Identification of Nectin-4 Oncoprotein As a Diagnostic and Therapeutic Target for Lung Cancer

Molecular Biology, Pathobiology, and Genetics

Identification of Nectin-4 Oncoprotein as a Diagnostic and Therapeutic Target for Lung Cancer

Atsushi Takano,1,2 Nobuhisa Ishikawa,1 Ryohei Nishino,1 Ken Masuda,1 Wataru Yasui,3 Kouki Inai,4 Hitoshi Nishimura,5 Hiroyuki Ito,6 Haruhiko Nakayama,6 Yohei Miyagi,7 Eiju Tsuchiya,7 Nobuoki Kohno,2 Yusuke Nakamura,1 and Yataro Daigo1

1Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Departments of 2Molecular and Internal Medicine, 3Molecular Pathology, and 4Pathology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan; 5Department of Thoracic Surgery, Saitama Cancer Center, Saitama, Japan; and 6Division of Thoracic Surgery and 7Molecular Pathology and Genetics Division, Kanagawa Cancer Center, Kanagawa, Japan

Abstract advanced NSCLC patients (5–9). However, issues of toxicity limit Gene expression profile analysis of lung cancers revealed the these treatment regimens to selected patients. transactivation of an immunoglobulin-like molecule Nectin-4 To identify new molecules involved in pathways of carcinogenesis, in the majority of non–small cell lung cancers (NSCLC). we had performed genome-wide expression profile analysis of 101 Immunohistochemical staining of 422 NSCLCs showed that a lung cancers with the cDNA microarray containing 27,648 genes and high level of Nectin-4 expression was associated with poor expressed sequence tags (ESTs) (10–15). To verify the biological and prognosis for NSCLC patients (P < 0.0001), and multivariate clinicopathologic significance of the respective gene products, we analysis confirmed its independent prognostic value (P < have been performing tumor-tissue microarray analysis of clinical 0.0001). We established an ELISA to measure serum Nectin-4 lung cancer materials as well as RNA interference assays (16–37). This systematic approach revealed that Nectin-4 (alias poliovirus receptor– and found that serum Nectin-4 levels were significantly higher related 4, PVRL4) was transactivated in the majority of NSCLCs. in NSCLC patients than in healthy volunteers. The proportion 2+ of the serum Nectin-4–positive cases was 88 of 164 (53.7%) The Nectin family is the Ca -independent immunoglobulin-like NSCLCs, whereas only 3 of 131 (2.3%) healthy volunteers were molecules consisting of four members (Nectin-1, Nectin-2, Nectin- falsely diagnosed as positive, which was superior to carci- 3, and Nectin-4), which are supposed to trans-interact homophili- noembryonic antigen (CEA) and cytokeratin 19-fragment cally and heterophilically, and play a role in cell-cell adhesion (38). (CYFRA21-1) in sensitivity and specificity. A combined ELISA Nectins 1, 2, and 3 are widely expressed in adult tissues, but Nectin- for both Nectin-4 and CEA increased sensitivity and classified 4 was expressed specifically in the embryo and placenta (39, 40). 65.0% of lung adenocarcinomas as positive with false-positive Recently, Nectin-4 was indicated to be overexpressed in breast rate of 4.6%. The use of both Nectin-4 and CYFRA21-1 carcinoma and have a soluble form that is released into the blood classified 68.3% of lung squamous cell carcinomas as positive (41, 42). In spite of the recent evidence of Nectin-4 overexpression with false-positive rate of 6.1%. Treatment of lung cancer cells in cancer, the biological significance of Nectin-4 activation in with small interfering RNAs against Nectin-4 suppressed its human cancer progression and its clinical potential as a therapeutic target were not fully described. expression and cell growth. In addition, exogenous expression of Nectin-4 increased the lamellipodia formation and the Here, we report that Nectin-4 plays a significant role in cancer invasive ability of mammalian cells through activation of cell growth and invasion, and could be a potential target for the small GTPase Rac1. Nectin-4 might play a significant role in development of therapeutic agents as well as a novel serum and lung carcinogenesis, and it should be a new candidate serum tissue biomarker for lung cancer. and tissue biomarker, as well as a therapeutic target. [Cancer Res 2009;69(16):6694–703] Materials and Methods Clinical tissue samples. Primary lung cancer samples as well as their Introduction corresponding normal tissues adjacent to resection margins from patients, who had no anticancer treatment before tumor resection, had been Lung cancer is the leading cause of cancer death worldwide (1). obtained earlier with informed consent (11). The histologic classification About 30% of patients who are diagnosed to have non–small cell of the tumor specimens was based on WHO criteria (43). All tumors lung cancer (NSCLC) are able to undergo curative resection, were staged on the basis of the pTNM pathologic classification of the whereas the remaining patients with an advanced disease are International Union Against Cancer (Table 1A; ref. 44). A total of 422 mainly treated with chemotherapy alone or in combination with a formalin-fixed samples of primary NSCLCs including 265 adenocarcinomas local treatment modality (2). In fact, the overall 5-year survival rate (ADC), 116 squamous cell carcinomas (SCC), 28 large cell carcinomas, 13 of NSCLC patients still remains at only 15% (3, 4). A number of adenosquamous cell carcinomas and adjacent normal lung tissues, had targeted therapies were shown to be effective for a subset of been obtained earlier along with clinicopathologic data from patients who had undergone surgery at Saitama Cancer Center (Saitama, Japan). This study and the use of all clinical materials were approved by the individual Note: Supplementary data for this article are available at Cancer Research Online Institutional Research Ethics Committees. (http://cancerres.aacrjournals.org/). Serum samples. Serum samples were obtained with informed consent Requests for reprints: Yataro Daigo, Institute of Medical Science, The University from 131 healthy volunteers as controls [88 males and 43 females; median of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 Japan. Phone: 81-3-5449-5457; age (F1 SD), 55.5 F 9.6 y; range, 31–83; Supplementary Table S1B] and from Fax: 81-3-5449-5406; E-mail: [email protected] I2009 American Association for Cancer Research. 86 nonneoplastic lung disease patients with chronic obstructive pulmonary doi:10.1158/0008-5472.CAN-09-0016 disease (COPD) enrolled as a part of the Japanese Project for Personalized

Cancer Res 2009; 69: (16). August 15, 2009 6694 www.aacrjournals.org

Table 1.

