Epigenetic Silencing of Cell Adhesion Molecule 1 in Different Cancer Progenitor Cells of Transgenic C-Myc and C-Raf Mouse Lung Tumors

Research Article

Epigenetic Silencing of Cell Adhesion Molecule 1 in Different Cancer Progenitor Cells of Transgenic c-Myc and c-Raf Mouse Lung Tumors

Stella Marie Reamon-Buettner and Juergen Borlak

Molecular Medicine and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany

Abstract exhibited atypical adenomatous hyperplasia as early as 3 months, Understanding molecular mechanisms underlying lung cancer postnatally. In the past, proteome mapping of c-Raf tumors has is a prerequisite toward treatment. To enable mechanistic been reported (8). investigations into the epigenetic regulation of the tumor Here, we developed 10 cell lines from single, spontaneously suppressor gene cell adhesion molecule 1 (Cadm1) in lung transformed lung tumor cells isolated from mice double-transgenic cancer progenitor cells, we developed 10 cell lines from single, for the proto-oncogenes c-Myc and c-Raf. These lines would enable spontaneously transformed lung tumor cells isolated from mechanistic investigations into the regulation of the gene encoding c-Myc and c-Raf double-transgenic mice. Specifically, we cell adhesion molecule 1 (Cadm1; Mouse Genome Informatics) in investigated Cadm1 promoter hypermethylation, which was lung cancer progenitor cells. Cadm1 is the mouse orthologue of significantly induced in transgenic transformed cells. Analysis the human gene CADM1 (TSLC1, IGSF4; for other synonyms, see Human Gene Nomenclature;1 ref. 9). CADM1 displays tumor of 69 CpGs displayed differential methylation pattern between and within progenitor cell lines, and the degree of methylation suppressor activity in human cancer, particularly non–small cell correlated well with transcriptional repression. Indeed, lung cancer (NSCLC; ref. 10). Indeed, loss or reduction of CADM1 restoration of Cadm1 gene expression was achieved by protein expression was observed in human lung adenocarcinomas treatment with the experimental demethylating drug 5-aza- and correlated with poor prognosis of patients, thus indicating 2¶-deoxycytidine. Furthermore, methylation of core CpGs in clinical significance (11–13). Nonetheless, this relationship may not be true for all types of lung tumors. After analysis of 47 primary the binding sites of Sp1, Sp3, and zinc finger 5 along the f promoter region of Cadm1 abrogated DNA-protein binding. lung adenocarcinoma of patients, only 40% had decreased Treatment with mithramycin A, an inhibitor of Sp1 or Sp3 CADM1 protein expression (12). Specifically, CADM1 was similar to binding, resulted in reduction of Cadm1 gene expression, healthy tissue in 16 bronchioloalveolar carcinomas (BAC) and 12 therefore suggesting a potential role of Sp1/Sp3 in Cadm1 adenocarcinomas other than BAC, but a further study on CADM1 regulation. Identifying molecular rules for the epigenetic protein in 93 lung adenocarcinomas evidenced loss of expression control of tumor suppressor genes enables mechanistic in 65% of the cases (13). This loss of expression correlated significantly with the non-BAC component and proliferative insights into lung cancer growth and opportunities for novel therapies. [Cancer Res 2008;68(18):7587–96] activity of the tumors. Lung adenocarcinomas with high CADM1 expression showed better prognosis than those without expression. When CADM1 protein was reexpressed in an NSCLC line A549, Introduction induction of apoptosis and inhibition of tumor growth were Lung cancer is a leading cause of death, but molecular observed, suggesting the potential of CADM1 for gene therapy (14). mechanisms underlying the disease are largely unknown. Thus, In general, the CADM1 gene encodes a 442-aa immunoglobin mouse models for human lung cancer have been developed in superfamily cell adhesion molecule and participates in cell-cell recent years to understand disease mechanisms (see reviews, interactions (15). The CADM1 protein is a transmembrane refs. 1, 2). These models include mouse strains with spontaneous glycoprotein consisting of an extracellular domain containing or carcinogen-induced tumors, as well as transgenic and knockout three immunoglobin-like C2-type domains, the transmembrane mice, in which lung tumors arise due to engineered genetic domain, and the cytoplasmic domain (10, 15). In vivo tumorigenesis modifications. There is evidence for the proto-oncogenes c-Myc in nude mice showed that deletion of the cytoplasmic domain and c-Raf to play a role in human lung malignancies (3, 4), and abrogates the tumor suppressor activity of Cadm1 (16), and transgenic mouse lines for these proto-oncogenes have been epigenetic silencing through promoter hypermethylation of established to study tumorigenesis of adenocarcinomas derived CADM1 was observed in lung cancer (10, 17, 18). from alveolar epithelium (5–7). Transgenic c-Myc mice developed Notably, epigenetic silencing of tumor suppressor genes is a within 10 to 14 months bronchioloalveolar carcinomas and frequent phenomenon in cancer (for instance, see reviews; papillary adenocarcinomas, whereas transgenic c-Raf mice refs. 19, 20), but the epigenetic mechanisms are still unknown. To gain more insights into the epigenetic control of Cadm1 resulting in transcriptional repression, we have undertaken a comprehensive Note: Supplementary data for this article are available at Cancer Research Online analysis of methylated CpGs along the promoter region of Cadm1. (http://cancerres.aacrjournals.org/). We also determined whether methylation of specific CpGs within Requests for reprints: Juergen Borlak, Molecular Medicine and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Strasse 1, D-30625 Hannover, Germany. Phone: 49-511-5350-559; Fax: 49-511-5350-573; E-mail: [email protected]. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-0967 1 http://www.genenames.org/ www.aacrjournals.org 7587 Cancer Res 2008; 68: (18). September 15, 2008

