The WNT5A/ROR2 signaling pathway in pancreatic ductal adenocarcinoma (PDAC).

PURPOSE: WNT5A/ROR2 signaling pathway has been involved in many human cancers. Its role in pancreatic ductal adenocarcinoma (PDAC) has not been clarified yet. The purpose of this study was to determine the prognostic value of WNT5A expression in conjunction with the ROR2 expression in the same PDAC human tissues. METHODS: We retrospectively analyzed by immunohistochemistry the WNT5A and ROR2 expression in117 paraffin-embedded PDAC specimens following surgical pancreatic resection. The prognostic value of WNT5A and ROR2 was assessed using Kaplan-Meier survival curves and multivariate Cox regression models. RESULTS: High ROR2 expression was detected in 65.8% (77/117) of PDAC tumors, in 28.2% (33/117) in tumor-stroma, and in 71.1% (65/90) of normal pancreatic tissue. High WNT5A expression was found in 76.9% (90/117) of tumors, in 59.0% (69/117) of tumor-stroma, and in 83.0% (73/88) of normal pancreatic tissue. Spearman's correlation coefficiency demonstrated weak association between ROR2 and WNT5A expression in tumor (r=0.184; p=0.047), and no association in stroma (r=0.036; p=0.699). Multivariate analysis showed that regional lymph node invasion and differentiation were independent prognostic factors of survival, while ROR2- and WNT5A expression were not. CONCLUSIONS: Variable expression patterns for ROR2 and WNT5A were demonstrated in PDAC and normal pancreatic tissues suggesting a role for WNT5A/ROR2 signalling pathway, not only in PDAC but also in the normal pancreatic tissue during inflammation. The lack of prognostic significance for ROR2 and WNT5A expression in our cohort, either alone or in subgroup analysis, underlines the complexity of their role in PDAC, which is highly dependent on the different molecular receptor-ligand tissue contexts.

Tyrosine kinases regulate chondrocyte hypertrophy: promising drug targets for Osteoarthritis.

Osteoarthritis (OA) is a major health problem worldwide that affects the joints and causes severe disability. It is characterized by pain and low-grade inflammation. However, the exact pathogenesis remains unknown and the therapeutic options are limited. In OA articular chondrocytes undergo a phenotypic transition becoming hypertrophic, which leads to cartilage damage, aggravating the disease. Therefore, a therapeutic agent inhibiting hypertrophy would be a promising disease-modifying drug. The therapeutic use of tyrosine kinase inhibitors has been mainly focused on oncology, but the Food and Drug Administration (FDA) approval of the Janus kinase inhibitor Tofacitinib in Rheumatoid Arthritis has broadened the applicability of these compounds to other diseases. Interestingly, tyrosine kinases have been associated with chondrocyte hypertrophy. In this review, we discuss the experimental evidence that implicates specific tyrosine kinases in signaling pathways promoting chondrocyte hypertrophy, highlighting their potential as therapeutic targets for OA.

Critical role of the Ror-family of receptor tyrosine kinases in invasion and proliferation of malignant pleural mesothelioma cells.

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with poor prognosis and closely related to exposure to asbestos. MPM is a heterogeneous tumor with three main histological subtypes, epithelioid, sarcomatoid, and biphasic types, among which sarcomatoid type shows the poorest prognosis. The Ror-family of receptor tyrosine kinases, Ror1 and Ror2, is expressed in various types of tumor cells at higher levels and affects their aggressiveness. However, it is currently unknown whether they are expressed in and involved in aggressiveness of MPM. Here, we show that Ror1 and Ror2 are expressed in clinical specimens and cell lines of MPM with different histological features. Studies using MPM cell lines indicate that expression of Ror2 is associated tightly with high invasiveness of MPM cells, whereas Ror1 can contribute to their invasion in the absence of Ror2. However, both Ror1 and Ror2 promote proliferation of MPM cells. We also show that promoted invasion and proliferation of MPM cells by Ror signaling can be mediated by the Rho-family of small GTPases, Rac1, and Cdc42. These findings elucidate the critical role of Ror signaling in promoting invasion and proliferation of MPM cells.

Mechanosensing Controlled Directly by Tyrosine Kinases.