A. Association between Nectin-4 positivity in NSCLC tissues and patients’ characteristics (n = 422)

TotalNectin-4 strong positive Nectin-4 weak positive Nectin-4 absent P: strong vs weak/absent

n = 422 n = 245 n =119 n =58

Gender Male 290 172 76 42 0.4577 Female 132 73 43 16 Age (y) <65 218 126 63 29 0.9217 z65 204 119 56 29 Histologic type c ADC 265 167 68 30 0.0059*, SCC 116 54 41 21 Others 41 24 10 7 pT factor c T1 143 69 51 23 0.0048 T2–T4 279 176 68 35 pN factor N0 259 142 81 36 0.105 N1+N2 163 103 38 22 Smoking Nonsmoker 127 70 40 17 0.4523 Smoker 295 175 79 41

B. Cox’s proportionalhazards modelanalysisof prognostic factors in patients with NSCLC

Variables Hazards ratio 95% CI Unfavorable/favorable P

Univariate analysis Nectin-4 2.116 1.551–2.887 Strong (2+)/Weak (1+) or () <0.0001b Age (y) 1.473 1.107–1.962 z65/< 65 0.0079b Gender 1.738 1.247–2.422 Male/Female 0.0011b Histologic type 1.402 1.053–1.867 Non-ADC/ADC 0.0208b pT factor 2.660 1.857–3.810 T2–T4/T1 <0.0001b pN factor 2.393 1.799–3.181 N1+N2/N0 <0.0001b Smoking 1.248 0.907–1.716 Smoker/nonsmoker 0.1734 Multivariate analysis Nectin-4 2.145 1.558–2.954 Strong (2+)/Weak (1+) or () <0.0001b Age (y) 1.679 1.250–2.254 z65/< 65 0.0006b Gender 1.437 0.996–2.072 Male/Female 0.0522 Histologic type 1.144 0.828–1.580 Non-ADC/ADC 0.4158 pT factor 1.916 1.319–2.785 T2–T4/T1 0.0006b pN factor 2.324 1.728–3.124 N1+N2/N0 <0.0001b

NOTE: Others, large-cell carcinoma (LCC) plus adenosquamous-cell carcinoma (ASC). *ADC vs non-ADC. cP < 0.05 (Fisher’s exact test). bP < 0.05.

Medicine (BioBank Japan) or admitted to Hiroshima University and its with precise follow-up record after treatments were also obtained. Group 1: affiliated Hospitals (76 males and 10 females; median age (F1 SD), 66.4 F serum samples obtained with informed consent before curative surgical 5.6 y; range, 54–73; Supplementary Table S1C). All of these COPD patients resection at Kanagawa Cancer Center Hospital from 95 early stage NSCLC were current and/or former smokers [the mean (F1 SD) of pack-year index patients [stage I, 48 males and 47 females; median age (F1 SD), 66.8 F 9.6 y; was 66.7 F 44.4; pack-year index was defined as the number of cigarette range, 38–84]. Group2: serum samplesobtained at diagnosis with informed packs (20 cigarette per pack) consumed a day multiplied by years]. Serum consent from 62 advanced stage NSCLC patients (stage IIIB–IV) treated samples were obtained with informed consent from 164 NSCLC patients later with an identical protocol of the first-line chemotherapy using both (123 ADCs and 41 SCCs) admitted to Hiroshima University Hospital, as well carboplatin and paclitaxel admitted to Hiroshima University Hospital [46 as Kanagawa Cancer Center Hospital [122 males and 42 females; median males and 16 females; median age (F1 SD), 61.7 F 10.2 y; range, 35–79]. age (F1 SD), 64.5 F 10.4 y; range, 30–85; Table 2]. To investigate the Patient samples were selected for the study on the basis of the following prognostic value of serum Nectin-4, two additional sets of serum samples criteria: (a) patients were newly diagnosed and previously untreated and (b) www.aacrjournals.org 6695 Cancer Res 2009; 69: (16). August 15, 2009

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2009 American Association for Cancer Research. Cancer Research their tumors were pathologically diagnosed as NSCLCs (stages I–IV). Serum color reaction was terminated by addition of 50 AL 2N sulfuric acid. Color was obtained at the time of diagnosis and stored at 150jC. intensity was determined by a photometer at a wavelength of 450 nm, with a Generation of murine anti–Nectin-4 monoclonal antibody. The anti– reference wave length of 570 nm. Standard curve was drawn for each plate Nectin-4 monoclonal antibodies (clones 19–33 and 66–97) were produced using recombinant soluble Nectin-4 proteins (Nectin-4 ectodomain). Serum using standard method after successive i.p. injections of 20 Ag soluble levels of Nectin-4 were calculated using dilutions of the recombinant recombinant protein (Nectin-4 ectodomain) to mice. Nectin-4 protein (raging from 0.1–300 ng/mL) as a reference. Levels of Flow cytometry and immunocytochemical analyses. Flow cytometric carcinoembryonic antigen (CEA) in serum were measured by ELISA with a and immunocytochemical analyses were performed using anti–Nectin-4 commercially available enzyme test kit (Hope Laboratories) according to the antibody (clone 19–33), as previously described (17). supplier’s recommendations. Levels of cytokeratin 19-fragment (CYFRA21-1) Immunohistochemistry and tissue microarray. Tumor-tissue micro- in serum were measured by ELISA with a commercially available kit (DRG). arrays were constructed using 422 formalin-fixed primary NSCLCs as Statistical analysis. We used contingency tables to analyze the described elsewhere (45–47). To investigate the status of the Nectin-4 relationshipof Nectin-4 levels in tissues or serum and clinicopathologic protein in clinical lung cancer samples that had been embedded in paraffin variables of NSCLC patients. Survival curves were calculated from the date blocks, we stained the sections using 16.25 Ag/mL of mouse anti–Nectin-4 of surgery or diagnosis to the time of death, or to the last follow-up antibody (clone 19–33) as previously described (34). Because the intensity of observation. Kaplan-Meier curves were calculated for each relevant variable, staining within each tumor tissue core was mostly homogenous, the and for Nectin-4 expression in lung tumors or for serum Nectin-4 levels; intensity of Nectin-4 staining was semiquantitatively evaluated by three differences in survival times among patient subgroups were analyzed using independent investigators without prior knowledge of clinicopathologic the log-rank test. Univariate and multivariate analyses were performed with data using following criteria: strong positive (scored as 2+), dark brown the Cox proportional hazard regression model to determine associations staining in >50% of tumor cells completely obscuring plasma membrane between clinicopathologic variables and cancer-related mortality. and cytoplasm; weak positive (1+), any lesser degree of brown staining In ELISA, differences in the serum levels of Nectin-4, CEA, and CYFRA21- appreciable in tumor cell plasma membrane and cytoplasm; absent (scored 1 between tumor groups and a healthy control were analyzed by Mann- as 0), no appreciable staining in tumor cells. Cases were defined as strongly Whitney U tests. positive if all of the three reviewers independently classified them as such. RNA interference assay. To evaluate the biological functions of Nectin-4 ELISA. Serum levels of Nectin-4 were measured by sandwich-type ELISA in lung cancer cells, we used small interfering RNA (siRNA) duplexes against that was originally developed using mouse anti–human Nectin-4 antibodies the target genes (Dharmacon) as described (37). The target sequences of the (please see above). In brief, for detection of Nectin-4 in serum, 96-well synthetic oligonucleotides for RNA interference were as follows: control 1 (a flexible microtiter plates (439454; NALGE NUNC International) were coated nonspecific control oligo); control 2 (Luciferase: Photinus pyralis luciferase with 5 Ag/mL of capturing monoclonal antibody to Nectin-4 (clone 19–33) gene), 5¶-CGUACGCGGAAUACUUCGA-3¶;siRNA-Nectin-4-#1, 5¶-ACA- overnight. Wells were blocked with 300 AL PBS (pH 7.4) containing 5% GUUACCACGUCUGAGGUU-3¶, siRNA-Nectin-4-#2, 5¶-AAUGGUUCAUGGC- bovine serum albumin (BSA) for 2 h and then incubated for 2 h with 6-fold CUGUUUUU-3¶. diluted serum samples in PBS (pH 7.4) containing 1% BSA. After washing Rac1 activation assay. Activation of Rac1 by Nectin-4 overexpression with PBS (pH 7.4) containing 0.05% Tween 20, the wells were incubated for was detected according to the supplier’s recommendations using Rac1 2 h with 10 ng/mL of biotin-conjugated monoclonal anti–Nectin-4 antibody activation assay kit (Cell Biolabs), and COS-7 and NIH-3T3 cells transfected (clone 66–97), followed by reaction with avidin-conjugated peroxidase with Nectin-4–expressing pcDNA3.1-myc/His plasmid or mock plasmid and (P347; Dako Cytomation) using a Substrate Reagent (R&D Systems). The further cultured for 48 h.

