Imaging, Diagnosis, Prognosis

Activation of KIF4A as a Prognostic Biomarker and Therapeutic Ta r g e t f o r L u n g C a n c e r MasayaTaniwaki,1, 2 Atsushi Takano,1Nobuhisa Ishikawa,1Wataru Yasui,3 Kouki Inai,4 Hitoshi Nishimura,5 EijuTsuchiya,6 Nobuoki Kohno,2 Yusuke Nakamura,1and Yataro Daigo1

Abstract Purpose and ExperimentalDesign: Toidentify molecules that might be useful as diagnostic/ prognosticbiomarkers and as targets for the development of new molecular therapies, we screened genes that were highly transactivated in a large proportion of101lung cancers by means of a cDNA microarray representing 27,648 genes. We found a gene encoding KIF4A, a kinesin family member 4A, as one of such candidates. Tumor tissue microarray was applied to examine the expression of KIF4A protein and its clinicopathologic significance in archival non ^ small cell lung cancer (NSCLC) samples from 357 patients. A role of KIF4A in cancer cell growth and/or survival was examined by small interfering RNA experiments. Cellular invasive activity of KIF4A on mammalian cells was examined using Matrigel assays. Results: Immunohistochemical staining detected positive KIF4A staining in 127 (36%) of 357 NSCLCs and 19 (66%) of 29 small-cell lung cancers examined. Positive immunostaining of KIF4A protein was associated with male gender (P = 0.0287), nonadenocarcinoma histology (P = 0.0097), and shorter survival for patients with NSCLC (P = 0.0005), and multivariate analysis confirmed its independent prognostic value (P = 0.0012).Treatment of lung cancer cells with small interfering RNAs for KIF4A suppressed growth of the cancer cells. Furthermore, we found that induction of exogenous expression of KIF4A conferred cellular invasive activity on mammalian cells. Conclusions: These data strongly implied that targeting the KIF4A molecule might hold a promise for the development of anticancer drugs and cancer vaccines as well as a prognostic biomarker in clinic.

Lung cancer is one of the most common and fatal cancers categorized as neuroendocrine tumors of the lung with certain in the world (1). A number of genetic alterations associated morphologic, ultrastructural, and immunohistochemical char- with development and progression of lung cancer have been acteristics (3, 4). In spite of applying surgical techniques reported; however, its molecular mechanisms still largely combined with various treatment modalities such as radiother- remain unclear (2). Two major histologically distinct types of apy and chemotherapy, the overall 5-year survival rate of lung lung cancer, non–small cell lung cancer (NSCLC) and small- cancer is still low at f15% (5). Patients with SCLC respond cell lung cancer (SCLC) have different pathophysiologic and favorably to the first-line multiagent chemotherapy; however, clinical features that suggest differences in the mechanisms of they often relapse in a short time. Hence, the only 20% of their carcinogenesis. NSCLC accounts for nearly 80% of lung patients with limited-stage disease can be cured with combined cancers, whereas SCLC accounts for 20% of them and is modality therapy and <5% of those with extensive disease are able to achieve 5-year survival after the initial diagnosis (6, 7). Therefore, new therapeutic strategies focusing on SCLC as

Authors’Affiliations: 1Laboratory of Molecular Medicine, Human Genome Center, well as NSCLC such as molecular-targeted agents are eagerly Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Departments awaited. of 2Molecular and Internal Medicine, 3Molecular Pathology, and 4Pathology, The genome-wide cDNA microarray analysis covering com- Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan; plete or near-complete set of genes enabled us to obtain 5Department of Thoracic Surgery, Saitama Cancer Center, Saitama, Japan; and 6Kanagawa Cancer Center Research Institute, Kanagawa, Japan comprehensive gene expression profiles and to compare the Received 5/30/07; revised 8/8/07; accepted 8/20/07. gene expression levels with clinicopathologic and biological The costs of publication of this article were defrayed in part by the payment of page information of cancers (8–14). This kind of approach is also charges. This article must therefore be hereby marked advertisement in accordance useful to identify unknown molecules involved in the with 18 U.S.C. Section 1734 solely to indicate this fact. carcinogenic pathway. Through the gene expression profile Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). analysis of 15 SCLCs and 86 NSCLCs coupled with purification Requests for reprints: Yataro Daigo, Laboratory of Molecular Medicine, Human of cancer cell population by laser microdissection on a cDNA Genome Center, Institute of Medical Science, The University of Tokyo, 4-6-1 microarray consisting of 27,648 genes, and their comparison Shirokanedai, Minato-Ward, Tokyo 108-8639, Japan. Phone: 81-3-5449-5457; with the expression profile data of 31 normal human tissues Fax: 81-3-5449-5406; E-mail: [email protected]. F 2007 American Association for Cancer Research. (27 adult and 4 fetal organs; refs. 15, 16), we identified a doi:10.1158/1078-0432.CCR-07-1328 number of potential molecular targets for diagnosis, treatment,

