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. 2009 May 15;15(10):3484-94.
doi: 10.1158/1078-0432.CCR-08-2904. Epub 2009 May 15.

Combined vascular endothelial growth factor receptor and epidermal growth factor receptor (EGFR) blockade inhibits tumor growth in xenograft models of EGFR inhibitor resistance

George N Naumov  1 , Monique B Nilsson, Tina Cascone, Alexandra Briggs, Oddbjorn Straume, Lars A Akslen, Eugene Lifshits, Lauren Averett Byers, Li Xu, Hua-Kang Wu, Pasi Jänne, Susumu Kobayashi, Balazs Halmos, Daniel Tenen, Xi M Tang, Jeffrey Engelman, Beow Yeap, Judah Folkman, Bruce E Johnson, John V Heymach
Affiliations

Combined vascular endothelial growth factor receptor and epidermal growth factor receptor (EGFR) blockade inhibits tumor growth in xenograft models of EGFR inhibitor resistance

George N Naumov et al. Clin Cancer Res. .

Abstract

Purpose: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) gefitinib and erlotinib benefit some non-small cell lung cancer (NSCLC) patients, but most do not respond (primary resistance) and those who initially respond eventually progress (acquired resistance). EGFR TKI resistance is not completely understood and has been associated with certain EGFR and K-RAS mutations and MET amplification.

Experimental design: We hypothesized that dual inhibition of the vascular endothelial growth factor (VEGF) and EGFR pathways may overcome primary and acquired resistance. We investigated the VEGF receptor/EGFR TKI vandetanib, and the combination of bevacizumab and erlotinib in vivo using xenograft models of EGFR TKI sensitivity, primary resistance, and three models of acquired resistance, including models with mutated K-RAS and secondary EGFR T790M mutation.

Results: Vandetanib, gefitinib, and erlotinib had similar profiles of in vitro activity and caused sustained tumor regressions in vivo in the sensitive HCC827 model. In all four resistant models, vandetanib and bevacizumab/erlotinib were significantly more effective than erlotinib or gefitinib alone. Erlotinib resistance was associated with a rise in both host and tumor-derived VEGF but not EGFR secondary mutations in the KRAS mutant-bearing A549 xenografts. Dual inhibition reduced tumor endothelial proliferation compared with VEGF or EGFR blockade alone, suggesting that the enhanced activity of dual inhibition is due at least in part to antiendothelial effects.

Conclusion: These studies suggest that erlotinib resistance may be associated with a rise in both tumor cell and host stromal VEGF and that combined blockade of the VEGFR and EGFR pathways can abrogate primary or acquired resistance to EGFR TKIs. This approach merits further evaluation in NSCLC patients.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest: J. V. Heymach, commercial reserch grant, AstraZeneca; consultant, AstraZeneca, Genentech.