Table 2. Association between serum Nectin-4 positivity and NSCLC patients’ characteristics (n = 164)

TotalSerum Nectin-4–positive Serum Nectin-4 absent P: positive vs absent

n = 164 n =88 n =76

Gender Male 122 69 53 0.215 Female 42 19 23 Age (y) <65 74 34 40 0.0845 z65 90 54 36 Histologic type ADC 123 66 57 >0.9999 SCC 41 22 19 pT factor T1 35 11 24 0.0039* T2–T4 129 77 52 pN factor N0 50 21 29 0.0612 N1–N3 114 67 47 Smoking Nonsmoker 50 24 26 0.3959 Smoker 114 64 50

*P < 0.05 (Fisher’s exact test).

Cancer Res 2009; 69: (16). August 15, 2009 6696 www.aacrjournals.org

Figure 1. Expression and cellular localization of Nectin-4 in lung cancers. A, flow cytometric analysis of the levels of endogenous Nectin-4 protein on the cell surface using anti-Nectin-4 monoclonal antibody and four lung cancer cell lines. Green peaks, the fluorescent intensities (Y-axis) detected by anti–Nectin-4 monoclonal antibody; black peaks, those of cells incubated with nonimmunized mouse IgG (control). B, subcellular localization of endogenous Nectin-4 protein, determined using anti–Nectin-4 monoclonal antibody. C, levels of secreted endogenous Nectin-4 protein detected with ELISA in culture medium from the four lung cancer cell lines.

Results (clone 19–33). This analysis indicated that Nectin-4 protein was Nectin-4 expression in lung tumors and normal tissues. To highly expressed and localized at the plasma membranes of NCI- search for novel target molecules for the development of H2170 and NCI-H358 cells, in which Nectin-4 protein had been therapeutic agents and/or diagnostic biomarkers for NSCLC, we detected at a high level by semiquantitative RT-PCR, but not in first screened genes that showed more than a 5-fold higher level of those of A549 and SBC-5 cells that did not express Nectin-4 expression in cancer cells than in normal cells, in the majority of (Fig. 1A). Immunocytochemical staining of the same set of lung NSCLCs analyzed by cDNA microarray. Among 27,648 genes and cancer cells with anti–Nectin-4 monoclonal antibody (clone 19–33) ESTs screened, we identified Nectin-4 to be a good candidate that indicated that Nectin-4 was detected at plasma membrane and showed more than a 5-fold higher level of expression in 87.5% of cytoplasm in NCI-H2170 and NCI-H358, but not in A549 and SBC-5 NSCLCs. We confirmed its transactivation by semiquantitative cells (Fig. 1B). Since the ectodomain of Nectin-4 (43.5 kDa) was reverse transcription-PCR (RT-PCR) experiments in 11 of 14 suggested to be secreted by cleavage of its extracellular portion additional NSCLC cases (6 of 7 ADCs; 5 of 7 SCCs), and in 7 of (41), we applied ELISA to examine its presence in the culture media 20 lung cancer cell lines (Supplementary Fig. S1). We subsequently of the lung cancer cell lines. The amounts of detectable Nectin-4 in attempted to generate mouse monoclonal antibodies specific to the culture media was concordant to the expression levels of human Nectin-4 and obtained two independent clones, 19-33 and Nectin-4 detected with RT-PCR, flow cytometry, and immunoflu- 66-97. As Nectin-4 was indicated to be a type I membrane protein, orescence analyses (Fig. 1C), further supporting the specific we examined Nectin-4 expression on the surface of lung cancer binding affinity of our monoclonal antibodies to Nectin-4. Attempts cells using flow cytometry with anti–Nectin-4 monoclonal antibody to measure endogenous Nectin-4 protein levels in the NCI-H2170 www.aacrjournals.org 6697 Cancer Res 2009; 69: (16). August 15, 2009

