Robinow syndrome is a skeletal dysplasia with both autosomal dominant and autosomal recessive inheritance patterns. It is characterized by short stature, limb shortening, genital hypoplasia, and craniofacial abnormalities. The etiology of dominant Robinow syndrome is unknown; however, the phenotypically more severe autosomal recessive form of Robinow syndrome has been associated with mutations in the orphan tyrosine kinase receptor, ROR2, which has recently been identified as a putative WNT5A receptor. Here, we show that two different missense mutations in WNT5A, which result in amino acid substitutions of highly conserved cysteines, are associated with autosomal dominant Robinow syndrome. One mutation has been found in all living affected members of the original family described by Meinhard Robinow and another in a second unrelated patient. These missense mutations result in decreased WNT5A activity in functional assays of zebrafish and Xenopus development. This work suggests that a WNT5A/ROR2 signal transduction pathway is important in human craniofacial and skeletal development and that proper formation and growth of these structures is sensitive to variations in WNT5A function. Developmental Dynamics 239:327-337, 2010. (C) 2009 Wiley-Liss...

The receptor tyrosine kinase Ror2 plays important roles in developmental morphogenesis. It has recently been shown that Ror2 mediates Wnt5a-induced noncanonical Wnt signaling by activating the Wnt–JNK pathway and inhibiting the β-catenin–TCF pathway. However, the function of Ror2 in noncanonical Wnt signaling leading to cell migration is largely unknown. We show, using genetically different or manipulated cultured cells, that Ror2 is critical for Wnt5a-induced, but not Wnt3a-induced, cell migration. Ror2-mediated cell migration requires the extracellular cysteine-rich domain (CRD), which is the binding site for Wnt5a, and the cytoplasmic proline-rich domain (PRD) of Ror2. Furthermore, Ror2 can mediate filopodia formation via actin reorganization, irrespective of Wnt5a, and this Ror2-mediated filopodia formation requires the actin-binding protein filamin A, which associates with the PRD of Ror2. Intriguingly, disruption of filopodia formation by suppressing the expression of either Ror2 or filamin A inhibits Wnt5a-induced cell migration, indicating that Ror2-mediated filopodia formation is essential for Wnt5a-induced cell migration.

Inappropriate kinase expression and subsequent promiscuous activity defines the transformation of many solid tumors including renal cell carcinoma (RCC). Thus, the expression of novel tumor-associated kinases has the potential to dramatically shape tumor cell behavior. Further, identifying tumor-associated kinases can lend insight into patterns of tumor growth and characteristics. Here, we report the identification of Ror2, a new tumor-associated kinase in RCC cell lines and primary tumors. Ror2 is an orphan receptor tyrosine kinase with physiological expression normally seen in the embryonic kidney. However, in RCC, Ror2 expression correlated with expression of genes involved at the extracellular matrix, including Twist and matrix metalloprotease-2 (MMP2). Expression of MMP2 in RCC cells was suppressed by Ror2 knockdown, placing Ror2 as a mediator of MMP2 regulation in RCC and a potential regulator of extracellular matrix remodeling. The suppression of Ror2 not only inhibited cell migration, but also inhibited anchorage independent growth in soft agar and growth in an orthotopic xenograft model. These findings suggest a novel pathway of tumor-promoting activity by Ror2 within a subset of renal carcinomas, with significant implications for unraveling the tumorigenesis of RCC.

Tyrosine kinase receptors represent targets of great interest for cancer therapy. Here we demonstrate, for the first time, the importance of the orphan tyrosine kinase receptor, ROR2, in melanoma progression. Using melanoma tissue microarrays we show that ROR2 is expressed predominantly in metastatic melanoma. Because ROR2 has been shown to specifically interact with the non-canonical Wnt ligand, Wnt5A, this corroborates our previous data implicating Wnt5A as a mediator of melanoma metastasis. We show here that increases in Wnt5A cause increases in ROR2 expression, as well as the PKC-dependent, clathrin-mediated internalization of ROR2. WNT5A knockdown by siRNA decreases ROR2 expression, but silencing of ROR2 has no effect on WNT5A levels. ROR2 knockdown does, however, result in a decrease in signaling downstream of Wnt5A. Using in vitro and in vivo metastasis assays we demonstrate that ROR2 is necessary for the Wnt5A-mediated metastasis of melanoma cells. These data imply that ROR2 may represent a novel target for melanoma therapy.

