Abstract
Abstract
The most common signature of aggressive cancers such as prostate cancer (PCa) involves metastasis. Tumor cell migration is a key step for formation of cancer metastasis and recent data suggest that mammalian target of rapamycin (mTOR) controls the metastatic landscape of PCa by regulating the expression of genes known to be involved in PCa metastasis. Also, current estimates suggest that PI3K/Akt/mTOR signaling is upregulated in 30-50% of PCa patients. We observed that the expression of a set of four signature genes YB-1, MTA1, CD-44 and vimentin, regulated by mTOR and known to be involved in metastasis, is significantly higher in PCa PC-3 and DU145 cells compared to normal prostate epithelial cells. siRNA mediated knockdown of YB-1 in both PC-3 and DU-145 cells resulted in inhibition of EMT as seen by a significant decrease in mesenchymal morphology associated with induction of E-cadherin and inhibition of YB-1. We recently showed that fisetin, a dietary flavonoid, is also a specific inhibitor of the mTOR pathway. We hypothesized that fisetin will inhibit epithelial to mesenchymal transition (EMT) in PCa cells by binding to and inhibiting the YB-1. Using in silico docking studies, we observed that fisetin interacts with the cold shock domain (CSD) of the YB-1 molecule. Fisetin binds to YB-1 protein located on residues from β1 and β4 strands of the CSD and shows consistent binding modes and tight affinity within the amino acid pocket. Calculated free binding energy for these conformations ranged from -11.9845 to -9.6273 kcal/mol suggesting strong binding affinity. Treatment of PCa PC-3 and DU145 cells with fisetin (0-80 μM) resulted in inhibition of cell motility that was associated with inhibition of phosphorylation of mTOR at Ser2481 and Ser2448 and YB-1. Using immuno-fluorescence we found that fisetin treatment induced E-cadherin re-expression and inhibited vimentin expression in both DU-145 and PC-3 cells suggesting a reversal of the EMT. This reversal of EMT phenotype was also associated with induction of occludin and claudin-1 and inhibition of fibronectin, N-cadherin, MTA1 and CD-44 expression. We also observed that fisetin treatment inhibited nuclear expression of repressors of EMT such as Snail, Slug and Twist. Because tumor cell migration is a key step for formation of cancer metastasis, we determined the effect of fisetin on cell migration using PC-3 cells. Fisetin treatment inhibited cell migration at both 40 and 80 μM that was associated with decrease in the expression of MMP2 and MMP9. In summary, we observed that YB-1 expression induces EMT in PCa cells and identified fisetin as a novel agent that binds to it and potentially interferes with its activity. Based on our observations we suggest that fisetin could be developed as a clinically relevant inhibitor of mTOR/YB-1 with therapeutic benefit for PCa metastasis and invasion.
Citation Format: Vaqar Mustafa Adhami, Mohammad Imran Khan, Rahul K. Lall, Imtiaz A. Siddiqui, Deeba N. Syed, Mario Sechi, Shah-Jahan M. Dodwad, Hasan Mukhtar. Role of YB-1, a regulator of epithelial to mesenchymal transition, in prostate cancer: inhibition by a dietary flavonoid fisetin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5551. doi:10.1158/1538-7445.AM2013-5551