Regulation of DNA Polymerase POLD4 Influences Genomic Instability in Lung Cancer

Published OnlineFirst September 22, 2010; DOI: 10.1158/0008-5472.CAN-10-0784

Molecular and Cellular Pathobiology Cancer Research Regulation of DNA Polymerase POLD4 Influences Genomic Instability in Lung Cancer

Qin Miao Huang1, Shuta Tomida1, Yuji Masuda5, Chinatsu Arima1, Ke Cao1, Taka-aki Kasahara1, Hirotaka Osada3, Yasushi Yatabe4, Tomohiro Akashi2, Kenji Kamiya5, Takashi Takahashi1, and Motoshi Suzuki1

Abstract Genomic instability is an important factor in cancer susceptibility, but a mechanistic understanding of how it arises remains unclear. We examined hypothesized contributions of the replicative DNA polymerase δ (pol δ) subunit POLD4 to the generation of genomic instability in lung cancer. In examinations of 158 lung cancers and 5 mixtures of 10 normal lungs, cell cycle- and checkpoint-related genes generally showed mRNA expression increases in cancer, whereas POLD4 showed reduced mRNA in small cell lung cancer (SCLC). A fraction of non–small cell lung cancer patients also showed low expression comparable with that in SCLC, which was associated with poor prognosis. The lung cancer cell line ACC-LC-48 was found to have low POLD4 expression, with higher histone H3K9 methylation and lower acetylation in the POLD4 promoter, as compared with the A549 cell line with high POLD4 expression. In the absence of POLD4, pol δ exhibited impaired in vitro DNA synthesis activity. Augmenting POLD4 expression in cells where it was attenuated altered the sensitivity to the chemical carcinogen 4-nitroquinoline-1-oxide. Conversely, siRNA-mediated reduction of POLD4 in cells with abundant expression resulted in a cell cycle delay, checkpoint activation, and an elevated frequency of chromosomal gap/break formation. Overexpression of an engineered POLD4 carrying silent mutations at the siRNA target site rescued these phenotypes, firmly establishing the role of POLD4 in these effects. Further- more, POLD4 overexpression reduced intrinsically high induction of γ-H2AX, a well-accepted marker of double-stranded DNA breaks. Together, our findings suggest that reduced expression of POLD4 plays a role in genomic instability in lung cancer. Cancer Res; 70(21); 8407–16. ©2010 AACR.

Introduction human cancers (3–8). Activation of DNA damage checkpoints, even in preneoplastic lesions, leads to cell cycle blockade or Lung cancer has become the leading cause of cancer death apoptosis (3, 6), and abrogation of that activation is thought in many industrialized countries, with small cell lung cancer to be critical during multistep transformation processes, (SCLC), a very aggressive subset formed by small cells with whereas fully malignant cells of overt cancers frequently carry scarce cytoplasm, molded nuclei, and neuroendocrine differ- various defects in checkpoint mechanisms (4, 5, 7, 8). entiation, accounting for about 15% to 20% of all lung cancer In lung cancer, it is conceivable that smoking plays a ma- cases (1). Due to its highly proliferative and metastatic poten- jor role in induction of DNA damage that may be detected by tial, as well as nearly certain recurrence after chemotherapy, checkpoint responses, although the intrinsic driving force be- the 5-year survival rate for SCLC is around 5% (2). hind this related genomic erosion has yet to be identified (1). It was recently reported that highly conserved DNA damage For many carcinogens, initiation of carcinogenesis requires response pathways are frequently altered at various phases in DNA replication, suggesting that genetic alterations are fixed in the genome during replication of damaged DNA. For this reason, a long-term hypothesis states that errors in DNA rep- Authors' Affiliations: Divisions of 1Molecular Carcinogenesis and lication and deficits in DNA repair account for multiple mu- 2Molecular Mycology and Medicine, Nagoya University Graduate School tations in cancer (9, 10). Herein, we report evidence showing of Medicine, 3Division of Molecular Oncology, Aichi Cancer Center δ δ Research Institute, and 4Department of Pathology and Molecular that polymerase (pol ), which is a core DNA replication Diagnosis, Aichi Cancer Center Hospital, Nagoya, Japan; and 5Research and repair protein, is frequently downregulated in the POLD4 Institute for Radiation Biology and Medicine, Hiroshima University, subunit in SCLC and a fraction of non–small cell lung cancer Hiroshima, Japan (NSCLC). Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Motoshi Suzuki, Nagoya University Graduate School of Medicine, Tsuruma-cho 65, Showa-ku, Nagoya 466-8550, Materials and Methods Japan. Phone: 81527442455; Fax: 81527442457; E-mail: msuzuki@ med.nagoya-u.ac.jp. Cell lines doi: 10.1158/0008-5472.CAN-10-0784 Cell lines with the prefix ACC-LC- were established in ©2010 American Association for Cancer Research. our laboratories. Calu6 and SK-LC-6 cells were generously

