Through genome-wide expression profile analysis for non–small cell lung cancers (NSCLC), we found overexpression of a Myc-associated protein with JmjC domain (MAPJD) gene in the great majority of NSCLC cases. Induction of exogenous expression of MAPJD into NIH3T3 cells conferred growth-promoting activity. Concordantly, in vitro suppression of MAPJD expression with small interfering RNA effectively suppressed growth of NSCLC cells, in which MAPJD was overexpressed. We found four candidate MAPJD target genes, SBNO1, TGFBRAP1, RIOK1, and RASGEF1A, which were the most significantly induced by exogenous MAPJD expression. Through interaction with MYC protein, MAPJD transactivates a set of genes, including kinases and cell signal transducers that are possibly related to proliferation of lung cancer cells. As our data imply that MAPJD is a novel member of the MYC transcriptional complex and its activation is a common feature of lung cancer, selective suppression of this pathway could be a promising therapeutic target for treatment of lung cancers. [Mol Cancer Ther 2007;6(2):542–51]

Lung carcinoma is one of the most common causes of cancer death worldwide, and non–small cell lung cancer (NSCLC) accounts for ∼80% of these cases (1). Although various cytotoxic agents offer multiple therapeutic choices for patients, each of the new regimens can provide only modest survival benefits to advanced NSCLC patients (24). Hence, development of novel therapeutic strategies, such as molecular targeted agents and antibodies as well as cancer vaccines, based on well-characterized molecular mechanisms involved in pulmonary carcinogenesis, are eagerly awaited.

Systematic analysis of expression levels of thousands of genes on a cDNA microarray is an effective approach to identify molecules involved in carcinogenic pathways (59); some of these genes or their products should be candidate targets for development of novel anticancer drugs and useful tumor markers. To isolate such molecules, we have been analyzing genome-wide expression profiles of lung cancers, using purified populations of tumor cells that were prepared by laser microbeam microdissection (59). To verify the biological and clinical significance of the respective gene products, we have established a screening system by a combination of the tumor tissue microarray analysis of clinical lung cancer materials as well as RNA interference (RNAi) technique (919). In the course of these systematic studies, we found Myc-associated protein with JmjC domain (MAPJD); C14orf169, chromosome 14 open reading frame 169; alias FLJ21802/NO66 protein to be overexpressed in the great majority of the NSCLCs. MAPJD was reported previously as a nuclear protein with a conserved JmjC domain that was commonly found in DNA- or chromatin-binding domains (20). JmjC domain-containing proteins are supposed to have the enzymatic activity that regulates chromatin remodeling and gene expressions. However, the role(s) of the members belonging to the JmjC-containing family in carcinogenic processes has not been clarified.

The MYC (c-Myc) oncogene is one of the most frequently overexpressed genes in human cancer (21). The expression level of MYC is tightly associated with cell proliferation in part by regulating genes involved in cell cycle control. MYC functions as a sequence-specific transcription factor belonging to the basic, helix-loop-helix, leucine zipper family. When dimerized with MYC-associated factor X, MYC binds to CACGTG (CANNTG) motifs (E-box) in the genome and activates the transcription of various target genes (22, 23). Recently, transcriptional activity of MYC/MYC-associated factor X heterodimers was linked to recruitment of cofactor complexes, including a transformation/transcription domain-associated protein (TRRAP) together with p300/CBP-associated factor (alias GCN5; ref. 24) or TIP60 histone acetyltransferases (HAT; ref. 25).

Here, we report that MAPJD plays a significant role in pulmonary carcinogenesis by activating various downstream target genes through interaction with MYC and suggest that this molecule represents a potential target for development of novel therapeutic drugs for lung cancer.

Cell Lines and Clinical Samples

The 30 human NSCLC cell lines used for this study were as follows: A427, A549, LC174, LC176, LC319, PC-3, PC-9, PC-14, SW900, SW1573, NCI-H23, NCI-H226, NCI-H358, NCI-H520, NCI-H522, NCI-H596, NCI-H647, NCI-H1373, NCI-H1435, NCI-H1650, NCI-H1666, NCI-H1703, NCI-H1781, NCI-H1793, NCI-H2170, RERF-LC-AI, SK-LU-1, SK-MES-1, EBC-1, and LX-1. All cells were grown in monolayer in appropriate medium supplemented with 10% FCS and maintained at 37°C in an atmosphere of humidified air with 5% CO2. Human small airway epithelial cells and bronchial epithelial cells were also included in the panel of the cells used in this study. Primary NSCLC samples had been obtained earlier with informed consent (5).

