Identification of the Sixth Ig-like Domain of VCAM-1 as a Novel Therapeutic Target in Lung Cancer Cell Invasion

Vascular cell adhesion molecule-1 (VCAM-1) is closely associated with tumor progression and metastasis. However, the relevance and role of VCAM-1 in lung cancer have not been clearly elucidated. In this study, we found that VCAM-1 was highly overexpressed in lung cancer tissue compared with that of normal lung, and high VCAM-1 expression correlated with poor survival of lung cancer patients. VCAM-1 knockdown reduced invasion in A549 human lung cancer cells, and competitive blocking experiments targeting the Ig-like domain 6 of VCAM-1 (VCAM-1-D6) demonstrated that the VCAM-1-D6 domain was critical for VCAM-1-mediated A549 cell invasion. Next, we developed a human monoclonal antibody specific to human and mouse VCAM-1-D6 (VCAM-1-D6 huMab), which was isolated from a human synthetic antibody library using phage display technology. Finally, we showed that VCAM-1-D6 huMab had a nanomolar affinity for VCAM-1-D6 and that it potently suppressed invasion in A549 and NCI-H1299 lung cancer cell lines. Taken together, these results suggest that VCAM-1-D6 is a novel therapeutic target in VCAM-1mediated lung cancer invasion and that our newly developed VCAM-1-D6 huMab will be a useful tool for inhibiting VCAM-1-expressing lung cancer cell invasion.


Introduction
Lung cancer is one of the more common types of cancer, and it is the leading cause of cancer death among men and the second leading cause of cancer death among women worldwide [1].Conventionally, patients with lung cancer are treated by surgical resection, platinum-based chemotherapy, and radiation therapy, alone or in combination [2].Moreover, recently, epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) have been determined to be the most effective molecules for targeted therapy in lung cancer.EGFR mutations and ALK gene rearrangements are successfully targeted with specific tyrosine kinase inhibitors, including erlotinib, gefitinib, and crizotinib [3,4].Furthermore, bevacizumab, a humanized anti-vascular endothelial growth factor (VEGF) antibody, is being used in clinics to treat lung cancer [5].However, despite the current availability of therapeutic regimens, the major obstacle to overcome in lung cancer treatment is lung cancer cell invasion and metastasis.During these processes, malignant tumors cells first intravasate into blood vessels and then extravasate into new tissues, where they can proliferate and produce a metastatic secondary tumor [6,7].Therefore, the identification of novel therapeutic targets in lung cancer cell invasion is critical for developing more effective therapeutic options for treating lung cancer patients.
Vascular cell adhesion molecule-1 (VCAM-1) is a 90-kDa glycoprotein that is predominantly expressed on activated endothelial cells in response to pro-inflammatory cytokines, including human tumor necrosis factor alpha (hTNFα) [8,9].VCAM-1 is a type I transmembrane protein that consists of an extracellular domain, with seven homologous immunoglobulin (Ig)-like domains, a transmembrane domain, and a cytosolic domain [10].During an inflammatory response, α4β1 integrin-expressing leukocytes adhere to VCAM-1-expressing endothelial cells, which promote their transmigration across the activated endothelium [11].Previously, we showed that the sixth Ig-like domain of VCAM-1 (VCAM-1-D6) is important for mediating this leukocyte transmigration, implying that it may play a role in VCAM-1-mediated inflammation [12].
Recently, some groups have also suggested roles for VCAM-1 in tumor progression and metastasis.VCAM-1 is overexpressed in several types of cancers, including renal, gastric, pancreatic, breast, and ovarian cancers [13][14][15][16][17][18].Moreover, VCAM-1 expression in breast cancer cells enhances their metastasis to the lungs by allowing them to interact with leukocytes that express α4 integrin counter-receptors [15].VCAM-1 expression is also associated with oncogenesis, tumor angiogenesis, and metastasis in gastric carcinoma [16].However, the role of VCAM-1 and its target domain for antibody therapy in lung cancer cell invasion have not yet been clearly identified.
In the present study, we showed that VCAM-1 expression is increased in lung cancer tissue compared with that of normal lung tissue, and that high VCAM-1 expression is associated with reduced survival of lung cancer patients.Moreover, siRNA-mediated VCAM-1 knockdown and competitive inhibition experiment using recombinant VCAM-1-D6 protein demonstrated that VCAM-1 is required for lung cancer cell invasion and that the VCAM-1-D6 domain of VCAM-1 is a key domain for regulating lung cancer cell invasion.Finally, by developing of a VCAM-1-blocking monoclonal antibody specific to VCAM-1-D6, we found that the antibody specifically inhibited the lung cancer cell invasion.In summary, this study provides proof-of-concept evidences showing a role for VCAM-1-D6 as a novel potential therapeutic target in lung cancer cell invasion.Furthermore, the antibody-based targeting of VCAM-1-D6 is an effective strategy for inhibiting the invasion of VCAM-1-expressing lung cancer cells.

