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

Gene expression profile analysis of lung cancers revealed the transactivation of an immunoglobulin-like molecule Nectin-4 in the majority of non–small cell lung cancers (NSCLC). Immunohistochemical staining of 422 NSCLCs showed that a high level of Nectin-4 expression was associated with poor prognosis for NSCLC patients (P < 0.0001), and multivariate analysis confirmed its independent prognostic value (P < 0.0001). We established an ELISA to measure serum Nectin-4 and found that serum Nectin-4 levels were significantly higher in NSCLC patients than in healthy volunteers. The proportion of the serum Nectin-4–positive cases was 88 of 164 (53.7%) NSCLCs, whereas only 3 of 131 (2.3%) healthy volunteers were falsely diagnosed as positive, which was superior to carcinoembryonic antigen (CEA) and cytokeratin 19-fragment (CYFRA21-1) in sensitivity and specificity. A combined ELISA for both Nectin-4 and CEA increased sensitivity and classified 65.0% of lung adenocarcinomas as positive with false-positive rate of 4.6%. The use of both Nectin-4 and CYFRA21-1 classified 68.3% of lung squamous cell carcinomas as positive with false-positive rate of 6.1%. Treatment of lung cancer cells with small interfering RNAs against Nectin-4 suppressed its expression and cell growth. In addition, exogenous expression of Nectin-4 increased the lamellipodia formation and the invasive ability of mammalian cells through activation of small GTPase Rac1. Nectin-4 might play a significant role in lung carcinogenesis, and it should be a new candidate serum and tissue biomarker, as well as a therapeutic target. [Cancer Res 2009;69(16):6694–703]


Introduction
Lung cancer is the leading cause of cancer death worldwide (1).About 30% of patients who are diagnosed to have non-small cell lung cancer (NSCLC) are able to undergo curative resection, whereas the remaining patients with an advanced disease are mainly treated with chemotherapy alone or in combination with a local treatment modality (2).In fact, the overall 5-year survival rate of NSCLC patients still remains at only 15% (3,4).A number of targeted therapies were shown to be effective for a subset of advanced NSCLC patients (5)(6)(7)(8)(9).However, issues of toxicity limit these treatment regimens to selected patients.
To identify new molecules involved in pathways of carcinogenesis, we had performed genome-wide expression profile analysis of 101 lung cancers with the cDNA microarray containing 27,648 genes and expressed sequence tags (ESTs) (10)(11)(12)(13)(14)(15).To verify the biological and clinicopathologic significance of the respective gene products, we have been performing tumor-tissue microarray analysis of clinical lung cancer materials as well as RNA interference assays .This systematic approach revealed that Nectin-4 (alias poliovirus receptorrelated 4, PVRL4) was transactivated in the majority of NSCLCs.
The Nectin family is the Ca 2+ -independent immunoglobulin-like molecules consisting of four members (Nectin-1, Nectin-2, Nectin-3, and Nectin-4), which are supposed to trans-interact homophilically and heterophilically, and play a role in cell-cell adhesion (38).Nectins 1, 2, and 3 are widely expressed in adult tissues, but Nectin-4 was expressed specifically in the embryo and placenta (39,40).Recently, Nectin-4 was indicated to be overexpressed in breast carcinoma and have a soluble form that is released into the blood (41,42).In spite of the recent evidence of Nectin-4 overexpression in cancer, the biological significance of Nectin-4 activation in human cancer progression and its clinical potential as a therapeutic target were not fully described.
Here, we report that Nectin-4 plays a significant role in cancer cell growth and invasion, and could be a potential target for the development of therapeutic agents as well as a novel serum and tissue biomarker for lung cancer.

Materials and Methods
Clinical tissue samples.Primary lung cancer samples as well as their corresponding normal tissues adjacent to resection margins from patients, who had no anticancer treatment before tumor resection, had been obtained earlier with informed consent (11).The histologic classification of the tumor specimens was based on WHO criteria (43).All tumors were staged on the basis of the pTNM pathologic classification of the International Union Against Cancer (Table 1A; ref. 44).A total of 422 formalin-fixed samples of primary NSCLCs including 265 adenocarcinomas (ADC), 116 squamous cell carcinomas (SCC), 28 large cell carcinomas, 13 adenosquamous cell carcinomas and adjacent normal lung tissues, had 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 Institutional Research Ethics Committees.
