings of
the National Academy of Sciences 107, no. 32 (August 10,
2010): 14182-14187.
As Published
http://dx.doi.org/10.1073/pnas.1001296107
Publisher
National Academy of Sciences (U.S.)
Version
Final published version
Accessed
Thu May 26 22:53:35 EDT 2016
Citable Link
http://hdl.handle.net/1721.1/84643
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and may be subject to US copyright law. Please refer to the
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Detailed Terms
HIF-2α deletion promotes Kras-driven lung
tumor development
Jolly Mazumdara,b, Michele M. Hickeya,c,1, Dhruv K. Panta,d, Amy C. Durhame, Alejandro Sweet-Corderof,
Anil Vachanig, Tyler Jacksh, Lewis A. Chodosha,d, Joseph L. Kissili, M. Celeste Simona,b,j,2, and Brian Keitha,d
a
Abramson Family Cancer Research Institute, cCell and Molecular Biology Graduate Group, dDepartment of Cancer Biology, and jDepartment of Cell and
Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; bHoward Hughes Medical Institute, University of
Pennsylvania, Philadelphia, PA 19104; eDepartment of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philidelphia, PA 19104;
f
Department of Pediatrics, Stanford University, Stanford, CA 94305; gAbramson Research Center, Philadelphia, PA 19104; hKoch Institute for Integrative
Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139; and iThe Wistar Institute, Philadelphia, PA 19104
Edited by Steven L. McKnight, University of Texas Southwestern, Dallas, TX, and approved June 21, 2010 (received for review February 1, 2010)
Non-small cell lung cancer (NSCLC) is the leading cause of cancer
deaths worldwide. The oxygen-sensitive hypoxia inducible factor
(HIF) transcriptional regulators HIF-1α and HIF-2α are overexpressed
in many human NSCLCs, and constitutive HIF-2α activity can promote murine lung tumor progression, suggesting that HIF proteins
may be effective NSCLC therapeutic targets. To investigate the consequences of inhibiting HIF activity in lung cancers, we deleted
Hif-1α or Hif-2α in an established KrasG12D-driven murine NSCLC
model. Deletion of Hif-1α had no obvious effect on tumor growth,
whereas Hif-2α deletion resulted in an unexpected increase in tumor
burden that correlated with reduced expression of the candidate
tumor suppressor gene Scgb3a1 (HIN-1). Here, we identify Scgb3a1
as a direct HIF-2α target gene and demonstrate that HIF-2α regulates
Scgb3a1 expression and tumor formation in human KrasG12D-driven
NSCLC cells. AKT pathway activity, reported to be repressed by
Scgb3a1, was enhanced in HIF-2α-deficient human NSCLC cells
and xenografts. Finally, a direct correlation between HIF-2α and
SCGB3a1 expression was observed in approximately 70% of human
NSCLC samples analyzed. These data suggest that, whereas HIF-2α
overexpression can contribute to NSCLC progression, therapeutic
inhibition of HIF-2α below a critical threshold may paradoxically
promote tumor growth by reducing expression of tumor suppressor
genes, including Scgb3a1.
|
hypoxia inducible mouse model
suppressor
| non-small cell lung cancer | tumor
D
espite the access of normal pulmonary epithelia to atmospheric O2 levels, tumor hypoxia has been observed in human
non-small cell lung cancer (NSCLC) and correlates with poor
prognosis and decreased disease-free survival (1, 2). Cells can
overcome hypoxic stress through multiple mechanisms, including
the stabilization of hypoxia inducible factor (HIF) transcriptional
regulators. HIFs consist of an oxygen labile α-subunit and a constitutively expressed β-subunit (also called ARNT), which together bind hypoxia response elements (HREs) to activate a large
number of target genes encoding proteins that mediate adaptive
responses to hypoxic stress (3, 4). Two distinct subunits, HIF-1
and HIF-2, have been demonstrated to regulate partly overlapping sets of target genes in hypoxic cells, although each protein
also fulfills unique essential functions (5–7).
Many HIF target genes encode proteins important for tumor
growth and progression, including glycolytic enzymes, VEGF,
matrix metalloproteinase-2 (MMP2), transforming growth factors
α and β (TGF-α and TGF-β), and numerous others (4, 8). Collectively, these factors modulate cancer cell metabolism and can
promote angiogenesis, invasion, and metastasis, suggesting that
HIF proteins may represent suitable targets for antitumor therapies (9–11). It is interesting to note that both HIF-1α and HIF-2α
proteins are overexpressed in approximately 50% of human
NSCLCs (12), further suggesting they may contribute directly to
NSCLC progression.
14182–14187 | PNAS | August 10, 2010 | vol. 107 | no. 32
In a recent report, ectopic expression of a nondegradable HIF2α promoted growth of KrasG12D-driven murine lung tumors (13).
Relative to KrasG12D controls, KrasG12D lung tumors expressing
stabilized HIF-2α were larger, displayed evidence of enhanced
angiogenesis and epithelial-mesenchymal transition (EMT), and
resulted in decreased survival (13). These phentoypes were correlated with increased expression of multiple HIF-2α target genes
including VEGFR2, Snail, and others (13). Collectively, these
results demonstrated that increased HIF-2α activity can drive
NSCLC growth and underscore the potential utility of developing
targeted therapies to inhibit HIF proteins in general, and HIF-2α
in particular, for NSCLC and renal cell carcinomas (RCC).
To determine the effects of eliminating HIF-1α and HIF-2α
expression in NSCLC, we used conditional “floxed” alleles to delete
HIF-1α or HIF-2α in lung tumors using the murine KrasG12D
NSCLC model (13). Deletion of HIF-1α in these tumor cells had no
detectable effect on tumor growth but, surprisingly, deletion of
HIF-2α actually increased tumor number and size. This phenotype
correlated with reduced expression of Scgb3a1 (14) also known as
high in normal-1 (HIN-1), a candidate tumor suppressor gene implicated in lung, breast, pancreatic, and other cancers (15–17). We
demonstrate here that Scgb3a1 is a direct HIF-2α target gene and
that HIF-2α deficient lung tumor xenografts are characterized by
enhanced AKT signaling, consistent with previous observations
that Scgb3a1 suppresses AKT activity in human breast cancer cells
(18). We further demonstrate that ectopic expression of Scgb3a1
suppresses the growth of HIF-2α–deficient lung tumor xenografts,
concomitant with reduced AKT signaling. Finally, a direct correlation between Hif-2α and Scgb3a1 expression was observed in
human NSCLC cells and primary NSCLC tumors. These data
suggest that, although HIF-2α overexpression can contribute to
NSCLC progression, therapeutic inhibition of HIF-2α below
a critical threshold may actually promote tumor growth by repressing Scgb3a1 and other HIF-2α target genes.
