[CANCER RESEARCH 64, 4137– 4147, June 15, 2004]
A Transcriptional Profiling Study of CCAAT/Enhancer Binding Protein Targets
Identifies Hepatocyte Nuclear Factor 3␤ as a Novel Tumor Suppressor
in Lung Cancer
Balazs Halmos,1 Daniela S. Bassères,1 Stefano Monti,4 Francesco D‘Aló,1 Tajhal Dayaram,1 Katalin Ferenczi,2
Bas J. Wouters,1 Claudia S. Huettner,5 Todd R. Golub,4 and Daniel G. Tenen3
1
Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston; 2Department of Dermatology, Brigham and Women’s Hospital, Boston; 3Harvard Institutes of
Medicine, Boston; 4Center for Genome Research, Whitehead Institute/Massachusetts Institute of Technology, Cambridge, Massachusetts; 5The Blood Center of SE Wisconsin,
Milwaukee, Wisconsin
ABSTRACT
We showed previously that CCAAT/enhancer binding protein ␣ (C/
EBP␣), a tissue-specific transcription factor, is a candidate tumor suppressor in lung cancer. In the present study, we have performed a transcriptional profiling study of C/EBP␣ target genes using an inducible cell
line system. This study led to the identification of hepatocyte nuclear
factor 3␤ (HNF3␤), a transcription factor known to play a role in airway
differentiation, as a downstream target of C/EBP␣. We found downregulation of HNF3␤ expression in a large proportion of lung cancer cell
lines examined and identified two novel mutants of HNF3␤, as well as
hypermethylation of the HNF3␤ promoter. We also developed a tetracycline-inducible cell line model to study the cellular consequences of
HNF3␤ expression. Conditional expression of HNF3␤ led to significant
growth reduction, proliferation arrest, apoptosis, and loss of clonogenic
ability, suggesting additionally that HNF3␤ is a novel tumor suppressor in
lung cancer. This is the first study to show genetic abnormalities of
lung-specific differentiation pathways in the development of lung cancer.
INTRODUCTION
Lung cancer remains a public health problem with ⬃170,000 cases
in the United States per year (1). It is the leading cause of cancer
deaths in both men and women with a 5-year survival rate of only
15%. A recent analysis of trials performed over the last 30 years
demonstrated clearly that only minimal progress has been made in the
treatment of this disease (2). The disappointing results of recent
studies have led to the realization that we have reached a “chemotherapy efficacy plateau” (3). Additional progress in the treatment of
lung cancer will depend critically on a better understanding of the
molecular events leading to the development of epithelial neoplasias
as well as the critical pathways sustaining the neoplastic, invasive
phenotype. Our understanding of the genetic abnormalities underlying
the development of lung cancer remains quite limited (4, 5). Both a
number of tumor suppressors, such as p53, p16, and retinoblastoma,
as well as several proto-oncogenes, such as k-ras and the epidermal
growth factor receptor, are known to play a role, but no abnormalities
of lung-specific tumor suppressors or proto-oncogenes have been
identified yet. A recent high-frequency allelotyping study demonstrated that in individual lung cancers, as many as 22 areas of loss of
Received 12/30/03; revised 3/19/04; accepted 4/9/04.
Grant support: NIH Grant Specialized Programs of Research Excellence in Lung
Cancer PA20-CA090578-01A1 (D. Tenen), American Association for Cancer Research/
Cancer Research Foundation of America/Astra-Zeneca Young Investigator Award for
Translational Lung Cancer Research (B. Halmos), and the Clinical Investigator Training
Program of Harvard/MIT (B. Halmos).
Note: Supplementary data for this article can be found at Cancer Research Online
(http://cancerres.aacrjournals.org).
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.
Requests for reprints: Daniel G. Tenen, Harvard Institutes of Medicine, Harvard
Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115. Phone: (617) 667-5561;
Fax: (617) 667-3299; E-mail: [email protected].
heterozygosity can be detected, suggesting that many tumor suppressor genes remain unidentified (6).
Aberrant differentiation is one of the hallmarks of cancers, and the
contribution of differentiation arrest to the multistep carcinogenesis
process has been accepted widely recently (7). In particular, it is
increasingly clear that aberrant regulation of transcriptional control
pathways of normal differentiation is one of the most common abnormalities in hematological malignancies, such as acute myeloid
leukemia (8). The transcriptional control of differentiation pathways
in airway epithelium is poorly understood, and its abnormalities in the
aberrant differentiation of lung cancers are largely unknown. A number of key transcription factors, such as thyroid transcription factor-1,
helix-loop-helix transcription factors, forkhead transcription factors,
such as hepatocyte nuclear factor 3␤ (HNF3␤), and CCAAT/enhancer
binding protein ␣ (C/EBP␣) are implicated in the complex developmental genetic instruction of lung morphogenesis and cell lineage
determination (9, 10).
C/EBP␣ is a leucine zipper transcription factor that serves as a
tissue-specific differentiation factor in a number of tissues, such as
hepatocytes, myeloid cells, and adipocytes (11). C/EBP␣ was also
identified recently as a novel tumor suppressor in acute leukemia (12).
Recurrent mutations of C/EBP␣ have been identified by a number of
groups in subtypes of acute leukemia (13, 14). C/EBP␣ is expressed
strongly in the lung, more specifically in both type II pneumocytes as
well as cells of the bronchial epithelium (15, 16). It also regulates the
expression of several genes, directly or indirectly, during lung differentiation, including surfactant B and uteroglobin (17, 18). Specific
lung abnormalities, such as an abnormal proliferation of type II
pneumocytes, have been described in C/EBP␣⫺/⫺ knockout mice
(19), suggesting that C/EBP␣ is important for normal lung development and the maintenance of normal alveolar structure. It is postulated
that this hyperproliferation is because in the absence of C/EBP␣, the
alveolar type II cells can continue to proliferate. In previous studies,
we have demonstrated that C/EBP␣ is down-regulated in a large
proportion of lung cancers (20). We also developed a tightly regulated, highly inducible cell line model system using a zinc-inducible
metallothionein promoter-based system. With the use of this stably
transfected cell line system, we have shown that the induction of
C/EBP␣ expression leads to growth arrest, apoptosis, and cellular
changes suggestive of differentiation, all supporting its role as a
candidate tumor suppressor gene.
To gain additional insight into the downstream effects of C/EBP␣
expression, we have performed transcriptional profiling studies on this
inducible cell line system. From the many C/EBP␣-regulated genes
identified, we have focused our additional studies on one, HNF3␤
(also known as Foxa2), given its known important role in airway
epithelial differentiation (21–23). Our studies demonstrate downregulation and novel mutations of HNF3␤ in lung cancer cell lines.
We also identified hypermethylation of the promoter region of the
HNF3B gene as a novel mechanism for epigenetic silencing of
HNF3␤ expression. To assess the functional consequences of HNF3␤
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HNF3␤ IS A TUMOR SUPPRESSOR IN LUNG CANCER
dilution of a monoclonal mouse anti-␤-tubulin (both from Sigma Chemicals)
were used, respectively. Detection was performed using enhanced chemiluminescence (Amersham Life Science, Piscataway, NJ).
