A Novel RET Kinase–B-Catenin Signaling Pathway Contributes to Tumorigenesis in Thyroid Carcinoma Taranjit S. Gujral, 1 Wendy van Veelen, 2 Douglas S. Richardson, 1 Shirley M. Myers, 1 Jalna A. Meens, 1 Dennis S. Acton, 2 Mireia Dun ˜ach, 3 Bruce E. Elliott, 1 Jo W.M. Ho ¨ppener, 2 and Lois M. Mulligan 1 1 Division of Cancer Biology and Genetics, Cancer Research Institute, Queen’s University, Kingston, Ontario, Canada; 2 Department of Metabolic and Endocrine Diseases, University Medical Center Utrecht, Utrecht, the Netherlands; and 3 Unitat de Biofı ´sica, Departament de Bioquı ´mica i Biologia Molecular, Facultat de Medicina, Universitat Auto `noma de Barcelona, Barcelona, Spain Abstract The RET receptor tyrosine kinase has essential roles in cell survival, differentiation, and proliferation. Oncogenic activa- tion of RET causes the cancer syndrome multiple endocrine neoplasia type 2 (MEN 2) and is a frequent event in sporadic thyroid carcinomas. However, the molecular mechanisms underlying RET’s potent transforming and mitogenic signals are still not clear. Here, we show that nuclear localization of B- catenin is frequent in both thyroid tumors and their metastases from MEN 2 patients, suggesting a novel mecha- nism of RET-mediated function through the B-catenin signaling pathway. We show that RET binds to, and tyrosine phosphorylates, B-catenin and show that the interaction between RET and B-catenin can be direct and independent of cytoplasmic kinases, such as SRC. As a result of RET- mediated tyrosine phosphorylation, B-catenin escapes cyto- solic down-regulation by the adenomatous polyposis coli/ Axin/glycogen synthase kinase-3 complex and accumulates in the nucleus, where it can stimulate B-catenin–specific transcriptional programs in a RET-dependent fashion. We show that down-regulation of B-catenin activity decreases RET-mediated cell proliferation, colony formation, and tumor growth in nude mice. Together, our data show that a B- catenin–RET kinase pathway is a critical contributor to the development and metastasis of human thyroid carcinoma. [Cancer Res 2008;68(5):1338–46] Introduction The RET receptor is required for development of urogenital and neural crest–derived cell types (1). Under normal cellular con- ditions, RET is activated by binding of both glial cell line–derived neurotrophic factor (GDNF) ligands and cell surface bound core- ceptors of the GDNF family receptor a (GFRa) proteins (2). How- ever, oncogenic activation of RET, by germline point mutations, leads to ligand-independent constitutive kinase activity, giving rise to the inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2). MEN 2 is characterized by medullary thyroid carcinoma (MTC), a tumor of thyroid C cells, and the adrenal tumor pheochromocytoma, as well as other less common tumor and developmental phenotypes (reviewed in ref. 3). MTC is the predominant disease feature, with early onset tumors and metastases to lymph nodes and distant organs (4). RET activation contributes to stimulation of RAS-ERK, c-Jun-NH 2 -kinase, phos- phoinositide 3-kinase, p38 mitogen-activated protein kinase (MAPK), SRC, STAT and ERK5 signaling cascades (reviewed in ref. 1). However, the identity of the critical secondary oncogenic signals involved in the broad and early metastatic pattern of RET- mediated MTC is still largely unknown. h-Catenin is an ubiquitously expressed multifunctional protein that plays important roles in cell adhesion and signal transduction (5). At the plasma membrane, h-catenin associates with E-cadherin and a-catenin in linking the cytoskeleton and adherens junctions, whereas in the nucleus, it acts as a mediator of transcription through other DNA-binding proteins, such as TCF/LEF family members (reviewed in ref. 6). Cytosolic-free h-catenin interacts with the adenomatous polyposis coli (APC) and axin proteins to form a complex, which in turn recruits glycogen synthase kinase-3 (GSK3) and casein kinase, to form a destruction complex that serine/threonine phosphorylates h-catenin and targets it to the proteosome (reviewed in ref. 7). Abnormal expression or localization of h-catenin has been reported in many tumor types (5, 8), and h-catenin–mediated loss of cell-cell adhesion has been implicated in anchorage-independent cell growth and cancer metastasis (8). The best-characterized mechanism leading to h-catenin–mediated signal transduction is through activation of the WNT pathway by binding of WNT proteins to frizzled or LRP family cell surface receptors (9). However, h-catenin signaling can also be induced in response to overexpression or activation of tyrosine kinases in a WNT- independent fashion, and both these pathways converge to target h-catenin to the nucleus and induce expression of a similar set of h-catenin–specific target genes (10–12). h-Catenin tyrosine phosphorylation causes its dissociation from membrane-associated E-cadherin, leading to accumulation of a pool of free cytoplasmic h-catenin (13). This can, in turn, increase the amount of h-catenin reaching the nucleus, where it acts as a transcription factor, up-regulating expression of genes involved in cell migration, growth, differentiation, and survival (8). Tyrosine phosphoryla- tion of h-catenin, followed by functional down-regulation of E-cadherin–mediated cell-cell contact, is potentially critical in initiating cell migration in both normal physiologic processes and in tumor metastasis (13). Loss of membrane-associated h-catenin, often with an accom- panying relative increase in cytosolic or nuclear expression, has been noted in anaplastic and poorly differentiated thyroid carci- nomas and in thyroid papillary microcarcinoma (14–16). However, h-catenin had not been previously investigated in RET-mediated tumor development and metastasis. Here, we report that RET Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Lois M. Mulligan, Cancer Research Institute, Botterell Hall Room 329, Queen’s University Kingston, Ontario, Canada K7L 3N6. Phone: 1-613-533- 6000, ext. 77475; Fax: 1-613-533-6830; E-mail: mulligal@queensu.ca. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-6052 Cancer Res 2008; 68: (5). March 1, 2008 1338 www.aacrjournals.org Research Article