Clin Cancer Res 2007;13(22) November 15, 2007 6624 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2007 American Association for Cancer Research. KIF4A Activation in Lung Carcinogenesis and/or choice of therapy (8–12). To verify the biological and reactions were optimized for the number of cycles to ensure product clinicopathologic significance of the respective gene products, intensity within the logarithmic phase of amplification. we have also been performing high-throughput screening of Northern blot analysis. Human multiple-tissue blots (BD Biosciences 32 loss-of-function effects by means of the RNA interference Clontech) were hybridized with a P-labeled PCR product of KIF4A. The cDNA probe of KIF4A was prepared by RT-PCR using the primers technique as well as tumor tissue microarray analysis of clinical described above. Prehybridization, hybridization, and washing were lung cancer materials (17–30). This systematic approach done according to the supplier’s recommendations. The blots were revealed that kinesin family member 4A (KIF4A) was frequently autoradiographed at room temperature for 30 h with intensifying overexpressed in the great majority of lung cancers and was BAS screens (Bio-Rad). essential to growth or progression of lung cancers. Western blotting. Cells were lysed in lysis buffer: 50 mmol/L Tris- Kinesin superfamily proteins, such as KIF4A, are microtubule- HCl (pH 8.0), 150 mmol/L NaCl, 0.5% NP40, 0.5% deoxycholate-Na, based motor proteins that generate directional movement 0.1% SDS, plus protease inhibitor (Protease Inhibitor Cocktail Set III; along microtubules. KIFs are key players or central proteins in Calbiochem/Merck KGaA). We used an enhanced chemiluminescence the intracellular transport system, which is essential for cellular Western blotting analysis system (GE Healthcare Biosciences), as function and morphology, including cell division (31). The previously described (19, 20). A commercially available goat polyclonal kinesin superfamily is also the first large protein family in anti-KIF4A antibody was purchased from Abcam, Inc., and was proved to be specific to human KIF4A, by Western blot analysis using lysates of mammals whose constituents have been completely identified lung cancer cell lines. and confirmed both in silico and in vivo (32). A large portion of Immunocytochemistry. Cultured cells were washed twice with PBS(-), human KIF4A is associated with the nuclear matrix during the fixed in 4% formaldehyde solution for 30 min at 37jC, and rendered interphase, whereas a small portion of them is found in the permeable by treatment for 3 min with PBS(-) containing 0.1% Triton cytoplasm. During mitosis, it is associated with chromosomes X-100. Cells were covered with CAS-BLOCK (Zymed) for 7 min to block throughout the entire process (33). A previous microarray study nonspecific binding before the primary antibody reaction. Then, the disclosed the elevated expression of KIF4A mRNA in human cells were incubated with polyclonal antibody to human KIF4A protein cervical cancer (34), although no report has clarified the (Abcam). The immunocomplexes were stained with a donkey anti-goat significance of KIF4A transactivation in human cancer progres- secondary antibody conjugated to Alexa 488 (Molecular Probes) and sion and its potential as a therapeutic target. viewed with a laser confocal microscope (TCS SP2 AOBS: Leica We here report the identification of KIF4A to be a promising Microsystems). Immunohistochemistry and tissue microarray analysis. To investigate target as a prognostic biomarker as well as for development of the significance of KIF4A overexpression in clinical lung cancers, we therapeutic agents and cancer vaccines, and also describe stained tissue sections using ENVISION+ kit/horseradish peroxidase possible biological roles of KIF4A protein in progression of (DakoCytomation). Anti-KIF4A antibody (Abcam) was added after lung cancer. blocking of endogenous peroxidase and proteins, and each section was incubated with horseradish peroxidase–labeled anti-goat IgG as the secondary antibody. Substrate chromogen was added and the speci- Materials and Methods mens were counterstained with hematoxylin. Tumor tissue microarrays were constructed as published previously, using formalin-fixed NSCLCs Lung cancer cell lines and tissue samples. The human lung cancer cell (35–37). Tissue areas for sampling were selected based on visual lines used in this study were as follows: lung adenocarcinomas A427, alignment with the corresponding H&E-stained sections on slides. A549, LC319, and NCI-H1373; lung squamous cell carcinomas (SCC) Three, four, or five tissue cores (diameter 0.6 mm; height 3-4 mm) RERF-LC-AI, SK-MES-1, NCI-H226, NCI-H520, and NCI-H2170; and taken from donor-tumor blocks were placed into recipient paraffin SCLCs DMS114, DMS273, SBC-3, and SBC-5. All cells were grown in blocks using a tissue microarrayer (Beecher Instruments). A core of monolayers in appropriate medium supplemented with 10% FCS and normal tissue was punched from each case. Five-micrometer sections of were maintained at 37jC in atmospheres of humidified air with 5% the resulting microarray block were used for immunohistochemical

