Carcinogenesis vol.35 no.10 pp.2232–2243, 2014 doi:10.1093/carcin/bgu129 Advance Access publication June 18, 2014
Transcriptional and posttranslational regulation of insulin-like growth factor binding protein-3 by Akt3 Quanri Jin1,†, Hyo-Jong Lee2,†, Hye-Young Min3,†, John Kendal Smith1, Su Jung Hwang2, Young Mi Whang1, Woo-Young Kim1,4, Yeul Hong Kim5 and Ho-Young Lee1,3,* 1
Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA, 2College of Pharmacy, Inje University, Gimhae, Gyungnam 621-749, Republic of Korea, 3 College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Republic of Korea, 4Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women’s University, Seoul 140-745, Republic of Korea and 5Department of Internal Medicine, Korea University College of Medicine, Seoul 136-705, Republic of Korea
Insulin-like growth factor (IGF)-dependent and -independent antitumor activities of insulin-like growth factor binding protein-3 (IGFBP-3) have been proposed in human non-small cell lung cancer (NSCLC) cells. However, the mechanism underlying regulation of IGFBP-3 expression in NSCLC cells is not well understood. In this study, we show that activation of Akt, especially Akt3, plays a major role in the mRNA expression and protein stability of IGFBP-3 and thus antitumor activities of IGFBP-3 in NSCLC cells. When Akt was activated by genomic or pharmacologic approaches, IGFBP-3 transcription and protein stability were decreased. Conversely, suppression of Akt increased IGFBP-3 mRNA levels and protein stability in NSCLC cell lines. Characterization of the effects of constitutively active form of each Akt subtype (HA-Akt-DD) on IGFBP-3 expression in NSCLC cells and a xenograft model indicated that Akt3 plays a major role in the Akt-mediated regulation of IGFBP-3 expression and thus suppression of Akt effectively enhances the antitumor activities of IGFBP-3 in NSCLC cells with Akt3 overactivation. Collectively, these data suggest a novel function of Akt3 as a negative regulator of IGFBP-3, indicating the possible benefit of a combined inhibition of IGFBP-3 and Akt3 for the treatment of patients with NSCLC.
Introduction Insulin-like growth factor binding protein-3 (IGFBP-3), the most abundant IGFBP in human serum (1), regulates the activation of the insulin-like growth factor (IGF)-1R pathway by sequestering free IGF-I and thus modulating IGF-I bioavailability (2). Beyond its direct role in modulating the action of IGF, IGFBP-3 also plays a role in an IGF-independent manner, in which it induces G1 cell cycle arrest and apopotosis in several human cancer cells (3–6). Several factors regulate the expression and stability of IGFBP-3. For instance, growth hormone and insulin are considered as inducers of IGFBP-3 (7). Expression of IGFBP-3 is also mediated by stimulation with a variety of proapoptotic and growth-inhibitory factors, such as transforming growth factor-β, retinoic acid, tumor necrosis factor-α, vitamin Abbreviations: ATRA, all-trans-retinoic acid; DNMT1, DNA methyltransferase I; EV, empty vector; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HA, hemagglutinin; IGF, insulin-like growth factor; IGFBP-3, insulin-like growth factor binding protein-3; NSCLC, non-small cell lung cancer; PTEN, phosphatase and tensin homolog; RT–PCR, reverse transcriptionpolymerase chain reaction; TGF-β, transforming growth factor-β. †
These authors contributed equally to this work.
Materials and methods Reagents Phosphate-buffered saline and cell culture media were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum was purchased from Gemini Bio-Products (West Sacramento, CA). Penicillin-streptomycin and trypsin-ethylenediaminetetraacetic acid were purchased from Invitrogen (Carlsbad, CA). Hygromycin B was purchased from Roche Applied Science (Indianapolis, IN). The adenoviral constructs expressing kinase-inactive Akt (Ad-Akt-KM), phosphatase and tensin homolog (PTEN) (Ad-PTEN) and empty vector (AdEV) were amplified as described previously (15). HA-Akt1, HA-Akt2 and HA-Akt3 (T308D/S473D) expression vectors (HA-Akt1DD, HA-Akt2DD and HA-Akt3DD) were kindly provided by Dr Gordon Mills (University of Texas M. D. Anderson Cancer Center, Houston, TX). IGF was purchased from R&D Systems (Minneapolis, MN). Perifosine was purchased from Selleckchem (Houston, TX) or LC Laboratories (Woburn, MA). Recombinant human IGFBP-3 (rBP3) was obtained from R&D Systems. LY294002 was purchased from EMD Chemicals (Gibbstown, NJ). Reagents unless otherwise indicated were purchased from Sigma–Aldrich (St Louis, MO). Cell culture The human NSCLC lines (A549, H460, H226B, H1299, H226Br, H322, H358 and H292) were purchased from the American Type Culture Collection or kindly provided by Dr Jack A. Roth (MD Anderson Cancer Center, Houston, TX). They were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics. TSC2-knockout (TSC2–/–) mouse embryonic fibroblast immortalized by p53 knockout (kindly provided by Dr D.J.Kwiatkowski at Brigham and Women’s Hospital, Boston, MA) were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. TSC2–/– and H226B cells expressing constitutively active Akt1, Akt2 or Akt3 were established by introduction of the pBABE retrovirus expressing hemagglutinin (HA)-tagged constitutively active Akts (HA-Akt1DD, HA-Akt2DD or HA-Akt3DD) (16) and selection by hygromycin B (50 μg/ ml). To analyze the effects of Akt activity on IGFBP-3 expression, H322 cells infected with Ad-PTEN, Ad-Akt (KM), or Ad-EV and H226B cells stably transfected with HA-Akt3DD were treated with all-trans-retinoic acid (ATRA) prior to cycloheximide treatment (10 μg/ml) or with recombinant IGFBP-3
