Activation of WD Repeat and High-Mobility Group Box DNA Binding Protein 1 in Pulmonary and Esophageal Carcinogenesis

Published OnlineFirst December 22, 2009; DOI: 10.1158/1078-0432.CCR-09-1405

Clinical Imaging, Diagnosis, Prognosis Cancer Research Activation of WD Repeat and High-Mobility Group Box DNA Binding Protein 1 in Pulmonary and Esophageal Carcinogenesis

Nagato Sato1,2, Junkichi Koinuma1, Masahiro Fujita3, Masao Hosokawa3, Tomoo Ito4, Eiju Tsuchiya5, Satoshi Kondo2, Yusuke Nakamura1, and Yataro Daigo1,6

Abstract Purpose: We attempted to identify novel biomarkers and therapeutic targets for lung and esophageal cancers. Experimental Design: We screened for genes that were overexpressed in a large proportion of lung and esophageal carcinomas using a cDNA microarray representing 27,648 genes or expressed sequence tags. A gene encoding WDHD1, a WD repeat and high-mobility group box DNA binding protein 1, was selected as a candidate. Tumor tissue microarray containing 267 archival non–small cell lung cancers and 283 esophageal squamous cell carcinomas (ESCC) was used to investigate the clinicopathologic significance of WDHD1 expression. The role of WDHD1 in cancer cell growth and/or survival was examined by small interfering RNA experiments and cell growth assays. The mechanism of WDHD1 activation through its phosphorylation in cancer cells was examined by immunoprecipitation and kinase assays. Results: Positive WDHD1 immunostaining was associated with a poor prognosis for patients with non–small cell lung cancer (P = 0.0403) as well as ESCC (P = 0.0426). Multivariate analysis indicated it to be an independent prognostic factor for ESCC (P = 0.0104). Suppression of WDHD1 expression with small interfering RNAs effectively suppressed lung and esophageal cancer cell growth. In addition, induc- tion of the exogenous expression of WDHD1 promoted the growth of mammalian cells. AKT1 kinase seemed to phosphorylate and stabilize the WDHD1 protein in cancer cells. Conclusions: WDHD1 expression is likely to play an important role in lung and esophageal carcinogenesis as a cell cycle regulator and a downstream molecule in the phosphoinositide 3-kinase/AKT pathway, and that WDHD1 is a candidate biomarker and a promising therapeutic target for cancer. Clin Cancer Res; 16(1); 226–39. ©2010 AACR.

Aerodigestive tract cancer (including carcinomas of lung, them consists of the squamous cell carcinoma. In spite of esophagus, oral cavity, pharynx, and larynx) accounts for the use of modern surgical techniques combined with var- one third of all cancer deaths in the United States and is ious treatment modalities, such as radiotherapy and che- the most common cancer in some parts of the world (1). motherapy, lung cancer and esophageal squamous cell Lung and esophageal cancers are the two major thoracic carcinoma (ESCC) are known to have the worst prognosis cancers that share common clinicopathologic characteris- of all malignant tumors; 5-year survival rates for lung can- tics: (a) embryologically, both of them are derived from cer patients including all disease stages still remain as low endoblast; (b) epidemiologically, there is a risk factor such as 15% and those for ESCC patients are much the same at as smoking; and (c) pathologically, a certain proportion of 16% (2, 3). A better understanding of the molecular basis of cancer has led to novel targeted strategies that inhibit specific key molecules in tumor growth, however currently Authors' Affiliations: 1Laboratory of Molecular Medicine, Human Genome available new targeted therapies are expected to improve Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; 2Department of Surgical Oncology, Hokkaido University Graduate School the prognosis of a small number of patients (4, 5). There- of Medicine; 3Keiyukai Sapporo Hospital; and 4Department of Surgical fore, novel strategies such as the development of molecu- Pathology, Hokkaido University, Sapporo, Japan; 5Kanagawa Cancer lar-targeted agents and cancer vaccines, which target Center Research Institute, Yokohama, Japan; and 6Department of Medical Oncology, Shiga University of Medical Science, Otsu, Japan molecules essential for the growth/survival of cancer cells, are eagerly awaited. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). We have been attempting to isolate potential molecu- Corresponding Author: Yataro Daigo, Institute of Medical Science, The Univer- lar targets for diagnosis and/or treatment of lung cancer sity of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639 Japan. Phone: and ESCC by analyzing genome-wide expression profiles 81-3-5449-5457; Fax: 81-3-5449-5406; E-mail: [email protected]. of 101 lung cancer and 19 ESCC tissue samples on a doi: 10.1158/1078-0432.CCR-09-1405 cDNA microarray containing 27,648 genes or expressed © 2010 American Association for Cancer Research. sequence tags (6–12). To verify the biological and clinical