Electrophoretic Mobility Shift Assay. Nuclear extracts were prepared
from untreated or 25 ␮M CdSO4-treated H358 ppc18-transfected or ppc22transfected cells after 16 h of induction as described previously (24). The gel
shift assay consisted of a binding reaction allowing the formation of DNAprotein complexes, which were then separated from unbound probe by native
gel electrophoresis. 50 ng of double-stranded DNA probe containing the
MATERIALS AND METHODS
C/EBP binding site of the HNF3␤ promoter was end-labeled with [␥-32P]ATP
Cell Lines and Cell Culture. The following lung cancer cell lines were and T4 polynucleotide kinase (New England Biolabs). The sequences of the
used in our study: squamous cell cancer, Calu-1, SK-MES-1, H157, H520, oligonucleotides used to generate the probes were as described previously
SW900, U1752, and EPLC103H; adenocarcinoma, A427, SK-LU-1, Calu-3, (25): sense, 5⬘-AATTCCCTGTTTGTTTTAGTTACGAAATGCGTTG-3⬘; and
H23, and H441; adenocarcinoma, bronchoalveolar type, H358, A549, and antisense, 5⬘-AATTCAACGCATTTCGTAACTAAAACAAACAGGG-3⬘. BindH322; adenosquamous cancer, H125, H292, and H596; large cell cancer, H460 ing reactions were performed by incubating 5 ␮g of nuclear extracts with 50,000
and H661; anaplastic, Calu-6; and small cell lung cancer, H526, H187, H69, cpm of the double-stranded probe in 20 ␮l of reaction mixtures consisting of ⫻1
H345, H211, H60, H82, N417, H128, and UMC19. All of the non-small cell binding buffer [2 mM HEPES-KOH (pH 7.9), 10 mM KCl, 0.5 mM MgCl2, 0.2 mM
lung cancer cell lines were grown in RPMI 1640 supplemented with 10% fetal DTT, and 2% glycerol] in the presence of 50 ng/␮l BSA and 25 ng/␮l
bovine serum, whereas all of the small cell lung cancer cell lines were grown poly(deoxyinosinic-deoxycytidylic acid) for 20 min at room temperature. For the
in RPMI 1640 supplemented by HITES medium (final concentration of 2.5% supershift assays, 2 ␮g of a rabbit polyclonal anti-C/EBP␣ antibody (sc-61X;
fetal bovine serum, 2.8 mM glutamine, 10⫺8 M hydrocortisone, 10⫺8 M estra- Santa Cruz Biotechnology) was added to the reaction mixture. The binding
diol, and 1% insulin/transferrin/Na-selenite (Sigma Chemical Co., St. Louis, reactions were separated on 4% acrylamide gel at 150 V. Subsequently, the gel
MO).
was dried under vacuum at 80°C for 1 h and submitted to autoradiography with an
Oligonucleotide Array Analysis. Ppc22-transfected H358 cells were intensifying screen for 6 h at ⫺80°C.
grown to 60% confluence in RPMI containing 10% fetal bovine serum.
Chromatin Immunoprecipitation Assays. Mock-transfected and ppc22Triplicate plates were induced by the addition of 100 ␮M of ZnSO4 for 6 and transfected cells were treated with 25 ␮M CdSO for 48 h. Chromatin immu4
12 h. Control cells (0 h of induction) were grown without the addition of
noprecipitation assay was performed as described previously (26). Briefly,
ZnSO4 to the medium. Cells were collected at the same time, and total cellular 1 ⫻ 108 cells were cross-linked by addition of formaldehyde to the medium at
RNA was isolated using the TRIzol method. RNA specimens were then a final concentration of 0.37%. Nuclear lysates were prepared, and before
processed and hybridized to Affymetrix Hu95 microarrays and scanned. The immunoprecipitation, 20% of each lysate was removed for analysis of input
expression value for each gene was calculated using Affymetrix GeneChip chromatin DNA. Immunoprecipitation was performed with 5 ␮g of normal
software.
rabbit IgG (Santa Cruz Biotechnologies) or 5 ␮g of rabbit polyclonal C/EBP␣
Preprocessing, Rescaling, and Filtering. The raw expression data con- antibody (sc-61; Santa Cruz Biotechnology) at 4°C for 6 h with rotation.
sisted of the scanner “signal” units as obtained from the GeneChip MAS5
Immune complexes were collected by incubating with protein A-agarose beads
software of Affymetrix. These raw data were rescaled to account for different
overnight at 4°C with rotation. Cross-links of the immunoprecipitated samples
chip intensities. Each chip in the data set was multiplied by the factor
and input samples were reversed by heating at 67°C in the presence of NaCl
constant/chip㛭intensity, where chip㛭intensity denotes the average intensity of
and RNase A (Sigma) for 5 h followed by proteinase K (Roche Diagnostics)
the chip (i.e., the expression level of the sample averaged across all of the
digestion. The DNA was recovered by phenol-chloroform extraction followed
probe sets in the chip), and constant is the same quantity for all of the chips
by ethanol precipitation and resuspended in distilled sterile water. Binding of
(chosen to be the average intensity of the median chip). From the initial set of
C/EBP␣ to the HNF3␤ promoter was assessed by PCR with the following
12,626 genes, a final set of 4,984 genes was obtained as follows: (a) setting the
primers: sense, 5⬘-GCCTCCACATCCAAACACC-3⬘; and antisense, 5⬘minimum signal to 10 and the maximum signal to 20,000; (b) excluding genes
CTCTCCGACTCCTCAGACACC-3⬘, which amplify a region that spans bps
for which the fold change (i.e., the maximum:minimum threshold value) was
⫺75 to ⫺104 (containing the C/EBP binding site) of the HNF3␤ promoter.
⬍3; and (c) excluding those genes for which the ␦ change (i.e., the difference
Sequencing. The promoter region and three exons of HNF3␤ were sebetween the maximum and minimum threshold values) was ⬍100. For the
quenced by the use of seven primer sets as described previously (27). PCR
identification of differentially expressed genes, a paired t-score was used.
When analyzing the pooled data, the “0 versus 6 h” pairwise differences and products were sequenced by the use of both the sense and antisense primers.
the “0 versus 12 h” pairwise differences were pooled together for the compu- Both the actual sequences and the traces were compared with that of wild-type
tation of the score. For the analysis, GeneCluster software and scripts written HNF3␤ using DNAStar software. All of the abnormal sequences were reamplified twice and resequenced in both directions. In cases where the abnormal
in R (an open-source statistical package) were used.
Northern Blotting. Total cellular RNA from cell lines was isolated using sequence was confirmed, the PCR product was subcloned into pGEM-T
TRIzol reagent. RNA (20 ␮g/lane) was separated on 1% agarose/4-morpho- vector, and at least five subclones were sequenced.
Deoxyazacytidine Treatment. The 5⬘-aza, 2⬘ deoxycytidine was a kind
linepropanesulfonic acid/formaldehyde gels and transferred to MagnaGraph
membranes (Osmonics, Westborough, MA). Hybridization was performed gift of Dr. Stephen Baylin (Johns Hopkins Oncology Center, Baltimore, MD).
with [32P]dCTP-labeled probes using Church-Gilbert hybridization solution. Cells were grown to 20% confluence, and then deoxyazacytidine was added at
The following probes were used: rat C/EBP␣-350 bp fragment flanking the rat 0.2 ␮M or 1 ␮M concentration to duplicate specimens. Medium was changed,
C/EBP␣/SV40 polyadenylic acid junction from plasmid ppc22; HNF3␤, 1.6 kb and fresh drug was added every day. RNA was collected using the TRIzol
full-length cDNA of rat HNF3␤ (kind gift of Dr. Robert Costa, University of method after 96 h of treatment. Real-time reverse transcription-PCR was
Illinois, Chicago, IL); cyclooxygenase-2 (COX-2), 700-bp fragment of the 3⬘ performed as described below.
Promoter Methylation Studies. Bisulfite sequencing was performed acuntranslated region of the human COX-2 mRNA subcloned into pGEM-T, cut
with NcoI-SacI; and interleukin 8, 500-bp EcoRI insert from PCDNA3 (kind cording to established methods (28). In brief, 2 ␮g of genomic DNA was
bisulfite treated and purified (Promega Wizard DNA Clean-UP System; Progift of Dr. Isaiah Fidler, M. D. Anderson Cancer Center, Houston, TX).
