A Transcriptional Profiling Study of CCAAT/Enhancer Binding Protein Targets Identifies Hepatocyte Nuclear Factor 3 As a Novel Tu

[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. Basse`res,1 Stefano Monti,4 Francesco D‘Alo´,1 Tajhal Dayaram,1 Katalin Ferenczi,2 Bas J. Wouters,1 Claudia S. Huettner,5 Todd R. Golub,4 and Daniel G. Tenen3 1Division 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 heterozygosity can be detected, suggesting that many tumor suppressor genes remain unidentified (6). ␣ We showed previously that CCAAT/enhancer binding protein (C/ Aberrant differentiation is one of the hallmarks of cancers, and the ␣ EBP ), a tissue-specific transcription factor, is a candidate tumor sup- contribution of differentiation arrest to the multistep carcinogenesis pressor in lung cancer. In the present study, we have performed a tran- process has been accepted widely recently (7). In particular, it is scriptional profiling study of C/EBP␣ target genes using an inducible cell line system. This study led to the identification of hepatocyte nuclear increasingly clear that aberrant regulation of transcriptional control factor 3␤ (HNF3␤), a transcription factor known to play a role in airway pathways of normal differentiation is one of the most common ab- differentiation, as a downstream target of C/EBP␣. We found down- normalities in hematological malignancies, such as acute myeloid regulation of HNF3␤ expression in a large proportion of lung cancer cell leukemia (8). The transcriptional control of differentiation pathways lines examined and identified two novel mutants of HNF3␤, as well as in airway epithelium is poorly understood, and its abnormalities in the hypermethylation of the HNF3␤ promoter. We also developed a tetracy- aberrant differentiation of lung cancers are largely unknown. A num- cline-inducible cell line model to study the cellular consequences of ber of key transcription factors, such as thyroid transcription factor-1, HNF3␤ expression. Conditional expression of HNF3␤ led to significant helix-loop-helix transcription factors, forkhead transcription factors, growth reduction, proliferation arrest, apoptosis, and loss of clonogenic such as hepatocyte nuclear factor 3␤ (HNF3␤), and CCAAT/enhancer ␤ ability, suggesting additionally that HNF3 is a novel tumor suppressor in binding protein ␣ (C/EBP␣) are implicated in the complex develop- lung cancer. This is the first study to show genetic abnormalities of mental genetic instruction of lung morphogenesis and cell lineage lung-specific differentiation pathways in the development of lung cancer. 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 INTRODUCTION hepatocytes, myeloid cells, and adipocytes (11). C/EBP␣ was also Lung cancer remains a public health problem with ϳ170,000 cases identified recently as a novel tumor suppressor in acute leukemia (12). in the United States per year (1). It is the leading cause of cancer Recurrent mutations of C/EBP␣ have been identified by a number of deaths in both men and women with a 5-year survival rate of only groups in subtypes of acute leukemia (13, 14). C/EBP␣ is expressed 15%. A recent analysis of trials performed over the last 30 years strongly in the lung, more specifically in both type II pneumocytes as demonstrated clearly that only minimal progress has been made in the well as cells of the bronchial epithelium (15, 16). It also regulates the treatment of this disease (2). The disappointing results of recent expression of several genes, directly or indirectly, during lung differ- studies have led to the realization that we have reached a “chemo- entiation, including surfactant B and uteroglobin (17, 18). Specific therapy efficacy plateau” (3). Additional progress in the treatment of lung abnormalities, such as an abnormal proliferation of type II ␣Ϫ Ϫ lung cancer will depend critically on a better understanding of the pneumocytes, have been described in C/EBP / knockout mice ␣ molecular events leading to the development of epithelial neoplasias (19), suggesting that C/EBP is important for normal lung develop- as well as the critical pathways sustaining the neoplastic, invasive ment and the maintenance of normal alveolar structure. It is postulated ␣ phenotype. Our understanding of the genetic abnormalities underlying that this hyperproliferation is because in the absence of C/EBP , the the development of lung cancer remains quite limited (4, 5). Both a alveolar type II cells can continue to proliferate. In previous studies, ␣ number of tumor suppressors, such as p53, p16, and retinoblastoma, we have demonstrated that C/EBP is down-regulated in a large as well as several proto-oncogenes, such as k-ras and the epidermal proportion of lung cancers (20). We also developed a tightly regulated, highly inducible cell line model system using a zinc-inducible growth factor receptor, are known to play a role, but no abnormalities metallothionein promoter-based system. With the use of this stably of lung-specific tumor suppressors or proto-oncogenes have been transfected cell line system, we have shown that the induction of identified yet. A recent high-frequency allelotyping study demon- C/EBP␣ expression leads to growth arrest, apoptosis, and cellular strated that in individual lung cancers, as many as 22 areas of loss of changes suggestive of differentiation, all supporting its role as a candidate tumor suppressor gene. Received 12/30/03; revised 3/19/04; accepted 4/9/04. ␣ Grant support: NIH Grant Specialized Programs of Research Excellence in Lung To gain additional insight into the downstream effects of C/EBP Cancer PA20-CA090578-01A1 (D. Tenen), American Association for Cancer Research/ expression, we have performed transcriptional profiling studies on this Cancer Research Foundation of America/Astra-Zeneca Young Investigator Award for inducible cell line system. From the many C/EBP␣-regulated genes Translational Lung Cancer Research (B. Halmos), and the Clinical Investigator Training ␤ Program of Harvard/MIT (B. Halmos). identified, we have focused our additional studies on one, HNF3 Note: Supplementary data for this article can be found at Cancer Research Online (also known as Foxa2), given its known important role in airway (http://cancerres.aacrjournals.org). epithelial differentiation (21–23). Our studies demonstrate down- 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 regulation and novel mutations of HNF3␤ in lung cancer cell lines. 18 U.S.C. Section 1734 solely to indicate this fact. We also identified hypermethylation of the promoter region of the Requests for reprints: Daniel G. Tenen, Harvard Institutes of Medicine, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115. Phone: (617) 667-5561; HNF3B gene as a novel mechanism for epigenetic silencing of Fax: (617) 667-3299; E-mail: [email protected]. HNF3␤ expression. To assess the functional consequences of HNF3␤ 4137

expression, we also generated a tetracycline-regulated inducible cell dilution of a monoclonal mouse anti-␤-tubulin (both from Sigma Chemicals) line system for the conditional expression of HNF3␤. With the use of were used, respectively. Detection was performed using enhanced chemilumi- this system, we demonstrate that induced expression of HNF3␤ in the nescence (Amersham Life Science, Piscataway, NJ). H358 lung cancer cell line leads to growth reduction, proliferation Electrophoretic Mobility Shift Assay. Nuclear extracts were prepared ␮ arrest, apoptosis, and loss of clonogenic ability, supporting its iden- from untreated or 25 M CdSO4-treated H358 ppc18-transfected or ppc22- tification as a novel tumor suppressor in lung cancer. transfected cells after 16 h of induction as described previously (24). The gel shift assay consisted of a binding reaction allowing the formation of DNA- protein complexes, which were then separated from unbound probe by native MATERIALS AND METHODS gel electrophoresis. 50 ng of double-stranded DNA probe containing the 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, 5Ј-AATTCCCTGTTTGTTTTAGTTACGAAATGCGTTG-3Ј; and H23, and H441; adenocarcinoma, bronchoalveolar type, H358, A549, and antisense, 5Ј-AATTCAACGCATTTCGTAACTAAAACAAACAGGG-3Ј. Bind- H322; 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 ϫ1

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; Ϫ8 Ϫ8 fetal bovine serum, 2.8 mM glutamine, 10 M hydrocortisone, 10 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 for6hatϪ80°C. grown to 60% confluence in RPMI containing 10% fetal bovine serum. Chromatin Immunoprecipitation Assays. Mock-transfected and ppc22- ␮ Triplicate plates were induced by the addition of 100 M of ZnSO4 for 6 and ␮ transfected cells were treated with 25 M CdSO4 for 48 h. Chromatin immu- 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 ϫ 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࿝intensity, where chip࿝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, 5Ј-GCCTCCACATCCAAACACC-3Ј; and antisense, 5Ј- minimum signal to 10 and the maximum signal to 20,000; (b) excluding genes CTCTCCGACTCCTCAGACACC-3Ј, which amplify a region that spans bps for which the fold change (i.e., the maximum:minimum threshold value) was Ϫ75 to Ϫ104 (containing the C/EBP binding site) of the HNF3␤ promoter. Ͻ3; and (c) excluding those genes for which the ␦ change (i.e., the difference Sequencing. The promoter region and three exons of HNF3␤ were se- between the maximum and minimum threshold values) was Ͻ100. 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 6h” 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 ream- in R (an open-source statistical package) were used. plified twice and resequenced in both directions. In cases where the abnormal 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. Ј Ј linepropanesulfonic acid/formaldehyde gels and transferred to MagnaGraph Deoxyazacytidine Treatment. The 5 -aza, 2 deoxycytidine was a kind 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 3Ј performed as described below. untranslated region of the human COX-2 mRNA subcloned into pGEM-T, cut Promoter Methylation Studies. Bisulfite sequencing was performed ac- 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 gift of Dr. Isaiah Fidler, M. D. Anderson Cancer Center, Houston, TX). bisulfite treated and purified (Promega Wizard DNA Clean-UP System; Pro- 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, 5Ј-TTGGAA- phoresed in 10 or 12% polyacrylamide minigels. A 1:2000 dilution of a GATAGAGAGGATAGA-3Ј; and antisense, 5Ј-CCCCTCCCTATTAC- polyclonal rabbit anti-C/EBP␣ antibody and a 1:2000 dilution of a polyclonal CAATTCAA-3Ј. The amplified PCR product was subcloned into pGEM-T goat anti-HNF3␤ antibody (both from Santa Cruz Biotechnology, Santa Cruz, vector, and clones were sequenced with the use of Sp6 antisense primer. CA), and a 1:1000 dilution of a monoclonal mouse anti-␤-actin and a 1:100 Peripheral blood mononuclear cell DNA served as negative control, whereas 4138