VCAM-1 expression is increased in lung cancer and is associated with reduced survival
To investigate VCAM-1 expression in normal lung and lung cancer patient tissue, we performed immunohistochemistry with a commercially available anti-VCAM-1 antibody.VCAM-1 expression was higher in lung cancer (n=9) compared with that in normal lung (n=10) tissue (Figures 1a and 1b).To further examine VCAM-1 expression in normal lung and lung cancer patient tissue, we analyzed lung cancer patient gene expression profiling data (GSE31210) obtained from the National Cancer for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database.VCAM-1 expression was significantly increased in lung cancer tissue (n=226) compared with that in normal lung tissue (n=20) (P=0.00015, Figure 1c).Taken together, these expression data suggest that increased levels of VCAM-1 play a role in lung cancer.
To elucidate the relationship between VCAM-1 expression and survival of lung cancer patients, we performed bioinformatics-based survival analysis.The 5-year overall survival rate was 91% in the low-VCAM-1-expression group (n=102), whereas this rate was significantly reduced to 80% in the high-VCAM-1-expression group (n=102) (P=0.0493, Figure 1d).These data demonstrate an association between high VCAM-1 expression and poor survival and further suggest that increased VCAM-1 expression is closely associated with lung cancer.

The VCAM-1-D6 domain of VCAM-1 plays a key role in regulating A549 lung cancer cell invasion
To examine the role of VCAM-1 in lung cancer cell invasion, we performed siRNA-mediated knockdown of VCAM-1 in the human lung cancer cell line A549, which was cultured in the presence of hTNFα to induce VCAM-1.First, we confirmed the reduced levels of VCAM-1 in VCAM-1 siRNA-, compared with scrambled siRNA-, transfected cells using immunoblot analysis (Figure 2a).Importantly, VCAM-1 knockdown significantly decreased A549 cell invasion compared with that of control knockdown cells (P<0.001,Figures 2b and 2c).Collectively, these results suggest that VCAM-1 regulates lung cancer cell invasion.
To investigate the functional role of VCAM-1-D6 in lung cancer cell invasion, we prepared an Fc-fusion protein of VCAM-1-D6 (VCAM-1-D6-Fc) and performed a competitive blocking experiment by treating A549 cells, grown in the presence of hTNFα with VCAM-1-D6-Fc, or Fc alone as a negative control.Importantly, A549 cell invasion was inhibited in a dose-dependent manner by the addition of VCAM-1-D6-Fc, but not by Fc, compared with that of mock-treated cells (Figures 2d  and 2e), suggesting that the VCAM-1-D6 domain is crucial for VCAM-1-mediated lung cancer cell invasion.