Serum samples.Serum samples were obtained with informed consent from 131 healthy volunteers as controls [88 males and 43 females; median age (F1 SD), 55.5 F 9.6 y; range, 31-83; Supplementary Table S1B] and from 86 nonneoplastic lung disease patients with chronic obstructive pulmonary disease (COPD) enrolled as a part of the Japanese Project for Personalized Medicine (BioBank Japan) or admitted to Hiroshima University and its affiliated Hospitals (76 males and 10 females; median age (F1 SD), 66.4 F 5.6 y; range, 54-73; Supplementary Table S1C).All of these COPD patients were current and/or former smokers [the mean (F1 SD) of pack-year index was 66.7 F 44.4; pack-year index was defined as the number of cigarette packs (20   their tumors were pathologically diagnosed as NSCLCs (stages I-IV).Serum was obtained at the time of diagnosis and stored at À150jC.Generation of murine anti-Nectin-4 monoclonal antibody.The anti-Nectin-4 monoclonal antibodies (clones 19-33 and 66-97) were produced using standard method after successive i.p. injections of 20 Ag soluble recombinant protein (Nectin-4 ectodomain) to mice.
Immunohistochemistry and tissue microarray.Tumor-tissue microarrays were constructed using 422 formalin-fixed primary NSCLCs as described elsewhere (45)(46)(47).To investigate the status of the Nectin-4 protein in clinical lung cancer samples that had been embedded in paraffin blocks, we stained the sections using 16.25 Ag/mL of mouse anti-Nectin-4 antibody (clone 19-33) as previously described (34).Because the intensity of staining within each tumor tissue core was mostly homogenous, the intensity of Nectin-4 staining was semiquantitatively evaluated by three independent investigators without prior knowledge of clinicopathologic data using following criteria: strong positive (scored as 2+), dark brown staining in >50% of tumor cells completely obscuring plasma membrane and cytoplasm; weak positive (1+), any lesser degree of brown staining appreciable in tumor cell plasma membrane and cytoplasm; absent (scored as 0), no appreciable staining in tumor cells.Cases were defined as strongly positive if all of the three reviewers independently classified them as such.
Statistical analysis.We used contingency tables to analyze the relationship of Nectin-4 levels in tissues or serum and clinicopathologic variables of NSCLC patients.Survival curves were calculated from the date of surgery or diagnosis to the time of death, or to the last follow-up observation.Kaplan-Meier curves were calculated for each relevant variable, and for Nectin-4 expression in lung tumors or for serum Nectin-4 levels; differences in survival times among patient subgroups were analyzed using the log-rank test.Univariate and multivariate analyses were performed with the Cox proportional hazard regression model to determine associations between clinicopathologic variables and cancer-related mortality.
In ELISA, differences in the serum levels of Nectin-4, CEA, and CYFRA21-1 between tumor groups and a healthy control were analyzed by Mann-Whitney U tests.
RNA interference assay.To evaluate the biological functions of Nectin-4 in lung cancer cells, we used small interfering RNA (siRNA) duplexes against the target genes (Dharmacon) as described (37).The target sequences of the synthetic oligonucleotides for RNA interference were as follows: control 1 (a nonspecific control oligo); control 2 (Luciferase: Rac1 activation assay.Activation of Rac1 by Nectin-4 overexpression was detected according to the supplier's recommendations using Rac1 activation assay kit (Cell Biolabs), and COS-7 and NIH-3T3 cells transfected with Nectin-4-expressing pcDNA3.1-myc/Hisplasmid or mock plasmid and further cultured for 48 h.

