Adrenomedullin Is a Cross-Talk Molecule that Regulates Tumor and Mast Cell Function during Human Carcinogenesis
2006; Elsevier BV; Volume: 168; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2006.050291
ISSN1525-2191
AutoresEnrique Zudaire, Alfredo Martı́nez, Mercedes Garayoa, Rubén Pı́o, Gurmeet Kaur, Michael R. Woolhiser, Dean D. Metcalfe, W. Alexander Van Hook, Reuben P. Siraganian, Theresa A. Guise, John M. Chirgwin, Frank Cuttitta,
Tópico(s)Receptor Mechanisms and Signaling
ResumoWe have previously demonstrated that adrenomedullin (AM) plays a critical role as an autocrine/paracrine tumor cell survival factor. We now present evidence that AM is an important regulator of mast cell (MC) function and that this modulation is potentially involved in tumor promotion. AM induced histamine or β-hexosaminidase release from rat and human MCs through a receptor-independent pathway. AM was chemotactic for human MCs and stimulated mRNA expression of vascular endothelial growth factor, monocyte chemoattractant protein-1, and basic fibroblast growth factor in this cell type. Differentiated but not undifferentiated human MCs responded to hypoxic insult with elevated AM mRNA/protein expression. Using confocal microscopy, we identified AM-producing MCs in tumor infiltrates of human breast and lung cancer patients. In mixed culture assays the AM-producing human MC line HMC-1 augmented both anchorage-dependent and -independent growth of human lung cancer A549 cells, an effect that was suppressed by MC-targeted siRNA AM knockdown. Finally, HMC-1 cells induced in vivo angiogenesis as assessed by directed in vivo angiogenesis assay analysis; neutralizing anti-AM monoclonal antibody blocked this ability. Our collective data suggest a new role for AM as a cross-talk molecule that integrates tumor and MC communication, underlying a unique promotion mechanism of human cancers. We have previously demonstrated that adrenomedullin (AM) plays a critical role as an autocrine/paracrine tumor cell survival factor. We now present evidence that AM is an important regulator of mast cell (MC) function and that this modulation is potentially involved in tumor promotion. AM induced histamine or β-hexosaminidase release from rat and human MCs through a receptor-independent pathway. AM was chemotactic for human MCs and stimulated mRNA expression of vascular endothelial growth factor, monocyte chemoattractant protein-1, and basic fibroblast growth factor in this cell type. Differentiated but not undifferentiated human MCs responded to hypoxic insult with elevated AM mRNA/protein expression. Using confocal microscopy, we identified AM-producing MCs in tumor infiltrates of human breast and lung cancer patients. In mixed culture assays the AM-producing human MC line HMC-1 augmented both anchorage-dependent and -independent growth of human lung cancer A549 cells, an effect that was suppressed by MC-targeted siRNA AM knockdown. Finally, HMC-1 cells induced in vivo angiogenesis as assessed by directed in vivo angiogenesis assay analysis; neutralizing anti-AM monoclonal antibody blocked this ability. Our collective data suggest a new role for AM as a cross-talk molecule that integrates tumor and MC communication, underlying a unique promotion mechanism of human cancers. Our general concept of cancer has dramatically changed throughout the past 2 decades from the model of a single transformational event to one of a multistaged process involving complex interactions with the surrounding cellular microenvironment.1Hanahan D Weinberg RA The hallmarks of cancer.Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22527) Google Scholar, 2Vogelstein B Kinzler KW The multistep nature of cancer.Trends Genet. 1993; 9: 138-141Abstract Full Text PDF PubMed Scopus (1511) Google Scholar Encompassed in these newly proposed cancer dynamics, chronic inflammation has been implicated as the driving force in many human malignancies.3Shacter E Weitzman SA Chronic inflammation and cancer.Oncology (Huntingt). 2002; 16: 217-230PubMed Google Scholar, 4O'Byrne KJ Dalgleish AG Chronic immune activation and inflammation as the cause of malignancy.Br J Cancer. 