Epidermal Growth Factor and Hypoxia-induced Expression of CXC Chemokine Receptor 4 on Non-small Cell Lung Cancer Cells Is Regulated by the Phosphatidylinositol 3-Kinase/PTEN/AKT/Mammalian Target of Rapamycin Signaling Pathway and Activation of Hypoxia Inducible Factor-1α
2005; Elsevier BV; Volume: 280; Issue: 23 Linguagem: Inglês
10.1074/jbc.m500963200
ISSN1083-351X
AutoresRoderick J. Phillips, Javier Mestas, Mehrnaz Gharaee‐Kermani, Marie D. Burdick, Antonio Sica, John A. Belperio, Michael P. Keane, Robert M. Strieter,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoNon-small cell lung cancer (NSCLC) expresses a particularly aggressive metastatic phenotype, and patients with this disease have a poor prognosis. CXC chemokine receptor 4 (CXCR4) is a cell surface receptor that has been shown to mediate the metastasis of many solid tumors including lung, breast, kidney, and prostate. In addition, overexpression of the epidermal growth factor receptor (EGFR) is associated with the majority of NSCLC and has been implicated in the process of malignant transformation by promoting cell proliferation, cell survival, and motility. Here we show for the first time that activation of the EGFR by EGF increases CXCR4 expression and the migratory capacity of NSCLC cells. Furthermore, many solid tumors are associated with low oxygen tension, and when NSCLC cells were cultured with EGF under hypoxic conditions, CXCR4 expression was dramatically enhanced. A molecular analysis of these events indicated that augmented CXCR4 expression was regulated by the phosphatidylinositol 3-kinase/PTEN/AKT/mammalian target of rapamycin signal transduction pathway, activation of hypoxia inducible factor (HIF) 1α, and ultimately HIF-1-dependent transcription of the CXCR4 gene. Thus, a combination of low oxygen tension and overexpression of EGFR within the primary tumor of NSCLC may provide the microenvironmental signals necessary to upregulate CXCR4 expression and promote metastasis. Non-small cell lung cancer (NSCLC) expresses a particularly aggressive metastatic phenotype, and patients with this disease have a poor prognosis. CXC chemokine receptor 4 (CXCR4) is a cell surface receptor that has been shown to mediate the metastasis of many solid tumors including lung, breast, kidney, and prostate. In addition, overexpression of the epidermal growth factor receptor (EGFR) is associated with the majority of NSCLC and has been implicated in the process of malignant transformation by promoting cell proliferation, cell survival, and motility. Here we show for the first time that activation of the EGFR by EGF increases CXCR4 expression and the migratory capacity of NSCLC cells. Furthermore, many solid tumors are associated with low oxygen tension, and when NSCLC cells were cultured with EGF under hypoxic conditions, CXCR4 expression was dramatically enhanced. A molecular analysis of these events indicated that augmented CXCR4 expression was regulated by the phosphatidylinositol 3-kinase/PTEN/AKT/mammalian target of rapamycin signal transduction pathway, activation of hypoxia inducible factor (HIF) 1α, and ultimately HIF-1-dependent transcription of the CXCR4 gene. Thus, a combination of low oxygen tension and overexpression of EGFR within the primary tumor of NSCLC may provide the microenvironmental signals necessary to upregulate CXCR4 expression and promote metastasis. Non-small cell lung cancer (NSCLC) 1The abbreviations used are: NSCLC, non-small cell lung cancer; CXCR4, CXC chemokine receptor 4; HIF-1, hypoxia inducible factor-1; VHL, von Hippel-Lindau; PI 3-kinase, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog deleted on chromosome 10; EGF, epidermal growth factor; EGFR, EGF receptor; mTOR, mammalian target of rapamycin; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. is one of the leading causes of malignancy-related mortality in the United States; indeed fewer than 15% patients survive beyond 5 years after diagnosis. The virulence of this cancer is mediated in part by the specific and aggressive metastatic pattern of primary neoplastic cells to regional lymph nodes, liver, adrenal glands, contralateral lung, brain, and the bone marrow (1Fidler I.J. Cancer Res. 1990; 50: 6130-6138PubMed Google Scholar, 2Smith W. Khuri F.R. Semin. Oncol. 