Published OnlineFirst March 14, 2013; DOI: 10.1158/0008-5472.CAN-12-3191
Cancer
Research
Molecular and Cellular Pathobiology
Dachshund Binds p53 to Block the Growth of Lung
Adenocarcinoma Cells
Ke Chen1,3, Kongming Wu1,3, Shaoxin Cai1,3, Wei Zhang1,3, Jie Zhou1,3, Jing Wang1,3,
Adam Ertel1,3, Zhiping Li1,3, Hallgeir Rui1, Andrew Quong1, Michael P. Lisanti2,3, Aydin Tozeren3,4,5,
Ceylan Tanes3,4,5, Sankar Addya1,3, Michael Gormley1,3, Chenguang Wang1,2,3, Steven B. McMahon1,3, and
Richard G. Pestell1,3
Abstract
Hyperactive EGF receptor (EGFR) and mutant p53 are common genetic abnormalities driving the
progression of non–small cell lung cancer (NSCLC), the leading cause of cancer deaths in the world. The
Drosophila gene Dachshund (Dac) was originally cloned as an inhibitor of hyperactive EGFR alleles.
Given the importance of EGFR signaling in lung cancer etiology, we examined the role of DACH1
expression in lung cancer development. DACH1 protein and mRNA expression was reduced in human
NSCLC. Reexpression of DACH1 reduced NSCLC colony formation and tumor growth in vivo via p53.
Endogenous DACH1 colocalized with p53 in a nuclear, extranucleolar location, and shared occupancy of
15% of p53-bound genes in ChIP sequencing. The C-terminus of DACH1 was necessary for direct p53
binding, contributing to the inhibition of colony formation and cell-cycle arrest. Expression of the stem
cell factor SOX2 was repressed by DACH1, and SOX2 expression was inversely correlated with DACH1
in NSCLC. We conclude that DACH1 binds p53 to inhibit NSCLC cellular growth. Cancer Res; 73(11);
3262–74. 2013 AACR.
Introduction
Lung cancer is the leading cause of cancer mortality worldwide with approximately 160,000 deaths reported each year in
the United States (1). Non–small cell lung cancer (NSCLC)
constitutes approximately 80% of lung tumors and includes
adenocarcinoma and squamous cell carcinoma, which are
histologically distinct from small cell lung cancer (SCLC; ref. 2).
Several oncogenes are important in lung cancer including KRas and EGF receptor (EGFR), which are mutated in 15% and
10% of NSCLCs. Other activating genetic changes include
overexpression of SOX2 and fusion proteins, including ALK
and ROS and other translocations, which induce tyrosine
kinase activity (3–5). Inactivation of tumor suppressors occur,
including the inactivation of p53, in which loss of heterozygosity (LOH) is observed in 60% and mutations arise in 50%
(6, 7).
p53 functions as a transcription factor that senses the
favorability of the local microenvironment, regulating gene
Authors' Affiliations: 1Departments of Cancer Biology, 2Stem Cell Biology
and Regenerative Medicine, 3Kimmel Cancer Center, Thomas Jefferson
University; 4Center for Integrated Bioinformatics; and 5School of Biomedical Engineering, Systems and Health Sciences, Drexel University, Philadelphia, Pennsylvania
Corresponding Authors: Richard G. Pestell, Departments of Cancer
Biology, Kimmel Cancer Center, Thomas Jefferson University, 233 S. 10th
Street, Philadelphia, PA 19107. Phone: 215-503-5692; Fax: 215-503-9334;
E-mail: [email protected]; and Kongming Wu, E-mail:
[email protected]
doi: 10.1158/0008-5472.CAN-12-3191
2013 American Association for Cancer Research.
3262
expression and, thereby, a variety of functions, including
senescence, energy metabolism, DNA repair, cell-cycle
arrest, and apoptosis (8). The function of p53 is mediated
through binding proteins, altered cellular localization, and
posttranslational modification (9). p53 activation in vivo
involves stress-induced stabilization via loss of binding to
its negative regulators, including Mdm2 and Mdmx, with
sequential recruitment of cointegrator complexes. The
mechanisms enhancing p53 function are fundamental to
the biology of p53 and tumor suppression.
The Drosophila Dac gene is a key member of the retinal
determination gene network (RDGN), including eyes absent
(eya), ey, twin of eyeless (toy), teashirt (tsh), and sin oculis (so),
which specify eye tissue identity (10, 11). Initially cloned as a
dominant inhibitor of the hyperactive EGFR, Ellipse, in
Drosophila, the mammalian DACH1 gene regulates gene
expression of target genes, in part, through interacting with
DNA-binding transcription factors (c-Jun, Smads, Six, and
ERa), and in part through intrinsic DNA-sequence specific
binding properties via Forkhead binding sequences (12–15).
Clinical studies have shown a correlation between poor
prognosis breast cancer and reduced DACH1 expression
(15) and loss of DACH1 expression has been observed in
prostate and endometrial cancer (14, 16). Given that Dac was
identified as a dominant inhibitor of Ellipse and the importance of hyperactive EGFR in human lung cancer, we examined the role of DACH1 in lung cancer cellular growth. These
studies identified DACH1 as a novel p53 binding partner that
participates in p53-mediated induction of p21CIP1 and cellcycle arrest.
Cancer Res; 73(11) June 1, 2013
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2013 American Association for Cancer Research.
Published OnlineFirst March 14, 2013; DOI: 10.1158/0008-5472.CAN-12-3191
Dachshund Binds p53
Materials and Methods
Cell culture, plasmid construction, reporter genes,
expression vectors, DNA transfection, and luciferase
assays
Cell culture, DNA transfection, and luciferase assays using
the Rad51-Luc, p21-Luc, and SOX2-Luc reporter genes were
conducted as previously described (17, 18). The H1299,
HEK293T, H460, and HCT116 cells were cultured in Dulbecco's
Modified Eagle Medium (DMEM) supplemented with 10%
fetal calf serum, 1% penicillin, and 1% streptomycin as previously described (15). The expression plasmids encoding an
N-terminal FLAG peptide linked to the DACH1, DACH1 C-terminal domain alone (C-term), or DACH1 C-terminal domain
deleted (DC) were previously described (13–15). The DACH1
C-terminal (C-term) and DACH1 C-terminal deleted (DC) were
subcloned into the MSCV-IRES-GFP retrovirus vector. GFPpositive cells were selected by FACS. Cells were plated at a
density of 1 105 cells in a 24-well plate on the day before
transfection with Superfect according to the manufacturer's
instructions (Qiagen). For reporter gene assays, a dose–
response was determined in each experiment with 50 and
200 ng of expression vector and the promoter reporter plasmids (0.5 mg). Luciferase activity was normalized for transfection efficiency using b-galactosidase reporters as an internal
control. The fold-change effect of expression vector was determined in comparison with the effect of the empty expression
vector cassette, and statistical analyses were carried out using
the t test.
Cell-cycle analysis
Cell-cycle parameters were determined using laser scanning
cytometry. Cells were processed by standard methods using
propidium iodide staining of cellular DNA. Each sample was
analyzed by flow cytometry with a FACScan Flow Cytometer
(Becton-Dickinson Biosciences) using a 488 nm laser. Histograms were analyzed for cell-cycle compartments using ModFit version 2.0 (Verity Software House). A minimum of 20,000
events were collected to maximize statistical validity of the
compartmental analysis. Annexin V staining was conducted as
described previously (19).
