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Ampka2 Regulates Bladder Cancer Growth Through SKP2-Mediated Degradation of P27 Stavros Kopsiaftis1,2, Katie L

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Ampka2 Regulates Bladder Cancer Growth Through SKP2-Mediated Degradation of P27 Stavros Kopsiaftis1,2, Katie L Published OnlineFirst September 16, 2016; DOI: 10.1158/1541-7786.MCR-16-0111 Cell Cycle and Senescence Molecular Cancer Research AMPKa2 Regulates Bladder Cancer Growth through SKP2-Mediated Degradation of p27 Stavros Kopsiaftis1,2, Katie L. Sullivan1,2, Isha Garg1,2, John A. Taylor III3, and Kevin P. Claffey1,2,4 Abstract AMP-activated protein kinase (AMPK) is the central metabolic proliferation and decreased p27 protein. The reduced p27 protein regulator of the cell and controls energy consumption based upon was determined to be dependent upon SKP2. Additionally, loss of nutrient availability. Due to its role in energy regulation, AMPK AMPKa2 in a xenograft and a chemical carcinogen model of has been implicated as a barrier for cancer progression and is bladder cancer resulted in larger tumors with less p27 protein suppressed in multiple cancers. To examine whether AMPK and high SKP2 levels. Consistent with the regulation observed regulates bladder cancer cell growth, HTB2 and HT1376 bladder in the bladder cancer model systems, a comprehensive survey cells were treated with an AMPK activator, 5-aminoimidazole-4- of human primary bladder cancer clinical specimens revealed carboxamide ribonucleotide (AICAR). AICAR treatment reduced low levels of AMPKa2 and p27 and high levels of SKP2. proliferation and induced the expression of p27Kip1 (CDKN1B), which was mediated through an mTOR-dependent mechanism. Implications: These results highlight the contribution of Interestingly, AMPKa2 knockdown resulted in reduced p27 AMPKa2 as a mechanism for controlling bladder cancer growth levels, whereas AMPKa1 suppression did not. To further deter- by regulating proliferation through mTOR suppression and mine the exact mechanism by which AMPKa2 regulates p27, induction of p27 protein levels, thus indicating how AMPKa2 HTB2 and HT1376 cells were transduced with an shRNA targeting loss may contribute to tumorigenesis. Mol Cancer Res; 14(12); AMPKa2. Stable knockdown of AMPKa2 resulted in increased 1182–94. Ó2016 AACR. Introduction Due to its role in responding to cellular energy and controlling the switch from anabolic to catabolic processes in response to Bladder cancer is currently the fifth most commonly diagnosed low nutrients, AMPK has long been thought to function to cancer with average age of onset of 71 years old. It is also one of inhibit tumorigenesis. In many cancers, the activation of AMPK the most expensive cancers to treat due to lifelong surveillance results in decreased proliferation and survival of cancer cells and invasive procedures (1, 2). Adenosine monophosphate (10–12). Additionally, it has been reported that AMPK activa- activated protein kinase (AMPK) is a critical protein in the cell tion and/or AMPKa2 protein or mRNA is diminished in breast that signals to control proliferation and protein translation under cancer, hepatocellular carcinoma, and gastric cancer (13–17). cellular stress (3, 4). Once activated by an upstream kinase such Although there are little data on the selective functions of the as STK11, AMPK signaling negatively regulates MTOR (mTOR) AMPKa isoforms, emerging evidence suggests that the proteins to halt protein translation and proliferation when energy avail- may indeed have some selective functions. For example, ability is low (5–7). AMPK is composed of a, b, and g subunits. AMPKa2 has been shown to localize to the nucleus and can The a subunit, which is composed of two isoforms (PRKAA1/ phosphorylate nuclear targets, whereas AMPKa1 does not AMPKa1orPRKAA2/AMPKa2), functions as the catalytic sub- (18, 19). Additionally, Phoenix and colleagues demonstrated unit, while the b and g subunits function as a regulatory and À À that AMPKa2 / mouse embryonic fibroblasts that were trans- AMP-sensing subunit, respectively (8, 9). formed with H-RasV12 formed tumors in a xenograft, while À À the AMPKa1 / mouse embryonic fibroblasts did not (20). À/À 1Center for Vascular Biology, University of Connecticut Health Center, AMPKa2 vascular smooth muscle cells also displayed Farmington Connecticut. 2Department of Cell Biology, University of increased proliferation and a reduction in CDKN1B (p27) Connecticut Health Center, Farmington Connecticut. 3Department of Surgery, University of Connecticut Health Center, Farmington, Con- protein (21). All of these data demonstrate that AMPKa2may necticut. 