2001; 85: 473-483Crossref PubMed Scopus (457) Google Scholar Recently, the mast cell (MC) has emerged as a primary candidate among the infiltrating cell population responsible for mediating tumor promotion.5Ribatti D Vacca A Nico B Crivellato E Roncali L Dammacco F The role of mast cells in tumour angiogenesis.Br J Haematol. 2001; 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221: 1-92Crossref PubMed Scopus (136) Google Scholar AM is elevated over normal levels in a variety of human malignancies of both neural and epithelial origin, including cancers of the brain, lung, colon, breast, ovary, uterus, prostate, skin, kidney, and eye.23Cuttitta F Pio R Garayoa M Zudaire E Julian M Elsasser TH Montuenga LM Martinez A Adrenomedullin functions as an important tumor survival factor in human carcinogenesis.Microsc Res Tech. 2002; 57: 110-119Crossref PubMed Scopus (54) Google Scholar Hypoxic insult and the resulting increase of hypoxia-inducible factor-1 have been implicated as one of the underlying pathways leading to AM overexpression in human tumors.24Garayoa M Martinez A Lee S Pio R An WG Neckers L Trepel J Montuenga LM Ryan H Johnson R Gassmann M Cuttitta F Hypoxia-inducible factor-1 (HIF-1) up-regulates adrenomedullin expression in human tumor cell lines during oxygen deprivation: a possible promotion mechanism of carcinogenesis.Mol Endocrinol. 2000; 14: 848-862Crossref PubMed Scopus (238) Google Scholar, 25Oehler MK Norbury C Hague S Rees MC Bicknell R Adrenomedullin inhibits hypoxic cell death by upregulation of Bcl-2 in endometrial cancer cells: a possible promotion mechanism for tumour growth.Oncogene. 2001; 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Hence our investigative interest in the role of AM as a cross-talk molecule regulating tumor and MC function as it relates to human carcinogenesis. Enhancing our understanding of the biological mechanisms surrounding cancer cells, their associated MCs infiltrates, and AM as a cross-talk communicator peptide between these cell populations may help to define new therapeutic targets for the clinical management of human malignancies. The human (H) MC leukemia line, HMC-1,35Butterfield JH Weiler D Dewald G Gleich GJ Establishment of an immature mast cell line from a patient with mast cell leukemia.Leuk Res. 1988; 12: 345-355Abstract Full Text PDF PubMed Scopus (638) Google Scholar was a gracious gift from Dr. J.H. Butterfield, donated through a Material Transfer Agreement between the National Institutes of Health (NIH) (Bethesda, MD) and the Mayo Clinic (Rochester, MN). Cells were routinely grown in 175-cm2 flasks in Iscove's medium containing 10% fetal bovine serum (Life Technologies, Gaithersburg, MD) and 1.2 mmol/L monothioglycerol (Sigma-Aldrich, St. Louis, MO). For all HMC-1 experimental evaluations cited in this text, semiconfluent cells were fed 2 days before testing and then seeded at 107 cells/175-cm2 flask for procedure implementation. When indicated, HMC-1 cells were exposed to the differentiation reagent phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) at 20 ng/ml for specified time periods.36Nilsson G Blom T Kusche-Gullberg M Kjellen L Butterfield JH Sundstrom C Nilsson K Hellman L Phenotypic characterization of the human mast-cell line HMC-1.Scand J Immunol. 1994; 39: 489-498Crossref PubMed Scopus (228) Google Scholar For the remainder of this study, PMA-treated HMC-1 cells will be considered mature (differentiated) and untreated HMC-1 cells will be considered immature (undifferentiated). The mammalian expression vector pSEC(neo) (Ambion, Austin, TX) was used for expression of an AM-specific siRNA in lung carcinoma A549 and MC HMC-1 cell lines. An insert containing nucleotides 511 to 531 (5′-AAGCTGGCACACCAGATCTAC-3′) of the AM gene was ligated into the pSEC(neo) backbone, and the final plasmid was referred to as pSEC(neo)-511. A control vector containing a scrambled sequence (5′-AAGGCGCCACTCGCCCAAATAAT-3′) served as a nonsilencing control (referred to as pSEC(neo)-SCR). All vectors were sequenced, in the forward and reverse directions, around the cloning boxes to verify the insertion of the AM sequences into the pSEC(neo) backbone. A549 and HMC-1 cells were transfected with plasmids pSEC(neo)-511 (A549-511 and HMC-1-511) and