Enhancing antiangiogenic therapy by blocking “anti-antiangiogenic” phenotype

Proc Amer Assoc Cancer Res, Volume 45, 2004 49 While antiangiogenic therapy is emerging as a potentially promising anticancer modality, how tumor vascular endothelial cells (EC) respond to antiangiogenic drugs is still poorly understood. Optimization of antiangiogenic therapy requires a better understanding of the molecular pathways regulating the biological response to antiangiogenic therapy. Our previous studies have found that while endothelium-targeting antiangiogenic drugs, such as endostatin, angiostatin or SU6668, inhibited vascularization and growth of squamous cell carcinoma (SCC), the inhibition was only partial. One possibility is that these tumors might develop mechanisms that counteract the antiangiogenic therapies. In the present study, we began to test this hypothesis by addressing whether SCC cells can confer EC resistance to antiangiogenic drugs and investigating the cellular/molecular mechanisms involved in regulating such resistant response. We found that conditioned medium of cultured SCC protected EC from apoptosis induced by endostatin, SU6668 or camptothecin, reducing EC apoptosis 17-, 14- and 10-fold, respectively, suggesting an “anti-antiangiogenic” phenotype of the SCC cells. Since the EGFR pathway plays important roles in sustaining the malignant phenotype in SCC, including angiogenesis, we further examined whether the “anti-antiangiogenic” phenotype could be attributed to EGFR signaling. Our data demonstrated that inhibition of EGFR signaling in SCC cells, using either selective tyrosine kinase inhibitor or antisense oligonucleotides, reversed the aforementioned endothelium protective phenotype, resulting in robust increases of endothelial apoptosis induced by the antiangiogenic drugs. This suggests that the EGFR signaling might be a key pathway that confers SCC cells the “anti-antiangiogenic” phenotype. Gene array profile showed that EGFR inhibitors greatly downregulated multiple angiogenic factors including VEGF, IL-8, MMp-2 and MMP-9. ELISA assays further confirmed reduction of these proteins in conditioned medium of SCC cells treated with EGFR inhibitors. However, transfection of VEGF to SCC cells only partially compensated EGFR inhibition, indicating that other molecules downstream to EGFR signaling also contribute to the EGFR-mediated “anti-antiangiogenic” phenotype. In vivo experiments demonstrated that when EGFR signaling was inhibited, SCC tumors responded more profoundly to antiangiogenic therapies (endostatin or SU6668), leading to almost complete eradication of SCC xenografts. Thus, our data has uncovered that solid tumors can counteract antiangiogenic therapy and that in SCC, the EGFR signaling pathway is key to such an “anti-antiangiogenic” phenotype. Therefore, combinations of a direct antiangiogenic therapy and an approach inhibiting the “anti-antiangiogenic” phenotype, such as EGFR targeting approaches, might enhance the overall antiangiogenic efficacy.