Protein kinase D1 mitigation against etoposide induced DNA damage in prostate cancer is associated with increased α-Catenin

BackgroundThe E-cadherin, alpha- and beta-Catenin interaction at the cell adherens junction plays a key role in cell adhesion; alteration in the expression and function of these genes are associated with disease progression in several solid tumors including prostate cancer. The membranous beta-Catenin is dynamically linked to the cellular cytoskeleton through interaction with alpha-Catenin at amino acid positions threonine 120 (T120) to 151 of beta-Catenin. Nuclear presence of alpha-Catenin modulates the sensitivity of cells to DNA damage. The objective of this study is to determine the role of alpha-Catenin and protein kinase D1 (PrKD1) in DNA damage response.MethodsProstate cancer cells; LNCaP, LNCaP (Sh-PrKD1; silenced PrKD1), C4-2 and C4-2 PrKD1 were used for various sets of experiments to determine the role of DNA damage in PrKD1 overexpression and silencing cells. These cells were treated with compound-10 (100 nM) and Etoposide (30 mu M), total cell lysates, cytosolic and nuclear fractions were prepared to observe various protein expressions. We performed single cell gel electrophoresis (COMET assay) to determine the etoposide induce DNA damage in C4-2 and C4-2 PrKD1 cells. The animal experiments were carried out to determine the tolerability of compound-10 by mice and generate preliminary data on efficacy of compound-10 in modulating the alpha-Catenin and PrKD1 expressions in inhibiting tumor progression.ResultsPrKD1, a novel serine threonine kinase, phosphorylates beta-Catenin T120. In silico analysis, confirmed that T120 phosphorylation alters beta- to alpha-Catenin binding. Forced expression of PrKD1 in prostate cancer cells increased beta- and alpha-Catenin protein levels associated with reduced etoposide induced DNA damage. Downregulation of alpha-Catenin abrogates the PrKD1 mitigation of DNA damage. The in vitro results were corroborated in vivo using mouse prostate cancer patient derived xenograft model by inhibition of PrKD1 kinase activity with compound-10, a selective PrKD inhibitor, demonstrating decreased total beta- and alpha-Catenin protein levels, and beta-Catenin T120 phosphorylation.ConclusionsAlteration in DNA damage response pathways play major role in prostate cancer progression. The study identifies a novel mechanism of alpha-Catenin dependent DNA damage mitigation role for PrKD1 in prostate cancer.