Cell proliferation assays
H1299 cells infected with MSCV-IRES-GFP, MSCVDACH1-IRES-GFP, MSCV-DACH1 C-term-IRES-GFP, or MSCVDACH1DC-IRES-GFP were seeded into 96-well plates in normal
growth medium, and cell growth was measured daily by MTT
assays using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide. The tet-inducible p53 cell line H1299 was plated
in growth medium in either the presence or absence of doxycycline (2 mg/mL; ref. 20).
Immunoprecipitation and Western blot
Immunoprecipitation (IP) and Western blot assays were
conducted in H1299, HCT116, and HEK293T cells as indicated.
Cells were pelleted and lysed in buffer (50 mmol/L HEPES, pH
7.2, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1
mmol/L DTT, and 0.1% Tween 20) supplemented with a protease inhibitor cocktail (Roche Diagnostics). Antibodies used for
www.aacrjournals.org
IP and Western blot were: anti-p53 (DO-I) (SC-126), anti-FLAG
(M2 clone, Sigma), and DACH1 (Proteintech, cat:10914-1-AP).
Protein–protein interaction by GST–pull-down assays
In vitro protein–protein interactions were done as described
previously (21). In vitro translated proteins were prepared by
coupled transcription–translation with a TNT-coupled reticulocyte lysate kit (Promega) using plasmid DNA (1.0 mg) in a
total of 50 mL. GST fusion proteins were prepared from E. coli.
In vitro translated protein (15 mL) was added to 5 mg GST fusion
protein of GST as control in 225 mL binding buffer [50 mmol/L
Tris-HCl, 120 mmol/L NaCl, 1 mmol/L DTT, 0.5% NP40, 1
mmol/L EDTA, 2 mg/mL leupeptin, 2 mg/mL aprotinin, 1
mmol/L phenylmethylsulfonyl fluoride (PMSF), and 2 mg/mL
pepstatin] and rotated for 2 hours at 4 C. Glutathione-Sepharose bead slurry (50 mL) was added and the mixture was rotated
for a further 30 minutes at 4 C. Beads were washed 5 times with
washing buffer (1 mL), and binding buffer (30 mL) was added
after the final wash. Sepharose beads were washed 5 times with
lysis buffer and boiled in SDS sample buffer, and the proteins
released were resolved by SDS-PAGE followed by Western blot.
Colony-forming assays
A total of 4.0 103 H1299 cells were plated in triplicate in 3
mL of 0.3% agarose (sea plaque) in complete growth medium in
the presence or absence of 2 mg/mL doxycycline overlaid on a
0.5% agarose base, also in complete growth medium. Two
weeks after incubation, colonies more than 50 mm in diameter
were counted using an Omnicon 3600 image analysis system.
The colonies were visualized after staining with 0.04% crystal
violet in methanol for 1 to 2 hours.
Immunohistochemistry
Immunohistochemical (IHC) analysis of human lung cancer
was conducted using a polyclonal DACH1 antibody from
Proteintech using the same approach as previously described
(15). Human lung cancer tissue arrays were from Biomax.
Microarray, cluster, and chromatin
immunoprecipitation analysis
DNA-free total RNA isolated from H1299 cells expressing
p53 and/or DACH1 were used to probe human OneArray
(Phalanx). RNA quality was determined by gel electrophoresis. Probe synthesis and hybridization were carried out as
described previously (21). Analysis of the arrays was conducted using GeneSpring. Arrays were normalized using robust
multi-array analysis, and the P value of 0.05 was applied as
statistical criteria for differentially expressed genes. These
genes were then grouped using hierarchical clustering with
"complete" agglomeration, and each cluster was further analyzed on the basis of the known function of the genes contained in the cluster. Expression profiles are displayed using
Treeview. Classification and clustering for pathway-level analysis were done by using gene sets Analysis of Sample Set
Enrichment Scores (ASSESS), available online. ASSESS provides a measure of enrichment of each gene set in each sample.
Gene set enrichment was dependent on a concordance of
at least 2 samples within the replicates that was opposite
between phenotypes (15).
Cancer Res; 73(11) June 1, 2013
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2013 American Association for Cancer Research.
3263
Published OnlineFirst March 14, 2013; DOI: 10.1158/0008-5472.CAN-12-3191
Chen et al.
Binding sites for p53 in human lung fibroblasts were
obtained from a chromatin immunoprecipitation sequencing
(ChIP-Seq) analysis of chromatin occupancy by p53 following
activation by 5-fluorouracil (5-FU; ref. 22). High-confidence
ChIP-Seq peaks were identified as described previously (22).
Genes regulated by p53 were identified by mapping ChIP-Seq
peaks to gene coding regions (ChIP-Seq peak was located inside
the transcribed region of the gene or within 20 kb of either end).
Gene identifiers of p53-regulated genes were translated from
gene symbol to ensembl gene identifier for comparison with
DACH1 regulated genes. DACH1 binding sites were identified
from ChIP-Seq of a breast cancer cell line stably expressing
DACH1. ChIP-Seq peaks from DACH1 ChIP-Seq were mapped
to the nearest proximal Ensembl gene identifier. Significant
overlap in p53 and DACH1 regulated genes was tested using the
hypergeometric distribution with all ensembl gene identifiers
in Homo sapiens used as a reference set.
Nude mice study
About 1 106 H1299 cells expressing p53 and/or DACH1
were implanted subcutaneously into 4- to 6-week-old athymic female nude mice purchased from the National Cancer
Institute, NIH. The tumor growth was measured every 5 days
for 32 days by using a digital caliper. Tumor weight was
measured when mice were sacrificed on day 35 after cell
implantation (15).
Results
DACH1 abundance is reduced in NSCLC
IHC staining was conducted to detect DACH1 abundance in
human lung cancer samples using an antibody from Proteintech, according to a previously described protocol (15). Quantitation of the nuclear DACH1 staining among distinct human
NSCLC tumors (n ¼ 97) and in 10 normal control samples
showed an approximately 50% decrease in DACH1 immunopositivity (Fig. 1A). DACH1 abundance was not reduced in
SCLC or carcinoid tumors (Fig. 1A).
In a separate cohort, the abundance of DACH1 was examined and shown to be reduced in cancer tissues (Fig. 1B).
DACH1 levels were decreased further in stage III versus stage I
cancer (Fig. 1B). In normal lung, DACH1 was identified within
the lung epithelial cells (Fig. 1C). Representative examples of
IHC for DACH1 in different subtypes of lung cancer are shown
in Fig. 1D.
In order to examine the mRNA expression of DACH1 in
human lung cancer, Oncomine databases were interrogated
(Supplementary Fig. S1). The relative abundance of DACH1
was reduced 2- to 3-fold in NSCLC, compared with normal
lung, in each of the databases. In contrast, DACH1 mRNA was
increased in carcinoid tumors compared with normal lung
(Supplementary Fig. S1E; n ¼ 144, normal; n ¼ 157, NSCLC
patients).
DACH1 binds to p53 via its carboxyl terminus
In view of the importance of p53 in NSCLC growth (7, 23),
we considered the possibility that DACH1 may associate
with p53. IHC staining was conducted of a tet-inducible p53
3264
Cancer Res; 73(11) June 1, 2013
H1299 NSCLC cell line. The parental H1299 cell line is p53
null due to a homozygous partial deletion of the TP53 gene.
Induction of p53 by tetracycline in the p53 H1299 line
restored p21CIP1 gene expression and cell-cycle arrest (24).
IHC staining of cells induced to express p53, showed nuclear
p53 colocalized with DACH1 (Fig. 2A; Supplementary Fig.
