Regulation of Hypoxia-Inducible Factor-1α, Vascular Endothelial Growth Factor, and Angiogenesis by an Insulin-Like Growth Factor-I Receptor Autocrine Loop in Human Pancreatic Cancer
2003; Elsevier BV; Volume: 163; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)63460-8
ISSN1525-2191
AutoresOliver Stoeltzing, Wenbiao Liu, Niels Reinmuth, Fan Fan, Alexander A. Parikh, Corazon D. Bucana, Douglas B. Evans, Gregg L. Semenza, Lee M. Ellis,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoActivation of the insulin-like growth factor-I receptor (IGF-IR) was recently shown to modulate angiogenesis by up-regulating the expression of vascular endothelial growth factor (VEGF). We hypothesized that inhibiting IGF-IR function would inhibit angiogenesis and growth of pancreatic cancer in vivo and sought to identify major signaling pathways regulated by IGF-IR in pancreatic cancer cells. Human pancreatic cancer cells (L3.6pl) were stably transfected with a dominant-negative form of IGF-IR (IGF-IR DN) or an empty vector (pcDNA). In vitro, IGF-IR DN cells exhibited a decrease in both constitutive and inducible phosphorylation of IGF-IR and Erk1/2. Constitutive expression of nuclear hypoxia-inducible factor-1α and secreted VEGF (P < 0.01) protein levels also were significantly lower in IGF-IR DN cells than in pcDNA cells. In vivo, IGF-IR inhibition led to decreases in pancreatic tumor volume and weight, vessel density, and tumor cell proliferation (P < 0.01 for all) and increases in tumor cell apoptosis (P < 0.02). Our results suggest that autocrine activation of the IGF-IR system significantly affects VEGF expression and angiogenesis in human pancreatic cancer. Thus, IGF-IR may be a valid target in the treatment of pancreatic cancer. Activation of the insulin-like growth factor-I receptor (IGF-IR) was recently shown to modulate angiogenesis by up-regulating the expression of vascular endothelial growth factor (VEGF). We hypothesized that inhibiting IGF-IR function would inhibit angiogenesis and growth of pancreatic cancer in vivo and sought to identify major signaling pathways regulated by IGF-IR in pancreatic cancer cells. Human pancreatic cancer cells (L3.6pl) were stably transfected with a dominant-negative form of IGF-IR (IGF-IR DN) or an empty vector (pcDNA). In vitro, IGF-IR DN cells exhibited a decrease in both constitutive and inducible phosphorylation of IGF-IR and Erk1/2. Constitutive expression of nuclear hypoxia-inducible factor-1α and secreted VEGF (P < 0.01) protein levels also were significantly lower in IGF-IR DN cells than in pcDNA cells. In vivo, IGF-IR inhibition led to decreases in pancreatic tumor volume and weight, vessel density, and tumor cell proliferation (P < 0.01 for all) and increases in tumor cell apoptosis (P < 0.02). Our results suggest that autocrine activation of the IGF-IR system significantly affects VEGF expression and angiogenesis in human pancreatic cancer. Thus, IGF-IR may be a valid target in the treatment of pancreatic cancer. 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In the current study, we hypothesized that inhibition of IGF-IR function in human pancreatic cancer cells would decrease VEGF expression and angiogenesis and in turn inhibit tumor growth in vivo. We further sought to elucidate the role of the IGF-I receptor/ligand system in the constitutive activation of intracellular pathways that mediate VEGF expression in pancreatic cancer cells. We found that constitutive activation of IGF-IR-mediated VEGF expression in pancreatic cancer cells occurred in part through increasing the nuclear translocation of hypoxia-inducible factor-1α (HIF-1α). Moreover, inhibition of IGF-IR function inhibited angiogenesis and growth of pancreatic tumors, suggesting that the IGF-IR system is a valid molecular target in the treatment of human pancreatic cancer. The human pancreatic cancer cell line L3.6pl32Bruns CJ Harbison MT Kuniyasu H Eue I Fidler IJ In vivo selection and characterization of metastatic variants from human pancreatic adenocarcinoma by using orthotopic implantation in nude mice.Neoplasia. 