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Materials and Methods Translational Relevance Cell lines and tissue samples. The human lung cancer cell Because there is a correlation between WDHD1 ex- lines used in this study were as follows: lung adenocarcino- pression and poor prognosis for patients with lung and mas (ADC) NCI-H1781, NCI-H1373, LC319, A549, and esophageal carcinomas, and multivariate analysis indi- PC14; lung squamous cell carcinomas (SCC) SK-MES-1, cated it to be an independent prognostic factor for NCI-H2170, NCI-H520, NCI-H1703, and LU61; a lung esophageal carcinomas, WDHD1 positivity in resected large-cell carcinoma LX1; and small-cell lung cancers specimens could be an index that provides informa- (SCLC) SBC-3, SBC-5, DMS273, and DMS114 (Supple- tion useful to physicians in applying adjuvant therapy mentary Table S1). The human esophageal carcinoma cell and intensive follow-up to the cancer patients who are lines used in this study were as follows: 10 SCC cell lines likely to suffer a relapse. Because WDHD1 should (TE1, TE2, TE3, TE4, TE5, TE6, TE8, TE9, TE10, and TE11) properly be classified as a typical cancer testis antigen and one ADC cell line (TE7; ref. 42). All cells were grown in and is likely to play an important role as a key compo- monolayer in appropriate medium supplemented with nent of the phosphoinositide 3-kinase/AKT pathway in 10% FCS and maintained at 37°C in humidified air with cancer proliferation, the selective inhibition of 5% CO2. Human small airway epithelial cells (SAEC) used WDHD1 expression and/or the targeting of enzymatic as a normal control were grown in optimized medium interaction between AKT1 and WDHD1 by small mol- (small airway growth medium) from Cambrex Bioscience, ecule compounds could be a promising therapeutic Inc. Primary non-SCLC (NSCLC) and ESCC tissue samples strategy that is expected to have a powerful biological as well as their corresponding normal tissues adjacent to re- effect on cancer with a minimal risk of adverse events. section margins from patients having no anticancer treatment before tumor resection had been obtained earlier with informed consent (7, 11, 12, 15). All tumors were significance of the gene products, we have established a staged on the basis of the pathologic tumor-node-metasta- screening system by combining the tumor tissue microar- sis classification of the International Union Against Cancer ray analysis of clinical lung and ESCC with RNA interfer- (43). Formalin-fixed primary lung tumors and adjacent ence technique and cell growth assays (13–37). In the normal lung tissue samples used for immunostaining on course of these systematic studies, we found that WD re- tissue microarrays had been obtained from 267 patients un- peat and high-mobility group box DNA binding protein dergoing curative surgery at Hokkaido University and its af- 1(WDHD1; alias acidic nucleoplasmic DNA-binding pro- filiated hospitals (Sapporo, Japan; please see tein, AND-1) was overexpressed in the great majority of Supplementary Table S2A). A total of 283 formalin-fixed lung cancers and ESCCs. primary ESCCs and adjacent normal esophageal tissue sam- WDHD1 is a human homologue of Ctf4 in Saccharomyces ples near to resection margins had also been obtained from cerevesiae, which was originally identified by screening for patients undergoing curative surgery at Hokkaido Universi- mutant genes affecting chromosome transmission fidelity ty Hospital and Keiyukai Sapporo Hospital (Sapporo, Ja- (38). Later studies indicate that Ctf4 is required for sister pan; Supplementary Table S2B). To be eligible for this chromatid cohesion and interacts with DNA polymerase study, tumor samples were selected from patients who ful- α in yeasts (39). Mcl1, a homologue of CTF4 in Schizosac- filled all of the following criteria: (a) patients suffered pri- charomyces pombe, is essential for the viability, maintenance mary NSCLC or ESCC with histologically confirmed stage of genome integrity, and regulation of telomere replication (only pT1 to pT3,pN0 to pN2, and pM0); (b) patients under- in fission yeast (40). Mcl1/Ctf4 is functionally related with went curative surgery, but did not receive any preoperative molecules involved in lagging strand DNA synthesis such as treatment; (c) among them, NSCLC patients with positive Rad2, Dna2, and Fen1 that plays a role in processing Oka- lymph node metastasis (pN1,pN2) were treated with plati- zaki fragments and physically interacts with DNA polymer- num-based adjuvant chemotherapies after surgery, and ase α to promote the chromatin association of DNA ESCC patients with pN positive were treated with adjuvant polymerase α (40), but its exact role in DNA replication chemotherapy using both platinum and 5-fluorouracil after has not yet been defined. Recently, it has been shown that surgery, whereas patients with pN0 did not receive adjuvant WDHD1 could interact with Mcm10 and is required for the chemotherapies; and (d) patients whose clinical follow-up proper loading of DNA polymerase α and replication initi- data were available. This study and the use of all clinical ma- ation in osteosarcoma U2OS cells (41). However, no report terials mentioned were approved by individual institution- has yet clarified the significance of WDHD1 transactivation al ethics committees. in clinical cancer progression. Semiquantitative reverse transcription-PCR. Atotalof3 We report here the identification of WDHD1 as a pre- μg aliquot of mRNA from each sample was reversely tran- dictive cancer biomarker in the clinic and as a potential scribed to single-stranded cDNAs using random primer therapeutic target, and also provide evidence for its in- (Roche Diagnostics) and SuperScript II (Invitrogen). Semi- volvement as a downstream target of phosphoinositide quantitative reverse transcription-PCR (RT-PCR) experi- 3-kinase (PI3K)/AKT pathway in human pulmonary and ments were carried out with the following sets of esophageal carcinogenesis. synthesized primers specific to WDHD1 or with β-actin

