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

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.

Pseudophosphorylated αB-crystallin is a nuclear chaperone imported into the nucleus with help of the SMN complex.

The human small heat shock protein αB-crystallin (HspB5) is a molecular chaperone which is mainly localized in the cytoplasm. A small fraction can also be found in nuclear speckles, of which the localization is mediated by successional phosphorylation at Ser-59 and Ser-45. αB-crystallin does not contain a canonical nuclear localization signal sequence and the mechanism by which αB-crystallin is imported into the nucleus is not known. Here we show that after heat shock pseudophosphorylated αB-crystallin mutant αB-STD, in which all three phosphorylatable serine residues (Ser-19, Ser-45 and Ser-59) were replaced by negatively charged aspartate residues, is released from the nuclear speckles. This allows αB-crystallin to chaperone proteins in the nucleoplasm, as shown by the ability of αB-STD to restore nuclear firefly luciferase activity after a heat shock. With the help of a yeast two-hybrid screen we found that αB-crystallin can interact with the C-terminal part of Gemin3 and confirmed this interaction by co-immunoprecipitation. Gemin3 is a component of the SMN complex, which is involved in the assembly and nuclear import of U-snRNPs. Knockdown of Gemin3 in an in situ nuclear import assay strongly reduced the accumulation of αB-STD in nuclear speckles. Furthermore, depletion of SMN inhibited nuclear import of fluorescently labeled recombinant αB-STD in an in vitro nuclear import assay, which could be restored by the addition of purified SMN complex. These results show that the SMN-complex facilitates the accumulation of hyperphosphorylated αB-crystallin in nuclear speckles, thereby creating a chaperone depot enabling a rapid chaperone function in the nucleus in response to stress.

αB-crystallin expression is correlated with phospho-ERK1/2 expression in human breast cancer.

αB-crystallin is regarded as a biomarker for triple-negative and/or basal-like breast cancer. In normal breast cells, overexpression of αB-crystallin leads to neoplastic-like changes, which likely relate to enhanced expression of phosphorylated ERK1/2 (pERK1/2). In this study, we investigated whether αB-crystallin expression is correlated to pERK1/2 expression in breast cancer. In a balanced tissue microarray the expression of αB-crystallin and pERK1/2 kinase were determined immunohistochemically, together with the triple-negativity and basal-like markers CK5/6 and SMA and the signaling molecules pAKT, pmTOR, EGFR, and IGF-1R. αB-crystallin expression significantly correlated with triple negativity and basal-like markers CK5/6 and SMA (Pearson Chi-square test p=0.004, p=0.001, and p<0.001, respectively). A significant correlation was also observed with pERK1/2 expression (p=0.002). siRNA-mediated knockdown of αB-crystallin in the triple-negative breast cell line MDA-MB468 did not show an effect on pERK1/2 expression levels, indicating that lowering the level of αB-crystallin does not reduce pERK1/2 expression. Our results confirm that αB-crystallin can be used as a biomarker for triple-negative and/or basal-like breast cancer. The expression of αB-crystallin correlates with pERK1/2 expression in breast cancer tissue suggesting that therapies targeting αB-crystallin might be considered for treatment of triple-negative or basal-like breast cancer.

αB-crystallin stimulates VEGF secretion and tumor cell migration and correlates with enhanced distant metastasis in head and neck squamous cell carcinoma.

BACKGROUND: αB-crystallin is able to modulate vascular endothelial growth factor (VEGF) secretion. In many solid tumors VEGF is associated with angiogenesis, metastasis formation and poor prognosis. We set out to assess whether αB-crystallin expression is correlated with worse prognosis and whether this is related to VEGF secretion and cell motility in head and neck squamous cell carcinoma (HNSCC). METHODS: αB-crystallin expression was determined immunohistochemically in tumor biopsies of 38 HNSCC patients. Locoregional control (LRC) and metastasis-free survival (MFS) of the patients were analyzed in relation to αB-crystallin expression. Additionally, the effects of αB-crystallin knockdown on VEGF secretion and cell motility were studied in vitro. RESULTS: Patients with higher staining fractions of αB-crystallin exhibited a significantly shorter MFS (Log-Rank test, p < 0.005). Under normoxic conditions αB-crystallin knockdown with two different siRNAs in a HNSCC cell line reduced VEGF secretion 1.9-fold and 2.1-fold, respectively. Under hypoxic conditions, a similar reduction of VEGF secretion was observed, 1.9-fold and 2.2-fold, respectively. The effect on cell motility was assessed by a gap closure assay, which showed that αB-crystallin knockdown decreased the rate by which HNSCC cells were able to close a gap by 1.5- to 2.0-fold. CONCLUSIONS: Our data suggest 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 and stimulating cell migration.