KDR Amplification Is Associated with VEGF-Induced Activation of the mTOR and Invasion Pathways but does not Predict Clinical Benefit to the VEGFR TKI Vandetanib

Abstract

Purpose: VEGF pathway inhibitors have been investigated as therapeutic agents in the treatment of non-small cell lung cancer (NSCLC) because of its central role in angiogenesis. These agents have improved survival in patients with advanced NSCLC, but the effects have been modest. Although VEGFR2/KDRis typically localized to the vasculature, amplification ofKDRhas reported to occur in 9% to 30% of the DNA from different lung cancers. We investigated the signaling pathways activated downstream ofKDRand whetherKDRamplification is associated with benefit in patients with NSCLC treated with the VEGFR inhibitor vandetanib.

Methods: NSCLC cell lines with or withoutKDRamplification were studied for the effects of VEGFR tyrosine kinase inhibitors (TKI) on cell viability and migration. Archival tumor samples collected from patients with platinum-refractory NSCLC in the phase III ZODIAC study of vandetanib plus docetaxel or placebo plus docetaxel (N= 294) were screened forKDRamplification by FISH.

Results: KDRamplification was associated with VEGF-induced activation of mTOR, p38, and invasiveness in NSCLC cell lines. However, VEGFR TKIs did not inhibit proliferation of NSCLC cell lines withKDRamplification. VEGFR inhibition decreased cell motility as well as expression of HIF1α inKDR-amplified NSCLC cells. In the ZODIAC study,KDRamplification was observed in 15% of patients and was not associated with improved progression-free survival, overall survival, or objective response rate for the vandetanib arm.

Conclusions: Preclinical studies suggestKDRactivates invasion but not survival pathways inKDR-amplified NSCLC models. Patients with NSCLC whose tumor hadKDRamplification were not associated with clinical benefit for vandetanib in combination with docetaxel.

Figure 1

VEGFR-2 signaling pathways in NSCLC cell lines with KDR CNGs. A, NSCLC cell lines with KDR CNG express elevated levels of VEGFR-2 compared with NSCLC cell lines without KDR CNG as determined by Western blot analysis. B, RPPA analysis shows increased expression of p38, mTOR, and p70s6K in NSCLC cell lines with KDR CNG compared to cell lines without KDR CNG. C, stimulation of H23 cells (KDR amplified) with VEGF (50 ng/mL) for 15 minutes results in activation of p38 and p70S6K. This effect is inhibited with VEGFR TKIs, sorafenib (SORA), vandetanib (VAN), or axitinib (AXIT). VEGF did not induce Akt activation in H23 cells. A431 cells treated with EGF serve as a positive control for activated Akt. D, treatment of A549 cells (KDR normal) with VEGF (50 ng/mL for 15 minutes) resulted in activation of Akt but not p38.

Figure 2

Direct effects of VEGFR TKIs on NSCLC cells with or without KDR CNG. A, the effect of VEGFR TKIs (sorafenib and cediranib) on viability of NSCLC cell lines with KDR CNG (copy number ≥4) or without KDR CNG (copy number = 2) was evaluated. There was no significant difference in IC₅₀ values between cell lines with or without KDR CNG. B, VEGFR TKIs inhibit the migration of NSCLC cells with KDR CNGs. H23 (KDR amplified), Calu-1 (KDR amplified), and A549 (KDR normal) NSCLC cells were plated in Boyden chambers in media containing 1 µmol/L cediranib (a pan-VEGFR inhibitor), sunitinib (TKI targeting VEGFR, PDGFR, and KIT), or imatinib (TKI targeting KIT, PDGFR, but not VEGFR) for 24 hours. Number of migrating cells was counted under 100× magnification. Data are graphed as mean ± SE; *, P < 0.05. C, the VEGFR TKI nintedanib inhibits the migration of H23 (KDR amplified) but not A549 (KDR normal) cells.

Figure 3

VEGFR TKIs modulate HIF1α and p-Met levels in NSCLC cells with KDR CNGs. A, H23 (KDR amplified) and A549 (KDR normal) NSCLC cells were treated with 1 µmol/L sunitinib (TKI targeting VEGFR, PDGFR, and KIT) or imatinib (TKI targeting KIT, PDGFR, but not VEGFR) for 24 hours. HIF1α levels were evaluated by ELISA. Sunitinib significantly reduced HIF1α levels, but imatinib did not. Sunitinib did not affect HIF1α levels in A549 cells. Data are graphed as mean ± SE; *, P < 0.05. B, H23 and Calu-1 (both KDR amplified) cells were treated with complete media containing bevacizumab (BEV), cediranib (CED), or vandetanib (VAN) for 24 hours. Western blot analysis shows p-Met levels were reduced following VEGFR pathway inhibition. C, Met is not constitutively activated in A549 (KDR normal) cells. D, Calu-1 cells were serum starved for 24 hours and then treated with VEGF or HGF (50 ng/mL) for 15 minutes to evaluate direct cross-talk between these receptors. HGF, but not VEGF, induced c-Met activation. E, H23 cells were treated with VEGF (50 ng/mL) alone or in combination with the p38 inhibitor SB203580 (1 µmol/L) or the c-Met inhibitor, EMD1214063 (1 µmol/L), and cell migration was evaluated by Boyden chamber assay. Inhibition of p38 or c-Med blocked VEGF-induced tumor cell migration.

Figure 4

Kaplan–Meier plots of PFS in the vandetanib plus docetaxel (A) or placebo plus docetaxel (B) treatment groups by KDR CNG status. Kaplan–Meier plots of OS in the vandetanib plus docetaxel (C) or placebo plus docetaxel (D) treatment groups by KDR CNG status.