A total of 300 formalin-fixed primary NSCLCs and adjacent normal lung tissue samples used for immunostaining on tissue microarrays had been obtained with informed consent from patients undergoing surgery earlier at Hokkaido University and its affiliated hospitals (Sapporo, Japan). The use of clinical materials in this study was approved by each of individual institutional ethical committees.

Semiquantitative Reverse Transcription-PCR

We prepared appropriate dilutions of each single-stranded cDNA prepared from mRNAs of clinical lung cancer samples and normal tissues (heart, liver, lung, bone marrow, testis, ovary, and placenta), using the level of β-actin (ACTB) expression as a quantitative control. The primer sets for amplification were as follows: ACTB-F (5′-GAGGTGATAGCATTGCTTTCG-3′) and ACTB-R (5′-CAAGTCAGTGTACAGGTAAGC-3′) for ACTB; MAPJD-F (5′-AGGAGAAGTTGGAGGTGGAAA-3′) and MAPJD-R (5′-CAGATGAAAGATCCAAATTCCAA-3′) for MAPJD; SBNO-F (5′-CTGACAGTGCATGTCTTTGG-3′) and SBNO-R (5′-TTCTGCAGCACACATTAGGA-3′) for sno, strawberry notch homologue 1 (SBNO; AK001563); TGFBRAP1-F (5′-GGGCTAACAATAAAGGCAGTC-3′) and TGFBRAP1-R (5′-CAACACATGGATGTTTTTGC-3′) for transforming growth factor-β receptor–associated protein 1 (TGFBRAP1; NM_004257); RIOK1-F (5′-GAAGACAGCCAAGACGAAAA-3′) and RIOK1-R (5′-TCCTCTGTCAACACCAGACA-3′) for RIO kinase 1 (RIOK1; yeast; NM_031480); and RASGEF1A-F (5′-TTTCCCATGTCTGACTTCGT-3′) and (5′-CAATGTCTTCAGGCTCTTCC-3′) for RasGEF domain family, member 1A (RASGEF1A; NM_145313). All reactions involved initial denaturation at 94°C for 2 min followed by 21 (for ACTB) or 30 (for others) cycles of 95°C for 30 s, 58°C to 62°C for 30 s, and 72°C for 45 s on a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA).

Northern Blot Analysis

Human multiple-tissue blots (23 normal tissues, including brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung, peripheral blood leukocyte, adrenal gland, bladder, bone marrow, lymph node, prostate, spinal cord, stomach, thyroid, tongue, trachea, and uterus; BD Biosciences Clontech, Palo Alto, CA) were hybridized with a 32P-labeled PCR product of MAPJD. The cDNA probe of MAPJD was synthesized by reverse transcription-PCR (RT-PCR) using a set of primers: MAPJD-F2 (5′-CTGGAAACAAGGCAGTAGTGATT-3′) and MAPJD-R2 (5′-GTACACTGAAGCCTGAAGGTGAT-3′). Prehybridization, hybridization, and washing were done according to the supplier's recommendations. The blots were autoradiographed with intensifying screens at room temperature for 14 days.

Western Blotting

Rabbit antibodies specific for MAPJD were raised by immunizing rabbits with recombinant human MAPJD protein and purified using standard protocols. Anti-myc antibody (9E10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-Flag antibody (M2; Santa Cruz Biotechnology) were used for detecting exogenously expressed proteins. We used an enhanced chemiluminescence Western blotting analysis system (GE Healthcare Biosciences, Piscataway, NJ). Cells were lysed in appropriate amounts of lysing buffer [150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 8.0), 1% NP40, 0.1% SDS, 0.5% sodium deoxycholate, plus protease inhibitor]. Proteins separated by SDS-PAGE were electroblotted onto nitrocellulose membranes (GE Healthcare Biosciences) and incubated with antibodies. A sheep anti-mouse IgG-horseradish peroxidase antibody (GE Healthcare Biosciences) and a goat anti-rabbit IgG-horse-radish peroxidase antibody (GE Healthcare Biosciences) were served as the secondary antibodies for the experiments.

