Signal Transduction HIF1a Regulates mTOR Signaling and Viability of Prostate Cancer Stem Cells Maximilian Marhold 1 , Erwin Tomasich 1 , Ahmed El-Gazzar 1 , Gerwin Heller 1 , Andreas Spittler 2 , Reinhard Horvat 3 , Michael Krainer 1 , and Peter Horak 1 Abstract Tumor-initiating subpopulations of cancer cells, also known as cancer stem cells (CSC), were recently identified and characterized in prostate cancer. A well-characterized murine model of prostate cancer was used to investigate the regulation of hypoxia-inducible factor 1a (HIF1A/HIF1a) in CSCs and a basal stem cell subpop- ulation (Lin  /Sca-1 þ /CD49f þ ) was identified, in primary prostate tumors of mice, with elevated HIF1a expression. To further analyze the consequences of hypoxic upregulation on stem cell prolifera- tion and HIF1a signaling, CSC subpopulations from murine TRAMP-C1 cells (Sca-1 þ /CD49f þ ) as well as from a human prostate cancer cell line (CD44 þ /CD49f þ ) were isolated and characterized. HIF1a levels and HIF target gene expression were elevated in hypoxic CSC-like cells, and upregulation of AKT occurred through a mechanism involving an mTOR/S6K/IRS-1 feedback loop. Inter- estingly, resistance of prostate CSCs to selective mTOR inhibitors was observed because of HIF1a upregulation. Thus, prostate CSCs show a hypoxic deactivation of a feedback inhibition of AKT signaling through IRS-1. In light of these results, we propose that deregulation of the PI3K/AKT/mTOR pathway through HIF1a is critical for CSC quiescence and maintenance by attenuating CSC metabolism and growth via mTOR and promoting survival by AKT signaling. We also propose that prostate CSCs can exhibit primary drug resistance to selective mTOR inhibitors. Implications: This work contributes to a deeper understanding of hypoxic regulatory mechanisms in CSCs and will help devise novel therapies against prostate cancer. Mol Cancer Res; 13(3); 1–9. Ó2014 AACR. Introduction On the basis of the cell-of-origin hypothesis, prostate cancer is one of the tumors known to harbor a distinct subpopulation of cancer stem (or initiating) cells (CSC), which are thought to confer resistance to common therapeutic measures, be it chemo- therapy or radiotherapy (1, 2). The exact origin of prostate CSCs is widely discussed, as basal and luminal prostate epithelial cells are both capable of developing into prostate CSCs and play a role in tumorigenesis (3–8). On the basis of their expression of specific surface markers, several subsets of human prostate CSCs, either of basal or luminal origin, were identified (9–11). Prostate CSCs have also been isolated from the mouse prostate, based on the expression of integrin a6/CD49f and stem cell antigen 1 (Sca-1; refs. 12–14). The jury is still out on which of these models best represents human disease (15). Recently, HIF1a has been shown to maintain the stemness of hematopoietic stem cells (HSC) in the hypoxic niche of the bone marrow (16). In parallel, it seems that prostate cancer cells target this HSC niche, leading to metastatic disease (17). Conversely, hypoxia leads to an increased metastatic potential of prostate cancer cells (18). Low oxygen levels, as present in the tumor microenvironment of solid tumors, result in the stabilization of the HIF1a protein by posttranslational mechanisms. HIF1a forms a heterodimeric transcriptional complex with HIF1b, trans- locates to the nucleus, and binds hypoxia-responsive elements found in the promoter regions of multiple downstream target genes, activating various adaptive pathways. Among many others, the PI3K/mTOR signaling pathway is regulated by hypoxic sig- naling (19). This pathway integrates growth factor signaling, cell metabolism, as well as diverse cellular stressors and modulates the adaptation of cell proliferation, apoptosis, autophagy, and pro- tein translation (20) and has a central role in prostate carcino- genesis (21). mTOR functions as a nutrient/hypoxia sensor and controls protein synthesis by phosphorylation of its two main targets, p70-S6 kinase 1 (S6K1) and the eIF4E-binding protein 1 (4E-BP1). Its central role makes it a bona fide target for molecular therapy, and its inhibition has been shown to be effective in renal cell and breast cancer (22, 23). We hypothesized that prostate CSCs may exhibit differential hypoxic signaling. Given the complex regulation of mTOR in hyp- oxia through multiple mechanisms and feedback loops, this might in turn affect their viability, stemness, and metastatic potential. Materials and Methods Cell culture and conditions The DU145 and TRAMP-C1 cancer cell lines were obtained from ATCC. DU145 cells were cultivated in RPMI-1640 growth 1 Department of Internal Medicine I and Comprehensive Cancer Center, Medical University of Vienna,Vienna, Austria. 2 Department of Surgery and Core Facility Flow Cytometry, Medical University of Vienna, Vienna, Austria. 3 Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). E. Tomasich and A. El-Gazzar contributed equally to this article. Corresponding Author: Peter Horak, Department of Internal Medicine I and Comprehensive Cancer Center, Medical University of Vienna, Waehringer Guer- tel 18-20, A-1090 Vienna, Austria. Phone: 43-1-40400-73792; Fax: 43-1-40400- 1685; E-mail: peter.horak@meduniwien.ac.at doi: 10.1158/1541-7786.MCR-14-0153-T Ó2014 American Association for Cancer Research. Molecular Cancer Research www.aacrjournals.org OF1