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(ACTB)–specific primers as an internal control: WDHD1, placed into a recipient paraffin block with a tissue micro- 5′-AGTGAAGGAACTGAAGCAAAGAAG-3′ and 5′-ATC- arrayer (Beecher Instruments). A core of normal tissue was CATTACTTCCCTAGGGTCAC-3′;ACTB,5′-GAGGTGATA‐ punched from each case, and 5-μm sections of the result- GCATTGCTTTCG-3′ and 5′-CAAGTCAGTGTACAGG- ing microarray block were used for immunohistochemical TAAGC-3′. PCRs were optimized for the number of cycles analysis. Three independent investigators semiquantita- to ensure product intensity to be within the linear phase of tively assessed WDHD1 positivity without prior knowl- amplification. edge of clinicopathologic data. Because the intensity of Northern blot analysis. Human multiple tissue blots cov- staining within each tumor tissue core was mostly homog- ering 23 tissues (BD Bioscience) were hybridized with an enous, the positivity of WDHD1 staining was recorded by [α-32P]-dCTP–labeled, 535-bp PCR product of WDHD1 following criteria: negative (no appreciable staining in tu- that was prepared as a probe using primers 5′-CTCTGATTC- mor cells) and positive (brown staining appreciable in the CAAAGCCGAAG-3′ and 5′-ATCCATTACTTCCCTAGGGT- nucleus and cytoplasm of tumor cells). Cases were accept- CAC-3′. Prehybridization, hybridization, and washing ed as positive only if all reviewers independently defined were done following the manufacturer's specifications. them as such. The blots were autoradiographed with intensifying screens Statistical analysis. Statistical analyses were done using at −80°C for 7 d. the StatView statistical program (SaS). Survival curves were Western blotting. Cells were lysed in lysis buffer [50 calculated from the date of surgery to the time of death mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.5% related to NSCLC or ESCC, or to the last follow-up obser- NP40, 0.5% sodium deoxycholate, and Protease Inhibitor vation. Kaplan-Meier curves were calculated for each rele- Cocktail Set III (EMD Biosciences, Inc.)]. We used an en- vant variable and for WDHD1 expression; differences in hanced chemiluminescence (Amersham) Western blotting survival times among patient subgroups were analyzed us- analysis system (GE Healthcare Bio-sciences), as previous- ing the log-rank test. Univariate and multivariate analyses ly described (15). A commercially available rabbit poly- were done with the Cox proportional hazard regression clonal antibody to human WDHD1 was purchased from model to determine associations between clinicopatholog- ATLAS Antibodies AB and was proved to be specific to hu- ic variables and cancer-related mortality. First, we analyzed man WDHD1 by Western blot analysis using lysates of associations between death and possible prognostic fac- lung and esophageal cancer cell lines. tors including age, gender, pT classification, and pN clas- Immunocytochemical analysis. Immunocytochemical sification, taking into consideration one factor at a time. analyses were done as previously described (30), using Second, multivariate Cox analysis was applied on back- 0.5 μg/mL of a rabbit polyclonal anti-WDHD1 antibody ward (stepwise) procedures that always forced strong (ATLAS Antibodies AB) for detecting endogenous WDHD1 WDHD1 expression into the model, along with any and as a primary antibody and an Alexa 488–conjugated don- all variables that satisfied an entry level of a P value of key anti-rabbit secondary antibody (Molecular Probe). <0.05. As the model continued to add factors, indepen- Each stained specimen was mounted with Vectashield dent factors did not exceed an exit level of P < 0.05. (Vector Laboratories, Inc.) containing 4′,6-diamidino-2- Two-tailed P value of <0.05 were considered statistically phenylindole and visualized with Spectral Confocal Scan- significant. ning Systems (TSC SP2 AOBS; Leica Microsystems). Cell growth assay. COS-7 cells were plated at densities of Immunohistochemistry and tissue microarray. To investi- 1×106 cells/100-mm dish, transfected with plasmids de- gate the WDHD1 protein in clinical samples that had been signed to express WDHD1 or mock plasmids. Cells were embedded in paraffin blocks, we stained the sections using selected in medium containing 0.4 mg/mL of geneticin ENVISION+ kit/horseradish peroxidase (DakoCytoma- (Invitrogen) for 7 d, and cell viability was assessed by tion) in the following manner. Briefly, slides were im- MTT assay (cell counting kit-8 solution; Dojindo Labora- mersed in Target Retrieval Solution and boiled at 108°C tories). The number of colonies stained with Giemsa was for 15 min in an autoclave for antigen retrieval. Rabbit also counted by colony formation assay using colony polyclonal anti-WDHD1 antibodies (1.6 μg/mL; ATLAS counting software (ImageJ software 1.42, NIH).6 Antibodies AB) were added to each slide after blocking RNA interference assay. Small interfering RNA (siRNA) of endogenous peroxidase and proteins, and the sections duplexes (Dharmacon, Inc.; 100 nmol/L) were transfected were incubated with horseradish peroxidase–labeled anti- into a NSCLC cell line A549 and an esophageal cancer cell rabbit IgG as the secondary antibody. Substrate-chromo- line TE9, using 30 μL of Lipofectamine 2000 (Invitrogen) gen was added, and the specimens were counterstained as described (33). The transfected cells were cultured for with hematoxylin. 7 d and cell growth was evaluated by MTT and colony Tumor tissue microarrays were constructed with forma- formation assays. siRNA duplexes against human WDHD1 lin-fixed 267 primary lung cancers and 283 primary and AKT1 used were as follows: si-WDHD1-#1, siGenome esophageal cancers as described elsewhere (44–46). The duplexes 1 [D-019780-01], 5′-GAUCAGACAUGUG- tissue area for sampling was selected based on visual align- CUAUUA-3′; si-WDHD1-#2: siGenome duplexes 2 ment with the corresponding H&E-stained section on a slide. Three, four, or five tissue cores (diameter, 0.6 mm; depth, 3-4 mm) taken from a donor tumor block were 6 http://rsb.info.nih.gov/ij/index.html

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Fig. 1. Expression of WDHD1 in lung and esophageal cancers and normal tissues. A, expression of WDHD1 in a normal lung tissue, 15 clinical lung cancer samples (lung ADC, lung SCC, and SCLC; top), and 15 lung cancer cell lines (bottom) detected by semiquantitative RT-PCR analysis. B, expression of WDHD1 in a normal esophagus and 10 clinical ESCC tissue samples and 11 ESCC cell lines detected by semiquantitative RT-PCR analysis. C, expression of WDHD1 protein in eight lung cancer and six esophageal cancer cell lines examined by Western blot analysis. D, subcellular localization of endogenous WDHD1 protein in lung cancer LC319 cells. WDHD1 was localized abundantly in the nucleus and weakly in cytoplasm during G0-G1 to S phase, distributed mainly in cytoplasm during mitotic phase, and partly located in chromatin during anaphase to telophase.

