Abstract
Purpose :
Crystallin proteins are major constituents of the vertebrate eye lens and are divided into two primary classes, α-crystallins and β,γ-crystallins. αB-crystallin (CRYAB) is a small heat shock protein and molecular chaperone. CRYAB has been studied extensively in other metastatic cancers, but less is known about its role in ocular melanoma. We provide the first expression patterning of CRYAB and other crystallins in ocular melanoma to help provide insight into its signaling, apoptotic, and autophagic implications in ocular melanoma development.
Methods :
OCM3 malignant melanoma cells derived from human eye tissue were grown to 80% confluence. Different OCM3 cell cultures were treated with either 1ug/mL CRYAB siRNA, 10uM AKT inhibitor IV, or 10uM Rapamycin, an mTOR inhibitor. Western blotting was performed on these cell lysates for phospho-AKT, cleaved-caspase-9, and CRYAB and results were analyzed with ImageJ using retinal pigmented epithelial (RPE) cells as a control. RPE cells were grown and treated with 1ug/mL CRYAB siRNA and chloroquine and then assayed for LC3-II and p62 protein levels by western blot. All results were analyzed via a one-tailed Student’s T-test.
Results :
CRYAB protein was present in both RPE and OCM3 cells with a 68 fold higher concentration observed in OCM3 cells (p<0.01). AKT and mTOR inhibition downregulated CRYAB expression sevenfold. However, phospho-AKT levels in similar experiments were not significantly changed. Cleaved-caspase-9 was upregulated when CRYAB was suppressed. Autophagy flux was downregulated in RPE cells in the presence of suppressed CRYAB (p<0.05) and p62 expression was elevated when CRYAB was decreased (p<0.01).
Conclusions :
The findings shed light into the role of αB-crystallin in ocular melanoma. The elevated concentration of CRYAB observed suggests that it has an effect on the cellular processes active in cancer. The data also suggest CRYAB is acting through the AKT/mTOR signaling axis and is active downstream of the proteins comprising the axis. The suppression of CRYAB could lead to apoptosis, and elevated CRYAB expression could be helping ocular melanoma bypass apoptosis. Also, CRYAB expression is regulating both LC3-II and p62 proteins and thus plays an active role in maintaining proper cellular autophagy. These findings raise questions about how CRYAB could be a potential target for helping halt ocular melanoma metastasis.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.