2004; 31: 11-15Crossref PubMed Scopus (51) Google Scholar, 3Fidler I.J. Cancer Chemother. Pharmacol. 1999; 43: 3-10Crossref PubMed Scopus (157) Google Scholar, 4Devesa S.S. Blot W.J. Stone B.J. Miller B.A. Tarone R.E. Fraumeni Jr., J.F. J. Natl. Cancer Inst. 1995; 87: 175-182Crossref PubMed Scopus (390) Google Scholar). In this respect, we and others have now demonstrated that the metastatic propensity of tumors from several different types of cancer including lung, breast, ovarian, renal, and prostate is related to the expression of the chemokine receptor CXCR4 (5Muller A. Homey B. Soto H. Ge N. Catron D. Buchanan M.E. McClanahan T. Murphy E. Yuan W. Wagner S.N. Barrera J.L. Mohar A. Verastegui E. Zlotnik A. Nature. 2001; 410: 50-56Crossref PubMed Scopus (4566) Google Scholar, 6Belperio J.A. Phillips R.J. Burdick M.D. Lutz M. Keane M. Strieter R. Chest. 2004; 125: 156Abstract Full Text Full Text PDF PubMed Google Scholar, 7Zlotnik A. Semin. Cancer Biol. 2004; 14: 181-185Crossref PubMed Scopus (249) Google Scholar, 8Taichman R.S. Cooper C. Keller E.T. Pienta K.J. Taichman N.S. McCauley L.K. Cancer Res. 2002; 62: 1832-1837PubMed Google Scholar, 9Strieter R.M. Belperio J.A. Phillips R.J. Keane M.P. Semin. Cancer Biol. 2004; 14: 195-200Crossref PubMed Scopus (187) Google Scholar, 10Staller P. Sulitkova J. Lisztwan J. Moch H. Oakeley E.J. Krek W. Nature. 2003; 425: 307-311Crossref PubMed Scopus (755) Google Scholar, 11Phillips R.J. Burdick M.D. Lutz M. Belperio J.A. Keane M.P. Strieter R.M. Am. J. Respir. Crit. Care Med. 2003; 167: 1676-1686Crossref PubMed Scopus (425) Google Scholar, 12Scotton C.J. Wilson J.L. Milliken D. Stamp G. Balkwill F.R. Cancer Res. 2001; 61: 4961-4965PubMed Google Scholar). In fact, in human NSCLC-SCID mouse chimera we have observed that the neoplastic cells present at the sites of the secondary metastases express dramatically up-regulated levels of this chemokine receptor in comparison with the cancerous cells present in the primary tumor (11Phillips R.J. Burdick M.D. Lutz M. Belperio J.A. Keane M.P. Strieter R.M. Am. J. Respir. Crit. Care Med. 2003; 167: 1676-1686Crossref PubMed Scopus (425) Google Scholar). Furthermore, in both NSCLC and breast cancer it has been shown that the ligand for CXCR4, CXCL12, exhibited peak levels of expression in organs that were the preferred destination for their respective metastases (5Muller A. Homey B. Soto H. Ge N. Catron D. Buchanan M.E. McClanahan T. Murphy E. Yuan W. Wagner S.N. Barrera J.L. Mohar A. Verastegui E. Zlotnik A. Nature. 2001; 410: 50-56Crossref PubMed Scopus (4566) Google Scholar, 11Phillips R.J. Burdick M.D. Lutz M. Belperio J.A. Keane M.P. Strieter R.M. Am. J. Respir. Crit. Care Med. 2003; 167: 1676-1686Crossref PubMed Scopus (425) Google Scholar). Moreover, when the CXCR4/CXCL12 biological axis was perturbed in these systems using either neutralizing anti-CXCR4 or neutralizing anti-CXCL12 antibodies, the host metastatic burden was significantly reduced, whereas the size of the primary tumor was unaffected (5Muller A. Homey B. Soto H. Ge N. Catron D. Buchanan M.E. McClanahan T. Murphy E. Yuan W. Wagner S.N. Barrera J.L. Mohar A. Verastegui E. Zlotnik A. Nature. 