Figure 1. Characterization of lung cancer cell lines derived from c-Myc and c-Raf transgenic mice. A, histopathology of lung tumors from double-transgenic c-Myc and c-Raf mice. B, isolated alveolar epithelial cells at culture days 7 and 10 and cell lines gA7 and B3. C, the transgenes c-Raf and c-Myc were detected by PCR in donor animals (23.2, 23.4) and cell lines. D, gene expression analysis by RT-PCR of Vim, Eno2, and the housekeeping gene Actb. Positive controls, DNA from a normal lung and a tumor; negative control, no DNA.

putative binding sites of transcription factors in the promoter both semiquantitative and quantitative RT-PCR, the primers used for region of Cadm1 would lead to abrogation of binding and Cadm1 consisted of forward primer 5¶-caactatccctcctcccaca-3¶ and reverse ¶ ¶ confirmed results by electrophoretic mobility shift assay (EMSA) primer 5 -tagctgtgtctgcgtctgct-3 . ¶ Â 6 supershift functional assays. Identifying molecular rules for the 5-Aza-2 -deoxycytidine treatment of cells. Cells (3 10 ) were seeded in T25 cell culture flasks containing 5 mL of DMEM with 10% FCS, epigenetic control of tumor suppressor genes in lung cancer 2Â L-glutamine, and 2Â penicillin/streptomycin. Cells were cultured 48 h, progenitor cells of transgenic c-Myc and c-Raf mouse lung tumors treated with fresh 2 Amol/L 5-aza-2¶-deoxycytidine (5-aza-dC; Sigma) enables mechanistic insights into lung cancer growth and dissolved in medium for 3 d, and allowed to recover for 2 d. opportunities for novel therapies. Prediction of promoter region and DNA methylation analysis. To predict for the promoter region of mouse Cadm1 and to identify CpG island Materials and Methods in this region, we used the 2,000 nt (À2,000 bp) of the translation start site ATG. We used publicly available prediction programs, such as Promoter Lung tumors and lung cancer cell lines. 3 4 5 We analyzed 6 lung tumors 2.0, Promoter Scan, and CpG island searcher. Genomic DNA was isolated and 10 cancer cell lines from c-Myc and c-Raf transgenic mice. The in tissue samples and cell lines using Nucleo Spin Tissue (Macherey-Nagel). development of the c-Myc and c-Raf double-transgenic mice and the Bisulfite treatment was undertaken on 1 Ag genomic DNA using CpGenome histopathology of lung tumors resulting from the overexpression of these DNA Modification kit (Chemicon International) or EpiTect Bisulfite kit proto-oncogenes form part of a related study on global gene expression (Qiagen) using manufacturer’s instructions, but treatment at 65jC instead analysis.2 Cells were isolated from lung tumors of 8-month-old transgenic of 60jC. Primers for methylation assays (Supplementary Table S1) were mice using our protocol for the isolation and primary culture of rodent designed with MethPrimer.6 PCRfragments were directly sequenced using respiratory epithelial cells (21). With this protocol, by day 4, mRNA for the BigDyeTerminator v3.1 kit and ABI 3100 Genetic Analyzer (Applied pulmonary surfactant-associated protein C (Sp-C), which is specifically Biosystems), or PCRfragments were cloned using TOPOTA Cloning kit activated in alveolar epithelium, can be detected in nontransgenic cells. (Invitrogen) before sequencing. Sequences were analyzed using SeqMan Normal lungs from three nontransgenic mice were used as control. (Lasergene 7.0). Differential methylation was determined in at least five Gene expression analysis. Total RNA was isolated from frozen mouse clones. lung tissues or cell lines with RNeasy Mini kit, and reverse transcription– Isolation of protein extracts, Western blot analysis, and EMSA. PCR (RT-PCR) was undertaken with Omniscript RT kit (Qiagen). Procedures for the isolation of protein extracts, Western blot analysis, and Semiquantitative RT-PCR was performed on T3 thermocyclers (Biometra), EMSA have been described previously (22). Protein extracts were isolated whereas quantitative RT-PCR on Light Cycler (Roche) using Thermostart from mouse lung tissues or cell lines. For binding experiments, 2 to 10 Ag Taq polymerase (ABgene). of nuclear proteins were used. Sp1 antibody (Sc-59Â) and Sp3 antibody Typically, 25 to 50 ng of cDNA were used for template. Reaction components and cycling variables are according to standard procedure. For