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2004 American Association for Cancer Research. HNF3␤ IS A TUMOR SUPPRESSOR IN LUNG CANCER universally methylated DNA (CpGenome Universally Methylated DNA; In- transcriptional profiling studies to identify downstream targets of tergen) was used as positive control. C/EBP␣ and gain additional functional insights into the mechanisms Plasmid Generation. The full-length cDNA of rat HNF3␤ was a kind gift leading to C/EBP␣-induced growth arrest and differentiation. of Robert Costa (University of Illinois, Chicago, IL). The 1.6-kb rat HNF3␤ To perform oligonucleotide array analysis, ppc22-transfected H358 cDNA was released from the pGEM-T vector backbone by digestion with cells were induced to express C/EBP␣ by the addition of zinc for 6 EcoRI. The fragment was blunt ended and subsequently subcloned into the and 12 h, respectively. Control cells (0 h induction) were grown dephosphorylated PvuII site of the multiple cloning site of vector pTRE2puro (Clontech, Palo Alto, CA) to generate plasmid pTRE2HNF3B. without the addition of zinc to the medium. Conditional expression of ␣ Generation of Inducible Cell Lines. H358 cells were transfected with the C/EBP was confirmed by Northern as well as Western blotting (Fig. transactivator Tet-off plasmid using lipofectamine transfection (Lipo- 1, A and C). Transcriptional profiling was then performed on total fectAMINE PLUS; Invitrogen Life Technologies, Inc., Carlsbad, CA) accord- cellular RNA using Affymetrix Hu-95 chips. The raw expression data ing to the manufacturer’s instructions. Clones were selected on the basis of were preprocessed, rescaled, and filtered as described in “Materials G418 resistance (G418 concentration of 500 ␮g/ml). Highly repressible clones and Methods.” Analysis of the results was performed by GeneCluster were identified by transient transfection with a TRE-luciferase reporter plas- software (data on the entire gene set is available as Supplementary mid (pTRE2pur-luc; Clontech). Clones were screened by performing luciferase Data). Experiments were carried out in triplicates, with three time assays after the cells were grown for 24 h with or without addition of points for each experiment (0, 6, and 12 h). We were interested in doxycycline (1 ␮g/ml). Clone 6 showed the highest level of repressibility after identifying those genes manifesting a differential expression between doxycycline withdrawal and was selected for additional studies. The second round of transfection with the pTRE2HNF3B plasmid was performed using 0hand6htoidentify the earliest wave of transcriptional changes 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 Se- quence 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, 5Ј-AAGATGGAAGGGCACGAGC-3Ј; reverse primer, 5Ј-TG- TACGTGTTCATGCCGTTCA-3Ј; and probe, 5Ј-TCCGACTGGAGCAGC- TACTATGCAGAGC-3Ј. Rat HNF3␤: forward primer, 5Ј-CTGAAGC- CCGAGCACCAT-3Ј; reverse primer, 5Ј-GCTGCTCGGAGGGACATGA-3Ј; and probe, 5Ј-TCCGACTGGAGCAGCTAC-3Ј. 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.

RESULTS Transcriptional Profiling of C/EBP␣-Inducible H358 Cells. As described previously (20), we have generated a stably transfected cell Fig. 1. A, Northern blot analysis confirms the gene chip results. Experiments were line from H358 adenocarcinoma cells using a mammalian expression performed with ppc18 and ppc22-transfected H358 cells. Cells were induced by the ␣ ␮ vector construct (ppc22; Ref. 29) harboring the rat C/EBP gene addition of 100 M of ZnSO4 to the medium for 6 and 12 h, respectively. Control cells under the control of the zinc-inducible metallothionein prom (MT-C/ 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 EBP ). We also generated control cell lines by stable transfection of binding protein ␣ (C/EBP␣), interleukin 8, cyclooxygenase-2, and hepatocyte nuclear H358 cells with a control vector (ppc18). H358 cells have practically factor 3␤ (HNF3␤). No induction is seen in ppc18-transfected cells (MOCK), whereas ␣ no detectable native C/EBP␣ expression either on the RNA or protein 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 level. In this cell line system, marked increase in C/EBP␣ mRNA can demonstrate equal loading. B, TaqMan assay confirms the induction of HNF3␤ mRNA by be detected as early as 3 h after induction by addition of zinc or 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 cadmium sulfate to the medium. The tight regulation and very strong medium. Western blots on 20 ␮g of whole cell lysate demonstrate an ϳ3-fold induction inducibility of this cell line system appeared to be ideally suited for of HNF3␤ protein by 24 h of induction. 4139