2001; 410: 50-56Crossref PubMed Scopus (4566) Google Scholar, 11Phillips R.J. Burdick M.D. Lutz M. Belperio J.A. Keane M.P. Strieter R.M. Am. J. Respir. Crit. Care Med. 2003; 167: 1676-1686Crossref PubMed Scopus (425) Google Scholar). Thus, it appears that the normal physiology of CXCR4 and CXCL12 has been usurped by several different types of cancer to promote the specific metastasis of neoplastic cells to distant organs. Tumors such as NSCLC typically require neovascularization to mediate growth and promote metastasis, yet paradoxically the most malignant tumors have been found to prosper under conditions of low oxygen tension or hypoxia (13Vogelstein B. Kinzler K.W. Nat. Med. 2004; 10: 789-799Crossref PubMed Scopus (3408) Google Scholar, 14Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5503) Google Scholar, 15Semenza G.L. Respir. Res. 2000; 1: 159-162Crossref PubMed Scopus (137) Google Scholar, 16Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1703) Google Scholar, 17Ryan H.E. Lo J. Johnson R.S. EMBO J. 1998; 17: 3005-3015Crossref PubMed Scopus (1353) Google Scholar, 18Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6172) Google Scholar). This paradox occurs because the tumor vasculature is structurally and functionally abnormal, resulting in perfusion that is characterized by marked spatial and temporal heterogeneity. Thus tumor progression requires an increased adaptation to hypoxia, and the master switch that appears to regulate this phenomenon is the transcription factor, hypoxia inducible factor-1 (HIF-1) (14Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5503) Google Scholar, 15Semenza G.L. Respir. Res. 2000; 1: 159-162Crossref PubMed Scopus (137) Google Scholar, 19Wang G.L. Semenza G.L. J. Biol. Chem. 1995; 270: 1230-1237Abstract Full Text Full Text PDF PubMed Scopus (1766) Google Scholar, 20Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2272) Google Scholar). Indeed, an extensive body of work has already shown that HIF-1 regulates the transcription of several gene clusters that are crucial to tumor progression including angiogenesis, cell survival, glucose metabolism, and invasion/metastasis (14Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5503) Google Scholar, 18Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6172) Google Scholar, 21Wood S.M. Wiesener M.S. Yeates K.M. Okada N. Pugh C.W. Maxwell P.H. Ratcliffe P.J. J. Biol. Chem. 1998; 273: 8360-8368Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). HIF-1 is a heterodimer comprising a constitutively expressed HIF-1β subunit and a highly regulated HIF-1α subunit (14Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5503) Google Scholar, 15Semenza G.L. Respir. Res. 2000; 1: 159-162Crossref PubMed Scopus (137) Google Scholar, 16Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1703) Google Scholar). Classically, ambient oxygen tension regulates the rate at which HIF-1α protein is degraded; under normoxic conditions specific proline residues in the HIF-1α protein are hydroxylated, facilitating the binding of the von Hippel-Lindau (VHL) tumor suppressor protein (22Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4572) Google Scholar, 23Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3982) Google Scholar). VHL is the recognition component of the E3 ubiquitin-protein ligase, and ubiquination of HIF-1α targets the protein for rapid degradation by the 26 S proteasome. By contrast, under hypoxic conditions the rates of proline hydroxylation decreases, thus preventing the binding of VHL to HIF-1α and promoting HIF-1-mediated transcription of target genes (13Vogelstein B. Kinzler K.W. Nat. Med. 2004; 10: 789-799Crossref PubMed Scopus (3408) Google Scholar, 14Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5503) Google Scholar, 24Leung S.K. Ohh M. J. Biomed. Biotechnol. 2002; 2: 131-135Crossref PubMed Scopus (6) Google Scholar). Oxygen-independent regulation of HIF-1α has also been shown to occur, although this is thought to be cell type-specific. Here, growth factors stimulate HIF-1α synthesis via activation of the phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinase pathways (14Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5503) Google Scholar, 25Fukuda R. Hirota K. Fan F. Jung Y.D. Ellis L.M. Semenza G.L. J. Biol. 