3 http://www.cbs.dtu.dk/services/Promoter/ 4 http://www-bimas.cit.nih.gov/molbio/proscan/ 5 http://cpgislands.usc.edu/cpg.aspx 2 F. Weltmeier, R. Halter, R. Spanel, J. Borlak. Unpublished data. 6 http://www.urogene.org/methprimer/index1.html

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(Sc-644Â; Santa Cruz Biotechnology) were used for the supershift experi- we isolated spontaneously transformed tumor cells from mice ments. Oligonucleotides were purchased from MWG Biotech and summa- double-transgenic for c-Myc and c-Raf (see Fig. 1A and B). After 4 rized in Supplementary Table S2. EMSA probes were generated by annealing to 7 days of culture, cells from the transgenic mice exhibited the complementary oligonucleotides using standard procedures. For Western typical cuboidal phenotype (Fig. 1B). By day 10, some cells began to blot analysis, total (100 Ag) or nuclear (30 Ag) protein extracts isolated from attain different morphologic characteristics (Fig. 1B), indicating mouse lung tissues or cell lines were used. Sp1 and Sp3 antibodies were described above, whereas Cadm1 antibody was purchased from Abnova individual phenotypes of tumor cells. From single cuboidal cells, (Igsf4, H00023705-M01, clone 2A5). 10 cell lines could be established. Double-transgenicity for c-Raf Mithramycin A treatment. Cells (1 Â 106) were seeded in T75 cell and c-Myc in the cell lines were confirmed by PCRdetection of the culture flasks containing 5 mL of DMEM with 10% FCS, 2Â L-glutamine, transgenes (Fig. 1C). The 10 cell lines no longer expressed the and 2Â penicillin/streptomycin for 5 d (f90% confluence), treated with mRNA for surfactant proteins (data not shown), which tend to 500 ng/mL mithramycin A for 24 h. Cells were pelleted for isolation of disappear in culture by day 7 (23). Nonetheless, transcripts of nuclear extracts and RNA. Vim and Eno2, which are markers of epithelial tumors, were strongly expressed (Fig. 1D). Because genetic alterations are known to occur in cancer cells, Results we determined by sequencing the presence of any hotspots Characterization of lung cancer cell lines from c-Myc and mutations in Kras and Trp53 two genes implicated in lung cancer, c-Raf double-transgenic mice. To enable mechanistic studies, particularly in smokers (24). We did not detect sequence alterations

Figure 2. Gene and protein expression analyses. A, Cadm1 gene expression in normal lungs from nontransgenic mice, as well as tumors and cell lines from double-transgenic c-Myc and c-Raf mice, as measured by quantitative RT-PCR. Compared with normal lungs, Cadm1 expression was reduced in tumors and cell lines. In seven cell lines, Cadm1 expression was minimal or absent. B, Western blot analysis of Cadm1 showed reduction of expression in lung tumors compared with a normal lung used as control (arrow, left) and loss in cancer cell lines (arrow, right). C, restoration of Cadm1 gene expression in two cell lines after treatment with 2 Amol/L of the demethylating agent 5-aza-dC (top) and corresponding Dal1 expression (bottom). D, Dal1 gene expression in normal lungs from nontransgenic mice, as well as tumors and cell lines from double-transgenic c-Myc and c-Raf mice as measured by quantitative RT-PCR. Dal1 expression was reduced in tumors, but in contrast to Cadm1 expression, much more elevated in cell lines. For comparison of Dal1 expression in samples, logarithmic values were computed due to a wide span in values obtained. www.aacrjournals.org 7589 Cancer Res 2008; 68: (18). September 15, 2008

