Regulation of Mammalian Target of Rapamycin Activity in PTEN-Inactive Prostate Cancer Cells by IKB Kinase A Han C. Dan, Mazhar Adli, and Albert S. Baldwin Lineberger Comprehensive Cancer Center, Department of Biology, University of North Carolina School of Medicine, Chapel Hill, North Carolina Abstract The mammalian target of rapamycin (mTOR) is a mediator of cell growth, survival, and energy metabolism at least partly through its ability to regulate mRNA translation. mTOR is activated downstream of growth factors, insulin, and Akt- dependent signaling associated with oncoprotein expression or loss of the tumor-suppressor PTEN. In this regard, mTOR activity is associated with cancer cell growth and survival. Here, we have explored an involvement of the IKB kinase (IKK) pathway, associated with nuclear factor-KB activation, in controlling mTOR activity. The experiments show that IKKA controls mTOR kinase activity in Akt-active, PTEN-null prostate cancer cells, with less involvement by IKKB. In these cells, IKKA associates with mTOR, as part of the TORC1 complex, in an Akt-dependent manner. Additionally, IKKA is required for efficient induction of mTOR activity downstream of constitutively active Akt expression. The results indicate a novel role for IKKA in controlling mTOR function in cancer cells with constitutive Akt activity. [Cancer Res 2007;67(13):6263–9] Introduction The mammalian target of rapamycin (mTOR) is a Ser/Thr kinase that is activated downstream of increases in amino acid levels, by exposure of cells to growth factors or insulin, in response to hypoxia, by expression/activation of oncoproteins and by the loss of the PTEN tumor suppressor (1–4). mTOR controls cell growth, at least partly, through its ability to phosphorylate S6K and 4EBP1, key regulators of mRNA translation. The control of mTOR downstream of insulin-dependent signaling involves the Ser/Thr kinase Akt, which regulates mTOR through the ability of this kinase to phosphorylate tuberous sclerosis complex 2 (TSC2; refs. 5, 6). TSC1 and 2 form a complex that exhibits GTPase activity, blocking the activity of Rheb, a GTPase that controls mTOR activation (7). Mutations in the TSC genes lead to tuberous sclerosis, a disease characterized by the appearance of benign tumors. mTOR interacts with the regulatory protein Raptor to comprise a rapamycin-sensitive complex (TORC1) that controls mTOR downstream functions (8, 9). A second mTOR complex (TORC2) contains the protein Rictor and this complex has been shown to function as the Pdk2 activity to phosphorylate Akt on Ser 473 (10). Importantly, although Raptor has been shown to be required for mTOR function in the TORC1 complex, a strong association between Raptor and mTOR has been proposed to negatively regulate mTOR activity (8). An additional protein termed GhL associates with mTOR to control mTOR kinase activity via stabilization of mTOR-Raptor interaction (11). The serine-threonine kinase Akt is widely activated in human cancers, where it controls suppression of apoptosis, cell growth and proliferation, and energy metabolism (4). In cancers, Akt is constitutively activated downstream of growth factor receptor signaling, through activating mutations in phosphatidylinositol 3-kinase (PI3K), or after PTEN loss of expression or mutation (4, 12–15). The initiating step in Akt activation is the binding of PIP3 to the PH domain of Akt, leading to translocation of Akt to the cell membrane where it is activated by phosphorylation through PDK1 (16, 17) and by a second activity termed PDK2 (10). Two important downstream effectors of Akt are the FOXO family of transcription factors and mTOR (4). Thus, mTOR activation is associated with a number of cancers, including those that are Akt active (15, 18). The nuclear factor-nB (NF-nB) pathway is activated downstream of a variety of inflammatory mediators and growth factor pathways (19, 20). NF-nB activation in most signal transduction cascades uses the IKK complex, containing IKKa, IKKh, and IKKg. IKKa and IKKh drive the catalytic activity of IKK, with IKKh appearing to be dominant in inflammatory-mediated pathways and IKKa func- tioning in the so-called alternative pathway (19, 21). The IKK complex phosphorylates the inhibitory InB proteins leading to their ubiquitination and subsequent proteasome-dependent degrada- tion. This allows efficient NF-nB nuclear accumulation and binding to target sequences in the promoters and regulatory sequences of genes encoding cytokines, chemokines, and regulators of apoptosis. The dysregulation of NF-nB activity is associated with numerous cancers and with the inflammatory process that promotes cancer (see refs. 20, 22, 23). Interestingly, it has been reported that Akt can activate NF-nB transcriptional activity (24, 25) and nuclear accumulation (26) through a pathway involving IKK. Here, we have explored a relationship between the IKK family of proteins and the regulation of mTOR activation downstream of cancer cell–associated, constitutively active Akt. Expression of IKKa promotes mTOR activity and knockdown of IKKa inhibits mTOR in PTEN-inactive prostate cancer cells. The ability of Akt to effectively activate mTOR requires IKKa as shown using IKKaÀ/À mouse embryonic fibroblast (MEF). Our data indicate that the IKKa subunit interacts with mTOR strongly in PTEN null prostate cancer cells in a manner that is dependent on Akt activity. In this setting, IKKa is required for mTOR activity, controlling its kinase activity. IKKa also regulates protein synthesis rates, paralleling its effects on mTOR function. The results show a new and unexpected role for IKKa in controlling the phenotype of cancer cells with constitutively active Akt. Materials and Methods Antibodies and reagents. Antibodies were obtained from the following sources. Antibodies against IKKa, IKKh, and mTOR were obtained from Upstate Biotechnology. Raptor and Rictor antibodies were obtained from Note: A. Baldwin is an investigator of the Samuel Waxman Cancer Research Foundation. Requests for reprints: Albert S. Baldwin, Lineberger Comprehensive Cancer Center, CB7295, University of North Carolina, Chapel Hill, NC 27599. Phone: 919-966- 3652; Fax: 919-966-0444; E-mail: abaldwin@med.unc.edu. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-1232 www.aacrjournals.org 6263 Cancer Res 2007; 67: (13). July 1, 2007 Research Article Research. on January 3, 2016. © 2007 American Association for Cancer cancerres.aacrjournals.org Downloaded from