Results
HIF-2α Deletion Promotes KrasG12D-Induced Lung Tumor Growth. The
inducible LSL-KrasG12D murine genetic model generates lung
tumors that faithfully model human lung adenocarcinoma initi-
Author contributions: J.M., M.M.H., A.S.-C., T.J., M.C.S., and B.K. designed research; J.M.,
M.M.H., A.S.-C., and B.K. performed research; A.V. provided human NSCLC samples; J.M.,
D.K.P., A.C.D., J.L.K, L.A.C., and B.K. analyzed data; and J.M. and B.K. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE22575).
1
Present address: Department of Cell Biology, Harvard Medical School, Boston, MA 02115.
2
To whom correspondence should be addressed. E-mail: celeste2@mail.med.upenn.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1001296107/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1001296107
A
B
12 week
+/
D
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10
5
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24 week
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45
40
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12 wk 24 wk
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12 wk 24 wk
% Ki67+ cells per field
Ki67
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8
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16
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0
+/ fl/
24 wk
18
16
14
12
*
10
8
6
4
2
0
+/
fl/
Fig. 1. Hif-2α deletion increases tumor burden. (A–F) KrasG12D/Hif-2α+/Δ
(designated +/Δ), or KrasG12D/Hif-2αfl/Δ (designated fl/Δ) mice were infected
with Adeno-Cre virus and killed 12 (A) or 24 (B) wk postinfection (w.p.i.). H&E
staining of murine lung sections indicate increased tumor burden in Hif-2α
deleted cohorts. Tumor number and area were measured using an automated
Axiovision system (Materials and Methods). (C–F) At 12 and 24 w.p.i, fl/Δ
animals presented significantly increased tumor number (C and E) and size (D
and F) compared with +/Δ controls. (n = 9–11 in each group). Error bars (C–F)
represent SEM. *P < 0.05.
Mazumdar et al.
1
0.8
0.6
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0
12 wk 24 wk
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40
30
20
10
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10
*
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TUNEL
12
12 wk
6
F
Tumor area/field (µm2)
*
14
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Tumor no. per mouse
12 wk
16
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Tumor area/field (µm2)
Tumor no. per mouse
C
F
1.2
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10
CELL BIOLOGY
mice display distinct types of progressive
ated in LSL-Kras
lesions including epithelial hyperplasia, adenomas, and adenocarcinomas (19). HIF-2α deficient KrasG12D lung tumors displayed a significant increase in hyperplastic lesions at 24 wk (Fig.
A
Adenoarcinomas /mouse
G12D
Identification of Scgb3a1 as a HIF-2α Target. To investigate molecular mechanisms underlying HIF-2α’s tumor suppressive effects,
we conducted global gene expression profiling on individual
tumors from KrasG12D/Hif-2afl/Δ or control KrasG12D/Hif-2α+/Δ
Adenomas /mouse
Hif-2α Deletion Promotes Tumor Progression. Lung tumors gener-
2A), and a similar trend at 12 wk. Similarly, KrasG12D/Hif-2αfl/Δ
animals displayed significantly increased numbers of adenomas
(Fig. 2B) at 24 wk compared with control animals. A similar trend
toward increased numbers of adenocarcinomas was observed at
24 wk but did not achieve statistical significance (Fig. 2C). Collectively, these data indicate that loss of HIF-2α accelerates tumor
progression in this context. Interestingly, deletion of HIF-1α in
parallel experiments did not alter disease kinetics (Fig. S2 G–I),
underscoring the differential effects of HIF-1α and HIF-2α in
these tumors.
The increased size of HIF-2α–deficient tumors correlated with
elevated cell proliferation (Fig. 2 D and E) and decreased apoptosis (Fig. 2 F and G), which was not observed in HIF-1α–
deficient tumors (Fig. S2 J and K). We also noted increased
numbers of infiltrating leukocytes in HIF-2α–deleted tumors
(Fig. S3 A and B), and granulocytes in particular (Fig. S3 C and
D), suggesting that inflammatory responses may differ between
KrasG12D/Hif-2αΔ/Δ, and KrasG12D/Hif-2α+/Δ lung tumors.
Hyperplasias /mouse
ation and progression (19). To evaluate the effect of HIF-2α lossof-function in lung tumor progression, we crossed mice carrying
conditional floxed Hif-2α fl or null Hif-2αΔ alleles (20) to mice
carrying the LSL-KrasG12D allele. The resulting experimental
KrasG12D/Hif-2α fl/Δ and control KrasG12D/Hif-2α+/Δ animals were
treated with adenovirus encoding the Cre recombinase by intranasal instillation. Cre-mediated deletion of the Lox-Stop-Lox (LSL)
gene cassette in the LSL-KrasG12D allele produced KrasG12D-driven
lung tumors as previously described (19) and in this case also
deleted the conditional Hif-2α fl allele. As the control KrasG12D/
Hif-2α+/Δ animals harbor a wild-type Hif-2α allele, HIF-2α function
was consequently retained in tumor tissue (Fig. S1A). Southern
blot analysis confirmed efficient deletion of the Hif-2αfl allele specifically in tumors but not surrounding lung tissue (Fig. S1B).
To evaluate the effect of HIF-2α deletion on lung tumor
progression, animals were analyzed 12 wk and 24 wk after adenoviral Cre delivery (Fig. S1C). Based on the recent report that
HIF-2α gain-of-function promotes lung tumor progression and
correlates with poor survival in this model (12, 13), we predicted
that deletion of HIF-2α would reduce tumor burden and progression. Surprisingly, KrasG12D/Hif-2αfl/Δ mutant mice displayed
a significant increase in tumor number and size at 12 wk (Fig. 1
A, C, and D) and 24 wk (Fig. 1 B, E, and F) postinfection, as
compared with their control littermates. Notably, deletion of
a floxed Hif-1αfl allele (21) in parallel experiments had no detectable effect on tumor number or volume in KrasG12D lung
tumors (Fig. S2 A–F).
8
6
4
2
0
24 wk
Fig. 2. Hif-2α deletion promotes tumor proliferation and progression. H&E
stained lung sections prepared from KrasG12D/Hif-2α+/Δ (+/Δ) or KrasG12D/Hif2αfl/Δ (fl/Δ) animals were graded for hyperplastic lesions (A), adenomas (B),
and adenocarcinomas (C). (D and E) Control and mutant tumors were
assessed 24 w.p.i. for proliferation by Ki67 staining (n = 6). (F and G) Enhanced cell death in +/Δ tumors as compared with Δ/Δ mutant tumors (n = 6)
as assessed by a TUNEL assay. Sections were imaged at 20×. Error bars represent SEM. *P < 0.05.