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2009 American Association for Cancer Research. Cancer Research and NCI-H358 cell lines by Western blotting using the two Association of Nectin-4 expression with poor clinical monoclonal antibodies (clones 19–33 and 66–97) failed to detect outcomes for NSCLC patients. To further verify the biological any bands on Western blots, indicating that these antibodies can be and clinicopathologic significance of Nectin-4, we examined the used for recognition of a native form of Nectin-4. We further expression of Nectin-4 protein by means of tissue microarrays confirmed that the two monoclonal antibodies (19–33 and 66–97) consisting of NSCLC tissues from 422 patients who underwent could specifically immunoprecipitate Nectin-4 protein in myc- surgical resection. We classified a pattern of Nectin-4 expression on tagged Nectin-4–overexpressing COS-7 cells (Supplementary Fig. the tissue array ranging from absent/weak (scored as 0–1+) to S2, left), but not in mock-transformant COS-7 cells (Supplementary strong (2+; Fig. 2B). Of the 422 NSCLC cases examined, Nectin-4 Fig. S2, right), suggesting that these antibodies have an ability to was strongly stained in 245 cases (58.1%; score 2+), weakly stained specifically recognize the native Nectin-4. in 119 cases (28.2%; score 1+), and not stained in 58 cases (13.7%; Northern blotting using Nectin-4 cDNA as a probe identified the score 0), whereas their adjacent normal lung tissues were not 3.7-kb transcript as a very faint signal in placenta and to a lesser stained (Fig. 2C). We then evaluated the association between degree in trachea among the 23 normal human tissues examined Nectin-4 status and clinicopathologic parameters. As shown in (data not shown), which was concordant with previous report (39). Table 1A, histological type (higher in ADC; P = 0.0059 by Fisher’s We subsequently examined expression of Nectin-4 protein in six exact test) and pT factor (higher in T2–T4; P = 0.0048 by Fisher’s normal tissues (heart, lung, liver, kidney, trachea, and placenta), as exact test) were significantly associated with the strong Nectin-4 well as lung cancers using anti–Nectin-4 monoclonal antibody, and positivity (score 2+). NSCLC patients whose tumors showed strong found that it was hardly detectable in the former five tissues, Nectin-4 expression revealed shorter survival periods compared whereas positive Nectin-4 staining appeared in placenta and lung with those with absent/weak Nectin-4 expression (P < 0.0001 by tumor tissues. The expression levels of Nectin-4 protein in lung log-rank test; Fig. 2D). We also applied univariate analysis to cancer were significantly higher than those in placenta (Fig. 2A). evaluate associations between patient prognosis and other factors

Figure 2. Expression of Nectin-4 in normaltissues and lungtumors, and its association with poor prognosis for NSCLCs. A, immunohistochemicalevaluationof Nectin-4 protein using anti–Nectin-4 antibody (clone 19–33) in lung adenocarcimona and six normal tissues (200). B, immunohistochemicalstaining pattern of Nectin-4 protein in representative lung adenocarcimonas using anti–Nectin-4 antibody on tissue microarrays (100). C, immunohistochemicalstaining of Nectin-4 protein using anti–Nectin-4 antibody in eight representative paired lung tumors and adjacent normal lung tissues (200). D, Kaplan-Meier analysis of survival in patients with NSCLC according to Nectin-4 expression.

Cancer Res 2009; 69: (16). August 15, 2009 6698 www.aacrjournals.org

Figure 3. Serologic concentration of Nectin-4 in NSCLC patients and its association with poor prognosis for NSCLCs. A, left, Serum Nectin-4 levels from patients with lung ADC or SCC determined by ELISA. Right, distribution of Nectin-4 in sera from patients at various clinical stages of lung cancer. B, left, ROC curve analysis of Nectin-4 (red), CEA (yellow), and CYFRA21-1 (green) as serum tumor biomarkers for NSCLC. Right, Serum Nectin-4 levels before and after surgical treatment in patients with NSCLC. C, serum Nectin-4 levels and the expression levels of Nectin-4 in primary tumor tissues in the same NSCLC patients. D, Kaplan-Meier analysis of survival after surgery in 95 patients with stage I NSCLC (top) or after the first-line chemotherapy with a combination of two drugs (carboplatin and paclitaxel) in 62 patients with stage IIIB to IV NSCLC (bottom), according to serum Nectin-4 positivity. including age (<65 versus z65), gender (female versus male), mean (F1 SD) serum levels of Nectin-4 in 131 healthy volunteers histologic type (ADC versus non-ADC), pT classification (T1 versus were 0.6 F 0.4 ng/mL, and those in 86 patients with COPD, who T2-4), pN classification (N0 versus N1+N2), smoking history were current and/or former smokers, were 0.6 F 0.9 ng/mL. The (nonsmoker versus smoker), and Nectin-4 expression status (score difference in the levels of serum Nectin-4 protein between NSCLC 0+, 1+ versus 2+). Among those parameters, strong Nectin-4 patients and healthy volunteers was significant with P value of positivity (P < 0.0001), elderly (P = 0.0079), male (P = 0.0011), non- <0.0001 (Mann-Whitney U test). The difference between healthy ADC (P = 0.0208), advanced pT stage (P < 0.0001), and advanced volunteers and COPD patients was not significant (P = 0.103 by pN stage (P < 0.0001) were significantly associated with poor Mann-Whitney U test). When classified according to histologic prognosis. Multivariate analysis of these prognostic factors types, the serum levels of Nectin-4 were 2.0 F 2.5 ng/mL in ADC revealed that strong Nectin-4 expression, elderly, larger tumor size, patients and 2.0 F 2.3 ng/mL in SCC patients (Fig. 3A, left). Serum and lymph node metastasis were independent prognostic factors Nectin-4 was detected even in patients with operative stage tumors for NSCLC patients (P < 0.0001, 0.0006, 0.0006, and <0.0001, (stages I–IIIA; Fig. 3A, right). Using receiver operating characteristic respectively; Table 1B). (ROC) curves drawn with the data of these 164 NSCLC patients and Serum levels of Nectin-4 in NSCLC patients. As the in vitro 131 healthy donors (Fig. 3B, left), the cutoff level in this assay was findings had suggested a possibility of application of Nectin-4 as a set to provide optimal diagnostic accuracy and likelihood ratios serum biomarker (Fig. 1C), we investigated whether the Nectin-4 is (minimal false-negative and false-positive results) for Nectin-4, secreted into sera of patients with NSCLC. ELISA experiments i.e., 1.0 ng/mL with a sensitivity of 53.7% [88 of 164 NSCLC; 6 of detected Nectin-4 in serologic samples from the majority of the 164 24 (25%) stage I, 10 of 20 (50%); stage II–IIIA; and 72 of 120 (60%) patients with NSCLC (Fig. 3A); serum levels of Nectin-4 in NSCLC stage IIIB–IV tumors] and a specificity of 97.7% (128 of 131; patients were 2.0 F 2.5 ng/mL (mean F 1 SD). In contrast, the Supplementary Table S1A). To evaluate the potential of serum www.aacrjournals.org 6699 Cancer Res 2009; 69: (16). August 15, 2009