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provided by L.J. Old (Memorial Sloan-Kettering Cancer (Supplementary Table S1). Results were normalized to the in- Center, New York, NY) and PC-10 cells by Y. Hayata (Tokyo put control. SDs are also presented. Medical University, Tokyo, Japan), whereas the NCI-H460, SK-MES-1, HCT116, and A549 cell lines were purchased Immunoprecipitation of pol δ complexes from the American Type Culture Collection. Cells were cul- Cells were lysed in Nonidet P40 (NP40) lysis buffer tured in RPMI 1640 supplemented with 5% fetal bovine [20 mmol/L HEPES (pH 7.8), 300 mmol/L NaCl, 1 mmol/L serum (Sigma-Aldrich). Plat-E cells were kindly provided by EDTA, and 1% NP40] supplemented with cOmplete (Roche Dr. T. Kitamura (University of Tokyo, Tokyo, Japan). HCT116 Diagnostics Gmbh), then centrifuged at 4°C. The anti-POLD1 and Plat-E cells were cultured in DMEM supplemented with antibody was added to the supernatant fraction, mixed 5% fetal bovine serum. gently, and incubated overnight at 4°C. After Protein G Sepharose (GE Healthcare) was incubated with the mixture Antibodies for 1 hour, it was precipitated and washed four times with A polyclonal anti-POLD4 antibody was raised against a pu- NP40 lysis buffer. The pol δ complex was recovered from rified protein preparation (see Fig. 3B) using a New Zealand the Sepharose by boiling in SDS sample buffer and was White rabbit (Fig. 6A), whereas another polyclonal antibody analyzed using Western blotting. was raised against a glutathione S-transferase (GST)-tagged POLD4 preparation, followed by removal of the anti-GST an- Purification and characterization of pol δ tibody (CycLex; Fig. 3D). Other antibodies were anti-POLD4 All proteins used in this study were overproduced in Escher- (POLD4/p12 subunit of pol δ) ascites (2B11, Abnova), POLD1 ichia coli and purified as previously described (13). The purity (p125 subunit of pol δ), Chk1 (Santa Cruz Biotechnology), of the proteins, including 3- and 4-subunit pol δ, was moni- NBS1-pSer343, Chk2-pTyr68, Chk1-pSer317 (Cell Signaling tored by SDS-PAGE and Coomassie brilliant blue R-250 stain- Technology), NBS1 (Oncogene Research/Calbiochem/ ing. Protein concentrations were determined using Bio-Rad Merk), ATM-pSer1981 (Rockland Immunochemicals), ATM protein assays with bovine serum albumin (Bio-Rad) as the (2C1, GeneTex, Inc.), H2AX (BL552), SMC1, SMC1-pSer966 standard. DNA polymerase activity was measured in a reaction (Bethyl laboratories), FLAG (Sigma-Aldrich), Alexa- mixture (25 μL) containing 20 mmol/L HEPES-NaOH (pH 7.5), Fluoro647–conjugated γ-H2AX, p21, p27 (BD), Chk2 (MBL), 50 mmol/L NaCl, 0.2 mg/mL bovine serum albumin, 1 mmol/L anti-trimethyl- histone H3 (Lys9), and anti-acetyl-histone dithiothreitol, 10 mmol/L MgCl2, 1 mmol/L ATP, 0.1 mmol/L H3 (Millipore/Upstate). each of dGTP, dATP, dCTP, and [α-32P]dTTP, 33 fmol (240 pmol for nucleotides) of singly primed ss-mp18 DNA Patients (Supplementary Table S1), 1.0 μg(9.1pmol)ofRPA,86ng Prior to obtaining patient samples, requisite approval (1.0 pmol as a trimer) of proliferating cell nuclear antigen from the review board of Aichi Cancer Center, Nagoya, (PCNA), 75 ng (260 fmol) of RFC, and 11 to 88 ng (46 to 372 fmol) Japan, and written informed consent from the patients were of pol δ. After incubation at 30°C for 10 minutes, the products obtained. were electrophoresed on 0.7% alkaline-agarose gels (13).