Development and characterization of a human monoclonal antibody specific to VCAM-1-D6 of human and mouse VCAM-1
To isolate human monoclonal antibodies to human and mouse VCAM-1-D6, a human synthetic single-chain variable fragment (scFv) library was pre-cleared to remove Fc binders.Phage ELISAs confirmed that approximately 90% of the scFvs binding to Fc were removed (data not shown).The library was alternately biopanned with human (hVCAM-1-D6-Fc) or mouse (mVCAM-1-D6-Fc) VCAM-1-D6 fusion proteins using VCAM-1-D6-Fc-coated magnetic beads.Ninety-six phage clones were randomly selected, rescued by helper phage infection, and tested for reactivity to human and mouse VCAM-1-D6 using phage enzyme immunoassays.Following DNA sequencing, two clones, recognizing both human and mouse VCAM-1-D6, with different complementarity determining region sequences were selected and converted to IgGs.They were expressed in human embryonic kidney (HEK) 293F cells and purified.SDS-PAGE and Coomassie staining showed that all IgG clones were greater than 90% pure (data not shown).

VCAM-1-D6 huMab specifically inhibits the invasion of VCAM-1-expressing lung cancer cells
Prior to performing functional assays, we verified VCAM-1 expression in the human lung cancer cell lines A549 and NCI-H1299 that were cultured in the absence or presence of hTNFα.Immunoblot analysis showed that VCAM-1 expression was dramatically increased in the A549 and NCI-H1299 cells in response to hTNFα (Figures 4a and 4d).Next, to investigate the effect of VCAM-1-D6 huMab on lung cancer cell invasion, we treated these cells cultured in the absence or presence of hTNFα with VCAM-1-D6 huMab, or IgG as a negative control, and then, we performed invasion assays.The invasion of A549 (Figures 4b and 4c) and NCI-H1299 cells (Figures 4e and 4f) was specifically inhibited by VCAM-1-D6 huMab, but not by the IgG control antibody.To further confirm the specific inhibitory effect of VCAM-1-D6 huMab on lung cancer cell invasion, we treated A549 cells, cultured in the absence or presence of hTNFα with VCAM  -1-D6 huMab or a commercially available VCAM-1-D1 blocking antibody, and then, we performed invasion assays.A549 cell invasion was more potently inhibited by the VCAM-1-D6 huMab than by the VCAM-1-D1 blocking antibody (Figures 5a and 5b).These data further demonstrated the specific role of the VCAM-1-D6 domain in regulating VCAM-1-mediated lung cancer cell invasion, and suggest that the VCAM-1-D6 huMab can be used to suppress the invasion of VCAM-1-expressing lung cancer cells in vivo.