1999; 1: 50-62Abstract Full Text PDF PubMed Scopus (293) Google Scholar was kindly provided by I. J. Fidler, Ph.D., D.V.M. (The University of Texas M. D. Anderson Cancer Center, Houston, TX). Cells were cultured and maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2 U/ml of a penicillin-streptomycin mixture (Flow Laboratories, Rockville, MD), vitamins (Life Technologies, Inc., Grand Island, NY), 1 mmol/L sodium pyruvate, 2 mmol/L l-glutamine, and nonessential amino acids and incubated in 5% CO2-95% air at 37°C. To clarify the influence of IGF-IR inhibition on VEGF regulation and angiogenesis, we stably transfected L3.6pl cells with a mutated dominant-negative (DN) form of IGF-IR (truncated at position 952 in the β-subunit transmembrane region), which was the generous gift of D. Prager M.D. (Cedars-Sinai Medical Center-UCLA School of Medicine, Los Angeles, CA),33Prager D Yamasaki H Weber MM Gebremedhin S Melmed S Human insulin-like growth factor I receptor function in pituitary cells is suppressed by a dominant negative mutant.J Clin Invest. 1992; 90: 2117-2122Crossref PubMed Scopus (34) Google Scholar, 34Prager D Li HL Asa S Melmed S Dominant negative inhibition of tumorigenesis in vivo by human insulin-like growth factor I receptor mutant.Proc Natl Acad Sci USA. 1994; 91: 2181-2185Crossref PubMed Scopus (214) Google Scholar or an empty vector (pcDNA3) as a control. Cell were transfected by using FuGENE 6 transfection reagent (Roche Diagnostics Corporation, Indianapolis, IN) according to the manufacturer's protocol as described elsewhere.19Reinmuth N Fan F Liu W Parikh AA Stoeltzing O Jung YD Bucana CD Radinsky R Gallick GE Ellis LM Impact of insulin-like growth factor receptor-I function on angiogenesis, growth, and metastasis of colon cancer.Lab Invest. 2002; 82: 1377-1389Crossref PubMed Scopus (162) Google Scholar, 20Reinmuth N Liu W Fan F Jung YD Ahmad SA Stoeltzing O Bucana CD Radinsky R Ellis LM Blockade of insulin-like growth factor I receptor function inhibits growth and angiogenesis of colon cancer.Clin Cancer Res. 2002; 8: 3259-3269PubMed Google Scholar Cells were then grown and expanded in selective medium containing neomycin (G418, Life Technologies). Successful transfection was verified by changes in insulin receptor substrate-1 (IRS-1) phosphorylation on stimulation with recombinant human IGF-I (rhIGF-I) (R&D Systems Inc., Minneapolis, MN), which was reduced in IGF-IR DN cells.19Reinmuth N Fan F Liu W Parikh AA Stoeltzing O Jung YD Bucana CD Radinsky R Gallick GE Ellis LM Impact of insulin-like growth factor receptor-I function on angiogenesis, growth, and metastasis of colon cancer.Lab Invest. 2002; 82: 1377-1389Crossref PubMed Scopus (162) Google Scholar, 20Reinmuth N Liu W Fan F Jung YD Ahmad SA Stoeltzing O Bucana CD Radinsky R Ellis LM Blockade of insulin-like growth factor I receptor function inhibits growth and angiogenesis of colon cancer.Clin Cancer Res. 2002; 8: 3259-3269PubMed Google Scholar Paraffin-embedded tissues from primary human pancreatic adenocarcinomas (n = 11) [confirmed by a pathologist on hematoxylin and eosin (H&E)-stained slides] or normal pancreas (n = 10) were stained for IGF-IR expression and IGF-IR activation by using antibodies to IGF-IR-α (Santa Cruz Biotechnology, Santa Cruz, CA) or phosphorylated IGF-IR (Biosource International, Camarillo, CA), respectively. Patients did not receive neoadjuvant therapy before surgery. Specimens were collected under a protocol approved by the Institutional Review Board of the M.D. Anderson Cancer Center. Tissue sections were deparaffinized in xylene, followed by treatment with a graded series of alcohol washes [100%, 95%, 80% ethanol/ddH2O (v/v)], rehydration in phosphate-buffered saline (PBS) (pH 7.5), and microwaving for 10 minutes in 100 mmol/L of citrate solution (pH 6.0) for antigen retrieval. Slides were then washed in PBS and incubated for 20 minutes in a protein-blocking solution consisting of PBS supplemented with 1% normal goat serum and 5% normal horse serum. Primary antibody was diluted in protein-blocking solution (1:200 for anti-IGF-IR-α, 1:50 for anti-phospho-IGF-IR) and applied for 18 hours at 4°C. Antibodies were then washed off, protein-block applied for 20 minutes, followed by 1 hour of incubation with