RNA interference Plasmid construction and isolation of stable clones Transfection was carried out using 50 nmol/L of a siRNA Using an IMAGE clone (ID: 6450262, Invitrogen) and the (Supplementary Table S1) duplex (Sigma-Aldrich) targeting primer sets (Supplementary Table S1), the POLD4 open read- each mRNA or negative control 1 (Ambion) with Lipofectamine- ing frame (ORF) was PCR-cloned, and inserted into the large 2000 (Invitrogen). HindIII-EcoRI fragment of pcDNA3 (FLAGPOLD4pcDNA3). ACC-LC-172 cells were transfected by FLAGPOLD4pcDNA3 Quantitative real-time reverse transcriptase-PCR and treated with G418 for establishment of stable clones. Total RNA and cDNA were prepared using an RNeasy Mini For construction of an siD4-resistant construct, silent Kit (QIAGEN) and SuperScript II Reverse Transcriptase (In- mutations were introduced by synthetic oligomers (Supple- vitrogen), respectively. Quantitative reverse transcriptase- mentary Table S1). The PCR fragments were inserted into a PCR (RT-PCR) was performed using a 7500 Fast Real-Time pGFP-MSCV vector (pMSCVpold4). Plat-E cells were trans- PCR System and Power CYBR Green PCR master mix, ac- fected using pMSCVpold4 and a VSV-G expression vector, cording to the manufacturer's instructions (Applied Biosys- and cultured for two days. The retrovirus was recovered from tems). The primer sets are described in Supplementary the culture medium and used for infection. After two days, Table S1. Ct values for POLD4 or POLD1 were normalized green fluorescent protein–positive cells were isolated using to those of 18S (ΔCt). The average ΔΔCt values were then FACS Vantage or Area2 (BD). calculated by normalization to the ΔCt values of A549 cells. MTT assay Chromatin immunoprecipitation ACC-LC-172 cells and siRNA-treated (48 h) or non-treated Chromatin immunoprecipitation samples were prepared cells were separately cultured in 200 μL of culture medium in from A549 or ACC-LC-48 cells logarithmically grown on 96-well plates at a density of 8,000/well. The next day, the 10-cm dishes, as previously described (11, 12). The genomic medium was replaced with that containing 4-nitroquinoline- DNA contents were determined by quantitative RT-PCR 1-oxide (4NQO) or vinorelbine, followed by incubation for

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Figure 1. Low expression levels of POLD4 in SCLC. A, microarray analysis of 149 NSCLC, 9 SCLC, and 5 normal lung mixture samples showed that the mRNA expression level of POLD4, but not of other pol δ subunits, was low in SCLC. Quartile data are shown in graphs. *, P < 0.05; **, P < 0.01, t-test. B, mRNA expression levels of POLD4 and POLD1 in 48 clinical tissue samples, independent of the microarray cohort, were determined by quantitative RT-PCR. The number of each sample class is in parentheses. Results were corrected with 18S expression values and are presented as relative values to that of A549, which was given a value of 1. Bars, SD. *, P < 0.05; **, P < 0.01, t-test. C, Western blotting analyses of POLD4 and POLD1 were carried out using a panel of lung cancer cell lines. D, the 149 NSCLC patients were classified into two groups according to POLD4 expression level, with the threshold set at −1 SD of the average. Kaplan-Meier survival curves for these groups showed a significant difference in both overall and relapse-free survival. P values were determined using a log-rank test.