Nectin-4 level as a cancer-specific biomarker, we next examined the patients, in the same set of serum samples from cancer patients relationship between serum Nectin-4 positivity and patients’ or and control individuals. ROC analyses (Fig. 3B, left) determined the healthy individuals’ characteristics (Supplementary Table S1B and cutoff value of CEA for NSCLC detection to be 2.5 ng/mL with a C; Table 2). Serum Nectin-4 positivity was significantly associated sensitivity of 42.7% [70 of 164 NSCLC; 4 of 24 (16.7%) for stage I, 3 of with pT factor (higher in T2–T4; P = 0.0039 by Fisher’s exact test) in 20 (15%) for stage II–IIIA, and 63 of 120 for (52.5%) stage IIIB–IV NSCLC patients (Table 2). Importantly, serum Nectin-4 positivity tumors] and a specificity of 97.7%, and also determined the cutoff was not associated with smoking history, gender, and age in value of CYFRA21-1 to be 2.0 pg/mL with a sensitivity of 39.0% individual groups of lung cancer, COPD, and healthy volunteer [64 of 164 NSCLC; 1 of 24 (4.2%) for stage I, 3 of 20 (15%) for stage (Supplementary Table S1B and C; Table 2). In addition, there was II–IIIA, and 60 of 120 (50%) for stage IIIB–IV tumors] and a no significant association between its positivity and respiratory specificity of 96.2% (please see also Supplementary Table S1A). The function in COPD patients (Supplementary Table S1C). To evaluate sum of the area under the ROC curve for serum Nectin-4 value was the feasibility of using serum Nectin-4 level as a tumor detection larger than that for serum CEA or CYFRA21-1, suggesting that biomarker, we also measured by ELISA serum levels of two Nectin-4 is likely to be a better diagnostic biomarker for NSCLC in conventional tumor markers (CEA and CYFRA21-1) for NSCLC the aspect of specificity and likelihood.

Figure 4. Inhibition of growth of NSCLC cells by siRNA against Nectin-4, and effect of Nectin-4 overexpression on activation of Rac1. A, expression of Nectin-4 in NCI-H2170 and NCI-H358 cells treated with si-Nectin-4 or controlsiRNAs analyzedby semiquantitative RT-PCR. B, MTT assay of the cells transfected with Nectin-4–specific siRNAs. All assays were performed thrice, and in triplicate wells. C, subcellular localization of exogenous Nectin-4 and F-actin in COS-7 and NIH-3T3 cells, detected with Alexa488-conjugated secondary antibodies to detect myc/His-tagged Nectin-4 and Alexa594-conjugated phalloidin. D, the elevated levels of GTP-Rac1 in COS-7 and NIH-3T3 cells transfected with Nectin-4–expressing plasmid.

Cancer Res 2009; 69: (16). August 15, 2009 6700 www.aacrjournals.org

We then evaluated the feasibility of using serum Nectin-4 level Effect of Nectin-4 on the growth of NSCLC cells. To assess as a tumor detection biomarker in combination with CEA or whether up-regulation of Nectin-4 plays a significant role in the CYFRA21-1. Measuring both Nectin-4 and CEA in serum can growth and/or survival of lung cancer cells, we transfected siRNAs improve overall sensitivity for detection of lung ADC to 65.0% (for for Nectin-4 to lung cancer cell lines NCI-H2170 and NCI-H358 diagnosing ADC, the sensitivity of CEA alone is 42.3% and that of that overexpressed endogenous Nectin-4. The levels of Nectin-4 Nectin-4 is 54.5%). False-positive results for either of the two tumor expression in the cells transfected with siRNA for Nectin-4 markers among 131 healthy volunteers (control group) amounted (si-Nectin-4-#1 and -#2) were significantly reduced compared to 4.6% (6 of 131), whereas the false-positive rates for CEA and with those transfected with any of the two control siRNAs Nectin-4 in the same control groupwere 2.3% (3 of 131) each. On (Fig. 4A). In accordance with the suppressive effect of the si- the other hand, Nectin-4 and CYFRA21-1 in serum can improve Nectin-4-#1, and -#2 on Nectin-4 expression, colony numbers and overall sensitivity for detection of lung SCC to 68.3% (for cell viability measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe- diagnosing SCC, the sensitivity of CYFRA21-1 alone is 53.7% and nyltetrazolium bromide (MTT) assays were significantly reduced that of Nectin-4 is 51.2%). False-positive results for either of the two (Supplementary Fig. S4A and B), but no such effect was observed tumor markers among 131 healthy volunteers (control group) by two control siRNAs. amounted to 6.1% (8 of 131), whereas the false-positive rates for To further examine the effect of Nectin-4 on the growth of lung CYFRA21-1 and Nectin-4 in the same control groupwere 3.8% (5 of cancer cells, we transfected plasmids designed to express Nectin-4 131) and 2.3% (3 of 131), respectively. or mock plasmids into PC-14 cells that did not express endogenous We then performed ELISA experiments using paired preopera- Nectin-4, and established two independent PC-14 cell lines tive and postoperative (2 months after the surgery) serum samples overexpressing exogenous Nectin-4 (PC-14-Nectin-4-#A and PC- from NSCLC patients to monitor the levels of serum Nectin-4 in the 14-Nectin-4-#B) and two control cells (PC-14-Mock-#A and PC-14- same patients. The concentration of serum Nectin-4 was decreased Mock-#B). MTT assay revealed that growth of the two PC-14- after surgical resection of primary tumors (Fig. 3B, right). We Nectin-4 cells was promoted at a significant degree compared with further compared the serum levels of Nectin-4 with the expression control PC-14-Mock cells (Supplementary Fig. S3A). To investigate levels of Nectin-4 in primary tumors in the same set of 12 NSCLC a potential role of Nectin-4 in vivo tumor growth, we s.c. cases whose serum had been collected before surgery (six patients transplanted either PC-14-Nectin-4-#B cells or PC-14-Mock-#B with Nectin-4–positive tumors and six with Nectin-4–negative cells into BALB/cAJcl-nu/nu mice. During the 20-day observation, tumors). The serum levels of Nectin-4 showed good correlation all three mice that were individually transplanted with PC-14- with the expression levels of Nectin-4 in primary tumors (Fig. 3C), Nectin-4-#B cells showed significantly more rapid tumor growth further suggesting that serum Nectin-4 was secreted from lung compared with three independent mice transplanted with PC-14- tumor. Mock-#B cells (Supplementary Fig. S3B). These findings imply an Association of serum Nectin-4 positivity with poor clinical in vivo and in vitro growth promoting effect of Nectin-4. outcomes for NSCLC patients. We next examined by ELISA the Enhancement of cellular invasion by overexpression of serum Nectin-4 levels before curative surgery in additional 95 Nectin-4. As the immunohistochemical analysis on tissue micro- patients with stage I NSCLC and those at diagnosis in 62 patients array had indicated that NSCLC patients with Nectin-4–strong with advanced NSCLC (stage IIIB–IV) who were newly diagnosed positive tumors showed shorter survival period than those with and previously untreated, and whose clinicopathologic background Nectin-4–negative or weak positive tumors, we examined a affecting prognosis was mostly identical, and found the correlation possible role of Nectin-4 in cellular migration and invasion by of serum Nectin-4 positivity with clinical outcomes. The median Matrigel assays using mammalian COS-7 and NIH-3T3 cells. As survival time of serum Nectin-4–positive patients with stage I shown in Supplementary Fig. S4B, cells transfected with myc/His- NSCLC after curative surgery or advanced NSCLC treated with an tagged Nectin-4–expressing plasmids showed significant invasive identical protocol of chemotherapy was shorter than those of activity through Matrigel compared with those transfected with patients with serum Nectin-4 negative (P = 0.0219 and 0.0269, mock vector. respectively, by log-rank test; Fig. 3D, top and bottom). We also Next, we examined the effect of Nectin-4 on the cell morphology applied univariate analysis to evaluate associations between by transfecting myc/His-tagged Nectin-4–expressing plasmid into patient prognosis and other factors including age, gender, COS-7 and NIH-3T3 cells. Immunocytochemical analysis using anti- histologic type, disease stage, smoking history, and serum Nectin- myc antibody for exogenous Nectin-4 and phalloidin for filamen- 4 positivity. Univariate analysis indicated that only serum Nectin-4 tous actin (F-actin) clearly detected that the membrane protru- positive was significantly associated with poor prognosis for early sions, which were strongly stained with phalloidin, was significantly stage NSCLC patients after surgery (P = 0.0301; Supplementary increased after transfection of Nectin-4 (Fig. 4C, top and bottom). Table S2A). In advanced NSCLCs, univariate analysis indicated No such effect was observed in the cells transfected with mock that serum Nectin-4 positive (P = 0.036) and poorer Performance vector. Interestingly, exogenous Nectin-4 was strongly stained and Status (P = 0.0004) were significantly associated with poor partly colocalized with F-actin at these protrusions. These results prognosis for newly diagnosed advanced NSCLC patients suggest that overexpression of Nectin-4 induced lamellipodia (Supplementary Table S2B). Multivariate analysis revealed that formation in these cells. both serum Nectin-4 positivity and poorer Performance Status Activation of Rac1 by overexpression of Nectin-4. The were independent prognostic factors for the NSCLC patients (P = extension of protrusions, such as lamellipodia, is essential for cell 0.0394 and 0.0004, respectively; Supplementary Table S2B). These motility, and the formation of lamellipodia requires activation of results independently support the high specificity and the great small GTPases Rac1 (48). We therefore examined whether Rac1 potentiality of serum Nectin-4 as a biomarker for detection of activity could be involved in Nectin-4–dependent enhancement of cancer at an early stage and for predicting the early progression the cell motility. Like other small GTPases, Rac1 regulates of the disease. molecular events by cycling between an inactive GDP-bound form www.aacrjournals.org 6701 Cancer Res 2009; 69: (16). August 15, 2009