CO2. Human small airway epithelial cells were grown in optimized analysis. Positivity for KIF4A was assessed semiquantitatively by three medium purchased from Cambrex Bio Science, Inc. Primary lung cancer independent investigators without prior knowledge of the clinical tissue samples had been obtained with informed consent as described follow-up data, each of whom recorded staining intensity as either previously (8, 12). A total of 357 NSCLCs and adjacent normal lung negative (no appreciable staining in tumor cells) or positive (brown tissue samples for immunostaining on tissue microarray were also staining appreciable in the nucleus and cytoplasm of tumor cells). Cases obtained from Saitama Cancer Center (Saitama, Japan). These patients were accepted as positive only if reviewers independently defined them received resection of their primary cancers, and among them only as such. patients with positive lymph node metastasis were treated with Statistical analysis. Statistical analyses were done using the StatView cisplatin-based adjuvant chemotherapies after their surgery. Twenty- statistical program (SaS). We used contingency tables to analyze the nine SCLC samples obtained from Hiroshima University (Hiroshima, relationship between KIF4A expression and clinicopathologic variables Japan) and Saitama Cancer Center were also used in this study. This in NSCLC patients. Tumor-specific survival curves were calculated from study and the use of all clinical materials were approved by individual the date of surgery to the time of death related to NSCLC, or to the institutional ethical committees. last follow-up observation. Kaplan-Meier curves were calculated for Semiquantitative reverse transcription-PCR. Total RNA was extracted each relevant variable and for KIF4A expression; differences in survival from cultured cells using the TRIzol reagent (Life Technologies, Inc.) times among patient subgroups were analyzed using the log-rank test. according to the manufacturer’s protocol. Extracted RNAs were treated Univariate and multivariate analyses were done with the Cox propor- with DNase I (Nippon Gene) and reversely transcribed using oligo(dT) tional hazard regression model to determine associations between primer and SuperScript II. Semiquantitative reverse transcription-PCR clinicopathologic variables and cancer-related mortality. First, we ana- (RT-PCR) experiments were carried out with the following synthesized lyzed associations between death and possible prognostic factors, KIF4A-specific primers or with h-actin (ACTB)–specific primers as an including age, gender, histologic type, pT classification, pN classifica- internal control: KIF4A,5¶-CAAAAACCAGCTTCTTCTCTGG-3¶ and tion, and smoking history, taking into consideration one factor at a 5¶-CAGGAAAGATCACAACCTCATTC-3¶; ACTB,5¶-GAGGTGAT- time. Second, multivariate Cox analysis was applied on backward AGCATTGCTTTCG-3¶ and 5¶-CAAGTCAGTGTACAGGTAAGC-3¶. PCR (stepwise) procedures that always forced KIF4A expression into the