S2). Association of p53 with DACH1 was shown by IPWestern blot analysis (Fig. 2B). IP with an antibody directed
to the DACH1 N-terminal FLAG tag coprecipitated p53.
Similarly, IP with a p53 antibody and subsequent Western
blot for DACH1 evidenced coprecipitated DACH1 (Fig. 2B).
In order to determine whether endogenous p53 associated
with DACH1, a cell line expressing p53 wild-type (wt) and
DACH1 was assessed. DACH1 expression is reduced in
NSCLC and many tumor cell lines. The human colon cancer
HCT116 was found to express p53-wt and DACH1 (Fig. 2C;
Supplementary Fig. S2B). IHC staining showed a colocalization of DACH1 with p53 in a nuclear, extranucleolar distribution (Fig. 2C). IP with an antibody directed toward endogenous p53 coprecipitated DACH1 (Fig. 2D). In view of the
reduced abundance of DACH1 in H1299 NSCLC, we investigated possible mechanisms governing the reduced expression. Cells were treated with the DNA methylase inhibitor 5
azacytidine (AZT) for 96 hours, either alone or with the
addition of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA; Supplementary Fig. S3). DACH1 abundance was induced by the addition of 5 azacytidine, TSA, and
a combination of both, indicating that endogenous DACH1
abundance is reduced in H1299 NSCLC because of DNA
methylation and histone deacetylation.
In order to determine whether DACH1 bound directly to p53,
the DACH1 protein was purified as a 6-His-GST protein and
p53 was expressed from in vitro translation (IVT). GST pulldown was conducted upon coincubation of DACH1 with p53.
Comparison was made between the binding of DACH1-wt and
the DACH1 DBD (183–293). p53-bound 6-His DACH1 (Fig. 2E),
but failed to bind the DACH1 DBD. The binding of DACH1-wt
to p53 was greater despite the significantly lower abundance of
6-His DACH1-wt compared with DACH1 DBD by Western
blotting to the 6-His epitope (Fig. 2E).
To identify the domains of DACH1 required for binding to
p53, a series of DACH1 mutant expression vectors were transfected into HEK293T cells (Fig. 2F). p53 localized with DACH1
in the nucleus of HEK293T cells (Fig. 2G). The N-terminal FLAG
epitope was used to immunoprecipitate DACH1. Transfection
of HEK293T cells with a series of DACH1 mutants showed that
DACH1 bound to p53 and that the DACH1 C-terminus was
sufficient for binding to p53 (Fig. 2H). Deletion of the DACH1 Cterminus abolished binding to p53 (Fig. 2H). These studies
indicated that the C-terminus of DACH1 is required for binding
to p53.
The DACH1 C-terminus is required for the enhancement
of p53-mediated inhibition of contact-independent
growth and cell-cycle arrest
In order to determine the functional significance of
DACH1 binding to p53 via its carboxyl terminus, colonyforming assays and cell proliferation studies were conducted.
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2013 American Association for Cancer Research.
Published OnlineFirst March 14, 2013; DOI: 10.1158/0008-5472.CAN-12-3191
Dachshund Binds p53
0.4
LUNG * P < 0.05 ** P > 0.05
0.35
0.3
**
**
0.25
0.2
0.15
*
*
*
*
0.1
0.05
0
0) 8) 1) (4) (4) (6) (5)
l (1 o (4 us (4 cell lar cell oid
a
rm den o ge veo all cin
No A uam Lar loal Sm Car
io
Sq
ch
on
r
B
C
B
0.3
DACH1 expression
DACH1 expression
A
0.25
NSCLC * P < 0.05 ** P < 0.05
0.2
0.15
*
0.1
*
**
*
0.05
0
)
3)
2)
7)
(10
,3
,4
,2
al
1
0
0
M
M
0–
m
r
M
N0
N1
2
No
2
2
N 1–
1–
1–
4
I (T
I (T
I
2–
T
(
e
g
ge
III
Sta
Sta tage
S
Normal tissue
D
Lung cancer tissue
Adeno
Squamous
Bronchioloalveolar
Large cell
Small cell
Carcinoid
Figure 1. Reduced DACH1 abundance in non–small cell lung cancer (NSCLC). A and B, immunohistochemical staining for DACH1 in lung cancer samples
was quantitated for each subtype of lung cancer and the data is shown as mean SEM for N as indicated in the figure in parentheses. C and D, representative
examples of immunohistochemical staining for DACH1 in each of the histologically distinct types of lung cancer as indicated.
Either DACH1-wt, DACH1DC, or the DACH1 C-terminus
were stably introduced into the tet-inducible p53 H1299
cell line. Tetracycline was used to induce p53. p53 expres-
www.aacrjournals.org
sion inhibited colony formation, reducing both the number
and size of colonies (Fig. 3A). DACH1-wt expression inhibited colony formation to a similar level as p53 (Fig. 3B).
Cancer Res; 73(11) June 1, 2013
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2013 American Association for Cancer Research.
3265
Published OnlineFirst March 14, 2013; DOI: 10.1158/0008-5472.CAN-12-3191
Chen et al.
A
B
H1299 cells
DACH1
Lysates
p53
Merge
FLAG IP
p53 IP
H1299
-
+
-
+
-
+
p53
+
+
+
+
+
+
DACH1
α-DACH1
DAPI
α-p53
Bar 10 μm
D
DAPI
DACH1
Merge
Vector
p53
DACH1
HCT116 cells (p53 endogenous)
Lysates
p53 IP
DACH1
Vector
HCT116 cells
C
α-DACH1
α-p53
Bar 10 μm
F
E
WT
-
Supernatant
DBD
WT
DBD
GST- HIS IP GST
-
3xFLAG tag
DNA Binding Domain
Trans-repression Domain
DACH1
DACH1
α-HIS (DACH1)
1
α-p53
183 293
α-HIS (DACH1-DBD)
G
p53
binding
H
Lysates
565 706
+
ΔDBD
ΔC
+
C-term +
FLAG IP IgG
293T cells
+ + + + +
Vector
WT
ΔDBD
ΔC
C-term
WT
p53
WT
ΔDBD
ΔC
C-term
Vector
293T
DACH1
Figure 2. DACH1 binds p53 via the
carboxyl terminus. Confocal
microcopy for DACH1 and p53
in the NSCLC lines H1299 cells (A)
and HCT116 cells (C). B,
immunoprecipitation for either
DACH1 (FLAG) or p53 and
sequential Western blotting for
either p53 or DACH1 in H1299
cells. D, IP-Western blotting in
HCT116 cells. IP was conducted
with a p53 antibody to detect
endogenous p53. E, GST
pull-down conducted with 6-His
DACH1 proteins (either wt or DBD
encoding AA 183-293) and IVTp53. The relative abundance of
pull-down is shown on the left
and remaining protein shown in
the supernatant (right). F,
schematic representation of
DACH1 expression vectors with
relative binding to p53 as
indicated. G, confocal microscopy
of HEK293T cells transfected with
expression vectors for p53 and
DACH1, showing colocalization
of p53 and DACH1 in the nucleus.
H, immunoprecipitation Western
blot of HEK293T cells transduced
with expression vectors encoding
either DACH1, DACH1 mutants,
or p53.
