[CANCER RESEARCH 63, 1138 –1143, March 1, 2003] Regulation of Colon Carcinoma Cell Invasion by Hypoxia-Inducible Factor 1 1 Balaji Krishnamachary, Shannon Berg-Dixon, Brian Kelly, Faton Agani, David Feldser, Gloria Ferreira, Narayan Iyer, Jessica LaRusch, Brian Pak, Panthea Taghavi, and Gregg L. Semenza 2 McKusick-Nathans Institute of Genetic Medicine and Departments of Pediatrics and Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287- 3914 ABSTRACT Hypoxia-inducible factor 1 (HIF-1) transactivates genes the products of which mediate tumor angiogenesis and glycolytic metabolism. Over- expression of the HIF-1 subunit, resulting from intratumoral hypoxia and genetic alterations, has been demonstrated in common human cancers and is correlated with tumor angiogenesis and patient mortality. Here we demonstrate that hypoxia or HIF-1 overexpression stimulates Matrigel invasion by HCT116 human colon carcinoma cells, whereas this process is inhibited by a small interfering RNA directed against HIF-1. We show that HIF-1 regulates the expression of genes encoding cathepsin D; matrix metalloproteinase 2; urokinase plasminogen activator receptor (uPAR); fibronectin 1; keratins 14, 18, and 19; vimentin; transforming growth factor ; and autocrine motility factor, which are proteins that play established roles in the pathophysiology of invasion. Neutralizing anti- bodies against uPAR block tumor cell invasion induced by hypoxia or HIF-1 overexpression. These results provide a molecular basis for pro- motion of the invasive cancer phenotype by hypoxia and/or HIF-1 overexpression. INTRODUCTION Genetic alterations promote tumor cell proliferation and survival by inducing physiological alterations within tumor cells, e.g., dysregula- tion of apoptosis, cell cycle, and growth factor signaling pathways, as well as in stromal cells, e.g., stimulation of angiogenesis (1). The resulting pathological increase in cell number defines a tumor. In contrast, cancer is defined by the ability to penetrate the ECM 3 of basement membrane and underlying stroma and to invade into sur- rounding tissue (2). Important properties of invasive cancer cells include decreased cell-cell adhesion, cytoskeletal remodeling, in- creased motility, increased production of ECM proteases, and synthe- sis of new ECM components (ECM remodeling). A consequence of increased cell number within a tumor is a corresponding increase in O 2 consumption. Tumor progression and patient mortality are correlated with both microvascular density (3– 6) and intratumoral hypoxia (7). The basis for this apparent paradox is that although angiogenesis is stimulated within tumors, the resulting vessels are structurally and functionally abnormal, resulting in a failure to deliver adequate O 2 . Tumor cell survival is thus dependent on the stimulation of angiogenesis and the metabolic adaptation of tumor cells to hypoxia. HIF-1 is a transcriptional activator, composed of O 2 -regulated HIF-1 and constitutively expressed HIF-1 subunits (8), that func- tions as a master regulator of O 2 homeostasis (9). Four lines of evidence indicate that HIF-1 plays important roles in tumor progres- sion. First, immunohistochemical analyses indicate that HIF-1 is overexpressed in primary and metastatic human cancers and that the level of expression is correlated with tumor angiogenesis and patient mortality (10 –17). Second, in addition to intratumoral hypoxia, ge- netic alterations in tumor suppressor genes (p53, VHL, PTEN) and oncogenes (SRC, HER2 neu , H-RAS) induce HIF-1 activity (18 –25). Third, in mouse xenograft assays, genetic manipulations that increase or decrease HIF-1 activity are associated with increased or decreased tumor growth and angiogenesis, respectively (19, 23, 26 –28). Fourth, HIF-1 controls the