[CANCER RESEARCH 64, 6127– 6136, September 1, 2004] The Ras/Mitogen-Activated Protein Kinase Pathway Inhibitor and Likely Tumor Suppressor Proteins, Sprouty 1 and Sprouty 2 Are Deregulated in Breast Cancer Ting Ling Lo, 1 Permeen Yusoff, 1 Chee Wai Fong, 1 Ke Guo, 2 Ben J. McCaw, 1 Wayne A. Phillips, 5 He Yang, 4 Esther Sook Miin Wong, 1 Hwei Fen Leong, 1 Qi Zeng, 2 Thomas Choudary Putti, 3 and Graeme R. Guy 1 1 Signal Transduction Laboratory and 2 Histology Unit, Institute of Molecular and Cell Biology, Proteos, Singapore; 3 Department of Pathology, National University Hospital, Singapore; 4 Bioinformatics Institute, Matrix, Singapore; and 5 Surgical Oncology Research Laboratory, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia ABSTRACT Sprouty (Spry) proteins were found to be endogenous inhibitors of the Ras/mitogen-activated protein kinase pathway that play an important role in the remodeling of branching tissues. We investigated Spry expression levels in various cancers and found that Spry1 and Spry2 were down- regulated consistently in breast cancers. Such prevalent patterns of down- regulation may herald the later application of these isoforms as tumor markers that are breast cancer specific and more profound than currently characterized markers. Spry1 and 2 were expressed specifically in the luminal epithelial cells of breast ducts, with higher expression during stages of tissue remodeling when the epithelial ducts are forming and branching. These findings suggest that Sprys might be involved as a modeling counterbalance and surveillance against inappropriate epithelial expansion. The abrogation of endogenous Spry activity in MCF-7 cells by the overexpression of a previously characterized dominant-negative mu- tant of Spry, hSpry2 Y55F resulted in enhanced cell proliferation in vitro. The hSpry2 Y55F stably expressing cells also formed larger and greater number of colonies in the soft-agar assay. An in vivo nude mice assay showed a dramatic increase in the tumorigenic potential of hSpry2 Y55F stable cells. The consistent down-regulation of Spry1 and 2 in breast cancer and the experimental evidence using a dominant-negative hSpry2 Y55F indicate that Spry proteins may actively maintain tissue in- tegrity that runs amok when their expression is decreased below normal threshold levels. This alludes to a previously unrecognized role for Sprys in cancer development. INTRODUCTION Growth factor signaling by receptor tyrosine kinases regulates important processes in target cells, including activation of the Ras/ mitogen-activated protein (Ras/MAP) kinase cascade, which has been shown to be central to both proliferation and differentiation of cells (1, 2). Since the discovery of the Ras/MAP kinase pathway over a decade ago, various genes or proteins that modulate the activity of the basic pathway components have been discovered (3, 4). However, given the putatively vital role of the Ras/MAP kinase pathway in such key processes, it would be expected that dysfunction of any of these key components would result in developmental disorders and inappropri- ate proliferation of cells, which are seen in various cancers (5, 6). Such deregulation has been documented with the inappropriate acti- vation or overexpression of various receptor tyrosine kinases (e.g., epidermal growth factor receptor and platelet-derived growth factor receptor) in cancers (7). Oncogenic forms of Ras proteins that have been constitutively activated by a point mutation are also expressed frequently in tumors. This enables them to cause hyperactivation of the MAP kinase pathway, independent of any upstream ligand stim- ulation (7). Ras has been shown to be hyperactivated in 90% of pancreatic, 60% of thyroid, and 45% of colorectal cancers (8). Ras/MAP kinase signaling can be down-regulated in a number of ways: (a) feed-back phosphorylation of the receptors on serine and threonine residues by MAP kinases; (b) dephosphorylation of key receptor tyrosine residues by tyrosine phosphatases; (c) competition with the binding of growth factors; and (d) by selective tagging and endosomal destruction of growth factor/receptor complexes (3, 4). Recently, a specialist Ras/MAP kinase inhibitor protein was dis- covered in a Drosophila genetic screen designed to identify compo- nents in fibroblast growth factor (FGF)-induced tracheal branching (9). The Drosophila Sprouty (dSpry) protein is induced by activation of the Ras/MAP kinase pathway and acts back on the pathway to inhibit it by an unspecified mechanism (9 –12). Later work indicated that dSpry is also an inhibitor of the Ras/MAP kinase pathway induced by other receptor tyrosine kinases, including the epidermal growth factor receptor during eye development and oogenesis (10 –12). Four mammalian Sprouty genes have since been identified, based on sequence similarities to dSpry (13, 14). Mammalian Sprys were found to be highly expressed within localized domains in the embryo, which overlapped with or are immediately adjacent to known expres- sion domains of one or more Fgf genes (14 –17). Conserved functions were found between dSpry and the mammalian Sprys in negative regulation of organogenesis. Mammalian Sprys have been shown to particularly function as negative regulators in FGF signaling during vertebrate embryonic development. The overexpression of mouse Spry2 or Spry4 results in the repression of FGF-mediated limb de- velopment in the chick (15) and inhibition of lung branching mor- phogenesis (14, 18, 19). In addition, the overexpression of mSpry4 in mouse embryos inhibited branching and sprouting of small blood vessels (20). The overexpression of Spry proteins in vitro was also found to inhibit FGF- and vascular endothelial growth factor-induced proliferation and migration by repressing pathways that lead to MAP kinase activation (21–23). However, Spry proteins did not affect the EGF- or phorbol ester-induced MAP kinase activation (21, 23). This data suggests that mammalian Spry proteins are not general inhibitors of the receptor tyrosine kinase-induced extracellular signal-regulated kinase signaling but rather selective inhibitors of particular receptor tyrosine kinase signaling. Currently, it is not known whether this mammalian specificity is because the target of the Sprys is exclusive to the FGF pathway, or because there is a tight spatial or temporal regulation of functionally related genes. The four mammalian Spry proteins are truncated in comparison to dSpry. However, they have a highly conserved COOH-terminal cys- teine-rich domain. Several short sequences in the NH 2 -termini of all of the Spry proteins distributed throughout the protein family are also conserved (24). There is good evidence that the cysteine-rich domain is a targeting domain that locates Spry proteins to membranes in Received 4/7/04; revised 5/28/04; accepted 6/30/04. Grant support: The Agency for Science, Technology and 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. Note: T. Lo, P. Yusoff, and C. W. Fong contributed equally to this work. Supplemen- tary data for this article can be found at Cancer Research Online (http://cancerres.aacrjour- nals.org). Requests for reprints: Graeme R. Guy, Signal Transduction Laboratory, Institute of Molecular and Cell Biology, 61 Biopolis Drive, 06-01 Proteos, Singapore 138673. Phone (65) 68743737; Fax: (65) 67791117; E-mail: mcbgg@imcb.a-star.edu.sg. ©2004 American Association for Cancer Research. 6127 Research. on July 6, 2015. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from