DESCRIPTION (provided by applicant): Akt is widely activated in a variety of cancers via (i) mutation in the PTEN tumor suppressor, (ii) activating mutations in PI3K, and (ii) through growth factor receptor upregulation/stimulation. Promotion of oncogenic mechanisms downstream of Akt involve stimulation of cellular proliferation and survival through phosphorylation of key substrates. For example, Akt is known to phosphorylate TSC2 to upregulate mTOR, to phosphorylate and inactivate pro-apoptotic Forkhead proteins, and to activate the transcription factor NF-kB (which functions in cell proliferation and survival). A key effector of the Akt pathway is mTOR, itself a kinase that functions in the TORC1 complex. Importantly, Akt is required for the development and progression of several animal models of cancer, and inhibition of mTOR with rapamycin blocks tumor growth induced by Akt. Additionally, a distinct mTOR complex (TORC2) containing the protein Rictor has been demonstrated to be the PDK2 activity that controls Akt activation via phosphorylation on ser473. Rapamycin, which blocks mTOR activity, has shown efficacy in certain cancers, but has failed in others due to a loss of a negative feedback pathway on IRS-1 and Akt activation. Thus, it is of extreme importance to understand potential regulatory mechanisms that could control both mTOR and Akt activation in cancers. We have published that IKK?, an upstream regulator of NF-kB pathway which is associated with oncogenesis, controls TORC1 activity in PTEN-null/inactive prostate cancers. A recently published paper from our group demonstrates that this interaction, reciprocally, leads to an mTOR/Raptor-dependent control of NF-kB activation controlling the expression of anti-apoptotic genes. Additionally, our preliminary data shows that IKK? associates with the TORC2 complex in PC3 prostate cancer cells to control Akt S473 phosphorylation and kinase activity. Thus, IKK? functions to regulate mTOR activity in both TORC1 and TORC2, raising the potential that IKK? inhibition could function to inhibit both TORC1 and TORC2, circumventing problems associated with rapamycin and its effects on feedback control of Akt activation. We propose to: (i) characterize mechanisms whereby IKK? controls TORC2 activity, determining if a specific IKK? inhibitor will block both TORC1 and TORC2 activity, (ii) determine if IKK??inhibition is cytotoxic/cytostatic in cancer cells and if this will block the loss of feedback control on IRS-1/Akt, and (iii) determine if genetic and pharmacologic inhibition of IKK? and/or IKK? will block progression and development of cancer in a PTEN-loss model of prostate cancer. As a human correlate, studies will be performed on primary human glioblastoma explants. The studies may identify a single mechanism to suppress mTOR and Akt activity in cancer. The studies are also the first to use a specific IKK? inhibitor for cancer studies.

PUBLIC HEALTH RELEVANCE: Deregulated of Akt is implicated in the pathogenesis of a number of cancers. Based on this, the Akt pathway has emerged as a key regulator of oncogenesis and cancer therapy resistance in a variety of tumors. Akt functions to promote growth and survival through upregulation of mTOR and through mTOR-independent pathways, which include phosphorylation of targets which control apoptosis. Targeting mTOR with rapamycin, or analogs, is efficacious in some tumors but ineffective in many others due, in part, to loss of a negative feedback control on IRS-1 which then leads to Akt activation. Based on these observations, the development of inhibitors that blocks both mTOR and Akt activation may prove effective in a number of cancers. We have recently published that IKK? is a key regulator of mTOR (TORC1) in Akt-active cancer cells. Additionally, we find that mTOR (TORC1) regulates IKK/NF-kB activity to induce expression of genes associated with cancer therapy resistance, demonstrating a novel function for the rapamycin-sensitive form of mTOR. We have now shown that IKK? associates with the TORC2 complex to regulate its ability to control Akt activity. Experiments are proposed to dissect the mechanism whereby IKK? regulates TORC2 activity in cancer cells and potentially downstream of growth factor-induced signaling. We hypothesize that inhibition of IKK? (using genetic and pharmacologic approaches) will block progression of cancers that depend on Akt and mTOR and will overcome cancer therapy resistance to rapamycin. Cell-based and animal model studies are proposed to test our hypotheses. The work is clinically relevant based on the following points: (i) we propose the use of a PTEN fl/fl model for prostate cancer which mimics the loss of PTEN seen in human cancers, including prostate cancer, (ii) the studies may reveal a mechanism to significantly and broadly enhance the efficacy of rapamycin by suppressing the downstream effects associated with the loss of feedback control on IRS-1 when TORC1 is inhibited, (iii) these studies will be the first to use a highly specific IKK? inhibitor in cancer models, (iv) we analyze primary human glioblastoma explants for therapeutic and mechanistic studies to test/validate our hypotheses, and (v) the results may reveal a single mechanism to simultaneously suppress TORC1 activity and Akt activation (via TORC2) in cancer.