Results
Nectin-4 expression in lung tumors and normal tissues.To search for novel target molecules for the development of therapeutic agents and/or diagnostic biomarkers for NSCLC, we first screened genes that showed more than a 5-fold higher level of expression in cancer cells than in normal cells, in the majority of NSCLCs analyzed by cDNA microarray.Among 27,648 genes and ESTs screened, we identified Nectin-4 to be a good candidate that showed more than a 5-fold higher level of expression in 87.5% of NSCLCs.We confirmed its transactivation by semiquantitative reverse transcription-PCR (RT-PCR) experiments in 11 of 14 additional NSCLC cases (6 of 7 ADCs; 5 of 7 SCCs), and in 7 of 20 lung cancer cell lines (Supplementary Fig. S1).We subsequently attempted to generate mouse monoclonal antibodies specific to human Nectin-4 and obtained two independent clones, 19-33 and 66-97.As Nectin-4 was indicated to be a type I membrane protein, we examined Nectin-4 expression on the surface of lung cancer cells using flow cytometry with anti-Nectin-4 monoclonal antibody (clone [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33].This analysis indicated that Nectin-4 protein was highly expressed and localized at the plasma membranes of NCI-H2170 and NCI-H358 cells, in which Nectin-4 protein had been detected at a high level by semiquantitative RT-PCR, but not in those of A549 and SBC-5 cells that did not express Nectin-4 (Fig. 1A).Immunocytochemical staining of the same set of lung cancer cells with anti-Nectin-4 monoclonal antibody (clone 19-33) indicated that Nectin-4 was detected at plasma membrane and cytoplasm in NCI-H2170 and NCI-H358, but not in A549 and SBC-5 cells (Fig. 1B).Since the ectodomain of Nectin-4 (43.5 kDa) was suggested to be secreted by cleavage of its extracellular portion (41), we applied ELISA to examine its presence in the culture media of the lung cancer cell lines.The amounts of detectable Nectin-4 in the culture media was concordant to the expression levels of Nectin-4 detected with RT-PCR, flow cytometry, and immunofluorescence analyses (Fig. 1C), further supporting the specific binding affinity of our monoclonal antibodies to Nectin-4.Attempts to measure endogenous Nectin-4 protein levels in the NCI-H2170 and NCI-H358 cell lines by Western blotting using the two monoclonal antibodies (clones 19-33 and 66-97) failed to detect any bands on Western blots, indicating that these antibodies can be used for recognition of a native form of Nectin-4.We further confirmed that the two monoclonal antibodies (19-33 and 66-97) could specifically immunoprecipitate Nectin-4 protein in myctagged Nectin-4-overexpressing COS-7 cells (Supplementary Fig. S2, left), but not in mock-transformant COS-7 cells (Supplementary Fig. S2, right), suggesting that these antibodies have an ability to specifically recognize the native Nectin-4.
Northern blotting using Nectin-4 cDNA as a probe identified the 3.7-kb transcript as a very faint signal in placenta and to a lesser degree in trachea among the 23 normal human tissues examined (data not shown), which was concordant with previous report (39).We subsequently examined expression of Nectin-4 protein in six normal tissues (heart, lung, liver, kidney, trachea, and placenta), as well as lung cancers using anti-Nectin-4 monoclonal antibody, and found that it was hardly detectable in the former five tissues, whereas positive Nectin-4 staining appeared in placenta and lung tumor tissues.The expression levels of Nectin-4 protein in lung cancer were significantly higher than those in placenta (Fig. 2A).
Association of Nectin-4 expression with poor clinical outcomes for NSCLC patients.To further verify the biological and clinicopathologic significance of Nectin-4, we examined the expression of Nectin-4 protein by means of tissue microarrays consisting of NSCLC tissues from 422 patients who underwent surgical resection.We classified a pattern of Nectin-4 expression on the tissue array ranging from absent/weak (scored as 0-1+) to strong (2+; Fig. 2B).Of the 422 NSCLC cases examined, Nectin-4 was strongly stained in 245 cases (58.1%; score 2+), weakly stained in 119 cases (28.2%; score 1+), and not stained in 58 cases (13.7%; score 