To understand how cells form tissues, we need to understand how the tyrosine kinases are involved in controlling cell mechanics, whether they act directly as parts of mechanosensing machines or indirectly. Cells test the critical parameter of matrix rigidity by locally contracting ("pinching") matrices and measuring forces, and the depletion of contractile units causes transformation. We report here that knocking down the receptor tyrosine kinases (RTKs), AXL, and ROR2, alters rigidity sensing and increases the magnitude or duration of local contraction events, respectively. Phospho-AXL and ROR2 localize to contraction units and bind major contractile components, tropomyosin 2.1 (AXL), myosin IIA (AXL), and filamin A (ROR2). At a molecular level, phosphorylated AXL localizes to active myosin filaments and phosphorylates tropomyosin at a tyrosine critical for adhesion formation. ROR2 binding of ligand is unnecessary, but binding filamin A helps function. Thus, AXL and ROR2 alter rigidity sensing and consequently morphogenic processes by directly controlling local mechanosensory contractions without ligands.

[Down-regulation of receptor-tyrosine-kinase-like orphan receptor 1 suppresses cell growth and enhances apoptosis in human colorectal carcinoma].

OBJECTIVE: To investigate the expression of receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) and its role in cell growth in human colorectal carcinoma (CRC). METHODS: Real-time quantitative PCR (qRT-PCR) and immunohistochemistry (IHC) were used to detect the expression of ROR1 in CRC (n=60) and matched tumor-adjacent tissues. The relationship between the expression of ROR1 and clinical features was analyzed by Pearson chi-square test. siRNA was used to down-regulate the expression of ROR1 in SW480 cells in vitro. The qRT-PCR and Western blotting were performed to confirm the down-regulation of ROR1 expression. The effects of down-regulated ROR1 on cell proliferation and apoptosis were measured by MTT assay and flow cytometry combined with annexin V-FITC/PI staining, respectively. RESULTS: Both the mRNA and protein expression of ROR1 were significantly up-regulated in CRC tissues than in tumor-adjacent tissues, and the expression of ROR1 was positively correlated with tumor size (≥5 cm), lymphatic metastasis and TNM stage (III and IV). siRNA could significantly down-regulate the expression of ROR1 in SW480 cells, then suppressed proliferation and enhanced apoptosis in SW480 cells. CONCLUSION: The expression of ROR1 was significantly higher in CRC tissues than in tumor-adjacent tissues. Down-regulation of ROR1 could play an anti-cancer role through inhibiting proliferation and inducing apoptosis in CRC.

Wnt5a/Ror2 mediates temporomandibular joint subchondral bone remodeling.

Increased subchondral trabecular bone turnover due to imbalanced bone-resorbing and bone-forming activities is a hallmark of osteoarthritis (OA). Wnt5a/Ror2 signaling, which can derive from bone marrow stromal cells (BMSCs), takes a role in modulating osteoblast and osteoclast formation. We showed previously that experimentally unilateral anterior crossbites (UACs) elicited OA-like lesions in mice temporomandibular joints (TMJs), displaying as subchondral trabecular bone loss. Herein, we tested the role of BMSC-derived Wnt5a/Ror2 signaling in regulating osteoclast precursor migration and differentiation in this process. The data confirmed the decreased bone mass, increased tartrate-resistant acid phosphatase (TRAP)-positive cell number, and enhanced osteoclast activity in TMJ subchondral trabecular bone of UAC-treated rats. Interestingly, the osteoblast activity in the tissue of TMJ subchondral trabecular bone of these UAC-treated rats was also enhanced, displaying as upregulated expressions of osteoblast markers and increased proliferation, migration, and differentiation capabilities of the locally isolated BMSCs. These BMSCs showed an increased CXCL12 protein expression level and upregulated messenger RNA expressions of Rankl, Wnt5a, and Ror2. Ex vivo data showed that their capacities of inducing migration and differentiation of osteoclast precursors were enhanced, and these enhanced capabilities were restrained after blocking their Ror2 signaling using small interfering RNA (siRNA) assays. Reducing Ror2 expression in the BMSC cell line by siRNA or blocking the downstream signalings with specific inhibitors also demonstrated a suppression of the capacity of the BMSC cell line to promote Wnt5a-dependent migration (including SP600125 and cyclosporine A) and differentiation (cyclosporine A only) of osteoclast precursors. These findings support the idea that Wnt5a/Ror2 signaling in TMJ subchondral BMSCs enhanced by UAC promoted BMSCs to increase Cxcl12 and Rankl expression, in which JNK and/or Ca(2+)/NFAT pathways were involved and therefore were engaged in enhancing the migration and differentiation of osteoclast precursors, leading to increased osteoclast activity and an overall TMJ subchondral trabecular bone loss in the UAC-treated rats.

Ror2 regulates branching, differentiation, and actin-cytoskeletal dynamics within the mammary epithelium.