www.aacrjournals.org 6625 Clin Cancer Res 2007;13(22) November 15, 2007 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2007 American Association for Cancer Research. Imaging, Diagnosis, Prognosis model, along with any and all variables that satisfied an entry level of compared KIF4A protein expressions in five normal tissues P < 0.05. As the model continued to add factors, independent factors (liver heart, kidney, lung, and testis) with those in lung cancers did not exceed an exit level of P < 0.05. using anti-KIF4A polyclonal antibodies by immunohisto- RNA interference assay. We had previously established a vector- chemistry. KIF4A expressed abundantly in testis (mainly in based RNA interference system, psiH1BX3.0, that was designed to cytoplasm and/or nucleus of primary spermatocytes) and lung synthesize small interfering RNAs (siRNA) in mammalian cells (17–27, cancers; however, its expression was hardly detectable in the 29, 30). Ten micrograms of siRNA expression vector were transfected remaining four normal tissues (Fig. 2A). using 30 AL of LipofectAMINE 2000 (Invitrogen) into lung cancer cell Association ofKIF4A overexpression with poor prognosis of lines SBC-3, SBC-5, LC319, and A549. The transfected cells were cultured for 7 days in the presence of appropriate concentrations of NSCLC. In SCLC and NSCLC tissues, immunohistochemical geneticin (G418); the number of colonies was counted by Giemsa analysis with anti-KIF4A polyclonal antibodies showed that staining; and viability of cells was evaluated by 3-(4,5-dimethylthiazol- KIF4A localized in the cytoplasm and nucleus (Fig. 2B). 2-yl)-2,5-diphenyltetrazolium bromide assay at 7 days after the Interestingly, the invasive border of the tumor adjacent to the treatment. Briefly, cell counting kit-8 solution (Dojindo) was added noncancerous areas showed the tendency of stronger staining. to each dish at a concentration of 1/10 volume, and the plates were Using tissue microarrays prepared from 357 NSCLCs and 29 incubated at 37jC for additional 2 h. Absorbance was then measured SCLCs, we did immunohistochemical analysis with anti-KIF4A at 490 nm, and at 630 nm as a reference, with a Microplate Reader polyclonal antibodies and found positive staining in 127 550 (Bio-Rad). To confirm suppression of KIF4A mRNA expression, (36%) of the 357 NSCLC cases and 19 (66%) of the 29 SCLCs semiquantitative RT-PCR experiments were carried out with the (Fig. 2C), whereas no staining was observed in any of following synthesized KIF4A-specific primers according to the standard protocol. The target sequences of the synthetic oligonucleotides for corresponding normal lung tissues examined. Of these KIF4A- RNA interference were as follows: control 1 (luciferase/LUC: Photinus positive NSCLC cases, 68 were adenocarcinomas (30% of 223), pyralis luciferase gene), 5¶-CGTACGCGGAATACTTCGA-3¶; control 2 37 were SCCs (39% of 94 cases), 14 were LCCs (52% of (scramble/SCR: chloroplast Euglena gracilis gene coding for 5S and 27 cases), and 8 were adenosquamous cell carcinomas (62% 16S rRNAs), 5¶-GCGCGCTTTGTAGGATTCG-3¶;siRNA-KIF4A-1, of 13). We then examined correlations of the KIF4A expression 5¶-GGAAGAATTGGTTCTTGAA-3¶;siRNA-KIF4A-2, 5¶-GATGTGGCT- in surgically resected NSCLCs with various clinicopathologic CAACTCAAAG-3¶. variables. The sample size of SCLCs was too small to be Matrigel invasion assay. We cloned the entire coding sequence evaluated further. Statistical analysis revealed that gender into the appropriate site of p3XFLAG-CMV-10 plasmid vector (Sigma). (higher in male; P = 0.0287 by m2 test) and histology (higher COS-7 and NIH3T3 cells transfected either with plasmids expressing in nonadenocarcinomas; P =0.0097bym2 test) were KIF4A or with mock plasmids were grown to near confluence in DMEM containing 10% FCS. The cells were harvested by trypsinization, washed significantly associated with the KIF4A positivity (Table 1). in DMEM without addition of serum or proteinase inhibitor, and The Kaplan-Meier method indicated significant association suspended in DMEM at 5  105/mL. Before preparing the cell sus- between KIF4A status (positive versus negative) in NSCLCs pension, the dried layer of Matrigel matrix (Becton Dickinson Labware) and tumor-specific survival rate (shorter survival periods in was rehydrated with DMEM for 2 h at room temperature. DMEM KIF4A-positive cases; P = 0.0005 by the log-rank test; Fig. 2D, (0.75 mL) containing 10% FCS was added to each lower chamber in left). Positive immunostaining of KIF4A protein was associated  5 24-well Matrigel invasion chambers, and 0.5 mL (2.5 10 cells) of cell with shorter survival for patients with lung adenocarcinoma suspension were added to each insert of the upper chamber. The plates (P = 0.005 by the log-rank test), whereas KIF4A expression also j of inserts were incubated for 22 h at 37 C. After incubation, the tended to be an unfavorable prognostic factor for patients with chambers were processed; cells invading through the Matrigel were lung SCC or LCC (P = 0.05 by the log-rank test; Supplementary fixed and stained by Giemsa as directed by the supplier (Becton Dickinson Labware). Figs. S1, left and right). By univariate analysis, histology (adenocarcinomas versus nonadenocarcinomas), tumor size Results (pT1 versus pT2-4), lymph node metastasis (pN0 versus pN1-3), age (<65 years versus z65 years), gender (female versus Expression ofKIF4A in lung cancers and normal tissues. Using male), and KIF4A positivity (negative versus positive) were all a cDNA microarray to screen for genes that were highly significantly related to poor tumor-specific survival of NSCLC transactivated in a large proportion of lung cancers, we patients (Table 2). Furthermore, multivariate analysis using identified KIF4A gene as a good candidate. This gene showed the Cox proportional hazard model indicated that pT stage, a 5-fold or higher level of expression in the majority of SCLC pN stage, age, and positive KIF4A staining were independent cases and in f40% of NSCLCs we examined. Subsequently, we prognostic factors for NSCLC (Table 2). To further analyze the confirmed its transactivation by semiquantitative RT-PCR prognostic value of KIF4A in more homogeneous populations experiments in all of eight SCLC cases and in 5 of 10 NSCLC of patients, we validated the relationship between KIF4A cases (Fig. 1A). We further confirmed a high level of KIF4A in expression and survival by subgroup analysis based on tumor 10 of 12 lung cancer cell lines by semiquantitative RT-PCR stage and the status of adjuvant treatment. We divided the and Western blot analyses using anti-KIF4A antibody (Fig. 1B, 357 NSCLC cases into two subgroups with or without the top and bottom). To determine the subcellular localization of adjuvant therapy: group 1 for node-negative cases (pN0; 212 endogenous KIF4A in lung cancer cells, we did immunocyto- patients) who had no adjuvant treatment and group 2 for chemical analysis using anti-KIF4A polyclonal antibodies; node-positive cases (pN1-3; 145 patients) who were treated with KIF4A protein was localized in the cytoplasm and nucleus of cisplatin-based adjuvant chemotherapy after surgery. We con- DMS273 cells (Fig. 1C). firmed that KIF4A expression was significantly associated with Northern blot analysis using KIF4A cDNA as a probe poor prognosis in the group 1 patients (P = 0.03 by the log-rank identified a 5.0-kb transcript specifically in testis among the test; Fig. 2D, middle), as well as with that in the group 2 patients 23 normal human tissues examined (Fig. 1D). Furthermore, we (P = 0.003 by the log-rank test; Fig. 2D, right).