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*To whom correspondence should be addressed. College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Republic of Korea. Tel: +82 2 880 9277; Fax: +82 2 6280 5327; Email:
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D, antiestrogens, antiandrogens and tumor suppressors (4,7). Several proteases have been involved in the non-responsiveness of cancer cells to IGFBP-3, including matrix metalloproteinases, cathepsins, neutrophil elastase and other serine proteases; these proteases represent a potential hurdle for the use of IGFBP-3 in lung cancer therapy (8–10). However, most of the studies involving these proteases were focused on the role of IGFBP-3 as a reservoir of IGF-I and little is known about the mechanisms underlying regulation of cellular IGFBP-3. We have previously demonstrated that treatment with the farnesyltransferase inhibitor SCH66336, a pharmacologic approach to inhibit Ras activation, decreases Akt activity in H1299 non-small cell lung cancer (NSCLC) cells (11). Recent reports have suggested that Akt, a serine/threonine protein kinase that serves as a key player in the control of cell transformation, proliferation, survival and metabolism (12), has an effect on the stability of several proteins, including BRCA1 (13) and the L-type subunits of Ca2+ channels (14). Based on these previous findings, we hypothesized that Akt may counteract IGFBP-3’s antitumor actions through regulating the expression and/ or stability of IGFBP-3 in NSCLC cells. This study was performed to investigate the role of Akt in the growth-inhibitory function of IGFBP-3 and the detailed mechanisms responsible for the effects of Akt on IGFBP-3 function. Here we show that Akt, especially Akt3, regulates cellular IGFBP-3 function by modulating its transcription and protein stability. Our data demonstrate that the antiproliferative and proapoptotic effects of IGFBP-3 are enhanced by inactivation of Akt, implying that one way to enhance the therapeutic potential of IGFBP-3 in NSCLC cells is to inhibit Akt activity. Our findings indicate a potential benefit to using Akt inhibitors in combined treatments with IGFBP-3 or other drugs that induce IGFBP-3 expression.
Akt3-mediated regulation of IGFBP-3
(rBP3). Cell lines used in this study were authenticated and validated prior to performing experiments. These cell lines were tested for authentication at Genetic Resources Core Facility of Johns Hopkins University in 2010 or at the Korean Cell Line Bank using AmplFLSTR identifiler PCR Amplification kit (Applied Biosystems, Foster, CA; cat. No. 4322288) in 2013.
Metabolic labeling Cells were incubated in a methionine- and cysteine-free RPMI medium (Sigma) for 1 h and pulse-labeled with trans-35S label (0.5 mCi; ICN Radiochemicals, Irvine, CA) for the indicated time periods. For the pulse-chase experiment, cells pulse labeled for 1 h were chased in fresh RPMI containing methionine (150 mg/l) and cysteine (150 mg/l) for the indicated time periods. Equal amounts of proteins from the total cell lysates were immunoprecipitated using an antibody against Flag or IGFBP-3 and then analyzed as described previously (11,17). Two independent experiments were performed with similar results; representative results from one experiment are presented. Western blot analysis Whole cell extracts were prepared as described previously (11,15). The antibodies used in this study include IGFBP-3 (Diagnostic Systems Laboratories, Webster, TX); Akt, pAkt (Ser473), pAkt (Thr308), PTEN, TSC2, pS6 ribosomal protein, S6 ribosomal protein, cleaved caspase-3 (Cell Signaling Technology, Danvers, MA); and Akt1, Akt2, Akt3, α-tubulin, β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), HA- and FLAG tags (Santa Cruz Biotechnology, Santa Cruz, CA). Reverse transcription-polymerase chain reaction Total RNA was reverse-transcribed using the Superscript® first strand cDNA system (Invitrogen) and further analyzed by quantitative real-time polymerase chain reaction (RT–PCR) (7500 ABI) using the SYBR® Green PCR Master Mix kit (Applied Biosystems, Foster, CA) and Applied Biosystems 7500 RealTime PCR System (Applied Biosystems). The primer sequences used for the quantitative RT–PCR are as follows: Mouse IGFBP-3 forward, 5′-CCA GGA AAC ATC AGT GAG TCC-3′; mouse IGFBP-3 reverse, 5′-GGA TGG AAC TTG GAA TCG GTC A-3′; mouse GAPDH forward, 5′-TGC ACC ACC AAC TGC TTA GC-3′; mouse GAPDH reverse, 5′-GGC ATG GAC TGT GGT CAT GAG-3′; human IGFBP-3 forward, 5′-TCT GCG TCA ACG CTA GTG C-3′; human IGFBP-3 reverse, 5′-GCT CTG AGA CTC GTA GTC AAC T-3′; human β-actin forward, 5′-GCG AGA AGA TGA CCC AGA TC-3′; human β-actin reverse, 5′-GGA TAG CAC AGC CTG GAT AG-3′. Quantification of mRNA expression was performed by the comparative cycle threshold method and normalized to the amount of GAPDH or β-actin mRNA. Cell viability and colony formation assays (anchorage-independent and -dependent) For the analysis of cell viability, NSCLC cells (1−2 × 103 cells/well) seeded in 96-well plates were treated with rhIGFBP-3 and/or LY294002 and incubated for 3−5 days. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed as described previously (11). For the colony formation analysis, Akt3DD was overexpressed in H226B cells through the transfection with recombinant pcDNA3-Akt3DD plasmid. For the anchorage-independent colony forming analysis, cells were suspended in a 0.35% agar/RPMI mixture (1 × 103/ml) and plated in six-well plates precoated with 0.6% bottom agar. Cells were then allowed to propagate for 14 days. Colonies