Immunohistochemistry and Tissue Microarray

Tumor tissue microarrays prepared from formalin-fixed lung cancers were constructed as published previously (2628). The staining pattern of MAPJD was assessed semiquantitatively as absent or positive by three independent investigators without prior knowledge of the clinical follow-up data. Cases with <20% of nuclear MAPJD-stained tumor cells were judged as MAPJD negative. Cases were accepted as positive only if reviewers independently defined them as such. We confirmed that the antibody was specific to MAPJD, on Western blots using lysates from cell lines that had been transfected with MAPJD expression vector as well as by immunocytochemical staining of cell lines that either expressed MAPJD endogenously or not.

To investigate the presence of MAPJD protein in tissue microarrays of clinical samples, we stained the sections using EnVision+ Kit/horseradish peroxidase (DakoCytomation, Glostrup, Denmark). Rabbit antibody to human MAPJD was added after blocking endogenous peroxidase and proteins, and the tissue sections were incubated with horseradish peroxidase–labeled anti-rabbit IgG as the secondary antibody. Substrate chromogen was added and the specimens were counterstained with hematoxylin.

RNAi Assay

We had established a vector-based RNAi system, psiH1BX3.0, to direct the synthesis of small interfering RNAs (siRNA) in mammalian cells, as reported elsewhere (10, 12, 13, 1519). Ten micrograms of aliquots of siRNA expression vector were transfected into NSCLC cell lines LC319 and A549, using 30 μL LipofectAMINE 2000 (Invitrogen, Carlsbad, CA). The transfected cells were cultured for 5 days in the presence of appropriate concentrations of geneticin (G418). Viable cell numbers were measured by Giemsa staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays in triplicate. The target sequences of the synthetic oligonucleotides for RNAi were as follows: control (LUC: luciferase gene from Photinus pyralis), 5′-CGTACGCGGAATACTTCGA-3′; scramble (SCR: gene coding for 5 S and 16 S rRNAs in chloroplasts of Euglena gracilis), 5′-GCGCGCTTTGTAGGATTCG-3′; siRNA-MAPJD-1 (si-MAPJD-1: 5′-CGCAGCTGCGAAGTGTTGTA-3′); and siRNA-MAPJD-2 (si-MAPJD-2: 5′-GATACGAAAGCAGCTGCGA-3′). To validate our RNAi system, down-regulation of MAPJD expression by functional siRNAs, but not by controls, was also confirmed in the cell lines used for this assay.

Flow Cytometry

Cells were plated at densities of 5 × 105/100-mm dish, transfected with siRNA expression vectors, and cultured in the presence of appropriate concentrations of geneticin. Cells were trypsinized 5 days after the transfection, collected in PBS, and fixed in 70% cold ethanol for 30 min. After treatment with 100 μg/mL RNase (Sigma-Aldrich Co., St. Louis, MO), the cells were stained with 50 μg/mL propidium iodide (Sigma-Aldrich) in PBS. Flow cytometry was done on a Becton Dickinson FACScan and analyzed by ModFit software (Verity Software House, Inc., Topsham, ME). The cells selected from at least 20,000 ungated cells were analyzed for DNA content. Cells were also prepared using Annexin V-FITC Apoptosis Detection kit (BioVision, Inc., Mountain View, CA) for Annexin V binding assay according to the supplier's protocol.

MAPJD-Expressing NIH3T3 Transfectants

MAPJD-expressing stable transfectants were established according to a standard protocol. The entire coding region of MAPJD was amplified by RT-PCR using the primer sets MAPJD-F3 (5′-CCGGAATTCCGGGCCACCATGGATGGGCTCCAGGCCAGTGCAGG-3′) and MAPJD-R3 (5′-CCGCTCGAGCGGATTTAGGGCTAGAGGCATCTTAG-3′). The product was digested with EcoRI and XhoI and cloned into appropriate sites of a pcDNA3.1-myc/His A(+) vector (Invitrogen) that contained c-myc/His epitope sequences (LDEESILKQE-HHHHHH) at the COOH terminus of the MAPJD protein. Using Fugene 6 transfection reagent (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions, we transfected NIH3T3 cells, which do not express endogenous MAPJD, with plasmids expressing either MAPJD (pcDNA3.1-MAPJD-myc/His) or mock plasmids (pcDNA3.1). Transfected cells were cultured in DMEM containing 10% FCS and geneticin (0.4 mg/mL) for 14 days, and then, 50 individual colonies were trypsinized and screened for stable transfectants by a limiting dilution assay. Expression of MAPJD was determined in each clone by RT-PCR, Western blotting, and immunostaining. NIH3T3 transfectants that stably expressed MAPJD were seeded onto six-well plates (5 × 104 cells per well) and maintained in medium containing 10% FCS and 0.4 mg/mL geneticin for 24, 48, 72, and 96 h. At each time point, cell proliferation was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in triplicate.