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Liu Z.G. Neckers L. Biochem. J. 2003; 370: 1011-1017Crossref PubMed Scopus (172) Google Scholar). In an effort to address the mechanisms governing the upregulation of CXCR4 in NSCLC we have used an in vitro model system to study this pivotal chemokine receptor. Our data indicate that exposure of NSCLC cells to hypoxia or EGF results in a significant up-regulation of CXCR4 expression and chemotactic behavior. In addition, both hypoxia and EGF activate HIF-1α, and this in turn increases transcription at the CXCR4 promoter. The PI 3-kinase inhibitors wortmannin and LY294002 and the mTOR inhibitor, rapamycin, inhibit activation of HIF-1α and, hence, up-regulation of CXCR4 expression. Moreover, introduction of wild type PTEN into NSCLC cells also inhibits hypoxia-induced up-regulation of CXCR4 expression. Taken together, therefore, these data suggest that dys-regulated signal transduction through the PI 3-kinase pathway in NSCLC leads to activation of HIF-1α, up-regulation of CXCR4, and increased metastatic potential. Human NSCLC Cell Lines—The H157 and A549 non-small cell lung cancer cell lines were obtained from the ATCC. These cell lines were cultured in RPMI 1640 media (Whitaker Biomedical Products, Whitaker, CA) together with 1 mm l-glutamine, 25 mm HEPES buffer, 100 units/ml penicillin, 100 ng/ml streptomycin, and 10% FCS (RPMI complete media). Before assay the cells were transferred to RPMI starvation media, which comprises 1 mm l-glutamine, 25 mm HEPES buffer, 100 units/ml penicillin, 100 ng/ml streptomycin, and 1% FCS or 0.25% human serum albumin. Where applicable the PI 3-kinase inhibitors, LY294002 (20–50 mm), wortmannin (100–250 nm), and the mTOR inhibitor rapamycin (10 ng/ml) were preincubated with cells for 2 h before exposure to hypoxia and stimulation with EGF (20 ng/ml). RNA Isolation and Real-time PCR—Total RNA was isolated from both A549 and H157 cells using TRIzol (Invitrogen) and by following the manufacturer's instructions. Briefly, cells were lysed in TRIzol and then mixed with chloroform. The lysate was then centrifuged to separate RNA, DNA, and protein. Total RNA was recovered, precipitated with isopropanol, washed in 75% ethanol to remove impurities and finally dissolved in water. Next, 1.5 μg of RNA was taken and DNase-treated to remove contaminating DNA before reverse transcription to cDNA using a ProSTAR first strand reverse transcription-PCR kit (Stratagene) and by following the manufacturer's instructions. Subsequently, the cDNA was assayed for changes in CXCR4 expression by real-time PCR using the ABI Prism 7700 sequence detector and SDS analysis software (Applied Biosystems, Foster City, CA) as previously described (34Belperio J.A. Dy M. Burdick M.D. Xue Y.Y. Li K. Elias J.A. Keane M.P. Am. J. Respir. Cell Mol. Biol. 2002; 27: 419-427Crossref PubMed Scopus (197) Google Scholar). Antibody Staining and Fluorescence-activated Cell Sorter Analysis— Cells from each cell line were taken and resuspended in ice-cold staining buffer (phosphate-buffered saline plus 2% FCS plus 0.1% sodium azide) and incubated with Fc block for 5 min at 4 °C. Subsequently, the cells were stained with fluorescein isothiocyanate-conjugated anti-CXCR4 antibodies or the appropriate isotype control at 4 °C for 20 min, after which time they were washed twice with staining buffer. Samples were finally analyzed on a FACScan flow cytometer (BD Biosciences) using Cellquest 3.2.1f1 software. Hypoxia Treatment and Extract Preparation—Cells were cultured to a density of ∼80% in complete media and then transferred to starvation media. Next, A549 and H157 cells were exposed to either normoxia (ambient oxygen tension) or hypoxia (94% nitrogen, 5% carbon dioxide, and 1% oxygen) in Modular Incubator Chambers (Billups-Rothenberg, Inc., Del Mar, CA) for the times indicated. Subsequently, whole cell extracts or nuclear and cytoplasmic extracts of A549 and H157 cells were prepared. Briefly, whole cell extract lysis