PNAS | August 10, 2010 | vol. 107 | no. 32 | 14183
mice. Specifically, RNA was isolated from tumors from experimental and control animals (n = 7 for each group) 28 wk after
infection and analyzed independently. Comparisons between the
genotypes identified a small set of genes (Table S1) whose expression was significantly and reproducibly reduced in HIF-2α–
deficient tumors, whereas expression levels of most genes were
unchanged. Subsequent quantitative RT-PCR (qRT-PCR) analysis on the same tumor RNAs revealed dramatic down-regulation
of multiple transcripts, including those encoding Scgb3a1, lactotransferrin, aquaporin 4, and ceruloplasmin (Fig. 3A). In contrast,
transcript levels of the HIF-1α target gene aconitase (Aco1) were
unaffected by Hif-2α deletion (Fig. 3A). The reduced expression
of Scgb3a1 was particularly intriguing as (i) Scgb3a1 is expressed
primarily in epithelial organs including lung, mammary gland,
trachea, prostate, pancreas, and salivary gland (22); (ii) Scgb3a1
expression is silenced in a variety of human cancers including
lung, breast, pancreas, and prostate (15, 16); and (iii) downregulation of Scgb3a1 is a significant independent predictor of
poor clinical outcome in early stage NSCLC (17).
1
+/
** ** ** *
fl/
** ** ** *
*
0.8
0.6
0.4
0.2
B
HIF-2 KD
VC VC C1 C2
VC VC C1 C2
HIF-1
HIF-2
actin
Hypoxic induction of mRNA
D
DFX
N
E
*
4
*
*
3
2
1
0
Scgb3a1 Aqp4
CP
3
2
0.5
1
0
8
6
** *
5
4
3
2
1
0
Scgb3a1
14184 | www.pnas.org/cgi/doi/10.1073/pnas.1001296107
Ac
x3
4
1
0
VC
HIF-2 KD C1
HIF-2 KD C2
HIF-2 KD C1R
7
o1
6
1
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o
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7a
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5
**
mHIF-2
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VC
HIF-2 KD
HIF-1 KD
6
5
DFX
Hypoxic/normoxic mRNA
N
HIF-2 KD C1
VC
A549
HIF-2 KD C2
HIF-2 KD C1R
2
6
F
Relative promoter occupancy
HIF-1 KD
Fold change in mRNA
C
Sl
c
2
1a
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ab
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Fold change in mRNA
(normalized to18s)
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gb
A
To extend our studies to human NSCLC, we introduced
a retroviral shRNA gene construct targeting human Hif-2α
mRNA (23) into human A549 lung adenocarcinoma cells, which
harbor an activating Kras mutation (24). Despite extensive
screening, only partial HIF-2α knockdown was observed in multiple cell clones, as revealed by Western blot and qRT-PCR
analyses (Fig. 3 B and C). Nevertheless, pooled HIF-2α knockdown cells (Fig. 3D), as well as independent knockdown cell
clones HIF-2α KD C1 and HIF-2α KD C2 (Fig. 3E), exhibited
a significant reduction in Scgb3a1 transcript levels (approximately
2.5-fold), consistent with our previous observations. A similar
reduction was observed for transcripts encoding aquaporin 4
(Aqp4), ceruloplasmin (CP), and VEGF (Fig. 3D and Fig. S4 A
and B) but not the HIF-1α specific target PGK1 (Fig. S4B). As
a control for functional specificity, we introduced a murine cDNA
encoding HIF-2α into the human HIF-2α KD C1 knockdown cells
(designated HIF-2α KD C1R, Fig. 3C), which in turn restored
Scgb3a1 transcript levels (Fig. 3E). Interestingly, HIF-1α depletion in A549 cells (Fig. 3B) did not alter transcript levels of
N
9
8
H
**
7
6
5
**
4
3
2
1
0
H4 H5 H6
Fig. 3. HIF-2α regulates Scgb3a1 tumor suppressor
gene expression. (A) A qRT-PCR analysis of mRNAs from
KrasG12D/Hif-2α+/Δ (+/Δ) or KrasG12D/Hif-2αfl/Δ (fl/Δ)
tumors (n = 7) harvested 28 w.p.i. Probe sets were selected from microarray analysis (Table S1). (B and C)
A549 human NSCLC cells transduced with retroviral
shRNA constructs targeting HIF-1α (HIF-1α KD) or HIF-2α
(HIF-2α KD) or with an empty viral control (VC). (B) Independent clones (C1 and C2) were assessed for HIF-1α
and HIF-2α protein levels. n = normoxia (21% O2). DFX =
deferoxamine (100 μM) treatment to stabilize HIF-α
subunits. (C). Endogenous human HIF-2α transcript levels were reduced in HIF-2α KD cells (hHIF-2α, Left). HIF-2α
rescue cells (HIF-2α KD C1R) were engineered through
stable expression of murine HIF-2α mRNA in HIF-2α KD
C1 cells (mHIF-2α, Right). (D) Hypoxic induction of
Scgb3a1, Aqp4, and CP transcript levels was reduced in
pooled HIF-2α KD cells but not HIF-1α KD cells. (E)
Scgb3a transcript levels were reduced in hypoxic HIF-2α
KD cell clones C1 and C2 (0.5% O2, 16 h) but restored in
hypoxic HIF-2α KD C1R cells. (F) Nuclear extracts (n = 3)
were isolated from uninfected hypoxic A549 cells (0.5%
O2, 16 h), sonicated, and immunoprecipitated with HIF2α antibodies. DNA was amplified using primers for HRE
regions 4, 5, and 6 (designated H4, H5, and H6) and
normalized to isotype matched IgG antibody. Error bars
(C–F) represent SD. *P < 0.05; **P < 0.005.
Mazumdar et al.
HIF-2α Levels Correlate with SCGB3a1 in Human NSCLC. The role of
HIF-2α in regulating Scgb3a1 led us to investigate whether HIF2α and SCGB3a1 transcript levels correlate in human NSCLC.
The qRT-PCR analysis of 15 independent human adenocarcinoma biopsies revealed that in 73% (11/15), HIF-2α mRNA
levels were significantly reduced relative to normal tissue, which
correlated directly with reduced SCGB3a1 mRNA expression
(Fig. 5A). In contrast, tumors that retained normal HIF-2α
mRNA levels did not display statistically significant changes in
SCGB3a1 mRNA levels. To further investigate the connection
between HIF-2α and SCGB3a1 expression, we evaluated lung
cancer datasets available through the Oncomine dataset repository (www.oncomine.org). Filtering specifically for NSCLC
tumor datasets containing HIF-2α and SCGB3a1 probes, we
found significant correlation between HIF-2α and SCGB3a1
expression in three of four datasets (Fig. 5B).
Mazumdar et al.