0), whereas their adjacent normal lung tissues were not stained (Fig. 2C).We then evaluated the association between Nectin-4 status and clinicopathologic parameters.As shown in Table 1A, histological type (higher in ADC; P = 0.0059 by Fisher's exact test) and pT factor (higher in T2-T4; P = 0.0048 by Fisher's exact test) were significantly associated with the strong Nectin-4 positivity (score 2+).NSCLC patients whose tumors showed strong Nectin-4 expression revealed shorter survival periods compared with those with absent/weak Nectin-4 expression (P < 0.0001 by log-rank test; Fig. 2D).We also applied univariate analysis to evaluate associations between patient prognosis and other factors
Serum levels of Nectin-4 in NSCLC patients.As the in vitro findings had suggested a possibility of application of Nectin-4 as a serum biomarker (Fig. 1C), we investigated whether the Nectin-4 is secreted into sera of patients with NSCLC.ELISA experiments detected Nectin-4 in serologic samples from the majority of the 164 patients with NSCLC (Fig. 3A); serum levels of Nectin-4 in NSCLC patients were 2.0 F 2.5 ng/mL (mean F 1 SD).In contrast, the mean (F1 SD) serum levels of Nectin-4 in 131 healthy volunteers were 0.6 F 0.4 ng/mL, and those in 86 patients with COPD, who were current and/or former smokers, were 0.6 F 0.9 ng/mL.The difference in the levels of serum Nectin-4 protein between NSCLC patients and healthy volunteers was significant with P value of <0.0001 (Mann-Whitney U test).The difference between healthy volunteers and COPD patients was not significant (P = 0.103 by Mann-Whitney U test).When classified according to histologic types, the serum levels of Nectin-4 were 2.0 F 2.5 ng/mL in ADC patients and 2.0 F 2.3 ng/mL in SCC patients (Fig. 3A, left).Serum Nectin-4 was detected even in patients with operative stage tumors (stages I-IIIA; Fig. 3A, right).Using receiver operating characteristic (ROC) curves drawn with the data of these 164 NSCLC patients and 131 healthy donors (Fig. 3B, left), the cutoff level in this assay was set to provide optimal diagnostic accuracy and likelihood ratios (minimal false-negative and false-positive results) for Nectin-4, i.e., 1.0 ng/mL with a sensitivity of 53.7% [88 of 164 NSCLC; 6 of 24 (25%) stage I, 10 of 20 (50%); stage II-IIIA; and 72 of 120 (60%) stage IIIB-IV tumors] and a specificity of 97.7% (128 of 131; Supplementary Table S1A).To evaluate the potential of serum Nectin-4 level as a cancer-specific biomarker, we next examined the relationship between serum Nectin-4 positivity and patients' or healthy individuals' characteristics (Supplementary Table S1B and C; Table 2).Serum Nectin-4 positivity was significantly associated with pT factor (higher in T2-T4; P = 0.0039 by Fisher's exact test) in NSCLC patients (Table 2).Importantly, serum Nectin-4 positivity was not associated with smoking history, gender, and age in individual groups of lung cancer, COPD, and healthy volunteer (Supplementary Table S1B and C; Table 2).In addition, there was no significant association between its positivity and respiratory function in COPD patients (Supplementary Table S1C).To evaluate the feasibility of using serum Nectin-4 level as a tumor detection biomarker, we also measured by ELISA serum levels of two conventional tumor markers (CEA and CYFRA21-1) for NSCLC patients, in the same set of serum samples from cancer patients and control individuals.ROC analyses (Fig. 3B, left) determined the cutoff value of CEA for NSCLC detection to be 2.5 ng/mL with a sensitivity of 42.7% [70 of 164 NSCLC; 4 of 24 (16.7%) for stage I, 3 of 20 (15%) for stage II-IIIA, and 63 of 120 for (52.5%) stage IIIB-IV tumors] and a specificity of 97.7%, and also determined the cutoff value of CYFRA21-1 to be 2.0 pg/mL with a sensitivity of 39.0% [64 of 164 NSCLC; 1 of 24 (4.2%) for stage I, 3 of 20 (15%) for stage II-IIIA, and 60 of 120 (50%) for stage IIIB-IV tumors] and a specificity of 96.2% (please see also Supplementary Table S1A).The sum of the area under the ROC curve for serum Nectin-4 value was larger than that for serum CEA or CYFRA21-1, suggesting that Nectin-4 is likely to be a better diagnostic biomarker for NSCLC in the aspect of specificity and likelihood.

