Epidermal Growth Factor Receptor Inhibition Reduces Angiogenesis via Hypoxia-Inducible Factor-1α and Notch1 in Head Neck Squamous Cell Carcinoma

Angiogenesis, a marker of cancer development, affects response to radiotherapy sensibility. This preclinical study aims to understand the receptor tyrosine kinase-mediated angiogenesis in head neck squamous cell carcinoma (HNSCC). The receptor tyrosine kinase activity in a transgenic mouse model of HNSCC was assessed. The anti-tumorigenetic and anti-angiogenetic effects of cetuximab-induced epidermal growth factor receptor (EGFR) inhibition were investigated in xenograft and transgenic mouse models of HNSCC. The signaling transduction of Notch1 and hypoxia-inducible factor-1α (HIF-1α) was also analyzed. EGFR was overexpressed and activated in the Tgfbr1/Pten deletion (2cKO) mouse model of HNSCC. Cetuximab significantly delayed tumor onset by reducing tumor angiogenesis. This drug exerted similar effects on heterotopic xenograft tumors. In the human HNSCC tissue array, increased EGFR expression correlated with increased HIF-1α and micro vessel density. Cetuximab inhibited tumor-induced angiogenesis in vitro and in vivo by significantly downregulating HIF-1α and Notch1. EGFR is involved in the tumor angiogenesis of HNSCC via the HIF-1α and Notch1 pathways. Therefore, targeting EGFR by suppressing hypoxia- and Notch-induced angiogenesis may benefit HNSCC therapy.


Cell culture and conditional medium collection
The CAL27 cell line was obtained from the American type culture collection (ATCC,VA,USA) and maintained in Dulbecco's modified eagle medium (DMEM) supplemented with 10% FBS, in a humidified atmosphere of 95% air / 5% CO 2 at 37°C. When CAL27 cells were grown to 80% confluence after overnight incubation, cells were serum-deprived for 12 h and then treated with or without 10ug/ml cetuximab in serum-deprived DMEM for a indicated time(12h) in Anoxomat chambers (Mart Microbiology, Lichtenvoorde, the Netherlands) with appropriate oxygen concentrations for hypoxia(1% O 2 ) or normoxia (21% O 2 ). The cells were continue grow in serum-deprived endothelial basic medium (EBM, Lonza, Walkersville, MD, USA) medium for another 24h, and then the cleared supernatants were collected as conditional medium (CM) and stored at-80°C. Pooled human umbilical vein endothelial cells (HUVECs) were purchase from Lonza. HUVECs were grew in endothelial growth medium 2 (EGM-2, Lonza) and passage 3 to 8 were used in experiment, as previously described [1].

Wound-healing assay, Boyden chamber migration assay and tube formation
HUVECs cells were suspended in six-well culture plates, and grown to 90% confluence. A scratch was made across the cell layer using a 20μl sterile pipette tip. Then cells were added the CM.
After 12 h, the cells were fixed and stained with acridine orange. Then the plates were observed under microscope and cells migrated into the gap were counted. Transwell Boyden chamber (Corning Life Sciences) were used to measure migration of endothelial cells. Transwell chamber contained a 6.5-mm-diameter polycarbonate filter (8μm pore size). Starved HUVECs cells (5×10 4 cells/well) incubated in 100μl of ECM were placed in the upper wells, whereas CM, as a chemo-attractant, was added to the lower wells. Then cells incubated for 12 h at 37°C. Using a cotton swab, HUVECs on the upper surface were carefully scrubbed off, and cells adhered to the lower membrane were fixed with 4% formaldehyde, stained by crystal violet, and observed under microscope. For capillary-like tube formation assay, HUVECs were seeded into 6-cm culture dishes coated with Matrigel (BD Biosciences, San Jose, CA) in CM. then the cell was cultured for 24 h at 37°C.Cells were fixed and observed under microscope. The formation of capillary-like structures was counted.

Cell immunofluoresence and confocal microscopy
Immunofluorescence was performed as previous described [1]. When CAL 27 cells were grown to 80% confluence after overnight incubation, they were incubated in serum-deprived DMEM with or without cetuximab under hypoxic condition. After 12 h, cells were fixed with 4% formaldehyde in PBS (pH 8.0) for 10 minutes. After washed with PBS three times, cells were permeabilized for 5 min with 0.5% Triton X-100 in PBS and washed thoroughly three times with PBS, and 10% FCS in PBS were used to block nonspecific binding for 60 min, then cells were incubated with HIF1α antibody (1:50) at 4°C overnight. Then cells were washed thoroughly again three times with PBS. Primary antibodies were detected by incubation with goat anti-rabbit antibody IgG Dylight 594 (1:200) at room temperature for 60 min and cells were washed three times with PBS.

Western blot analysis
Western blot were performed as previously described [2]. Briefly, CAL27 cells were treated with the indicated concentrations of cetuximab in DMEM containing 2% FBS for 24 h. Then the cells were lysed, and the total protein was separated using 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA). The immunoblots were cultured overnight at 4°C with the corresponding primary antibodies in blocking solution, followed by incubation with horseradish peroxidase-conjugated secondary antibody (Pierce Chemical, Rockford, IL). Then blots were developed by a West Pico ECL kit.