DACH1
+ + + + + + p53
α-FLAG (DACH1)
Merge
DAPI
α-p53
α-FLAG (DACH1)
Bar 10 μm
The DACH1 C-terminus was required for inhibition of colony
formation in the presence of p53 (Fig. 3A). DACH1 enhanced p53-dependent inhibition of colony size and number
(Fig. 3B–E). The DACH1 C-terminus was sufficient to
enhance a p53-mediated inhibition of colony formation. In
the presence of p53, the expression of DACH1DC was defective in reducing colony formation, compared with DACH1-wt
(Fig. 3C), despite a relatively greater abundance of the DC
protein expressed, based on Western blotting (data not
shown).
3266
Cancer Res; 73(11) June 1, 2013
DACH1 inhibits cell proliferation and enhances p53dependent cell-cycle arrest
In order to determine the mechanism by which DACH1
enhanced p53-mediated inhibition of colony formation, we
assessed cell proliferation, cell-cycle changes, and cellular
apoptosis. p53 expression inhibited H1299 cell proliferation
assessed using the MTT assay (Fig. 3F). DACH1 expression
inhibited cell proliferation and enhanced p53-mediated inhibition of cell proliferation (Fig. 3F). The p53 binding-defective
DACH1 mutant (DACH1DC) exhibited reduced p53-dependent
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2013 American Association for Cancer Research.
Published OnlineFirst March 14, 2013; DOI: 10.1158/0008-5472.CAN-12-3191
Dachshund Binds p53
A
H1299 cells
Vector
DACH1-ΔC
DACH1
DACH1-C term
p53 (-)
p53 (+)
250
Colony number
P < 0.01
300
200
100
200
150
100
50
0
p53 DACH1 -
+
-
+
+
+
F
0
p53
DACH1
ΔC
C-term
+
+
-
+
+
-
+
+
G
Fold change
10
P < 0.001
8
6
4
2
0
p53
DACH1
-
+
-
+
+
+
Day 4
inhibition of cell proliferation (Fig. 3G). In H460 cells with
endogenous expression of p53, DACH1 inhibited cellular proliferation. However, the inhibition of cell proliferation by
DACH1 was abolished when p53 expression was knocked down
by p53 shRNA (Fig. 3H). The C-terminal deletion mutant of
DACH1 failed to repress cell proliferation either in the presence
or absence of p53 (Fig. 3I).
The effect of DACH1 on p53-dependent cell-cycle function
was determined by fluorescence-activated cell sorting
(FACS) analysis. Induction of p53 increased the proportion
of cells in G0–G1 (Fig. 4A). DACH1 expression enhanced p53-
E
H1299
300
200
P < 0.01
200
100
0
p53
DACH1
-
+
-
+
+
+
H
H1299 (MTT)
Repression rate (100%)
12
www.aacrjournals.org
P < 0.01 P < 0.01
400
1.4
1.2
1
0.8
0.6
7
4
3
0.2
1
+
+
P < 0.01
5
2
+
+
Day 4
120
80
40
0
p53 +
DACH1 +
ΔC C-term -
H460 (MTT)
6
0.4
0
p53 +
DACH1 +
ΔC C-term -
P < 0.01
P < 0.01
+
+
+
+
-
I
H1299 (MTT)
P < 0.01 P < 0.01
H1299
160
Repression rate (100%)
400
D
H1299
Colony size (10-5pixel2)
C
H1299
Colony size (10-5pixel2)
500
Fold change
B
Colony number
Figure 3. DACH1 inhibition of
NSCLC colony formation and cell
proliferation requires the DACH1
p53-binding domain. A, H1299
colony-forming assays comparing
DACH1-wt and DACH1 mutants are
shown. B–E, colony size and number
are shown as mean SEM for n > 5
separate experiments using stably
transduced cell lines. DACH1
expression vectors were stably
transduced into tet-inducible p53
H1299 cells. Colony number (B and
C) and colony size (D and E), are
shown as mean SEM for n > 5
separate experiments. F–I, H1299
cells and H460 cells were assessed
for cellular proliferation using the
MTT assay.
0
DACH1 -
+
Vector
-
+
shp53
Day 4
1.2
H460 (MTT)
P < 0.01
1
0.8
0.6
0.4
0.2
0
DACH1
ΔC
- +
+ -
- +
+ -
Vector
shp53
Day 4
dependent induction of G0–G1 but deletion of the DACH1
C-terminus abolished this effect (Fig. 4A). In the absence of
p53, DACH1 had no effect on S phase but, in the presence
of p53, it was associated with an inhibition of S phase. The
induction of p53 reduced the proportion of cells in the S
phase of the cell cycle at 24 hours (Fig. 4B). DACH1 expression in the presence of p53 enhanced the inhibition of
S phase. Deletion of the DACH1 C-terminus abolished the
p53-dependent inhibition of S phase (Fig. 4B). These findings
suggest that DACH1 and p53 both inhibit S-phase cell-cycle
progression and that DACH1 expression with p53 enhanced
Cancer Res; 73(11) June 1, 2013
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2013 American Association for Cancer Research.
3267
Published OnlineFirst March 14, 2013; DOI: 10.1158/0008-5472.CAN-12-3191
Chen et al.
A
B
H1299 cells
H1299 cells
100
90
80
70
60
50
40
30
20
10
0
DACH1
-
+
-
-
+
-
0
DACH1
-
+
-
-
DACH1-ΔC
-
-
+
-
-
+
DACH1-ΔC
-
-
+
-
25
P < 0.01
S (%)
G0–G1 (%)
20
P < 0.01
10
5
0h
24 h
Tet
0h
24 h
p53
-
+
p53
-
+
D
H1299 cells
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
+
H1299 cells
Early apoptosis (%)
P < 0.01
8
7
6
5
4
3
2
1
0
Annexin V/ 7-AAD
P < 0.01
DACH1
-
+
-
-
+
-
DACH1
-
+
-
-
+
-
DACH1-ΔC
-
-
+
-
-
+
DACH1-ΔC
-
-
+
-
-
+
Tet
0h
24 h
Tet
0h
24 h
p53
-
+
p53
-
+
E
F
H460 cells
25
P < 0.01
S (%)
20
15
10
5
0
DACH1
ΔC
-
+
-
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
DACH1
ΔC
+
Vector
-
+
-
+
shp53
a G0–G1 cell-cycle accumulation, requiring the DACH1 carboxyl terminus.
p53 induces apoptosis in H1299 cells when assessed by the
sub-G1 fraction of the cell cycle by FACS or by Annexin V
staining (25). Expression of p53 increased the proportion of
cells in the sub-G1 fraction at 24 hours, which was enhanced by
Cancer Res; 73(11) June 1, 2013
Figure 4. DACH1 inhibition of S
phase and induction of apoptosis
in NSCLC is enhanced in the
presence of p53, requiring the
DACH1 C-terminus. A–D, H1299
cells stably expressing tetinducible p53 were transduced
with a retroviral expression vector
encoding DACH1-wt or mutants
and treated with tetracycline. Cells
were analyzed for cell cycle by
FACS (A and B) and apoptosis by
FACS or Annexin V staining (C and
D). Cells were analyzed at 0 and 24
hours after p53 induction. Data are
mean SEM for n > 5 separate
experiments. E and F, H460 cells
expressing DACH1 or the
DACH1DC mutant were assessed
in the presence of p53 shRNA for
S-phase distribution (E) or Annexin
V staining (F). Data are mean SEM for n > 5 separate
experiments.