Identification of Downstream Genes of MAPJD by cDNA Microarray

LC319 cells were transfected with either siRNA against MAPJD (si-MAPJD-2) or luciferase (control siRNA). mRNAs were extracted 12, 18, and 24 h after the transfection, labeled with Cy5 or Cy3 dye, and subjected to cohybridization onto cDNA microarray slides containing 23,040 genes as described (17). After normalization of the data, genes with signals higher than the cutoff value were analyzed further. Genes whose intensity was significantly decreased in accordance with the reduction of MAPJD expression were initially selected using self-organizing map cluster analysis (29). Validation of candidate downstream genes of MAPJD was done with semiquantitative RT-PCR experiments of the same mRNAs from LC319 cells used for microarray hybridization, with gene-specific primers.

Reporter Gene Assay

The fragment of each downstream gene was amplified by PCR using the following primers: SBNO-F3 (5′-CGACGCGTCGCTCTAACCATTCATCAGCTC-3′) and SBNO-R2 for SBNO; TGFBRAP1-F3 (5′-CGACGCGTCGAGACATTCTGACCATAGCACC-3′) and TGFBRAP1-R2 for TGFBRAP1; RIOK1-F3 (5′-CGACGCGTCGTTCCTACAATGTCTTCAGTC-3′) and RIOK1-R2 for RIOK1; and RASGEF1A-F3 (5′-CGACGCGTCGCAAGACCTTCACCATGTGG-3′) and RASGEF1A-R2 for RASGEF1A. The fragment of each downstream gene was also cloned into the pGL3 basic vector. Luciferase assays were done using a Dual-Luciferase Reporter Assay System according to the manufacturer's instructions (Promega, Madison, WI).

Immunoprecipitation Assays

Lung cancer LC319 cells (5 × 106), transfected with plasmids expressing MAPJD or c-MYC (p3XFLAG-MAPJD or MYC), or the mock vector (control) were incubated in 1 mL lysis buffer (0.5% NP40, 50 mmol/L Tris-HCl, 150 mmol/L NaCl) in the presence of proteinase inhibitor. Cell extracts were precleared by incubation at 4°C for 1 h with 100 μL protein G-Agarose beads, in final volumes of 2 mL lysis buffer in the presence of proteinase inhibitor. Immunoprecipitation and additional Western blotting using antibodies specific for endogenous MAPJD, MYC (Santa Cruz Biotechnology), TRRAP (sc-11411), and TIP60 (sc-5725) were done as described elsewhere (12, 15, 18).

Chromatin Immunoprecipitation Assay

Cells were cross-linked in 1% formaldehyde for 10 min. The fixed chromatin samples were subjected to immunoprecipitation using chromatin immunoprecipitation (ChIP) assay kit according to the manufacturer's instructions (Upstate, Charlottesville, VA). The sets of primers used for ChIP assay are SBNO-F2 (5′-CGACGCGTCGTCTGTTCTGAGCTTCCATAC-3′) and SBNO-R2 (5′-CCGCTCGAGCGGACTAATTCCACTCTCAC-3′) for SBNO; TGFBRAP1-F2 (5′-CGACGCGTCGGAAAGTCTCACTTCCAATGG-3′) and TGFBRAP1-R2 (5′-CCGCTCGAGCGGATCATGTCTACTGGCTGATC-3′) for TGFBRAP1; RIOK1-F2 (5′-CGACGCGTCGATAGATGTTCCAGAGACATTC-3′) and RIOK1-R2 (5′-CCGCTCGAGCGGTTCAGAAGCCAACAGTGGC-3′) for RIOK1; and RASGEF1A-F2 (5′-CGACGCGTCGCACTGAAGTAATCATGGCAAC-3′) and RASGEF1A-R2 (5′-CCGCTCGAGCGGAGACAACGGACGTCTGCG-3′) for RASGEF1A.

Electrophoretic Mobility Shift Assay

Electrophoretic mobility shift assay was done by incubating immunoprecipitated or recombinant proteins (MAPJD or MYC) with 32P-labeled oligonucleotide using a standard protocol. The sequences of oligonucleotides for the dsDNA probe were 5′-CCCGTCGCACGTGGTGGCCA-3′ and 5′-TGGCCACCACGTGCGACGGG-3′.