48 hours. Viable cells were measured in triplicate using then the cells were released by three washes in PBS, incubated TetraColor One (Seikagaku) with reference to the viability in normal culture medium for various times, and harvested of mock-treated cells. for flow-cytometric and Western blotting analyses.

Cell cycle synchronization Chromosomal analysis Calu6 was synchronized according to the method of Colcemid at 0.1 μg/mL was added to the culture medium Nakagawa et al. (8), with some modifications. Briefly, 24-hour for 2 hours, then HCT116 cells were harvested, washed with treatment with 2 mmol/L thymidine was used to arrest ex- PBS, and incubated at 37°C in 75 mmol/L KCl for 30 minutes, ponentially proliferating cells in the S phase. The cells were followed by centrifugation at 1,500 rpm for 5 minutes. The then released from arrest by three washes in PBS, then cells were further incubated in Carnoy's solution (methanol: siCTRL- and siD4-treated cells were grown in fresh medium acetate = 3:1) for 30 minutes, collected by centrifugation at for 12 and 15 hours, respectively. The release times were not 800 rpm for 5 minutes, resuspended in 0.5 mL of Carnoy's the same due to a delayed cell cycle progression by siD4. Aphi- solution, dropped onto a wet (75% ethanol) slide glass from dicolin at 1 μmol/L was used for the second block for 24 hours, a height of 30 cm, and flamed with a burner. The slide glass

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Figure 2. Detection of histone modifications. A, after treating cells with 0, 0.25, or 1 μg/mL of trichostatin A (TSA) for 48 (A549 and ACC-LC-48) or 16 (ACC-LC-172) hours (left), or with 0, 0.5, 1, or 2 μmol/L of valproic acid (VPA) for 16 hours (right), total RNA was extracted and quantitative RT-PCR was performed. The results were corrected by 18S expression values and are presented as relative to that without drugs, which was set at 1. Bars, SD. *, P < 0.05; **, P < 0.01, t-test. B, ACC-LC-48 and A549 cells were treated with 0, 0.25, or 1 μg/mL of TSA for 16 or 48 hours, then Western blotting analysis was performed for detection of POLD4 and POLD1, with α-tubulin used as the control (left). Western blotting analysis was also performed for POLD4 with or without 2 mmol/L VPA. C, chromatin immunoprecipitation samples were prepared as described in Materials and Methods. Input controls represent samples not subjected to immunoprecipitation (IP). In the promoter region of POLD4 of the NSCLC cell line A549, histone H3 was acetylated at a level 5.6 ± 2.4-fold greater as compared with the SCLC cell line ACC-LC-48. The trimethylation level of Lys9 of histone H3 in ACC-LC-48 was 3.6 ± 2.2-fold greater than that in A549. In a control experiment to analyze histone H3 modifications in the POLD1 promoter, similar amounts of acetylation and methylation were detected in the two cell lines.

was left overnight at room temperature, then dipped into units had expression levels in the order of SCLC > NSCLC > 4% Giemsa solution for 15 minutes, rinsed with water from normal lung (Fig. 1A), whereas the majority of growth- the opposite side of the sample, air-dried, and shielded by a related mRNAs had increased levels of expression in the lung coverslip. Mitotic chromosomes were analyzed using a Bx 60 cancer specimens (Supplementary Fig. S1A). In hierarchical microscope (Olympus), and the Invision V 4.07 (Biovision clustering analysis, POLD4 was found in a distant small Technology) and Photoshop (Adobe) software packages. branch (Supplementary Fig. S2), suggesting a unique control mechanism of POLD4 expression. RT-PCR findings from a Results second panel of clinical samples confirmed our results (Fig. 1B). In addition, distinctive downregulation of POLD4 was Low expression of POLD4 mRNA and POLD4 protein observed in another database set (Supplementary Fig. S1B). in SCLC The low level of POLD4 expression seen is unlikely attribut- Genomic instability is thought to underlie the pathogene- able to the detection of splicing variants (Supplementary sis of lung cancer. To elucidate the underlying mechanisms, Fig. S3).Western blotting analysis showed that POLD4 (alter- we screened for genes involved in DNA metabolism (Dataset natively called p12) was detected at the level of the back- S1) using our previous profiling data set consisting of 149 ground in ACC-LC-76 and -172 (Fig. 1C). In addition, two cases of NSCLC, 9 cases of SCLC, and 5 normal lung mixture other SCLC cell lines showed marginal expression levels, specimens (14). We found that POLD4 expression was dis- whereas apparent expressions were also observed in the tinctly reduced in SCLC, whereas a small fraction of NSCLC NSCLC cell lines. In contrast, both panels of cell lines ex- specimens also had a low expression of POLD4 that was com- pressed a comparable amount of POLD1 (p125). It was also parable with SCLC (Fig. 1A). In contrast, other DNA pol δ sub- noted that a fraction of NSCLC specimens with low POLD4