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2009 American Association for Cancer Research. Cancer Research and an active GTP-bound form. In its active (GTP-bound) state, cell surface and/or extracellular Nectin-4. Based on the data, the Rac1 binds specifically to the p21-binding domain of p21-activated production of monoclonal antibodies targeting cell surface Nectin- protein kinase to control downstream signaling cascades. Based on 4 that could induce antibody-dependent cytotoxicity/complement- this mechanism, we attempted to investigate a possible activation dependent cytotoxicity activity and/or block oncogenic receptor of Rac1 by Nectin-4 overexpression, using lysate from COS-7 and signals, or those neutralizing the secreted Nectin-4 would be NIH-3T3 cells transfected with Nectin-4–expressing plasmid or warranted. mock plasmid, which were mixed with p21-activated protein We established our original ELISA system by the two mouse kinase-p21-binding domain agarose beads to affinity-precipitate monoclonal antibodies to measure serum levels of Nectin-4 and the protein complex containing GTP-Rac1 (active form). The levels showed that serum Nectin-4 levels were significantly higher in of GTP-Rac1 were elevated in COS-7 and NIH-3T3 cells transfected lung cancer patients than in healthy volunteers. This is a great with Nectin-4–expressing plasmid, compared with those trans- advantage to developpracticaland standardized diagnostic kits in fected with mock vector (Fig. 4D). These results indicate that the clinic. Extracellular part of Nectin-4 is reported to be processed Nectin-4 could enhance the lamellipodia formation and cellular by TACE/ADAM-17 and a soluble Nectin-4 is shed from the cell invasion possibly through activation of Rac1. surface and released into the extracellular space (41). Metal- loprotease-mediated cleavage of some single-pass type I trans- Discussion membrane proteins, such as L1CAM released by ADAM10- mediated membrane-proximal cleavage, has been shown to Nectin family members are both homophilic and heterophilic stimulate cellular migration and neurite outgrowth (50). The cell adhesion molecules that bind afadin, an actin filament ectodomain shedding of Nectin-4 might exert some signals (F-actin)–binding protein through their cytoplasmic tails and important for cancer progression. Importantly, serum Nectin-4 associate with the actin cytoskeleton, and could regulate many levels measured by our ELISA system showed higher sensitivity and other cellular activities such as movement, differentiation, specificity (53.7% and 97.7%, respectively) than conventional serum polarization, and the entry of viruses, in cooperation with other tumor markers for NSCLC (CEA or CYFRA21-1) that are being used cell adhesion molecules and cell surface membrane receptors in clinical practice (Supplementary Table S1A). Furthermore, serum (38, 39). In our study, we have shown that activation of Nectin-4 is Nectin-4 in patients with operable stages of NSCLC also showed likely to be an essential contributor to the cell growth and highly higher sensitivity (25% in stage I; 50% in stage II–IIIA) than serum malignant phenotype of tumors through the Rac1-signaling CEA (16.7% in stage I; 15% in stage II–IIIA) or CYFRA21-1 (4.2% in pathway. Although detailed functional association between Nec- stage I; 15% in stage II–IIIA). Although further validation using a tin-4 transactivation and lung carcinogenesis remains to be larger set of serum samples covering various clinical stages will be clarified, targeting the Nectin-4 pathway provides a large possibility required, our data presented here also indicate a potential clinical for developing new types of therapeutic drugs such as nucleic acid usefulness of Nectin-4 as a serologic biomarker for NSCLC that drugs, monoclonal antibodies, and cancer vaccines that are could be widely used in clinical practice, such as detection of expected to have a powerful biological activity against cancer with cancer, prediction of the malignant potential of tumor, and a minimal risk of adverse events in patients. monitoring the disease control condition after any anticancer Possible mechanisms of antibody-based cancer therapy can be treatment. Finally, it should be noted that we observed activation of classified into two categories (49). One is the direct action, which Nectin-4 in more than half of a series of other types of cancers such can be subcategorized into three modes, (a) blocking function, as bladder and cervical carcinomas we examined (data not shown), (b) stimulating function, and (c) targeting function. The other suggesting its diagnostic and therapeutic application to a wide- mechanism of antibody therapy is the indirect action by the range of tumors. immune system, including complement-dependent cytotoxicity and antibody-dependent cytotoxicity (49). We confirmed that two Disclosure of Potential Conflicts of Interest mouse monoclonal antibodies raised against the ectodomain of Nectin-4 (clones 19–33 and 66–97) for ELISA had no antitumor No potential conflicts of interest were disclosed. activity in vitro when treated Nectin-4–overexressing lung cancer Acknowledgments cells only with the antibodies (data not shown). Because Nectin-4 was highly expressed on the surface of lung cancer cells and the Received 1/4/09; revised 6/2/09; accepted 6/15/09. The costs of publication of this article were defrayed in part by the payment of page ectodomain of Nectin-4 was secreted (Fig. 1A), we need to clarify charges. This article must therefore be hereby marked advertisement in accordance in the future study the mechanism of carcinogenesis involving the with 18 U.S.C. Section 1734 solely to indicate this fact.