Western Blotting. Whole cell lysates were isolated using radioimmuno- mega). The resultant bisulfite-modified DNA was amplified using a primer set
precipitation assay lysis buffer and protease inhibitors (phenylmethylsulfonyl amplifying a 369-bp fragment of the HNF3␤ promoter and part of exon 1
fluoride, pepstatin, and leupeptin), and 20 ␮g of protein/lane were electro- (positions 192–540). The following primers were used: sense, 5⬘-TTGGAAGATAGAGAGGATAGA-3⬘; and antisense, 5⬘-CCCCTCCCTATTACphoresed in 10 or 12% polyacrylamide minigels. A 1:2000 dilution of a
CAATTCAA-3⬘. The amplified PCR product was subcloned into pGEM-T
polyclonal rabbit anti-C/EBP␣ antibody and a 1:2000 dilution of a polyclonal
goat anti-HNF3␤ antibody (both from Santa Cruz Biotechnology, Santa Cruz, vector, and clones were sequenced with the use of Sp6 antisense primer.
Peripheral blood mononuclear cell DNA served as negative control, whereas
CA), and a 1:1000 dilution of a monoclonal mouse anti-␤-actin and a 1:100
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expression, we also generated a tetracycline-regulated inducible cell
line system for the conditional expression of HNF3␤. With the use of
this system, we demonstrate that induced expression of HNF3␤ in the
H358 lung cancer cell line leads to growth reduction, proliferation
arrest, apoptosis, and loss of clonogenic ability, supporting its identification as a novel tumor suppressor in lung cancer.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 2004 American Association for Cancer
Research.
HNF3␤ IS A TUMOR SUPPRESSOR IN LUNG CANCER
universally methylated DNA (CpGenome Universally Methylated DNA; Intergen) was used as positive control.
Plasmid Generation. The full-length cDNA of rat HNF3␤ was a kind gift
of Robert Costa (University of Illinois, Chicago, IL). The 1.6-kb rat HNF3␤
cDNA was released from the pGEM-T vector backbone by digestion with
EcoRI. The fragment was blunt ended and subsequently subcloned into the
dephosphorylated PvuII site of the multiple cloning site of vector pTRE2puro
(Clontech, Palo Alto, CA) to generate plasmid pTRE2HNF3B.
Generation of Inducible Cell Lines. H358 cells were transfected with the
transactivator Tet-off plasmid using lipofectamine transfection (LipofectAMINE PLUS; Invitrogen Life Technologies, Inc., Carlsbad, CA) according to the manufacturer’s instructions. Clones were selected on the basis of
G418 resistance (G418 concentration of 500 ␮g/ml). Highly repressible clones
were identified by transient transfection with a TRE-luciferase reporter plasmid (pTRE2pur-luc; Clontech). Clones were screened by performing luciferase
assays after the cells were grown for 24 h with or without addition of
doxycycline (1 ␮g/ml). Clone 6 showed the highest level of repressibility after
doxycycline withdrawal and was selected for additional studies. The second
round of transfection with the pTRE2HNF3B plasmid was performed using
identical methods. Cells were grown continuously in the presence of doxycycline to suppress transresponder gene induction, and clones were selected on
the basis of dual resistance to G418 and puromycin (1 ␮g/ml). Clones were
screened for inducibility of HNF3␤ expression after 24 – 48 h of doxycycline
withdrawal.
Real-Time PCR Assay. TRIzol-extracted RNA was DNase treated, reverse transcribed, and subsequently amplified using an ABIPrism 7700 Sequence Detector (Applied Biosystems) by the following parameters: 50°C (30
min); 95°C (15 min) followed by 40 cycles of 94°C (15 s); and 60°C (60 s).
Primers and probe (FAM-labeled) were as follows: human HNF3␤ forward
primer, 5⬘-AAGATGGAAGGGCACGAGC-3⬘; reverse primer, 5⬘-TGTACGTGTTCATGCCGTTCA-3⬘; and probe, 5⬘-TCCGACTGGAGCAGCTACTATGCAGAGC-3⬘. Rat HNF3␤: forward primer, 5⬘-CTGAAGCCCGAGCACCAT-3⬘; reverse primer, 5⬘-GCTGCTCGGAGGGACATGA-3⬘;
and probe, 5⬘-TCCGACTGGAGCAGCTAC-3⬘. Primers and probe (VIC-labeled) for 18 S rRNA were from Applied Biosystems.
Bromodeoxyuridine Proliferation Assay. Proliferation assays were performed with the use of BrdU Flow kit (Becton Dickinson PharMingen, San
Diego, CA). In brief, bromodeoxyuridine was added to medium to achieve a
final concentration of 10 ␮M for 45 min, then cells were trypsinized and treated
according to the manufacturer’s instructions. Flow cytometry was performed
on a fluorescence-activated cell scan cytometer (Becton Dickinson).
Annexin/Propidium Iodide Apoptosis Assay. Cells were collected after
trypsinization, washed with PBS, and stained with annexin/propidium iodide
according to the manufacturer’s instructions (Roche Diagnostics, Mannheim,
Germany). Samples were analyzed on a fluorescence-activated cell scan cytometer (Becton Dickinson).
Clonogenic Assays. One thousand each of H358 pTRE2HNF3B/4 and
pTRE2HNF3B/31 cells were mixed with 1.5 ml of 1.25% methylcellulose/
Iscove’s modified Dulbecco’s medium/tetracycline-free fetal bovine serum/
puromycin with or without 1 ␮g/ml of doxycycline and plated onto 20-mm cell
culture plates. Doxycycline was added every 2–3 days to the appropriate
plates. Six plates/condition were seeded. The number of colonies was counted
on day 14.
transcriptional profiling studies to identify downstream targets of
C/EBP␣ and gain additional functional insights into the mechanisms
leading to C/EBP␣-induced growth arrest and differentiation.
To perform oligonucleotide array analysis, ppc22-transfected H358
cells were induced to express C/EBP␣ by the addition of zinc for 6
and 12 h, respectively. Control cells (0 h induction) were grown
without the addition of zinc to the medium. Conditional expression of
C/EBP␣ was confirmed by Northern as well as Western blotting (Fig.
1, A and C). Transcriptional profiling was then performed on total
cellular RNA using Affymetrix Hu-95 chips. The raw expression data
were preprocessed, rescaled, and filtered as described in “Materials
and Methods.” Analysis of the results was performed by GeneCluster
software (data on the entire gene set is available as Supplementary
Data). Experiments were carried out in triplicates, with three time
points for each experiment (0, 6, and 12 h). We were interested in
identifying those genes manifesting a differential expression between
0 h and 6 h to identify the earliest wave of transcriptional changes
RESULTS
Transcriptional Profiling of C/EBP␣-Inducible H358 Cells. As
described previously (20), we have generated a stably transfected cell
line from H358 adenocarcinoma cells using a mammalian expression
vector construct (ppc22; Ref. 29) harboring the rat C/EBP␣ gene
under the control of the zinc-inducible metallothionein prom (MT-C/
EBP␣). We also generated control cell lines by stable transfection of
H358 cells with a control vector (ppc18). H358 cells have practically
no detectable native C/EBP␣ expression either on the RNA or protein
level. In this cell line system, marked increase in C/EBP␣ mRNA can
be detected as early as 3 h after induction by addition of zinc or
cadmium sulfate to the medium. The tight regulation and very strong
inducibility of this cell line system appeared to be ideally suited for
Fig. 1. A, Northern blot analysis confirms the gene chip results. Experiments were
performed with ppc18 and ppc22-transfected H358 cells. Cells were induced by the
addition of 100 ␮M of ZnSO4 to the medium for 6 and 12 h, respectively. Control cells
were grown without the addition of zinc (0 h of induction). Total cellular RNA was
collected, and Northern blots were performed with specific probes for CCAAT/enhancer
binding protein ␣ (C/EBP␣), interleukin 8, cyclooxygenase-2, and hepatocyte nuclear
factor 3␤ (HNF3␤). No induction is seen in ppc18-transfected cells (MOCK), whereas
very strong inducibility is observed in ppc22-transfected cells (MT-CEBP␣) consistent
with the gene chip findings. Ethidium-bromide staining of the RNA gel is shown to
demonstrate equal loading. B, TaqMan assay confirms the induction of HNF3␤ mRNA by
C/EBP␣. C, induction of HNF3␤ protein by C/EBP␣. Stably transfected H358 cells
(MOCK and MT-CEBP␣) were induced by the addition of 100 ␮M of ZnSO4 to the
medium. Western blots on 20 ␮g of whole cell lysate demonstrate an ⬃3-fold induction
of HNF3␤ protein by 24 h of induction.