Cancer Research
Cancer Res 2009; 69: (16).August 15, 2009 We then evaluated the feasibility of using serum Nectin-4 level as a tumor detection biomarker in combination with CEA or CYFRA21-1.Measuring both Nectin-4 and CEA in serum can improve overall sensitivity for detection of lung ADC to 65.0% (for diagnosing ADC, the sensitivity of CEA alone is 42.3% and that of Nectin-4 is 54.5%).False-positive results for either of the two tumor markers among 131 healthy volunteers (control group) amounted to 4.6% (6 of 131), whereas the false-positive rates for CEA and Nectin-4 in the same control group were 2.3% (3 of 131) each.On the other hand, Nectin-4 and CYFRA21-1 in serum can improve overall sensitivity for detection of lung SCC to 68.3% (for diagnosing SCC, the sensitivity of CYFRA21-1 alone is 53.7% and that of Nectin-4 is 51.2%).False-positive results for either of the two tumor markers among 131 healthy volunteers (control group) amounted to 6.1% (8 of 131), whereas the false-positive rates for CYFRA21-1 and Nectin-4 in the same control group were 3.8% (5 of 131) and 2.3% (3 of 131), respectively.
We then performed ELISA experiments using paired preoperative and postoperative (2 months after the surgery) serum samples from NSCLC patients to monitor the levels of serum Nectin-4 in the same patients.The concentration of serum Nectin-4 was decreased after surgical resection of primary tumors (Fig. 3B, right).We further compared the serum levels of Nectin-4 with the expression levels of Nectin-4 in primary tumors in the same set of 12 NSCLC cases whose serum had been collected before surgery (six patients with Nectin-4-positive tumors and six with Nectin-4-negative tumors).The serum levels of Nectin-4 showed good correlation with the expression levels of Nectin-4 in primary tumors (Fig. 3C), further suggesting that serum Nectin-4 was secreted from lung tumor.
Association of serum Nectin-4 positivity with poor clinical outcomes for NSCLC patients.We next examined by ELISA the serum Nectin-4 levels before curative surgery in additional 95 patients with stage I NSCLC and those at diagnosis in 62 patients with advanced NSCLC (stage IIIB-IV) who were newly diagnosed and previously untreated, and whose clinicopathologic background affecting prognosis was mostly identical, and found the correlation of serum Nectin-4 positivity with clinical outcomes.The median survival time of serum Nectin-4-positive patients with stage I NSCLC after curative surgery or advanced NSCLC treated with an identical protocol of chemotherapy was shorter than those of patients with serum Nectin-4 negative (P = 0.0219 and 0.0269, respectively, by log-rank test; Fig. 3D, top and bottom).We also applied univariate analysis to evaluate associations between patient prognosis and other factors including age, gender, histologic type, disease stage, smoking history, and serum Nectin-4 positivity.Univariate analysis indicated that only serum Nectin-4 positive was significantly associated with poor prognosis for early stage NSCLC patients after surgery (P = 0.0301; Supplementary Table S2A).In advanced NSCLCs, univariate analysis indicated that serum Nectin-4 positive (P = 0.036) and poorer Performance Status (P = 0.0004) were significantly associated with poor prognosis for newly diagnosed advanced NSCLC patients (Supplementary Table S2B).Multivariate analysis revealed that both serum Nectin-4 positivity and poorer Performance Status were independent prognostic factors for the NSCLC patients (P = 0.0394 and 0.0004, respectively; Supplementary Table S2B).These results independently support the high specificity and the great potentiality of serum Nectin-4 as a biomarker for detection of cancer at an early stage and for predicting the early progression of the disease.
Effect of Nectin-4 on the growth of NSCLC cells.To assess whether up-regulation of Nectin-4 plays a significant role in the growth and/or survival of lung cancer cells, we transfected siRNAs for Nectin-4 to lung cancer cell lines NCI-H2170 and NCI-H358 that overexpressed endogenous Nectin-4.The levels of Nectin-4 expression in the cells transfected with siRNA for Nectin-4 (si-Nectin-4-#1 and -#2) were significantly reduced compared with those transfected with any of the two control siRNAs (Fig. 4A).In accordance with the suppressive effect of the si-Nectin-4-#1, and -#2 on Nectin-4 expression, colony numbers and cell viability measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were significantly reduced (Supplementary Fig. S4A and B), but no such effect was observed by two control siRNAs.