Expression of MAPJD in Lung Cancers and Normal Tissues

We previously screened 27,648 genes on a cDNA microarray to screen transcripts that showed 3-fold or higher expression in cancer cells than in normal control cells in more than half of NSCLCs analyzed. Among the up-regulated genes, we identified the C14orf169 (later termed to MAPJD, due to evidences shown below) transcript and confirmed its increased expression in 9 of 10 representative NSCLC cases by semiquantitative RT-PCR experiments (Fig. 1A). We also observed high levels of MAPJD expression in 27 of the 30 lung cancer cell lines, whereas a very weak band of the PCR product was detectable in cells derived from normal airway epithelia (small airway epithelial cells and bronchial epithelial cells; Fig. 1B). Northern blotting using MAPJD cDNA as a probe identified a very weak 2.5-kb band ubiquitously in 23 normal human tissues examined, and additional semiquantitative RT-PCR experiments using the same primers as above detected MAPJD transcript in lung cancer cells, much more abundantly than normal tissues examined (heart, liver, lung, bone marrow, testis, ovary, and placenta; data not shown).

Immunohistochemical analysis with anti-MAPJD polyclonal antibody using tissue microarrays consisting of 300 NSCLCs revealed positive staining of the nucleus in 80% of adenocarcinomas (132 of 164 cases examined), 81% of squamous cell carcinomas (85 of 105 cases), 62% of large cell carcinomas (13 of 21 cases), and 90% of bronchioloalveolar cell carcinomas (9 of 10 cases), whereas, no detectable staining was observed in any of their adjacent normal lung tissues (Fig. 1C).

Effects of MAPJD on Cell Growth

To assess whether MAPJD is essential for growth or survival of lung cancer cells, we designed and constructed plasmids to express siRNA against MAPJD (si-MAPJD-1 and si-MAPJD-2) and two control siRNAs (for luciferase or scramble) and transfected each of them into LC319 and A549 cells (representative data of LC319 was shown in Fig. 2A). The amount of MAPJD transcript in the lung cancer cells transfected with each of si-MAPJD-1 and si-MAPJD-2 was significantly decreased in comparison with those transfected with either of the two control siRNAs (Fig. 2A, left, top); transfection of si-MAPJD-1 or si-MAPJD-2 also resulted in significant decreases in colony numbers and cell viability measured by colony formation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays (si-2 versus luciferase, P = 1.1 × 10−7, unpaired t test; Fig. 2A, left, bottom; right). To clarify the mechanisms of this phenotype further, we did flow cytometrical analysis using LC319 cells that had been transfected with si-MAPJD-2 and found that the sub-G1 proportion of cells treated with si-MAPJD-2 was significantly higher than those treated with control siRNA against LUC (Fig. 2B). Moreover, we confirmed increased number of apoptotic cells transfected with si-MAPJD-2 compared with those transfected with luciferase using Annexin V binding assay (Fig. 2C).

To further verify a potential role of MAPJD in tumorigenesis, we established NIH3T3 cells that stably expressed exogenous MAPJD. The growth rate of NIH3T3-derivative cells that stably expressed MAPJD was much higher than that of cells transfected with mock plasmid in a MAPJD dose-dependent manner (Fig. 2D).

Identification of MAPJD Target Genes

Because MAPJD was present in nucleus and includes a JmjC domain, which is suggested to play an important role in the transcriptional regulation, we first attempted to identify downstream genes specifically regulated by MAPJD in cancer cells. siRNA-MAPJD-2 or siRNA-LUC (control siRNA) was transfected into LC319 cells, in which MAPJD was expressed at a high level, and alterations in gene expression at various time points were monitored using a cDNA microarray consisting of 23,040 genes. Among hundreds of genes that had been down-regulated by this approach, we selected 53 genes whose expression were significantly decreased in accordance with the reduction of MAPJD expression by doing the self-organizing map clustering analysis (29). Semiquantitative RT-PCR analysis confirmed time-dependent reduction of these 53 candidate transcripts in LC319 cells transfected with si-MAPJD-2 (data not shown). We also evaluated the transactivation of these genes in accordance with introduction of exogenous MAPJD expression in lung cancer cell lines (data not shown) and finally selected four top candidate MAPJD target genes, SBNO, TGFBRAP1, RIOK1, and RASGEF1A, which were the most significantly induced by MAPJD expression.