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HNF3␤ IS A TUMOR SUPPRESSOR IN LUNG CANCER
most likely highly enriched in direct targets of C/EBP␣. We also
performed a comparison between the uninduced control and induced
“pooled” specimens (6 samples, 3 at 6 h and 3 at 12 h). This
comparison yields a set of genes that is up- or down-regulated consistently between 6 and 12 h, therefore is less likely to harbor
false-positive candidate genes and might also identify important upor down-regulated genes that might not be significantly changed at 6 h
but become evident by 12 h. We felt the two comparisons would
provide complementary information and would also serve as internal
controls for the validity of our findings. We used paired t-statistic to
rank genes. From the ranked list of genes, we excluded genes with a
t-statistic ⬍1 (representing no association). To arrive at a biologically
relevant set of genes, we used two additional sets of criteria. We
selected only those genes where the level of induction or repression
was a minimum 3-fold (mean difference using means of triplicate
experiments). We also excluded genes where the absolute difference
between induced/uninduced was ⬍500 fluorescence intensity signal
units, thereby removing genes where the changes might appear significant based on fold-differences, but this is likely to be biologically
irrelevant or spurious secondary to low levels of expression. The top
genes obtained this way are shown in Tables 1 and 2. Of note is that
a very strong overlap was noted between the 0 versus 6 h and the 0
versus pooled comparisons (34 of the top 45 up-regulated and 23 of
the top 35 down-regulated genes from the 0 versus 6 h comparison
Table 1 Upregulated genes
This table shows the list of the top upregulated genes based on an analysis comparing 0 versus 6 h of C/EBP␣ induction (first 45 genes) as well as additional genes from “pooled
analysis” of uninduced versus “pooled” induced specimens (i.e. including data from both 6 as well as 12 h of induction). Of note is that 34 of the top 45 genes from the first analysis
were identified in the pooled analysis as well. A large number of the identified genes fell into three main clusters: (a) acute phase reactant genes; (b) genes involved in terminal
metabolism; and (c) adipocyte/lipid metabolism-associated genes. Fold-change was determined by the fold-difference between the means of grouped specimens. Ranking was based
on t-score.
a
Rank
Score
Fold
change
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
20
16.683
9.771
9.027
8.375
8.087
7.777
7.137
6.623
6.23
5.859
5.644
5.527
5.451
5.301
5.273
5.189
5.071
5.017
4.828
4.467
4.354
4.273
4.203
4.117
4.06
4.02
3.702
3.513
3.386
3.215
3.189
3.136
2.95
25.48
15.85
3.4
8.63
3.78
9.94
4.56
6.61
3.52
6
3.21
13.96
4.62
5.99
4.55
5.03
3.86
4.72
3.84
32.06
5.98
39.7
3.33
3.27
4.41
3.62
6.06
10.65
3.37
6.02
4.23
91.14
4.86
7.141
35
36
37
38
39
40
41
42
43
44
45
2.912
2.814
2.77
2.644
2.589
2.518
2.393
2.344
1.949
1.778
1.439
37.19
18.18
19.05
4.77
4.61
8.85
88.66
8.11
144.7
4.01
9.99
Pooled analyses
1
2
3
4
5
6
7
8
9
5.024
3.867
3.448
2.467
2.273
2.148
2.045
1.975
1.874
3.18
3.26
3.17
3.39
3.65
3.16
4.49
3.7
4.43
Description
Accession no.
Tumor necrosis factor-inducible gene 14 (TSG-14)
Cyclooxygenase-2 (hCox-2)
BTG1
Folate receptor ␣
E4BP4
Integrin, ␣ subunit
Semaphorin E
Hepatocyte nuclear factor-3 ␤
Cell adhesion kinase ␤ (CAK␤)
RGP4 mRNA
hbc647
Protease inhibitor 12 (PI12; neuroserpin)
Insulin-like growth factor-binding protein-3
Zinc finger protein (clone 647)
Glutaredoxin
HM74
Helix-loop-helix basic phosphoprotein (G0S8)
MEGF9
Interleukin 4 receptor
Chemokine exodus-1
GC-Box binding protein BTEB2
Interleukin 8 (IL8)
Low-Mr GTP-binding protein (RAB32)
Putative endothelin receptor type B-like protein
Cytokine (GRO-␤)
HRIHFB2017
Monocarboxylate transporter 2 (hMCT2)
␤-Thromboglobulin-like protein
Jak2 kinase
Receptor protein-tyrosine kinase (HEK8)
Bone morphogenetic protein 2A (BMP-2A)
Spliceosomal protein (SAP 62)
Erythroblastosis virus oncogene homolog 2 (ets-2)
1-Phosphatidylinositol-4,5-Bisphosphate
Phosphodiesterase ␥
Heme Oxygenase 1
Cytokine (GRO-␥)
Hepatic dihydrodiol dehydrogenase gene
Adipophilin
Tyrosine kinase
Ets transcription factor PDEF (PDEF)
Cyritestin protein
Apolipoprotein apoC-IV (APOC4)
HOX11 homeodomain {homeobox}
Wiskott-Aldrich syndrome protein (WASP)
Fas (Apo-1, CD95)
M31166
U04636
X61123
U78793
X64318
X68742
AB000220
AB028021
U43522
U27768
U68494
Z81326
M35878
X16282
X76648
D10923
L13463
AB011542
X52425
U64197
D14520
M28130
U59878
U87460
M36820
AB015331
AF058056
M17017
AF058925
L36645
M22489
L21990
J04102
AL022394
ADP-ribosylation factor-like protein 4
Ceramide glucosyltransferase
p63 mRNA for transmembrane protein
Small GTP-binding protein
Interleukin 1 receptor
UDP-N-acetylglucosamine pyrophosphorylase
RDC-1 POU domain containing protein
MDC-3.13 isoform 2 mRNA
Quinone oxidoreductase2 (NQO2)
U73960
D50840
X69910
U57094
M27492
AB011004
X64624
AF099935
U07736
Z82244
M36821
U05861
X97324
Cluster
Acute phasea
Acute phasea
a
Metabolisma
a
a
Acute phasea
a
a
a
Metabolisma
a
a
a
Acute phasea
Acute phase
a
Acute phasea
a
a
Acute phasea
a
Metabolisma
Acute phasea
a
Acute phasea
a
a
a
Metabolism
Acute phasea
Metabolisma
Lipida
a
AF071538
X89654
U32576
S38742
U12707
X89101
a
Lipid
a
Lipid
Lipid
Acute phase
Metabolism
Metabolism
Also identified in pooled analysis.
