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Published OnlineFirst March 10, 2009; DOI: 10.1158/0008-5472.CAN-08-2905 Published Online First on March 10, 2009 as 10.1158/0008-5472.CAN-08-2905 Research Article Identification of Ligand-Induced Proteolytic Cleavage and Ectodomain Shedding of VEGFR-1/FLT1 in Leukemic Cancer Cells Nader Rahimi,1 Todd E. Golde,2 and Rosana D. Meyer1 1Departments of Pathology and Ophthalmology, Boston University Medical School, Boston, Massachusetts and 2Department of Neuroscience, Mayo Clinic, Mayo Clinic College of Medicine, Jacksonville, Florida Abstract endothelial cells as it is poorly tyrosine phosphorylated (4–7) and Vascular endothelial growth factor receptor-1/fms-related lacks the capacity to induce gene expression (8) or to stimulate tyrosine kinase 1 (VEGFR-1/FLT1)is expressed as a mem- cellular responses such as proliferation and migration in cells with brane-bound receptor tyrosine kinase and as an alternatively endothelial origin (6, 7, 9, 10). spliced soluble protein (sVEGFR-1)containing the 1-6 IgG-like Interestingly, the expression and activation of VEGFR-1 in domain of its ectodomain. sVEGFR-1 is known as a naturally nonendothelial cells, in particular cancer cells, are linked to cell occurring inhibitor of angiogenesis and as a surrogate marker proliferation (11, 12), and a recent study indicates that prevent- for cancer progression; it is also linked to pregnancy-induced ing VEGFR-1 signaling by a VEGFR-1–specific ligand, PlGF-blocking hypertension called preeclampsia and to avascularity of nor- antibody, inhibits tumor growth in mice (13), suggesting a functional mal cornea. It remains an open question whether alternative role for VEGFR-1 signaling in tumor growth. The potential role of mRNA splicing is the only mechanism by which sVEGFR-1 VEGFR-1 in cancer was recently highlighted by the observation that is generated. In this study, we show that in leukemic cancer up to 76% of patients with leukemic cancer were identified as cells, PlGF and VEGF-A both induce tyrosine phosphoryla- positive for VEGFR-1 (14, 15). Moreover, the expression and tion of VEGFR-1 and render it susceptible to ectodomain activation of VEGFR-1 in leukemic cancer cells are reported to shedding, resulting in the generation of sVEGFR-1 and an promote cell proliferation and tumor growth (16). intracellular cytoplasmic fragment. Activation of protein VEGFR-1 is expressed as a membrane-bound receptor tyrosine kinase C and tumor necrosis factor-A–converting enzyme kinase and as a soluble form (sVEGFR-1) due to an alternative family metalloproteases are critically required for the occur- splicing of VEGFR-1 mRNA, which encodes its extracellular domain rence of sVEGFR-1. Following the removal of the ectodomain, containing 1-6 immunoglobulin-like domains of VEGFR-1 (17). the remnant of VEGFR-1 remains attached to the membrane, sVEGFR-1 is considered to play a key role in a number of physiologic and the activity of ;-secretase/presenilin is required for its and pathologic conditions; it acts as a surrogate marker for acute release from the cell membrane. We propose that sVEGFR-1 myelogenous leukemia (AML) cancer progression (18) and has been produced via ectodomain shedding plays a prominent role identified as an endogenous inhibitor of angiogenesis in certain in the VEGF receptor system by antagonizing VEGF receptor cancers (19, 20). The presence of sVEGFR-1 is also required for signaling by acting as a dominant-negative form and/or form- avascularity of normal cornea (21) and its expression is linked to ing a nonsignaling dimerizing complex with VEGF receptors. pregnancy-induced hypertension characterized as preeclampsia (22). [Cancer Res 2009;69(6):2607–14] Despite the clear physiologic importance of sVEGFR-1, little is known about the source of circulating sVEGFR-1 and the molecular mechanism involved in its generation. The soluble ectodomain Introduction of cell surface receptors is produced either via alternative mRNA splicing as it has been shown for VEGFR-1 (17) or via proteolytic Vascular endothelial growth factor receptor-1 [VEGFR-1, also cleavage of the ectodomain from the cell surface following called fms-related tyrosine kinase 1 (FLT-1)] is described to play a ligand-induced down-regulation as it has been shown for various negative role in angiogenesis mainly by acting as a decoy receptor receptor tyrosine kinases (RTK), including TIE2, c-kit, HER2, and in endothelial cells. The negative role of VEGFR-1 in angiogenesis c-Met (reviewed in ref. 23). In this study, we show that VEGFR-1 is was initially suggested based on the observation that loss of highly active in leukemic cancer cells, undergoes tyrosine phos- VEGFR-1 in mice causes an increase in the number of endothelial phorylation, and activates a number of key signaling pathways. progenitors, resulting in vascular disorganization (1, 2), and further Tyrosine phosphorylation predisposes VEGFR-1 for ectodomain by a gene-targeting study in which the entire cytoplasmic region of shedding and proteolytic cleavage via a protein