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expression levels comparable with SCLC were associated with δ complex is required to exclude the possibility that POLD4 poor prognosis (Fig. 1D). In accord with the low level of mRNA expression in SCLC and a fraction of NSCLC is sufficient for expression (Supplementary Fig. S4), POLD4 was induced by tri- constituting the 4-subunit structure of the pol δ complex. chostatin A or valproic acid, class I and II mammalian histone POLD4 was identified in the immunoprecipitated pol δ com- deacetylase inhibitors, in ACC-LC-48 and -172 (Fig. 2A and B). plex using the anti-POLD1 antibody in the A549 cell line with ACC-LC-48 was further shown to be associated with higher a high expression of POLD4, but not in ACC-LC-172 cells with histone H3K9 methylation and lower acetylation patterns in a low level of POLD4 (Fig. 3A), which strongly suggests that the POLD4 promoter region than was A549 (Fig. 2C). POLD4 most of pol δ in ACC-LC-172 cells lack POLD4. In the pres- expression in ACC-LC-76 was induced by 5 aza-2' deoxycyti- ence of pol δ accessory proteins, including PCNA, RPA, and dine, although the promoter methylations were not dense RFC, POLD4-deficient pol δ showed DNA replication activity and their contribution to POLD4 expression is yet to be stud- that was lower than that of intact pol δ (Fig. 3B and C), which ied (Supplementary Fig. S5A). On the other hand, proteasome- supports a previous finding (17, 18). We also examined the dependent protein degradation pathways did not seem to play functional consequences of reduced POLD4 expression a major role (Supplementary Fig. S5B). in lung cancer pathogenesis. To this end, we attempted to establish SCLC cell lines with ectopic POLD4 expression, POLD4-deficient pol δ impairs pol and nucleotide although most of the SCLC cells did not allow POLD4 over- excision DNA repair activities expression (data not shown); such intolerance has also been Because pol δ is an essential protein complex for DNA rep- reported for another replicative DNA polymerase subunit, lication and repair (15, 16), a subunit stoichiometry of the pol POLA1 (19). For this reason, we chose the NSCLC cell line

Figure 3. Effects of POLD4 depletion on in vitro pol δ and NER activities. A, after immunoprecipitation was performed using the anti-POLD1 antibody (IP), POLD4 and POLD1 were detected by Western blotting analysis. A whole cell extract (WCE) was used as the control. B, the purity of the recombinant pol δ complex with or without the POLD4 subunit was analyzed by SDS gel electrophoresis. Each subunit is indicated on the right. C, DNA replication activity between the two structures of pol δ was compared in the presence of accessory proteins using primed M13 DNA as a template primer. The length of the synthesized DNA is indicated on the right. The amounts of pol δ complexes in the reactions varied from 0 to 88 ng, as described in Materials and Methods. D, expression levels of POLD4 in Calu6, SK-LC-6, and PC-10 cells. Depletion of POLD4 was monitored in Calu6 and PC-10 cells treated with either siCTRL or siD4. Below, results of MTT assays to determine cell growth two days after treatment with various concentrations of drugs. OD values with no drug were regarded as 100%. Clones and siRNA treatments are indicated. D4O/E and VC represent clones with POLD4 overexpression and the vector control, respectively.