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2004 American Association for Cancer Research. HNF3␤ IS A TUMOR SUPPRESSOR IN LUNG CANCER most likely highly enriched in direct targets of C/EBP␣. We also relevant set of genes, we used two additional sets of criteria. We performed a comparison between the uninduced control and induced selected only those genes where the level of induction or repression “pooled” specimens (6 samples, 3 at 6 h and 3 at 12 h). This was a minimum 3-fold (mean difference using means of triplicate comparison yields a set of genes that is up- or down-regulated con- experiments). We also excluded genes where the absolute difference sistently between 6 and 12 h, therefore is less likely to harbor between induced/uninduced was Ͻ500 fluorescence intensity signal false-positive candidate genes and might also identify important up- units, thereby removing genes where the changes might appear sig- or down-regulated genes that might not be significantly changed at 6 h nificant based on fold-differences, but this is likely to be biologically but become evident by 12 h. We felt the two comparisons would irrelevant or spurious secondary to low levels of expression. The top provide complementary information and would also serve as internal genes obtained this way are shown in Tables 1 and 2. Of note is that controls for the validity of our findings. We used paired t-statistic to a very strong overlap was noted between the 0 versus 6 h and the 0 rank genes. From the ranked list of genes, we excluded genes with a versus pooled comparisons (34 of the top 45 up-regulated and 23 of t-statistic Ͻ1 (representing no association). To arrive at a biologically 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. Fold Rank Score change Description Accession no. Cluster 1 20 25.48 Tumor necrosis factor-inducible gene 14 (TSG-14) M31166 Acute phasea 2 16.683 15.85 Cyclooxygenase-2 (hCox-2) U04636 Acute phasea 3 9.771 3.4 BTG1 X61123 a 4 9.027 8.63 Folate receptor ␣ U78793 Metabolisma 5 8.375 3.78 E4BP4 X64318 a 6 8.087 9.94 Integrin, ␣ subunit X68742 a 7 7.777 4.56 Semaphorin E AB000220 Acute phasea 8 7.137 6.61 Hepatocyte nuclear factor-3 ␤ AB028021 a 9 6.623 3.52 Cell adhesion kinase ␤ (CAK␤) U43522 a 10 6.23 6 RGP4 mRNA U27768 11 5.859 3.21 hbc647 U68494 12 5.644 13.96 Protease inhibitor 12 (PI12; neuroserpin) Z81326 13 5.527 4.62 Insulin-like growth factor-binding protein-3 M35878 a 14 5.451 5.99 Zinc finger protein (clone 647) X16282 15 5.301 4.55 Glutaredoxin X76648 Metabolisma 16 5.273 5.03 HM74 D10923 a 17 5.189 3.86 Helix-loop-helix basic phosphoprotein (G0S8) L13463 a 18 5.071 4.72 MEGF9 AB011542 a 19 5.017 3.84 Interleukin 4 receptor X52425 Acute phasea 20 4.828 32.06 Chemokine exodus-1 U64197 Acute phase 21 4.467 5.98 GC-Box binding protein BTEB2 D14520 a 22 4.354 39.7 Interleukin 8 (IL8) M28130 Acute phasea 23 4.273 3.33 Low-Mr GTP-binding protein (RAB32) U59878 a 24 4.203 3.27 Putative endothelin receptor type B-like protein U87460 a 25 4.117 4.41 Cytokine (GRO-␤) M36820 Acute phasea 26 4.06 3.62 HRIHFB2017 AB015331 a 27 4.02 6.06 Monocarboxylate transporter 2 (hMCT2) AF058056 Metabolisma 28 3.702 10.65 ␤-Thromboglobulin-like protein M17017 Acute phasea 29 3.513 3.37 Jak2 kinase AF058925 a 30 3.386 6.02 Receptor protein-tyrosine kinase (HEK8) L36645 31 3.215 4.23 Bone morphogenetic protein 2A (BMP-2A) M22489 Acute phasea 32 3.189 91.14 Spliceosomal protein (SAP 62) L21990 a 33 3.136 4.86 Erythroblastosis virus oncogene homolog 2 (ets-2) J04102 a 34 2.95 7.141 1-Phosphatidylinositol-4,5-Bisphosphate AL022394 a Phosphodiesterase ␥ 35 2.912 37.19 Heme Oxygenase 1 Z82244 Metabolism 36 2.814 18.18 Cytokine (GRO-␥) M36821 Acute phasea 37 2.77 19.05 Hepatic dihydrodiol dehydrogenase gene U05861 Metabolisma 38 2.644 4.77 Adipophilin X97324 Lipida 39 2.589 4.61 Tyrosine kinase a 40 2.518 8.85 Ets transcription factor PDEF (PDEF) AF071538 41 2.393 88.66 Cyritestin protein X89654 a 42 2.344 8.11 Apolipoprotein apoC-IV (APOC4) U32576 Lipid 43 1.949 144.7 HOX11 homeodomain {homeobox} S38742 a 44 1.778 4.01 Wiskott-Aldrich syndrome protein (WASP) U12707 45 1.439 9.99 Fas (Apo-1, CD95) X89101 Pooled analyses 1 5.024 3.18 ADP-ribosylation factor-like protein 4 U73960 Lipid 2 3.867 3.26 Ceramide glucosyltransferase D50840 Lipid 3 3.448 3.17 p63 mRNA for transmembrane protein X69910 4 2.467 3.39 Small GTP-binding protein U57094 5 2.273 3.65 Interleukin 1 receptor M27492 Acute phase 6 2.148 3.16 UDP-N-acetylglucosamine pyrophosphorylase AB011004 Metabolism 7 2.045 4.49 RDC-1 POU domain containing protein X64624 8 1.975 3.7 MDC-3.13 isoform 2 mRNA AF099935 9 1.874 4.43 Quinone oxidoreductase2 (NQO2) U07736 Metabolism a Also identified in pooled analysis. 4140