pSEC(neo)-SCR (A549-SCR and HMC-1-SCR) using FuGENE6 (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. After 72 hours, cells were exposed to the appropriate media containing 800 μg/ml of geneticin (Invitrogen). Stable transfectants were screened for AM expression by quantitative real-time polymerase chain reaction (PCR). Conditioned medium from appropriate test cells was aspirated from culture flasks, centrifuged, snap-frozen on dry-ice and stored at −80°C until used for quantitative AM radioimmunoassay or methylthiazoletetrazolium cell proliferation assays. Cells were harvested by washing once in serum-free RPMI 1640 (Life Technologies), trypsinizing for 3 minutes, pelleting cells by low-speed centrifugation, lysing cells in RLT buffer (RNeasy mini kit; Qiagen Inc., Valencia, CA) containing 2-mercaptoethanol (Sigma-Aldrich), snap-freezing lysate on dry-ice, and storing frozen sample at −80°C until ready for RNA extraction. Total RNA (3.5 μg) from all samples was reverse-transcribed in a final volume of 21 μl using the SuperScript First-Strand Synthesis system (Invitrogen) following the manufacturer's instructions. The quantitative real-time PCR reaction was run in an Opticon cycler (MJ Research, Waltham, MA) using Sybr Green PCR master mix (Applied Biosystems, Foster City, CA) following the manufacturer's instructions. Thermocycling was performed in a final volume of 25 μl containing 2 μl of cDNA (1:10 dilution) and 400 nmol/L of primers (18S forward: 5′-ATGCTCTTAGCTGAGTGTCCCG-3′, 18S reverse: 5′-ATTCCTAGCTGCGGTATCCAGG-3′; AM forward: 5′-TTGGATGTCGCGTCGGAG-3′, AM reverse: 5′-TAGCTGCTGGACATCCGCA-3′; vascular endothelial growth factor (VEGF) forward: 5′-TTCATGGATGTCTATCAGCGCA-3′, VEGF reverse: 5′-CCGCATAATCTGCATGGTGA-3′; basic fibroblast growth factor (bFGF) forward: 5′-CGACCCTCACATCAAGCTACAAC-3′, bFGF reverse: 5′-CCAGTTCGTTTCAGTGCCACAT-3′; monocyte chemoattractant protein-1 (MCP-1) forward: 5′-AGATGCAATCAATGCCCCAGT-3′, MCP-1 reverse: 5′-TGGCCACAATGGTCTTGAAGAT-3′). 18S rRNA was run for every sample as a normalizing housekeeping gene. For every sample, both the housekeeping gene and target gene were amplified in triplicates in the same run using the following cycle scheme: after initial denaturation of the samples at 95°C for 2 minutes, 46 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 45 seconds. Fluorescence was measured in every cycle and a melting curve was performed after the PCR by increasing temperature from 50 to 96°C (0.5°C increments). A defined single peak was obtained for all amplicons, thus confirming the specificity of the amplification. Rat peritoneal MCs were prepared according to our previously established protocol under approved NIH institutional review board guidelines.37Hook WA Tsuji S Siraganian RP Magainin-2 releases histamine from rat mast cells.Proc Soc Exp Biol Med. 1990; 193: 50-55Crossref PubMed Scopus (18) Google Scholar Briefly, male Sprague-Dawley rats (240 to 270 g) were euthanized and the peritoneal cavity lavaged with 35 ml of PIPES AC buffer [119 mmol/L NaCl, 5 mmol/L KCl, 25 mmol/L piperazine-N,N′-bis-2-ethanesulfonic acid (NaOH to pH 7.3), 0.3 mmol/L CaCl2, 5.6 mmol/L glucose, and 0.1% bovine serum albumin]. Harvested cells were purified to 95% MC homogeneity via Percoll gradient centrifugation and used for histamine release experiments. Human peripheral blood mononuclear cells were collected from normal donors after informed consent on an approved NIH institutional review board protocol and subsequently enriched to 85% CD34+ cells using affinity column apheresis as previously described.38Woolhiser MR Brockow K Metcalfe DD Activation of human mast cells by aggregated IgG through FcgammaRI: additive effects of C3a.Clin Immunol. 