Discussion
Lung cancer is one of the leading causes of cancer death worldwide [1].Despite the remarkable advances in lung cancer therapeutics, lung cancer cell invasion and metastasis remain the major obstacles to overcome to improve patient survival.Thus, increasing attention is focused on identifying novel molecules that are differentially expressed and/or play a key role in lung cancer cell invasion.Although the roles of VCAM-1 have been reported in several types of cancer [13][14][15][16][17][18], the roles of VCAM-1 in lung cancer have not yet been elucidated.More specifically, the role of the VCAM-1-D6 domain and its potential as a therapeutic target in lung cancer have not been explored.
In the present study, we propose, for the first time, that the VCAM-1-D6 domain may be used as a novel therapeutic target in VCAM-1-mediated lung cancer cell invasion.Several lines of evidence support our proposal.First, compared with normal lungs, VCAM-1 was highly overexpressed at the mRNA and protein level in lung cancer tissue.Second, high VCAM-1 expression correlated with poor survival of lung cancer patients, implying that VCAM-1 has a key role in lung cancer progression.Third, siRNA-mediated knockdown of VCAM-1 in hTNFα-treated A549 human lung cancer cells greatly reduced cell invasion, suggesting VCAM-1 is critical for this process.Fourth, using competitive inhibition assays with recombinant VCAM-1-D6, we identified VCAM-1-D6 as a key domain regulating the A549 cell invasion.Fifth, by developing a VCAM-1-D6 blocking antibody, VCAM-1-D6 huMab, we demonstrated that antibody-based inhibition of VCAM-1-D6 effectively reduced VCAM-1-mediated invasion of A549 and NCI-H1299 lung cancer cell lines.Sixth, we confirmed that the A549 cell invasion was more potently inhibited by the VCAM-1-D6 huMab than by a blocking antibody to the VCAM-1-D1 domain (51-10C9), demonstrating the specificity of the role of VCAM-1-D6 in lung cancer cell invasion.Our study focused on elucidating the role of the VCAM-1-D6 domain in VCAM-1-mediated lung cancer cell invasion using domain-specific recombinant protein and antibodies; however, we cannot exclude the possibility that other domains of VCAM-1 and other associated regulatory molecules including metalloproteinases also contribute to this process.
In this study, we generated a human antibody to VCAM-1-D6, which is the functionally critical domain for VCAM-1-mediated lung cancer cell invasion.Conventional antibody screening uses the whole extracellular domain of a protein as a target antigen; thus, most selected antibodies bind to the target protein but are not necessarily functional.Therefore, the identification of functional domains would drastically facilitate the time-consuming and labor-intensive functional antibody screening process.Here, we demonstrated a more effective strategy to develop a functional antibody by first identifying VCAM-1-D6 as a key domain regulating VCAM-1-mediated lung cancer cell invasion and subsequently isolating a VCAM-1-D6-specific antibody from a human synthetic antibody library.The antibody isolated using this strategy, VCAM-1-D6 huMab, efficiently inhibited VCAM-1-mediated lung cancer cell invasion.Moreover, VCAM-1-D6 huMab is a human antibody, which makes it is likely to be less immunogenic in vivo, and it has a nanomolar affinity.Both of these qualities may make VCAM-1-D6 huMab an attractive candidate therapeutic for potential use in combination with existing chemotherapies to suppress the invasion of VCAM-1-expressing lung cancer cells in human lung cancer patients.
VCAM-1 regulates many distinct cell processes by specifically interacting with various proteins, including α4β1 integrin, CD44, moesin, ezrin, and secreted protein acidic and rich in cysteine (SPARC), depending on the spatiotemporal environment of a particular cell [23][24][25][26].Here, we showed that lung cancer cell invasion was specifically and prominently inhibited by VCAM-1-D6 huMab but not by a VCAM-1-D1 blocking antibody.These data strongly suggest that the VCAM-1-D1 domain, which interacts with α4β1 integrin expressed on leukocytes to regulate leukocyte recruitment during the initiation and progression of inflammation [8,24], does not play a role in VCAM-1-mediated lung cancer cell invasion.Moreover, these data imply that the VCAM-1-D6 domain is required for the interaction of VCAM-1 with specific cellular binding proteins that regulate VCAM-1-mediated lung cancer cell invasion.Furthermore, VCAM-1-D6 huMab likely acts as a VCAM-1-D6-blocking antibody by interrupting the molecular interactions between VCAM-1-D6 and its binding partners, resulting in inhibition of lung cancer cell invasion.Thus, VCAM-1-D6 huMab may be used as a valuable research tool for identifying the interacting partners of VCAM-1 via the VCAM-1-D6 domain, and for elucidating the detailed molecular mechanisms of VCAM-1-mediated lung cancer cell invasion.In summary, this study provides proof-of-concept evidence of the utility of developing a domain-specific antibody, following the identification of a key functional protein domain, for antibody-based targeting in cancer therapy.Here, we identified VCAM-1-D6 as a novel potential therapeutic target in lung cancer cell invasion, and we demonstrated the efficacy of domain-specific, antibody-based targeting of VCAM-1-D6 to inhibit the invasion of VCAM-1-expressing human lung cancer cells.Based on our data, we propose a mode of action whereby VCAM-1-D6 huMab binds to VCAM-1 expressed on the surface of lung cancer cells and specifically blocks the interaction between VCAM-1-D6 and its binding proteins, which is necessary for lung cancer cell invasion (Figure 6).In future studies, we will investigate the precise mechanism of the VCAM-1-D6 domain in VCAM-1mediated cell invasion and evaluate the in vivo efficacy of VCAM-1-D6 huMab in lung cancer animal models.CA, USA) at 4°C overnight, and then with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5,000; Cell Signaling Technology, Danvers, MA, USA) or horse anti-mouse IgG (1:5,000; Cell Signaling Technology).Following several washes with TBST, protein bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA) according to the manufacturer's instructions.