[D-019780-02], 5′-GGUAAUACGUGGACUCCUA-3′; and with 50 μg/mL propidium iodide (Sigma/Aldrich) in PBS. si-AKT1: siGenome duplexes 8 [D-003000-08], 5′- Flow cytometry was done on a Becton Dickinson FACScan GACCGCCUCUGCUUUGUCA-3′. We also designed and analyzed by ModFit software (Verity Software House, siRNA oligonucleotides against control-1 [EGFP, enhanced Inc.). The cells selected from at least 20,000 ungated cells green fluorescent protein (GFP) gene, a mutant of Aequorea were analyzed for DNA content. victoria GFP], 5′-GAAGCAGCACGACUUCUUC-3′,and Live cell imaging. Cells were grown on a 35-mm glass- control-2 (LUC, luciferase gene from Photinus pyralis), 5′- bottom dish in phenol red–free DMEM containing 10% CGUACGCGGAAUACUUCGA-3′. FCS. Cells were transfected with siRNA and subjected to time Flow cytometry. Cells were collected in PBS and fixed lapse imaging using a computer-assisted fluorescence micro- in 70% cold ethanol for 30 min. After treatment with scope (Olympus, LCV100) equipped with an objective lens 100 μg/mL RNase (Sigma/Aldrich), the cells were stained (Olympus, UAPO 40×/340 numerical aperature = 0.90),

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Fig. 2. Expression of WDHD1 in normal tissues and association of WDHD1 overexpression with poor prognosis for NSCLC and ESCC patients. A, Northern blot analysis of the WDHD1 transcript in 23 normal adult human tissues. A strong signal was observed in testis. B, immunohistochemical analysis of WDHD1 protein expressions in five normal tissues (liver, heart, kidney, lung, and testis) with those in lung SCC. WDHD1 was expressed abundantly in testis (mainly in nucleus and/or cytoplasm) and lung cancers, but its expression was hardly detectable in the remaining four normal tissues. C and D, association of WDHD1 expression with poor prognosis. Kaplan-Meier analysis of survival of patients with NSCLC (C, P = 0.0403 by the log-rank test) and ESCC (D, P = 0.0426 by the Log-rank test) according to expression of WDHD1.

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a halogen lamp, a red LED (620 nm), a CCD camera precipitated using Flag-M2 agarose. The immunoprecipi- (Olympus, DP30), differential interference contrast optical tant was confirmed to be WDHD1 by Western blotting components, and interference filters. For differential inter- using a mouse monoclonal anti–Flag-M2 antibody. The ference contrast imaging, the red LED was used with a filter Flag-tagged WDHD1 protein was incubated with recombi- cube containing an analyzer. Image acquisition and analy- nant human AKT1 protein (rhAKT1; Invitrogen) in kinase sis were done by using MetaMorph 6.13 software (Univer- buffer [20 mmol/L Tris (pH 7.5), 10 mmol/L MgCl2, 2 sal Imaging). mmol/L MnCl2, 1 mmol/L phenylmethylsulfonyl fluo- In vitro kinase assay. Flag-tagged WDHD1 protein that ride, 1 mmol/L DTT] supplemented with a mixture of pro- was exogenously expressed in COS-7 cells was immuno- tease inhibitors, 10 mmol/L NaF, 5 nmol/L microcystin

Table 1. WDHD1 positivity and NSCLC patients' characteristics

A. Association between WDHD1 positivity in NSCLC tissues and patients' characteristics (N = 267) Total WDHD1 positive WDHD1 negative P: positive vs negative N = 267 n = 136 (50.9%) n = 131 (49.1%)

Gender Female 89 (33.3%) 27 (10.1%) 62 (23.2%) <0.0001* Male 178 (66.7%) 109 (40.8%) 69 (25.8%) Age (y) <65 132 (49.4%) 58 (21.7%) 74 (27.7%) 0.0277* ≥65 135 (50.6%) 78 (29.2%) 57 (21.3%) Smoking Nonsmoker 88 (33.0%) 31 (11.6%) 57 (21.3%) 0.0004* Smoker 179 (67.0%) 105 (39.3%) 74 (27.7%) Histologic type ADC 157 (58.8%) 59 (22.1%) 98 (36.7%) <0.0001* Non-ADC 110 (41.2%) 77 (28.8%) 33 (12.4%) pT factor

T1 114 (42.7%) 43 (16.1%) 71 (26.6%) 0.0002*

T2+T3 153 (57.3%) 93 (34.8%) 60 (22.5%) pN factor

N0 208 (77.9%) 98 (36.7%) 110 (41.2%) 0.0264*

N1+N2 59 (22.1%) 38 (14.2%) 21 (7.9%)

