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Aurora Kinase Inhibition Induces PUMA Via NF-Kb to Kill Colon Cancer Cells

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Aurora Kinase Inhibition Induces PUMA Via NF-Kb to Kill Colon Cancer Cells Published OnlineFirst February 21, 2014; DOI: 10.1158/1535-7163.MCT-13-0846 Molecular Cancer Cancer Biology and Signal Transduction Therapeutics Aurora Kinase Inhibition Induces PUMA via NF-kB to Kill Colon Cancer Cells Jing Sun1,2,4, Kyle Knickelbein1,2, Kan He1,3, Dongshi Chen1,2, Crissy Dudgeon1,2, Yongqian Shu4, Jian Yu1,3, and Lin Zhang1,2 Abstract Aurora kinases play a key role in mitosis and are frequently overexpressed in a variety of tumor cells. Inhibition of aurora kinases results in mitotic arrest and death of cancer cells, and has been explored as an anticancer strategy. However, how aurora inhibition kills cancer cells is poorly understood. In this study, we found that inhibition of aurora kinases by siRNA or small-molecule inhibitors led to induction of p53 upregulated modulator of apoptosis (PUMA), a BH3-only Bcl-2 family protein, in colorectal cancer cells irrespective of p53 status. Deficiency in PUMA increased polyploidy, improved cell survival, and abrogated mitochondria-mediated apoptosis induced by aurora kinase inhibitors. In response to aurora kinase inhibition, PUMA was directly activated by p65 through the canonical NF-kB pathway following AKT inhibition. Furthermore, PUMA was necessary for the chemosensitization and in vivo antitumor effects of aurora kinase inhibitors in colon cancer cells. These results suggest that PUMA induction mediates the apoptotic response to mitotic arrest imposed by aurora kinase inhibition, and may be a useful indicator for the anticancer activity of aurora kinase inhibitors. Mol Cancer Ther; 13(5); 1298–308. Ó2014 AACR. Introduction critical initiator of apoptosis in cancer cells (7). It is Aurora kinases are a group of highly conserved serine/ transcriptionally activated by p53 in response to DNA threonine kinases that play a key role in mitosis. They are damage, and is indispensable for p53-dependent apopto- frequently overexpressed in a variety of tumors and sis induced by radiation and cytotoxic chemotherapeutic promote cell-cycle progression (1). There are three aurora drugs (8). PUMA can also be induced in a p53-indepen- kinases in mammalian cells, including aurora A, B, and C, dent manner by a variety of nongenotoxic stimuli, such as among which aurora A and B are most abundantly kinase inhibitors, growth factor deprivation, and inflam- expressed in cancer cells (2). Small-molecule inhibitors matory cytokines (9–14). p53-independent PUMA induc- of aurora kinases have been developed and shown pro- tion can be mediated by different transcription factors, mises in recent preclinical studies (3). In response to including the p53 homologue p73, Forkhead Box O3a k aurora kinase inhibition, cancer cells undergo mitotic (FoxO3a), and NF- B (9–14). Upon its induction, PUMA arrest and eventually, cell death (4). Accumulating evi- potently induces apoptosis in cancer cells by acting upon dence suggests that aurora kinase inhibitors kill cancer other Bcl-2 family members such as Bax, Bcl-2, and Bcl-XL, cells by inducing apoptosis via mitochondrial dysfunction resulting in mitochondrial outer membrane permeabili- (5). However, the mechanisms by which mitotic arrest zation and activation of the caspase cascade (15–17). imposed by aurora kinase inhibitors triggers apoptotic PUMA was previously shown to be induced by anti- cell death are poorly understood (6). mitotic drugs such as microtubule-targeting agents (18), p53 upregulated modulator of apoptosis (PUMA) is a and upregulated in response to polyploidy (19, 20), sug- BH3-only Bcl-2 family member, which functions as a gesting it may play a role in initiating the apoptotic response to mitotic arrest. In this study, we found that PUMA is transcriptionally activated through the canon- ical NF-kB pathway following aurora kinase inhibition, Authors' Affiliations: 1University of Pittsburgh Cancer Institute; Depart- ments of 2Pharmacology and Chemical Biology and 3Pathology, University which contributes to the in vitro and in vivo anticancer of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; 4Department activities of aurora kinase inhibitors. Our results suggest of Medical Oncology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, PR China that PUMA induction may be a useful indicator for the therapeutic effects of aurora kinase inhibitors. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Materials and Methods Corresponding Author: Lin Zhang, UPCI Research Pavilion, Room 2.42a, Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh, PA 15213. Phone: Cell culture and drug treatment 412-623-1009; Fax: 412-623-7778; E-mail: [email protected] The human colorectal cancer cell lines, including doi: 10.1158/1535-7163.MCT-13-0846 HCT116, DLD1, RKO, HT29, SW480, and SW48 were Ó2014 American Association for Cancer Research. obtained from the American Type Culture Collection. Cell 1298 Mol Cancer Ther; 13(5) May 2014 Downloaded from mct.aacrjournals.org on October 3, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst February 21, 2014; DOI: 10.1158/1535-7163.MCT-13-0846 PUMA-Mediated Anticancer Effect of Aurora Kinase Inhibitors lines were last tested and authenticated for genotypes, Analysis of NF-kB nuclear translocation drug response, morphology, and absence of mycoplasma HCT116 cells pretreated with BAY 11-7082 were sub- in October 2012. p53-knockout (KO) and PUMA-KO colon jected to ZM-447439 or TNF-a for 3 hours. NF-kB nuclear cancer cell lines and wild-type (WT) and PUMA-KO translocation was analyzed by nuclear fractionation. mouse embryonic fibroblasts (MEF) cells were previously Briefly, nuclear extracts were isolated from cells plated described (13). All cell lines were maintained at 37Cin5% and treated in 75 cm2 flasks using the NE-PER Nuclear/ CO2. Colon cancer cell lines were cultured in McCoy’s 5A Cytoplasmic Extraction Kit (Thermo Fisher) according to modified media (Invitrogen), and MEF cells were cultured the manufacturer’s instructions, and probed by Western in DMEM media (Invitrogen). Cell culture media were blotting for p65. supplemented with 10% defined FBS (HyClone), 100 units/mL penicillin, and 100 mg/mL streptomycin (Invi- Luciferase assays trogen). For drug treatment, cells were plated in 12-well PUMA luciferase reporter constructs have been previ- plates at 20% to 30% density 24 hours before treatment. ously described (9). Mutations were introduced into the The dimethyl sulfoxide (DMSO; Sigma) stocks of agents p65 binding sites of Fragment A using QuickChange XL used, including ZM-447439 (Selleck Chemicals), VX-680, Site-Directed Mutagenesis Kit (Agilent Technologies) as Gefinitib (LC Laboratories), 5-fluorouracil (5-FU; Sigma), previously described (13). Cells were transfected with BAY 11-7082 (Merck Chemicals), GX15-070 (Cayman PUMA reporters containing either wild-type or mutant Chemical), were diluted to appropriate concentrations p65 binding sites (13), along with the transfection control with the cell culture medium. Human TNF-a (R&D Sys- b-galactosidase reporter pCMVb (Promega), and treated tems) was diluted with PBS. For NF-kB inhibition, cells with 15 mmol/L ZM-447439 for 24 hours. Cell lysates were pretreated with BAY 11-7082 for 1 hour before ZM- were collected and luciferase activities were measured 447439 treatment. Transfection of expression constructs as previously described (13). All reporter experiments of wild-type and constitutive AKT was performed as were done in triplicate and repeated 3 times. described (12). Chromatin immunoprecipitation Real-time reverse transcription-PCR Chromatin immunoprecipitation (ChIP) was done Total RNA was isolated from cells using the Mini RNA using the ChIP Assay kit (Millipore) with p65 (Santa Cruz) Isolation II Kit (Zymo Research) according to the manu- antibody for chromatin precipitation as described (13). facturer’s protocol. Total RNA (1 mg) was used to generate The precipitates were analyzed by PCR using primers 50- cDNA using SuperScript II reverse transcriptase (Invitro- GTCGGTCTGTGTACGCATCG-30 and 50-CCCGCGTGA- gen). Real-time PCR was carried out for PUMA and glyc- CGCTACGGCCC-30 as previously described (13). eraldehyde-3-phosphate dehydrogenase as described (13). Apoptosis assays Western blotting Adherent and floating cells were harvested, stained Antibodies used for Western blotting included those with Hoechst 33258 (Invitrogen), and analyzed for apo- against active caspase-3, p-ERK (T202/Y204), IkB, p-IkB ptosis by nuclear staining assay. A minimum of 300 cells (S32/S36), p-GSK3b, p65 (total), p-p65 (S536), FoxO3a were analyzed for each treatment. For colony formation (total), AKT (total), p-AKT (S473; Cell Signaling Technol- assays, equal numbers of cells were subjected to various c a ogy), cytochrome , -tubulin, Bcl-XL (BD Biosciences), treatments and plated into 12-well plates at different caspase-9 (Stressgen Bioreagents), cytochrome oxidase dilutions. Colonies were visualized by crystal violet (Sig- subunit IV (Cox IV; Invitrogen), Bcl-2 (Dako), Flag (Sig- ma) staining 14 days after plating as previously described ma), PUMA (17), p53, Bim, Bid, Noxa, and b-actin (EMD (13). Each experiment was performed in triplicate and Millipore). Western blotting analysis was performed as repeated at least twice. previously described (13). The release of cytochrome c was detected in the cytosol following subcellular fractio- Xenograft tumors nations as described (13). All animal experiments were approved by the Institu- tional Animal
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