kinase C (PKC)– VEGFR-1, including its kinase domain, was deleted. The knockin dependent pathway. Ligand-induced down-regulation and ectodo- truncated VEGFR-1 mice developed normally with no apparent main shedding contributes to formation of sVEGFR-1, which may defect in vasculogenesis (3). Additional observations further antagonize VEGF receptor signaling by acting as a dominant- showed that VEGFR-1 lacks a significant signaling capacity in negative form and/or forming a nonsignaling dimerizing complex with VEGF receptors. Requests for reprints: Nader Rahimi, Boston University Medical Campus, Room 344, 650 Albany Street, Boston, MA 02118. Phone: 617-638-5011; Fax: 617-638-5337; Materials and Methods E-mail: [email protected]. I2009 American Association for Cancer Research. Reagents and antibodies. Recombinant VEGF, recombinant PlGF, doi:10.1158/0008-5472.CAN-08-2905 and ELISA kit, including anti–VEGFR-1 antibody that specifically recognizes www.aacrjournals.org 2607 Cancer Res 2009; 69: (6). March 15, 2009 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst March 10, 2009; DOI: 10.1158/0008-5472.CAN-08-2905 Cancer Research the ectodomain of VEGFR-1 for detection of soluble VEGFR-1, were Results purchased from R&D. Mouse anti-phosphotyrosine antibody (PY-20) was VEGFR-1 is expressed in human leukemic cancer cells, purchased from Transduction Laboratories. Mouse anti-phosphotyrosine antibody (4G10) was purchased Upstate Biotechnology, Inc. The following undergoes tyrosine phosphorylation, and activates multiple antibodies were purchased from Santa Cruz Biotechnology, Inc.: rabbit and signaling pathways. Despite its expression in endothelial cells, goat polyclonal anti–VEGFR-1 antibodies, which specifically recognize the VEGFR-1 is poorly tyrosine phosphorylated in response to ligand cytoplasmic domain of VEGFR-1; goat anti-rabbit antibody; anti-PLCg1 stimulation and lacks proliferative signaling capability in endothe- antibody; goat anti-mouse antibody; and donkey anti-goat antibody lial cells (4, 5, 18, 26). In contrast, VEGFR-1 in certain cancer cells, antibodies. Anti–phospho-PLCg1 (pY783) was purchased from Biosource including leukemic cancer cells such as SKI-DLBCL (a diffuse large International. GF-109203X was purchased from Calbiochem. B-cell lymphoma) cells, promotes cell proliferation (24). Our initial Cell lines and constructs. Construction and expression of wild- observation indicates that in SKI-DLBCL and HT (diffuse large type VEGFR-1 antibodies and mutant VEGFR-1, D1050/VEGFR-1, were B-cell lymphoma) cells, VEGFR-1 is expressed significantly at lower described elsewhere (9). The following lymphoma cell lines, including levels compared with HUVEC (primary human umbilical vein HT, SKI-DLBCL (a diffuse large B cell), and Hs602 (a diffuse large cell endothelial) cells (Fig. 1A). VEGFR-1, however, is highly tyrosine lymphoma), were grown in RPMI medium as described (24) and were phosphorylated in response to PIGF (Fig. 1A) and VEGF stim- kindly provided by Dr. Malcolm A.S. Moore (Memorial Sloan-Kettering B Cancer Center, New York, NY). ulation (Fig. 1 ). Because VEGFR-1 undergoes tyrosine phosphor- Cell proliferation assay. Cell proliferation was measured by the uptake ylation in response to PlGF, we also analyzed the PlGF-dependent of [3H]thymidine as described before (9). Cells were plated at 5 Â 104/mL in activation of key signaling pathways in SKI-DLBCL and HT cells. RPMI containing 10% fetal bovine serum (FBS) in 24-well plates and The results showed that PlGF treatment of SKI-DLBCL and HT cells incubated at 37jC for 12 h. Cells were then washed twice in PBS and stimulates the activation of AKT, PLCg1, and mitogen-activated growth-arrested in RPMI for 12 h at 37jC. Depending on the experimental protein kinase (MAPK; Fig. 1C). The data indicate that the VEGFR- conditions as indicated in the figure legends, VEGF was added and 1 signaling machinery system is fully operational in leukemic cells incubated for 18 to 20 h at 37jC. Cells were pulsed for 4 h with and may play a pivotal role in proliferation and survival of leukemic 3 [ H]thymidine (0.2 ACi/mL) and harvested and measured by a scintillation cancer cells. counter. Quadruplicate samples were performed for each group. Three PlGF stimulates ectodomain shedding and proteolytic independent experiments were performed. cleavage of VEGFR-1. ThepresenceofsolubleVEGFR-1 Immunoprecipitation, cell fractionation, and Western blotting. (sVEGFR-1, herein referred to as ectodomain of VEGFR-1) is Equal numbers of cells from the indicated cell lines were grown in 10-cm culture dishes until 80% to 90% confluent. After overnight serum starvation, elevated in human lymphoma cell lines and in patients with AML cells were left either resting or stimulated with 100 ng/mL of PlGF or VEGF and myelodysplastic syndromes (11, 18, 24). The elevated level of at 37jC as indicated in the figure legends. Cells were prepared and lysed as sVEGFR-1 is also suggested to be an independent prognostic factor described (7, 9).

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