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HNF3␤ IS A TUMOR SUPPRESSOR IN LUNG CANCER
Table 2 Downregulated genes
This table shows the list of most highly downregulated genes after conditional expression of C/EBP␣ as derived from Affymetrix transcriptional profiling analysis. The first group
of genes is based on an analysis between 0 versus 6 h of induction, while the list of genes from the “pooled analysis” were derived from analyzing control uninduced versus pooled
induced specimens (including data from both 6 as well as 12 h of induction). Of note is that 23 of the 35 genes identified in the first analysis were also identified in the “pooled” analysis.
a
Rank
Score
Fold
change
Description
Accession No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
13.371
11.877
11.574
8.82
8.06
6.982
6.967
6.457
6.239
6.224
6.067
5.615
5.562
5.429
5.052
4.86
4.635
4.614
4.426
4.21
3.755
3.717
3.695
3.485
3.375
3.327
3.237
2.941
2.918
2.76
2.469
2.297
2.25
2.241
2.111
6.66
4.55
5.54
3.06
4.51
7.59
10.31
4.44
3.47
3.11
3.43
12.75
7.74
3.51
6.35
3.27
3.06
77.25
9.02
7.63
8.53
16.34
3.63
3.7
3.51
5.25
38.22
4.34
3.01
3.56
49.75
6.43
6.29
3.28
3.06
Kidney ␥-glutamyl transpeptidase type II
Stanniocalcin-related protein
Dual specificity phosphatase MKP-5
Growth arrest and DNA-damage-inducible protein (gadd45)
Proteinase activated receptor-2
Connective tissue growth factor
CPE-receptor
CYR61
Breast cancer antiestrogen resistance 3 protein (BCAR3)
Cisplatin resistance associated alpha protein (hCRA alpha)
Id-2H
Tyrosine hydroxylase type 4
ADP ribosylation factor-like protein
Protein kinase, Dyrk2
Putative tetraspan transmembrane protein L6H (TM4SF5)
Tob
GAR22 protein
High-sulphur keratin
Transmembrane receptor (ror1)
Testicular inhibin beta-B-subunit
FGF-9
RGP3
Transcriptional activator
HRY gene
MAL gene exon 1
Smad6
Human uromodulin (Tamm-Horsfall glycoprotein)
Frizzled-7
Ets-related transcription factor (ERT)
MAD-related gene SMAD7 (SMAD7)
Ras-related rho mRNA
CCCAAT/enhancer binding protein ␣
SH3 binding protein
Transcription factor ERF-1
Epidermal growth factor receptor (HER3)
M30474
AF098462
AB026436
M60974
U67058
X78947
AB000712
Y11307
U92715
U78556
D13891
M17589
AB016811
Y09216
AF027204
D38305
Y07846
X63755
M97675
M31682
D14838
U27655
U49857
L19314
X76220
AF035528
M15881
AB017365
AF017307
AF010193
M12174
Y11525
AB000462
U85658
M34309
Pooled analyses
1
2
3
4
5
6
7
8
9
10
11
12
6.926
5.294
3.209
3.132
2.364
2.017
1.997
1.805
1.777
1.702
1.651
1.291
3.17
3.24
4.7
3.29
34.77
3.55
3.63
7.74
3.07
91.91
3.54
4.77
Interferon-inducible 56 Kd protein
uPA gene
Lung amiloride sensitive Na⫹ channel protein
PTPL1-associated RhoGAP
Thyroid transcript factor 1
RBP-MS/type 4
Trio
Leucine zipper protein
CD97 gene exon 1
Choline kinase
Luteinizing hormone, ␤ subunit
Squamous cell carcinoma of esophagus mRNA for GRB-7
SH2 domain protein
M24594
X02419
X76180
U90920
X82850
D84110
U42390
Z50781
X94630
D10704
Cluster
a
a
a
a
a
Proliferationa
a
Proliferationa
a
a
a
a
a
a
a
a
a
Proliferationa
a
a
a
a
a
Proliferation
Proliferation
D43772
Also identified in pooled analysis.
appeared in the 0 versus pooled most highly induced/repressed gene
list); therefore, the pooled analysis contributed only a few genes to the
lists. Such strong overlap does suggest that the gene set arrived at this
way represents an enriched set of genes regulated directly or indirectly
by C/EBP␣ in H358 cells.
To confirm the findings of the oligonucleotide array analysis, the
induction/repression of several genes was confirmed by Northern blot
analysis (Fig. 1A). As control, RNA was collected from H358 cells
stably transfected with empty vector (ppc18) and treated in an identical fashion to cells collected for the oligonucleotide array analysis.
Fig. 1 shows representative results for interleukin 8, COX-2, and
HNF3␤ genes. These studies confirmed the gene chip results in that
all of the examined genes showed consistent findings with our gene
chip data, and none of the examined genes appeared regulated by zinc
itself, as demonstrated by the absence of induction in the control
(ppc18) cell line.
An analysis of the highly up-regulated genes demonstrated three
major clusters of genes in this set. First, many of the up-regulated
genes are acute phase reactants, such as interleukin 8, COX-2,
tumor necrosis factor-inducible gene 14, and exodus-1 among
others. A second group of genes comprises genes of metabolic
pathways, such as hepatic dihydrodiol dehydrogenase, folate receptor, glutaredoxin, monocarboxylate transporter 2, and quinone
oxidoreductase. The third group of genes includes genes known to
be involved in fat metabolism or adipocyte differentiation, such as
adipophilin, ADP-ribosylation factor-like protein-4, and ceramide
glucosyltransferase. Lastly, one of the most highly induced genes
is HNF3␤. This finding is of particular importance because HNF3␤
is known to play a major role in the transcriptional control of
cellular differentiation, including airway epithelial differentiation
(21, 30), thereby suggesting that C/EBP␣ might indeed act as
master regulator of airway epithelial differentiation.
Our analysis identified also a set of highly repressed genes by
C/EBP␣. Many of the repressed genes are growth factors, growth
factor receptors, or proangiogenic molecules, such as connective
tissue growth factor, Cyr61 (also called CCN1), fibroblast growth
factor-9, epidermal growth factor receptor, and urokinase-plas-
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HNF3␤ IS A TUMOR SUPPRESSOR IN LUNG CANCER
ing to this promoter element of the HNF3␤ promoter in vivo, suggesting that the induction identified through our transcriptional profiling studies is indeed due to direct regulation by C/EBP␣.
Abnormalities of HNF3␤ in Lung Cancer Cells. Given our finding of C/EBP␣-mediated induction of HNF3␤ and the established role
of HNF3␤ in airway development and differentiation, a process also
regulated by C/EBP␣, we decided to focus our additional studies on
dissecting the role of HNF3␤ in lung cancer. We have determined the
expression of HNF3␤ by Northern blotting in 25 lung cancer cell lines
representing all of the histological subtypes of lung cancer. Although
HNF3␤ is strongly expressed in normal lung, its expression is undetectable or very weak in 15 of 25 cell lines examined (Fig. 3A).
Western blotting demonstrated strong correlation of HNF3␤ expression between mRNA and protein levels (Fig. 3B). Of the 10 cell lines
that expressed HNF3␤ mRNA at substantial levels, 5 had transcripts
of sizes different from that observed in normal lung, most likely
representing products of alternative splicing. We have performed
Fig. 2. The hepatocyte nuclear factor 3␤ (HNF3␤) promoter is directly bound by
CCAAT/enhancer binding protein ␣ (C/EBP␣). A, electrophoretic mobility shift assay.
Oligonucleotides containing C/EBP recognition sequences from the HNF3␤ promoter
were used for electrophoretic mobility shift assay experiments with nuclear extracts from
H358 cells stably transfected with a mock construct (H358-MOCK) or with the MT-C/
EBP␣ construct. The cells were treated with 25 ␮M CdSO4 for 16 h before nuclear extract
preparation to induce C/EBP␣ expression as described previously. A rabbit polyclonal
antibody specific for C/EBP␣ was included in the reaction as indicated to promote
supershifting of the C/EBP␣ complex. B, chromatin immunoprecipitation. Chromatin
immunoprecipitations were performed from CdSO4-induced (25 ␮M) mock-transfected or
MT-C/EBP␣-transfected H358 cells using an antibody specific for C/EBP␣ or normal
rabbit IgG. A 20% input control from each chromatin sample as well as the precipitated
chromatin were analyzed by PCR using primers specific for the C/EBP site in the human
HNF3␤ promoter. NC, No chromatin control.
minogen activator. This is in line with the established role of
C/EBP␣ in proliferation arrest (29, 31, 32). C/EBP␣ is identified as
one of the down-regulated genes. This is not surprising, because
the ppc22 plasmid construct RNA does not hybridize with the
oligonucleotide on the Affymetrix chip.