B. Cox's proportional hazards model analysis of prognostic factors in patients with NSCLCs Variables HazardsHazard ratio ratio (95% (95% CI) CI) Unfavorable/favorableUnfavorable/favorableP P Univariate analysis UnivariateWDHD1 analysis 1.668 (1.017-2.734) Positive/negative 0.0425† † WDHD1Age (y) 1.6682.171 (1.017-2.734) (1.313-3.590) ≥65/65> Positive/negative0.0025† 0.0425 † GenderAge (y) 2.1712.135 (1.313-3.590) (1.198-3.803) Male/Female0.0 ≥65/65> 100† 0.0025 † SmokingGender 2.1351.870 (1.198-3.803) (1.075-3.253) Smoker/non-SmokerMale/Female0.0 0.0267† 100 † HistologicSmoking type 1.8702.493 (1.075-3.253) (1.516-4.101) Non-ADC/ADCSmoker/non-Smoker0.0003† 0.0267 † † pTHistologic factor type 2.4933.722 (1.516-4.101) (2.026-6.838) T2+T3/T1 Non-ADC/ADC<0.0001 0.0003 † † pNpT factor factor 3.7224.430 (2.026-6.838) (2.718-7.220) N1+N2/N0 T2+T3/T1 <0.0001 <0.0001 † MultivariatepN factor analysis 4.430 (2.718-7.220) N1+N2/N0 <0.0001 MultivariateWDHD1 analysis 0.816 (0.467-1.424) Positive/negative 0.4741 AgeWDHD1 (y) 0.8161.806 (0.467-1.424) (1.084-3.011) ≥65/65> Positive/negative0.0233† 0.4741 † AgeGender (y) 1.8061.263 (1.084-3.011) (0.568-2.811) Male/female ≥65/65> 0.5669 0.0233 SmokingGender 1.2631.290 (0.568-2.811) (0.619-2.685) Smoker/nonsmokerMale/female 0.4966 0.5669 HistologicSmoking type 1.6281.290 (0.891-2.973no) (0.619-2.685) Non-ADC/ADCSmoker/nonsmoker0.1127 0.4966 † pTHistologic factor type 1.6282.291 (0.891-2.973no) (1.199-4.380) T2+T3/T1 Non-ADC/ADC 0.0121 0.1127 † † pNpT factor factor 2.2913.723 (1.199-4.380) (2.254-6.148) N1+N2/N0 T2+T3/T1 <0.0001 0.0121 † pN factor 3.723 (2.254-6.148) N1+N2/N0 <0.0001

(Continued on the following page)

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Table 1. WDHD1 positivity and NSCLC patients' characteristics (Cont'd)

C. Multivariate logistic regression analysis for the correlation between WDHD1 expression and clinicopathologic characteristics Characteristics‡ Odds ratio P

Gender (male/female) 2.130 0.0339§ Age (≥65 y/<65 y) 1.335 0.2914 Smoking (smoker/nonsmoker) 1.285 0.4739 Histologic type (non-ADC/ADC) 2.437 0.0033§

pT factor (T2-3/T1) 1.397 0.2690 § pN factor (N1-2/N0) 1.971 0.0487

Abbreviations: Non-ADC, squamous cell carcinoma plus large-cell carcinoma and adenosquamous cell carcinoma; 95% CI, 95% confidence interval. *P < 0.05 (Fisher's exact test). †P < 0.05. ‡Selected factors from Table 1A that were significantly associated with WDHD1 positivity. §P < 0.05.

LR, and 50 μmol/L ATP. The reaction was terminated by plasm in the cells during G0-G1 to S phase and in chroma- the addition of a 0.2 volume of 5× protein sample buffer tin during anaphase to telophase (Fig. 1D). and the proteins were analyzed by Western blotting using Northern blot analysis using a WDHD1 cDNA fragment phospho-AKT substrate (PAS) antibody (Cell Signaling). A as a probe identified about 5-kb transcript exclusively in reblotting of the same membrane with anti–Flag-M2 anti- testis (Fig. 2A). Immunohistochemical analysis with antibody was done to confirm that the phosphorylated band WDHD1 polyclonal antibodies using five normal tissues was WDHD1. (liver heart, kidney, lung, and testis) as well as lung cancer tissues revealed strong staining of WDHD1 in testis and lung cancer cells (mainly in nucleus and weakly in cyto- Results plasm), but its expression was hardly detectable in the remaining four normal tissues (Fig. 2B). WDHD1 expression in lung and esophageal cancers and Association of WDHD1 expression with poor prognosis for normal tissues. To identify novel molecules such as cancer patients with NSCLC or ESCC. To investigate the biological testis antigens that were highly transactivated in a large and clinicopathologic significance of WDHD1 in pulmo- proportion of lung and esophageal cancers, but scarcely nary and esophageal carcinogenesis, we carried out immu- expressed in normal tissues, we had applied cDNA micro- nohistochemical staining on tissue microarray containing array analysis, and identified elevated expression (3-fold tissue sections from 267 NSCLC and 283 ESCC cases that or higher) of the WDHD1 transcript in the majority of underwent surgical resection. WDHD1 staining with poly- lung cancer and ESCC tissue samples examined. Moreover, clonal antibody specific to WDHD1 was mainly observed WDHD1 showed testis-specific expression in normal tis- in the nucleus as well as the cytoplasm of tumor cells, but sues. We confirmed its overexpression by means of semi- not detected in their adjacent normal lung tissue cells quantitative RT-PCR experiments in 14 of 15 lung cancer (Supplementary Fig. S1B and C). Of the 267 NSCLCs ex- tissues, in all of 15 lung cancer cell lines, in 6 of 10 ESCC amined, WDHD1 was stained positively in 136 cases tissues, and in 8 of 11 ESCC cell lines (Fig. 1A and B). We (50.9%) and negatively in 131 cases (49.1%; Table 1A). also confirmed its elevated expression in another set of We then examined the association of WDHD1 expression primary tumor tissues (six of six NSCLCs and five of six with clinical outcomes, and found that expression of ESCCs); however, its expression was hardly detectable in WDHD1 in NSCLCs was significantly associated with male their adjacent normal lung or esophagus tissues (Supple- gender (P < 0.0001, Fisher's exact test; Table 1A), older mentary Fig. S1A). We subsequently confirmed by Western age (≥65 years; P = 0.0277), smoker (current or exsmokers; blotting analysis overexpression of WDHD1 protein (126 P = 0.0004), non-ADC histology (P < 0.0001), larger tu- kDa) in lung and esophageal cancer cell lines using anti- mor size (pT2-3; P = 0.0002), presence of lymph node me- WDHD1 polyclonal antibody (Fig. 1C). To examine the tastasis (pN1-2; P = 0.0264), and with tumor-specific subcellular localization of endogenous WDHD1 in cancer 5-year survival after the resection of primary tumors cells, we performed immunocytochemical analysis of lung (P = 0.0403 by log-rank test; Supplementary Table S3A; cancer LC319 cells that overexpressed WDHD1 protein Fig. 2C). We also applied univariate analysis to evaluate using anti-WDHD1 polyclonal antibody. WDHD1 was associations between patient prognosis and several factors detected abundantly in the nucleus and weakly in cyto- including age, gender, smoking history, pT stage (tumor