Construction and preparation of Fc fusion proteins
The DNA sequences encoding human VCAM-1-D6 (amino acids 511-595) and mouse VCAM-1-D6 (amino acids 511-595) were amplified by polymerase chain reaction (PCR).The PCR products were subcloned using two asymmetric SfiI sites into a modified pCEP4 mammalian expression vector (Invitrogen, Carlsbad, CA, USA) that contains the hinge and CH2-CH3 domains of human IgG1 at the 3' end of the cloning site.The constructs were verified by DNA sequencing.The Fc fusion protein constructs were transfected into Expi293F™ cells using the ExpiFectamine™ Transfection Kit (Gibco).After 7 days, the culture supernatants were collected, and the fusion proteins were purified using affinity chromatography with Protein A-Sepharose beads (GenScript, Piscataway, NJ, USA).After quantifying the protein concentrations using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and performing dialysis in phosphate-buffered saline (PBS), the sample purity was analyzed by SDS-PAGE and Coomassie brilliant blue staining.The final pooled fractions were aliquoted and stored at -80°C.

Selection of human antibodies specific to VCAM-1-D6 using phage display technology
Biopanning to select human antibodies specific to VCAM-1-D6 from a synthetic human scFv antibody library was performed as described previously [21].Briefly, following pre-clearing of Fc binders, 1 × 10 7 magnetic beads (Dynabeads M-270 epoxy; Invitrogen) were coated with 4 µg recombinant human or mouse VCAM-1-D6-Fc for six rounds of biopanning.After the final round, 96 individual phage clones that displayed scFv were randomly selected from colonies grown on output plates.They were tested for their reactivity to human or mouse VCAM-1-D6-Fc using a phage enzyme immunoassay.The final enzyme-linked immunosorbent assay (ELISA)-positive scFv clones were analyzed by DNA sequencing, and two unique scFv clones with different complementarity determining region sequences were identified.

Preparation of VCAM-1-D6 huMabs
The variable heavy chain (VH) and variable light chain (VL) genes of the selected scFv clones (clone 1 and 2) were amplified using PCR.The VH genes were engineered with a 5' EcoRI site and a 3' ApaI site, and the VL with a 5' HindIII site and a 3' BsiWI site.PCR fragments were digested with the appropriate restriction enzymes (New England BioLabs, Beverly, MA, USA) and cloned into the bicistronic mammalian expression vector pcDNA3.1 (Invitrogen), which encodes the CH1/hinge/CH2/CH3 domains of human IgG1 in the VH cloning site and the constant kappa chain in the VL cloning site.The VCAM-1-D6 huMabs were expressed and purified as described previously [21].

Transwell invasion assays
Cell invasion assays were performed as described previously with minor modifications [20].Briefly, 1.5 × 10 5 A549 or NCI-H1299 cells were plated in 6-well plates, allowed to attach overnight, and treated with 20 ng/ml hTNFα (R&D systems, Minneapolis, MN, USA) for 24 h at 37°C.For all cell invasion assays, 5 × 10 4 A549 or NCI-H1299 cells were loaded onto the upper part of the transwell insert pre-coated with 100 µl Matrigel (Corning, Tewksbury, MA, USA), and the lower chamber was filled with complete medium.To investigate the effects of VCAM-1 knockdown on cell invasion, transwell invasion assays were performed using scrambled siRNA-and VCAM-1 siRNA-transfected A549 cells.To verify the effects of the Fc fusion proteins on cell invasion, transwell invasion assays were performed in the presence of 0, 10, 20, or 40 µg/ml VCAM-1-D6-Fc fusion protein, or 40 µg/ml

Figure 1 .
Figure 1.Expression and survival analyses of VCAM-1 in lung cancer patient samples.(a) Representative images are shown of the immnohistochemical examination of VCAM-1 in normal lung (n=10) and lung cancer (n=9) tissues using a commercially available anti-VCAM-1 antibody (scale bar=200 µM); (b) A dot plot shows quantification of the immunohistochemical analyses of VCAM-1.Each dot represents the average of VCAM-1 expression levels quantified from three independent areas/sample.The values represent the mean ± SEM of the VCAM-1 expression calculated from each sample (*P<0.05);(c) A box plot illustrates the results from VCAM-1 expression analyses that was performed with lung cancer patient gene expression profiling data obtained from the NCBI Gene Expression Omnibus database; (d) A Kaplan-Meier plot shows the overall survival of lung cancer patients classified by their VCAM-1 expression level (red: high expression group, n=102; blue: low expression group, n=102).