To examine the possible promoter-specific transactivation of these target genes by MAPJD, we cotransfected into LC319 cells reporter plasmids containing a 1 kb upstream region of the putative transcription start site of each of the four genes fused to a luciferase reporter gene and MAPJD-expressing plasmids. MAPJD-transfected cells displayed higher luciferase activity than mock-transfected cells (Fig. 3A).

Interaction of MAPJD with MYC and Their Transcriptional Regulation

The reporter gene assay suggested that MAPJD could act as a transcription factor or regulate gene expressions together with some transcription factor(s). Hence, we searched for candidate consensus binding motif of transcription factors common to regulatory regions of these four candidate MAPJD target genes and found that all of the four candidate genes contained several copies of E-box (CANNTG) in their possible transcriptional regulatory regions. Because MYC is one of the proteins binding to the E-box, we then examined and confirmed by immunoprecipitation assay an association of exogenously expressed MAPJD or MYC with endogenous ones in LC319 cells (Fig. 3B). To examine the association of MAPJD and/or MYC with promoter sites of the four genes, we did ChIP assay with anti-MAPJD antibody or anti-MYC antibodies using extracts of LC319 cells. Genomic segments corresponding to the 1 kb upstream region containing the putative transcription start sites were confirmed to be associated with both MAPJD and MYC proteins (Fig. 3C). To further confirm transcriptional activity of MAPJD and MYC on the MAPJD target genes, we did luciferase assay. LC319 cells transfected with MYC or MAPJD induced higher luciferase activity than those transfected with mock vector, indicating that MAPJD and MYC are responsible for the transactivation of these four genes (Fig. 3D). Moreover, cotransfection of both MAPJD and MYC expression plasmids further increased luciferase activity of reporter plasmids (Fig. 3D). These data suggest that MAPJD and MYC could form a complex and synergistically regulate the transcriptional activity of the candidate MAPJD target genes.

We subsequently focused on the E-box motifs in the possible promoter region of the RIOK1 gene, which has eight E-box motifs from −1504 to +35 (Fig. 4A). ChIP assay with anti-MAPJD and anti-MYC antibodies using nuclear extracts of LC319 cells detected that only the genomic segment-7 containing an E-box motif seemed to be most significantly associated with endogenous MAPJD, whereas MYC almost equally bound to all of the seven segments (data not shown). These data suggest that MAPJD was possibly associated with the E-box of segment-7 in the RIOK1 gene. We also did an electrophoretic mobility shift assay using a double-stranded oligonucleotide probe that corresponds to the possible binding sequence and myc-tagged MAPJD protein purified by immunoprecipitation. A shifted band was detected in the presence of MAPJD, whereas no shifted band was observed after cold competitive oligonucleotides were added, supporting the specific interaction between the oligonucleotide probe and MAPJD (Fig. 4B, left). The band was supershifted after the addition of anti-MAPJD antibody, but not that of normal rabbit IgG, thus independently confirming the specific interaction (Fig. 4C, left). MYC also interacted with this probe specifically (Fig. 4B and C, right).

MAPJD Recruits MYC-Related HAT Complex to Regulatory Regions of Its Target Genes

MYC is known to activate gene transcription through binding to E-boxes in regulatory regions of its target genes and subsequent recruiting of a protein complex, including the TRRAP protein together with GCN5/p300/CBP-associated factor or TIP60 HATs that preferentially acetylate histones H3 and H4 (24, 25). Therefore, we investigated whether MAPJD could recruit the MYC-regulated HAT complex to the four MAPJD target genes (SBNO1, TGFBRAP1, RIOK1, and RASGEF1A) and increase the level of histone acetylation. We first confirmed by immunoprecipitation assay a cognate association of FLAG-tagged MAPJD with endogenous MYC-HAT complex proteins (TRRAP and TIP60) as well as endogenous MYC in LC319 cells (Fig. 5A). We then did ChIP assay with antibodies to TRRAP and triacetylated histone H4 using nuclear extracts of LC319 cells transfected with the MAPJD expression vector. We observed enhancement of the association between TRRAP and each of the gene regulatory regions as well as the increase in triacetylated histone H4 levels in the cells transfected with MAPJD expression plasmid (Fig. 5B). Although the precise mechanism should be further investigated, these data suggest that the MAPJD-MYC complex might bind to the E-box site and recruit chromatin-modifying factors to this site, as similar to an example of MYC/MYC-associated factor X (Fig. 6; refs. 23, 30).

