ARTICLES 594 VOLUME 10 | NUMBER 6 | JUNE 2004 NATURE MEDICINE Inactivation of the tumor-suppressor gene PTEN occurs in glioblas- toma multiforme, endometrial cancer and prostate cancer, among others. The tumor-suppressor function of PTEN is linked to its lipid phosphatase activity; loss of this activity leads to accumulation of its substrate, phosphatidylinositol 3,4,5-trisphosphate, and activation of the PI3K signaling pathway 1 . One consequence of PTEN loss is hyper- activation of the oncogenic serine/threonine kinase AKT and phos- phorylation of downstream AKT substrates, including BAD 2 , FOXO proteins 3,4 and GSK3 (ref. 5). Phosphorylation and inactivation of these proteins can lead to enhanced cell survival, increased cell prolif- eration and altered cellular metabolism. PI3K signaling has been implicated in the regulation of mTOR (in mouse, encoded by the gene Frap1) and S6K. In Drosophila melanogaster, loss of Tor is epista- tic to loss of Pten, and in mouse and cell-based models loss of PTEN sensitizes cells to mTOR inhibition 6–10 . This pathway has been further elucidated through studies in D. melanogaster and mammalian cells showing that tuberin, the protein product of Tsc2, regulates Tor and is an AKT substrate. Thus, AKT-dependent phosphorylation inhibits tuberin, leading to activation of mTOR and S6K 11–15 . On the basis of these cumulative observations, derivatives of the mTOR inhibitor rapamycin are being tested in clinical trials in patients with cancer 16 . We previously showed that a probasin promoter-myr-HA-AKT1 transgene directs production of activated AKT1 spatially restricted to the luminal epithelial cells of the mouse ventral prostrate and, as a result, these mice develop a highly penetrant prostatic intraepithelial neoplasia (PIN) phenotype 17 . The phenotype bears many of the hall- marks of mTOR activation including increased cell number, increased cell size, and activation of the downstream kinase S6K, together suggesting that activation of mTOR downstream of AKT may be linked to the development of PIN in these mice. Here, we show that the Akt-induced PIN phenotype is completely dependent on mTOR. Specifically, treatment with the mTOR inhibitor RAD001 led to a rapid loss of intraluminal epithelial cells marked by the induc- tion of apoptosis, and reversed the PIN phenotype within 14 d. Mice carrying both AKT1 and BCL2 transgenes were resistant to RAD001- induced apoptosis and had a PIN phenotype partially resistant to RAD001. Further analysis of this partial resistance showed that Hif- 1α target genes, including those encoding enzymes essential for gly- colysis, were the principal constituents of the transcriptional response to elevated Akt activity and of the response to mTOR inhibition. These data suggest that the response to mTOR inhibition is mediated through independent apoptotic and Hif-1α regulatory pathways. 1 Departments of Medical Oncology, Pediatric Oncology and Biostatistical Sciences, Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA. 2 Departments of Medicine and Pathology, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Street, Boston, Massachusetts 02115, USA. 3 Novartis Pharmaceutical Corporation, One Health Plaza, East Hanover, New Jersey 07936, USA. 4 Department of Molecular Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA. 5 The Broad Institute at Harvard and MIT, 320 Charles Street, Cambridge, Massachusetts 02142, USA. 6 Novartis Institute for Biomedical Research, Oncology, CH-4002 Basel, Switzerland. Correspondence should be addressed to W.R.S. (william_sellers@dfci.harvard.edu). Published online 23 May 2004; doi:10.1038/nm1052 mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways Pradip K Majumder 1,2 , Phillip G Febbo 1,2 , Rachel Bikoff 1,2 , Raanan Berger 1,2 , Qi Xue 1,2 , Louis M McMahon 3 , Judith Manola 1 , James Brugarolas 1,2 , Timothy J McDonnell 4 , Todd R Golub 1,2,5 , Massimo Loda 1,2 , Heidi A Lane 6 & William R Sellers 1,2,5 Loss of PTEN function leads to activation of phosphoinositide 3-kinase (PI3K) signaling and Akt. Clinical trials are now testing whether mammalian target of rapamycin (mTOR) inhibition is useful in treating PTEN-null cancers. Here, we report that mTOR inhibition induced apoptosis of epithelial cells and the complete reversal of a neoplastic phenotype in the prostate of mice expressing human AKT1 in the ventral prostate. Induction of cell death required the mitochondrial pathway, as prostate-specific coexpression of BCL2 blocked apoptosis. Thus, there is an mTOR-dependent survival signal required downstream of Akt. Bcl2 expression, however, only partially restored intraluminal cell growth in the setting of mTOR inhibition. Expression profiling showed that Hif-1α targets, including genes encoding most glycolytic enzymes, constituted the dominant transcriptional response to AKT activation and mTOR inhibition. These data suggest that the expansion of AKT-driven prostate epithelial cells requires mTOR-dependent survival signaling and activation of HIF-1α, and that clinical resistance to mTOR inhibitors may emerge through BCL2 expression and/or upregulation of HIF-1α activity. © 2004 Nature Publishing Group http://www.nature.com/naturemedicine