In the current study, we show that more αB-crystallin protein is present in hypoxic HNSCC tumor areas than in normoxic areas. Since an increased αB-crystallin expression might be the result of a stress-induced transcriptional upregulation, we investigated whether hypoxic stress is able to induce αB-crystallin expression in HNSCC cell lines. Remarkably, under hypoxic conditions αB-crystallin mRNA expression was found to be downregulated in UT-SCC-5 and UT-SCC-15 cells. It is not clear how αB-crystallin is downregulated, but this could be due to a general transcription shutdown caused by epigenetic modifications [38, 39]. Only after reoxygenation, a significant upregulation of αB-crystallin mRNA in the HNSCC cell lines was found. The same trend was observed at the protein level. The upregulation of αB-crystallin has also been observed in other cell types and by different forms of reoxygenation stress, such as chemical ischemic/recovery stress and ischemic/reperfusion injury [40, 41], although from those studies it appeared that the upregulation of αB-crystallin is not a general mechanism since not all cell types show this effect . The reoxygenation-induced upregulation of αB-crystallin also fits with the studies performed with the piglets, where hypoxia-induced αB-crystallin upregulation was detected [25, 26]. In these studies piglets were maintained in a hypoxic chamber for either 1 or 4 hours and were allowed to recover over periods of 1 to 68 hours under normoxic condition and thus underwent a period of reoxygenation.
The reoxygenation-induced upregulation of αB-crystallin mRNA is at least partially caused by ROS, based on the inhibitory effect of the ROS-scavenger NAC (Figure 7). Despite the low level of oxygen, significant levels of ROS can be present in hypoxic areas and thus can be responsible for the induction of αB-crystallin expression. ROS-levels in hypoxic areas can be increased by moments of reoxygenation due to intermittent, perfusion-limited hypoxia  or produced by necrotic cells which are often present in hypoxic tumor areas [34, 42–44]. Also ROS can be produced by a synergistic effect of oncogenic-induced stimulation of increased mitochondrial capacity and low oxygen levels, which causes an ineffective functioning of mitochondrial respiratory complexes . For this a hypoxia-induced downregulation of thioredoxin reductase 1 seems to be important in maintaining high levels of ROS under hypoxic conditions . As mentioned earlier, some normoxic areas may also contain significant levels of αB-crystallin. These local αB-crystallin expressions might be explained by intermittent hypoxia as well. As shown by Bennewith and colleagues a substantial proportion of tumor cells can go through cycles of hypoxia and normoxia [46, 47]. If the intervals of hypoxia are too short or if pimonidazole is not present at the hypoxic moments, erroneously no staining by this marker will be detected [46, 47]. Nevertheless, αB-crystallin induced by the ROS formed during the reoxygenation periods might still be present.
αB-crystallin is a stress protein which may enhance cell survival upon ROS exposure, as shown for H2O2 treated mouse retinal pigment epithelium cells . Based on knockdown experiments, we showed that αB-crystallin is able to play a role in the survival of cells coping with hypoxia and glucose-deprivation stress as well. It is thus possible that the αB-crystallin present in hypoxic tumor areas plays a role in tumor cell survival during hypoxic stress [34, 49]. This protective activity of αB-crystallin may further increase the number of αB-crystallin-positive cells in hypoxic tumor areas. In summary, the relative higher levels of αB-crystallin in HNSCC hypoxic tumor areas might be caused by a combination of ROS-induced αB-crystallin upregulation and an enhanced survival of αB-crystallin-positive cells exposed to this stress.
The enhanced expression of αB-crystallin in HNSCC may have a negative effect on the prognosis of the patient. We have recently found that αB-crystallin expression is associated with distant metastases formation in HNSCC patients . This association might relate to the chaperone function of αB-crystallin in mediating folding and secretion of VEGF. αB-crystallin is able to bind misfolded vascular endothelial growth factor (VEGF), leading to enhanced VEGF secretion [50, 51]. VEGF is specifically upregulated by hypoxia-inducible factor 1 (HIF1) and is important for tumor vascularization. VEGF induction is thus a mechanism to alleviate hypoxic circumstances [52, 53]. Cycling hypoxia-induced VEGF expression has been shown to increase pulmonary metastasis formation in mice . Since αB-crystallin can increase hypoxic cell survival and can help in the (re)folding of hypoxia-induced VEGF expression, αB-crystallin expression could ultimately increase the risk of hypoxic tumors to become metastasis-prone . Furthermore, by increasing hypoxic cell survival αB-crystallin may also decrease the sensitivity of a tumor to cancer treatments, such as radiation or other cancer treatments, as shown by the effect of αB-crystallin on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as well as cisplatin-induced apoptosis in human ovarian cancer cells . Because of its potential to interfere with anti-tumor therapies, αB-crystallin might be a promising target for anti-cancer treatment.