[CANCER RESEARCH 60, 5630 –5634, October 15, 2000] Advances in Brief Hyperinducibility of Hypoxia-responsive Genes without p53/p21-dependent Checkpoint in Aggressive Prostate Cancer 1 Konstantin Salnikow, 2 Max Costa, William D. Figg, 1 and Mikhail V. Blagosklonny 2 Nelson Institute of Environmental Medicine, and Kaplan Comprehensive Cancer Center, New, York University School of Medicine, New York, New York 10016 [K. S., M. C.], and Medicine Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892 [W. D. F. and M. V. B.] Abstract Hypoxia limits tumor growth but selects for higher metastatic potential. We tested the functional activity of hypoxia-inducible factor-1 (HIF-1) in prostate cell lines ranging from normal epithelial cells (PrEC), hormone- dependent LNCaP, hormone-independent DU145, PC-3 to highly meta- static PC-3M cancer cell lines. We found that HIF-1-stimulated transcrip- tion was the lowest in PrEC and LNCaP cells and the highest in PC-3M cells. The induction by hypoxia of the HIF-1 dependent genes Cap43 and GAPDH was the highest in the most aggressive PC-3M cancer cells. Because these advanced prostate cancer cell lines have lost p53 function, this further shifts a balance from p53 to HIF-1 transcriptional regulation, and a high ratio of HIF-1-dependent:p53-dependent transcription was a marker of the advanced malignant phenotype. Transient transfection of HIF-1 expression vector induced transcription from p21 promoter con- struct in prostate cancer cell lines. Furthermore, hypoxia slightly induced p21 mRNA in these cells. However, neither expression of p21 nor hypoxia caused growth arrest in PC-3M cells. Therefore, high inducibility of HIF-1-dependent genes, loss of p53 functions with high ratio of HIF-1- dependent:p53-dependent transcription, and loss of sensitivity to p21 inhibition is a part of hypoxic phenotype associated with aggressive cancer behavior. Introduction Tumor progression toward aggressive and metastatic potential is a fundamental process in neoplasia, but stimuli that drive this progres- sion are poorly understood. Hypoxia limits tumor growth, and tumors with poor vascularization fail to grow and form metastases (1, 2). On the other hand, hypoxia selects for more aggressive and metastatic cancer phenotypes that are associated with poor prognosis (3). Hy- poxia in tumors develops early because of inadequate vascularization of the tumor. The transcriptional response to hypoxia is mediated by HIF-1 3 (4, 5). Lack of HIF-1 retards solid tumor growth and vascu- larity because of the reduced capacity to produce VEGF during hypoxia (1, 2). Increased glycolysis may protect cells from hypoxia, and most glycolytic enzymes are HIF-1-dependent (5). Hypoxia in- hibits cell growth and may cause a p53-dependent apoptosis (6, 7). Taking into account that hypoxia, while limiting tumor growth, is inevitably associated with tumor progression, we envision the ability of cancer cells to survive hypoxia as a natural test that on successful completion allows further tumor progression. We propose that the adverse conditions associated with hypoxia provide a driving force for selection of aggressive, autonomous, and metastatic phenotypes. In- terestingly, hypoxia and carcinogenic nickel exert almost identical effects on gene expression. Furthermore, nickel, a potent nonmuta- genic carcinogen, induces gene expression, in part through HIF-1 transcription factor (8, 9). Previously, we have demonstrated an increase in HIF-driven tran- scription versus a p53-driven transcription in nickel-transformed cells (8). If hypoxia plays a significant role in tumor progression, we predict that not only nickel-transformed cells but also natural human cancer cells would have HIF-1:p53 alterations. In fact, Zhong et al. (10) have demonstrated that elevated amounts of HIF-1 protein exist in PC-3 prostate cancer cells under normoxic conditions linking HIF-dependent transcription under normoxia with tumor progression (11). Here we evaluated HIF-1- and p53-dependent transcription in a panel of prostate cell lines ranging from normal PrECs to the most aggressive PC-3M cells, previously selected for increased metastatic potential in mice. The comparison of PC-3M cells with less aggressive cells revealed more pronounced “hypoxic” features of the aggressive cancer phenotype. Because hypoxia already exists in primary prostate carcinomas (12), our data suggest that an increased inducibility of HIF-dependent genes may be a hallmark of the hypoxia-driven selec- tion. Furthermore, we have shown that rather high levels of HIF-1 are required for transcriptional activation of p21 waf1/cip1 . This activation occurs in prostate cancer cells in a p53-independent manner. The accumulation of p21 did not result in growth arrest in either PC-3M or DU-145 cells. Using flow cytometry, we have shown that prostate cancer cells lost their p21-dependent cell cycle control, whereas p53-dependent cell cycle control was still intact in these cells. Materials and Methods Cell Lines and Reagents. The human prostate cancer cell lines, LNCaP, DU-145, and PC-3 cells were obtained from American Type Culture Collec- tion (Manassas, VA). PC-3M cells, a highly metastatic clone of PC-3 cells, were described previously (13). PrEC, a nontransformed primary cell line, were obtained from Clonetics (San Diego, CA) and incubated in PrECM medium with supplements according to supplier’s instructions. MEF and MEF HIF-1-/- were obtained from Dr. R. Johnson (University of California San Diego) and were described previously (9). DFX was obtained from Sigma (St. Louis, MO) and prepared as a stock solution in water. Ad-p21, a wt p21- expressing adenovirus was obtained from Dr. W. S. El-Deiry (University of Pennsylvania, Philadelphia, PA), and viral titer was determined as described previously (14). Plasmids and Transient Transfection. WWP-Luc, a p21 promoter- luciferase construct, was obtained from Dr. W. S. El-Deiry (University of Pennsylvania). Bax-Luc, a Bax promoter-luciferase construct was obtained from K. Vousden (ABL Basic Research Program, NCI-FCRDC). pC53-SN3, containing wt p53 in a pCMV-Neo-Bam vector, was obtained from Dr. B. Vogelstein (Johns Hopkins University Baltimore, MD). pCM- Vb.HA-HIF-1a expression plasmid was obtained from Dr. D. Livingston (Dana-Farber Cancer Institute, Boston, MA). pCMV.-galactosidase was pur- chased from Clontech (Palo Alto, CA). GFP-expressing plasmid was obtained from Promega. Received 5/10/00; accepted 8/29/00. 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 Supported in part by NIH Grants ES05512, ES00260, and CA16087 ( to K. S. and M. C.). 2 To whom requests for reprints should be addressed, (to K. S.) at Nelson Institute of Environmental Medicine, Kaplan Comprehensive Cancer Center, New York University, New York, NY 10016. Fax: (914) 351-2118; E-mail:salnikow@env.med.nyu.edu; or (to M. V. B.) at Medicine Branch, Building 10, R 12N226, NIH, Bethesda, MD 20892. Fax: (301) 402-0172; E-mail: mikhailb@box-m.nih.gov. 3 The abbreviations used are: HIF-1, hypoxia-inducible factor; VEGF, vascular endo- thelial growth factor; PrEC, prostate epithelial cell; DFX, desferrioxamine; wt, wild type; GFP, green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CMV, cytomegalovirus. 5630 Research. on January 21, 2016. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from