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Fig. 1. KIF4A expression in lung cancers and normal tissues. A, expression of KIF4A in clinical samples of SCLC (top panels)andNSCLC (bottom panels), and normal lung tissues, analyzed by semiquantitative RT-PCR.We prepared appropriate dilutions of each single-stranded cDNA prepared from mRNAs of lung cancer samples, using the level of h-actin (ACTB)expressionasa quantitative control. B, expression of KIF4A in lung cancer cell lines, examined by semiquantitative RT-PCR (top panels) and Western blot analyses (bottom panels). IB, immunoblot. Expression of ACTB served as a quantity control. C, subcellular localization of endogenous KIF4A protein in DMS273 cells. KIF4A staining is observed at the cytoplasm and nucleus of the cells. DAPI, 4¶,6-diamidino-2-phenylindole. D, expression of KIF4A in normal human tissues, detected by Northern blot analysis.

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Inhibition ofgrowth oflung cancer cells by siRNA against SBC-3, SBC-5, and LC319 cells, which strongly expressed KIF4A KIF4A. To assess whether KIF4A is essential for growth or (Fig. 3; Supplementary Fig. S2). The KIF4A-mRNA levels in cells survival of lung cancer cells, we constructed plasmids to express transfected with si-KIF4A-1 or si-KIF4A-2 were significantly siRNAs against KIF4A (si-KIF4As) as well as control plasmids decreased in comparison with cells transfected with either (siRNAs for luciferase and scramble) and transfected them into control siRNAs (Fig. 3A; Supplementary Fig. S2). We observed

Fig. 2. Immunohistochemical and clinicopathologic evaluation of KIF4A protein expression in lung cancer tissues. A, expression of KIF4A in five normal human tissues as well as lung SCC, detected by immunohistochemical staining. Magnification, Â100. Positive staining appeared predominantly in the cytoplasm and nucleus of primary spermatocytes in the testis and lung cancer cells. B, expression of KIF4A in lung SCC, detected by immunohistochemical staining. Magnifications, Â10 0 (left)andÂ200 (right). Invasive border of the tumor adjacent area to the noncancerous area showed the tendency of stronger staining. C, KIF4A expression in SCLCs, lung adenocarcinomas (ADC), and lung SCCs. Its expression is not observed in normal lung. D, association of KIF4A overexpression with poor clinical outcomes for NSCLC patients. Left, Kaplan-Meier analysis of tumor-specific survival in patients with NSCLC according to KIF4A expression. Middle, Kaplan-Meier analysis of tumor-specific survival period among group 1patients with node-negative (pN0) NSCLC according to presence or absence of KIF4A. Right, Kaplan-Meier analysis of tumor-specific survival period among group 2 patients with node-positive (pN1-3) tumor according to presence or absence of KIF4A.

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Table 1. Association between KIF4A positivity in NSCLC tissues and patient characteristics

Total (n = 357) KIF4A positive (n = 127) KIF4A absent (n = 230) C2 P (positive vs absent)

Gender Female 10729 784.7840.0287* Male 250 98 152 Age (y) <65 177 69 108 1.78 NS z65 180 58 122 Histologic type ADC 223 68 155 6.692 0.0097* Non-ADC 134 59 75 pT factor

T1 113 36 77 0.996 NS T2-4 244 91 153 pN factor

N0 212 75 137 0.009 NS N1-3 145 52 Smoking history Never smoker 104 32 72 1.478 NS Smoker 253 95 158

Abbreviations: ADC, adenocarcinoma; non-ADC, SCC plus large cell carcinoma and adenosquamous cell carcinoma; NS, no significance. *P < 0.05 (m2 test). significant decreases in the numbers of viable cells (Figs. 3B-C; Discussion Supplementary Fig. S2). si-KIF4As revealed no significant effect on cell viability of A549 cells in which KIF4A expression was Several molecular-targeting drugs have been developed and hardly detectable (Supplementary Fig. S3). proved their efficacy in cancer therapy; however, the proportion Cellular invasive effect of KIF4A on mammalian cells. As the of patients showing good response is still limited (38). immunohistochemical analysis on tissue microarray had Therefore, further development of molecular-targeting drugs indicated that lung cancer patients with KIF4A-positive for cancer is urgently awaited. We have screened the therapeutic tumors revealed shorter cancer-specific survival periods than target molecules by the following strategy: (a) identifying those with KIF4A-negative tumors, we did Matrigel invasion up-regulated genes in lung cancer by genome-wide cDNA assays to determine whether KIF4A might play some role in microarray system (8–12); (b) verifying the candidate genes cellular invasive ability. Invasion of COS-7-KIF4A cells or for its no, or very low level of, expression in normal tissues by NIH3T3-KIF4A cells through Matrigel was significantly Northern blotting (15, 16); (c) validating clinicopathologic enhanced, compared with the control cells transfected with significance of their overexpression by means of tissue micro- mock plasmids, thus independently suggesting that KIF4A array containing hundreds of archived lung cancer samples could contribute to the highly malignant phenotype of lung (18–30); and (d) verifying whether the target gene is essential cancer cells (Fig. 4A-C). for growth or the survival of cancer cells by RNA interference