300
250
**
200
150
100
50
0
5 10 15 20 25 30 35
HIF-1
HIF-2
Scgb3a1
pAKT
HIF-2
KD C1
Scgb3a1
HIF-2
KD C1 R
A549
C
HIF-2
KD C2
d.p.i
HIF-2 KD C1
VC
HIF-2 KD C1R
HIF-2 KD C1 Scgb3a1
3
**
2.5
2
1.5
1
0.5
0
(1x106 cells)
HIF-2
KD C1
1
Fold change in tumor weight
VC
HIF-2 KD C1
HIF-2 KD C1R
350
D
AKT
GSK
4EBP-1
actin
KDT1
C2 KDT2
C3
KDT3
C4
S473
AKT
pGSK3
GSK
S473
pGSK3
C1
pAKT
KDT4
pAKT
S473
AKT
pGSK3
GSK
Fig. 4. HIF-2α suppresses A549 xenograft growth and AKT signaling. (A)
Nude (Nu/Nu) mice were injected s.c. with 1 × 106 HIF-2α KD C1 cells. A549
cells expressing the empty vector (VC) injected in contralateral flank served
as controls, and individual tumor volumes were measured (mm3) at indicated
days postimplantation (d.p.i). HIF-2α KD C1 cells (n = 10) formed significantly
larger tumors compared with control cells (n = 10). Tumors formed by mHIF2α expressing HIF-2α KD C1R cells (n = 6) were comparable to control tumors
(n = 6). Pooled VC tumor volumes from two simultaneously conducted independent experiments [(VC; HIF-2α KD C1) and (VC; HIF-2α KD C1R)] were
used for computation. (B) Ectopic expression of HIF-2α or Scgb3a1 in HIF-2α
KD C1 cells inhibits tumor growth. Xenografts (n = 10) were generated as in
A and tumor weights measured at 35 d. (C) A549 wild-type cells, HIF-2α KD
C1, HIF-2α KD C2, HIF-2α KD C1R, and HIF-2α KD C1 cells expressing Scgb3a1
were cultured under hypoxic conditions (0.5% O2) for 20 h and whole cell
extracts analyzed for AKT pathway activation by Western blot analysis. Actin
served as the loading control. (D) HIF-2α KD C1 [n = 4 (KD T1-4)] and contralateral A549 control [n = 4 (C1-4])] xenograft tumors were processed for
whole cell protein extraction and analyzed by Western blot. Error bars
represent SEM. **P < 0.005.
Discussion
Many human cancers display a direct correlation between HIF-α
overexpression and poor prognosis (4, 8), consistent with the
idea that HIF target genes positively regulate critical features of
tumor progression, including cancer cell metabolism, survival,
movement, and local angiogenesis. This correlation has spurred
the development of HIF inhibitors as therapeutic treatments for
cancer, as well as other diseases (11). The specific activities of HIF1α and HIF-2α in tumor progression are quite complex, however,
and appear to vary depending on cell type and oncogenic background. For example, although HIF-1α overexpression drives
murine mammary tumor progression and metastasis in a murine
model (25), it also inhibits the growth of certain von Hippel-Lindau
(VHL)-deficient RCCs (26, 27), in part by restricting the activity
of the c-Myc oncoprotein (28, 29). In contrast, HIF-2α expression
potentiates c-Myc signaling in RCCs and promotes tumor growth
(29). Furthermore, a recent report demonstrated that expression
of a stabilized (gain-of-function) Hif-2α allele similarly promotes
tumor progression in the LSL-KrasG12D murine NSCLC model
(13). These and other data suggest that inhibiting either HIF-1α
or HIF-2α (or both) may be of therapeutic value in specific
disease settings.
PNAS | August 10, 2010 | vol. 107 | no. 32 | 14185
CELL BIOLOGY
HIF-2α Deficiency Reduces AKT Signaling. Enforced Scgb3a1 expression was shown to inhibit AKT pathway signaling associated
with decreased AKT phosphorylation at Ser473 in human breast
cancer cells (18). We therefore hypothesized that HIF-2α
knockdown in A549 cells would increase AKT pathway activity.
Exposure of HIF-2α KD C1 and HIF-2α KD C2 cells to hypoxia
significantly increased levels of phopshorylated AKT at Ser473
(Fig. 4C). Moreover, levels of phosphorylated AKT downstream
effectors including pGSK-3β and 4EBP-1 were higher compared
with control cells (Fig. 4C). Notably, restoring either HIF-2α or
Scgb3a1 expression in the HIF-2α KD C1 cells reduced AKT,
GSK-3β, and 4EBP-1 phosphorylation to nearly control levels
(Fig. 4C). A similar trend was observed in the xenograft tumors,
as Western blot analysis revealed that HIF-2α KD C1 tumors
displayed increased pAKT and pGSK3β levels compared with
control tumors (Fig. 4D).
B
A
Tumor volume (mm3)
Scgb3a1 or other genes identified in the microarray experiment
(Fig. 3D and Fig. S4C).
Our results indicate that HIF-2α regulates Scgb3a1 expression
in lung adenocarcinoma cells in a cell autonomous manner and
that this activity is not shared by HIF-1α. We next investigated the
possibility that HIF-2α regulates Scgb3a1 directly. Analysis of
human and murine Scgb3a1 gene sequences revealed multiple
putative HREs spanning the upstream promoter and enhancer
regions (Fig. S4D). ChIP assays revealed HIF-2α occupation of
two HREs (H4 and H5) in the Scgb3a1 promoter in A549 cells,
which increased (4- to 7-fold) under hypoxic conditions (Fig. 3F).
Not all HREs in the Scgb3a1 promoter appear functional, as H6
fails to bind HIF-2α (Fig. 3F). Moreover, HIF-1α failed to bind
H4 and H5 (Fig. S4E). Collectively, these data demonstrate that
Scgb3a1 is a direct HIF-2α target gene.
To test the effects of HIF-2α knockdown in tumor formation by
the A549 cells, we implanted 5 × 106 HIF-2α KD C1 or HIF-2α KD
C2 cells s.c. in immunocompromised mice to generate xenograft tumors. Consistent with our results from the autochthonous
HIF-2α deficient lung tumors, xenograft tumors from HIF-2α KD
C1 cells displayed a modest but significant growth advantage over
A549 control tumors and a similar trend was also observed for
HIF-2α KD C2 cells (Fig. S5 A and B). As the recipient mice had to
be killed between 25 and 35 d due to the large size of these xenografts, the experiment was repeated using fewer (1 × 106) HIF-2α
KD C1 cells (Fig. 4A). In this experiment, HIF-2α KD C1 xenografts displayed a clear and significant growth advantage over the
vector control xenografts, and restored HIF-2α expression (HIF-2α
KD C1R cells) suppressed tumor growth to levels essentially indistinguishable from the vector control tumors (Fig. 4 A and B).
Importantly, ectopic expression of human Scgb3a1 in the HIF-2α
KD C1 cells (Fig. S5B) also effectively suppressed tumor growth,
implicating Scgb3a1 as a critical mediator of HIF-2α activity in this
setting (Fig. 4B).
Fold change in mRNA
C
B
1.6
*
1.4
T
C
*
Fold change in mRNA
A
1.2
1
0.8
0.6
0.4
0.2
0
HIF-2
5
4
3
2
1
0
Scgb3a1
T
6
HIF-2
Scgb3a1
HIF-2 /Scgb3a1 gene expression correlation in NSCLC datasets.
Dataset
C
HIF-2
HIF-2
Pearson Correlation
p-value
Bild (n=111)
0.437
1.60E-06
Kim (n=138)
0.377
5.10E-06
Ding (n= 68)
0.161
0.168
Bittner (n=73)
0.503
3.02E-06
Over
expressed
Deleted
Vegf
Vegfr-2
Snail
Angiogenesis,
EMT transition
Tumor
growth
Akt signaling
Scgb3a1
Lactotransferrin ?