4Neag Comprehensive Cancer Center, University of Connec- be more selective for controlling p27 protein levels and pro- ticut Health Center, Farmington Connecticut. liferation than AMPKa1. Note: Supplementary data for this article are available at Molecular Cancer The cell-cycle inhibitor protein p27 is a critical protein for Research Online (http://mcr.aacrjournals.org/). controlling cellular proliferation, and its loss has been heavily Corresponding Author: Kevin P. Claffey, University of Connecticut Health implicated in tumorigenesis. Low expression of p27 protein in Center, 263 Farmington Avenue, Farmington, CT 06030. Phone: 860-679- many different cancer tissues, including bladder cancer, has 8713; Fax: 860-679-1201 E-mail: [email protected] been shown to be associated with worse survival and invasive doi: 10.1158/1541-7786.MCR-16-0111 disease (22–24). Under normal conditions, most urothelial cells express p27 due to their slow proliferative rate. In a Ó2016 American Association for Cancer Research. À À chemical carcinogenesis model of bladder cancer, p27 / mice 1182 Mol Cancer Res; 14(12) December 2016 Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst September 16, 2016; DOI: 10.1158/1541-7786.MCR-16-0111 AMPKa2 Regulates Bladder Cancer Growth through p27 develop bladder cancer at a much earlier time point than their staining solution (50 mg/mL PI and 100 mg/mL RNase A diluted wild-type counter parts due to the critical role of p27 in in PBS) for 30 minutes before analysis by flow cytometry. controlling urothelial proliferation (25). These experiments demonstrate how crucial p27 is in controlling bladder cancer AICAR, compound C, and rapamycin treatments and cell growth. The exact mechanism for loss of p27 function immunoblot analysis in bladder cancer is currently unknown. One of the major HTB2 and HT1376 cells were plated and 18 hours later sub- mechanisms governing p27 regulation is S-phase kinase-asso- sequently re-fed and treated with 0.5 mmol/L AICAR, 5 nmol/L ciated protein 2 (SKP2) mediated degradation (26). Although rapamycin or 5 mmol/L compound C for a total of 24 hours. there are no studies correlating the expression status of SKP2 Whole-cell lysates were then harvested and analyzed by immu- and p27 in bladder cancer, independent studies have shown noblot as described previously (14). Immunoblots were visual- that p27 is downregulated in invasive bladder cancer and SKP2 ized using ECL reagents (Millipore) and developed on a Kodak is upregulated (27). Therefore, this study seeks to determine Multimodal Imager (2000MM). Signal was quantified by select- theimpactofAMPKa activation on bladder cancer prolifera- ing each band and measuring total photon counts using the tion and whether this is dependent on or mediated in part by Kodak imaging software. Immunoblots are representative of at SKP2-mediated degradation and control of p27 protein. least three distinct experiments. All blots signals are normalized to the b-actin representative for that experiment. Materials and Methods Immunofluorescence Cell lines and reagents HTB2 and HT1376 cells transduced with either shGFP or Cell lines were purchased and maintained according to the shAMPKa2 were plated in a 6-well dish containing glass cover ATCC. Cells obtained from ATCC were frozen down within 5 slips and allowed to grow for 48 hours. Cells were then fixed and passages of being received, and aliquots were not cultured for stained using a p27 antibody. Nuclei were visualized by staining more than 15 passages. Rapamycin and 5-aminoimidazole-4- with 40,6-diamidino-2-phenylindole (DAPI). Cover slips were carboxamide ribonucleotide (AICAR) were obtained from mounted with Prolong gold antifade reagent (Invitrogen) and Cayman Chemical. Compound C was obtained from EMD Milli- visualized on a Zeiss Axio Observer.Z1 with a axiocam MRm pore. Propidium iodide and RNase A were obtained from Invi- camera using Zen software at 400Â magnification. trogen. Antibodies targeting AMPKa1 and AMPKa2 were Thr172 obtained from U.S. Biological, total AMPKa, pAMPKa , siRNA transfection SKP2, and pS6 were obtained from Cell Signaling Technology, Lipofectamine 2000 (Life Technologies) was used to transfect p27 was obtained from BD Pharmingen, KI67 was obtained from HTB2 or HT1376 cells with 100 nmol/L of either siLuciferase, DAKO and b-actin was obtained from Abcam. siAMPKa1, siAMPKa2 (Dharmacon), or siSKP2 (Cell Signaling Technology). Cells were lysed 72 hours post transfection and HTB2 and HT1376 stable cell transfection, selection, and further analyzed by immunoblot. maintenance HTB2 and HT1376 cells were transduced with lentivirus-con- Quantitative RT-PCR taining shGFP and shAMPKa2 in the LKO.1-puromycin vector RNA isolation and cDNA synthesis were carried out using (Sigma) as described previously (20). Briefly, HEK-293 FT cells the RNeasy mini kit (Qiagen) and iScript cDNA synthesis kit were plated and transfected the next day with the LKO.1 vector (Bio-Rad), respectively, according to the manufacturer pro- (shGFP or shAMPKa2) and vectors containing RRE, REV, and tocol. Primers are as follows: AMPKa1forward(50-AGGAG-
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