1685P - Prostate Cancer Stem Cells Display Lowered Activity of the mTOR Pathway and Resistance against mTOR Inhibition Caused by Elevated Hypoxic Signaling

Abstract

ABSTRACT Tumor-initiating subpopulations of cancer cells, also known as cancer stem cells (CSCs), were recently identified and characterized in Prostate cancer (PCa). We theorized that the PI3K/AKT pathway and mTOR, one of its main effectors, play a role in CSC maintenance. Moreover, hypoxia was known to be involved in CSC maintenance and to have a regulating effect on mTOR signaling. In our study, we therefore evaluated the effects of mTOR inhibitors on proliferation, assessed the activity of the mTOR pathway and measured the mitochondrial activity as well as levels of radical oxygen species (ROS) of human as well as murine CSCs in vitro. Further, we examined basal levels of mediators of hypoxic signaling such as HIF1α and its effectors.We isolated CSC like cells from the human PCa cell line DU145 and the murine PCa cell line TRAMPC1 using FACS according to their expression of the stem cell markers CD44, CD49f and Sca-1. We used sphere formation assays to confirm stem and progenitor cell properties. Next, we treated the CSC and non-CSC cell populations with mTOR inhibitors under normoxic and hypoxic conditions in order to determine their cell viability at time points up to 72h. Mitochondrial activity was measured using extracellular flux (XF) analysis and levels of ROS were obtained using ROS-specific fluorescent dyes and flow cytometry. Our results suggest that CSC like PCa cells are more resistant to mTOR inhibitors when compared to their non-CSC counterparts. Most interestingly, while the non-CSC population displays higher sensitivity to mTOR inhibitors under hypoxic conditions, the viability of CSCs remains unaffected. Immunoblotting uncovers generally lower mTOR activity in CSCs compared to non-CSCs. Hypoxia leads to a decrease in mTOR activity in both subpopulations, though this effect is stronger in CSC like subpopulations through higher HIF1α-mediated negative feedback in both cell lines studied. At the same time we do not observe alterations in signaling through AR or PI3K downstream effectors besides mTOR, such as AKT. We propose that prostate CSCs might be resistant against mTOR inhibition and that inhibitors targeting multiple kinases along the PI3K/AKT/mTOR axis would be more effective. Our findings could be helpful to assess the impact of mTOR targeted therapy in prostate cancer in light of ongoing clinical trials. Disclosure All authors have declared no conflicts of interest.