HNF3␤ Is Regulated by C/EBP␣. Our transcriptional profiling
studies showed an ⬃6-fold induction of HNF3␤ mRNA as early as 6 h
after C/EBP␣ induction. This finding is particularly interesting given
the established role of HNF3␤ in lung development as well as cellular
differentiation (10, 21–23). Therefore, we decided to establish
whether this regulation was direct or indirect and also focused in our
additional studies on dissecting the role of HNF3␤ in lung cancer as
a possible critical target of the changes induced by C/EBP␣. We
confirmed the up-regulation of HNF3␤ as a result of the conditional
expression of C/EBP␣ by Northern blotting, Real-time reverse transcription-PCR assay, as well as Western blotting and showed that zinc
itself does not induce the expression of HNF3␤ (Fig. 1, B and C). The
degree of induction on the protein level is ⬃3-fold. Although the peak
induction on the RNA level occurs by 6 h, on the protein level the
peak expression is somewhat delayed and occurs between 24 and 48 h.
The HNF3␤ promoter does have a putative C/EBP-binding site, and
one report did show previously indirect evidence of C/EBP proteins
regulating the HNF3␤ promoter (25). In electrophoretic mobility shift
assays, we demonstrated a strong increase in specific binding activity
upon induction of C/EBP␣ (Fig. 2A). The specificity of binding was
confirmed by supershift assays using a C/EBP␣ antibody. We confirmed the same finding by chromatin immunoprecipitation assays
using primers flanking the putative C/EBP-binding site of the HNF3␤
promoter (Fig. 2B). These assays demonstrated direct C/EBP␣ bind-
Fig. 3. Hepatocyte nuclear factor 3␤ (HNF3␤) is down-regulated and mutated in lung
cancer cell lines. A, Northern blot analysis of HNF3␤ expression in lung cancer cell lines.
A representative Northern blot is shown demonstrating strong expression in total RNA
from normal lung as compared with weak or absent expression in a number of cell lines
examined. Ethidium bromide staining of 28S RNA is shown as loading control. Lane 1,
normal lung; Lane 2, SKMES-1; Lane 3, H520; Lane 4, U1752; Lane 5, A427; Lane 6,
SKLU-1; Lane 7, H23; Lane 8, H441; Lane 9, SW900; Lane 10, H292; Lane 11, H596;
Lane 12, H460; Lane 13, H526; and Lane 14, H187. B, Western blot analysis of HNF3␤
expression in lung cancer cell lines. Twenty of 22 cell lines examined had identical
expression levels on the mRNA and protein level. A representative Western blot is shown.
Lane 1, SK-MES-1; Lane 2, Calu-1; Lane 3, U1752; Lane 4, A427; Lane 5, SKLU-1;
Lane 6, H23; Lane 7, H441; Lane 8, SW900; Lane 9, H292; Lane 10, H596; Lane 11,
H460; Lane 12, Calu-6; and Lane 13, H520. C, sequencing of the HNF3␤ gene. The
diagram shows the adopted sequencing strategy using seven primer sets to sequence the
promoter, all of the coding sequences, and parts of the 3⬘ untranslated region of the
HNF3␤ gene in 31 lung cancer cell lines. Also shown is a bar diagram of the structural
motifs of the HNF3␤ protein and the structure of the two mutants found. A heterozygous
mutation in the H60, small cell lung cancer cell line introduces a G-D amino acid change
at position 92, whereas in the SKLU-1 cell line, a homozygous base insertion leads to a
frameshift and a predicted premature termination at amino acid 218.
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HNF3␤ IS A TUMOR SUPPRESSOR IN LUNG CANCER
Fig. 4. Demethylating treatment leads to up-regulation of hepatocyte nuclear factor 3␤
(HNF3␤). Real-time PCR study showing up-regulation of HNF3␤ mRNA in 3 of 4 cell
lines after 5⬘ deoxy 2⬘ azacytidine treatment. Cells were treated with 0.2 or 1 ␮M
deoxyazacytidine for 96 h. SKLU-1 cells had no detectable expression of HNF3␤,
whereas H322, A427, and H596 cells demonstrated significant up-regulation of HNF3␤
mRNA on treatment.
genomic sequencing of the HNF3␤ gene to exclude the possibility that
these cell lines carry mutant forms of HNF3␤. The HNF3␤ gene is
located on chromosome 20p11 and has 3 exons. A sequencing strategy
was adopted (27) using a set of seven primers to sequence a portion
of the HNF3␤ promoter, exons 1 and 2, and all of the coding regions
of exon 3, as well as the first 68 bps of the 3⬘-untranslated region in
31 lung cancer cell lines. Besides a number of single-base polymorphisms, two mutant forms of HNF3␤ were found (Fig. 3C). One cell
line, H60 (small cell lung cancer), harbors a heterozygous G-A
mutation at position 2916 (GenBank accession no. AF176110) resulting in a G-D amino acid change at codon 92 inside the NH2-terminal
activation domain (TAD II). A homozygous C deletion at codon 194
(position 3220 in AF176110) in the middle of the forkhead domain
was found in the SKLU-1 cell line (adenocarcinoma) leading to a
frameshift and a truncated 218-amino acid protein. Interestingly, in
neither cell line is HNF3␤ detectable at either the mRNA or at the
protein level, suggesting that the resulting mRNAs are unstable and/or
that the expression of HNF3␤ is silenced by another mechanism (e.g.,
promoter methylation). None of the cell lines with aberrant-sized
transcripts carried any mutations.
Promoter Methylation of the HNF3␤ Promoter. To establish
whether promoter methylation could play a role in silencing HNF3␤
expression, 4 HNF3␤ nonexpressor cell lines, A427, H596, SKLU-1,
and H322, were treated for 96 h with two different concentrations
(200 nM and 1 ␮M) of deoxyazacytidine, a demethylating agent.
Significant up-regulation of HNF3␤ mRNA was observed in 3 of the
4 cell lines examined (H322, A427, and H596; Fig. 4). SKLU-1 cells
had no detectable HNF3␤ mRNA expression regardless of treatment.
The promoter of HNF3␤ is very rich in CpG dinucleotides and meets
the criteria of a CpG island. We performed bisulfite sequencing of a
369-bp segment of the promoter as well as the 5⬘ untranslated region
of the first exon of the HNF3␤ gene (positions ⫹192 to 560). This
region contains 25 CpG dinucleotides that are putative targets for
promoter methylation. After bisulfite treatment, products were subcloned into pGEM-T vector, and multiple clones were sequenced. All
4 of the above cell lines showed evidence of methylation with the
densest methylation observed in the case of SKLU-1 (9 of 9 clones
sequenced had methylated Cs; 32–96% of all of the Cs methylated/
clone). Two of 6 clones of H596 (28 –52% Cs methylated), 5 of 8
clones of H322 (32– 40%), and 2 of 3 clones of A427 (16 –76% Cs
methylated) had evidence of methylation. These results strongly suggest that promoter hypermethylation could be a putative mechanism
for silencing of HNF3␤ expression.