2004; 110: 172-180Crossref PubMed Scopus (107) Google Scholar Cells were placed in serum-free media (StemPro-34 SFM, Life Technologies) supplemented with 2 mmol/L l-glutamine, 50 mg/ml streptomycin, 100 IU/ml penicillin, and fortified with a human recombinant cytokine mixture (Pepro Tech, Rocky Hill, NJ) containing 100 ng/ml of stem cell factor, 100 ng/ml of IL-6, and 30 ng/ml of IL-3 (first week only of in vitro culture). Cultures were maintained at 37°C and 5% CO2, and half the culture media was replaced every 7 days. Greater than 95% of the cells were confirmed to be human cultured mast cells (HCMCs) after 10 weeks of in vitro incubation when assessed via Kimura staining. Histamine release was assayed by an automated fluorometric method previously described.39Siraganian RP Hook WA Histamine release and assay methods for the study of human allergy.in: Rose NR de Macario EC Fahey JL Friedman H Penn GM Manual of Clinical Laboratory Immunology. ASM Press, Washington DC1992: 709-716Google Scholar In brief, histamine was extracted from cell samples and coupled to o-phthalaldehyde under alkaline conditions to give a fluorescent product. The histamine-o-phthalaldehyde complex was stabilized and fluorescent intensity increased by HCl addition. Fluorometric analysis of AM-induced histamine released into the media was determined by comparing observed fluorescence for each test sample to values generated by histamine standard curve. Spontaneous release in all experiments was <5% of the total histamine. Results were expressed as the percentage of maximum release obtained with MCs treated with 3% perchloric acid. For all histamine quantitations, given replicates of each sample showed <8% variation between values. The luteinizing hormone-releasing hormone (LHRH) and its peptide antagonist [N-Ac-d-Nal(2)1,d-pF-Phe2,d-Trp3, d-Arg6]LHRH (Nal-Arg) were obtained from Dr. J. Rivier (Salk Institute, La Jolla, CA). Substance P (SP), AM, and AM peptide fragments (AM1-12, AM34-52, and AM22-52) were obtained from Phoenix Pharmaceutical, Inc. (Belmont, CA). The AM serum-binding protein human complement factor H was purchased from Quidel Corp. (San Diego, CA). Anti-AM monoclonal antibody MoAb-G6 was developed in-house and was previously shown to block a variety of AM-mediated biological effects.28Miller MJ Martinez A Unsworth EJ Thiele CJ Moody TW Elsasser T Cuttitta F Adrenomedullin expression in human tumor cell lines. Its potential role as an autocrine growth factor.J Biol Chem. 1996; 271: 23345-23351Crossref PubMed Scopus (305) Google Scholar, 40Martinez A Weaver C Lopez J Bhathena SJ Elsasser TH Miller MJ Moody TW Unsworth EJ Cuttitta F Regulation of insulin secretion and blood glucose metabolism by adrenomedullin.Endocrinology. 1996; 137: 2626-2632Crossref PubMed Scopus (159) Google Scholar Analysis of peptide-induced β-hexosaminidase (β-hex) release from human MCs was accomplished following our previously established protocol.38Woolhiser MR Brockow K Metcalfe DD Activation of human mast cells by aggregated IgG through FcgammaRI: additive effects of C3a.Clin Immunol. 2004; 110: 172-180Crossref PubMed Scopus (107) Google Scholar HCMCs (104 cells) were suspended in HEPES-Tyrodes buffer (10 nmol/L HEPES, 137 mmol/L NaCl, 2.7 mmol/L KCl, 0.4 mmol/L Na2HPO4, 5.6 mmol/L d-glucose, 1.8 mmol/L CaCl2, 1.3 mmol/L MgSO4) containing 0.025% bovine serum albumin. Appropriate molar concentrations of AM were added to MC suspensions and peptide/cell mixtures incubated for 30 minutes at 37°C to stimulate release of β-hex. Reactions were stopped by centrifugation at 4°C, supernatant removed, snap-frozen on dry ice, and stored at −80°C until ready for β-hex analysis. Deionized water (150 μl) was added to cell pellets, frozen on dry ice, and either thawed for immediate β-hex quantitation or stored at −80°C for future measurements. Supernatant or cell lysate samples (50 μl) were incubated in 0.04 mol/L citric acid with 0.02 mol/L Na2HPO4 containing 10 mmol/L p-nitrophenyl N-acetyl-α-d-glucosaminide (pNAG, β-hex substrate; Sigma) for 90 minutes at 37°C. The reaction was developed using 0.4 mol/L glycine and absorbance was determined at 405 nm. Resulting β-hex values (mean ± SD) were represented as: [(β-hex in supernatant X 2)/(β-hex in supernatant + {total β-hex in cell pellet × 4})]100%. A 96-well ChemoTx microplate (NeuroProbe, Inc., Gaithersburg, MD) was used to study MC motility. Upper and lower chambers of the microplate were separated by a porous membrane (8 μm in diameter) precoated with 10 μg/ml of fibronectin (Sigma-Aldrich). Varying concentrations (0.01 nmol/L to 500 nmol/L) of synthetic AM diluted in RPMI 1640 plus 10% fetal calf serum (Life