Ror2, a Developmentally Regulated Kinase, Promotes Tumor Growth Potential in Renal Cell Carcinoma

Oncogene (2009) 28, 2513–2523 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE Ror2, a developmentally regulated kinase, promotes tumor growth potential in renal cell carcinoma TM Wright1,2, AR Brannon1,2, JD Gordan3, AJ Mikels4,5, C Mitchell6, SChen 2, I Espinosa7, M van de Rijn7, R Pruthi2,8, E Wallen2,8, L Edwards6, R Nusse4,5 and WK Rathmell1,2,9 1Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 2Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 3Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA, USA; 4Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA, USA; 5Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA; 6Department of Biostatistics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 7Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; 8Department of Urology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA and 9Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Inappropriate kinase expression and subsequent promis- Introduction cuous activity defines the transformation of many solid tumors including renal cell carcinoma (RCC). Thus, the Although tumor biology is tightly linked in many ways expression of novel tumor-associated kinases has the to the processes of embryonic development, a largely potential to dramatically shape tumor cell behavior. untapped area of solid tumor oncology is the specific Further, identifying tumor-associated kinases can lend examination of growth promoting pathways that are insight into patterns of tumor growth and characteristics. unique and critical to early developmental patterning. Here, we report the identification of the RTK-like orphan One such signaling pathway, Wnt, is a process common receptor 2 (Ror2), a new tumor-associated kinase in RCC to both developmental and malignant states (Coombs cell lines and primary tumors. Ror2 is an orphan receptor et al., 2008). The identification of these signaling tyrosine kinase with physiological expression normally molecules that play a physiologic role in early develop- seen in the embryonickidney. However, in RCC, Ror2 mental processes, with potential to promote tumor expression correlated with expression of genes involved at growth or other pathologic characteristics in the adult, the extracellular matrix, including Twist and matrix provides an opportunity to define biological cause for metalloprotease-2 (MMP2). Expression of MMP2 in various tumor attributes (Nakagawara, 2001). Further, RCC cells was suppressed by Ror2 knockdown, placing identifying such molecules with restricted tissue expres- Ror2 as a mediator of MMP2 regulation in RCC and a sion in the adult organism provides a valuable chance potential regulator of extracellular matrix remodeling. for diagnostic or therapeutic implementation. The suppression of Ror2 not only inhibited cell migration, Epithelial cells require modulations in diverse signal- but also inhibited anchorage-independent growth in soft ing pathways to support the numerous cellular changes agar and growth in an orthotopicxenograft model. These necessary to attain metastatic potential. Specifically, findings suggest a novel pathway of tumor-promoting receptor tyrosine kinase (RTK) activation causes activity by Ror2 within a subset of renal carcinomas, with deregulated signaling and can define tumor subsets significant implications for unraveling the tumorigenesis across a broad spectrum of tumors (Stommel et al., of RCC. 2007). Such variations in kinase activation have the Oncogene (2009) 28, 2513–2523; doi:10.1038/onc.2009.116; potential to facilitate the cellular adaptations that published online 18 May 2009 determine patterns of tumor growth and can provide tremendous insight into the cell biology of that Keywords: renal cell carcinoma; Ror2; tyrosine kinase; transformative process (De Luca et al., 2008; de cancer; cell signaling Castro-Carpeno et al., 2008). Among the cellular changes associated with tumor progression is tissue remodeling compatible with the extensive migration necessary for the progression of metastatic disease. This cellular transition is shared by the developmentally regulated processes, which direct limb formation or other tissue patterning events. Tissue remodeling is driven by matrix metalloproteases Correspondence: Dr WK Rathmell, Lineberger Cancer Center, (MMPs), a family of enzymes that degrade the extra- University of North Carolina at Chapel Hill, 450 West Drive CB cellular matrix (ECM), and is integral to both normal 7295, Chapel Hill, NC 27599-7295, USA. E-mail: [email protected] tissue development and cancer progression (Liotta