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. Cancer Research in 6 lung tumors and 10 cell lines from the c-Myc and c-Raf double- thymidine, which can then be detected by sequencing. PCRis transgenic mice. Furthermore, sequencing of the entire Cadm1 performed using a pair of primers not containing CpGs, thereby gene amplified from these lung tumors and cell lines did not reveal amplifying both methylated and unmethylated fragments (bisulfite any mutations. sequencing) or a pair of primers that specifically amplify only Cadm1 is repressed in lung tumors and cancer cell lines methylated fragments (MSP-PCR). To search for methylated CpGs from c-Myc and c-Raf double-transgenic mice. We then associated with loss of Cadm1 expression, especially those which examined the gene expression of Cadm1 in three normal lungs may abrogate binding of transcription factors, we developed five from nontransgenic mice, as well as 6 lung tumors and 10 cancer assays covering a total of 69 CpGs, including five CpGs in the cell lines from c-Myc and c-Raf double-transgenic mice. Cadm1 coding region (Fig. 3B). expression was markedly reduced in tumors and cell lines Assay 1 (6 CpGs, À944 to À837) and assay 2 (10 CpGs, À682 to compared with the three normal lungs from nontransgenic mice À531) are based on bisulfite sequencing, whereas assay 3 (14 CpGs, (Fig. 2A). In 7 of 10 cell lines, Cadm1 expression was basically À456 to À341), assay 4 (27 CpGs, À396 to À180), and assay 5 (37 absent. This loss of Cadm1 gene expression correlated with CpGs, À302 to +41) are based on MSP-PCRand sequencing. Due to corresponding protein expression, which was also reduced in lung high frequency of CpGs, no PCRprimers for bisulfite sequencing tumors and absent in all cancer cell lines except one (Fig. 2B). For could be obtained to investigate methylation status nearer the the cell line B3, which displayed the highest Cadm1 expression translation start site. The three MSP-PCRassays overlap each among the cell lines, a faint protein expression was observed (data other. After bisulfite treatment and direct sequencing of the PCR not shown). The Cadm1 repression pointed to a possible epigenetic product, a CpG site was scored methylated if the C (methylated) regulation, especially because two cell lines responded to treatment allele was f20% of the T (not methylated) allele. But for assay 4, with the demethylating agent 5-aza-dC, thereby restoring Cadm1 PCRproducts were cloned before sequencing due to high CG expression (Fig. 2C). content, causing difficulties in obtaining clear direct sequencing For comparison with Cadm1, we also investigated the gene electropherograms. Examples and results of the five different expression of Dal1 (differentially expressed in adenocarcinoma of assays are shown in Fig. 4A–C and summarized in Supplementary the lung), a gene encoding an actin-binding protein and with Table S3. which Cadm1 directly associates (25). Dal1 expression was Overall, we observed methylation in 69 CpGs, including five reduced in the tumors than in controls, but much elevated in the CpGs after the translation start site (see Fig. 4A and C). cell lines (Fig. 2D). This relationship remained essentially true Methylation pattern in seven cell lines lacking or with minimal even when the cell lines were cultured at 50% confluency. Cadm1 gene expression was denser than those lines with gene Nevertheless, Dal1 expression seemed to be less at 50%, especially expression (Fig. 4C). Furthermore, after cloning and sequencing of with hD10, which showed the lowest among the cell lines (data not clones, differential methylation pattern was observed between and shown). Indeed, whereas Cadm1 expression decreased to nonex- within cell lines (Fig. 4B and C). The degree of methylation as istent in the cell lines, Dal1 expression remarkably increased determined by methylation index (MI), which is the number of (Fig. 2A versus Fig. 2D). Interestingly though, in the two cell lines methylated CpGs over total CpGs, correlated highly with the loss of where Cadm1 was restored after 5-aza-dC treatment, Dal1 gene expression (Fig. 5). Regardless of assay, this correlation was increased correspondingly (Fig. 2C). clearly observed in cell lines than in tumors, which may be caused Aberrant hypermethylation in the promoter region of by a mixture of normal and tumor cells. Cadm1 correlates with loss of gene expression. The absence Methylated CpGs within the core binding sequence of of Cadm1 mutations in the lung tumors and cell lines and transcription factors lead to abrogation of binding. Methyla- restoration of the Cadm1 expression through 5-aza-dC treatment tion of CpGs in the promoter region of genes can lead to prompted us to investigate the epigenetic silencing of this gene abrogation of binding of transcription factors. Using Transfac,7 we through CpG methylation of the promoter region. In the past, analyzed for putative binding sites of transcription factors in the CpG methylation of the Cadm1 promoter has been investigated in promoter region of Cadm1, in particular those containing CpGs rat and human genomes, but not in mouse. Initially, we annotated within the predicted core sequence. Such analysis revealed three the promoter region of mouse Cadm1 and used the 2,000 nt Sp1, an Sp3, and a zinc finger 5 (Zf5) binding sites (Fig. 2B and (À2,000 bp) upstream of the translation start site ATG of Cadm1 to Supplementary Fig. S1). The Sp1 binding sites would affect CpGs identify CpG islands in this region. We used several programs À224 (Sp1-3), À211 (Sp1-2), and À164 (Sp1-1), whereas Sp3 that of to predict the CpG island (see Materials and Methods); essentially, CpG À437. The Zf5 binding site would involve CpGs À192, À190, the same results were obtained. For instance, a CpG island was and À188. Methylation of these CpGs sites was observed especially predicted starting À808 ATG (i.e., length, 809 bp; %GC, 60; in cell lines with markedly reduced Cadm1 expression. For Sp1 site, ObsCpG/ExpCpG, 0.846). In human CADM1, the predicted TATA CpG À164 (Sp1-1) was more heavily methylated than CpGs À224 Box is located À374 to À371 from ATG (see ref. 17) and six CpGs (Sp1-3) and À211 (Sp1-2; Supplementary Fig. S1). (À458, À440, À433, À397, À394, À381) are routinely analyzed We designed oligonucleotide probes and carried out EMSA on for hypermethylation in this gene. Using this information from Sp1 using nuclear extracts from the cell line A2C12, in which human CADM1, we performed alignment using human and mouse Cadm1 was not expressed. The EMSA probes consisted of the sequences to determine homologous regions (Fig. 3A). There is very following: wild type, a probe containing two mutated nucleotides high similarity between human and mouse sequences, especially in the core sequence, a probe with methylated CpG in the core within the 200 nt before translation start site. sequence, and a probe with two methylated CpGs outside the Most techniques used in identifying methylated CpGs are based on bisulfite treatment of genomic DNA, which converts cytosine to uracil, but methylated cytosines remain unaltered in this process. During PCRamplification, uracil will be converted to 7 http://www.gene-regulation.com/pub/databases.html