Molecular targeted drugs are expected to be highly specific to malignant cells and have minimal adverse reactions due to their well-defined mechanisms of action. Toward identification of appropriate molecular targets for development of such drugs, we combined genome-wide expression analysis for selecting genes that were overexpressed in lung cancer cells with high-throughput screening of loss-of-function effects by means of the RNAi technique (519). Using this systematic approach, we found MAPJD to be overexpressed commonly in clinical NSCLC samples as well as lung cancer cell lines and showed that overexpression of this gene product plays an indispensable role in the growth of lung cancer cells. MAPJD was initially identified in Xenopus as a dual location protein in the nucleolus and in a special type of synchronously replicating chromatin (20). The human MAPJD encodes a 641-amino acid protein with a JmjC domain. JmjC domain was found commonly in proteins bound to DNA or chromatin, and JmjC domain-containing proteins are thought to regulate chromatin structure and/or gene expressions by regulating modification of histones (20).

Many studies have shown that cell cycle progression rates are closely related to MYC levels (21). Modest increase in the levels of MYC was supposed to be an initiating event in the various forms of human cancer (21). MYC regulates the transcription of downstream target genes by recruiting the acetyltransferase complexes to the target genes. In this process, it was suggested that MYC may work through the modification of HAT activities, in concert with other chromatin remodeling enzymes, some of which may not be identified yet, and that different combinations of HATs and chromatin remodeling factors might be applied in different cellular conditions (2125). We here showed that MAPJD transactivated a set of genes possibly related to lung cancer cell proliferation, by interacting with MYC. We identified four candidate downstream genes of MAPJD, SBNO1, TGFBRAP1, RIOK1, and RASGEF1A. Our data obtained by the reporter gene and ChIP assays using the E-box motifs in the possible promoter region of each of the four genes suggested that MYC and MAPJD might synergistically have the transactivating activity through their association with promoter sites of the target genes. Importantly, it should be noted that TGFBRAP1 has only one E-box in the predicted promoter region examined (70 bp upstream of the transcription initiation site). This independently supports our hypothesis that MYC and MAPJD could synergistically bind to the same promoter and activate transcription of downstream genes, although future detailed studies with mutant promoter constructs could be required to clarify this mechanism. Interestingly, all of the four candidate MAPJD target genes have been supposed to be involved in oncogenic signalings. SBNO1 is a downstream component of the Notch signaling pathway that is known to function in the oncogenic process and is required during embryogenesis and oogenesis in Drosophila (31, 32). TGFBRAP1 binds to TGFB receptors that were reported to be related to tumorigenesis (33, 34). RIOK1 is overexpressed in colon cancers (35). RASGEF1A contains a RasGEF domain, which might play an important role in the Ras signaling pathway. Although, the precise function of these downstream genes in human carcinogenesis and even their physiologic function in mammalian cells remain to be elucidated, our data suggest that MAPJD could be one of the key regulators to selectively activate the transcription of several genes through the recruitment of HAT complex to chromatin and subsequent histone acetylation, along with MYC oncoprotein.

We confirmed that MAPJD localized at nucleolus and nucleoplasm in cancer cells (data not shown). The nucleolus is known to function in ribosome biogenesis, a complicated process that includes the transcription of rRNA genes, the processing and modification of these transcripts, and their assembly with both ribosomal proteins as well as nonribosomal ones to guide the formation of preribosomal particles (36, 37). In addition, it was reported recently that nucleolus also harbors diverse functions that involved the assembly of various other ribonucleoprotein particles, the modification of small RNAs, the control of the cell cycle, the sequestration of regulatory molecules, and nuclear export process (38, 39). It was reported recently that MYC bound to human rDNA in nucleoli and stimulated transcription of rRNA by associating with the RNA polymerase I–specific factor SL1 (40). The combined evidence suggests that MAPJD might also play an important role at nucleolus in cancer cells, by interacting with other nucleolar proteins as well as MYC.

In summary, we showed that MAPJD is involved in the gene transcription as a possible member of the MYC transcriptional complex. Because MAPJD is likely to be essential for promotion of growth of lung cancer, we suggest that it could be a novel therapeutic target for development of anticancer drugs.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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