buffer was composed of 20 mm HEPES, pH 7.9, 25% glycerol, 420 mm NaCl, 1.5 mm MgCl2, and 0.2 mm EDTA plus a panel of protease and phosphatase inhibitors (phenylmethylsulfonyl fluoride, dithiothreitol, and NaF at 1 mm; aprotinin, leupeptin, pepstatin, and β-glycerophosphate at 10 μg/ml). Buffer A for extraction of cytoplasmic fractions was composed of 10 mm HEPES, pH 7.9, 10 mm KCl, and 0.1 mm EDTA (plus the above panel of protease and phosphatase inhibitors), and buffer C for extraction of the nuclear fraction was composed of 20 mm HEPES, pH 7.9, 400 mm NaCl, and 1 mm EDTA (plus the protease and phosphatase inhibitors described above). Western Blotting—Immunoblotting was performed on 40 μg of total protein from either whole cell or nuclear and cytoplasmic extracts. After SDS-PAGE the proteins were electrophoretically transferred to a polyvinylidene difluoride membrane at 100 V for 1 h at room temperature and then blocked in BLOTTO for 30 min. Subsequently, the membranes were incubated overnight at 4 °C with either a mouse anti-human HIF1-α (1:500; BD Biosciences), rabbit anti-human CXCR4 (1:500; Oncogene Research Products, Cambridge, MA), or rabbit anti-human phospho-AKT (1:1000; Cell Signaling Technology, Beverly, MA). Subsequently, the blots were washed in Tween-Tris buffered saline and then incubated with either donkey anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated secondary antibodies for 45 min at room temperature. After washing in Tween-Tris buffered saline (×3, 15 min each wash), the immunoreactive proteins were finally visualized using ECL Plus (Amersham Biosciences) and by following the manufacturer's instructions. To demonstrate equal loading of each lane, the membranes were then reprobed with a GAPDH antibody (1:500; Abcon) or total AKT antibody (1:1000; Cell Signaling Technology, Beverly, MA). Chemotaxis—A549 cells and H157 cells previously exposed to either hypoxia or normoxia for 24 h were harvested by trypsinization, counted, and resuspended in RPMI 1640 media containing 10% FCS at a concentration of 106/ml. Neuroprobe filters (5-μm diameter) pretreated with 5 μg/ml fibronectin and 12-well chemotaxis chambers were used for these assays. CXCL12 (30 ng/ml; Peprotech, Rocky Hill, NJ) was added to the lower wells, and 105 cells were added to each of the upper wells. The chemotaxis chambers were then incubated for 6 h at 37 °C. After fixing in methanol and staining in 2% toluidine blue, the number of cells that had migrated through to the underside of the filters was calculated by counting the total number of cells in 5 separate fields of view under 400× magnification. In similar experiments A549 cells were either left untreated or pretreated with the PI 3-kinase inhibitors wortmannin (100 nm; Upstate Biotechnology) and LY294002 (20 μm; Cell Signal Technology) for 2 h before chemotactic analysis. Electrophoretic Mobility Shift Assay—A549 cells were either exposed to hypoxia or normoxia for 6 h, and then nuclear extracts were prepared. Oligonucleotide probes for HIF-1α were generated by 5′ end labeling of the sense strand with [γ-32P]ATP (Amersham Biosciences) and T4 polynucleotide kinase. Subsequently, labeled wild type (WT; 5′-agcttGCCCTACGTGCTGTCTCAg-3′) and mutant (M; 5′-agcttGCCCTAAAAGCTGTCTCAg-3′) probes (20Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2272) Google Scholar) were purified using the MER-maid kit (Bio 101 Systems, Irvine, CA) and by following the manufacturer's instructions. Binding reactions were performed in a total volume of 20 μl containing 10 μg of nuclear extract and 0.5 μg of poly(dI-dC)·(dI-dC) in 10 mm Tris-HCl, pH 7.5, 50 mm KCl, pH 7.5, 50 mm NaCl, 1 mm MgCl2, 1 mm EDTA, 5 mm dithiothreitol, and 5% glycerol. Probe (5 × 104 cpm) was then added to the reaction mixture and incubated for 10 min at room temperature before loading onto a 5% non-denaturing polyacrylamide gel. Electrophoresis was performed at 175 volts in 0.5× Tris-buffered