Table 2. Cox proportional hazards model analysis of prognostic factors in patients with NSCLCs

Variables Hazards ratio (95% confidence interval) Unfavorable/favorable P

Univariate analysis KIF4A 1.690 (1.254-2.276) Positive/negative 0.0006* Age (y) 1.572 (1.171-2.112) z65/65> 0.0026* Gender 1.690 (1.203-2.372) Male/female 0.0025*

pT factor 2.708 (1.857-3.951) T2-4/T1 <0.0001* pN factor 2.369 (1.769-3.171) N1-3/N0 <0.0001* Histologic type 1.407(1.050-1.884) Non-ADC/ADC 0.0222* Smoking history 1.193 (0.862-1.652) Smoker/never smoker NS Multivariate analysis KIF4A 1.657(1.221-2.248) Positive/negative 0.0012* Age (y) 1.754 (1.300-2.365) z65/65> 0.0002* Gender 1.368 (0.942-1.986) Male/female NS

pT factor 2.130 (1.446-3.138) T2-4/T1 0.0001* pN factor 2.429 (1.800-3.279) N1-3/N0 <0.0001* Histologic type 0.99 (0.717-1.367) Non-ADC/ADC NS

*P < 0.05.

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Fig. 3. Inhibition of growth of lung cancer cells that overexpressed KIF4A by siRNA against KIF4A. A, expression of KIF4A in response to si-KIF4A (si-#1 or si-#2) or control siRNAs (LUC or SCR) in SBC-5 cells, analyzed by semiquantitative RT-PCR. B, colony formation assays of SBC-5 cells transfected with specific siRNAs or control plasmids. C, viability of SBC-5 cells evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in response to si-KIF4A (si-#1 or si-#2), si-LUC, or si-SCR. All assays were done thrice, and in triplicate wells.

assay (17, 19, 20, 22–27, 29, 30). Using this approach, we have also shown to be involved in neuronal survival (40). shown here that KIF4A is frequently overexpressed in clinical In this study, the treatment of NSCLC cells with specific lung cancer samples and cell lines, and that its gene products siRNA to knockdown KIF4A expression resulted in suppres- play indispensable roles in the growth and progression of lung sion of cancer cell growth. We also showed additional cancer cells. evidences supporting the significance of this pathway in Kinesins constitute a superfamily of microtubule-based carcinogenesis; for example, the expression of KIF4A also motor proteins with 45 members in mice and humans that resulted in the significant promotion of the cellular invasion represent diverse functions, including the transport of vesicles, in in vitro assays. Moreover, clinicopathologic evidence organelles, chromosomes, protein complexes, and mRNA obtained through our tissue microarray experiments indicated (39–42). The inhibition of the mitotic kinesin Eg5 by small that NSCLC patients with KIF4A-positive tumors had shorter molecules such as monastrol is being evaluated as an approach cancer-specific survival periods than those with KIF4A-negative to develop a novel class of antiproliferative drugs for the tumors. The results obtained by in vitro and in vivo assays treatment of malignant tumors (43, 44). The KIF4 subfamily strongly suggested that KIF4A is likely to be an important consists of KIF4A, KIF4B, KIF21A, and KIF21B (39). KIF4A is a growth factor and might be associated with a highly novel component of the chromosome condensation and malignant phenotype of lung cancer cells, although the segregation machinery functioning in multiple steps of mitotic molecular mechanisms underlying increased KIF4A expression division and plays essential roles in regulating anaphase spindle levels in many cancer cells remains to be clarified. Because dynamics and the completion of cytokinesis (45, 46). KIF4 is KIF4A should be classified as one of the typical cancer testis

Fig. 4. Enhancement of cellular invasiveness by KIF4A introduction into mammalian cells. A, transient expression of KIF4A in COS-7 and NIH3T3 cells, detected by Western blot analysis. B and C, assays demonstrating the invasive nature of NIH3T3 and COS-7 cells in Matrigel matrix after transfection with expression plasmids for human KIF4A. Giemsa staining (B; magnification, Â100), and the relative number of cells migrating through the Matrigel-coated filters (C). Assays were done thrice and in triplicate wells.

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antigens, selective inhibition of KIF4A activity by molecular In summary, activation of KIF4A has a specific functional role targeted agents could be a promising therapeutic strategy for growth and/or malignant phenotype of cancer cells. KIF4A that is expected to have a powerful biological activity against overexpression in resected specimens may be a useful index for cancer with a minimal risk of adverse events. Moreover, application of adjuvant therapy to the patients who are likely to KIF4A antigens may be used as HLA-restricted epitope have poor prognosis. In addition, the data strongly imply the peptides for cancer vaccines that can induce specific immune possibility of designing new anticancer drugs to specifically responses by cytotoxic T cells against cancer cells with KIF4A target the oncogenic activity of KIF4A for treatment of lung expression. cancer patients.