Technologies) were added to assigned wells of the lower chamber. HMC-1 cells were diluted in RPMI 1640 plus 10% fetal calf serum to a concentration of 5 × 105 cells/ml and 25 μl of this suspension (12,500 cells) was added to each well of the upper chamber. VEGF (R&D Systems, Minneapolis, MN), at a concentration of 10 nmol/L, was used as a positive control for MC migration. After a 4-hour incubation at 37°C, the membrane was fixed and stained with Hema3 (Biochemical Sciences, Inc., Swedesboro, NJ) as recommended by the manufacturer. HMC-1 cells that had traversed the membrane and were attached to the side facing the lower well were photographed through a ×25 microscope objective, and the number of cells per photographic field were counted. For each test measurement four fields were examined and resulting values reported as the mean ± SD. HMC-1 cells were incubated under hypoxic conditions according to a previously described protocol.24Garayoa M Martinez A Lee S Pio R An WG Neckers L Trepel J Montuenga LM Ryan H Johnson R Gassmann M Cuttitta F Hypoxia-inducible factor-1 (HIF-1) up-regulates adrenomedullin expression in human tumor cell lines during oxygen deprivation: a possible promotion mechanism of carcinogenesis.Mol Endocrinol. 2000; 14: 848-862Crossref PubMed Scopus (238) Google Scholar In brief, cells were subjected to reduced oxygen tension using a seamless hypoxia chamber maintained at 37°C and flushed to equilibration with a gas mixture containing 1% O2, 5% CO2, and 94% N2 (Roberts Oxygen, Co., Gaithersburg, MD). At appropriate incubation times, cells were removed from the chamber and the chamber re-equilibrated with the hypoxia gas mixture through repeated flushing. In parallel studies using an iron chelator/hypoxia mimetic, cells were exposed to 260 μmol/L desferrioxamine mesylate (Sigma-Aldrich) and incubated for appropriate time schedules at 37°C under normal atmospheric conditions (21% O2). Ferrous ethylenediammonium sulfate (300 μmol/L) (Sigma-Aldrich) was added to desferrioxamine mesylate cultures as a negative control to saturate iron chelate conditions and restore hypoxia-inducible factor-1 levels to normoxic values. The concentrations of AM found in conditioned media of test samples were determined using a commercially available radioimmunoassay kit (Phoenix Pharmaceuticals, Inc., Mountain View, CA). Samples (1 ml) were initially diluted in an equal volume of 0.1% alkali-treated casein in phosphate-buffered saline (pH 7.4) and applied to prewashed reverse-phase Sep-Pak C-18 cartridges (Waters Corp., Milford, MA). The peptide fraction was eluted from the C18 matrix with 3 ml of 80% isopropanol containing 0.125 N HCl and freeze-dried overnight. AM levels found in lyophilized extracts were then determined by radioimmunoassay following the manufacturer's instructions. Data were statistically evaluated by a two-tailed Student's t-test using Prism 3.0 software. Differences were regarded as significant when P < 0.05. HMC-1 was treated with PMA (20 ng/ml) for 3 days and its proliferation capability compared to untreated HMC-1 by MTT assay throughout a time course. In brief, a single cell suspension of 2 × 105 cells/ml (50 μl/well) was seeded into 96-well polyvinylchloride plates. The assay was performed in RMPI 1640 with 10% fetal calf serum. Cells grew at 37°C, 5% CO2, in a humid incubator and the dye and solubilization solutions (Promega Proliferation Assay; Promega, Madison, WI) were added every day for 5 days to separate plates. The Spectra Rainbow (Tecan, Raleigh, NC) plate reader and software was used to determine changes in the number of viable cells from dye reduction measured by absorbance at 570 nm. To assess the influence of HMC-1-secreted AM on the anchorage-independent growth of tumor cells, A549-511 (1 × 105) was grown in the presence of 3-day-old conditioned media from HMC-1-SCR or HMC-1-511 (4 × 106 cells/175-cm2 flask). Growth capabilities were evaluated as indicated above. The anchorage-independent growth of A549-511 in the presence of HMC-1-SCR or HMC-1-511 was examined by soft agar clonogenic assay. Both HMC-1-SCR and HMC-1-511 were exposed to PMA (20 ng/ml) for 3 day
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