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Table 2. WDHD1 positivity and ESCC patients’ characteristics

A. Association between WDHD1-positivity in ESCC tissues and patients' characteristics (N = 283) Total WDHD1 positive WDHD1 negative P: positive vs negative

N = 283 n = 175 (61.8%) n = 108 (38.2%)

Gender Female 27 (9.5%) 15 (5.3%) 12 (4.2%) 0.5343 Male 256 (90.5%) 160 (56.5%) 96 (33.9%) Age (y) <65 176 (62.2%) 116 (41.0%) 60 (21.2%) 0.0781 ≥65 107 (37.8%) 59 (20.8%) 48 (17.0%) pT factor

T1 73 (25.8%) 43 (15.2%) 30 (10.6%) 0.5775

T2+T3 210 (74.2%) 132 (46.6%) 78 (27.6%) pN factor

N0 91 (32.2%) 58 (20.5%) 33 (11.7%) 0.6954

N1+N2 192 (67.8%) 117 (41.3%) 75 (26.5%)

B. Cox's proportional hazards model analysis of prognostic factors in patients with ESCCs B. Cox's proportional hazards model analysis of prognostic factors in patients with ESCCs P Variables Hazards ratioHazards (95% CI) ratio (95% Unfavorable/Favorable CI) Unfavorable/FavorableP Univariate analysis Univariate analysis WDHD1 1.1317 (1.009-1.863) Positive/negative 0.0435* WDHD1 1.1317 (1.009-1.863) Positive/negative 0.0435* Age (y) 0.957 (0.710-1.290) ≥65/65> 0.7746 Age (y) 0.957 (0.710-1.290) ≥65/65> 0.7746 Gender 3.117 (1.593-6.101) Male/Female 0.0009* Gender 3.117 (1.593-6.101) Male/Female 0.0009* pT factor 2.702 (1.833-3.983) T2+T3/T1 <0.0001* pT factor 2.702 (1.833-3.983) T2+T3/T1 <0.0001* pN factor 3.428 (2.347-5.009) N1+N2/N0 <0.0001* pN factor 3.428 (2.347-5.009) N +N /N <0.0001* Multivariate analysis 1 2 0 Multivariate analysis WDHD1 1.496 (1.099-2.036) Positive/negative 0.0104* WDHD1 1.496 (1.099-2.036) Positive/negative 0.0104* Gender 3.031 (1.546-5.940) Male/female 0.0012* Gender 3.031 (1.546-5.940) Male/female 0.0012* pT factor 2.048 (1.373-3.053) T2+T3/T1 0.0004* pT factor 2.048 (1.373-3.053) T2+T3/T1 0.0004* pN factor 2.865 (1.936-4.239) N1+N2/N0 <0.0001* pN factor 2.865 (1.936-4.239) N1+N2/N0 <0.0001* *P < 0.05. *P < 0.05.

size, T1 versus T2+T3), pN stage (node status, N0 versus N1 cells in 175 cases (61.8%) and negatively in 108 cases +N2), histologic type (non-ADC versus ADC), and WDHD1 (38.2%), whereas their adjacent normal esophagus tissues expression (positive versus negative). All those parameters were not stained (Supplementary Fig. S1D and E; Table 2A). were significantly associated with poor prognosis (Table ESCC patients whose tumors showed WDHD1 expression re- 1B). In multivariate analysis, WDHD1 status did not reach vealed shorter tumor-specific survival periods compared the statistically significant level as an independent prognos- with those with absent WDHD1 expression (P =0.0426by tic factor for surgically treated lung cancer patients enrolled log-rank test; Supplementary Table S3B; Fig. 2D). We also ap- in this study (P = 0.4741), whereas pT and pN stages as well plied univariate analysis to evaluate associations between as age did so (Table 1B). The result might be due to the rel- ESCC patient prognosis and several factors including age, evance of WDHD1 expression mainly to the other prognos- gender, pT stage (tumor depth, T1 versus T2+T3), pN stage tic factors—male gender, non-ADC histology, and/or (node status, N0 versus N1+N2), and WDHD1 status (positive presence of lymph node metastasis in lung cancer—proba- versus negative). All those parameters except for age were sig- bly because multivariate logistic regression analysis for the nificantly associated with poor prognosis (Table 2B). Multi- six clinicopathologic parameters that were significantly as- variate analysis using a Cox proportional hazard factors sociated with WDHD1 positivity in lung cancer (Table 1A) determined that WDHD1 (P = 0.0104) as well as other three also determined that these three factors were independent factors (male gender, larger tumor size, and lymph node features associated with WDHD1 expression (P = 0.0339, metastasis) were independent prognostic factors for surgi- 0.0033, and 0.0487, respectively; Table 1C). cally treated ESCC patients (Table 2B). Of the 283 ESCC cases examined, WDHD1 was stained Effects of WDHD1 on cell growth and cell cycle progres- positively in the nucleus as well as the cytoplasm of tumor sion. To assess whether WDHD1 plays an essential role