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. Fold Rank Score change Description Accession No. Cluster 1 13.371 6.66 Kidney ␥-glutamyl transpeptidase type II M30474 a 2 11.877 4.55 Stanniocalcin-related protein AF098462 a 3 11.574 5.54 Dual specificity phosphatase MKP-5 AB026436 a 4 8.82 3.06 Growth arrest and DNA-damage-inducible protein (gadd45) M60974 a 5 8.06 4.51 Proteinase activated receptor-2 U67058 a 6 6.982 7.59 Connective tissue growth factor X78947 Proliferationa 7 6.967 10.31 CPE-receptor AB000712 a 8 6.457 4.44 CYR61 Y11307 Proliferationa 9 6.239 3.47 Breast cancer antiestrogen resistance 3 protein (BCAR3) U92715 a 10 6.224 3.11 Cisplatin resistance associated alpha protein (hCRA alpha) U78556 11 6.067 3.43 Id-2H D13891 12 5.615 12.75 Tyrosine hydroxylase type 4 M17589 a 13 5.562 7.74 ADP ribosylation factor-like protein AB016811 a 14 5.429 3.51 Protein kinase, Dyrk2 Y09216 a 15 5.052 6.35 Putative tetraspan transmembrane protein L6H (TM4SF5) AF027204 a 16 4.86 3.27 Tob D38305 17 4.635 3.06 GAR22 protein Y07846 a 18 4.614 77.25 High-sulphur keratin X63755 a 19 4.426 9.02 Transmembrane receptor (ror1) M97675 a 20 4.21 7.63 Testicular inhibin beta-B-subunit M31682 a 21 3.755 8.53 FGF-9 D14838 Proliferationa 22 3.717 16.34 RGP3 U27655 a 23 3.695 3.63 Transcriptional activator U49857 a 24 3.485 3.7 HRY gene L19314 25 3.375 3.51 MAL gene exon 1 X76220 26 3.327 5.25 Smad6 AF035528 27 3.237 38.22 Human uromodulin (Tamm-Horsfall glycoprotein) M15881 a 28 2.941 4.34 Frizzled-7 AB017365 29 2.918 3.01 Ets-related transcription factor (ERT) AF017307 30 2.76 3.56 MAD-related gene SMAD7 (SMAD7) AF010193 a 31 2.469 49.75 Ras-related rho mRNA M12174 a 32 2.297 6.43 CCCAAT/enhancer binding protein ␣ Y11525 33 2.25 6.29 SH3 binding protein AB000462 34 2.241 3.28 Transcription factor ERF-1 U85658 35 2.111 3.06 Epidermal growth factor receptor (HER3) M34309 Proliferation Pooled analyses 1 6.926 3.17 Interferon-inducible 56 Kd protein M24594 2 5.294 3.24 uPA gene X02419 Proliferation 3 3.209 4.7 Lung amiloride sensitive Naϩ channel protein X76180 4 3.132 3.29 PTPL1-associated RhoGAP U90920 5 2.364 34.77 Thyroid transcript factor 1 X82850 6 2.017 3.55 RBP-MS/type 4 D84110 7 1.997 3.63 Trio U42390 8 1.805 7.74 Leucine zipper protein Z50781 9 1.777 3.07 CD97 gene exon 1 X94630 10 1.702 91.91 Choline kinase D10704 11 1.651 3.54 Luteinizing hormone, ␤ subunit 12 1.291 4.77 Squamous cell carcinoma of esophagus mRNA for GRB-7 D43772 SH2 domain protein a Also identified in pooled analysis. appeared in the 0 versus pooled most highly induced/repressed gene tumor necrosis factor-inducible gene 14, and exodus-1 among list); therefore, the pooled analysis contributed only a few genes to the others. A second group of genes comprises genes of metabolic lists. Such strong overlap does suggest that the gene set arrived at this pathways, such as hepatic dihydrodiol dehydrogenase, folate re- way represents an enriched set of genes regulated directly or indirectly ceptor, glutaredoxin, monocarboxylate transporter 2, and quinone by C/EBP␣ in H358 cells. oxidoreductase. The third group of genes includes genes known to To confirm the findings of the oligonucleotide array analysis, the be involved in fat metabolism or adipocyte differentiation, such as induction/repression of several genes was confirmed by Northern blot adipophilin, ADP-ribosylation factor-like protein-4, and ceramide analysis (Fig. 1A). As control, RNA was collected from H358 cells glucosyltransferase. Lastly, one of the most highly induced genes stably transfected with empty vector (ppc18) and treated in an iden- is HNF3␤. This finding is of particular importance because HNF3␤ tical fashion to cells collected for the oligonucleotide array analysis. is known to play a major role in the transcriptional control of Fig. 1 shows representative results for interleukin 8, COX-2, and cellular differentiation, including airway epithelial differentiation HNF3␤ genes. These studies confirmed the gene chip results in that (21, 30), thereby suggesting that C/EBP␣ might indeed act as all of the examined genes showed consistent findings with our gene master regulator of airway epithelial differentiation. chip data, and none of the examined genes appeared regulated by zinc itself, as demonstrated by the absence of induction in the control Our analysis identified also a set of highly repressed genes by (ppc18) cell line. C/EBP␣. Many of the repressed genes are growth factors, growth An analysis of the highly up-regulated genes demonstrated three factor receptors, or proangiogenic molecules, such as connective major clusters of genes in this set. First, many of the up-regulated tissue growth factor, Cyr61 (also called CCN1), fibroblast growth genes are acute phase reactants, such as interleukin 8, COX-2, factor-9, epidermal growth factor receptor, and urokinase-plas- 4141

