We have previously shown that the anti-epidermal growth factor receptor monoclonal antibody cetuximab (C225; Erbitux), which was recently approved for the treatment of metastatic colorectal cancer, has anti-angiogenic properties, inhibiting vascular endothelial growth factor (VEGF) secretion in culture and in animal models. Here, we have furthered the study, demonstrating that cetuximab reduces the cellular levels of hypoxia-inducible factor-1 alpha (HIF-1α), a transcriptional regulator of VEGF expression, in A431 epidermoid carcinoma cells under both normoxic and hypoxic culture conditions. Expression of a constitutively active Ras in A431 cells rendered cellular resistance to the cetuximab-mediated reduction of the HIF-1α level. Cell lines with naturally occurring PTEN mutations were also resistant to cetuximab-mediated reduction of the HIF-1α level. Pharmacological inhibition of two major EGF receptor downstream signal transduction pathways, i.e., the phosphatidylinositol 3-kinase pathway with LY294002 and the mitogen-activated protein kinase kinase pathway with PD98059, showed that LY294002 reduced the HIF-1α level in both normoxic and hypoxic A431 cells, whereas PD98059 reduced the level of HIF-1α only in normoxic A431 cells. Further studies have shown that cetuximab reduced the cellular level of HIF-1α in the presence of a proteasome inhibitor, lactacystin, indicating that cetuximab is acting at the level of protein synthesis rather than degradation. The reduction of HIF-1α in response to cetuximab treatment was accompanied by transcriptional inhibition of VEGF expression, measured by a luciferase assay in A431 cells transfected with a vector containing the VEGF hypoxia response element. Taken together, our results indicate that the previously demonstrated inhibition of VEGF by cetuximab occurs at the level of transcription in response to a reduced level of HIF-1α and justify further testing of therapeutic strategies that combine cetuximab with approaches inhibiting the function of VEGF or the VEGF receptor.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]