The EphB4 Receptor Tyrosine Kinase Promotes Lung Cancer Growth: A Potential Novel Therapeutic Target

Despite progress in locoregional and systemic therapies, patient survival from lung cancer remains a challenge. Receptor tyrosine kinases are frequently implicated in lung cancer pathogenesis, and some tyrosine kinase inhibition strategies have been effective clinically. The EphB4 receptor tyrosine kinase has recently emerged as a potential target in several other cancers. We sought to systematically study the role of EphB4 in lung cancer. Here, we demonstrate that EphB4 is overexpressed 3-fold in lung tumors compared to paired normal tissues and frequently exhibits gene copy number increases in lung cancer. We also show that overexpression of EphB4 promotes cellular proliferation, colony formation, and motility, while EphB4 inhibition reduces cellular viability in vitro, halts the growth of established tumors in mouse xenograft models when used as a single-target strategy, and causes near-complete regression of established tumors when used in combination with paclitaxel. Taken together, these data suggest an important role for EphB4 as a potential novel therapeutic target in lung cancer. Clinical trials investigating the efficacy of anti-EphB4 therapies as well as combination therapy involving EphB4 inhibition may be warranted.</p

The EphB4 Receptor Tyrosine Kinase Promotes Lung Cancer Growth: A Potential Novel Therapeutic Target

<div><p>Despite progress in locoregional and systemic therapies, patient survival from lung cancer remains a challenge. Receptor tyrosine kinases are frequently implicated in lung cancer pathogenesis, and some tyrosine kinase inhibition strategies have been effective clinically. The EphB4 receptor tyrosine kinase has recently emerged as a potential target in several other cancers. We sought to systematically study the role of EphB4 in lung cancer. Here, we demonstrate that EphB4 is overexpressed 3-fold in lung tumors compared to paired normal tissues and frequently exhibits gene copy number increases in lung cancer. We also show that overexpression of EphB4 promotes cellular proliferation, colony formation, and motility, while EphB4 inhibition reduces cellular viability <i>in vitro</i>, halts the growth of established tumors in mouse xenograft models when used as a single-target strategy, and causes near-complete regression of established tumors when used in combination with paclitaxel. Taken together, these data suggest an important role for EphB4 as a potential novel therapeutic target in lung cancer. Clinical trials investigating the efficacy of anti-EphB4 therapies as well as combination therapy involving EphB4 inhibition may be warranted.</p></div

Tumor remission in mouse lung xenografts in response to EphB4 inhibition alone and in combination.

<p><i>A</i>. A549 cells were injected into flanks of nude mice, allowed to establish primary tumors, and treated with PBS (control), paclitaxel, soluble EphB4 (sEphB4-HSA), or paclitaxel plus soluble EphB4. Error bars indicate standard deviation among six mice per group. <i>B–C</i>. H1993 or H446 cells were injected into flanks of nude mice, allowed to establish primary tumors, and treated with PBS (control) or soluble EphB4 (sEphB4-HSA). Error bars indicate SEM among six mice per group. *, p<0.05; **, p<0.01; ****, p<0.0001.</p

Kaplan-Meier analysis of patient survival.

<p>The ordinate axis in each graph represents the fraction of living patients. <i>A</i>. Survival stratified by EphB4 expression. ACIS scores above and below 500 were chosen for stratification based on the median score of the overall cohort. Arrows represent median survival (50% living patients) in months. <i>B</i>. Survival stratified by race.</p

EphB4 modulation affects cell growth and migration <i>in vitro</i>.

<p><i>A</i>. H661 cells were transfected with either wild-type EphB4 empty-vector constructs, and their effect on cell growth was assessed with or without stimulation with ephrin-B2/Fc. The number of colonies formed in EV-transfected and EphB4-transfected stable clones is shown. Each condition was repeated in six replicates. Error bars indicate SEM. <i>B</i>. Representative colonies as assessed in Panel <i>A</i>. <i>C</i>. Quantification of average colony size of EphB4-harboring H661 cells versus control cells. Error bars indicate SEM. <i>D</i>. Cell proliferation with and without ephrin-B2 stimulation is shown. Data are represented as mean percent change relative to the initial time point among three replicates. Error bars indicate SEM normalized to percent proliferation. ***, p<0.001; ****, p<0.0001 relative to H661-EV. <i>E.</i> H446 and H526 cells were stimulated with ephrin-B2 ligand and assessed for cell viability. *, p<0.01; ns, not significant. <i>F</i>. Representative images of wounds created in confluent layers of cells in culture. <i>G</i>. Quantification of wound closure. Data are represented as average percent wound closure compared to the initial wound size among six independent replicates. Error bars indicate SEM normalized to percent closure. **, p<0.01; ***, p<0.001; ****, p<0.0001 relative to H661-EV.</p

Expression of tumor-associated biomarkers in NSCLC tumor xenografts treated with sEphB4-HSA and paclitaxel <i>in vivo</i>.

<p><i>A</i>. Ki-67 staining, TUNEL staining, phosphorylated S6 expression, and CD31 staining are demonstrated by immunofluorescence. DAPI was used as a nuclear counterstain. <i>B</i>. Quantification of fluorescence intensity normalized to DAPI. Values atop each bar represent percent intensity relative to the corresponding DAPI image. sEphB4-HSA, soluble EphB4; pS6, phosphorylated S6. <i>C</i>. Phosphorylated Akt and phosphorylated Src expression are demonstrated by immunohistochemistry.</p

EphB4 protein knockdown reduces SCLC cell viability.

<p><i>A.</i> EphB4 protein knockdown following siRNA transfection. GAPDH was used as a protein loading control. Scr, scrambled siRNA. <i>B.</i> Viability of cells following EphB4 knockdown and/or treatment with SN-38. Error bars indicate SD. **, p<0.01; ***, p<0.001. <i>C.</i> Representative images of topoisomerase I relaxation assays. Cells were left unstimulated (media) or stimulated for 15 minutes with HGF or ephrin-B2, and nuclear lysates were harvested for use in topoisomerase I relaxation assays. Nuclear lysates were assayed undiluted (neat) or serially diluted as indicated. R, relaxed conformations of plasmid DNA; SC, supercoiled conformation of plasmid DNA. <i>D.</i> The relative integrated density of the SC band was quantified and normalized to media for each condition. Data shown are representative of two independent experiments.</p