Ceruloplasmin ?
Fig. 5. HIF-2α and SCGB3a1 levels correlate in NSCLC samples. (A and B) RNA
was isolated from fresh frozen or formaldehyde/paraformaldehyde fixed
human NSCLC tumors (designated T) (n = 15), used to generate cDNA for
quantitative RT-PCR analysis. Uninvolved adjacent lung tissue (designated C)
served as control. Transcipt levels were normalized to 18s RNA; 73% (11 out of
15) NSCLC samples analyzed demonstrated significantly decreased levels of
both HIF-2α and SCGB3a1 mRNA compared with adjacent control tissue (Left).
Samples with normal SCGB3a1 expression (n = 4) did not display any significant change in HIF-2α level (Right). Error bars represent SEM. *P < 0.05. (B)
Significant correlation between HIF-2α and SCGB3a1 mRNA expression was
observed in three (of four) human NSCLC datasets obtained from Oncomine.
(C) Schematic representation of the opposing roles of HIF-2α in tumorigenesis,
wherein both HIF-2α overexpression through activation of protumorigenic
target genes, and HIF-2α deletion through loss of expression of candidate
tumor suppressor genes, including Scgb3a1, could promote tumor growth.
Other HIF-2α targets such as lactotransferrin and ceruloplasmin may also influence tumor outcome, but their role in tumor suppression is as yet unclear.
The data presented here indicate that HIF-2α has an unexpected tumor suppressive role in KrasG12D-driven lung tumors
and that reduced expression of the HIF-2α target Scgb3a1 contributes to this effect. This interpretation is supported by the
observation that restored Scgb3a1 expression inhibited the
growth of HIF-2α–deficient A549 xenograft tumors. Moreover,
elevated AKT signaling was observed in HIF-2α–deficient A549
cells and was inhibited by restored expression of either HIF-2α
or Scgb3a1. These observations are consistent with previous
reports that enforced Scgb3a1 expression inhibits AKT activity in
human breast cancer cells (18).
Scgb3a1 was originally identified as a candidate tumor suppressor through gene expression studies comparing human mammary carcinomas to normal mammary epithelial cells (14). It was
subsequently shown that Scgb3a1 expression is reduced in human
lung, prostate, pancreatic, and nasopharyngeal cancers, and corresponding hypermethylation of the Scgb3a1 promoter was
reported for multiple malignancies (15–17, 30). Moreover, decreased Scgb3a1 expression was identified as a primary independent predictor of poor clinical outcome in early stage human
NSCLCs (17). Our data indicate that Scgb3a1 expression is directly
regulated by HIF-2α in human lung adenocarcinoma cells: this
relationship is underscored by the direct correlation between HIF14186 | www.pnas.org/cgi/doi/10.1073/pnas.1001296107
2α and SCGB3a1 mRNA levels observed in approximately 70% of
human lung adenocarcinomas analyzed and in three of four
NSCLC datasets obtained from the Oncomine database.
Interestingly, our results contrast directly with those from
a recent report in which HIF-2α inhibition in A549 cells, as well as
in HCT116 colon carcinoma and U87MG glioblastoma cells, actually reduced xenograft tumor growth (31). The nature of these
disparate results is not clear, as identical shRNA sequences were
used; however, our data are supported by two important controls.
First, the phenotypes observed in our HIF-2α deficient A549
cells are reversed by enforced expression of a murine HIF-2α
mRNA that escapes shRNA inhibition, confirming the HIF-2α
dependent nature of these phenotypes. Second, the HIF-2α deficient A549 xenograft tumors phenocopy directly the behavior of
autochthonous tumors generated by Hif-2α genetic deletion in the
LSL-KrasG12D model.
The tumor suppressive activity of HIF-2α we observed was
surprising, as expression of a stabilized HIF-2α in the identical
LSL-KrasG12D model promoted tumor growth, and HIF-2α overexpression similarly promotes growth of RCCs (29). In particular,
it raises the question of how HIF-2α can behave as both a tumor
suppressor and an oncogene in the same NSCLC model. It appears
that entirely different sets of HIF-2α target genes mediate these
effects in a manner perhaps analogous to the proproliferative and
proapoptotic activities of c-Myc (32). Whereas increased HIF-2α
function elevates the expression of genes associated with angiogenesis and epithelial-to-mesenchymal transition (EMT) in LSLKrasG12D NSCLCs (13), HIF-2α deletion in the same model
diminishes the expression of a putative tumor suppressor Scgb3a1,
among other genes.
Collectively, these results strongly suggest that inhibition of
overexpressed HIF-2α in NSCLCs (and perhaps other carcinomas) might have beneficial effects but that reducing HIF-2α
function below a critical threshold might also drive tumor progression by inhibiting the expression of specific tumor suppressor
genes. This model (Fig. 5C) assumes that HIF-2α overexpression
either fails to increase Scgb3a1 function above normal levels or
that increased Scgb3a1 activity is offset by elevated expression of
other HIF-2α targets. It is also interesting to note that the time at
which HIF-2α function affects tumor progression differs between
the two models. In a previous study (13), expression of stabilized
HIF-2α was initiated at the earliest stage in lung tumorigenesis
(i.e., Ad-Cre infection). In contrast, HIF-2α protein stabilization in
the lung tumors of our control mice probably occurs subsequent to
Kras activation, either as a result of tumor hypoxia or additional
oncogenic events. The importance of this temporal difference in
expression is not yet clear. In general, however, our results caution
that inhibition of HIF complexes in cancer treatment may require
a narrower therapeutic window than initially imagined.
Our results also raise a number of interesting questions for
subsequent work, including (i) to what degree does the suppression of other putative HIF-2α target genes (lactotransferrin,
ceruloplasmin, etc.) contribute to the phenotypes we observe; (ii)
are there noncell-autonomous effects of HIF-2α deletion that
regulate LSL-KrasG12D NSCLC tumor progression (for example,
the increase in granulocyte infiltration); and (iii) is the relation
between HIF-2α and Scgb3a1 observed only in the context of
activating Kras mutations or does it extend to other oncogenic
backgrounds? Importantly, how does Scgb3a1 regulate AKT activity and are additional signaling pathways involved in its tumor
suppressive effects? To what degree does elevated AKT activity,
in concert with other direct and indirect HIF-2α targets, contribute to the proliferative and apoptotic phenotypes we observe
in KrasG12D-driven, HIF-2α deficient lung tumors?
In summary, we used both autochthonous and xenograft genetic tumors to reveal a tumor suppressive effect of HIF-2α in
NSCLC and identify its direct target gene Scgb3a1 as a downstream effector. This may be a more general phenomenon: for
Mazumdar et al.