To further examine the effect of Nectin-4 on the growth of lung cancer cells, we transfected plasmids designed to express Nectin-4 or mock plasmids into PC-14 cells that did not express endogenous Nectin-4, and established two independent PC-14 cell lines overexpressing exogenous Nectin-4 (PC-14-Nectin-4-#A and PC-14-Nectin-4-#B) and two control cells (PC-14-Mock-#A and PC-14-Mock-#B).MTT assay revealed that growth of the two PC-14-Nectin-4 cells was promoted at a significant degree compared with control PC-14-Mock cells (Supplementary Fig. S3A).To investigate a potential role of Nectin-4 in vivo tumor growth, we s.c.transplanted either PC-14-Nectin-4-#B cells or PC-14-Mock-#B cells into BALB/cAJcl-nu/nu mice.During the 20-day observation, all three mice that were individually transplanted with PC-14-Nectin-4-#B cells showed significantly more rapid tumor growth compared with three independent mice transplanted with PC-14-Mock-#B cells (Supplementary Fig. S3B).These findings imply an in vivo and in vitro growth promoting effect of Nectin-4.
Enhancement of cellular invasion by overexpression of Nectin-4.As the immunohistochemical analysis on tissue microarray had indicated that NSCLC patients with Nectin-4-strong positive tumors showed shorter survival period than those with Nectin-4-negative or weak positive tumors, we examined a possible role of Nectin-4 in cellular migration and invasion by Matrigel assays using mammalian COS-7 and NIH-3T3 cells.As shown in Supplementary Fig. S4B, cells transfected with myc/Histagged Nectin-4-expressing plasmids showed significant invasive activity through Matrigel compared with those transfected with mock vector.
Next, we examined the effect of Nectin-4 on the cell morphology by transfecting myc/His-tagged Nectin-4-expressing plasmid into COS-7 and NIH-3T3 cells.Immunocytochemical analysis using antimyc antibody for exogenous Nectin-4 and phalloidin for filamentous actin (F-actin) clearly detected that the membrane protrusions, which were strongly stained with phalloidin, was significantly increased after transfection of Nectin-4 (Fig. 4C, top and bottom).No such effect was observed in the cells transfected with mock vector.Interestingly, exogenous Nectin-4 was strongly stained and partly colocalized with F-actin at these protrusions.These results suggest that overexpression of Nectin-4 induced lamellipodia formation in these cells.
Activation of Rac1 by overexpression of Nectin-4.The extension of protrusions, such as lamellipodia, is essential for cell motility, and the formation of lamellipodia requires activation of small GTPases Rac1 (48).We therefore examined whether Rac1 activity could be involved in Nectin-4-dependent enhancement of the cell motility.Like other small GTPases, Rac1 regulates molecular events by cycling between an inactive GDP-bound form and an active GTP-bound form.In its active (GTP-bound) state, Rac1 binds specifically to the p21-binding domain of p21-activated protein kinase to control downstream signaling cascades.Based on this mechanism, we attempted to investigate a possible activation of Rac1 by Nectin-4 overexpression, using lysate from COS-7 and NIH-3T3 cells transfected with Nectin-4-expressing plasmid or mock plasmid, which were mixed with p21-activated protein kinase-p21-binding domain agarose beads to affinity-precipitate the protein complex containing GTP-Rac1 (active form).The levels of GTP-Rac1 were elevated in COS-7 and NIH-3T3 cells transfected with Nectin-4-expressing plasmid, compared with those transfected with mock vector (Fig. 4D).These results indicate that Nectin-4 could enhance the lamellipodia formation and cellular invasion possibly through activation of Rac1.

Discussion
Nectin family members are both homophilic and heterophilic cell adhesion molecules that bind afadin, an actin filament (F-actin)-binding protein through their cytoplasmic tails and associate with the actin cytoskeleton, and could regulate many other cellular activities such as movement, differentiation, polarization, and the entry of viruses, in cooperation with other cell adhesion molecules and cell surface membrane receptors (38,39).In our study, we have shown that activation of Nectin-4 is likely to be an essential contributor to the cell growth and highly malignant phenotype of tumors through the Rac1-signaling pathway.Although detailed functional association between Nectin-4 transactivation and lung carcinogenesis remains to be clarified, targeting the Nectin-4 pathway provides a large possibility for developing new types of therapeutic drugs such as nucleic acid drugs, monoclonal antibodies, and cancer vaccines that are expected to have a powerful biological activity against cancer with a minimal risk of adverse events in patients.
