VDR agonists down regulate PI3K/Akt/mTOR axis and trigger autophagy in Kaposi's sarcoma cells

Area of Science:

• Oncology
• Molecular Biology
• Cell Biology

Background:

• Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor (KSHV/vGPCR) drives Kaposi's sarcoma pathogenesis.
• 1α,25(OH)₂D₃ and its analog TX 527 inhibit vGPCR-expressing endothelial cell proliferation, NF-κB activity, and induce apoptosis via VDR.
• The role of PI3K/Akt/mTOR signaling and autophagy in the antineoplastic mechanism of these VDR agonists requires further investigation.

Purpose of the Study:

• To investigate whether 1α,25(OH)₂D₃ or TX 527 regulates the PI3K/Akt/mTOR axis.
• To determine if these VDR agonists induce autophagy as part of their antineoplastic mechanism.
• To elucidate the VDR and NF-κB dependence of these cellular processes.

Main Methods:

• Cell proliferation assays using LY294002 (PI3K/Akt inhibitor).
• Western blot analysis to assess Akt and mTOR phosphorylation, and LC3-II levels.
• Nuclear and cytoplasmic fractionation to localize phosphorylated Akt.
• Quantitative PCR or Western blot to analyze TNFAIP3/A20 and Beclin-1 expression.
• Autophagy flux assay using Chloroquine (CQ).

Main Results:

• 1α,25(OH)₂D₃ and TX 527 decreased vGPCR-expressing endothelial cell proliferation, similar to PI3K/Akt inhibition.
• Both compounds reduced Akt and mTOR phosphorylation in a dose- and time-dependent manner, with decreased nuclear Akt phosphorylation.
• TNFAIP3/A20 was downregulated, while Beclin-1 and the autophagy marker LC3-II were upregulated.
• Autophagy flux was confirmed using Chloroquine treatment.

Conclusions:

• VDR agonists 1α,25(OH)₂D₃ and TX 527 inhibit the PI3K/Akt/mTOR signaling pathway in vGPCR-expressing endothelial cells.
• These VDR agonists induce autophagy, contributing to their antineoplastic effects.
• The observed effects are mediated through a VDR-dependent mechanism, involving regulation of NF-κB target genes.