Materials and Methods
Generation of and Characterization of Inducible LSL-KrasG12D, Conditional Hifαfl Mice, and Lung Tumors. Mice carrying the previously described inducible
(LSL-KrasG12D) knock-in allele (19), the conditional floxed Hif-2αfl allele, and
the related null Hif-2αΔ allele (20) were intercrossed to generate experimental and control animals (SI Materials and Methods). Lung morphometry,
histology, and immunohistochemistry were performed as described in SI
Materials and Methods.
Cell Culture and Infection. A549 cells (ATCC) were grown in DMEM with 10%
FBS (Gemini), amino acids, and antibiotics (SI Materials and Methods); A549
cells were infected with retroviruses encoding shRNAs targeting HIF-1α (34),
HIF-2α (23) (designated HIF-1α KD and HIF-2α KD, respectively), or with the
retroviral backbone as the negative viral control (VC).
Western Blot Analysis. Protein extracts were prepared and analyzed as described in SI Materials and Methods. For hypoxic analysis, cells were cultured
under 0.5% O2 and 5% CO2 for 16–20 h in an InVivo2 400 hypoxia workstation
with O2 control (Ruskinn Technology Ltd.). Western blots were probed with
antibodies against HIF-1α (Cayman for mouse protein, R&D for human protein), HIF-2α (Novus Biologicals), Scgb3a1 (Abcam), AKT, pAKT, GSK-3β, pGSK3-α/β, p4EBP1, and β-actin (Cell Signaling).
lation kit (Ambion) and purified on RNeasy spin columns (Qiagen) as described in SI Materials and Methods; cDNA was synthesized using a high
capacity c-DNA transcription kit (Applied Biosystems). Transcript levels were
normalized to 18S rRNA using an Applied Biosystems 7900HT analyzer and
represented as fold change 2−ΔCT (normoxia)/ 2−ΔCT (hypoxia); mean normoxic value was set to 1.
cDNA Microarray and Oncomine Analysis. Microarray analysis was performed
by the University of Pennsylvania Microarray Core facility, and Oncomine data
were analyzed as described in SI Materials and Methods.
ChIP Analysis. A549 cells were cultured either under normoxia or hypoxia,
crosslinked with 1% formaldehyde for 20 min at 37 °C, and quenched with
1 M glycine. Nuclear lysates were analyzed as described in SI Materials
and Methods.
Xenograft Assays. We injected 5 × 106 or 1 × 106 (as indicated) viable HIF-2α KD
C1 or HIF-2α KD C2 cells in a 1:1 mix of DMEM:growth factor reduced matrigel
matrix (BD) s.c. into one flank of 6-wk-old nude (NU/NU) female mice (Charles
River Laboratories). The other flank was either injected with wild-type A549
cells or cells carrying the empty vector (VC). Tumor volume (mm3) was determined using a caliper every 5 d postimplantation (d.p.i). Animals were
killed 5 wk postimplantation, or earlier (3-4 wk) in case of ulcerating tumors.
Statistics. P values were determined using the Student’s t test; P < 0.05 was
considered significant.
RNA Purification, Reverse Transcription, and Real-Time PCR Amplification. RNA
was extracted with TRIzol (Invitrogen) or RecoverAll Total nucleic Acid Iso-
ACKNOWLEDGMENTS. We thank Shetal Patel and Ania Warczyk (University
of Pennsylvania) for assistance with immunohistochemistry and xenograft
assays, Hongwei Yu and Alan Wanicur (University of Pennsylvania) for
preparation of tissue sections, and William Y. Kim (University of North
Carolina) and the Simon laboratory for helpful discussions. This work was
supported by the Howard Hughes Medical Institute (M.C.S.) and National
Institutes of Health Grant HL66130 (to B.K. and M.C.S.).
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Mazumdar et al.
PNAS | August 10, 2010 | vol. 107 | no. 32 | 14187
CELL BIOLOGY
example, a previous report demonstrated that either HIF-1α or
HIF-2α expression can limit tumor growth in genetically manipulated GS9L glioma and murine embryonic stem cells by altering
angiogenesis and tumor cell apoptosis (33). Determining the
cellular conditions and molecular mechanisms that distinguish the
tumor promoting, or tumor suppressive, activities of HIF-1α and
HIF-2α proteins will clearly require additional investigation.
Supporting Information
Mazumdar et al. 10.1073/pnas.1001296107
SI Materials and Methods
Generation of Inducible LSL-KrasG12D and Conditional Hif-αfl Mice. To
generate experimental LSL-KrasG12D; Hif-2afl/Δ and control
LSL-KrasG12D; Hif-2α+/Δ cohorts, mice heterozygous for the
previously described inducible (LSL-KrasG12D) knock-in allele (1)
were crossed to animals carrying the conditional floxed Hif-2α fl
allele, or the related null Hif-2αΔ allele (2). Lung tumorigenesis
was initiated by infecting 6-wk-old mice with 2.5 × 107 pfu of
recombinant adenovirus expressing the Cre recombinase (University of Iowa Gene Transfer Vector Core) by intranasal instillation. Southern blot analysis of NcoI-digested genomic DNA
excised from tumors 24 w.p.i. revealed efficient deletion of the
conditional Hif-2αfl allele in tumors but not surrounding lung
tissue (2) (Fig. S1). A corresponding strategy was used to generate experimental LSL-KrasG12D; Hif-1αfl/Δ and control LSLKrasG12D; Hif-1α+/Δ cohorts. Mice were maintained on a mixed
129/Bl6 background, and analysis was largely restricted to littermates. Occasionally, multiple litters of the same age (within
approximately 1 wk) were used to provide sufficient numbers of
each genotype; however, these multiple litters were typically the
offspring of sisters mated to a single male. All mice were
maintained in microisolator cages and treated in accordance
with the National Institutes of Health and American Association
of Laboratory Animal Care Standards and consistent with the
animal care and use regulations of the University of Pennsylvania and Massachusetts Institute of Technology.
Lung Morphometry, Histology, and Immunohistochemistry. Lungs
from mice 12 or 24 w.p.i. were fixed in 3.6% paraformaldehyde and
all lobes embedded in paraffin. Sections (5 μm) were cut from
three distinct zones (top, middle, bottom) of each paraffin block,
stained with H&E and assessed for tumor type (hyperplasia, adenoma, adenocarcinoma), numbers, and areas using AxioVision
digital image processing software as per manufacturer’s protocol.
Briefly, the desired region of quantification was imaged, total
region area measured (μm2), pixels matching tumor cells selected,
and a binary mask created to compute tumor numbers and area.
The measurements were independently confirmed by manual
grading of tumor lesions. Tumor lesions were graded as described
previously (3). Immunohistochemistry was performed using the
MOM kit (Vector Labs) as per manufacturer’s protocol with
minor modifications. Ki67 (Novocastra) and Gr.1 (ebiosciences)
were used at a 1:100 dilution, and incubated with tissue sections
overnight (O/N) at 4 °C. Secondary antibody incubation time was
increased to 1 h (RT) and sections were counterstained with hematoxylin. Cell death was detected by TUNEL staining (Millipore), as per manufacturer’s protocol.
