Abstract 3944: In-vitro evaluation of the Hypoxia Inducible Factor (HIF) pathway in head and neck squamous cell carcinoma (HNSCC)
Authors: Darr Owen A.F. , Chinn Steven B. , Carey Thomas E. , Prince Mark E. 
Published: August 2012

Abstract
Objective: To evaluate the effect of hypoxia on the expression of genes regulated by oxygen (HIF1-α, HIF2-α, VEGF, cMET, OCT4 and EGFR) and on cell migration in UM-SCC-103 cells while controlling for mutations in the von Hippel-Lindau (VHL) gene, a regulator of HIF1. Methods: UM-SCC-103, a cell line derived from an oral cavity HNSCC, was used in all experiments. DNA and cDNA was sequenced to analyze UM-SCC-103 for mutations of VHL. Cells were cultured in normoxic (20% O2) and hypoxic (1% O2) conditions. RNA was isolated from each group at specific time points after onset of hypoxia (0, 6, 12 and 24 hrs), converted to cDNA and assessed by real-time qPCR for β-actin, HIF1-α, HIF2-α, VEGF, cMET, OCT4 and EGFR expression. A wound-healing assay was performed on cells grown to 90-95% confluence, then scratched with a plastic pipette tip, and incubated under normoxic or hypoxic conditions; measurements were taken at 0, 12, 16, and 20 hrs. Wound closure at each time point was quantified using ImageJ software by measuring the area of the wound. Mean percent change from baseline was calculated and analyzed with a paired t-test statistic. Results: Sequence analysis of VHL genomic DNA and cDNA revealed no mutations, indicating the presence of wild-type (wt) VHL. The HIF1-α transcript decreased to 35% below the normoxic control at 24 hours, while HIF2-α showed a transient 20% increase at 6 hours before returning to normoxic levels. Under hypoxic conditions, transcripts of VEGF steadily increased over time, reaching 61% over normoxic at 24 hrs. OCT4 was increased by 65% at 12 hrs and 36% at 24 hrs, relative to normoxic conditions. cMET increased by 10% at 24 hrs while EGFR expression level remained unchanged. The cell migration/wound-healing assay showed that cells incubated under hypoxic conditions had significantly increased migration at 12, 16 and 20 hrs compared to cells incubated under normoxic conditions (62% v. 20% wound closure p=0.0001, 86% v. 46% p=0.0001 and 97% v. 71% p=0.0015 respectively). Conclusions: UM-SCC-103 expresses wt-VHL, suggesting physiologic regulation of HIF under normoxic conditions. Under hypoxic conditions, decreased expression of HIF1-α message over time is consistent with a potential negative feedback loop inhibiting HIF1-α transcription. HIF2-α, thought to be associated with chronic hypoxia, is likely regulated differently, and showed no evidence of transcriptional down-regulation over 24 hrs. HIF downstream targets cMET, VEGF and OCT4 increased in hypoxia. Given the known association of cMET and VEGF with tumor cell migration and survival, respectively, and of OCT4 with the stem cell phenotype, our results are consistent with a role for hypoxia as a driver of a biologically aggressive phenotype in HNSCC. Wound-healing assays support this assertion, demonstrating increased cell migration in hypoxia. Hypoxia may also shift cells into the stem cell niche.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3944. doi:1538-7445.AM2012-3944