Glycodelin reduces breast cancer xenograft growth in vivo Laura C. Hautala 1,2 , Riitta Koistinen 1,2 , Markku Seppa ¨la ¨ 2 , Ralf B€ utzow 1,3 , Ulf-H ˚ akan Stenman 2 , Pirjo Laakkonen 4 and Hannu Koistinen 2 * 1 Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland 2 Department of Clinical Chemistry, Helsinki University Central Hospital, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland 3 Department of Pathology, Helsinki University Central Hospital, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland 4 Molecular Cancer Biology Research Program and Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Malignant growth is characterized by loss of cell differentiation, uncontrolled proliferation and resistance to apoptosis. Many tu- mor suppressor genes that protect cells against malignant trans- formation regulate cell differentiation. Here, we show for the first time that glycodelin, a differentiation-related protein, reduces breast cancer tumor growth in vivo. We found that glycodelin cDNA-transfected MCF-7 breast cancer cells showed a differenti- ated phenotype and produced smaller tumors in mouse mammary fat pads compared with control-transfected cells. Glycodelin- induced differentiation was associated with reduced expression of oncogenes and increased expression of tumor suppressor genes. Our results suggest that glycodelin acts as a tumor suppressor in breast cancer. This may explain its reported association with a more favorable prognosis in some cancers. ' 2008 Wiley-Liss, Inc. Key words: breast cancer; differentiation; glycodelin; tumor growth; xenograft tumor Cancers may arise through several mechanisms. The main fea- tures include loss of cell differentiation, uncontrolled proliferation and resistance to apoptosis. 1 In breast cancer cells, the malignant phenotype can be reversed back to normal by various mechanisms, such as blocking of b 1 integrin and epidermal growth factor recep- tor by antibodies, and inhibition of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase pathways. 2,3 In addition, many of the proteins that protect the cells against malignant transformation also regulate cell differentiation. 4 Glycodelin is a lipocalin protein mainly expressed in reproduc- tive tissues. 5 It is also expressed in the bone marrow and in many normal and malignant epithelial cells, including those of normal and malignant breast tissues. 6–11 In the reproductive and immune systems, glycodelin is involved in cell recognition. 12 The relation- ship of glycodelin with epithelial differentiation is well estab- lished in the normal endometrium, in which temporal glycodelin expression is progesterone regulated. 5 This is also the case in cul- tured endometrial carcinoma cell lines. 13,14 Recent reports suggest that glycodelin actually drives epithelial differentiation. 8,13,15 In ovarian serous carcinoma, glycodelin is more frequently expressed in well differentiated than in poorly differentiated carci- nomas, and it correlates with better survival. 9 Preliminary results suggest that glycodelin expression is associated with better prog- nosis also in breast cancer. 11 We investigated the molecular changes associated with glyco- delin-induced differentiation. Given that the surrounding microen- vironment plays a role in tumor development, we used various cell culture conditions and a mouse xenograft model to elucidate the effects of glycodelin on tumor growth in vivo. We found that, in experimental preclinical mouse model, glycodelin-producing human breast carcinoma cells form smaller tumors than the glyco- delin nonproducing control cells do. Material and methods The study was approved by the Institutional Review Board, Department of Obstetrics and Gynecology, University Central Hospital, Helsinki and by the State Provincial Office of Southern Finland. Transfection and cell culture The protein-encoding region of glycodelin cDNA was cloned in both antisense and sense orientations, 15 and transfected into MCF- 7 human breast carcinoma cells (HTB-22, American Type Culture Collection) and into phenotypically different MDA-MB-231 breast cancer cells (HTB-26, American Type Culture Collection ). The glycodelin concentration in cell culture medium was mea- sured by an immunofluorometric assay (the detection limit of the assay is 0.5 ng/ml). 16 For MCF-7 cells, 8 stable glycodelin cDNA- transfected cell clones (glycodelin-producing cells) and 8 cell clones into which glycodelin was transfected in antisense orienta- tion (glycodelin nonproducing control cells) were selected for fur- ther investigation. The cells were cultured on plastic/glass, or in or on Matrigel TM ; (Becton Dickinson) in RPMI-1640 supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 lg/ml strep- tomycin and 2 mM L-glutamine at 37°C in a humidified atmos- phere of 5% CO 2 in air. Live cell images of the cells cultured on Matrigel were obtained using an Axiovert 200 inverted light microscope connected to an AxioCam digital camera and visual- ized by AxioVision software (all from Zeiss). Cell proliferation assay Cell proliferation was assessed in plastic cell culture plates using BrdU Cell Proliferation ELISA (Boehringer-Mannheim). The cells were plated in triplicate at densities from 1,000 to 10,000 cells in a 96-well plastic cell culture plate and incubated for 24 hr before adding the BrdU-labeling reagent and incubat- ing for another 3 hr. Two different sense-transfected and antisense-transfected cell clones were tested at least in 2 separate experiments. To evaluate cell proliferation in Matrigel, equal amounts of sense and antisense cells (50,000 cells/200 ll, 3 cell clones for both) were grown inside Matrigel for 1 and 2 weeks, and cell pro- liferation was assessed by H 3 -thymidine (8 lCi/200 ll, Perkin Elmer Life Sciences) incorporation assay. Briefly, after 5 hr incu- bation with H 3 -thymidine, the cells were treated with cell recovery solution (Becton Dickinson) to dissolve cells from the gel, washed and counted in a beta-counter (1216 RackBeta, Wallac). Grant sponsors: University of Helsinki, the Academy of Finland, the Helsinki University Central Hospital Research Funds, the Finnish Cancer Foundation. *Correspondence to: Department of Clinical Chemistry, Biomedicum Helsinki, University of Helsinki, P.O. Box 63 (Haartmaninkatu 8), 00014 University of Helsinki, Finland. Fax: 1358-9-47171731. E-mail: hannu.k.koistinen@helsinki.fi Received 21 January 2008; Accepted after revision 26 May 2008 DOI 10.1002/ijc.23773 Published online 20 August 2008 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 123, 2279–2284 (2008) ' 2008 Wiley-Liss, Inc. Publication of the International Union Against Cancer