Richard H. Pin, Maura Reinblatt, Yuman Fong, Memorial Sloan-Kettering Cancer Center, New York, NY

INTRODUCTION: Hypoxia is a common feature of solid tumors that induces vascular endothelial growth factor (VEGF) expression, stimulating angiogenesis and tumor proliferation. The VEGF signaling pathway is inhibited by soluble VEGF receptors such as Flk-1, which bind VEGF and block its interaction with endothelial cells. We set out to attenuate angiogenesis in primary hepatocellular carcinoma (HCC) with hypoxia-induced Flk-1 expression. METHODS: SK-Hep-1 human HCC cells incubated in hypoxia (1% O₂) or normoxia (21% O₂) were assessed by ELISA for hypoxia-inducible factor 1 (HIF-1), the transcriptional regulator of hypoxia-inducible genes. A hypoxia-responsive enhancer (HRE), regulated by HIF-1, was constructed by multimerizing the hypoxia sensitive region of the VEGF promoter. HRE was cloned into a luciferase reporter vector (HRE/Luc) to assess enhancer function. HRE was then cloned upstream of the Flk-1 gene (HRE/Flk-1). SK-Hep-1 cells were transfected with HRE/Flk-1 or no DNA, and then incubated in hypoxia or normoxia. Capillary inhibition was determined by human umbilical vein endothelial cell (HUVEC) assay. Western blot demonstrated Flk-1 expression and ELISA assessed VEGF suppression. RESULTS: HIF-1 expression increased 6-fold in hypoxic SK-Hep-1 cells compared to normoxic cells (p<0.01). Hypoxic SK-Hep-1 cells transfected with HRE/Luc had 45-fold greater luciferase activity compared to controls (p<0.01), while normoxic SK-Hep-1 cells transfected with HRE/Luc had 2.5-fold greater luciferase activity (p<0.05). In the HUVEC assay, HRE/Flk-1 transfection of hypoxic SK-Hep-1 cells yielded a 78% reduction in capillary formation vs. non-transfected, hypoxic controls (p<0.01). HRE/Flk-1 transfection of normoxic SK-Hep-1 cells resulted in a 34% reduction in capillary formation vs. non-transfected, normoxic controls (p<0.01). Western blot of SK-Hep-1 cells transfected with HRE/Flk-1 revealed increased Flk-1 expression in hypoxia compared to normoxia. Western blot also demonstrated that SK-Hep-1 cells did not express Flk-1 without prior HRE/Flk-1 transfection. VEGF levels were reduced 70% with HRE/Flk-1 transfection in hypoxia (p<0.01) and 21% in normoxia (p<0.01). CONCLUSIONS: Pairing a hypoxia-responsive enhancer with a soluble VEGF receptor inhibits capillary formation, particularly at low oxygen concentrations. Tumor hypoxia can thus be used to induce anti-angiogenic factors like Flk-1, potentially reducing hepatocellular tumor growth and metastases.