H460 cells
Early apoptosis (%)
30
3268
+
-
Tet
C
Sub-G1 (%)
15
Annexin V/ 7-AAD
P < 0.01
-
+
-
+
Vector
-
+
-
+
shp53
DACH1 (Fig. 4C). Deletion of the DACH1 C-terminus abolished
DACH1-dependent enhancement of p53 apoptosis (Fig. 4C). A
similar observation was made when apoptosis was assessed
by Annexin V staining (Fig. 4D). In the distinct H460 NSCLC
line that expresses p53, DACH1 inhibited S phase, requiring
the DACH1 C-terminus. p53 short hairpin RNA (shRNA)
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2013 American Association for Cancer Research.
Published OnlineFirst March 14, 2013; DOI: 10.1158/0008-5472.CAN-12-3191
Dachshund Binds p53
abolished DACH1 inhibition of S phase showing the requirement of p53 for DACH1–cell-cycle inhibition (Fig. 4E). Similarly,
DACH1 induction of apoptosis assessed by Annexin V staining
required the DACH1 C-terminus and was abolished by p53
shRNA (Fig. 4F).
DACH1 inhibition of Rad51 and induction of p21CIP1
requires endogenous p53
We next examined the functional significance of DACH1 on
p53-dependent gene expression using the tet-H1299 cells and
the known p53 target genes, p21CIP1 and Rad51. An analysis of
gene expression was conducted upon induction of either p53,
DACH1, or the combination of both. The expression of p53
induced (Fig. 5A, in red) and repressed (Fig. 5A, in green)
clusters of genes (representative example Fig. 5A). DACH1
expression augmented p53-mediated induction of gene clusters
(Genes 1; Fig. 5A) and augmented repression of p53-repressed
genes (Genes 2; Fig. 5A). Thus, the combination of DACH1 and
p53 expression augmented expression of common target genes
shown. p21CIPI is an important target of p53-mediated cell-cycle
arrest (26). Induction of DACH1 increased p21CIP1 abundance
approximately 3-fold (Fig. 5B). Expression of DACH1 in the
presence of p53 enhanced p21CIP1 induction by p53 (Fig. 5B).
The expression of p53 enhanced p21CIP1 promoter activity,
which was augmented by the expression of DACH1, but not
DACH1DC (Fig. 5C). Expression of the DACH1-binding defective mutant p53-R248Q did not enhance p21CIP1 transcriptional
activity and DACH1 did not affect p21CIP1 promoter activity in
the presence of p53-R248Q, suggesting the effect of DACH1 on
p21CIP1 requires p53 association (Fig. 5D). DACH1 induced p53
activity in cells. In order to determine the mechanism by which
DACH1 induced p21CIP1 and reduced Rad51 abundance, we
examined the ability of DACH1 to regulate transcription using
the promoter-regulatory regions of these 2 target genes
assessed in luciferase reporter assays. DACH1 expression inhibited Rad51 promoter reporter activity in the presence of p53
(Fig. 5E). In addition, the DACH1 DBD mutant repressed Rad-51
Luc reporter activity (Fig. 5F). DACH1DC failed to repress Rad51
reporter activity (Fig. 5G). As DACH1 bound p53 and induced
p53 target expression, we considered the possibility that
DACH1 may enhance p53-dependent transcriptional activity.
We deployed a synthetic multimeric canonical p53 response
element reporter gene (Fig. 5H). In order to consider the
possibility that DACH1 and p53 may bind to common genes
in the context of chromatin, sets of genes binding p53 in IMR90
lung fibroblasts in ChIP Seq (22) were compared with genes
binding DACH1, in ChIP Seq (22). A total of 743 binding sites for
p53 and 1844 binding sites for DACH1 were identified. 112 genes
regulated by both DACH1 and p53 were identified (P < 1E16;
Supplementary Table S1). Collectively, these studies show that
DACH1 directly associates with the p53 tumor suppressor to
enhance p53-dependent apoptosis associated with increased
transcriptional activity at the p21CIP1 target gene.
DACH1 enhances p53-dependent suppression of NSCLC
tumor growth in vivo
In order to determine the significance of DACH1 inhibition
of NSCLC in vivo, H1299 cells were stably transduced with an
www.aacrjournals.org
expression vector encoding DACH1 and/or p53. Tumors in
nude mice were measured every 5 days, with terminal analysis
at day 32 (Fig. 6). Tumor weight was reduced by DACH1 or p53
and DACH1 expression further augmented p53-mediated
growth suppression (Fig. 6A and B). Expression of either
DACH1 or p53 inhibited tumor growth and DACH1 enhanced
p53-dependent tumor suppression (Fig. 6C and D). Immunohistochemistry showed the induction of DACH1 and p53 by the
inducible systems; however, DACH1 did not enhance p53
abundance. DACH1 expression enhanced p53-mediated induction of p21CIP1, and enhanced p53-mediated repression of
RAD51 and Ki67 (Fig. 6E–J).
DACH1 restrains SOX2 expression and transcription in
NSCLC
In order to further examine the potential mechanisms by
which DACH1 inhibited NSCLC, we considered SOX2 as an
additional target. We examined expression profiles of DACH1,
SOX2, and p53 in NSCLC (n ¼ 351) by creating a NSCLC metadatabase, as previously described, for breast cancer (21). A
comparison was made with expression in normal lung (n ¼ 74).
DACH1 expression was reduced in NSCLC (Fig. 7A and C)
compared with normal lung and SCLC (n ¼ 38). Reduced
DACH1 expression correlated with increased SOX2 in NSCLC.
Low DACH1 expression was found in NSCLC of all stages (Fig.
7B). In contrast DACH1 was increased in SCLC (Fig. 7A and B).
Reduced DACH1 correlated with increased EGFR expression in
a subset NSCLC patient samples (Supplementary Fig. S4).
Patients with Low DACH1 and high EGFR predicted an
approximately 1-year worse prognosis (Supplementary Fig.
S4E), compared with other tumors. In order to examine the
mechanism by which DACH1 may repress SOX2 expression, we
conducted transcriptional analysis using the SOX2 promoter
linked to a luciferase reporter gene in H1299 NSCLC cell lines.
DACH1 repressed SOX2, requiring the DACH1 C-terminus (Fig.
7D). p53 repressed SOX2 and DACH1 enhanced p53 repression
(Fig. 7D).
Discussion
The current studies provide evidence that the cell-fate–
determination factor DACH1 directly binds and enhances
several functions of p53 in NSCLC. The abundance of DACH1
was reduced in NSCLC, correlating with clinical stage. Reduced
DACH1 abundance was shown in NSCLC, as measured by both
RNA and protein abundance. Prior studies of DACH1 had
shown reduced abundance of DACH1 in human malignancies,
including breast cancer, prostate cancer, and endometrial
cancer (14–16). We examined expression profiles of DACH1
and p53 in NSCLC by creating a NSCLC meta-database,
as previously described, for breast cancer (21). A comparison
was made with expression in normal lung. DACH1 expression
was reduced in NSCLC (Fig. 7), confirming oncomine analysis
(Supplementary Fig. S1). The current studies extend the spectrum of human tumors in which DACH1 expression is reduced.
The molecular mechanisms by which DACH1 inhibits
contact-independent growth are poorly understood. The
current studies show the importance of p53 in DACH1mediated inhibition of NSCLC tumor formation in vivo and
Cancer Res; 73(11) June 1, 2013
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2013 American Association for Cancer Research.
3269
Published OnlineFirst March 14, 2013; DOI: 10.1158/0008-5472.CAN-12-3191
Chen et al.