Possible mechanisms of antibody-based cancer therapy can be classified into two categories (49).One is the direct action, which can be subcategorized into three modes, (a) blocking function, (b) stimulating function, and (c) targeting function.The other mechanism of antibody therapy is the indirect action by the immune system, including complement-dependent cytotoxicity and antibody-dependent cytotoxicity (49).We confirmed that two mouse monoclonal antibodies raised against the ectodomain of Nectin-4 (clones 19-33 and 66-97) for ELISA had no antitumor activity in vitro when treated Nectin-4-overexressing lung cancer cells only with the antibodies (data not shown).Because Nectin-4 was highly expressed on the surface of lung cancer cells and the ectodomain of Nectin-4 was secreted (Fig. 1A), we need to clarify in the future study the mechanism of carcinogenesis involving the cell surface and/or extracellular Nectin-4.Based on the data, the production of monoclonal antibodies targeting cell surface Nectin-4 that could induce antibody-dependent cytotoxicity/complementdependent cytotoxicity activity and/or block oncogenic receptor signals, or those neutralizing the secreted Nectin-4 would be warranted.
We established our original ELISA system by the two mouse monoclonal antibodies to measure serum levels of Nectin-4 and showed that serum Nectin-4 levels were significantly higher in lung cancer patients than in healthy volunteers.This is a great advantage to develop practical and standardized diagnostic kits in the clinic.Extracellular part of Nectin-4 is reported to be processed by TACE/ADAM-17 and a soluble Nectin-4 is shed from the cell surface and released into the extracellular space (41).Metalloprotease-mediated cleavage of some single-pass type I transmembrane proteins, such as L1CAM released by ADAM10mediated membrane-proximal cleavage, has been shown to stimulate cellular migration and neurite outgrowth (50).The ectodomain shedding of Nectin-4 might exert some signals important for cancer progression.Importantly, serum Nectin-4 levels measured by our ELISA system showed higher sensitivity and specificity (53.7% and 97.7%, respectively) than conventional serum tumor markers for NSCLC (CEA or CYFRA21-1) that are being used in clinical practice (Supplementary Table S1A).Furthermore, serum Nectin-4 in patients with operable stages of NSCLC also showed higher sensitivity (25% in stage I; 50% in stage II-IIIA) than serum CEA (16.7% in stage I; 15% in stage II-IIIA) or CYFRA21-1 (4.2% in stage I; 15% in stage II-IIIA).Although further validation using a larger set of serum samples covering various clinical stages will be required, our data presented here also indicate a potential clinical usefulness of Nectin-4 as a serologic biomarker for NSCLC that could be widely used in clinical practice, such as detection of cancer, prediction of the malignant potential of tumor, and monitoring the disease control condition after any anticancer treatment.Finally, it should be noted that we observed activation of Nectin-4 in more than half of a series of other types of cancers such as bladder and cervical carcinomas we examined (data not shown), suggesting its diagnostic and therapeutic application to a widerange of tumors.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.

Figure 1 .
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.

Figure 3 .
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.

Figure 4 .
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 control siRNAs analyzed by 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.
cigarette per pack) consumed a day multiplied by years].Serum samples were obtained with informed consent from 164 NSCLC patients (123 ADCs and 41 SCCs) admitted to Hiroshima University Hospital, as well as Kanagawa Cancer Center Hospital [122 males and 42 females; median age (F1 SD), 64.5 F 10.4 y; range, 30-85; Table 2].To investigate the prognostic value of serum Nectin-4, two additional sets of serum samples with precise follow-up record after treatments were also obtained.Group 1: serum samples obtained with informed consent before curative surgical resection at Kanagawa Cancer Center Hospital from 95 early stage NSCLC patients [stage I, 48 males and 47 females; median age (F1 SD), 66.8 F 9.6 y; range, 38-84].Group 2: serum samples obtained at diagnosis with informed consent from 62 advanced stage NSCLC patients (stage IIIB-IV) treated later with an identical protocol of the first-line chemotherapy using both carboplatin and paclitaxel admitted to Hiroshima University Hospital [46 males and 16 females; median age (F1 SD), 61.7 F 10.2 y; range, 35-79].Patient samples were selected for the study on the basis of the following criteria: (a) patients were newly diagnosed and previously untreated and (b)