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Figure 3. Bioinformatic analysis of the promoter region of Cadm1 in mouse and different assays used to search for methylated CpGs associated with loss of Cadm1 expression. A, alignment (MegaAlign, Lasergene 7.0) of human and mouse sequences to determine homologous regions in the promoter region of Cadm1. In the human sequence, 14 CpGs (double underlined) and putative TATA box (see ref. 17) in the predicted promoter region are shown. In the mouse sequence, CpGs are numbered by the location of the cytosine residue relative to the translation site ATG (+1). In putative binding sites of Sp1, Sp3, and Zf5, the core sequence is underlined. B, different assays and location of analyzed CpGs along the promoter region of Cadm1. Indicated also are the predicted promoter (À417 to À174 relative translation site ATG, +1) obtained with Promoter Scan, as well as the translation start site ATG. core sequence (see Supplementary Table S2). Compared with mentary Table S2; Fig. 6B). Binding was observed in the wild- the wild-type probe, binding was dramatically reduced in the type probe, and bound protein was supershifted with the Sp3 mutated probe, as well as in the probe with methylated CpG antibody. In the mutated probe, no binding was observed, whereas in the core sequence (Fig. 6A). Binding was not abolished in the in the methylated probe, binding was observed, but the bound probe where methylation occurred outside the core sequence. protein was not supershifted by the Sp3 antibody. After Western Similar results were obtained from nuclear extracts isolated blot analysis, the Sp3 antibody detected a protein of an expected from normal lung tissues, as well as from the cell line A2C12 size of 100 kDa in the nuclear extracts of the cell line A2C12 that was treated with 5-aza-dC which were used as controls (Fig. 6B). (data not shown). The Sp1 antibody could supershift the bound It is of considerable importance that mithramycin A, which is an protein and detect a protein of an expected size of 106 kDa in inhibitor of Sp1 or Sp3 binding, could block the expression of genes the nuclear extracts of the cell line A2C12 after Western blot regulated by these transcription factors (26). On the other hand, analysis (Fig. 6A). mithramycin A may also function as a demethylating agent on lung Similar experiments were conducted on Sp3, using three cancer cells (27). To determine such effects on the regulation of oligonucleotide probes consisting of a wild type, a probe containing Cadm1, we treated a cell line (B3) that still expressed Cadm1,as two mutated nucleotides in the core sequence, and a probe in well as a cell line (A2C12) with no Cadm1 expression, both with which the CpG in the core sequence was methylated (see Supple- 500 ng/mL mithramycin A for 24 h (Fig. 6C). We observed a www.aacrjournals.org 7591 Cancer Res 2008; 68: (18). September 15, 2008

Figure 4. Aberrant promoter hypermethylation of Cadm1.A, sequence electropherogram of a clone from cell line gD1 obtained with assay 5 (CpGs À302 to +41), in which 31 of 37 CpGs, including those in the coding sequence, were methylated. Arrows, methylated non-CpG cytosines; one of these cytosines is located in the core sequence of the putative binding site of Ap2 in the Cadm1 promoter region, the other non-CpG methylated cytosine is adjacent to the translation start site ATG. B, methylation-specific PCR (MSP-PCR) to analyze 27 CpGs at À396 to À180 in lung cancer cell lines. M, methylated; U, unmethylated. Amplification of methylated fragments corresponded well with Cadm1 expression (top). Fragments amplified with the methylation-specific primers were cloned and sequenced. Differential methylation of clones in seven cell lines with remarkably reduced or absent Cadm1 gene expression (bottom). Methylated CpGs are designated with solid boxes: green solid boxes, methylated CpGs within the primer sequence; blue solid boxes, methylated CpGs outside the primer sequence. Open boxes, nonmethylated CpGs. C, methylation patterns in the promoter region of Cadm1 in lung cancer cell lines, in four assays that could be analyzed by direct sequencing, and of clones in three cell lines. Methylated CpGs are designated with solid boxes: green, methylated CpGs within the primer sequence; dark blue, f40% to 100% methylation; light blue, f20% to 30% methylation. Open boxes, unmethylated. Dense methylation patterns corresponded with loss of Cadm1 expression.