References patient management of non-small cell lung cancer. Lung 9. Cesare G, Paolo M, Filomena G, et al. Sorafenib and Cancer 2007;57 Suppl 2:S18–23. Sunitinib in the Treatment of advanced non-small cell 1. Ahmedin J, Rebecca S, Elizabeth W, et al. Cancer 6. Sandler A, Gray R, Perry MC, et al. Paclitaxel- lung cancer. The Oncologist 2007;12:191–200. Statistics, 2007. CA Cancer J Clin 2007;57:43–66. carboplatin alone or with bevacizumab for non-small- 10. Daigo Y, Nakamura Y. From cancer genomics to 2. Parkin DM. Global cancer statistics in the year 2000. cell lung cancer. N Engl J Med 2006;355:2542–50. thoracic oncology: discovery of new biomarkers and Lancet Oncol 2001;2:533–43. 7. Shepherd FA, Rodrigues Pereira J, Ciuleanu T, et al. therapeutic targets for lung and esophageal carcinoma. 3. Naruke T, Tsuchiya R, Kondo H, Asamura H. Prognosis Erlotinib in previously treated non-small-cell lung Gen Thorac Cardiovasc Surg 2008;56:43–53. and survival after resection for bronchogenic carcinoma cancer. N Engl J Med 2005;353:123–32. 11. Kikuchi T, Daigo Y, Katagiri T, et al. Expression based on the 1997 TNM-staging classification: the 8. Thatcher N, Chang A, Parikh P, et al. Gefitinib plus profiles of non-small cell lung cancers on cDNA Japanese experience. Ann Thorac Surg 2001;71:1759–64. best supportive care in previously treated patients with microarrays: identification of genes for prediction of 4. Schiller JH, Harrington D, Belani CP, et al. Comparison refractory advanced non-small-cell lung cancer: results lymph-node metastasis and sensitivity to anti-cancer of four chemotherapy regimens for advanced non-small- from a randomised, placebo-controlled, multicentre drugs. Oncogene 2003;22:2192–205. cell lung cancer. N Engl J Med 2002;346:92–8. study (Iressa Survival Evaluation in Lung Cancer). 12. Kakiuchi S, Daigo Y, Tsunoda T, Yano S, Sone S, 5. Thatcher N. The place of targeted therapy in the Lancet 2005;366:1527–37. Nakamura Y. Genome-wide analysis of organ-preferen-