Cell Culture and Infection. The pcDNA3.0-HIF-2α gene construct
used to restore HIF-2α expression in HIF-2α KD C1R cells has
been described previously (4). To establish HIF-2α KD C1 cells
expressing Scgb3a1 (HIF-2α KD C1 Scgb3a1), human Scgb3a1
cDNA (MHS 1010–7430241; Open Biosystems) was subcloned
into pLK0-1 Neo lentiviral plasmid (13425; Addgene) using Age1
and EcoR1 restriction sites. The empty lentiviral backbone served
as a negative control.
Western Blot Analysis. Whole cell extracts were made using RIPA
buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1%
TritonX-100, 1% Sodium deoxycholate, 0.1% SDS, 5 mg/mL
Mazumdar et al. www.pnas.org/cgi/content/short/1001296107
Aprotinin, 5 mg/mL Leupeptin, 1 mM PMSF). Xenograft tumor
samples were homogenized (Euro Turrax T20b; Ika Labortechnik)
in RIPA buffer. For detection of HIF-α protein knockdown following retrovirus infection, cells were treated with the hypoxia
mimetic iron chelator deferoxamine (200 μM) for 5 h at 37 °C.
RNA Purification, Reverse Transcription, and Real-Time PCR Amplification.
RNA was extracted with TRIzol (Invitrogen) and purified on
RNeasy spin columns (Qiagen). Fresh frozen xenograft and NSCLC
tissue was mechanically disrupted in TRIzol using either a Euro
Turrax T20b (Ika Labortechnik) homogenizer or a tissue grinding
pestle (Kontes Glass Company). RNA from formaldehyde- or
paraformaldehyde-fixed, paraffin embedded (FFPE) sections
was harvested using RecoverAll Total nucleic Acid Isolation
kit (Ambion).
cDNA Microarray Analysis. Dye-coupled cDNA was hybridized to
MOE430A microarray gene chips (Affymetrix) (n = 7 in each
group). Quality analysis on each individual chip was performed
using affyPLM, available through Bioconductor (5). The normalized unscaled standard error (NUSE) boxplots were computed, and GCRMA software package used to obtain gene
expression values (6). A principal component analysis (PCA)
identified one outlier, which was removed for subsequent analysis.
Cyber-T analysis (7) was used to identify differentially regulated
genes based on a preselected false discovery rate (FDR) of 10%.
This generated a list of 677 genes; P values were computed using
the MAS5 algorithm. Genes that displayed significant (P < 0.05)
change between the samples in each group were selected for
further validation (Table S1).
Oncomine Analysis. The Oncomine database (www.oncomine.org)
was searched for Hif-2α and Scgb3a1 genes. This generated 48 lung
cancer datasets for Hif-2α and 16 datasets for Scgb3a1. We further
filtered for datasets derived from tumors, and identified four datasets (namely Bild lung, Ding lung, Kim lung, and Bittner lung)
with expression values for both Hif-2α and Scgb3a1 genes. All of
the datasets have Affymetrix U133 Plus 2.0 microarray platform
and have three probes corresponding to Hif-2α: 200878_at,
200879_as_at, and 241055_at, and one probe corresponding to
Scgb3a1: 230378_at.
For each dataset the following was performed: the complete data
were downloaded from the National Center for Biotechnology
Information’s Gene Expression Omnibus (GEO) repository and
processed to obtain the expression value matrix of the samples and
the probe sets. Only those samples corresponding to the NSCLC
categories were selected and the signal values of the probe sets
corresponding to the two genes were further selected. The data
were log2 transformed to approximate data symmetry. For Hif-2α,
boxplots of the probe sets were generated and the probe set with the
highest median intensity (200878_at) was used for further analysis.
ChIP Analysis. Nuclear lysate was harvested and sonicated (Sonic
Dismembrator Model 500; Fisher Scientific) to obtain 500-bp to
1,000-bp DNA fragments. Chromatin-protein complexes were
cleared by centrifugation at 14,000 rpm for 15 min. The soluble
chromatin was immunoprecipitated with polyclonal HIF-2α and
monoclonal HIF-1α antibodies (Novus Biologicals). DNA crosslinks were reversed by heating at 65 °C for 16 h with NaCl, treated
with RNase A and protease K, and analyzed by qRT-PCR.
1 of 5
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Natl Acad Sci USA 104:2301–2306.
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Cell Biol 23:9361–9374.
A
Hif-2
fl/fll
X LSL-KrasG12D/+
F2 LSL-KrasG12D/+, Hif-2
F3
LSL-KrasG12D/+, Hif-2
5. Gentleman RC, et al. (2004) Bioconductor: Open software development for
computational biology and bioinformatics. Genome Biol 5:R80.
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909–917.
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expression data: Regularized t-test and statistical inferences of gene changes.
Bioinformatics 17:509–519.
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encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem 269:23757–23763.
Hif-2
/
X LSL-KrasG12D/+
fl/+
X LSL-KrasG12D/+, Hif-2
fl/
X LSL-KrasG12D/+, Hif-2
/+
+/
Adeno-Cre
# KrasG12D/+, Hif-2
Mutant
X KrasG12D/+, Hif-2
+/
Control
Ea
r
N
l u orm
ng a
tis l
su
e
Tu
m
or
1
Tu
m
or
2
B
/
HIF-2 2loxP
HIF-2 1loxP
H&E staining
Masked profile
24 wk lung section
C
5X
Fig. S1. (A) Breeding scheme to generate KrasG12D/Hif-2α+/Δ and KrasG12D/Hif-2αfl/Δ mice. Mice carrying either a Hif-2α floxed allele (Hif-2α fl/fl) or Hif-2α null
(Hif-2αΔ/+) allele were crossed with LSL-KrasG12D animals to yield KrasG12D/Hif-2αfl/Δ (mutant) and Hif-2αfl/+ (control) mice. # indicates concomitant activation of
KrasG12D allele and Hif-2α deletion upon exposure to Cre recombinase. (B) Efficient recombination of Hif-2α indicated by the presence of 1loxP product in two
independent test tumors (designated T1 and T2) but not in adjacent uninfected lung tissue or ear as detected by Southern blot analysis. (C) A binary mask
(Right) of an H&E stained section (Left) generated using the imaging software Axiovision 4 correctly identifies the tumor lesions (arrowheads).