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Figure 5. Quantitative correlation of methylation status with loss of Cadm1 expression in normal lungs, tumors, and cell lines. Quantitative RT-PCR was plotted against MI, calculated as the number of methylated CpGs over total CpGs. MI was based on results of directly sequenced fragments from four assays spanning CpGs À944 to +41. Gene expression of Cadm1 decreased as MI increased.

decrease of Cadm1 gene expression in the cell line B3. In contrast, mouse lung tumors. These cell lines were established from single, there was no restoration of gene expression in A2C12. spontaneously transformed cells. We also analyzed six lung tumors To determine methylation of the putative binding site of Zf5 in from c-Myc and c-Raf double-transgenic mice, as well as three the promoter region of Cadm1, we used three oligonucleotide normal lungs from nontransgenic mice as controls. As compared probes for EMSA as follows: wild type, a probe with all three CpGs with the normal lungs, Cadm1 gene expression was markedly in the core sequence that were methylated, and a probe in which reduced or absent in lung tumors and cell lines, and this result the nucleotide C in the three CpGs of the core sequence were corresponded with loss of protein expression. Treatment with mutated to T (see Supplementary Table S2; Fig. 6D). With the wild- the demethylating agent 5-aza-dC restored Cadm1 expression type probe, binding was observed with 10 Ag of nuclear extracts in two cell lines, suggesting epigenetic regulation. Using five whether from normal lung tissues or from the cell line A2C12, different assays, we investigated the methylation status of treated or untreated with 5-aza-dC. This binding was not observed 69 CpGs, including five CpGs after the translation start site. in the methylated probe. In the mutated probe, binding was also Overall, methylation pattern in seven cell lines lacking or with observed, but this was different from the wild-type and the minimal Cadm1 gene expression was denser than those lines methylated probe. Because no Zf5 antibody is available commer- with gene expression (see Fig. 4). Furthermore, after cloning cially, supershift experiments and Western blot analysis could not and sequencing of clones, differential methylation pattern was be undertaken. observed between and within cell lines. The degree of methylation correlated highly with the loss of gene expression, and this correla- Discussion tion was clearly observed in the cell lines regardless of assay Recent developments in the field of ‘‘epigenetics,’’ i.e., heritable used. Thus, we show transcriptional repression and promoter changes in gene expression without accompanying alterations in hypermethylation of Cadm1 in lung cancer. Investigations into the the DNA sequence, show a much wider contribution of epigenetic different cancer progenitor cells of transgenic c-Myc and c-Raf alterations in the development of cancer (see reviews; refs. 19, mouse lung tumors bring mechanistic insights into the epigenetic 20, 28). Epigenetic changes, which include DNA methylation, control of Cadm1. histone modifications, and noncoding RNAs, can disrupt the Promoter hypermethylation of CADM1 (TSLC1, IGSF4) that normal stem-cell or progenitor-cell program leading to disease is associated with loss of gene expression is known in various (19, 29). This epigenetic progenitor model of cancer puts forward cancer in humans, including NSCLC (10, 17, 18). Analysis of that cancer first arises as an epigenetic alteration of stem/ methylation of CADM1 promoter region in primary NSCLCs progenitor cells within a given tissue, which is mediated by showed methylation in 21 of 48 tumors (17). Furthermore, aberrant regulation of tumor progenitor genes. Furthermore, tumor investigation of 103 primary NSCLCs for association of CADM1 cell heterogeneity is due in part to epigenetic variation in methylation with tobacco smoking and with the clinical character- progenitor cells, and epigenetic plasticity together with genetic istics of tumors found methylation in 45% cases (18). Methylation lesions drives tumor progression. Thus, in line with this view, was observed in all histologic subtypes of NSCLC, including epigenetically disrupted stem/progenitor cells might be a crucial adenocarcinoma, squamous cell carcinoma, adenosquamous target for cancer risk assessment and chemoprevention. carcinoma, and large cell carcinoma. In rats, the expression of We thus investigated the epigenetic control of Cadm1 in 10 the Cadm1 gene and its methylation pattern in lung adenocarci- different cancer progenitor cells of transgenic c-Myc and c-Raf nomas induced by N-nitrosobis (2-hydroxypropyl) amine (BHP) www.aacrjournals.org 7593 Cancer Res 2008; 68: (18). September 15, 2008