A
B
H1299 cells (p53 signal pathway )
Genes 1
H1299 cells
CDKN1A
MDM2
RRM2B
NOXA1
TP53TG1
PID1
THBS1
TGFA
CLCA2
TNFRSF10B
PLD1
FAS
GADD45A
TP53INP2
PIK3R1
PMAIP1
PERP
SERPINE2
TGFBI
CASP1
NRG1
IGF1
BRCA1
CHEK1
TOPBP1
CDK2
CDC25A
SNAI1
PLK1
CDC25C
CDC45
TOP2A
ECT2
E2F1
FOXM1
RAD51
MCM7
UHRF1
SUV39H1
BIRC5
Genes 2
+ - - + - DACH1
- + - - + DACH1-ΔC
- - + + + p53
-
α-FLAG
(DACH1)
Vector
α-p53
DACH1
P < 0.05
α-Rad51
p53
α-p21 (L.E.)
α-p21 (S.E.)
DACH1/ p53
α-Vinculin
-2.9
C
0
D
E
p21-Luc
P < 0.01
2
H1299-Tet p53+/+
1
15
10
0.8
0.6
0.4
0.2
+
-
+
+
+
+
-
-
-
-
+
+
0
p53
p53-R248Q
DACH1
- + - - +
- - -
G
1.4
3.5
H1299-Tet p53+/+
1.2
Fold induction
1
0.8
0.6
0.4
1
0.8
0.6
0.2
0
p53
+
ΔC
-
-
+
+
293T
3
2.5
P < 0.01
2
1.5
0.5
0
p53
ΔDBD
+
+
1
0.4
0.2
+
+
p53RE-luc
-1479
H1299-Tet p53+/+
+
+
DACH1 -
H
RAD51-Luc
-1479
0
p53
- + - - +
+ + +
Fold induction
-
RAD51-Luc
Fold induction
P < 0.01
5
1
1.2
1.2
H1299
Fold induction
Fold induction
Fold induction
H1299
P < 0.01
F
-1479
-2400
20
3
0
p53
DACH1
ΔC
ΔDBD
RAD51-Luc
p21-Luc
-2400
4
2.7
0
+
+
+
DACH1
C-term
ΔC
ΔDBD
-
+
-
+
-
+
-
+
CIP1
. Microarray-based gene expression analysis (A), Western
Figure 5. DACH1 enhances p53-dependent transcriptional regulation of Rad51 and p21
blot (B), and Luciferase reporter assays (C–H) were conducted in H1299 cells stably expressing p53 and DACH1 wt. In the microarray analysis,
expression direction is color coded (red, increased; blue, decreased). Genes 1 represent a cluster of genes that are induced by p53 and that are
further enhanced in their expression through expression of DACH1. Genes 2 is a cluster of genes that are repressed by p53 in which repression
is enhanced by expression of DACH1. B, Western blot analysis of cells that were transduced with the DACH1-expressing retrovirus and p53
abundance was induced by tetracycline. Western blot was conducted with antibodies as indicated, with longer exposure (L.E.) or shorter exposure
(S.E.) shown. C–H, in the reporter assays, DACH1, DACH1 DDBD, or the DACH1DC mutant were compared using either the Rad51 or
p21CIP1 promoter-luciferase reporters. The relative induction of luciferase activity was normalized to b-galactosidase and shown as fold change. Data
are mean SEM for n 5 separate transfections. Activity of the multimetric canonical p53 response element luciferase reporter gene was assessed
in transient expression studies after cotransfection with either DACH1-wt or the DACH1 mutants. Data are mean SEM for n > 5 separate
transfections.
3270
Cancer Res; 73(11) June 1, 2013
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2013 American Association for Cancer Research.
Published OnlineFirst March 14, 2013; DOI: 10.1158/0008-5472.CAN-12-3191
Dachshund Binds p53
P < 0.05
600
P < 0.05
400
200
0
DACH1
-
+
Vector
E
Vector
-
+
p53
1.2
P < 0.05
1
0.8
P < 0.05
0.6
0.4
0.2
0
DACH1
D
H1299 Tumor
Tumor size (fold change)
800
C
H1299 Tumor
Vector
1,600
DACH1
Vector/ p53
1,200
DACH1/ p53
800
400
0
- +
Vector
DACH1
- +
p53
p53
7
1.4
H1299 Tumor (day 32)
P < 0.05
1.2
P < 0.05
1
0.8
0.6
0.4
0.2
0
DACH1
12 17 22 27 32
Days
DACH1/p53
100
80
60
40
20
0
DACH1
p53
+
p53
DACH1
G
-
+
-
+
+
+
+
-
+
+
+
p53
Fold change
100
80
60
40
20
0
DACH1
p53
α-p53
Fold change
H
α-p21
* P < 0.01
6
*
4
2
1.4
1.2
1
0.8
0.6
0.4
0.2
0
DACH1
p53
+
RAD51
*
Fold change
I
p21
8
0
DACH1
p53
1.4
1.2
1
0.8
0.6
0.4
0.2
0
DACH1
p53
+
+
+
P < 0.01
*
Ki67
+
-
+
+
+
* P < 0.01
Fold change
J
α-Ki67
-
+
F
α-DACH1
α-RAD51
-
Vector
Fold change
P < 0.05
1,000
1.4
P < 0.05
B
H1299 Tumor
Tumor size (mm3)
Tumor weight (mg)
1,200
Tumor weight (fold change)
A
*
-
+
-
+
+
+
Figure 6. DACH1 enhances p53-dependent NSCLC growth suppression in mice. Serial measurements were conducted every 5 days of H1299 tumors
stably expressing either DACH1 and/or p53, injected into nude mice. The data are SEM for n ¼ 12 separate tumors for each group (total of 48 tumors
analyzed). The data are mean for tumor terminal weight, raw data (A), tumor weight fold change compared with vector control (B), tumor size (C), and
tumor size measured as fold change versus vector (D; data are shown as mean SEM). E–J, immunohistochemical analysis of extirpated tumors. Data
for immunohistochemical analysis are quantitated as mean SEM for n ¼ 4 tumors in each group.
www.aacrjournals.org
Cancer Res; 73(11) June 1, 2013
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2013 American Association for Cancer Research.
3271
Published OnlineFirst March 14, 2013; DOI: 10.1158/0008-5472.CAN-12-3191
Chen et al.
A
SOX2 versus DACH1
expression fold changes from the sample means
annotated with pathology information
2
ADC
LCC
SCC
SCLC
1.8
1.6
SOX2 fold change
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
B
0.5
1
DACH1 fold change
1.5
Figure 7. DACH1 constrains SOX2
expression. A, gene expression
values for DACH1 and SOX2 in
subjects with NSCLC (n ¼ 251) and
SCLC (n ¼ 38) divided by the
sample means. The tumor types
are shown as adenocarcinomas
(ADC), large cell carcinomas (LCC),
squamous cell carcinomas (SCC),
and small-cell lung cancer (SCLC).
B, SOX2 versus DACH1
expression in NSCLC and SCLC
patients divided by the sample
means annotated with cancer
stage information. C, correlation
between SOX2 and DACH1
expression by tumor types,
showing positive correlation
between reduced DACH1 and
increased SOX2 abundance. D,
SOX2 promoter luciferase reporter
assays conducted in H1299 cells.
H1299 cells were transfected with
SOX2-Luc and expression vectors
encoding DACH1 or DACH1
mutants. Data are mean SEM of
n > 5 separate transfection.