and Received 3 December 2008; revised 23 March 2009; accepted 8 April Kohn, 2001). These processes signal cellular alterations 2009; published online 18 May 2009 that allow single cells to detach from the epithelial Ror2 promotes invasive growth in RCC TM Wright et al 2514 tumor collective, interrupting cell–cell junctions and for kinases with a basal level of tyrosine phosphoryla- increasing cell motility. Taken together, this highly tion in unstimulated RCC lysates. Tyrosine kinases coordinated process is critical for the lethality of most expected to be activated in RCC such as the epithelial epithelial malignancies. growth factor receptor (EGFR) were detected on the One such lethal epithelial malignancy, renal cell array. Among the phosphorylated tyrosine kinases carcinoma (RCC), affects over 40 000 individuals in interrogated on the array, Ror2 was positively identified the United States annually (Cancer Facts and Figures, on multiple examinations of the RCC cell line, 786-0 2008). Nearly a third of these patients present with RC3, a control vector-transfected derivative of the unresectable or metastatic disease, for which antiangio- VHLÀ mutant RCC cell line 786-0 (Figure 1a). genic therapies are the mainstay of therapy (Rathmell Abundant expression of the endogenous protein in et al., 2005; Cowey and Rathmell, 2008). RCC describes 786-0 RC3 cells was confirmed by immunoblot analysis. a group of histologically and genetically distinct Immunoprecipitation of Ror2 with independent anti- neoplasms, the most common being clear cell RCC, bodies confirms high protein levels and basal autopho- with the remainder being papillary and chromophobe sphorylation of the tyrosine kinase (Supplementary RCCs (Linehan et al., 2003). The mechanisms that Figure 1A). On examination of other cell lines, we promote tumor invasiveness or the acquisition of observed Ror2 expression in the parental RCC cell line, metastatic potential for the subtypes of RCC remain 786-0, using two independent antibodies targeting Ror2, undefined. but not in the SV-40-immortalized human proximal ECM remodeling, a common step toward invasive- tubule cell line, HKC (Supplementary Figure 1B). ness, is promoted by tumor cells through changes Additionally, we demonstrated that Ror2 expression in intracellular signaling and commonly acquiring was present in an unrelated RCC cell line, RCC4 2-1 characteristics of less differentiated cellular precursors (Supplementary Figure 1B) further signifying Ror2’s (Liotta and Kohn, 2001). One developmentally regu- role in RCC tumorigenesis deserved further exploration. lated pathway inappropriately activated in many epithe- Ror2 is a developmentally regulated kinase, with lial tumors is Wnt protein signaling via the frizzled and expression primarily relegated to embryonic mesenchy- lipoprotein receptor-related protein coreceptors (Atkins mal tissues (Yoda et al., 2003). To examine a possible et al., 2004; van Amerongen et al., 2008). Here we report role in malignant tissue, Ror2 mRNA expression was the identification of a Wnt receptor of embryonic analysed by reverse transcription (RT)–PCR in 19 mesenchymal origin expressed in RCC cell lines and primary human RCC tumors. Four representative tumors that defines a subgroup of human tumors with tumor specimens are demonstrated in Figure 1b with an invasive growth profile of gene expression. The novel the HPRT gene used as a control for RNA abundance RTK-like orphan receptor 2 (Ror2) is part of a family of and integrity. The band detected as Ror2 in Figure 1b orphan RTKs (Deloukas et al., 1998; Oldridge et al., was purified and sequenced, confirming identity to the 2000). Ror2 is characterized by an intracellular tyrosine Ror2 CRD domain as predicted (data not shown). kinase domain (Masiakowski and Carroll, 1992) and an Overall, Ror2 transcript was detected in more than 55% extracellular frizzled-like cysteine-rich domain (CRD), of an unselected set of archival renal tumors (Figure 1b). shown to act as a receptor for Wnt ligands (Oishi et al., Ror2 mRNA was detected even in some tumors of non- 1997). Mouse Ror2 (mRor2) expression is normally clear cell histology, suggesting that various genetic observed in the developing heart, brain and lungs mechanisms may have the potential to promote the (Takeuchi et al., 2000; Matsuda et al., 2001; Yoda induction of Ror2 in renal cancers. et al., 2003; Schwabe et al., 2004), with the greatest Detecting Ror2 expression in a malignant context expression seen in migrating neural crest and mesench- prompted an evaluation of Ror2 expression in the ymal tissues (Yoda et al., 2003). In the

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