Figures

Fig. 1
Fig. 1
Vandetanib inhibits EGFR in a manner similar to gefitinib and erlotinib. NIH3T3 cells were transfected to express EGFR bearing exon 19 deletions (A) or L858R mutations (B) and treated with EGF and increasing concentrations of vandetanib or gefitinib. Expression of activated EGFR was evaluated by Western analysis. C, inhibition of EGFR activation in HCC827, A549, and H1975 NSCLC cell lines by erlotinib or vandetanib was evaluated by multiplex bead assay.
Fig. 2
Fig. 2
Effect of EGFR/VEGFR inhibitors on tumor cell viability in vitro. MTT assay was done to evaluate the effect of gefitinib (A), erlotinib (B), vandetanib (C), and bevacizumab (D) on the growth of NSCLC cell lines. These four compounds were tested on EGFR wild-type cell lines (A549 and Calu-6), cells with EGFR-activating mutations (HCC827, H3255), and H1975 cells that express both the EGFR-activating mutation and the T790M resistance mutation. Cells were treated with gefitinib, erlotinib, vandetanib (0.001-10 nmol/L), or bevacizumab (0.001-10 μg/mL) for 72 h. The percentage of viable cells is shown relative to that of untreated control. Results are shown as mean values with SD based on results from at least three replicate wells.
Fig. 3
Fig. 3
EGFR TKI sensitive xenograft model of NSCLC. A, HCC827 xenografts (wild-type K-RAS, EGFR exon 19 del.; n = 6-7 mice per group) were treated with either gefitinib (blue squares), erlotinib (green diamonds), or vandetanib (red triangles). All mice bearing HCC827 tumors (∼ 400 mm3) responded to gefitinib, erlotinib, and vandetanib treatment within 5 d, and tumors remained at a microscopic size for >60 d of continuous treatment. In contrast, tumor growth progressed in all vehicle-treated mice. B, Vandetanib effectively reduced even large (∼1,200 mm3) HCC827 tumors to a microscopic size within ∼10 d. C, HCC827 tumor xenografts from mice treated with vehicle, gefitinib, or vandetanib for 12, 24, or 72 h were isolated and protein lysates were evaluated for EGFR phosphorylation and downstream signaling by Western blot. D, HCC827 cells were transfected to express copies of mutated EGFR bearing the T790M resistance mutation, injected into nude mice and treated with either vehicle (black circles), erlotinib (blue squares), gefitinib (green diamonds), or vandetanib (red triangles). Vandetanib treatment effectively inhibited tumor growth by day 100, followed by gradual resistance to treatment by day 230. Results are shown as mean tumor volumes and SE at each time point.
Fig. 4
Fig. 4
Xenograft model of NSCLC primary resistance. A, animals bearing A549 (K-RAS mutant, EGFR wild-type) xenografts were treated with erlotinib (green diamonds), bevacizumab (pink upside-down triangles), erlotinib and bevacizumab combination (gray squares), and vandetanib (red triangles). A549 tumors responded moderately to EGFR TKI (erlotinib) and VEGF inhibition (bevacizumab) single-agent treatments. However, combined targeting of EGFR and VEGFR pathways using erlotinib and bevacizumab combination treatment or vandetanib inhibited tumor growth better than erlotinib or bevacizumab monotherapies. Erlotinib-treated mice were allowed to progress (i.e. to become erlotinib-resistant) to a mean tumor volume of ∼400 mm3 and were randomized into four new treatment groups: control (black circles), erlotinib (green diamonds), erlotinib/bevacizumab combination group (gray squares), and vandetanib (red triangles). Erlotinib-treated and control tumors continued growth with similar kinetics after treatment crossover. However, dual inhibition of EGFR and VEGFR (erlotinib/bevacizumab combination and vandetanib treatments) resulted in stable disease where tumors remained at ∼400 mm3 for approximately 40 d. Results are shown as mean tumor volumes and SE at each time-point. B, representative immunohistochemical images of VEGF staining in A549 vehicle-treated and erlotinib-resistant tumors. C and D, expression of human (C) and mouse (D) VEGF RNA in A549 tumors treated with vehicle or with erlotinib for 2 wk or after tumors became resistant to therapy. Data are graphed as the mean ± SD. * P < 0.05 (E) VEGF is elevated in the plasma of mice with tumors resistant to erlotinib. Data are graphed as the mean ± SE.
Fig. 5
Fig. 5
Xenograft model of NSCLC secondary resistance. H1975 NSCLC cells bearing an EGFR-activating mutation as well as the T790M mutation were injected into nude mice. Established tumors were treated with gefitinib (blue squares), erlotinib (green diamonds), vandetanib (red triangles), or bevacizumab as a single agent (pink upside-down triangles) or in combination with erlotinib (gray squares). Results are shown as mean tumor volumes and SE at each time point.
Fig. 6
Fig. 6
Differential expression of EGFR and VEGF in A549 and H1975 xenografts. Expression of phosphorylated EGFR on A549 and H1975 tumor cells and tumor-associated endothelial cells was evaluated by double-staining. In A549 tumors colocalization of pEGFR and CD34 was frequently observed (A). However, in H1975 tumors endothelium was weak or negative for pEGFR staining (B). Panel B also shows a representative vital pEGFR-positive zone comprising tumor cells (red) supported by CD34 positive (blue) vessels. To evaluate endothelial cell proliferation, A549 tumors were immunostained with antibodies directed against Ki67 (blue, mouse specific) and von Willebrand factor (red). Peritumoral vessels (C) and intratumoral vessels (D) showed positive endothelial cell nuclei, indicating active angiogenesis. E, nearly twice as much pEGFR was detected on endothelial cells within A549 tumors as compared with H1975 tumors. pEGFR staining in tumor cells did not differ significantly between the two cell types. Results are shown as mean ± SD. F, Ki67 and von Willebrand factor staining was quantified to evaluate endothelial cell proliferation within A549 tumors. Dual inhibition of EGFR/VEGFR pathways dramatically reduced the number of proliferating endothelial cells. Data are graphed as mean ± SD. G, in vitro VEGF secretion by A549 and H1975 cells was evaluated by ELISA assay and graphed as mean ± SD.
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