Conditional Expression of HNF3␤ Leads to Growth Arrest. To
analyze additionally the role of HNF3␤ in airway epithelium, we set
out to establish an inducible cell line system. We selected a tetracycline-inducible system requiring the establishment of doubly stably
transfected cell lines (33) to develop lines with tight control of
expression and to avoid potential toxicity from the heavy metal
inducers. The H358 cell line was initially transfected with the Tet-Off
transactivator plasmid, and stably transfected inducible transactivator
clones were selected. For the second transfection, a plasmid construct
was generated consisting of the HNF3␤ cDNA under the control of
the tTA-regulated TRE-driven promoter (pTRE2HNF3B). Doubletransfectant transresponder clones were selected based on G418 and
puromycin resistance. From 50 clones screened, 2 clones were selected for additional analysis. These clones (clones 4 and 31) demonstrated a 6 – 8-fold induction of HNF3␤ RNA on withdrawal of
doxycycline from the medium, which translated into an ⬃3– 4-fold
increase in HNF3␤ protein. It was noted that HNF3␤ expression could
be fully suppressed even using a 1000-fold lower concentration of
doxycycline (1 ng/ml as opposed to 1 ␮g/ml) than recommended
(Clontech). When these lower concentrations were used, the induction
of HNF3␤ occurred significantly earlier, most likely because even
several washes of the cells might not reduce the doxycycline concentration sufficiently to allow transresponder gene activation when the
higher suppressive concentrations are used. At the lower doxycycline
concentrations used, the induction of HNF3␤ mRNA occurred as
early as 2 h after the withdrawal of doxycycline as assessed by
real-time PCR assay (Fig. 5A), whereas on the protein level, the
Fig. 5. Characterization of tetracycline-inducible cell lines. The Tet-off system was
used to generate doubly stably transfected H358 cells with an inducible hepatocyte nuclear
factor 3␤ (HNF3␤) construct. Two clones, clones 4 and 31, were selected for additional
studies. A, quantitative reverse transcription-PCR assay (TaqMan) confirming inducibility
of HNF3␤ RNA as early as 2 h after withdrawal of doxycycline (Clone 4). Identical results
were obtained with clone 31. B, Western blots demonstrate strong inducibility of HNF3␤
on withdrawal of doxycycline from the medium. Induction occurs as early as 16 h after
doxycycline withdrawal.
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HNF3␤ IS A TUMOR SUPPRESSOR IN LUNG CANCER
induction is clearly detectable by Western blotting as early as 16 h
after the withdrawal of doxycyline from the medium (Fig. 5B). Of
note is that this degree of induction is very similar to the level of
HNF3␤ induction obtained after C/EBP␣ expression in the ppc22transfected H358 cell line.
In both of these clones, induction of HNF3␤ protein led to very
substantial growth reduction noticeable as early as within 7 days (Fig.
6A). In fact, in 1 of the clones (clone 31), after day 4 of HNF3␤
induction, no additional increase in cell numbers was noted, and by
day 14, no viable cells could be seen. In clone 4, the growth reduction
was very substantial, but slow cell proliferation did continue despite
induction of HNF3␤. As expected, no change in cell proliferation was
noted on the withdrawal of doxycycline from the medium in the
parental cell line.
Changes in cell proliferation and cell cycle profile were also assessed in a bromodeoxyuridine proliferation assay (Fig. 6B). Although
no change in cell cycle profile was noted on days 2 or 4 (data not
shown), by day 7, a very significant increase in the fraction of
apoptotic cells was noted. This was accompanied by a depletion of
cells in G0/G1 and S phase and an increase in the number of cells in
G2-M phase. These findings are suggestive of G2-M arrest and apoptosis as a result of HNF3␤ induction. The changes were slightly more
prominent in clone 31 than in clone 4. No change in the cell cycle
profile or in the rate of apoptosis was noted in the control cell line.
Changes in the rate of apoptosis were also determined by annexin/
propidium iodide flow cytometry. These studies have shown that by
day 7 of HNF3␤ induction, there was a highly significant increase in
the rate of cell death in both clones (from 3 to 20% apoptotic cells in
clone 31; Fig. 6C). No change in the rate of apoptosis was seen on
doxycyline withdrawal in the parental cell line.
We have also performed methylcellulose-based clonogenic assays
to assess the ability of H358 cells to form colonies with and without
the induction of HNF3␤ expression (Fig. 6D). In clone 4, we observed
a significant reduction in the colony-forming ability of the cells when
grown in the absence of doxycycline. Interestingly, clone 31 cells
were not able to form colonies even in the presence of doxycycline,
suggesting that possibly even a slight “leakiness” of HNF3␤ expression might lead to very substantial reduction of colony-forming ability.
DISCUSSION
We previously showed that C/EBP␣ is a tumor suppressor in lung
cancer and demonstrated strong growth-inhibitory activity of C/EBP␣
in lung cancer cell lines (20). In the present study, we have identified
transcriptional changes secondary to conditional expression of
C/EBP␣ in a C/EBP␣ nonexpressor lung adenocarcinoma cell line,
H358. The tight regulation and strong inducibility of C/EBP␣ expression proved to be optimal for this study. As shown in Tables 1 and 2,
the changes in gene expression levels were marked and highly consistent in-between triplicate samples but only for a very limited set of
genes, suggesting that we were able to identify the initial wave of
transcriptional changes as a result of C/EBP␣ expression.
The genes identified this way also correlate very well with prior
knowledge about the function of C/EBP␣. Three groups of genes
stand out from the list of up-regulated genes. The first is genes
involved in acute phase reaction, such as interleukin 8, COX-2, and
numerous tumor necrosis factor-inducible genes. It is well known that
C/EBP family members regulate the acute phase response (34 –36). In
fact, many acute phase genes have both C/EBP as well as nuclear
factor ␬B binding sites in their promoters, suggesting that C/EBP
proteins and nuclear factor ␬B cooperatively regulate the acute phase
response (37– 40). In addition, prior studies have also shown that
C/EBP␣ can directly regulate a number of acute phase genes in
hepatocytes (41). Secondly, many genes up-regulated by C/EBP␣
play a role in terminal metabolism, such as enzymes of metabolic
pathways (hepatic dihydrodiol dehydrogenase, folate receptor, and
quinone oxidoreductase) and are suggestive of the transcriptional
profile of a more differentiated cell. The third group of genes (such as
adipophilin and ceramide glucosyltransferase) is involved in lipid
metabolism. For instance, adipophilin, a gene up-regulated by
C/EBP␣, is a prominent protein component of lipid storage droplets
and is thought to be necessary for the formation and cellular function
of these structures (42, 43). It is very interesting to note that the
induction of C/EBP␣ in the H358 cell line did indeed lead to the
appearance of lipid droplets in the cytoplasm as determined by electron microscopy (20), a feature of more mature pneumocytes. C/EBP␣
plays a major role in the development of preadipocytes to adipocytes
and is known to regulate the expression of many genes involved in
lipid metabolism in adipose tissues (44 –50). It might not be surprising
that C/EBP␣ plays a similar role in alveolar cells, where the production of lipids in the form of surfactant is critical to the proper
functioning of the airway epithelium. Also, both of these sets of genes
appear to be markers of a more differentiated cellular state. C/EBP␣
is a critical differentiation factor in a number of cell types, such as
hepatocytes, adipocytes, and myeloid cells (44, 51–54). On the basis
of our findings, it is likely to play a similar role in airway epithelial
cells as well. A hyperproliferation of type II pneumocytes has been
observed in C/EBP␣ knockout mice supporting such a role (19). In
fact, in previous studies, we have described intracellular changes
detected by electron microscopy suggestive of airway epithelial differentiation in the identical cell line system, where these transcriptional profiling studies were done (20). Also, a strikingly large number of C/EBP␣-repressed genes are proangiogenic factors or growth
factors. These findings are consistent with the role of C/EBP␣ in
growth arrest (29, 31, 32, 55).
A validation of our hypothesis that using such an approach would
enable us to identify direct C/EBP␣ targets is that a number of genes
identified are known to be regulated directly by C/EBP members
(such as interleukin 8 and COX-2; Refs. 56 and 57). Additional
validation comes from a study (58) in which primary human CD34⫹
cells were transduced with a retroviral construct that expresses the
C/EBP␣ cDNA fused in-frame with the estrogen receptor ligandbinding domain. In these cells, the addition of estradiol leads to
granulocytic differentiation. This system was used to identify target
genes of C/EBP␣ in primary human hematopoietic cells by the use of
Affymetrix oligonucleotide arrays. Quite strikingly, many of the regulated genes (e.g., tumor necrosis factor-inducible gene 14, COX-2,
HM74, glutaredoxin, ARF-like protein, adipophilin, and p63) identified were common to the C/EBP␣ target genes identified in our study.