Cancer Res 2009; 69: (16). August 15, 2009 6702 www.aacrjournals.org

tial metastasis of human small cell lung cancer in mice. progression in patients with lung cancer. Clin Cancer 38. Takai Y, Miyoshi J, Ikeda W, Ogita H. Nectins and Mol Cancer Res 2003;1:485–99. Res 2007;13:434–42. nectin-like molecules: roles in contact inhibition of cell 13. Kakiuchi S, Daigo Y, Ishikawa N, et al. Prediction of 26. Suzuki C, Takahashi K, Hayama S, et al. Identification movement and proliferation. Nat Rev Mol Cell Biol 2008; sensitivity of advanced non-small cell lung cancers to of Myc-associated protein with JmjC domain as a novel 9:603–15. gefitinib (Iressa, ZD1839). Hum Mol Genet 2004;13: therapeutic target oncogene for lung cancer. Mol Cancer 39. Reymond N, Fabre S, Lecocq E, Adelaide J, Dubreuil P, 3029–43. Ther 2007;6:542–51. Lopez M. Nectin4/PRR4, a new afadin-associated 14. Kikuchi T, Daigo Y, Ishikawa N, et al. Expression 27. Yamabuki T, Takano A, Hayama S, et al. Dickkopf-1 as member of the nectin family that trans-interacts with profiles of metastatic brain tumor from lung adeno- a novel serologic and prognostic biomarker for lung and nectin1/PRR1 through V domain interaction. J Biol carcinomas on cDNA microarray. Int J Oncol 2006;28: esophageal carcinomas. Cancer Res 2007;67:2517–25. Chem 2001;276:43205–15. 799–805. 28. Hayama S, Daigo Y, Yamabuki T, et al. Phosphoryla- 40. Fabre S, Reymond N, Cocchi F, et al. Prominent role 15. Taniwaki M, Daigo Y, Ishikawa N, et al. Gene tion and activation of cell division cycle associated 8 by of the Ig-like V domain in trans-interactions of nectins. 00 expression profiles of small-cell lung cancers: molecular aurora kinase B plays a significant role in human lung Nectin3 and nectin 4 bind to the predicted C-C¶-C -D signatures of lung cancer. Int J Oncol 2006;29:567–75. carcinogenesis. Cancer Res 2007;67:4113–22. h-strands of the nectin1 V domain. J Biol Chem 2002; 16. Suzuki C, Daigo Y, Kikuchi T, Katagiri T, Nakamura Y. 29. Kato T, Sato N, Hayama S, et al. Activation of HJURP 277:27006–13. Identification of COX17 as a therapeutic target for non- (Holliday Junction-Recognizing Protein) involved in the 41. Fabre-Lafay S, Garrido-Urbani S, Reymond N, small cell lung cancer. Cancer Res 2003;63:7038–41. chromosomal stability and immortality of cancer cells. Goncalves A, Dubreuil P, Lopez M. Nectin-4, a new 17. Ishikawa N, Daigo Y, Yasui W, et al. ADAM8 as a novel Cancer Res 2007;67:8544–53. serological breast cancer marker, is a substrate for serological and histochemical marker for lung cancer. 30. Taniwaki M, Takano A, Ishikawa N, et al. Activation tumor necrosis factor-a-converting enzyme (TACE)/ Clin Cancer Res 2004;10:8363–70. of KIF4A as a prognostic biomarker and therapeutic ADAM-17. J Biol Chem 2005;280:19543–50. 18. Kato T, Daigo Y, Hayama S, et al. A novel human target for lung cancer. Clin Cancer Res 2007;13:6624–31. 42. Fabre-Lafay S, Monville F, Garrido-Urbani S, et al. tRNA-dihydrouridine synthase involved in pulmonary 31. Ishikawa N, Takano A, Yasui W, et al. Cancer-testis Nectin-4 is a new histological and serological tumor carcinogenesis. Cancer Res 2005;65:5638–46. antigen lymphocyte antigen 6 complex locus K is a associated marker for breast cancer. BMC Cancer 19. Furukawa C, Daigo Y, Ishikawa N, et al. Plakophilin 3 serologic biomarker and a therapeutic target for lung and 2007;7:73. oncogene as prognostic marker and therapeutic target esophageal carcinomas. Cancer Res 2007;67:11601–11. 43. Travis WD, Colby TV, Corrin B, Shimosato Y, for lung cancer. Cancer Res 2005;65:7102–10. 32. Mano Y, Takahashi, K, Ishikawa N, et al. Fibroblast Brambilla E. Histological Typing of Lung and Pleural 20. Ishikawa N, Daigo Y, Takano A, et al. Increases of growth factor receptor 1 oncogene partner as a novel Tumors: World Health Organization International His- amphiregulin and transforming growth factor-a in prognostic biomarker and therapeutic target for lung tological Classification of Tumors, 3rd edn. Berlin: serum as predictors of poor response to gefitinib among cancer. Cancer Sci 2007;98:1902–13. Springer; 1999. patients with advanced non-small cell lung cancers. 33. Suda T, Tsunoda T, Daigo Y, Nakamura Y, Tahara H. 44. Sobin L, Wittekind CH. TNM Classification of Cancer Res 2005;65:9176–84. Identification of human leukocyte antigen-A24-restrict- Malignant Tumours, 6th ed. New York: Wiley-Liss; 2002. 21. Suzuki C, Daigo Y, Ishikawa N, et al. ANLN plays a ed epitope peptides derived from gene products 45. Chin SF, Daigo Y, Huang HE, et al. A simple and critical role in human lung carcinogenesis through the upregulated in lung and esophageal cancers as novel reliable pretreatment protocol facilitates fluorescent activation of RHOA and by involvement in the targets for immunotherapy. Cancer Sci 2007;98:1803–8. in situ hybridisation on tissue microarrays of paraffin wax phosphoinositide 3-kinase/AKT pathway. Cancer Res 34. Kato T, Sato N, Takano A, et al. Activation of placenta embedded tumour samples. Mol Pathol 2003;56:275–9. 2005;65:11314–25. specific transcription factor distal-less homeobox 5 46. Callagy G, Cattaneo E, Daigo Y, et al. Molecular 22. Ishikawa N, Daigo Y, Takano A, et al. Characteriza- predicts clinical outcome in primary Lung cancer classification of breast carcinomas using tissue micro- tion of SEZ6L2 cell-surface protein as a novel prognostic patients. Clin Cancer Res 2008;14:2363–70. arrays. Diagn Mol Pathol 2003;12:27–34. marker for lung cancer. Cancer Sci 2006;97:737–45. 35. Mizukami Y, Kono K, Daigo Y, et al. Detection of 47. Callagy G, Pharoah P, Chin SF, et al. Identification 23. Takahashi K, Furukawa C, Takano A, et al. The novel cancer-testis antigen-specific T-cell responses in and validation of prognostic markers in breast cancer neuromedin u-growth hormone secretagogue receptor 1 TIL, regional lymph nodes, and PBL in patients with with the complementary use of array-CGH and tissue b/neurotensin receptor 1 oncogenic signaling pathway esophageal squamous cell carcinoma. Cancer Sci 2008; microarrays. J Pathol 2005;205:388–96. as a therapeutic target for lung cancer. Cancer Res 2006; 99:1448–54. 48. Takai Y, Sasaki T, Matozaki T. Small GTP-Binding 66:9408–19. 36. Harao M, Hirata S, Irie A, et al. HLA-A2-restricted CTL Proteins. Physiol Rev 2001;81:153–208. 24. Hayama S, Daigo Y, Kato T, et al. Activation of epitopes of a novel lung cancer-associated cancer testis 49. Imai K, Takaoka A. Comparing antibody and small- CDCA1-2, members of centromere protein complex, antigen, cell division cycle associated 1, can induce molecule therapies for cancer. Nat Rev Cancer 2006;6: involved in pulmonary carcinogenesis. Cancer Res 2006; tumor-reactive CTL. Int J Cancer 2008;123:2616–25. 714–27. 66:10339–48. 37. Hirata D, Yamabuki T, Ito T, et al. Involvement of 50. Fogel M, Gutwein P, Mechtersheimer S, et al. L1 25. Kato T, Hayama S, Yamabuki Y, et al. Increased epithelial cell transforming sequence 2 (ECT2) oncoan- expression as a predictor of progression and survival in expression of insulin-like growth factor-II messenger tigen in lung and esophageal cancer progression. Clin patients with uterine and ovarian carcinomas. Lancet RNA-binding protein 1 Is associated with tumor Cancer Res 2009;15:256–66. 2003;362:869–75.

www.aacrjournals.org 6703 Cancer Res 2009; 69: (16). August 15, 2009

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2009 American Association for Cancer Research. Identification of Nectin-4 Oncoprotein as a Diagnostic and Therapeutic Target for Lung Cancer

Atsushi Takano, Nobuhisa Ishikawa, Ryohei Nishino, et al.

Cancer Res 2009;69:6694-6703.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/69/16/6694

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2009/08/13/69.16.6694.DC1

Cited articles This article cites 48 articles, 23 of which you can access for free at: http://cancerres.aacrjournals.org/content/69/16/6694.full#ref-list-1

Citing articles This article has been cited by 18 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/69/16/6694.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/69/16/6694. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.