Mazumdar et al. www.pnas.org/cgi/content/short/1001296107
2 of 5
A
B
24 week
fl/
+/
12 week
20X
10
5
+/
fl/
+/
8
6
4
2
0
+/
fl/
H
fl/
+/
16
17.5
14
Adenomas/mouse
20
15
12.5
10
7.5
5
2.5
0
12 wk
12
10
8
6
4
2
0
24 wk
fl/
20
15
10
5
0
+/
fl/
I
Adenocarcinomas/mouse
0
10
25
12 wk
24 wk
+/
4
3.5
J
24 wk
40
35
30
25
20
15
10
5
0
+/
K
fl/
*
3
2
1.5
1
0.5
12 wk
fl/
75
60
45
30
15
0
fl/
2.5
0
+/
90
% Ki67+ cells per field
15
12
F
24 wk
30
24 wk
+/
fl/
16
% TUNEL+ cells per field
20
14
Tumor area/field (µm2)
25
E
12 wk
16
Tumor no. per mouse
30
G
Hyperplastic lesions/mouse
D
12 wk
35
Tumor area/field (µm2)
Tumor no. per mouse
C
14
12
10
8
6
4
2
0
24 wk
24 wk
fl/Δ
Fig. S2. (A–F) Kras
, Hif-1α (designated +/Δ) or Kras
, Hif-1α
(designated fl/Δ) mice were infected with Adeno-Cre virus and killed 12 (A) or 24 (B)
w.p.i, and tumor sections were treated with H&E stain. (C–F) Tumor numbers and volumes in each cohort (n = 5) were measured as described in Materials and
Methods. (G–I) Tumors from mutant and control mice were graded for hyperplastic lesions (G), adenomas (H), and adenocarcinomas (I). Tumors were also
assessed for Ki67 (J) and TUNEL (K) staining as measures of proliferation and apoptosis, respectively. Error bars (C–I) represent SEM. *P < 0.05.
+/Δ
G12D
+/
fl/
10X
C
B
+/
140
CD45
A
CD45+ cells per field
G12D
+/
100
80
60
40
20
D
Gr.1+ cells per field
Gr.1
24 wk
+/
300
20X
*
120
0
fl/
fl/
250
fl/
*
200
150
100
50
0
24 wk
Fig. S3. (A and B) Mutant tumors displayed significantly increased association with tumor infiltrating CD45 cells (n = 6), and in particular granulocytes (n = 5–6
for each group) (C and D). Error bars (B and D) represent SEM. *P < 0.05.
Mazumdar et al. www.pnas.org/cgi/content/short/1001296107
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4
3
5
2.5
4
3
1.5
2
*
1
0
*
* *
1
1
0
0
Scgb3a1
N
Aqp4
H
7
*
6
*
Ceruloplasmin
N
Pgk1
5
2
0.5
H
Vegf
5
*
*
*
4
3
4
3
2
2
1
1
0
0
A5
49
H
KD IF2
C
1
H
KD IF2
C
1
R
VC
HIF-1 KD C1
HIF-1 KD C2
3.5
4
6
3
3
5
2.5
4
2
2
Scgb3a1
E
2
1
1
0
3
1.5
0.5
1
0
0
Aqp4
Ceruloplasmin
Hs Scgb3a1
2000 5'
1000 5'
8
7
6 5 4 3
Exon 1
2
1
Relative promoter occupancy
Hypoxic induction of mRNA
(normalized to 18s)
*
2
C
D
HIF-2 KD C2
3
B
Hypoxic induction of mRNA
(normalized to 18s)
HIF-2 KD C1
VC
A5
49
H
KD IF2
C
H 1
KD IF2
C
1
R
Hypoxic induction of mRNA
(normalized to 18s)
A
N
H
8
7
6
*
5
4
3
2
1
0
H4 H5 H1-2
Scgb3a1 Pgk1
Fig. S4. (A) Hypoxic induction of Scgb3a1, aquaporin, and ceruloplasmin transcripts was reduced in HIF-2α KD C1 and HIF-2α KD C2 clones. (B) Expression of
the HIF-1α specific target gene Pgk1 was unaffected by HIF-2a expression levels in HIF-2α KD cells. In contrast, hypoxic induction of the previously characterized
HIF target gene Vegf (HIF-1α and HIF-2α shared target) was significantly reduced in HIF-2α KD C1 cells but restored in HIF-2α KD C1R cells. (C) Hypoxic induction
of Scgb3a1, aquaporin, and ceruloplasmin transcripts is not significantly affected in HIF-1α KD C1 and HIF-1α KD C2 clones. (n = 3). *P < 0.05. Error bars
represent SD. (D) Schematic representation of the human Scgb3a1 promoter region (2,000-bp 5′) demonstrating the presence of multiple putative HREs. (E)
Nuclear extracts were isolated from A549 cells cultured under hypoxia (0.5% O2, 16h), sonicated, and immunoprecipitated with HIF-1α antibody. DNA was
amplified using primers for human Scgb3a1 HRE regions 4 and 5 (designated H4 and H5), and normalized to mouse IgG antibody. Human Pgk1 HRE 1 and 2
served as a positive control (human Pgk1 5′18-bp region containing HREs 1 and 2 have been previously described) (8). Error bars represent SD. *P < 0.05.
Mazumdar et al. www.pnas.org/cgi/content/short/1001296107
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5x106 cells
Tumor volume (mm3)
A
A549
450
400
350
HIF-2 KD C1
**
300
200
100
0
1
5
10 15
20 25
Days post implantation
Relative gene expression
(normalized to 18s)
HIF-2 KD C2
500
400
300
250
200
150
100
50
0
B
A549
600
1
5
10
15
20
25
30
35
Days post implantation
HIF-2 KD C1 VC
HIF-2 KD C1 Scgb3a1
6
5
**
4
3
2
1
0
hScgb3a1
Fig. S5. (A) Nude (Nu/Nu) mice were injected s.c. with 5 × 106 HIF-2α KD C1 and C2 cells. A549 cells injected in contralateral flank served as controls, and
individual tumor volumes were measured (mm3). HIF-2α KD C1 cells (n = 6) showed a modest but significant increase in tumor size compared with control
tumors. **P < 0.005. HIF-2α KD C2 tumors did not reach statistical significance but showed a similar trend. (B) SCGB3a1 transcript levels in HIF-2α KD C1 cells
engineered to stably express a human SCGB3a1 cDNA. Error bars represent SD. *P < 0.05, **P < 0.005.
Table S1. Microarray detection of misregulated genes in HIF-2αΔ/fl lung tumors
Gene name
Description
Fold difference
P value
Ltf
Abp1
Aqp 4
Scgb3a1
Gabrp
Slco 1a5
Slc27a2
LOC672450
Fbxo36
Cp
Mocs1
Aco1
Lactotransferrin
Amiloride binding protein 1
Aquaporin 4
Secretoglobin, family 3a, member 1
Gamma-amnibutyric acid (GABA-A) receptor, pi
Solute carrier organic anion transporter family, member 1a5
Solute carrier family 27 (fatty acid transporter), member 2
V (kappa) gene product
F-box protein 36
Ceruloplasmin
Molybdenum cofactor synthesis 1
Aconitase 1
−6.51
−3.74
−3.53
−3.25
−2.83
−2.82
−2.52
−2.03
−2
−1.61
1.59
0.72
0.018
0.032
0.039
0.047
0.042
0.041
0.041
0.045
0.033
0.42
0.05
0.204
n = 7 (HIF-2α +/Δ tumors), n = 7 (HIF-2α fl/Δ tumors).
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