Figure 6. Analysis of methylated CpGs in putative binding sites of transcription factors in the promoter region of Cadm1.A, left, EMSA with Sp1 probes, wild type (Wt), a probe with two mutated nucleotides in the core sequence (Mut), a probe with the CpG in the core sequence methylated (Meth-1), and a probe with two CpGs outside the core sequence methylated (Meth-2). Binding was dramatically abolished in the mutated probe (Mut), as well as in the probe with the methylated CpG in the core sequence (Meth-1). Controls were included with no oligonucleotide probes (À). Arrows point to supershifted bands after incubation with Sp1 antibody. Right, Western blot analysis of Sp1 in nuclear extracts isolated from cell line A2C12 that was treated or not treated with 5-aza-dC (Aza) and used in the EMSA experiment. A protein of an expected size of 106 kDa (arrow) was detected. B, left, EMSA with Sp3 probes, wild type (Wt), a probe with two mutated nucleotides in the core sequence (Mut), and a probe with the CpG in the core sequence methylated (Meth). Binding in the wild-type probe (Wt) and bound protein was supershifted with the Sp3 antibody (arrow). No binding in the mutated probe (Mut); in the methylated probe (Meth), there was binding, but the bound protein was not supershifted by the Sp3 antibody. Right, Western blot analysis, the Sp3 antibody detected a protein of an expected size of 100 kDa in nuclear extracts isolated from cell line A2C12 that was treated or not treated with 5-aza-dC (Aza) and used in the EMSA experiment. C, Cadm1 expression after treatment with mithramycin A, an inhibitor of Sp1 or Sp3 binding. Presence of RNA was controlled by RT-PCR of the housekeeping gene Actb (not shown). Cell line B3 that still expressed Cadm1, as well as cell line A2C12 with no Cadm1 expression, were treated with 500 ng/mL mithramycin A for 24 h. Quantitative RT-PCR results show a decrease of Cadm1 expression in the cell line B3 after mithramycin A (MMA) treatment. Negative control, no DNA. D, EMSA with Zf5 probes, wild type (Wt), a probe with all three CpGs in the core sequence methylated (Meth), and a probe in which the nucleotide C in the three CpGs of the core sequence mutated to T (Mut). For each probe, 2, 5, and 10 Ag of nuclear extracts were used. In the wild-type probe, binding was observed with 10 Ag of nuclear extracts (arrow). No binding in the methylated probe (Meth); in the mutated probe, binding was also observed, but this was different from the wild-type and methylated probe.

has been studied as well (30). Cadm1 was highly methylated Results from methylation studies may be influenced by the in four lung adenocarcinomas, but unmethylated in two normal number and location of analyzed CpGs sites, as well as the assay lung tissues, suggesting aberrant Cadm1 methylation may be used. To obtain a comprehensive map of methylation in the involved in BHP-induced development of lung adenocarcinomas promoter region of Cadm1, we used five assays, in which four could in rats. be directly sequenced giving an average level of methylation

