Critical loss of phosphatase and tensin homolog (PTEN) beside increased insulin signaling often results in mTOR activation which is associated with progression of lethal prostate cancer (PCa). Recently mTOR was shown to control de novo pyrimidine synthesis via S6K which directly phosphorylates Ser1859 on CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotase), the enzyme that catalyzes the first three steps of de novo pyrimidine synthesis. We assessed the CAD expression and phosprylation at Ser1859 in different in-vitro and in-vivo models of PCa and also tested the potential efficacy of fisetin, an agent which we previously established as a dual PI3K/AKT and mTOR inhibitor. We observed upregulation of CAD both at transcriptional and translational levels in PCa cell lines when compared with the non-tumorigenic prostate cells. However, a highly significant upregulation of CAD phosphorylation at Ser1859 was evident in PTEN deficient cells when compared with wild type PTEN cells. To confirm, we assessed the CAD phosphorylation at Ser1859 in PTEN knockout mice samples and observed significant increase in the CAD phosphorylation at Ser1859 but not in total CAD levels, suggesting high demands of de novo pyrimidine synthesis in PTEN deficient tumors. In another model of spontaneous PCa (TRAMP) an inverse association between CAD phosphorylation at Ser1859 and PTEN was observed. Based on the facts that PTEN deficient cells have high mTOR activity that is prerequisite for CAD phosphorylation we tested the efficacy of fisetin to inhibit CAD phosphorylation at Ser1859 in different PCa models. Fisetin significantly suppressed the CAD phosphorylation in tumorigenic PCa cells in-vitro. In-vivo efficay of fisetin for inhibition of CAD phosphorylation was also established in the xenograft study. However, decrease in abundance of CAD mRNA and protein in xenograft samples suggested fisetin additionally suppressed other mechanism(s) important for CAD expression. Next we determined the effect of fisetin to inhibit the growth factors (insulin/IGF-1/EGF) stimulated CAD oligomerization in PCa cells important for CAD activity. Fisetin significantly suppressed the growth factors induced CAD oligomerization as observed by electron microscopy and density gradient based western blotting. However, we did not find significant S-phase blockage by fisetin under similar conditions. Based on these data we suggest that PTEN deficient PCa epithelial tumors possess heightened de novo pyrimidine requirements, satisfied by growth factor stimulated and mTOR regulated CAD phosphorylation and oligomerization. Fisetin inhibits both CAD phosphorylation at Ser1859 and oligomerization and thereby limits pyrimidine sufficiency in PTEN deficient PCa cells. Our results add additional mechanistic insight in the fisetin mediated inhibition of PCa and suggest the use of fisetin in PTEN deficient PCa incidents.

Citation Format: Mohammad Imran Khan, Vaqar Mustafa Adhami, Omar Mohammad Haidar, Bilal Bin Hafeez, Ajit Kumar Verma, Hasan Mukhtar. PTEN deficiency in prostate epithelial cells is associated with increased CAD phosphorylation and is inhibited by fisetin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1437. doi:10.1158/1538-7445.AM2014-1437