Alexa594 conjugated goat anti-rabbit secondary antibody (1:600) (Jackson ImmunoResearch Laboratories, West Grove, PA). Tissues were then counterstained with Hoechst dye (1:2000). Tumor and normal tissue regions were identified by parallel comparison to H&E-stained slides and images were obtained at ×100 magnification. Western blot analysis was done to detect secreted IGF-II protein in conditioned medium from parental L3.6pl cells.35Quinn KA Treston AM Unsworth EJ Miller MJ Vos M Grimley C Battey J Mulshine JL Cuttitta F Insulin-like growth factor expression in human cancer cell lines.J Biol Chem. 1996; 271: 11477-11483Crossref PubMed Scopus (177) Google Scholar IGF-I protein in conditioned medium was measured by using an enzyme-linked immunosorbent assay (ELISA) kit for human IGF-I (R&D Systems Inc.). Briefly, L3.6pl pancreatic cancer cells were incubated in 10% FBS-MEM for 48 hours and conditioned medium was harvested, centrifuged (360 × g, 5 minutes) and filtered through a 0.22-μm filter (Corning Inc., Corning, NY). For IGF-II detection, proteins in samples of conditioned media (100 μg/sample) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a denaturing 15% gel under reducing conditions. Western blotting was performed with a monoclonal primary antibody against human IGF-II (1:3000 in 5% milk) (Upstate Biotechnology, Lake Placid, NY) and a secondary antibody for chemiluminescent analysis (ECL; Amersham Biosciences, Piscataway, NJ) as described previously.19Reinmuth N Fan F Liu W Parikh AA Stoeltzing O Jung YD Bucana CD Radinsky R Gallick GE Ellis LM Impact of insulin-like growth factor receptor-I function on angiogenesis, growth, and metastasis of colon cancer.Lab Invest. 2002; 82: 1377-1389Crossref PubMed Scopus (162) Google Scholar IGF-IR phosphorylation was investigated using a rabbit anti-phospho-IGF-IR antibody at 1:1000 dilution (Biosource International) for Western blot analysis. Signaling pathways activated by IGF-I in IGF-IR DN- or pcDNA-transfected cells were investigated by Western blotting.20Reinmuth N Liu W Fan F Jung YD Ahmad SA Stoeltzing O Bucana CD Radinsky R Ellis LM Blockade of insulin-like growth factor I receptor function inhibits growth and angiogenesis of colon cancer.Clin Cancer Res. 2002; 8: 3259-3269PubMed Google Scholar, 36Jung YD Liu W Reinmuth N Ahmad SA Fan F Gallick GE Ellis LM Vascular endothelial growth factor is upregulated by interleukin-1 beta in human vascular smooth muscle cells via the P38 mitogen-activated protein kinase pathway.Angiogenesis. 2001; 4: 155-162Crossref PubMed Scopus (100) Google Scholar Briefly, cells were grown to 50 to 60% cell confluence, incubated overnight in 1% FBS-MEM, and treated thereafter for various times with rhIGF-I (100 ng/ml) in 1% FBS-MEM. To investigate constitutive phosphorylation levels of signaling intermediates in IGF-IR DN- and pcDNA-transfected cells, cells were incubated in 10% FBS-MEM for 48 hours and harvested in PBS. Protein was extracted from cell lysates with RIPA buffer, and 50-μg protein samples were subjected to Western blot analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a denaturing 10% gel. Activated signaling pathways were identified by using the following antibodies: anti-extracellular signal-regulated kinase (Erk)-1/2 (p42/44) (Oncogene Research Products, San Diego, CA); and anti-phosphospecific Erk-1/2 (p42/44), anti-Akt, anti-phosphospecific Akt, anti-phosphospecific c-jun amino-terminal kinase (JNK), anti-P38, and anti-phosphospecific P38 from Cell Signaling Technologies (Beverly, MA). For Northern blot analyses, total RNA was extracted from cells by using Tri Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. Northern blot analysis was performed as described previously.19Reinmuth N Fan F Liu W Parikh AA Stoeltzing O Jung YD Bucana CD Radinsky R Gallick GE Ellis LM Impact of insulin-like growth factor receptor-I function on angiogenesis, growth, and metastasis of colon cancer.Lab Invest. 