References 1. Greenlee RT,Hill-Harmon MB, Murray T,Thun M.Can- non-small cell lung cancer. Cancer Res 2003;63: formation and cytokinesis. Proc Nat Acad Sci U S A cer statistics, 2001.CACancerJClin 2001;51:15^ 36. 7038 ^ 41. 2005;102:343 ^ 8. 2. Sozzi G. Molecular biology of lung cancer. Eur J 18. Ishikawa N, DaigoY,YasuiW, et al. ADAM8 as a novel 32. Miki H, OkadaY, Hirokawa N. Analysis of the kinesin Cancer 2001;37 Suppl 7:S63 ^ 73. serological and histochemical marker for lung cancer. superfamily: insights into structure and function. 3. Morita T, Sugano H. A statistical analysis of lung Clin Cancer Res 2004;10:8363 ^ 70. Trends Cell Biol 2005;15:467 ^ 76. cancer registered in the Annual of Pathological 19. Kato T, Daigo Y, Hayama S, et al. A novel human 33. Lee YM, Kim W. Association of human kinesin su- Autopsy Cases inJapan between1958 and1987,with tRNA-dihydrouridine synthase involved in pulmonary perfamily protein member 4 with BRCA2-associated special reference to the characteristics of lung cancer carcinogenesis. Cancer Res 2005;65:5638 ^ 46. factor 35. Biochem J 2003;374:497 ^ 503. in Japan. Acta Pathol Jpn 1990;40:665 ^ 75. 20. Furukawa C, DaigoY, Ishikawa N, et al. Plakophilin 3 34. NarayanG,BourdonV,ChagantiS,etal.Genedos- 4. Simon GR, Wagner H. Small cell lung cancer. Chest oncogene as prognostic marker and therapeutic target age alterations revealed by cDNA microarray analysis 2003;123 Suppl 1:S259 ^ 71. for lung cancer. Cancer Res 2005;65:7102^ 10. in cervical cancer: identification of candidate ampli- 5. Parkin DM. Global cancer statistics in the year 2000. 21. Ishikawa N, Daigo Y,Takano A, et al. Increases of fied and overexpressed genes. Genes Chromosomes Lancet Oncol 2001;2:533 ^ 43. amphiregulinandtransforminggrowthfactor-a in se- Cancer 2007;46:373 ^ 84. rum as predictors of poor response to gefitinib among 35. Chin SF, DaigoY, Huang HE, et al. A simple and reli- 6. Chute JP, Chen T, Feigal E, Simon R, Johnson BE. Twenty years of phase III trials for patients with exten- patients with advanced non-small cell lung cancers. able pretreatment protocol facilitates fluorescent Cancer Res 2005;65:9176^ 84. in situ hybridisation on tissue microarrays of paraffin sive-stage small-cell lung cancer: perceptible prog- ress. J Clin Oncol 1999;17:1794 ^ 1801. 22. Suzuki C, Daigo Y, Ishikawa N, et al. ANLN plays a wax embedded tumour samples. Mol Pathol 2003; critical role in human lung carcinogenesis through the 56:275^9. 7. SandlerAB. Chemotherapy for small cell lung cancer. activation of RHOA and by involvement in the phos- 36. Callagy G, Cattaneo E, Daigo Y, et al. Molecular Semin Oncol 2003;30:9 ^ 25. phoinositide 3-kinase/AKT pathway. Cancer Res classification of breast carcinomas using tissue micro- 8. KikuchiT, DaigoY,KatagiriT, et al. Expression profiles 2005;65:11314^ 25. arrays. Diagn Mol Pathol 2003;12:27^ 34. of non-small cell lung cancers on cDNA microarrays: 23. Ishikawa N, DaigoY,Takano A, et al. Characterization 37. Callagy G, Pharoah P, Chin SF, et al. Identification identification of genes for prediction of lymph-node of SEZ6L2 cell-surface protein as a novel prognostic and validation of prognostic markers in breast cancer metastasis and sensitivity to anti-cancer drugs. Onco- marker for lung cancer. Cancer Sci 2006;97:737 ^ 45. with the complementary use of array-CGH and tissue gene 2003;22:2192 ^ 205. 24. Takahashi K, Furukawa C,Takano A, et al. The neuro- microarrays. JPathol 2005;205:388^96. 9. Kakiuchi S, Daigo Y, Tsunoda T, Yano S, Sone S, medin u-growth hormone secretagogue receptor 38. Ranson M, Hammond LA, Ferry D, et al. ZD1839, Nakamura Y. Genome-wide analysis of organ-prefer- 1b/neurotensin receptor 1 oncogenic signaling path- a selective oral epidermal growth factor receptor- ential metastasis of human small cell lung cancer in way as a therapeutic target for lung cancer. Cancer tyrosine kinase inhibitor, is well tolerated and active in mice. Mol Cancer Res 2003;1:485^99. Res 2006;66:9408 ^ 19. patients with solid, malignant tumors: results of a 10. Kakiuchi S, Daigo Y, Ishikawa N, et al. Prediction of 25. Hayama S, Daigo Y, Kato T, et al. Activation of phase I trial. J Clin Oncol 2002;20:2240 ^50. sensitivity of advanced non-small cell lung cancers to CDCA1-2, members of centromere protein complex, 39. Lawrence CJ, Dawe RK, Christie KR, et al. Astandard- gefitinib (Iressa, ZD1839). Hum Mol Genet 2004;13: involved in pulmonary carcinogenesis. Cancer Res ized kinesinnomenclature. JCell Biol 2004;167:19^22. 