Ror2 is an orphan receptor tyrosine kinase with expression normally restricted to early stages of development. However, emerging evidence has placed aberrantly expressed Ror2, leading to an invasive phenotype, in several cancers including renal cell carcinoma (RCC). Although Ror2 is currently identified as up-regulated in an assortment of cancers, neither the regulatory role or mechanism of action have been delineated. We sought to place Ror2 in the most commonly mutated pathway of RCC, the loss of the tumor suppressor von Hippel-Lindau (VHL), which causes hypoxia-inducible factor (HIF)-1α and -2α stabilization and the transcriptional activation of a broad repertoire of response genes. We found that Ror2 was indeed associated with the pVHL loss in RCC as well as with VHL somatic mutations tightly coordinated with the induction of RCC. Additionally, knockdown and rescue analysis of HIF expression suggests that Ror2 is dependent on pathologic stabilization of either HIF-1α or HIF-2α. Subsequent evaluation of the ROR2 promoter suggests that HIF-2α and its dimerization partner, aryl hydrocarbon nuclear transferase localize to the ROR2 promoter via a cryptic transcriptional element. This data substantiates a unique regulation pattern for Ror2 in the VHL-HIF axis that has the potential to be applied to other cancer etiologies.

The receptor tyrosine kinase Ror2 acts as a receptor or coreceptor for Wnt5a to mediate Wnt5a-induced activation of the Wnt/JNK pathway and inhibition of the β-catenin-dependent canonical Wnt pathway. However, little is known about how Ror2 cooperates with another receptor component(s) to mediate Wnt5a signaling. We show here that Ror2 regulates Wnt5a-induced polymerization of Dishevelled (Dvl) and that this Ror2-mediated regulation of Dvl is independent of the cytoplasmic region of Ror2. Ror2 can associate with Frizzled7 (Fz7) via its extracellular cysteine-rich domain to form a receptor complex that is required for the regulation of Dvl and activation of the AP-1 promoter after Wnt5a stimulation. Suppressed expression of Fz7 indeed results in the inhibition of Wnt5a-induced polymerization of Dvl and AP-1 activation. Interestingly, both the DIX and the DEP domains of Dvl are indispensable for Dvl polymerization and subsequent AP-1 activation after Wnt5a stimulation. We further show that polymerized Dvl is colocalized with Rac1 and that suppressed expression of Rac1 inhibits Wnt5a-induced AP-1 activation. Collectively, our results indicate that Ror2/Fz receptor complex plays an important role in the Wnt5a/Rac1/AP-1 pathway by regulating the polymerization of Dvl.

Regulation of expression of the intestinal epithelial actin-binding protein, villin, is poorly understood. The aim of this study was to determine whether Wnt5a stimulates Ror2 in intestinal epithelia caused transient increases in phospho-ERK1/2 (pERK1/2) and subsequently increased expression of villin transcript and protein. To demonstrate Wnt5a–Ror2 regulation of villin expression, we overexpressed wild-type, truncated, or mutant Ror2 constructs in HT29 adenocarcinoma cells and non-transformed fetally derived human intestinal epithelial cells, added conditioned media containing Wnt5a and measured changes in ERK1/2 phosphorylation, villin amplicons, and protein expression by RT-PCR and Western blot techniques. Wnt5a addition caused a transient increase in pERK1/2, which was maximal at 10 min but extinguished by 30 min. Transient transfection with a siRNA duplex against Ror2 diminished Ror2 amplicons and protein and reduced the extent of pERK1/2 activation. Structure–function analysis revealed that the deletion of the cysteine-rich, kringle, or tyrosine kinase domain or substitution mutations of tyrosine residues in the intracellular Ser/Thr-1 region of Ror2 prevented the Wnt5a stimulation of pERK1/2. Deletion of the intracellular proline and serine/threonine-rich regions of Ror2 had no effect on Wnt5a stimulation of pERK1/2. The increase in villin expression was blocked by pharmacological inhibition of MEK-1 and casein kinase 1...