2
SOX2 versus DACH1
expression fold changes from the sample means
annotated with stage information
2
1.8
SOX2 fold change
1.6
1.4
1.2
1
IA
IB
IIA
IIB
IIIA
IIIB
IV
SCLC
0.8
0.6
0.4
0.2
0
0
0.5
1
DACH1 fold change
1.5
C
Lung cancer
2
D
Correlation
1.4
P
Sox2-Luc
–422
H1299
Adeno
0.35
Large cell
0.56
3.65E-5
0.0043
Squamous
0.23
0.022
Small cell
–0.16
0.34
Fold induction
1.2
1
0.8
0.4
0.2
0
p53
DACH1
ΔC
3272
Cancer Res; 73(11) June 1, 2013
P < 0.01
0.6
-
+
-
+
-
+
+
-
+
+
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2013 American Association for Cancer Research.
Published OnlineFirst March 14, 2013; DOI: 10.1158/0008-5472.CAN-12-3191
Dachshund Binds p53
the inhibition of colony number and size. DACH1 bound
p53 to inhibit NSCLC cellular proliferation and colony
formation. The carboxyl terminus of DACH1 was required
for p53 binding, and was required for p53-dependent inhibition of colony formation and cellular proliferation.
DACH1 inhibited S phase requiring the presence of p53.
DACH1-induced p21CIP1 and inhibited Rad51, requiring the
p53 binding region of DACH1. Furthermore, DACH1 expression enhanced p53 signaling via a canonical p53 binding
site. Collectively, these studies are consistent with a model
in which DACH1 functions as an accessory tumor suppressor in the p53 pathway.
Recent studies showed the ability of DACH1 to inhibit the
expression of tumor-initiating cells (18, 27). Genome-wide
expression analysis of mammary tumors showed DACH1
expression repressed a gene signature associated with stem
cells, including Sox2, Nanog, and Klf4 (18). As SOX2 amplification is a feature of NSCLC, we examined the possibility that
DACH1 may repress SOX2, contributing to NSCLC tumor
suppression. SOX2 is an amplified lineage-survival oncogene
in lung squamous cell carcinoma (3) that is oncogenic in the
lung (28), with mutations identified in both NSCLC and SCLC.
SOX2 expression is essential for lung development, can reprogram cells to pluripotency, and collaborates with FoxG1 in
generating self-renewing neural precursor cells (29). DACH1
binds the SOX2 gene promoter in ChIP-Seq and inhibits SOX2
expression in breast cancer cells (18). DACH1 binds FOXO
binding sites, competing with endogenous FOXO proteins in
ChIP assays and antagonized FOXO signaling (12). Thus,
DACH1 inhibits 2 key pathways promoting stem cell renewal.
DACH1 and SOX2 expression were inversely correlated in
NSCLC but not SCLC and DACH1 repressed SOX2 via the
promoter of SOX2. The DACH1-mediated repression of SOX2
was p53-dependent. SOX2 contributes to the expansion of
several stem cell types. It has been proposed that a cancer
stem or progenitor cell contributes to the onset of NSCLC (30).
The repression of SOX2 by DACH1 may contribute a mechanism to inhibit NSCLC.
The loss of normal stem cells is largely due to p53-p21CIPI–
mediated proliferative arrest, senescence, and apoptotic elimination (31, 32). The diminished stem cell function observed in
certain strains of mice encoding germ line alleles of mutant p53
(the gene encoding p53, also known as Trp53; refs. 33, 34), and
the diminished stem cell pool in Dach1/ mice (27), is
consistent with the possibility that DACH1 and p53 may also
cooperate in stem cell function. It is known that p53 deletion
improves stem cell function in mice, in particular with animals
with short dysfunctional telomeres (35, 36). p53 may reduce
stem cell number through regulating the polarity of cell
division, with recent studies showing that loss of p53 favors
symmetric division of cancer stem cells (37). DACH1 has been
shown to regulate the polarity of mammary epithelial cells in
3D Matrigel (15). It is possible that DACH1 and p53 may
regulate stem cell signaling modules although common gene
pathways, as ChIP-Seq analysis of p53 binding sites in IMR90
cells (37) compared with DACH1 binding sites, indicated
approximately 15% of p53 binding sites also bound DACH1.
The functional significance of p53 in DACH1-dependent regulation of cell polarity remains to be determined, but may
provide an additional mechanism for coordinating progenitor
cell expansion.
Disclosure of Potential Conflicts of Interest
R.G. Pestell holds minor (<$10,000) ownership interests in and serves as the
CSO/Founder of the biopharmaceutical companies ProstaGene, LLC and AAA
Phoenix, Inc. In addition, R.G. Pestell holds ownership interests (value unknown)
for several submitted patent applications. No potential conflicts of interest were
disclosed by the other authors.
Disclaimer
The Pennsylvania Department of Health disclaims responsibility for any
analysis, interpretations, or conclusions.
Authors' Contributions
Conception and design: K. Chen, K. Wu, M.P. Lisanti, R.G. Pestell
Development of methodology: K. Chen, K. Wu, H. Rui, C. Wang, S.B. McMahon
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): K. Chen, S. Cai, J. Wang, H. Rui, S.B. McMahon
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): K. Chen, K. Wu, A. Ertel, H. Rui, A. Quong, A. Tozeren,
C. Tanes, S. Addya, M. Gormley, C. Wang, R.G. Pestell
Writing, review, and/or revision of the manuscript: K. Chen, A. Ertel, H. Rui,
A. Quong, M.P. Lisanti, A. Tozeren, C. Tanes, M. Gormley, R.G. Pestell
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Chen, W. Zhang, J. Zhou, Z. Li
Study supervision: K. Chen, K. Wu, Z. Li, R.G. Pestell
Acknowledgments
The authors thank Jeannine Nicole Moore for preparation of this manuscript.
Grant Support
This work was supported in part by grants no. R01CA70896, R01CA132115,
R01CA75503, R01CA86072, (R.G. Pestell), the Kimmel Cancer Center NIH
Cancer Center Core grant P30CA56036 (R.G. Pestell), a generous grant from
the Dr. Ralph and Marian C. Falk Medical Research Trust (R.G. Pestell), a
grant from the Pennsylvania Department of Health (R.G. Pestell), Margaret
Q. Landenberger Research Foundation, and the Department of Defense
Concept Award W81XWH-11-1-0303 (K. Wu). M.P. Lisanti and his laboratory
were supported via the resources of Thomas Jefferson University.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received August 10, 2012; revised February 1, 2013; accepted February 1, 2013;
published OnlineFirst March 14, 2013.
References
1.
2.
3.
Jemal A, Tiwari RC, Murray T, Ghafoor A, Samuels A, Ward E, et al.
Cancer statistics, 2004. CA Cancer J Clin 2004;54:8–29.
Travis WD. Pathology of lung cancer. Clin Chest Med 2002;23:65–81.
viii
Bass AJ, Watanabe H, Mermel CH, Yu S, Perner S, Verhaak RG,
et al. SOX2 is an amplified lineage-survival oncogene in lung
and esophageal squamous cell carcinomas. Nat Genet 2009;41:
1238–42.
www.aacrjournals.org
4.
5.
6.
Kwak EL, Bang YJ, Camidge DR, Shaw AT, Solomon B, Maki RG, et al.
Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.
N Engl J Med 2010;363:1693–703.
Takeuchi K, Soda M, Togashi Y, Suzuki R, Sakata S, Hatano S, et al.
RET, ROS1 and ALK fusions in lung cancer. Nat Med 2012;18:378–81.
Takahashi T, Nau MM, Chiba I, Birrer MJ, Rosenberg RK, Vinocour M,
et al. p53: a frequent target for genetic abnormalities in lung cancer.
Science 1989;246:491–4.
Cancer Res; 73(11) June 1, 2013
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2013 American Association for Cancer Research.
