Published OnlineFirst March 13, 2014; DOI: 10.1158/0008-5472.CAN-13-2946 Cancer Therapeutics, Targets, and Chemical Biology Research Maintaining Glycogen Synthase Kinase-3 Activity Is Critical for mTOR Kinase Inhibitors to Inhibit Cancer Cell Growth Junghui Koo1, Ping Yue1, Anthony A. Gal2, Fadlo R. Khuri1, and Shi-Yong Sun1 Abstract mTOR kinase inhibitors that target both mTORC1 and mTORC2 are being evaluated in cancer clinical trials. Here, we report that glycogen synthase kinase-3 (GSK3) is a critical determinant for the therapeutic response to this class of experimental drugs. Pharmacologic inhibition of GSK3 antagonized their suppressive effects on the growth of cancer cells similarly to genetic attenuation of GSK3. Conversely, expression of a constitutively activated form of GSK3b sensitized cancer cells to mTOR inhibition. Consistent with these findings, higher basal levels of GSK3 activity in a panel of human lung cancer cell lines correlated with more efficacious responses. Mechanistic investigations showed that mTOR kinase inhibitors reduced cyclin D1 levels in a GSK3b-dependent manner, independent of their effects on suppressing mTORC1 signaling and cap binding. Notably, selective inhibition of mTORC2 triggered proteasome-mediated cyclin D1 degradation, suggesting that mTORC2 blockade is responsible for GSK3-dependent reduction of cyclin D1. Silencing expression of the ubiquitin E3 ligase FBX4 rescued this reduction, implicating FBX4 in mediating this effect of mTOR inhibition. Together, our findings define a novel mechanism by which mTORC2 promotes cell growth, with potential implications for under- standing the clinical action of mTOR kinase inhibitors. Cancer Res; 74(9); 2555–68. Ó2014 AACR. Introduction with weak activity against mTORC2. Although some rapalogs The mTOR, a serine–threonine protein kinase related tightly (e.g., everolimus) are approved by the U.S. Food and Drug to the family of the phosphoinositide 3-kinase–related kinases, Administration for the treatment of advanced renal cell cancer exerts different biologic functions primarily through forming and pancreatic neuroendocrine tumors, the single-agent activ- two complexes with the essential partner protein raptor ity of rapalogs in most other tumor types has been modest at (mTOR complex 1; mTORC1) and rictor (mTOR complex 2; best (4). Hence, great efforts have been made to identify novel mTORC2; refs. 1, 2). Compared with the mTORC1, which is mTOR inhibitors that suppress both mTORC1 and mTORC2 involved in regulation of many key cellular processes, including activity. As a result, several ATP-competitive inhibitors of cell growth and metabolism primarily via regulating cap- mTOR kinase, including INK128 and PP242, have been devel- dependent protein translation initiation, relatively little is oped and are being tested in clinical trials (5, 6). These mTOR known about the biologic functions of the mTORC2 other kinase inhibitors (TORKinibs) in general more dramatically than its regulation of cytoskeleton and Akt-mediated cell inhibit protein synthesis, suppress Akt phosphorylation, and survival (2). Nonetheless, mTOR signaling is dysregulated in induce G1 arrest and/or apoptosis in some cancer cells than the – various types of human cancers and, hence, has emerged as an conventional allosteric mTOR inhibitor, rapamycin (7 10). A in vivo attractive cancer therapeutic target (3). robust anticancer activity of these inhibitors against The conventional mTOR inhibitors, rapamycin and its ana- certain types of cancers was also observed (8, 11, 12). Some logs (rapalogs), are specific allosteric inhibitors of mTORC1 TORKinibs have been tested in clinical trials (5, 6). Therefore, these TORKinibs not only represent novel potential cancer therapeutic agents, but also are valuable research tools for understanding the biology of mTORCs. fi 1 Authors' Af liations: Departments of Hematology and Medical Oncology Glycogen synthase kinase-3 (GSK3) is a ubiquitous serine/ and 2Pathology, Emory University School of Medicine and Winship Cancer Institute, Atlanta, Georgia threonine kinase that is present in mammals in two isoforms: a and b (13). GSK3 was initially identified as an enzyme involved Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). in the regulation of glycogen metabolism. Increasing evidence during the past decades indicates that GSK3 has a key role in Note: F.R. Khuri and S.