The trafficking of primordial germ cells (PGCs) across multiple embryonic structures to the nascent gonads ensures the transmission of genetic information to the next generation through the gametes, yet our understanding of the mechanisms underlying PGC migration remains incomplete. Here we identify a role for the receptor tyrosine kinase-like protein Ror2 in PGC development. In a Ror2 mouse mutant we isolated in a genetic screen, PGC migration and survival are dysregulated, resulting in a diminished number of PGCs in the embryonic gonad. A similar phenotype in Wnt5a mutants suggests that Wnt5a acts as a ligand to Ror2 in PGCs, although we do not find evidence that WNT5A functions as a PGC chemoattractant. We show that cultured PGCs undergo polarization, elongation, and reorientation in response to the chemotactic factor SCF (secreted KitL), whereas Ror2 PGCs are deficient in these SCF-induced responses. In the embryo, migratory PGCs exhibit a similar elongated geometry, whereas their counterparts in Ror2 mutants are round. The protein distribution of ROR2 within PGCs is asymmetric, both in vitro and in vivo; however, this asymmetry is lost in Ror2 mutants. Together these results indicate that Ror2 acts autonomously to permit the polarized response of PGCs to KitL. We propose a model by which Wnt5a potentiates PGC chemotaxis toward secreted KitL by redistribution of Ror2 within the cell.

Osteosarcoma, a common malignancy primarily affecting children, generally has a poor prognosis. Novel diagnostic, prognostic and therapeutic markers are required to ameliorate the negative outcomes of this disease. We investigated two potential markers, WNT-5a and ROR2, which are hypothesized to dysregulate WNT signaling pathways to promote tumorigenesis in other types of cancer. We investigated WNT-5a and ROR2 expression using immunohistochemistry in 42 osteosarcoma and 12 osteochondroma specimens, and compared the expression of these proteins with one another as well as with clinicopathological parameters. WNT-5a was detected in 34/42 (81.0%) cases and ROR2 was detected in 31/42 (73.8%) cases, significantly higher than in osteochondroma (16.7 and 25.0%, respectively; both P<0.05). Expression of these proteins was positively correlated (r=0.552, P<0.05). Furthermore, expression of WNT-5a and ROR2 was both correlated with Enneking surgical stage and tumor metastasis (P<0.05), but not with patient gender, age or pathological type. Thus, WNT-5a and ROR2 were more highly expressed in more severe disease states, and therefore may play a coordinated role in the occurrence and progression of osteosarcoma.

Soft-tissue sarcomas are a group of malignant tumours whose clinical management is complicated by morphological heterogeneity, inadequate molecular markers and limited therapeutic options. Receptor tyrosine kinases (RTKs) have been shown to play important roles in cancer, both as therapeutic targets and as prognostic biomarkers. An initial screen of gene expression data for 48 RTKs in 148 sarcomas showed that ROR2 was expressed in a subset of leiomyosarcoma (LMS), gastrointestinal stromal tumour (GIST) and desmoid-type fibromatosis (DTF). This was further confirmed by immunohistochemistry (IHC) on 573 tissue samples from 59 sarcoma tumour types. Here we provide evidence that ROR2 expression plays a role in the invasive abilities of LMS and GIST cells in vitro. We also show that knockdown of ROR2 significantly reduces tumour mass in vivo using a xenotransplantation model of LMS. Lastly, we show that ROR2 expression, as measured by IHC, predicts poor clinical outcome in patients with LMS and GIST, although it was not independent of other clinico-pathological features in a multivariate analysis, and that ROR2 expression is maintained between primary tumours and their metastases. Together, these results show that ROR2 is a useful prognostic indicator in the clinical management of these soft-tissue sarcomas and may represent a novel therapeutic target.