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in cell growth or survival, we carried out a colony forma- types, and this triggers a cascade of responses, from cell tion assay of COS-7 transfected with WDHD1 expression growth and proliferation to survival, motility, epithelial- plasmids and confirmed the growth-promoting activity mesenchymal transition, and angiogenesis (48). We there- of cells overexpressing WDHD1, compared with those fore examined whether WDHD1 could be involved in the transfected with mock vectors (Supplementary Fig. S2). PI3K and/or AKT pathway. We first immunoblotted, using We further constructed several siRNA expression oligonu- PAS antibody, the Flag-tagged WDHD1 protein that was cleotides specific to WDHD1 sequences and transfected exogenously expressed in COS-7 cells and immunopreci- them into lung cancer A549 cells as well as esophageal pitated using Flag-M2 agarose, and detected the positive cancer TE9 cells that endogenously expressed WDHD1 at band that represented possibly phosphorylation by endog- high levels. Knockdown effects were confirmed by semi- enous AKT (Fig. 4A). In vitro kinase assay using the Flag- quantitative RT-PCR when we used si-WDHD1-#1 and tagged WDHD1 immunoprecipitant as a substrate and si-WDHD1-#2 (Fig. 3A). MTT assays and colony forma- recombinant human AKT1 protein (rhAKT1) as a kinase tion assays revealed a drastic reduction in the number of with subsequent immunoblotting with PAS antibody also cells transfected with si-WDHD1-#1 and #2 (Supplemen- proved the direct phosphorylation of WDHD1 by AKT1 tary Fig. S3; Fig. 3B). Flowcytometric analysis at 24 to 72 (Fig. 4B). To examine the effect of AKT1 on WDHD1 pro- hours after the transfection of si-WDHD1 to the A549 and tein function in cancer cells, we measured the level of en- TE9 cells revealed the decrease of the number of cells at S dogenous WDHD1 protein after transfection of siRNA for phaseandtheincreaseofthatinG0-G1 phase (Supple- AKT1 to LC319 cells or after treatment of the cells with var- mentary Fig. S4A; Fig. 3C). To further investigate the effect ious concentrations of LY294002 (0-20 μmol/L for 24 of WDHD1 on the cell cycle progression, we synchronized hours), a specific inhibitor of the catalytic subunit of A549 and TE9 cells (5 × 105 cells/100-mm dish) trans- PI3K. Total amount of WDHD1 as well as phosphorylated fected with siRNA for si-WDHD1. The flow cytometric WDHD1 was significantly decreased by LY294002 treat- and Western blot analyses at 0, 4.5, and 9 hours after re- ment or introduction of siRNA for AKT1, indicating a pos- moval of aphidicolins showed that the number of the sibility that WDHD1 protein stability is regulated by the A549 and TE9 cells in G0-G1 phase was increased and PI3K/AKT signaling (Fig. 4C and D). the progression to S phase was delayed (Supplementary Fig. S4B and C). To further clarify the effect of WDHD1 Discussion knockdown on cellular morphology and cell cycle, we examined A549 cells transfected with siRNA for WDHD1 us- We performed a genome-wide expression profile analy- ing time lapse microscopy. Although the cell division was sis of 101 lung cancers and 19 ESCC cells after enrichment observed at about every 10 hours in control cells, the of cancer cells by laser microdissection, using a cDNA mi- WDHD1 knocked down cells started cell division slowly croarray containing 27,648 genes or expressed sequence and died shortly after cell division (Fig. 3D). tags (7–12). Through the analyses, we identified a number Phosphorylation of WDHD1. WDHD1 protein was de- of genes that could be potentially good candidates for the tected as double bands by Western blotting, indicating a development of novel diagnostic markers, therapeutic possible modification of the WDHD1 protein. Therefore, drugs, and/or immunotherapy (13–37). In this study, we we first incubated extracts from A549 cells that overex- selected WDHD1 as a good candidate for cancer biomark- pressed endogenous WDHD1 and also COS-7 cells trans- er(s) for lung cancer and/or ESCC, and provided evidences fected with WDHD1-expressing plasmids in the presence for its possible role in human carcinogenesis. or absence of protein phosphatase (New England Bio- WDHD1,ahomologueofCtf4/Mcl1 in Saccharomyces labs), and analyzed the molecular size of WDHD1 protein cerevesiae and Schizosaccharomyces pombe, encodes a puta- by Western blot analysis. The measured weight of the ma- tive 1129 amino acid protein with high-mobility group jority of both endogenous and exogenous WDHD1 pro- box domains and a WD-repeat domain. Ctf4/Mcl1 has tein in the extracts treated with phosphatase was smaller been described as a part of a replisome progression com- than that in the untreated cells. The data indicated that plex that associates with the GINS complex as well as with WDHD1 was possibly phosphorylated in cells (Supple- DNA polymerase/primase (49). To maintain the genome mentary Fig. S5A). Immunoprecipitation of WDHD1 with integrity, chromosomal DNA is precisely replicated only anti-WDHD1 antibody followed by immunoblotting with once per cell cycle. Initiation of chromosomal replication pan-phospho–specific antibodies indicated phosphoryla- starts by the binding of several factors to replication ori- tion of endogenous WDHD1 at its serine and tyrosine re- gins, and the interactions among these factors are crucial sidues (Supplementary Fig. S5B). for subsequent processes (50). Ctf4/Mcl1 physically inter- Involvement of WDHD1 in PI3K/AKT pathway. To eluci- acts with DNA pol α and induces the association of Pol α date the importance of WDHD1 phsphorylation in cancer to chromatin, and plays a critical role in lagging strand cells, we next screened the candidate kinases for WDHD1 synthesis and Okazaki fragment processing (40). Recent by referring possible phosphorylation sites on WDHD1 studies showed that WDHD1 interacts with MCM10 and protein, and found a consensus phosphorylation site for is essential for DNA synthesis, suggesting its role as a rep- AKT kinase (R-X-R-X-X-S374; ref. 47). PI3K/AKT pathway lication initiation factor (41). The function of Ctf4/Mcl1 is well known to be activated in a wide range of tumor in chromosome replication, cohesion, and segregation