EDTA (TBE), pH 8.3 (1× TBE composed of 89 mm Tris-HCl, 89 mm boric acid, and 5 mm EDTA), at 4 °C for 3 h. Gels were then vacuum-dried and exposed to film with intensifying screens for 24 h. Excess (100 fold) unlabeled WT oligonucleotide was preincubated with the reaction mixture for 15 min before the addition of [γ-32P]ATP WT probe. Transient Transfections—A549 and H157 cells were cultured to a density of ∼80% in complete media in 6-well plates. Transfections were performed with Lipofectamine 2000 and Opti-MEM media (Invitrogen) and by following the manufacturer's instructions. Briefly, 2.5 μg of either WT- or C124S-PTEN constructs (35Myers M.P. Stolarov J.P. Eng C. Li J. Wang S.I. Wigler M.H. Parsons R. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9052-9057Crossref PubMed Scopus (738) Google Scholar) were mixed with 4 μl of Lipofectamine and 1 ml of Opti-MEM media for 20 min at room temperature. Subsequently, this mixture was added to the NSCLC cells, which were then incubated at 37 °C for 90 min. Finally, the transfection mixture was removed and replaced with either Dulbecco's modified Eagle's medium plus 10% FCS (H157 cells) or RPMI1640 plus 10% FCS (A549 cells). The cells were then cultured for 24 h and 37 °C before exposure to normoxia or hypoxia for a further 24 h. Upon exposure to normoxia or hypoxia, cultures were returned to media containing only 1% FCS. Subsequently, RNA was prepared and subjected to real-time PCR analysis of CXCR4 expression as described above. In similar experiments A549 and H157 cells were cotransfected with a 2.6-kilobase sequence of either the WT-CXCR4 promoter (36Schioppa T. Uranchimeg B. Saccani A. Biswas S.K. Doni A. Rapisarda A. Bernasconi S. Saccani S. Nebuloni M. Vago L. Mantovani A. Melillo G. Sica A. J. Exp. Med. 2003; 198: 1391-1402Crossref PubMed Scopus (695) Google Scholar, 37Caruz A. Samsom M. Alonso J.M. Alcami J. Baleux F. Virelizier J.L. Parmentier M. Arenzana-Seisdedos F. FEBS Lett. 1998; 426: 271-278Crossref PubMed Scopus (100) Google Scholar) or a mutant form of CXCR4 promoter, where the HIF-1α binding site had been mutated upstream of a luciferase reporter together with 0.5 μg of the Renilla control construct (pRL-SV40; Promega, Madison, WI) and a HIF-1α construct. A GFP construct was used to equalize the DNA transfection load. The cells were then cultured for 48 h and 37 °C before analysis. After the 48-h incubation period, cell extracts were made using the luciferase reporter lysis buffer (Promega). Each lysate was subsequently assayed in the dual luciferase reporter assay (Promega) following the manufacturer's instructions; luciferase activity was determined using a Monolight series 2010 luminometer (Analytical Luminescence Laboratory) and then normalized to the Renilla control. Statistical Analysis—Comparisons were evaluated by Student's unpaired t test. Results were considered statistically significant if p values were 0.05 or less. Hypoxia Promotes Up-regulation of CXCR4 Expression in Non-small Cell Lung Cancer Cells—We have previously shown that up-regulation of CXCR4 expression is a key component in the metastasis of NSCLC cells in vivo (11Phillips R.J. Burdick M.D. Lutz M. Belperio J.A. Keane M.P. Strieter R.M. Am. J. Respir. Crit. Care Med. 2003; 167: 1676-1686Crossref PubMed Scopus (425) Google Scholar), but the mechanisms that regulate expression of this chemokine receptor are unclear. Initial evidence in other cancerous cells has indicated that hypoxia-induced HIF-1 activation may be involved (10Staller P. Sulitkova J. Lisztwan J. Moch H. Oakeley E.J. Krek W. Nature. 