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 unde- tectable 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 ϳ6-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 tran- Fig. 3. Hepatocyte nuclear factor 3␤ (HNF3␤) is down-regulated and mutated in lung scription-PCR assay, as well as Western blotting and showed that zinc cancer cell lines. A, Northern blot analysis of HNF3␤ expression in lung cancer cell lines. itself does not induce the expression of HNF3␤ (Fig. 1, B and C). The 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 degree of induction on the protein level is ϳ3-fold. Although the peak examined. Ethidium bromide staining of 28S RNA is shown as loading control. Lane 1, induction on the RNA level occurs by 6 h, on the protein level the 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; peak expression is somewhat delayed and occurs between 24 and 48 h. Lane 12, H460; Lane 13, H526; and Lane 14, H187. B, Western blot analysis of HNF3␤ The HNF3␤ promoter does have a putative C/EBP-binding site, and expression in lung cancer cell lines. Twenty of 22 cell lines examined had identical one report did show previously indirect evidence of C/EBP proteins 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; regulating the HNF3 promoter (25). In electrophoretic mobility shift Lane 6, H23; Lane 7, H441; Lane 8, SW900; Lane 9, H292; Lane 10, H596; Lane 11, assays, we demonstrated a strong increase in specific binding activity H460; Lane 12, Calu-6; and Lane 13, H520. C, sequencing of the HNF3␤ gene. The upon induction of C/EBP␣ (Fig. 2A). The specificity of binding was diagram shows the adopted sequencing strategy using seven primer sets to sequence the promoter, all of the coding sequences, and parts of the 3Ј untranslated region of the confirmed by supershift assays using a C/EBP␣ antibody. We con- HNF3␤ gene in 31 lung cancer cell lines. Also shown is a bar diagram of the structural firmed the same finding by chromatin immunoprecipitation assays 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 using primers flanking the putative C/EBP-binding site of the HNF3 at position 92, whereas in the SKLU-1 cell line, a homozygous base insertion leads to a promoter (Fig. 2B). These assays demonstrated direct C/EBP␣ bind- frameshift and a predicted premature termination at amino acid 218. 4142

of the first exon of the HNF3␤ gene (positions ϩ192 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). Double- transfectant 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) dem- 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 onstrated a 6–8-fold induction of HNF3 RNA on withdrawal of lines after 5Ј deoxy 2Ј azacytidine treatment. Cells were treated with 0.2 or 1 ␮M doxycycline from the medium, which translated into an ϳ3–4-fold deoxyazacytidine for 96 h. SKLU-1 cells had no detectable expression of HNF3␤, increase in HNF3␤ protein. It was noted that HNF3␤ expression could whereas H322, A427, and H596 cells demonstrated significant up-regulation of HNF3␤ mRNA on treatment. 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 genomic sequencing of the HNF3␤ gene to exclude the possibility that of HNF3␤ occurred significantly earlier, most likely because even these cell lines carry mutant forms of HNF3␤. The HNF3␤ gene is several washes of the cells might not reduce the doxycycline concen- located on chromosome 20p11 and has 3 exons. A sequencing strategy tration sufficiently to allow transresponder gene activation when the was adopted (27) using a set of seven primers to sequence a portion higher suppressive concentrations are used. At the lower doxycycline of the HNF3␤ promoter, exons 1 and 2, and all of the coding regions concentrations used, the induction of HNF3␤ mRNA occurred as of exon 3, as well as the first 68 bps of the 3Ј-untranslated region in early as 2 h after the withdrawal of doxycycline as assessed by 31 lung cancer cell lines. Besides a number of single-base polymor- real-time PCR assay (Fig. 5A), whereas on the protein level, the phisms, 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 Fig. 5. Characterization of tetracycline-inducible cell lines. The Tet-off system was 4 cell lines examined (H322, A427, and H596; Fig. 4). SKLU-1 cells 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 had no detectable HNF3␤ mRNA expression regardless of treatment. studies. A, quantitative reverse transcription-PCR assay (TaqMan) confirming inducibility The promoter of HNF3␤ is very rich in CpG dinucleotides and meets 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␤ the criteria of a CpG island. We performed bisulfite sequencing of a on withdrawal of doxycycline from the medium. Induction occurs as early as 16 h after 369-bp segment of the promoter as well as the 5Ј untranslated region doxycycline withdrawal. 4143

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2004 American Association for Cancer Research. HNF3␤ IS A TUMOR SUPPRESSOR IN LUNG CANCER induction is clearly detectable by Western blotting as early as 16 h C/EBP␣ can directly regulate a number of acute phase genes in after the withdrawal of doxycyline from the medium (Fig. 5B). Of hepatocytes (41). Secondly, many genes up-regulated by C/EBP␣ note is that this degree of induction is very similar to the level of play a role in terminal metabolism, such as enzymes of metabolic HNF3␤ induction obtained after C/EBP␣ expression in the ppc22- pathways (hepatic dihydrodiol dehydrogenase, folate receptor, and transfected H358 cell line. quinone oxidoreductase) and are suggestive of the transcriptional In both of these clones, induction of HNF3␤ protein led to very profile of a more differentiated cell. The third group of genes (such as substantial growth reduction noticeable as early as within 7 days (Fig. adipophilin and ceramide glucosyltransferase) is involved in