Leukemia Inhibitory Factor: A Newly Identified Metastatic Factor in Rhabdomyosarcomas Marcin Wysoczynski, 1 Katarzyna Miekus, 1 Kacper Jankowski, 1 JensWanzeck, 1 Salvatore Bertolone, 1 Anna Janowska-Wieczorek, 2 Janina Ratajczak, 1 and Mariusz Z. Ratajczak 1,3 1 James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky; 2 Department of Medicine, University of Alberta, Edmonton, Alberta, Canada; and 3 Department of Physiology, Pomeranian Medical University, Szczecin, Poland Abstract Rhabdomyosarcoma frequently infiltrates bone marrow and this process involves the stromal-derived factor-1 (SDF-1)– CXCR4 axis. Because leukemia inhibitory factor (LIF), like SDF- 1, is secreted by bone marrow stroma and directs the regeneration of skeletal muscles, we examined whether the LIF–LIF receptor (LIF-R) axis affects the biology of rhabdo- myosarcoma cells. We found that in rhabdomyosarcoma cells, LIF stimulates the following: ( a) phosphorylation of mitogen- activated protein kinase p42/44, AKT, and signal transducers and activators of transcription 3, (b) adhesion and chemotaxis, and ( c) increased resistance to cytostatics. To compare the biological effects of LIF versus SDF-1, we examined the RH30 cell line, which is highly responsive to both ligands, and found that the chemotaxis of these cells is significantly reduced when the inhibitors of both receptors (T140 for CXCR4 and gp190 blocking antibody for LIF-R) are added simultaneously. Subsequently, by using repetitive chemotaxis to LIF or SDF-1, we selected from the RH30 line subpopulations of cells that respond to LIF but not SDF-1 (RH30-L) or to SDF-1 but not LIF (RH30-S). We found that ( a) RH30-L cells seed better to the bone marrow, liver, and lymph nodes of immunodeficient mice than RH30-S cells and (b) mice inoculated i.m. with the RH30-L cells had more rhabdomyosarcoma cells in the bone marrow and lung after 6 weeks. Thus, we present the first evidence that the LIF–LIF-R axis may direct rhabdomyosarcoma metastasis. Further, because we showed that the in vivo metastasis of RH30 cells is inhibited by small interfering RNA against LIF-R, molecular targeting of this axis could become a new strategy to control the metastasis of rhabdomyosarcoma. [Cancer Res 2007;67(5):2131–40] Introduction Rhabdomyosarcomaisthemostcommonsoft-tissuesarcomaof adolescence and childhood and accounts for 5% of all malignant tumors in patients under 15 years of age (1–10). There are two major histologic subtypes of rhabdomyosarcoma, alveolar rhabdo- myosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). Clinical evidence indicates that ARMS is more aggressive, has a significantlyworseoutcomethanERMS,andinalmostallcasesis associated with bone marrow involvement. Moreover, ARMS is characterized by the translocation t(2;13)(q35;q14) in 70% of cases or the variant t(1;13)(p36;q14) in a smaller percentage of cases. These translocations disrupt the PAX3 and PAX7 genes on chro- mosome2and1,respectively,andthe FKHR geneonchromosome 13, and generate PAX3-FKHR and PAX7-FKHR fusion genes (7–10). These fusion genes encode the fusion proteins PAX3-FKHR and PAX7-FKHR, believed to play a role in cell survival and dysregula- tion of the cell cycle in ARMS cells (1–10). Why rhabdomyosarcoma cells metastasize to the bone marrow is still poorly understood. There is increasing evidence that bone marrow stroma secretes chemoattractants, which attract rhabdo- myosarcomacellsintothebonemarrowmicroenvironmentwhere they find conditions favorable for survival and expansion. We and others showed that the a-chemokine stromal-derived factor-1 (SDF-1) and hepatocyte growth factor/scatter factor (HGF/SF) are secreted by bone marrow stroma and, along with their respective receptors CXCR4 and c-met, are expressed in rhabdomyosarcoma cells and play an important role in the metastasis of rhabdomyo- sarcoma (11–14). Although both SDF-1 and HGF chemoattracted rhabdomyosarcoma cells and enhanced their metastatic behavior (11, 14), surprisingly, blockage of both the SDF-1–CXCR4 and the HGF–c-metaxesdidnotcompletelyinhibitthechemotaxisofthese cells to conditioned medium by bone marrow stroma or lymph nodes, suggesting the involvement of other chemoattractants as potential prometastatic factors in rhabdomyosarcoma. Based on observations that the gp190 signaling cytokine, leukemiainhibitoryfactor(LIF),stimulatesproliferationofskeletal muscle satellite cells and myocytes (15, 16), as well as cardiomyo- cytes(17),andthatnormalbonemarrowstromacellsexpressand secreteLIF(18),wehypothesizedthattheLIF–LIFreceptor(LIF-R) axis is such a prometastatic factor promoting the progression of rhabdomyosarcoma. Furthermore, because oncostatin M (OSM), anothercytokineinthegp190signalingfamily,alsobindstoLIF-R, we also examined the possibility of the involvement of this molecule. We looked at the biological responses of LIF-R–positive ARMS and ERMS cell lines to stimulation by exogenous LIF or OSM, such as phosphorylation of signaling proteins, cell prolifer- ation, survival of rhabdomyosarcoma cells exposed to chemother- apy, adhesion, chemotaxis, chemoinvasion, and expression of matrix metalloproteinases (MMP). Our findings provide evidence for first time that the LIF–LIF-R (cytokine–gp190 signaling receptor axis) regulates the metastatic behavior of rhabdomyosarcoma cells and their metastasis to the bone marrow and suggest that it could also contribute to their resistance to conventional methods of treatment. Materials and Methods Cell lines. We used human rhabdomyosarcoma cell lines (gift of Dr. Peter Houghton, St. Jude Children’s Research Hospital, Memphis, TN) comprising ARMS lines (RH1, RH28, RH30, and CW9019) and ERMS lines Requests for reprints: MariuszZ.Ratajczak,StemCellBiologyProgramatJames GrahamBrownCancerCenter,UniversityofLouisville,DeliaBaxterResearchBuilding, Room 118E, 580 South Preston Street, Louisville, KY 40202. Phone: 502-852-1788; Fax: 502-852-3032; E-mail: mzrata01@louisville.edu. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-1021 www.aacrjournals.org 2131 Cancer Res 2007; 67: (5). 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