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Gregory Konar, Peng Shang, Subrata Mishra, Sayan Ghosh, James T Handa, Debasish Sinha; αB-crystallin is active in Signaling, Autophagy, and Apoptosis in Ocular Melanoma. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3169.
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© ARVO (1962-2015); The Authors (2016-present)
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 (OM). The chaperone abilities of CRYAB make it a favorable target for studying autophagy. We provide the first expression patterning of CRYAB in OM cells to give insight into its autophagic, signaling, and apoptotic implications in OM progression in the eye.
OCM3 malignant melanoma cells derived from human eye tissue were grown to 80% confluence. Protein lysates were prepared and western blotting was performed for CRYAB with retinal pigmented epithelial (RPE) cells as a control. Different OCM3 cell cultures were treated with either 1ug/mL siRNAs against CRYAB, 10uM AKT inhibitor IV, or 10uM Rapamycin, an mTOR inhibition. Western blotting was performed on these cell lysates for AKT, mTOR, cleaved-caspase-9, and CRYAB. All blot results were analyzed with ImageJ. RPE cells were grown and treated with 10uM CRYAB siRNA and chloroquine and then assayed for LC3-II and p62 protein levels by Western Blot.
CRYAB protein expression was present in both RPE cells and OCM3 cells, with an upregulation observed in OCM3 cells (p < 0.01). AKT and mTOR inhibition both downregulated CRYAB expression. However, relative p-AKT and p-mTOR levels were not significantly changed. Cleaved-caspase-9 was upregulated (p < 0.05) when CRYAB was suppressed. Autophagy flux was downregulated in RPE cells in the presence of decreased CRYAB levels (p < 0.05) and p62 expression was elevated (p < 0.05) when CRYAB was decreased.
The findings shed light into the role of CRYAB in OM. CRYAB expression patterns show there is active molecular chaperone activity in OM. The data also suggest CRYAB is acting through the AKT/mTOR signaling axis and is active downstream of PI3K/AKT/mTOR. The expression of cleaved-caspase-9 indicates that suppressing CRYAB could lead to apoptosis, and heightened CRYAB expression could be helping OM bypass apoptosis. CRYAB expression is regulating both LC3-II and p62 protein expression and thus plays an active role in proper autophagy in a cellular system. These findings raise questions about how CRYAB could contribute to autophagic maintenance in OM.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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