Effect of hypoxia on the expression of αB-crystallin in head and neck squamous cell carcinoma

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. αB-crystallin (HspB5) is a small heat shock protein, which is also associated with metastasis formation in HNSCC. In this study, we investigated whether αB-crystallin protein expression is increased in hypoxic areas of HNSCC biopsies and analyzed whether hypoxia induces αB-crystallin expression in vitro and in this way may confer hypoxic cell survival.

Methods: In 38 HNSCC biopsies, the overlap between immunohistochemically stained αB-crystallin and pimonidazole-adducts (hypoxiamarker) was determined. Moreover, expression levels of αB-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 αB-crystallin on cell survival under hypoxic conditions.

Results: In all biopsies αB-crystallin was more abundantly present in hypoxic areas than in normoxic areas. Remarkably, hypoxia decreased αB-crystallin mRNA expression in the HNSCC cell lines. Only after reoxygenation, a condition that stimulates ROS formation, αB-crystallin expression was increased. αB-crystallin mRNA levels were also increased by extracellular ROS, and NAC abolished the reoxygenation-induced αB-crystallin upregulation. Moreover, it was found that decreased αB-crystallin levels reduced cell survival under hypoxic conditions.

Conclusions: We provide the first evidence that hypoxia stimulates upregulation of αB-crystallin in HNSCC. This upregulation was not caused by the low oxygen pressure, but more likely by ROS formation. The higher expression of αB-crystallin may lead to prolonged survival of these cells under hypoxic conditions.

Figure 1

Immunofluorescent staining of human HNSCC for αB-crystallin, pimonidazole-modified proteins and PAL-E. Shown is a representative biopsy section. The fluorescent grey scale images were binarized, resulting in black and white images for αB-crystallin staining (A) and pimonidazole-modified proteins staining, indicating the hypoxic areas (B). The merged image with αB-crystallin staining (assigned red), pimonidazole staining (assigned green) and PAL-E blood vessel staining (assigned blue) shows a substantial overlap between αB-crystallin and hypoxic regions. Hypoxic regions are mostly located in areas at greater distance from vessels (C).

Figure 2

αB-crystallin expression is increased in hypoxic areas. The symbols represent the relative amount of αB-crystallin staining in the normoxic areas and in the hypoxic areas for each individual HNSCC. Equal staining of αB-crystallin in the normoxic and hypoxic areas would be according to the grey line.

Figure 3

Relative αB-crystallin mRNA expression during hypoxia. αB-crystallin mRNA expression levels in UT-SCC-5 cells after incubation in a humidified 37CH35 hypoxystation at 0.1% O₂ for the indicated time points. αB-crystallin mRNA expression levels were assessed via RT-qPCR (N = 4) *** p < 0.001, ** 0.001 < p < 0.01.

Figure 4

Relative αB-crystallin mRNA levels after hypoxia and reoxygenation. αB-crystallin mRNA levels in UT-SCC-5 and UT-SCC-15 cells under 48 hours normoxia (N), hypoxia (H, 0.1% O₂) and after reoxygenation (R, 24 hours 0.1% O₂/24 hours normoxia). αB-crystallin mRNA expression levels were assessed via RT-qPCR (N = 4). *** p < 0.001, ** 0.001 < p < 0.01, * 0.01 < p < 0.05.

Figure 5

Relative αB-crystallin protein levels after hypoxia and reoxygenation. αB-crystallin protein expression levels in UT-SCC-5 cells under 48 hours normoxia (N), hypoxia (H, 0.1% O₂) and after reoxygenation (R, 24 hours 0.1% O₂/24 hours normoxia). αB-crystallin protein expression was analyzed of 3–4 independent incubations via western blotting (A) and quantified (B). ***p < 0.001, ** 0.001 < p < 0.01.

Figure 6

Relative αB-crystallin mRNA levels upon H₂O₂-incubation. Relative αB-crystallin mRNA levels in UT-SCC-5 cells after incubation with 0.0 mM (mock), 0.3 mM, 1.5 mM or 3.0 mM H₂O₂ for 1 hour and 7 hours of recovery. αB-crystallin mRNA expression levels were assessed via RT-qPCR (N = 4). *** p < 0.001.

Figure 7

Effect of the ROS-scavenger NAC on αB-crystallin mRNA levels during reoxygenation. UT-SCC-5 cells after incubation with mock or NAC under 48 hours normoxia (N), hypoxia (H, 0.1% O₂) and after reoxygenation (R, 24 hours 0.1% O₂/24 hours normoxia). ** 0.001 < p < 0.01, * 0.01 < p < 0.05.

Figure 8

Knockdown of αB-crystallin expression reduces hypoxia and hypoglycemia survival. Expression of αB-crystallin mRNA in UT-SCC-5 cells was reduced by three different αB-crystallin siRNAs (αB1, αB2 and αB3). LUC and EGFP were used as negative control siRNAs (A). Survival of siRNA-treated UT-SCC-5 cells under normoxic (N) and hypoxic (H, 0.1% O₂ for 24 hours) conditions in the presence of 5 mM or 0 mM glucose (B). Cell survival was assessed via a colorimetric assay using cell counting kit-8. The optical density (O.D.) of siEGFP-treated cells was set at 100%. *** p < 0.001, ** 0.001 < p < 0.01, * 0.01 < p < 0.05.