2003; 425: 307-311Crossref PubMed Scopus (755) Google Scholar, 36Schioppa T. Uranchimeg B. Saccani A. Biswas S.K. Doni A. Rapisarda A. Bernasconi S. Saccani S. Nebuloni M. Vago L. Mantovani A. Melillo G. Sica A. J. Exp. Med. 2003; 198: 1391-1402Crossref PubMed Scopus (695) Google Scholar). To address this phenomenon in non-small cell lung cancer, therefore, we exposed tumor cells (cultured in RPMI starvation media) to a hypoxic environment (94% N2, 5% CO2, 1% O2) and performed a kinetic analysis to examine changes in CXCR4 expression and function (Fig. 1). Using real-time PCR, our data revealed that the expression of CXCR4 mRNA was strongly elevated in hypoxia-exposed A549 and H157 NSCLC cells by 6 h when compared with the normoxic control (Fig. 1A). This expression remained elevated until at least 24 h (Fig. 1A). Next, we wanted to determine whether the increase in CXCR4 mRNA correlated with an increase in protein levels of CXCR4. We examined this both at the level of intracellular expression (Fig. 1B) and cell surface expression (Fig. 1C). Our results indicated that NSCLC cells exposed to hypoxia showed a significant increase in intracellular CXCR4 protein levels when compared with the normoxic control by 6 h, and these levels remained elevated until at least 24 h (Fig. 1B). Indeed, by 24 h both A549 cells and H157 cells showed significantly greater expression of CXCR4 at the cell surface (Fig. 1C). To determine whether this increased expression of CXCR4 was functional, we performed chemotaxis assays (Fig. 1D). Here, NSCLC cells were exposed to hypoxia or normoxia for 24 h and then treated with CXCL12 for 6 h. Although both A549 and H157 cells demonstrated chemotactic behavior in response to CXCL12 under normoxic conditions, the magnitude of these responses was dramatically enhanced in those cells exposed to hypoxia for 24 h (Fig. 1D). Thus, we have demonstrated that hypoxia not only increases expression of CXCR4 on NSCLC cells but also enhances the migratory ability of these cells in response to CXCL12. Hypoxia Activates HIF-1α Expression in NSCLC Cells and Promotes HIF-1-mediated Transcription at the CXCR4 Promoter—Having established that a physiological event such as hypoxia is capable of up-regulating CXCR4 expression in NSCLC cells, we next wanted to examine the underlying biochemistry that mediates this phenomenon. It has been well established that hypoxia regulates the expression of HIF-1α, which is a key component of the transcription factor HIF-1 (14Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5503) Google Scholar, 15Semenza G.L. Respir. Res. 2000; 1: 159-162Crossref PubMed Scopus (137) Google Scholar, 16Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1703) Google Scholar). And HIF-1 itself is thought to regulate the transcription of several gene clusters crucial to tumor progression including angiogenesis, cell survival, glucose metabolism, and invasion/metastasis (13Vogelstein B. Kinzler K.W. Nat. Med. 2004; 10: 789-799Crossref PubMed Scopus (3408) Google Scholar, 14Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5503) Google Scholar, 18Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6172) Google Scholar, 21Wood S.M. Wiesener M.S. Yeates K.M. Okada N. Pugh C.W. Maxwell P.H. Ratcliffe P.J. J. Biol. Chem. 1998; 273: 8360-8368Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Thus, we exposed A549 cells and H157 cells to normoxia or hypoxia for the times indicated and then examined intranuclear HIF-1α expression by Western analysis (Fig. 2A). Under normoxic conditions little or no intranuclear expression of HIF-1α was observed. This is in keeping with known data, which has suggested that under normal ambient conditions the tumor suppressor gene, VHL, binds to HIF-1α and targets it for degradation (22Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4572) Google Scholar, 23Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3982) Google Scholar). However, under hypoxic conditions, strong intranuclear expression of HIF-1α was observed within 2 h, and this expression remained elevated for a total of 6 h; by 24 h, HIF-1α expression had returned to background levels (Fig. 2A). Next, we wanted to determine whether hypoxia promoted an increase in t
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