Receptor tyrosine kinase-like orphan receptors (ROR) 1 and 2 are atypical members of the receptor tyrosine kinase (RTK) family and have been associated with several human diseases. The vertebrate RORs contain an ATP binding domain that deviates from the consensus amino acid sequence, although the impact of this deviation on catalytic activity is not known and the kinase function of these receptors remains controversial. Recently, ROR2 was shown to signal through a Wnt responsive, β-catenin independent pathway and suppress a canonical Wnt/β-catenin signal. In this work we demonstrate that both ROR1 and ROR2 kinase domains are catalytically deficient while CAM-1, the C. elegans homolog of ROR, has an active tyrosine kinase domain, suggesting a divergence in the signaling processes of the ROR family during evolution. In addition, we show that substitution of the non-consensus residues from ROR1 or ROR2 into CAM-1 and MuSK markedly reduce kinase activity, while restoration of the consensus residues in ROR does not restore robust kinase function. We further demonstrate that the membrane-bound extracellular domain alone of either ROR1 or ROR2 is sufficient for suppression of canonical Wnt3a signaling, and that this domain can also enhance Wnt5a suppression of Wnt3a signaling. Based on these data...

Development of the metanephric kidney begins with the induction of a single ureteric bud (UB) on the caudal Wolffian duct (WD) in response to GDNF (glial cell line-derived neurotrophic factor) produced by the adjacent metanephric mesenchyme (MM). Mutual interaction between the UB and MM maintains expression of GDNF in the MM, thereby supporting further outgrowth and branching morphogenesis of the UB, while the MM also grows and aggregates around the branched tips of the UB. Ror2, a member of the Ror family of receptor tyrosine kinases, has been shown to act as a receptor for Wnt5a to mediate noncanonical Wnt signaling. We show that Ror2 is predominantly expressed in the MM during UB induction and that Ror2- and Wnt5a-deficient mice exhibit duplicated ureters and kidneys due to ectopic UB induction. During initial UB formation, these mutant embryos show dysregulated positioning of the MM, resulting in spatiotemporally aberrant interaction between the MM and WD, which provides the WD with inappropriate GDNF signaling. Furthermore, the numbers of proliferating cells in the mutant MM are markedly reduced compared to the wild-type MM. These results indicate an important role of Wnt5a-Ror2 signaling in morphogenesis of the MM to ensure proper epithelial tubular formation of the UB required for kidney development.

Secreted molecules of the Wnt family regulate key decisions in embryogenesis and adult tissue homeostasis by activating a complex network of Wnt signaling pathways. Although the different branches of Wnt signaling have been studied for more than 25 years, fluorophore tagged constructs for live cell imaging of Wnt molecules activating the Wnt/β-catenin pathway have become available only recently. We have generated a fluorophore tagged Wnt construct of the Xenopus Wnt5a protein (Xwnt5A) with the enhanced green fluorescent protein (EGFP), Xwnt5A-EGFP. This construct activates non-canonical Wnt pathways in an endocytosis dependent manner and is capable of compensating for the loss of endogenous Xwnt5A in Xenopus embryos. Strikingly, non-canonical Wnt pathway activation was restricted to short-range signaling while an inhibitory effect was observed in transwell cell cultures taken as long-range signaling model sytem. We used our Xwnt5A-EGFP construct to analyze in vivo binding of Wnt5A to its co-receptor ROR2 on the microscopic and on the molecular level. On the microscopic level, Xwnt5A-EGFP clusters in the membrane and recruits ROR2-mCherry to these clusters. Applying dual-colour dual-focus line-scanning fluorescence correlation spectroscopy on dorsal marginal zone explants...