-Y. Sun are Georgia Research Alliance Distinguished Cancer Scientists. regulating a diverse range of cellular functions, including cell survival and death (13). Thus, GSK3 inhibition has been con- Corresponding Author: Shi-Yong Sun, Emory University School of Med- icine and Winship Cancer Institute, 1365-C Clifton Road NE, C3088, sidered an attractive therapeutic strategy for certain diseases Atlanta, GA 30322. Phone: 404-778-2170; Fax: 404-778-5520; E-mail: such as diabetes, neurodegenerative diseases, and mental dis- [email protected] orders (14, 15). GSK3 has been implicated in the regulation of doi: 10.1158/0008-5472.CAN-13-2946 oncogenesis with complex patterns: It acts paradoxically as a Ó2014 American Association for Cancer Research. tumor suppressor in some cancer types while potentiating www.aacrjournals.org 2555 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst March 13, 2014; DOI: 10.1158/0008-5472.CAN-13-2946 Koo et al. growth of cancer cells in others (16, 17). One well-known have not been authenticated. These cell lines were cultured important cancer-related function of GSK3 is to positively in RPMI-1640 or Dulbecco's Modified Eagle Medium con- regulate proteasomal degradation of several oncogenic pro- taining 5% fetal calf serum at 37C in a humidified atmo- – teins such as c-Myc, c-Jun, cyclin E, Mcl-1, and cyclin D (18 20). sphere of 5% CO2 and 95% air. Plasmid transfection was For example, GSK3-dependent cyclin D1 phosphorylation is conducted in H1299 or HEK-293T cells largely because of required for cyclin D1 degradation mediated by the E3 ubiqui- their high transfection efficiency. tin ligase FBX4 (20, 21). It has been suggested that GSK3 can inhibit the mTOR Cell growth assay pathway by phosphorylating TSC2 in a manner dependent on Cells were seeded in 96-well cell culture plates and treated AMPK-priming phosphorylation (22). A recent study has the next day with the given agents. Viable cell numbers were shown that GSK3 phosphorylates the turn motif of p70S6K determined using a sulforhodamine B (SRB) assay as described and cooperates with mTOR to control the activity of p70S6K previously (26). Combination index (CI) for drug interaction and cell proliferation (23), thus providing a rationale for (e.g., synergy) was calculated using the CompuSyn software cotargeting mTOR and GSK3 to treat diseases such as cancer. (ComboSyn, Inc.). The IC50, which represents the concentra- In a longstanding effort to identify strategies or agents that can tion required for 50% growth inhibition, was estimated from a potentially enhance the therapeutic efficacy of mTOR inhibi- concentration-dependent growth curve. tors in cancer therapy, we unexpectedly found that the activity of GSK3 is crucial for TORKinibs to exert their inhibitory effects Cell-cycle analysis on the growth of cancer cells. Thus, this work has focused on Cells were harvested after a given treatment and stained demonstrating the impact of GSK3 on the therapeutic activity with propidium iodide for cell-cycle analysis as described of TORKinibs against cancer cells and on understanding the previously (27). underlying mechanisms. Colony formation assay Materials and Methods The effects of the given drugs on colony formation on plates Reagents were measured as previously described (28). PP242, INK128, and AZD8055 were purchased from Active Biochem. Torin 1 was purchased from Tocris. The GSK3 inhib- Western blot analysis itor SB216763, the proteasome inhibitor MG132, and the protein Preparation of whole-cell protein lysates and Western blot synthesis inhibitor cycloheximide (CHX) were purchased from analysis was performed as described previously (28). Sigma Chemical Co. The NEDD8-activating enzyme inhibitor Gene knockdown by siRNA or small hairpin RNA MLN4924 was provided by Millennium Pharmaceuticals, Inc. 0 0 Cyclin D1, p-GSK3a/b (S21/9), p-AKT (S473), AKT, p-S6 (S235/ Rictor #1 (5 -AAGCAGCCTTGAACTGTTTAA-3 ), rictor #2 (50-AAACTTGTGAAGAATCGTATC-30), raptor #1 (50-AAGGC- 236), and S6 antibodies were purchased from Cell Signaling 0 0 Technology, Inc. GSK3a/b antibody was purchased from TAGTCTGTTTCGAAAT-3 ), raptor #2 (5 -AAGGACAACGGC- 0 a 0 Upstate/EMD Millipore. Polyclonal rictor and raptor antibodies CACAAGTAC-3 ), GSK-3 (5 -AAGTGATTGGCAATGGCT- 0 b 0 0 were purchased from Bethyl Laboratories, Inc. Both polyclonal CAT-3 ), and GSK-3 (5 -AAGTAATCCACCTCTGGCTAC-3 ) a b and monoclonal actin antibodies were purchased from Sigma siRNAs were synthesized by Qiagen. GSK3 / siRNA (#6301) Chemical Co. Myc-tagged constitutively active form of were purchased from Cell Signaling Technology, Inc. The GSK3b (GSK3bCA; ref. 24) was provided by Dr. Binhua P. nonsilencing control siRNA duplexes were described previous- Zhou (The University of Kentucky, College of Medicine, ly (29). Transfection of these siRNA duplexes was conducted in Lexington, Kentucky). Flag-cyclin D1 expression plasmid 6-well plates using the Lipofectamine 2000 transfection was provided by Dr. Alan Diehl (Abramson Family