3273
Published OnlineFirst March 14, 2013; DOI: 10.1158/0008-5472.CAN-12-3191
Chen et al.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
3274
Toyooka S, Tsuda T, Gazdar AF. The TP53 gene, tobacco exposure,
and lung cancer. Hum Mutat 2003;21:229–39.
Ko LJ, Prives C. p53: puzzle and paradigm. Genes Dev 1996;10:
1054–72.
Meek DW, Anderson CW. Posttranslational modification of p53: cooperative integrators of function. Cold Spring Harb Perspect Biol 2009;1:
a000950.
Popov VM, Wu K, Zhou J, Powell MJ, Mardon G, Wang C, et al. The
Dachshund gene in development and hormone-responsive tumorigenesis. Trends Endocrinol Metab 2010;21:41–9.
Jemc J, Rebay I. Targeting Drosophila eye development. Genome Biol
2006;7:226.
Zhou J, Wang C, Wang Z, Dampier W, Wu K, Casimiro MC, et al.
Attenuation of Forkhead signaling by the retinal determination factor
DACH1. Proc Natl Acad Sci U S A 2010;107:6864–9.
Popov VM, Zhou J, Shirley LA, Quong J, Yeow WS, Wright JA, et al. The
cell fate determination factor DACH1 is expressed in estrogen receptor-alpha-positive breast cancer and represses estrogen receptoralpha signaling. Cancer Res 2009;69:5752–60.
Wu K, Katiyar S, Witkiewicz A, Li A, McCue P, Song LN, et al. The cell
fate determination factor dachshund inhibits androgen receptor
signaling and prostate cancer cellular growth. Cancer Res 2009;69:
3347–55.
Wu K, Li A, Rao M, Liu M, Dailey V, Yang Y, et al. DACH1 is a cell fate
determination factor that inhibits Cyclin D1 and breast tumor growth.
Mol Cell Biol 2006;26:7116–29.
Nan F, Lu Q, Zhou J, Cheng L, Popov VM, Wei S, et al. Altered
expression of DACH1 and cyclin D1 in endometrial cancer. Cancer
Biol Ther 2009;8:1534–9.
Li Z, Jiao X, Wang C, Shirley LA, Elsaleh H, Dahl O, et al. Alternative
cyclin D1 splice forms differentially regulate the DNA damage
response. Cancer Res 2010;70:8802–11.
Wu K, Jiao X, Li Z, Katiyar S, Casimiro MC, Yang W, et al. Cell fate
determination factor Dachshund reprograms breast cancer stem cell
function. J Biol Chem 2011;286:2132–42.
Liu M, Ju X, Willmarth NE, Casimiro MC, Ojeifo J, Sakamaki T, et al.
Nuclear factor-kappaB enhances ErbB2-induced mammary tumorigenesis and neoangiogenesis in vivo. Am J Pathol 2009;174:
1910–20.
Sykes SM, Stanek TJ, Frank A, Murphy ME, McMahon SB. Acetylation
of the DNA binding domain regulates transcription-independent apoptosis by p53. J Biol Chem 2009;284:20197–205.
Casimiro MC, Crosariol M, Loro E, Ertel A, Yu Z, Dampier W, et al. ChIP
sequencing of cyclin D1 reveals a transcriptional role in chromosomal
instability in mice. J Clin Invest 2012;122:833–43.
Cancer Res; 73(11) June 1, 2013
22. Botcheva K, McCorkle SR, McCombie WR, Dunn JJ, Anderson CW.
Distinct p53 genomic binding patterns in normal and cancer-derived
human cells. Cell Cycle 2011;10:4237–49.
23. Meuwissen R, Berns A. Mouse models for human lung cancer. Genes
Dev 2005;19:643–64.
24. Srivenugopal KS, Shou J, Mullapudi SR, Lang FF Jr, Rao JS, AliOsman F. Enforced expression of wild-type p53 curtails the transcription of the O(6)-methylguanine-DNA methyltransferase gene in human
tumor cells and enhances their sensitivity to alkylating agents. Clin
Cancer Res 2001;7:1398–409.
25. Baptiste-Okoh N, Barsotti AM, Prives C. A role for caspase 2 and PIDD
in the process of p53-mediated apoptosis. Proc Natl Acad Sci U S A
2008;105:1937–42.
26. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature
2000;408:307–10.
27. Watanabe A, Ogiwara H, Ehata S, Mukasa A, Ishikawa S, Maeda D,
et al. Homozygously deleted gene DACH1 regulates tumor-initiating activity of glioma cells. Proc Natl Acad Sci U S A 2011;108:
12384–9.
28. Lu Y, Futtner C, Rock JR, Xu X, Whitworth W, Hogan BL, et al. Evidence
that SOX2 overexpression is oncogenic in the lung. PLoS One 2010;5:
e11022.
29. Lujan E, Chanda S, Ahlenius H, Sudhof TC, Wernig M. Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor
cells. Proc Natl Acad Sci U S A 2012;109:2527–32.
30. Sutherland KD, Berns A. Cell of origin of lung cancer. Mol Oncol
2010;4:397–403.
31. Nardella C, Clohessy JG, Alimonti A, Pandolfi PP. Pro-senescence
therapy for cancer treatment. Nat Rev Cancer 2011;11:503–11.
32. Velasco-Velazquez MA, Homsi N, De La Fuente M, Pestell RG. Breast
cancer stem cells. Int J Biochem Cell Biol 2012;44:573–7.
33. Maier B, Gluba W, Bernier B, Turner T, Mohammad K, Guise T, et al.
Modulation of mammalian life span by the short isoform of p53. Genes
Dev 2004;18:306–19.
34. Tyner SD, Venkatachalam S, Choi J, Jones S, Ghebranious N, Igelmann H, et al. p53 mutant mice that display early ageing-associated
phenotypes. Nature 2002;415:45–53.
35. Artandi SE, Chang S, Lee SL, Alson S, Gottlieb GJ, Chin L, et al.
Telomere dysfunction promotes non-reciprocal translocations and
epithelial cancers in mice. Nature 2000;406:641–5.
36. Sahin E, Depinho RA. Linking functional decline of telomeres, mitochondria and stem cells during ageing. Nature 2010;464:520–8.
37. Cicalese A, Bonizzi G, Pasi CE, Faretta M, Ronzoni S, Giulini B, et al.
The tumor suppressor p53 regulates polarity of self-renewing divisions
in mammary stem cells. Cell 2009;138:1083–95.
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2013 American Association for Cancer Research.
Published OnlineFirst March 14, 2013; DOI: 10.1158/0008-5472.CAN-12-3191
Dachshund Binds p53 to Block the Growth of Lung
Adenocarcinoma Cells
Ke Chen, Kongming Wu, Shaoxin Cai, et al.
Cancer Res 2013;73:3262-3274. Published OnlineFirst March 14, 2013.
Updated version
Supplementary
Material
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
doi:10.1158/0008-5472.CAN-12-3191
Access the most recent supplemental material at:
http://cancerres.aacrjournals.org/content/suppl/2013/03/14/0008-5472.CAN-12-3191.DC1
This article cites 37 articles, 16 of which you can access for free at:
http://cancerres.aacrjournals.org/content/73/11/3262.full.html#ref-list-1
This article has been cited by 4 HighWire-hosted articles. Access the articles at:
/content/73/11/3262.full.html#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at
[email protected].
To request permission to re-use all or part of this article, contact the AACR Publications Department at
[email protected].
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2013 American Association for Cancer Research.