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SK-LC-6, which has a low level of POLD4 expression. Al- others, siD4 altered the cell cycle of each population (Sup- though 4NQO produces a DNA adduct that is removed plementary Fig. S7A and B), and induced p21 and p27 through pol δ–dependent nucleotide excision DNA repair (Supplementary Fig. S7C). (NER), POLD4-introduced SK-LC-6 cells were more resistant We synchronized and released cells from the G1-S to 4NQO (Fig. 3D and Supplementary Fig. S6A). Similar re- boundary, and chased them. A fraction of the siD4-treated sults were obtained with the SCLC cell line ACC-LC-172, de- cells did not progress into the S-phase, but rather stayed spite the findings that this cell line is intrinsically resistant to at G1-S (Fig. 4A, filled arrows). Again, persistent induction 4NQO (Supplementary Fig. S6B). In a reciprocal experiment of p21 and p27 was detected (Fig. 4B). It was also ob- using the NSCLC cell lines Calu6 and PC-10, siRNA treatment served that the timing of entry into the G1 phase was against POLD4 (siD4) reduced the protein level to nearly one not the same between siCTRL and siD4 cells. In the latter, third of the original level, which resulted in increased sensi- G1 entry was seen 12 hours after release, which was tivity to 4NQO (Fig. 3D). 2 hours later than that with the siCTRL cells (Fig. 4A, open To examine whether POLD4 expression has any specificity arrows). In addition, the DNA damage checkpoint genes to the NER pathway, the effects of POLD4 expression on sen- NBS1, ATM, CHK2, SMC1, and CHK1 were phosphorylated sitivities to vinorelbine, a microtubule assembly inhibitor, throughout S to G2 (Fig. 4C), suggesting that the cell cycle were studied. For this experiment, we used PC-10 cells, as was delayed by activation of these checkpoint proteins. they have modest sensitivity to a variety of drugs, indicating These phenotypes were rescued in stable clones that car- that this cell line has a low possibility of having abnormal ried pold4 with silent mutations at the siD4 target site drug metabolic pathways (data not shown). Our results (Fig. 5). Additional knockdown of p27 resulted in efficient showed that siD4 did not alter the sensitivity to vinorelbine S-phase entry, which indicates that p27 induction inhibits (Fig. 3D). cell cycle progression at the G1-S boundary in Calu6 cells (Supplementary Fig. S8A–D). POLD4 required for cell cycle progression These results suggest that POLD4 downregulation acti- POLD4 A previous study showed that the ortholog of vates checkpoint proteins, induces G1-S arrest, and delays Cdm1 in Schizosaccharomyces pombe (S. pombe) is a non- the cell cycle from S to G2. Generation of double-stranded essential gene (20), although it may be required in mam- DNA breaks (DSB) was also assessed by a knockdown exper- malian cells (21, 22). To study human POLD4 functions, we iment of DNAPK, a nonhomologous end-joining kinase, depleted POLD4 using siD4 and analyzed the cell cycle which had a synergistic effect with siD4 on the G1-S popula- progression. In the NSCLC cell line Calu6 as well as in tion (Supplementary Fig. S8E and F).

Figure 4. POLD4 reduction induces checkpoint activation. A, Calu6 cells were synchronized at the G1-S boundary and released, then DNA contents were measured every two hours. Filled arrows, G1-S arrested population; open arrows, G1 re-entry. B, after treating Calu6 cells with siRNA for 48 hours, the cells were analyzed for the expression levels of G1-S checkpoint proteins. C, at each time point after release, Western blotting analysis was performed using the indicated antibodies.

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Figure 5. Ectopic overexpression of POLD4 with silent mutations rescues cell cycle phenotypes. A, Western blotting analysis using the rabbit polyclonal anti-POLD4 antibody was carried out to monitor overexpression in Calu6 pold4_1 and _2 clones (top), which were resistant to siD4 treatment (anti-FLAG antibody, bottom). B, Calu6 pold4_1 and _2 and the vector control clones of pMSCVvc_1 and _2 were synchronized and released, then monitored for DNA content. Filled arrows, G1-S arrested population; open arrows, G1 re-entry. C and D, at each time point after release, Western blotting analysis was performed using the indicated antibodies and Calu6 stable clones.