Ror2 is a Wnt ligand receptor that is overexpressed in a variety of tumors including clear cell renal cell carcinoma (ccRCC). Here we demonstrate that expression of wild type Ror2 results in increased tumorigenic properties in in vitro cell culture and in vivo xenograft models. In addition, Ror2 expression produced positive changes in both cell migration and invasion, which were dependent on matrix metalloprotease 2 (MMP2) activity. Mutations in key regions of the kinase domain of Ror2 resulted in the abrogation of increased tumor growth, cell migration, and cell invasion observed with expression of wild-type Ror2. Finally, we examined Ror2 expression as a prognostic biomarker for ccRCC utilizing the TCGA ccRCC dataset. High expression of Ror2 showed a significant correlation with higher clinical stage, nuclear grade, and tumor stage. Furthermore, high expression of Ror2 in ccRCC patients correlated with significant lower overall survival, cancer specific survival, and recurrence free survival. Together, these findings suggest that Ror2 plays a central role in influencing the ccRCC phenotype, and can be considered as a negative prognostic biomarker and potential therapeutic target in this cancer.

Aims: To investigate the clinical significance of receptor tyrosine kinase-like orphan receptor 2 (ROR2) in cervical cancer. Methods: We examined ROR2 levels in 8 pairs of surgically resected cervical cancer and adjacent normal cervical tissues by real-time PCR. Moreover, we performed immunohistochemistry to examine ROR2 expression in 94 paraffin-embedded cervical cancer samples and analyzed the association between ROR2 expression, clinicopathologic factors and prognosis. Results: ROR2 expression was up-regulated in cervical cancer tissues compared with adjacent normal cervix. In paraffin-embedded cervical cancer samples, high expression of ROR2 was shown in 40 (42.6%) of 94 cases, also, it was significantly associated with tumor stage (P = 0.018) and lymph nodes metastasis (P = 0.013). Moreover, survival analysis showed that ROR2 expression was an independent prognostic factor of poor overall and recurrent free survival (P = 0.045 and 0.001, respectively). Conclusion: These results indicate that ROR2 is significantly correlated with cancer progression and poor prognosis in cervical cancer.

Wnt5a, which regulates various cellular functions in Wnt signaling, is involved in inflammatory responses, however the mechanism is not well understood. We examined the role of Wnt5a signaling in intestinal immunity using conditional knockout mice for Wnt5a and its receptor Ror2. Removing Wnt5a or Ror2 in adult mice suppressed dextran sodium sulfate (DSS)-induced colitis. It also attenuated the DSS-dependent increase in inflammatory cytokine production and decreased interferon-γ (IFN-γ)-producing CD4+ Th1 cell numbers in the colon. Wnt5a was highly expressed in stromal fibroblasts in ulcerative lesions in the DSS-treated mice and inflammatory bowel disease patients. Dendritic cells (DCs) isolated from the colon of Wnt5a and Ror2 deficient mice reduced the ability to differentiate naïve CD4+ T cells to IFN-γ-producing CD4+ Th1 cells. In vitro experiments demonstrated that the Wnt5a-Ror2 signaling axis augmented the DCs priming effect of IFN-γ, leading to enhanced lipopolysaccharide (LPS)-induced interleukin (IL)-12 expression. Taken together, these results suggest that Wnt5a promotes IFN-γ signaling, leading to IL-12 expression in DCs, and thereby inducing Th1 differentiation in colitis.

RTK-like orphan receptor 2 (ROR2) is overexpressed in several cancers and has tumorigenic activity. However, the expression of ROR2 and its functional and prognostic significance have yet to be evaluated in pancreatic ductal adenocarcinoma (PDAC). Quantitative real-time polymerase chain reaction was used to characterize the expression of ROR2 mRNA in PDAC, corresponding peritumoral tissues, and PDAC cell lines. Immunohistochemical analysis with tissue microarrays was used to evaluate ROR2 expression in PDAC and to investigate the relationship of this expression to clinicopathological factors and prognosis. The expression of ROR2 mRNA and protein was significantly higher in PDAC than in normal pancreatic tissues. High cytoplasmic ROR2 expression in cancer cells was significantly associated with a primary tumor, distant metastasis, and TNM stage, and high stromal ROR2 expression was significantly associated with regional lymph node metastasis and TNM stage. The Kaplan–Meier method and Cox regression analyses showed that high ROR2 expression in tumor cytoplasm or stromal cells was significantly associated with malignant attributes and reduced survival in PDAC. We present strong evidence that ROR2 could be used as an indicator of poor prognosis and could represent a novel therapeutic target for PDAC.