lipid 6A). In fact, in 1 of the clones (clone 31), after day 4 of HNF3␤ metabolism. For instance, adipophilin, a gene up-regulated by induction, no additional increase in cell numbers was noted, and by C/EBP␣, is a prominent protein component of lipid storage droplets day 14, no viable cells could be seen. In clone 4, the growth reduction and is thought to be necessary for the formation and cellular function was very substantial, but slow cell proliferation did continue despite of these structures (42, 43). It is very interesting to note that the induction of HNF3␤. As expected, no change in cell proliferation was induction of C/EBP␣ in the H358 cell line did indeed lead to the noted on the withdrawal of doxycycline from the medium in the appearance of lipid droplets in the cytoplasm as determined by elec- parental cell line. tron microscopy (20), a feature of more mature pneumocytes. C/EBP␣ Changes in cell proliferation and cell cycle profile were also as- plays a major role in the development of preadipocytes to adipocytes sessed in a bromodeoxyuridine proliferation assay (Fig. 6B). Although and is known to regulate the expression of many genes involved in no change in cell cycle profile was noted on days 2 or 4 (data not lipid metabolism in adipose tissues (44–50). It might not be surprising shown), by day 7, a very significant increase in the fraction of that C/EBP␣ plays a similar role in alveolar cells, where the produc- apoptotic cells was noted. This was accompanied by a depletion of tion of lipids in the form of surfactant is critical to the proper cells in G0/G1 and S phase and an increase in the number of cells in functioning of the airway epithelium. Also, both of these sets of genes ␣ G2-M phase. These findings are suggestive of G2-M arrest and apo- appear to be markers of a more differentiated cellular state. C/EBP ptosis as a result of HNF3␤ induction. The changes were slightly more is a critical differentiation factor in a number of cell types, such as prominent in clone 31 than in clone 4. No change in the cell cycle hepatocytes, adipocytes, and myeloid cells (44, 51–54). On the basis profile or in the rate of apoptosis was noted in the control cell line. of our findings, it is likely to play a similar role in airway epithelial Changes in the rate of apoptosis were also determined by annexin/ cells as well. A hyperproliferation of type II pneumocytes has been propidium iodide flow cytometry. These studies have shown that by observed in C/EBP␣ knockout mice supporting such a role (19). In day 7 of HNF3␤ induction, there was a highly significant increase in fact, in previous studies, we have described intracellular changes the rate of cell death in both clones (from 3 to 20% apoptotic cells in detected by electron microscopy suggestive of airway epithelial dif- clone 31; Fig. 6C). No change in the rate of apoptosis was seen on ferentiation in the identical cell line system, where these transcrip- doxycyline withdrawal in the parental cell line. tional profiling studies were done (20). Also, a strikingly large num- We have also performed methylcellulose-based clonogenic assays ber of C/EBP␣-repressed genes are proangiogenic factors or growth to assess the ability of H358 cells to form colonies with and without factors. These findings are consistent with the role of C/EBP␣ in the induction of HNF3␤ expression (Fig. 6D). In clone 4, we observed growth arrest (29, 31, 32, 55). a significant reduction in the colony-forming ability of the cells when A validation of our hypothesis that using such an approach would grown in the absence of doxycycline. Interestingly, clone 31 cells enable us to identify direct C/EBP␣ targets is that a number of genes were not able to form colonies even in the presence of doxycycline, identified are known to be regulated directly by C/EBP members suggesting that possibly even a slight “leakiness” of HNF3␤ expres- (such as interleukin 8 and COX-2; Refs. 56 and 57). Additional sion might lead to very substantial reduction of colony-forming abil- validation comes from a study (58) in which primary human CD34ϩ ity. cells were transduced with a retroviral construct that expresses the C/EBP␣ cDNA fused in-frame with the estrogen receptor ligand- DISCUSSION binding domain. In these cells, the addition of estradiol leads to granulocytic differentiation. This system was used to identify target We previously showed that C/EBP␣ is a tumor suppressor in lung genes of C/EBP␣ in primary human hematopoietic cells by the use of cancer and demonstrated strong growth-inhibitory activity of C/EBP␣ Affymetrix oligonucleotide arrays. Quite strikingly, many of the reg- in lung cancer cell lines (20). In the present study, we have identified ulated genes (e.g., tumor necrosis factor-inducible gene 14, COX-2, transcriptional changes secondary to conditional expression of HM74, glutaredoxin, ARF-like protein, adipophilin, and p63) identi- C/EBP␣ in a C/EBP␣ nonexpressor lung adenocarcinoma cell line, fied were common to the C/EBP␣ target genes identified in our study. H358. The tight regulation and strong inducibility of C/EBP␣ expres- Our transcriptional profiling study identified also HNF3␤ (also sion proved to be optimal for this study. As shown in Tables 1 and 2, known as Foxa2) as one of the most highly induced downstream the changes in gene expression levels were marked and highly con- targets of C/EBP␣ in lung cancer cells. This finding is particularly sistent in-between triplicate samples but only for a very limited set of intriguing given the known function of HNF3␤ in foregut develop- genes, suggesting that we were able to identify the initial wave of ment and transcriptional regulation in the mature airway epithelium transcriptional changes as a result of C/EBP␣ expression. (10, 59, 60). HNF3␤ is a member of the forkhead family of transcrip- The genes identified this way also correlate very well with prior tion factors. The amino acid sequence of HNF3␤ is highly conserved knowledge about the function of C/EBP␣. Three groups of genes (97.8% homology with between the human and rat orthologue; Ref. stand out from the list of up-regulated genes. The first is genes 27). HNF3␤ binds DNA through a homologous winged helix motif involved in acute phase reaction, such as interleukin 8, COX-2, and common to a number of proteins known to be critical for determina- numerous tumor necrosis factor-inducible genes. It is well known that tion of events in embryogenesis, the forkhead box (61). Targeted C/EBP family members regulate the acute phase response (34–36). In disruption of HNF3B results in embryonic lethality with defective fact, many acute phase genes have both C/EBP as well as nuclear development of the foregut endoderm (62). In the adult, HNF3␤ factor ␬B binding sites in their promoters, suggesting that C/EBP regulates the transcription of numerous liver-enriched genes, and the proteins and nuclear factor ␬B cooperatively regulate the acute phase HNF3 proteins play a pivotal role in the regulation of metabolism and response (37–40). In addition, prior studies have also shown that in the differentiation of metabolic tissues such as the pancreas and 4144

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 4ϩ and clone 31ϩ) demonstrate exponential growth, the withdrawal of doxycycline from the medium (clone 4Ϫ and clone 31Ϫ) 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 accumula- ϭ tion of cells in the G2-M phase are noted (A apo- ϭ ϭ ϭ ptosis, 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 ϭ ϭ ϭ R1 S phase, R2 G2-M, R3 G0/G1, R4 ϭ 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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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2004 American Association for Cancer Research. HNF3␤ IS A TUMOR SUPPRESSOR IN LUNG CANCER liver (63, 64). HNF3␤ is known to be a key regulator of airway performing transcriptional profiling studies on the above-described epithelial differentiation (21, 23, 30, 65). It influences the expression cell line system to fully delineate the transcriptional changes after of a number of target genes in the respiratory epithelium, such as HNF3␤ induction. thyroid transcription factor-1, another master gene of airway epithelial In summary, our studies have identified the downstream targets of cell differentiation, surfactant protein-B, and Clara-cell secretory pro- C/EBP␣, a candidate tumor suppressor in lung cancer, in neoplastic tein (21, 22, 66). HNF3␤ is expressed at the onset of lung morpho- airway epithelial cells. These studies led to the identification of genesis (day 10 gestation) and throughout lung development (65, 67). HNF3␤, a known differentiation factor in airway epithelium as a It is expressed at highest levels in proximal bronchial and bronchiolar downstream target of C/EBP␣. Additional studies of HNF3␤ show epithelial cells, but it is also expressed in type II pneumocytes. It has down-regulation of its expression in lung cancer cell lines, identify been shown previously that members of the C/EBP family, including novel mutant forms of HNF3␤ in lung cancer, and demonstrate that C/EBP␣, bind and activate the LF-H3␤ site of the HNF3␤ promoter, promoter methylation is a putative mechanism for epigenetic silencing which might mediate cell-specific expression of HNF3␤ (25). Our of the HNF3␤ gene. Our studies also show strong growth-inhibitory studies using both in vitro electrophoretic mobility as well as in vivo and proapoptotic properties of HNF3␤ and suggest that HNF3␤ can chromatin immunoprecipitation assays clearly show direct binding of indeed act as a tissue-specific tumor suppressor in lung cancer. 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 REFERENCES characteristic of the differentiated alveolar pneumocytes but also by 1. Jemal A, Murray T, Samuels A, et al. Cancer statistics, 2003. CA - Cancer J Clin turning on a secondary wave of differentiation events by inducing the 2003;53:5–26. 2. Breathnach OS, Freidlin B, Conley B, et al. Twenty-two years of Phase III trials for expression of HNF3␤ itself. patients with advanced non-small cell lung cancer: sobering results. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2004 American Association for Cancer Research. 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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