2002; 82: 1377-1389Crossref PubMed Scopus (162) Google Scholar, 37Ahmad SA Liu W Jung YD Fan F Wilson M Reinmuth N Shaheen RM Bucana CD Ellis LM The effects of angiopoietin-1 and -2 on tumor growth and angiogenesis in human colon cancer.Cancer Res. 2001; 61: 1255-1259PubMed Google Scholar Briefly, probes for VEGF (human VEGF-specific 204-bp cDNA probe)19Reinmuth N Fan F Liu W Parikh AA Stoeltzing O Jung YD Bucana CD Radinsky R Gallick GE Ellis LM Impact of insulin-like growth factor receptor-I function on angiogenesis, growth, and metastasis of colon cancer.Lab Invest. 2002; 82: 1377-1389Crossref PubMed Scopus (162) Google Scholar or glyceraldehyde-3-phosphate dehydrogenase (American Type Culture Collection, Manassas, VA) were radiolabeled by the random primer technique with a commercially available kit (Rediprime II, Amersham Biosciences), and nylon membranes (Hybond-N+, Amersham Biosciences) were subsequently hybridized overnight (Rapid-hyb Solution, Amersham Biosciences) at 65°C. Autoradiography was performed thereafter in the linear range of the film (Hyperfilm MP, Amersham Biosciences). To investigate VEGF induction by IGF-I, parental and transfected cells were grown to 50 to 60% confluence in standard medium as described above and then incubated in 5% FBS-containing medium overnight. Cells were then incubated in the presence or absence of rhIGF-I (100 ng/ml) in 1% FBS-MEM for 24 hours because induction of VEGF mRNA expression in this cell line was maximal by this time. Total RNA was extracted, and VEGF mRNA expression was determined by Northern blot analysis. VEGF protein concentrations in conditioned media from IGF-IR DN- or pcDNA- transfected L3.6pl cells were measured by using an ELISA kit for human VEGF (Biosource International). Supernatant (3 ml) from the transfected cells was collected after 48 hours of incubation in 10% FBS-MEM. Cells were rinsed with cold PBS, trypsinized, and counted. VEGF ELISA was performed according to the manufacturer's protocol, and VEGF levels (in pg) were calculated per 1 × 106 cells. Experiments using the cyclooxygenase-2 (Cox-2) inhibitor celecoxib were performed by dissolving 200-mg celecoxib capsules (obtained from the Pharmacy at M. D. Anderson Cancer Center) in dimethyl sulfoxide (100 mmol/L stock solution). Preliminary experiments confirmed that dimethyl sulfoxide, at a final concentration of 0.01% in cell culture medium, did not induce VEGF expression. Western blot analyses for HIF-1α were performed from nuclear extracts from parental and transfected cells as described elsewhere.38Jung YD Fan F McConkey DJ Jean ME Liu W Reinmuth N Stoeltzing O Ahmad SA Parikh AA Mukaida N Ellis LM Role of P38 MAPK, AP-1, and NF-kappaB in interleukin-1beta-induced IL-8 expression in human vascular smooth muscle cells.Cytokine. 2002; 18: 206-213Crossref PubMed Scopus (84) Google Scholar, 39Fukuda R Hirota K Fan F Jung YD Ellis LM Semenza GL Insulin-like growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells.J Biol Chem. 2002; 277: 38205-38211Crossref PubMed Scopus (713) Google Scholar Briefly, IGF-IR DN- and pcDNA-transfected L3.6pl cells were incubated for 48 hours in 10% FBS-MEM, washed twice with ice-cold PBS, and harvested into cold PBS (1 ml). After centrifugation (400 × g for 5 minutes at 4°C), cell pellets were incubated with ice-cold lysis buffer (buffer A: 10 mmol/L HEPES, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L dithiothreitol, 0.2 mmol/L phenylmethyl sulfonyl fluoride, and 1× protein inhibitor) for 30 minutes on ice, centrifuged again (7150 × g for 3 minutes at 4°C), and the remaining cell pellets were washed twice with ice-cold buffer A. For nuclear protein extraction, washed pellets were incubated in ice-cold buffer C (20 mmol/L HEPES, 25% glycerol, 450 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L ethylenediaminetetraacetic acid, 0.5 mmol/L dithiothreitol, 0.5 mmol/L phenylmethyl sulfonyl fluoride, and 1× protein inhibitor) for 30 minutes on ice, and supernatant was collected after centrifugation (22,000 × g for 15 minutes at 4°C). Protein concentrations were then determined19Reinmuth N Fan F Liu W Parikh AA Stoeltzing O Jung YD Bucana CD Radinsky R Gallick GE Ellis LM Impact of insulin-like growth factor receptor-I function on angiogenesis, growth, and metastasis of colon cancer.Lab Invest. 2002; 82: 1377-1389Crossref PubM
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