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. Epigenetic Silencing of Cadm1 in Lung Cancer of a CpG site in the mixture of amplified fragments. Bisulfite the consensus Sp1 element induced a significant decrease in Sp1/ sequencing, when feasible, is the most reliable method. Amplifica- Sp3 binding. tion with methylation-specific primers, which relies heavily on We determined whether methylation of potential Sp1 or Sp3 primer design, may lead to false negatives or positives and could binding sites in the promoter region of Cadm1 could affect binding. be biased unless followed by sequencing (see Supplementary Our results on Sp1 showed that binding was dramatically reduced Table S3). This can be exemplified in assay 5 to interrogate 37 CpGs in the mutated EMSA probe, as well as in the probe with (À302 to +41). This assay was carried out using a pair of methylated CpG in the core sequence compared with the wild-type methylation-specific primers, in which three CpGs (À302, À298, probe. However, binding was not abolished in the probe wherein À296) are contained in the forward primer and two CpGs in methylation occurred outside the core sequence. With Sp3, the reverse primer (+38, +41). We were able to amplify fragments in mutation in the core consensus site led to no binding, but in the all samples, whether they are normal lungs, tumors, or cell lines. methylated probe, binding was observed, but the bound protein But after sequencing, in contrast to the cell lines, no other was no longer supershifted by the Sp3 antibody. These findings methylated CpGs were observed in the normal lungs and tumors, suggest that Sp1 or Sp3 may be involved in the transcriptional except those CpGs contained in the primers (see Supplementary regulation of Cadm1 in mouse and that promoter hypermethyla- Fig. S1). The degree of methylation obtained with this assay tion can lead to abrogation of binding. Furthermore, we analyzed for CpGs À302 to +41 seems to correlate highly with the loss the effects of mithramycin A, a drug that modifies GC-rich regions of Cadm1 expression (see Fig. 4C). Similar finding was obtained of the DNA and blocks Sp1 or Sp3 binding on the expression of in the homologous region in human CADM1. After methylation Cadm1. Our treatment of a cell line (B3) that still expressed Cadm1 mapping of CADM1 in human cervical cancer, a 180-bp un- with 500 ng/mL mithramycin A for 24 h led to a decrease of gene methylated core immediately upstream of the ATG site was expression, thus further documenting the importance of Sp1 and found to correlate most strongly with the active expression of this Sp3 in causing transcriptional activation. Further studies are gene (31). required to determine quantitatively the role of Sp1 and Sp3 in the CADM1 directly associates with DAL1, a gene encoding an actin- transcriptional regulation of Cadm1. binding protein likewise implicated in the suppression of growth in Additionally, Zf5 is a ubiquitously expressed protein that lung cancer cells. DAL1 anchors CADM1 to the actin cytoskeleton; contains five COOH terminal zinc fingers and a conserved NH2 thus, loss of DAL1 expression could lead to decreased cell adhesion terminal ZiN/POZ domain. Originally cloned as a transcriptional (25). Inactivation of DAL1 due to promoter hypermethylation has repressor from the mouse c-Myc promoter, Zf5 can act as an been observed in human lung cancer as well (32–34). We, therefore, activator, as well as a repressor of transcription (39, 40). The POZ also determined Dal1 expression in our study samples. We found domain contributes to the repressor activity, whereas the active that Dal1 expression was reduced in c-Myc and c-Raf mouse lung function results from the DNA-binding ability of the zinc finger tumors than in normal lungs, but surprisingly, much more elevated domain. We also investigated whether methylation of the putative in the cell lines. Indeed, whereas Cadm1 expression decreased to binding site of Zf5 in the promoter region of Cadm1 could affect nonexistent in the cell lines, Dal1 expression remarkably increased. binding. Compared with the wild-type probe, binding was not A previous study generated mice deficient for Dal1 gene to observed in the methylated probe. This suggests a possible role of elucidate its function in development and tumorigenesis (35). Zf5 in the regulation of Cadm1, but further studies on its activation Dal1 null mice developed normally and were fertile. Rates of are necessary, although a previous report suggested a direct cellular proliferation and apoptosis in brain, mammary, and lung relationship between intact Sp1 sites and Zf5 in promoter tissues from the 4.1B/Dal-1 null mice were indistinguishable activation (39). Indeed, activation of the HIV-1 long terminal from those seen with wild-type mice. Aging studies indicate that repeat by Zf5 depends on Zf5 binding to DNA, three intact Sp1 these mice did not have a propensity to develop tumors. These sites, and the Zf5 ZiN/POZ domain. findings indicate that the Dal1 gene is not required for normal In conclusion, we investigated the consequences of c-Myc and development and that Dal1 may not function as a tumor sup- c-Raf overexpression on the epigenetic regulation of the tumor pressor gene. suppressor gene Cadm1. Indeed, tumor progenitor cells isolated DNA methylation in the promoter of certain genes is from lung tumors can be used in determining the molecular basis associated with transcriptional silencing. Such methylation can for response and resistance to drug therapies or to even predict affect gene expression by interfering with transcription factor which patients are likely to respond to treatment. Unlike studies binding or by recruiting histone deacetylases through methyl- with DNA isolated from entire tumors, where results are rather DNA–binding proteins. Indeed, many of these transcription difficult to interpret due to contamination of normal cells, the use factors bind to sequences containing CpGs, and binding can be of spontaneously transformed cancer cell lineages enable mecha- hindered by methylation. Our analysis of potential binding sites nistic investigations of Cadm1 gene expression. Notably, the cell of transcription factors in the promoter region of Cadm1 lines exhibited heterogeneity of DNA methylation pattern resulting revealed those of Sp1, Sp3, and Zf5 in which CpGs are present in differential repression of Cadm1 expression and response to in the core site. The transcription factors Sp1 and Sp3 are 5-aza-dC, a demethylating drug in clinical development for the ubiquitously expressed and bind to GC-rich promoter region. treatment of leukemia. Several studies show methylation-dependent disruption of Sp1 A further point of consideration is the plasticity seen in the or Sp3 binding leading to reduced expression of targeted gene lung tumor cell lines. Upon treatment with 5-aza-dC and despite (36–38). This disruption may even involve methylated CpGs out- repeated treatments at different dose levels (e.g., 10 Amol/L) and side the consensus binding site, such as in p21(Cip1) promoter prolonged treatment regiments, Cadm1 expression was restored (36). In p21(Cip1) promoter, by using an electrophoretic mobility in two cell lines only, whereas the remaining eight lines from the shift assay, methylation within the consensus Sp1-binding site same tumors did not respond to this treatment. This shows did not reduce Sp1/Sp3 binding, but methylation outside of epigenetic plasticity in response to treatment with 5-aza-dC. www.aacrjournals.org 7595 Cancer Res 2008; 68: (18). September 15, 2008

Knowledge of the precise epigenetic control of tumor suppressor Acknowledgments genes is crucial for their controlled reactivation in lung cancer Received 3/25/2008; revised 5/13/2008; accepted 6/23/2008. cells by epigenetic drugs, which represents a new class of Grant support: Ministry of Science and Culture, Lower Saxony, Germany grant therapeutic strategy to hinder tumor growth and subsequent 25A.5-7251-99-3/00. The costs of publication of this article were defrayed in part by the payment of page progression. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Annika Roskowetz, Wiebke Goettmann, Antje Schulmeyer, Stefanie Disclosure of Potential Conflicts of Interest Marschke, and Andreas Hiemisch for the technical support; Jessica Decke for her contribution in the establishment of the cell lines; Roman Halter for the mouse lung No potential conflicts of interest were disclosed. tissues; and Susanne Reymann for support in Transfac analysis.

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Cancer Res 2008; 68: (18). September 15, 2008 7596 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. Epigenetic Silencing of Cell Adhesion Molecule 1 in Different Cancer Progenitor Cells of Transgenic c-Myc and c-Raf Mouse Lung Tumors

Stella Marie Reamon-Buettner and Juergen Borlak

Cancer Res 2008;68:7587-7596.

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