Our transcriptional profiling study identified also HNF3␤ (also
known as Foxa2) as one of the most highly induced downstream
targets of C/EBP␣ in lung cancer cells. This finding is particularly
intriguing given the known function of HNF3␤ in foregut development and transcriptional regulation in the mature airway epithelium
(10, 59, 60). HNF3␤ is a member of the forkhead family of transcription factors. The amino acid sequence of HNF3␤ is highly conserved
(97.8% homology with between the human and rat orthologue; Ref.
27). HNF3␤ binds DNA through a homologous winged helix motif
common to a number of proteins known to be critical for determination of events in embryogenesis, the forkhead box (61). Targeted
disruption of HNF3B results in embryonic lethality with defective
development of the foregut endoderm (62). In the adult, HNF3␤
regulates the transcription of numerous liver-enriched genes, and the
HNF3 proteins play a pivotal role in the regulation of metabolism and
in the differentiation of metabolic tissues such as the pancreas and
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HNF3␤ IS A TUMOR SUPPRESSOR IN LUNG CANCER
Fig. 6. Induction of hepatocyte nuclear factor 3␤
(HNF3␤) leads to growth arrest, apoptosis, and loss
of clonogenic potential. A, growth curves of clones
4 and 31 of HNF3␤-inducible H358 cells are
shown. While cells grown in the presence of doxycycline (clone 4⫹ and clone 31⫹) demonstrate
exponential growth, the withdrawal of doxycycline
from the medium (clone 4⫺ and clone 31⫺) leads
to growth arrest noticeable by day 7 of culture. B,
bromodeoxyuridine (BrdU) proliferation assays
performed on day 7 of induction demonstrate no
changes in the control H358 cell line (a), whereas
in both clones 4 and 31 (b and c), an increase in the
number of apoptotic cells as well as an accumulation of cells in the G2-M phase are noted (A ⫽ apoptosis, G1 ⫽ G0/G1, G ⫽ G2-M, and S ⫽ S phase).
Representative histograms of uninduced (d) and
induced (e) clone 31 cells are shown (the gates are
R1 ⫽ S phase, R2 ⫽ G2-M, R3 ⫽ G0/G1,
R4 ⫽ apoptotic cells). C, annexin/propidium iodide
flow cytometry studies performed on day 7 of culture confirm marked increase in the number of
apoptotic cells after the withdrawal of doxycyline
(a, clone 4, and b, clone 31). Doxycycline treatment has no effect on the rate of apoptosis in the
parental H358 cell line (c). Representative histograms of uninduced (d) and induced (e) H358
pTRE2HNF3B/4 cells. D, methylcellulose clonogenic assays were performed by plating 1000 cells/
plate in 1.25% methylcellulose. Experiments were
done six times. The number of colonies was
counted after 14 days. H358 6108/4 cells readily
formed colonies in the presence of doxycycline,
whereas significantly fewer colonies were formed
in the absence of doxycycline.
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HNF3␤ IS A TUMOR SUPPRESSOR IN LUNG CANCER
liver (63, 64). HNF3␤ is known to be a key regulator of airway
epithelial differentiation (21, 23, 30, 65). It influences the expression
of a number of target genes in the respiratory epithelium, such as
thyroid transcription factor-1, another master gene of airway epithelial
cell differentiation, surfactant protein-B, and Clara-cell secretory protein (21, 22, 66). HNF3␤ is expressed at the onset of lung morphogenesis (day 10 gestation) and throughout lung development (65, 67).
It is expressed at highest levels in proximal bronchial and bronchiolar
epithelial cells, but it is also expressed in type II pneumocytes. It has
been shown previously that members of the C/EBP family, including
C/EBP␣, bind and activate the LF-H3␤ site of the HNF3␤ promoter,
which might mediate cell-specific expression of HNF3␤ (25). Our
studies using both in vitro electrophoretic mobility as well as in vivo
chromatin immunoprecipitation assays clearly show direct binding of
this promoter element by C/EBP␣. This finding further suggests that
C/EBP␣ might act as a master regulator of airway epithelial differentiation not only by controlling the expression of sets of genes
characteristic of the differentiated alveolar pneumocytes but also by
turning on a secondary wave of differentiation events by inducing the
expression of HNF3␤ itself.
Although several other members of the forkhead family of proteins,
such as FoxO family members, have been implicated in carcinogenesis (68, 69), to our knowledge, no studies have ever investigated the
role of HNF3␤ in any type of cancer. Our results show that whereas
HNF3␤, as expected, is expressed strongly in normal lung, its expression is lost in more than half of all of the lung cancer cell lines.
Genomic sequencing of 31 lung cancer cell lines also identified two
mutant forms of HNF3␤. To our knowledge, these are the first mutant
forms of HNF3␤ ever described in cancer. Treatment of HNF3␤
nonexpressor cell lines with the demethylating agent deoxyazacytidine also leads to up-regulation of HNF3␤ expression in 3 of 4 cell
lines examined, suggesting that promoter methylation might be a
mechanism of silencing. Indeed, bisulfite sequencing of these HNF3␤
nonexpressing cell lines revealed evidence of promoter hypermethylation in a CpG-rich segment of the promoter/exon 1 junction in all of
the 4 cell lines examined. These results do suggest both point mutations and promoter hypermethylation as genetic/epigenetic mechanisms leading to aberrations of the HNF3␤ transcriptional program of
airway epithelial differentiation. It is interesting to note that a recent
high-frequency allelotyping study did find an ⬃40% rate of loss of
heterozygosity of the chromosomal region of HNF3␤, 20p11 in nonsmall cell lung cancers suggestive of the presence of an as yet
unidentified tumor suppressor gene in this region (6). Our results
suggest that HNF3␤ is a strong candidate as the 20p11 tumor suppressor. We are conducting additional studies to define the full spectrum of mutations, loss of heterozygosity, and promoter hypermethylation in primary non-small cell lung cancer specimens to fully
establish the role of HNF3␤ as a novel tumor suppressor.
We have also generated a doubly stably transfected, tetracyclineregulatable cell line system for the conditional expression of HNF3␤
to study its cellular effects. This cell line system allowed us to show
that conditional expression of HNF3␤ leads to proliferation arrest,
apoptosis, and loss of clonogenic ability, further corroborating our
hypothesis that HNF3␤ might act as a tissue-specific tumor suppressor in the development of lung cancer. Although HNF3␤ has a
well-established role in driving maturation of certain cell types, its
role in growth control or apoptosis has not been demonstrated as of
yet. On the other hand, other forkhead family members are known
mediators of cell signaling pathways in control of cell cycle progression (70, 71). This inducible model system should also allow us to
define the transcriptional program of differentiation of airway epithelial cells as well as the pathways involved in cell cycle arrest and
apoptosis as result of HNF3␤ induction. We are in the process of
performing transcriptional profiling studies on the above-described
cell line system to fully delineate the transcriptional changes after
HNF3␤ induction.
In summary, our studies have identified the downstream targets of
C/EBP␣, a candidate tumor suppressor in lung cancer, in neoplastic
airway epithelial cells. These studies led to the identification of
HNF3␤, a known differentiation factor in airway epithelium as a
downstream target of C/EBP␣. Additional studies of HNF3␤ show
down-regulation of its expression in lung cancer cell lines, identify
novel mutant forms of HNF3␤ in lung cancer, and demonstrate that
promoter methylation is a putative mechanism for epigenetic silencing
of the HNF3␤ gene. Our studies also show strong growth-inhibitory
and proapoptotic properties of HNF3␤ and suggest that HNF3␤ can
indeed act as a tissue-specific tumor suppressor in lung cancer.
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A Transcriptional Profiling Study of CCAAT/Enhancer
Binding Protein Targets Identifies Hepatocyte Nuclear Factor
3 β as a Novel Tumor Suppressor in Lung Cancer
Balazs Halmos, Daniela S. Bassères, Stefano Monti, et al.
Cancer Res 2004;64:4137-4147.
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