Induction of chromosome breaks and gaps by numbers of chromosomal gaps and breaks to twice as many POLD4 reduction as observed with siCTRL treatment, whereas this phenotype Activation of the ATM, ATR, and DNA-PK pathways suggests was readily suppressed in cells expressing pold4 with silent that both DSB and single-stranded breaks were generated by mutations at the siRNA target site (Fig. 6A, C, and D). POLD4 reduction. These results are in accord with our recent observation that SCLC cells are associated with constitutive DSB (5). Notably, POLD4 overexpression reduced the γ-H2AX Discussion intensity (Fig. 6A and B). Furthermore, we investigated whether POLD4 reduction also induces any chromosome aberrations. To date, despite the premise that deficits in DNA replica- For this experiment, most lung cancer cell lines were not appli- tion machinery account for multiple mutations in cancer cable to this particular purpose because they exhibit aneuploidy (10), and findings showing that laboratory-born animals in association with chromosomal instability (23). We used and strains defective in DNA replication machineries exhibit HCT116 cells where POLD4 expression levels were sufficiently genomic instability (24, 25), core DNA replication proteins high (Fig. 6A). siD4 treatment significantly increased the have rarely been identified as a frequent target for alterations

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Figure 6. Chromosomal gap/break formation in cells. A, Western blotting analysis was performed to confirm the overexpression of POLD4 in the stable clones (ACC-LC-172, D4 O/E1, and O/E2). Shown are representative results of Western blotting analysis using HCT116 and the monoclonal antibody (left), HCT116 stable clones and the rabbit polyclonal anti-POLD4 antibody (middle), and a mixture of 7 stable clones with pold4 overexpression and the anti-FLAG antibody (right), which were used to monitor silencing efficiency, overexpression, and resistance against siD4 treatment, respectively. B, S-phase cells from ACC-LC-172 D4 O/E and control clones were quantitated in regard to γ−H2AX induction, as indicated. C, representative photograph of mitotic chromosomes. Arrows, chromosomal breaks; bar, 10 μmol/L. D, chromosomal gaps and breaks were quantitated by counting 100 mitotic cells. The numbers of cells with ≥2 gaps/breaks were plotted for mock, vector control (VC), and mD4-overexpressed HCT116 cells (pold4). Stable VC and mD4 clones were mixtures of 7 independent clones. *, P < 0.05; **, P < 0.01, Fisher's exact test). To acquire sufficient numbers of high-quality images, the sums of aberrations from five independent experiments were used for comparison. The numbers of total gaps/breaks are also shown in Supplementary Fig. S9.

in human cancers. The present results show that the pol δ (<1 kb) with similar base substitution spectra (26, 27), where- subunit POLD4 is frequently reduced in some lung cancer as the results of recent next-generation sequencing studies specimens and cell lines, whereas overexpression of POLD4 that utilized one sample each of SCLC and NSCLC showed complements part of the pathologic phenotypes of lung frequent transversions in both types of cancer, and also indi- cancer cell lines. cated that SCLC carried significantly more frequent deletions It is interesting to note that although the POLD4 reduction (53 of 65) than NSCLC (93 of 424; P =1.28×10-20, Fisher's was most evident in SCLC, which is a very aggressive form of exact test, denominators include all small insertions and de- lung cancer, a small fraction of NSCLC cases also showed low letions; refs. 28, 29). Impaired activity in replicative DNA expression in association with poor prognosis. In in vitro ex- polymerases is also known to increase the possibility of large periments, we found that POLD4 is required to suppress DSB deletions and duplications of chromosomes and arms (30, 31). and subsequent checkpoint activation. Together with our Using datasets made from 74 lung adenocarcinoma patients previous findings that clinically obtained SCLC specimens for gene expression levels as well as copy number variations were frequently associated with discrete γ-H2AX foci forma- (32, 33), we noted that low POLD4 expression was significantly tion (5), these results suggest that reduced POLD4 expression associated with 8p, 9q, and 13q deletions, and 5p, 7p, 8q, and may play a role in lung cancer progression in association 14q amplifications, which are shared by both NSCLC and with the genomic instability phenotypes. SCLC (refs. 34, 35; Supplementary Table S2). In addition, we Along this line, cells with impaired activity of replicative observed that low POLD4 expression in primary tumors was DNA polymerases induce deletions with short nucleotides associated with high expression of HDAC2 (P = 0.0036; see

