Abstract LB-201: Prostate cancer stem cells display low mTOR activity and resistance to mTOR inhibitors in hypoxia
Authors: Marhold Maximilian , Tomasich Erwin , Hofbauer Günther , Krainer Michael , Horak Peter 
Published: August 2012

Abstract
Tumor-initiating subpopulations of cancer cells, also known as cancer stem cells (CSCs), were recently identified and characterized in prostate cancer (PCa). We hypothesized that one of the frequently altered molecular signaling pathways in PCa, the PI3K/AKT pathway, and mammalian target of rapamycin (mTOR) in particular, play a role in PCa stem cell maintenance. In addition, hypoxia is known to be involved in cancer stem cell maintenance and to have a regulating effect on mTOR signaling. In our study, we evaluated the effects of mTOR inhibitors on proliferation of human as well as murine CSCs in hypoxic conditions in vitro. We isolated CSC like subpopulations from the androgen independent human prostate cancer cell line DU145 using fluorescence activated cell sorting according to their expression of the stem cell marker CD44. Further, we sorted the murine prostate cancer cell line TRAMP-C1 based on the expression of the stem cell markers Sca-1 and CD49f. We used sphere formation assays to confirm the stem and progenitor cell properties of the sorted subpopulations. Next, we treated the CSC and non-CSC cell populations with mTOR inhibitors rapamycin and everolimus under normoxic (20% O2) and hypoxic (3% O2) conditions in order to determine their viability at time points up to 72 hours. Our results suggest that CSC like prostate cancer cells with elevated expression of stem cell markers, such as CD44, Sca-1 or CD49f, are more resistant to mTOR inhibitors when compared to their non-CSC counterparts. Most interestingly, while the non-CSC subpopulation of prostate cancer cells displays increased sensitivity to mTOR inhibitors under hypoxic conditions, the viability of CSCs remains essentially unaffected. Immunoblotting reveals lower mTOR activity in prostate cancer stem cells compared to non-CSCs. Hypoxia in this context leads, as expected, to a decrease in mTOR activity in both subpopulations, though this effect is significantly pronounced in CSCs like subpopulations of both cancer cell lines in our study. At the same time, we do not observe alterations in signaling through other PI3K downstream effectors, such as AKT. We propose that prostate cancer stem cells might be resistant against small molecule inhibitors of mTOR and that inhibitors targeting multiple kinases along the PI3K/AKT/mTOR axis would be more effective in this setting. We also hypothesize that intratumoral hypoxia might contribute to the mTOR resistance of prostate cancer stem cells by inhibiting mTOR signaling and promoting cell growth through other, PI3K dependent pathways. These findings could be helpful to assess the impact of mTOR targeted therapy in prostate cancer, notably in light of ongoing clinical trials of mTOR inhibitors in this entity.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-201. doi:1538-7445.AM2012-LB-201