Regulation of expression of the intestinal actin-binding protein, villin, a marker of intestinal epithelial differentiation, is poorly understood. Activation of the extracellular calcium-sensing receptor (CaSR) on sub-epithelial myofibroblasts stimulated the secretion of Wnt5a, while activation of the CaSR on intestinal epithelia increased expression of Ror2, a Wnt-family co-receptor. Immunocytochemistry has localized Ror2 expression in the epithelia lining the small intestine from the crypt base to the villus tip. The aim of this study was to determine whether Wnt5a binding Ror2 in intestinal epithelia stimulated transient increases in phospho-ERK1/2 (pERK1/2) which lead to increased expression of villin transcript and protein. To examine Wnt5a-Ror2 regulation of villin expression, we transgenically overexpressed wild-type, truncated, or mutant Ror2 constructs in HT-29 adenocarcinoma cells and nontransformed fetally-derived human intestinal epithelial cells (HIECs), added conditioned media containing Wnt5a and measured changes in ERK1/2 phosphorylation, villin amplicons and protein expression by RT-PCR and Western blot techniques. Wnt5a addition caused a transient increase in pERK1/2, which was maximal at 10 min but diminished by 30 min. Transient transfection with a siRNA duplex against Ror2 diminished Ror2 amplicons and protein and reduced the extent of pERK1/2 activation. Structure-function analysis revealed that deletion of the cysteine-rich...

The transmembrane receptor ‘ROR2’ resembles members of the receptor tyrosine kinase family of signalling receptors in sequence but its' signal transduction mechanisms remain enigmatic. This problem has particular importance because mutations in ROR2 are associated with two human skeletal dysmorphology syndromes, recessive Robinow Syndrome (RS) and dominant acting Brachydactyly type B (BDB). Here we show, using a constitutive dimerisation approach, that ROR2 exhibits dimerisation-induced tyrosine kinase activity and the ROR2 C-terminal domain, which is deleted in BDB, is required for recruitment and activation of the non-receptor tyrosine kinase Src. Native ROR2 phosphorylation is induced by the ligand Wnt5a and is blocked by pharmacological inhibition of Src kinase activity. Eight sites of Src-mediated ROR2 phosphorylation have been identified by mass spectrometry. Activation via tyrosine phosphorylation of ROR2 receptor leads to its internalisation into Rab5 positive endosomes. These findings show that BDB mutant receptors are defective in kinase activation as a result of failure to recruit Src.

The Wnts include a large family of secreted proteins that serve as important signals during embryonic development and adult homeostasis. In the most well understood Wnt signaling pathway, Wnt binding to Frizzled and low density lipoprotein receptor-related protein induces β-catenin protein stabilization and entry into the nucleus, resulting in changes in target gene transcription. Emerging evidence suggests that Wnt5a can inhibit Wnt/β-catenin signaling through interaction with the receptor Ror2. The Ror2 protein belongs to the receptor tyrosine kinase superfamily and contains several recognizable structural motifs. However, limited information is available regarding which specific domains are required for the inhibitory signaling activity of Wnt5a. Through mutation and deletion analysis, we have analyzed which specific domains and residues, including those necessary for tyrosine kinase activity, mediate the Wnt5a signal. To determine whether Ror2 can inhibit canonical Wnt signaling in vivo, we examined the effect of Ror2 loss on the expression of the Wnt reporter Axin2LacZ, finding increased reporter activity in Ror2 null mice, demonstrating that Ror2 can also inhibit Wnt/β-catenin signaling in the context of intact tissues.