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POLD4 Reduction and Genomic Instability in Lung Cancer

Supplementary Information), which was in accord with our expression. This notion is also in line with a recent hypoth- in vitro data showing that POLD4 expression is regulated by esis that DNA replication stress leads to DSB, genomic insta- histone modifications. bility, and selective pressure for later p53 mutations (3, 5, 6). SCLC exhibits aggressive cell growth leading to very poor It is important to note, however, that involvement of POLD4 clinical prognosis, although it is also characterized by large as a crucial effecter of an unidentified primary target for areas of necrosis (36) and cell lines are difficult to establish genetic/epigenetic alterations in lung cancer cannot be for- (37). The putative growth disadvantage due to low POLD4 merly precluded. Future studies, such as with genetically might be partially compromised by the characteristics of engineered mice, are required to determine whether POLD4 SCLC. First, G1-S transition may be supplemented by factors downregulation is involved in the early stage of carcinogen- that are highly expressed in SCLC. With regard to this esis or if it contributes to the late progression stage. speculation, it has been reported that CDC6 and CDT1, which Our results indicate that POLD4 is required for maintenance are recruited at G1-S for licensing the DNA replication, are ab- of genomic stability of human cells. With low expression levels normally overexpressed in the early stage of carcinogenesis of POLD4, lung cancer may grow with accumulation of DNA (38). In our analysis of SCLC, >10-fold expressions of CDC6 damage. Publicly available datasets show that POLD4 expres- and CDT1 were observed in the specimens (Supplementary sion levels vary widely in other types of cancer, although most Fig. S1A). The G1-S transition may be thereby facilitated by datasets do not contain information for corresponding normal forced entry into the S phase in SCLC. Second, nearly all SCLC tissues. It would be interesting to study whether POLD4 tumors are deficient of Rb and p53, which are critical regulators reduction is specific to lung cancer or may be involved in the for G1-S transition (1, 23). Checkpoint impairment may benefit development of other types of cancer. SCLC cell proliferation, survival, increased genomic instability, and tumor progression, as well as suppress the growth defect Disclosure of Potential Conflicts of Interest induction caused by POLD4 downregulation. Third, the POLD4 ortholog of Cdm1 is a nonessential gene in S. pombe (20). Al- No potential conflicts of interest were disclosed. though the complexity of the DNA replication mechanisms and amounts of chromosomal DNA are not the same between Grant Support yeast and human cells, siD4-treated cells were able to prolifer- A Grant-in-Aid for Scientific Research on Innovative Areas, a Grant-in-Aid ate throughout the cell cycle, as shown in Fig. 4 (22). for Scientific Research on Priority Areas from the Ministry of Education, Tobacco smoke contains benzo(a)pyrene, one of the most Culture, Sports, Science, and Technology of Japan, a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, and the Person- potent carcinogens, whereas DNA adducts of benzo(a)pyrene alized Medicine Project of the Japan Science and Technology Agency. are removed by pol δ–dependent NER as are those of 4NQO. The costs of publication of this article were defrayed in part by the payment Along this line, previous epidemiologic studies have indi- of page charges. This article must therefore be hereby marked advertisement in cated a strong association of lung cancer with tobacco smok- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ing (1). Benzo(a)pyrene adducts might remain in DNA and Received 03/05/2010; revised 08/23/2010; accepted 09/07/2010; published drive genomic instability in lung cells with reduced POLD4 OnlineFirst 09/22/2010.

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8416 Cancer Res; 70(21) November 1, 2010 Cancer Research

Regulation of DNA Polymerase POLD4 Influences Genomic Instability in Lung Cancer

Qin Miao Huang, Shuta Tomida, Yuji Masuda, et al.

Cancer Res 2010;70:8407-8416. Published OnlineFirst September 22, 2010.

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