[CANCER RESEARCH 64, 6310 – 6318, September 1, 2004] Expression of Complement Factor H by Lung Cancer Cells: Effects on the Activation of the Alternative Pathway of Complement Daniel Ajona, 1 Zafira Castan ˜o, 1 Mercedes Garayoa, 1,3 Enrique Zudaire, 4 Maria J. Pajares, 1,3 Alfredo Martinez, 4 Frank Cuttitta, 4 Luis M. Montuenga, 1,3 and Ruben Pio 1,2 1 Division of Oncology, Center for Applied Medical Research, Pamplona, Spain; 2 Departments of Biochemistry and 3 Histology and Pathology, School of Medicine, University of Navarra, Pamplona, Spain; and 4 Cell and Cancer Biology Branch, National Cancer Institute, NIH, Bethesda, Maryland ABSTRACT The complement system is important in immunosurveillance against tumors. However, malignant cells are usually resistant to complement- mediated lysis. In this study, we examine the expression of factor H, an inhibitor of complement activation, and factor H-like protein 1 (FHL-1), its alternatively spliced form, in lung cancer. We also evaluate the poten- tial effect of factor H/FHL-1 in the protection of lung cancer cells against the activation of the complement cascade. By Northern blot analysis we demonstrate a high expression of factor H and FHL-1 in most non-small cell lung cancer cell lines, although neuroendocrine pulmonary tumors (small cell lung carcinoma and carcinoid cell lines) had undetectable levels. Western blot analysis of conditioned medium showed the active secretion of factor H and FHL-1 by cells that were positive by Northern blot. Expression of factor H/FHL-1 mRNA was also shown in a series of non-small cell lung cancer biopsies by in situ hybridization. Interestingly, many cultured lung cancer cells were able to bind fluorescence-labeled factor H to their surfaces. Deposition of C3 fragments from normal human serum on H1264, a lung adenocarcinoma cell line, was more efficient when factor H/FHL-1 activity was blocked by specific antibodies. Blocking factor H/FHL-1 activity also enhanced the release of anaphyla- toxin C5a and moderately increased the susceptibility of these cells to complement-mediated cytotoxicity. In summary, we demonstrate the ex- pression of factor H and FHL-1 by some lung cancer cells and analyze the contribution of these proteins to the protection against complement acti- vation. INTRODUCTION Lung cancer is the leading cause of cancer deaths throughout the world (1). The 5-year survival rates for lung cancer are 15% in all developed countries and 5% in many developing countries. These poor survival rates demand new strategies for early detection and major improvements in therapy. New biological and molecular knowl- edge of lung carcinogenesis has led to the proposal of new therapeutic strategies against lung cancer. Some of these strategies are based on monoclonal antibodies targeted to tumor-associated antigens that, among other mechanisms, can initiate complement-dependent cell lysis (2, 3). However, tumor cells seem to have mechanisms to circumvent this complement-mediated immune response (4). It has been suggested that human non-small cell lung cancer cells are especially resistant to complement-mediated lysis as compared with normal cells (5). The complement system consists of a cascade of functionally related proteins capable of causing cell lysis. This system plays a key role in the elimination of non-self cells and the initiation of inflam- matory response (6). The activation of the complement cascade is highly controlled by several regulatory proteins that prevent comple- ment activation. The alternative pathway of complement is spontane- ously activated and requires specific protection mechanisms to restrict the destructive effects of an uncontrolled activated system (7). Com- plement factor H is a key inhibitor of the activation of the alternative pathway. Factor H is a 150-kDa glycoprotein present in human plasma, which is constitutively produced by the liver but is also synthesized extrahepatically by mononuclear phagocytes, fibroblasts, endothelial cells, mesangial cells, astrocytes, oligodendrocytes, and neurons (8 –10). Factor H is composed of 20 repetitive domains termed short consensus repeats, each 60 amino acids in length. Additionally, a 42-kDa protein produced by alternative splicing from the factor H mRNA has also been found. This protein, named factor H-like protein 1 (FHL-1), contains the first 7 domains of factor H and 4 additional amino acids at the COOH-terminal end (11). FHL-1 shares the complement regulatory functions of factor H. Both factor H and FHL-1 bind to C3b, the key component for complement activa- tion, destroying the C3 convertase. Factor H is also a necessary cofactor for the inactivation of C3b by factor I. The ultimate result of factor H activity is the inhibition of the alternative pathway of com- plement (12–14). In addition to its regulation of complement activa- tion, other functions have been found for factor H. It is a ligand for L-selectin (15) and also binds to the integrin Mac-1 (CD11b/CD18), enhancing the activation response of human neutrophils (16). Factor H also induces the secretion of interleukin 1 by monocytes (17) and acts as a chemotactic protein for these cells (18). Factor H binds to cell surface components of several pathogens (19 –23), inhibiting the alternative pathway of complement and thus enhancing their patho- genicity. Finally, factor H binds and increases the receptor-mediated activity of adrenomedullin, a vasodilator and tumor-promoting pep- tide (24). Expression of factor H and FHL-1 has been demonstrated in cell lines from several malignancies: glioblastomas, myosarcomas, neuro- blastomas, and carcinomas from nasopharynge, ovary, cervix, blad- der, and kidney (25–30). Ovarian tumors also produce factor H and FHL-1 (29). Finally, a clinically approved immunoassay for the detection of bladder cancer in urine is based in the quantification of factor H or a factor H-related molecule (30). The objective of our study was to determine whether factor H is expressed in lung cancer, and if so, to investigate the contribution of factor H to the resistance of the lung cancer cells to complement- mediated cytotoxicity. We found that factor H and FHL-1 are ex- pressed in many non-small cell lung cancer cell lines and non-small cell lung cancer tumors. Factor H is also secreted to the extracellular milieu and is able to bind to the tumor cell surface. Our results also demonstrate that factor H and/or FHL-1 limit the activation of com- plement on lung cancer cell membranes, although, in our experimental conditions, not to the extent to fully justify the resistance observed in lung cancer cells to complement-mediated lysis. Received 7/29/03; revised 5/17/04; accepted 6/24/04. Grant support: Funded through the “UTE project CIMA,” and partially supported by Red Tema ´tica de Investigacio ´n Cooperativa de Centros de Ca ´ncer del Ministerio de Sanidad y Consumo (#C03/10). R. Pio was the recipient of the 2004 –2006 AACR-Cancer Research and Prevention Foundation Career Development Award in Translational Lung Cancer Research. 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. Requests for reprints: Ruben Pio, Department of Biochemistry, School of Medicine, University of Navarra, Irunlarrea 1, Pamplona 31080, Spain. Phone: 34-948-425600; Fax: 34-948-425649; E-mail: rpio@unav.es. ©2004 American Association for Cancer Research. 6310 Research. on November 3, 2015. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from