3029^ 43. 2006;66:10339 ^ 48. 40. Midorikawa R,TakeiY,Hirokawa N. KIF4motorregu- 11. Kikuchi T, DaigoY, Ishikawa N, et al. Expression pro- 26. Kato T, Hayama S, Yamabuki Y, et al. Increased ex- lates activity-dependentneuronalsurvivalbysuppress- files of metastaticbrain tumor from lung adenocarci- pression of insulin-like growth factor-II messenger ing PARP-1enzymaticactivity.Cell 2006;125:371 ^ 83. nomas on cDNA microarray. Int J Oncol 2006;28: RNA-binding protein 1 is associated with tumor pro- 41. Hirokawa N. Kinesin and dynein superfamily 799 ^ 805. gression in patients with lung cancer. Clin Cancer proteins and the mechanism of organelle transport. 12. Taniwaki M, DaigoY, Ishikawa N, et al. Gene expres- Res 2007;13:434^42. Science 1998;279:519 ^ 26. sion profiles of small-cell lung cancers: molecular sig- 27. SuzukiC,TakahashiK,HayamaS,etal.Identification 42. Miki H, Setou M, Kaneshiro K, Hirokawa N. All natures of lung cancer. Int J Oncol 2006;29:567 ^ 75. of Myc-associated protein with JmjC domain as a kinesin superfamily protein, KIF, genes in mouse and 13. Yamabuki T, Daigo Y, Kato T, et al. Genome-wide novel therapeutic target oncogene for lung cancer. human. ProcNatl Acad SciU S A 2001;98:7004 ^ 11. gene expression profile analysis of esophageal squa- Mol CancerTher 2007;6:542 ^551. 43. Muller C, Gross D, Sarli V, et al. Inhibitors of kinesin mous-cell carcinomas. IntJ Oncol 2006;28:1375 ^84. 28.YamabukiT,Takano A, Hayama S, et al. Dickkopf-1as Eg5: antiproliferative activity of monastrol analogues 14. Ochi K, Daigo Y, Katagiri T, et al. Prediction of re- a novel serologicand prognosticbiomarker for lung against human glioblastoma cells. Cancer Chemother sponse to neoadjuvant chemotherapy for osteosarco- and esophageal carcinomas. Cancer Res 2007;67: Pharmacol 2007;59:157 ^ 64. ma by gene-expression profiles. Int J Oncol 2004;24: 2517^25. 44. Koller E, Propp S, Zhang H, et al. Use of a chemically 647^55. 29. Hayama S, Daigo Y,Yamabuki T, et al. Phosphoryla- modified antisense oligonucleotide library to identify and 15. Saito-HisaminatoA, KatagiriT,Kakiuchi S, Nakamura tion and activation of cell division cycle associated validate Eg5 (kinesin-like 1) as a target for antineoplastic T, TsunodaT, NakamuraY. Genome-wide profiling of 8 by aurora kinase B plays a significant role in human drugdevelopment. Cancer Res 2006;66:2059^66. gene expression in 29 normal human tissues with a lung carcinogenesis. Cancer Res 2007;67:4113^ 22. 45. Zhu C, Zhao J, Bibikova M, et al. Functional anal- cDNA microarray.DNARes 2002;9:35^45. 30. Kato T, Sato N, Hayama S, et al. Activation of ysis of human microtubule-based motor proteins, the 16. Ochi K, DaigoY, Katagiri T, et al. Expression profiles HJURP (Holliday junction-recognizing protein) in- kinesins and dyneins, in mitosis/cytokinesis using of two types of human knee joint cartilage. J Hum volved in the chromosomal stability and immortality RNA interference. Mol Biol Cell 2005;16:3187 ^ 99. Genet 2003;48:177^ 82. of cancer cells. Cancer Res 2007;67:8544 ^ 53. 46. Mazumdar M, Sundareshan S, Misteli T.Human chro- 17. Suzuki C, Daigo Y, Kikuchi T, Katagiri T, Nakamura Y. 31. Zhu C, Jiang W. Cell cycle-dependent translocation mokinesin KIF4A functions in chromosome condensa- Identification of COX17 as a therapeutic target for of PRC1on the spindle by Kif4 is essential for midzone tionand segregation. JCell Biol 2004;166:613^20.

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Masaya Taniwaki, Atsushi Takano, Nobuhisa Ishikawa, et al.

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