Abstract
BACKGROUND: The presence of hypoxia in head and neck squamous cell carcinoma (HNSCC) is associated with therapeutic resistance and increased risk of metastasis formation. alphaB-crystallin (HspB5) is a small heat shock protein, which is also associated with metastasis formation in HNSCC. In this study, we investigated whether alphaB-crystallin protein expression is increased in hypoxic areas of HNSCC biopsies and analyzed whether hypoxia induces alphaB-crystallin expression in vitro and in this way may confer hypoxic cell survival. METHODS: In 38 HNSCC biopsies, the overlap between immunohistochemically stained alphaB-crystallin and pimonidazole-adducts (hypoxiamarker) was determined. Moreover, expression levels of alphaB-crystallin were analyzed in HNSCC cell lines under hypoxia and reoxygenation conditions and after exposure to reactive oxygen species (ROS) and the ROS scavenger N-acetylcysteine (NAC). siRNA-mediated knockdown was used to determine the influence of alphaB-crystallin on cell survival under hypoxic conditions. RESULTS: In all biopsies alphaB-crystallin was more abundantly present in hypoxic areas than in normoxic areas. Remarkably, hypoxia decreased alphaB-crystallin mRNA expression in the HNSCC cell lines. Only after reoxygenation, a condition that stimulates ROS formation, alphaB-crystallin expression was increased. alphaB-crystallin mRNA levels were also increased by extracellular ROS, and NAC abolished the reoxygenation-induced alphaB-crystallin upregulation. Moreover, it was found that decreased alphaB-crystallin levels reduced cell survival under hypoxic conditions. CONCLUSIONS: We provide the first evidence that hypoxia stimulates upregulation of alphaB-crystallin in HNSCC. This upregulation was not caused by the low oxygen pressure, but more likely by ROS formation. The higher expression of alphaB-crystallin may lead to prolonged survival of these cells under hypoxic conditions.