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WDHD1 in Pulmonary and Esophageal Carcinogenesis

Fig. 3. Growth-promoting effect of WDHD1. A and B, inhibition of growth of a lung cancer cell line A549 (left) and an esophageal cancer cell line TE9 (right) by siRNAs against WDHD1. A, WDHD1 gene knockdown effect in A549 and TE9 cells by two si-WDHD1 (si-WDHD1-#1 and si-WDHD1-#2) and two control siRNAs (si-EGFP and si-LUC), analyzed by semiquantitative RT-PCR. B, MTT assays of A549 and TE9 cells transfected with si-WDHD1s or control siRNAs. Columns, relative absorbance of triplicate assays; bars, SD. C, flow cytometric analysis of lung cancer cells transfected with si-WDHD1. A549 cells were transfected with si-WDHD1-#1 or si-LUC, and collected at 24, 48, and 72 h after transfection for flow cytometry. The numbers besides the panels indicate the percentage of cells at each phase.

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Fig. 3. Continued. D, time lapse imaging analysis of A549 cells transfected with si-WDHD1. A549 cells were transfected with si-WDHD1-#1 or si-LUC and their microscopic images were captured every 30 min. Representative microscopic images of cells at every 12 h (from 24-108 h; left) and at every 30 min (from 90-92.5 h; right) are shown.

was reported to date, but the overexpression of WDHD1 in various human cancers, and has been recognized as a risk human cancer cells and its possible role in human carcino- factor for early disease recurrence and poor prognosis genesis have not been described. (48). Our data indicated that inhibition of PI3K/AKT In this study, we obtained important clinicopathologic pathway using LY294002 or siRNA for AKT1 decreased and biological evidences supporting the significance of the level of total and phosphorylated WDHD1 in lung WDHD1 transactivation in human carcinogenesis. We cancer cells. Because these results indicate that WDHD1 showed through our tissue microarray experiments that plays a significant role in cancer cell growth/survival as NSCLC and ESCC patients with WDHD1-positive tumors one of the components of the PI3K/AKT pathway, selec- had shorter cancer-specific survival periods than those tive targeting of functional interaction between AKT1 with WDHD1-negative tumors. In addition, the enhanced and WDHD1 could be a promising therapeutic strategy. expression of WDHD1 significantly promoted growth of Further analyses of the mechanism of growth suppression mammalian cells. Moreover, WDHD1 knockdown by by specific inhibition of WDHD1 phosphorylation by siRNA in cancer cells delayed S-phase entry and progres- AKT1 may be of the great benefit toward the development sion, and resulted in cell death right after cell division. of new types of anticancer agents. The data imply the importance of WDHD1 in cancer cell FromtheresultofNorthernblotandimmunohisto- cycle progression, although further detailed analyses of chemical analyses, WDHD1 was expressed only in testis WDHD1 function in coordinated maintenance of the and cancer cells. Cancer testis antigens have been recog- cancer genome integrity are required. nized as a group of highly attractive targets for cancer vac- Our study also described the involvement of WDHD1 cine treatment (34–37). Although other factors, such as activation in cancer cell signaling. WDHD1 seemed to the in vivo spontaneous immunogenicity of the protein, be phosphorylated and stabilized by AKT1. PI3K/AKT sig- are also important and further examination will be neces- naling is important for cell proliferation and survival (48). sary, WDHD1 oncoantigen may be useful for screening In addition, AKT1 phosphorylation frequently occurs in of HLA-restricted epitope peptides for cancer vaccine that

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WDHD1 in Pulmonary and Esophageal Carcinogenesis

Fig. 4. Possible regulation of WDHD1 stability by its phosphorylation through PI3K/AKT signaling. A, immunoprecipitant of Flag-tagged WDHD1 in COS-7 cells transfected with Flag-tagged WDHD1-expressing plasmid was detected with anti-PAS antibody or anti–Flag-M2 antibody. B, in vitro phosphorylation of Flag-tagged WDHD1 protein by recombinant human AKT1 (rhAKT1). C, reduction of WDHD1 protein by PI3K inhibition with LY294002. LC319 were treated with PI3K inhibitor LY294002 in concentrations of 0 or 20 μmol/L for 24 h and served for Western blot analysis. D, reduction of WDHD1 protein by AKT1 inhibition with siRNA against AKT1. LC319 were transfected with siRNA for AKT1 or EGFP and served for Western blot analysis.

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can induce specific immune responses by cytotoxic T cells Disclosure of Potential Conflicts of Interest against WDHD1-positive cancer cells. Because WDHD1 expression could have pivotal functions in cancer cell No potential conflicts of interest were disclosed. survival, vaccination with the peptides from this protein should reduce the risk of the emergence of immune escape Acknowledgments variant tumors that have lost their antigen expression. In conclusion, WDHD1 was overexpressed in the great The costs of publication of this article were defrayed in majority of lung and esophageal cancer tissues, and it part by the payment of page charges. This article must is likely to play significant roles in cancer cell growth therefore be hereby marked advertisement in accordance and/or survival. The data indicate WDHD1 to be a poten- with 18 U.S.C. Section 1734 solely to indicate this fact. tial therapeutic target and a candidate biomarker for Received 6/2/09; revised 8/27/09; accepted 8/29/09; patients with lung and esophageal cancers. published OnlineFirst 12/22/09.

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Activation of WD Repeat and High-Mobility Group Box DNA Binding Protein 1 in Pulmonary and Esophageal Carcinogenesis

Nagato Sato, Junkichi Koinuma, Masahiro Fujita, et al.

Clin Cancer Res 2010;16:226-239. Published OnlineFirst December 22, 2009.

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