Figure 2 .
Figure 2. Modulation of VCAM-1 levels and function affect A549 lung cancer cell invasion.(a) Representative immunoblots show the expression of VCAM-1 from scrambled siRNA-or VCAM-1 siRNA-transfected A549 cells cultured in the presence of hTNFα; (b) Representative images depict the invasion of the scrambled siRNA-or VCAM-1 siRNA-transfected A549 cells cultured in the presence of hTNFα (200 × magnification, scale bar=200 µM); (c) Invading cells were quantified in the scrambled siRNA-or VCAM-1 siRNA-transfected A549 cell groups and expressed as a percentage of the control values.All values represent the mean ± SEM of triplicate measurements from one of three independent experiments (***P<0.001);(d) Representative images illustrate the invasion of VCAM-1-D6-Fc-or Fc-treated A549 cells cultured in the presence of hTNFα (200 × magnification, scale bar=200 µM); (e) The numbers of invading cells in the VCAM-1-D6-Fc-or Fc-treated A549 cell groups were counted and expressed as a percentage of the control values.All values represent the mean ± SEM of triplicate measurements from one of three independent experiments (**P<0.01,***P<0.001).

Figure 4 .
Figure 4. Effects of VCAM-1-D6 huMab on A549 and NCI-H1299 lung cancer cell invasion.Representative immunoblots show the expression of VCAM-1 in A549 (a) and NCI-H1299 (d) lung cancer cells in the absence or presence of hTNFα; Representative images highlight the effects of VCAM-1-D6 huMab treatment on A549 (b) and NCI-H1299 (e) cell invasion, in the absence or presence of hTNFα, compared with those of control IgG treatment (200 × magnification, scale bar=200 µM); The numbers of A549 (c) and NCI-H1299 (f) invading cells in the presence of control IgG or VCAM-1-D6 huMab are expressed as a percentage of the mock control values.The values represent the mean ± SEM of triplicate measurements from one of three independent experiments (***P<0.001).

PreprintsFigure 5 .
Figure 5.The specificity of the VCAM-1-D6 huMab on A549 lung cancer cell invasion.(a) Representative images highlight the specific effects of VCAM-1-D6 huMab on A549 cell invasion, in the absence or presence of hTNFα, compared with those of a VCAM-1-D1 blocking antibody or control IgG (200 × magnification, scale bar=200 µM); (b) The numbers of invading cells in the presence of control IgG, VCAM-1-D1 blocking antibody, and VCAM-1-D6 huMab are expressed as a percentage of the mock control values.The values represent the mean ± SEM of triplicate measurements from one of two independent experiments (***P<0.001).

Figure 6 .
Figure 6.Schematic representation of the inhibitory mechanism of VCAM-1-D6 huMab in lung cancer cell invasion.(a) A schematic diagram illustrates the primary structure of VCAM-1, which is comprised of an extracellular domain with seven Ig-like domains (D1~D7), a transmembrane domain, and a cytosolic domain; (b) A proposed model of the inhibitory mechanism of VCAM-1-D6 huMab on lung cancer cell invasion is shown.Under stressful conditions, such as the abrupt elevation of TNFαlevels within the tumor microenvironment, VCAM-1 levels are dramatically increased on the surface of lung cancer cells.The VCAM-1-D6 domain of VCAM-1 interacts with an unknown binding protein, which promotes lung cancer cell invasion; however, the addition of VCAM-1-D6 huMab specifically blocks this interaction and inhibits VCAM-1-mediated lung cancer cell invasion.