expression of gene products that stimulate angio- genesis, such as VEGF, and that promote metabolic adaptation to hypoxia, such as glucose transporters and glycolytic enzymes, pro- viding a molecular basis for its effects on tumor growth and angio- genesis (9, 29 –31). Intratumoral hypoxia is correlated with an increased risk of inva- sion in human cancer (7) and rodent xenografts (32), indicating that the hypoxic tumor microenvironment may select for mutations that promote survival (33) and invasion. An alternate, but not mutually exclusive, hypothesis is that hypoxia acts as a physiological stimulus to induce expression of genes the products of which promote invasion. This model is supported by studies demonstrating that tumor cells that are transiently subjected to hypoxia manifest increased rates of inva- sion through basement membrane ex vivo (34). HIF-1 overexpres- sion was observed in human brain and colon cancer biopsies at the invading tumor margin (16, 17). We hypothesized that HIF-1 over- expression, induced either by intratumoral hypoxia or by genetic alterations, activates programs of gene expression controlling invasion by cancer cells. MATERIALS AND METHODS Cell Culture and Transfection. HCT-116 cells were cultured in McCoy’s 5A medium with 10% FBS and 1% penicillin-streptomycin (Life Technolo- gies, Inc.). Hif1a +/+ and Hif1a -/- ES cells were maintained in high-glucose DMEM with 15% FBS, 1% penicillin-streptomycin, nonessential amino acids, sodium pyruvate, and 200 g/ml of G418 (9). 786-0 RCCs and the WT-8 subclone expressing VHL (provided by William Kaelin, Harvard Medical School, Boston, MA) were cultured in DMEM with 10% FBS, 1% penicillin- streptomycin and 1 mg/ml of G418 (35). Five  10 5 HCT116 cells were plated per 6-cm dish and transfected with 2 g of pCEP4 or pCEP4/HIF-1 (23) using LipofectAMINE-Plus (Life Technologies, Inc.). To generate siRNA HIF-1 , two oligonucleotides consisting of ribonucleosides except for the presence of 2'-deoxyribonucleosides (dTdT) at the 3' end, 5'-AGAGGUG- GAUAUGUGUGGGdTdT-3' and 5'-CCCACACAUAUCCACCUCUdTdT- 3', were synthesized and annealed (Dharmacon Research, Inc.). HCT116 cells were exposed to 100 nM siRNA HIF-1 in the presence of Oligofectamine (Invitrogen) for 4 h (36). Control experiments were performed using siRNA directed against UFP3B mRNA (provided by Josh Mendell and Hal Dietz, Johns Hopkins University, Baltimore, MD). Invasion Assays. For invasion assays, 12-mm-diameter Transwell polycar- bonate filters (12-m pore size, Costar) in a modified Boyden chamber were coated with 100 l of Matrigel (Sigma) at 1:20 dilution in serum-free medium and air-dried for 24 h. Five  10 4 HCT116 cells in 200 l of complete medium were seeded into the inner chamber. Six hundred l of medium were added to the lower chamber, and the plate was incubated at 37°C in a 5% CO 2 /95% air Received 10/24/02; accepted 12/27/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the Children’s Brain Tumor Foundation and NIH (P20-CA86346 and R01-HL55338). 2 To whom requests for reprints should be addressed, at The Johns Hopkins University School of Medicine, CMSC-1004, 600 North Wolfe Street, Baltimore, MD 21287-3914. Phone: (410) 955-1619; Fax: (410) 955-0484; E-mail: gsemenza@jhmi.edu 3 The abbreviations used are: ECM, extracellular matrix; HIF-1, hypoxia-inducible factor 1; VEGF, vascular endothelial growth factor; FBS, fetal bovine serum; RCC, renal carcinoma cell; VHL, von Hippel-Lindau protein; uPA, urokinase plasminogen activator; uPAR, uPA receptor; MMP2, matrix metalloprotease 2; ES, embryonic stem; TGF, transforming growth factor; K19, keratin 19; AMF, autocrine motility factor; siRNA, small interfering RNA. 1138 Research. on November 7, 2015. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from