Wnt signaling encompasses ß-catenin-dependent and -independent networks. How receptor context provides Wnt specificity in vivo to assimilate multiple concurrent Wnt inputs throughout development remains unclear. Here, we identified a refined expression pattern of Wnt/receptor combinations associated with the Wnt/ß-catenin-independent pathway in mammary epithelial subpopulations. Moreover, we elucidated the function of the alternative Wnt receptor Ror2 in mammary development and provided evidence for coordination of this pathway with Wnt/ß-catenin-dependent signaling in the mammary epithelium. Lentiviral short hairpin RNA (shRNA)-mediated depletion of Ror2 in vivo increased branching and altered the differentiation of the mammary epithelium. Microarray analyses identified distinct gene level alterations within the epithelial compartments in the absence of Ror2, with marked changes observed in genes associated with the actin cytoskeleton. Modeling of branching morphogenesis in vitro defined specific defects in cytoskeletal dynamics accompanied by Rho pathway alterations downstream of Ror2 loss. The current study presents a model of Wnt signaling coordination in vivo and assigns an important role for Ror2 in mammary development.

ROR1, an embryonic protein with an emerging role in cancer biology.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a member of the ROR family consisting of ROR1 and ROR2. RORs contain two distinct extracellular cysteine-rich domains and one transmembrane domain. Within the intracellular portion, ROR1 possesses a tyrosine kinase domain, two serine/threonine-rich domains and a proline-rich domain. RORs have been studied in the context of embryonic patterning and neurogenesis through a variety of homologs. These physiologic functions are dichotomous based on the requirement of the kinase domain. A growing literature has established ROR1 as a marker for cancer, such as in CLL and other blood malignancies. In addition, ROR1 is critically involved in progression of a number of blood and solid malignancies. ROR1 has been shown to inhibit apoptosis, potentiate EGFR signaling, and induce epithelial-mesenchymal transition (EMT). Importantly, ROR1 is only detectable in embryonic tissue and generally absent in adult tissue, making the protein an ideal drug target for cancer therapy.

Hematopoietic stem cell fate decisions are regulated by Wnt antagonists: comparisons and current controversies.

Wingless and int (Wnt) proteins are secreted proteins that are important for regulating hematopoietic stem cell self-renewal and differentiation in the bone marrow microenvironment in mice. The mechanisms by which Wnt signaling regulates these hematopoietic cell fate decisions are not fully understood. Secreted Wnt antagonists, which are expressed in bone and bone marrow stromal cells, either bind to Wnt ligands directly or block Wnt receptors and co-receptors to halt Wnt-mediated signal transduction in both osteolineage and hematopoietic cell types. Secreted frizzled related proteins-1 and -2, Wnt inhibitory factor-1, Dickkopf-1, and Sclerostin are Wnt antagonists that influence hematopoietic cell fate decisions in the bone marrow niche. In this review, we compare and contrast the roles of these Wnt antagonists and their effects on hematopoietic development in mice, and also discuss the clinical significance of targeting Wnt antagonists within the context of hematopoietic disease.

The dual role of the novel Wnt receptor tyrosine kinase, ROR2, in human carcinogenesis.

The Wnt signaling pathway is involved in the development and progression of many human cancers, yet attempts to target the pathway therapeutically have been disappointing to date. The recent discovery that the ROR2 receptor tyrosine kinase (RTK) is a novel Wnt receptor provides the potential to target the non-canonical Wnt pathway for cancer treatments. As a member of the RTK superfamily of surface receptors ROR2 appears to possess dual roles as a tumor suppressor or activator depending on tumor type. This review will explore the dual role of ROR2 in tumorigenesis and provide an up to date analysis of current literature in this rapidly expanding field.

Crosstalk between ROR1 and the Pre-B cell receptor promotes survival of t(1;19) acute lymphoblastic leukemia.

We report that t(1;19) ALL cells universally exhibit expression of and dependence on the cell surface receptor ROR1. We further identify t(1;19) ALL cell sensitivity to the kinase inhibitor dasatinib due to its inhibition of the pre-B cell receptor (pre-BCR) signaling complex. These phenotypes are a consequence of developmental arrest at an intermediate/late stage of B-lineage maturation. Additionally, inhibition of pre-BCR signaling induces further ROR1 upregulation, and we identify distinct ROR1 and pre-BCR downstream signaling pathways that are modulated in a counterbalancing manner-both leading to AKT phosphorylation. Consistent with this, AKT phosphorylation is transiently eliminated after dasatinib treatment, but is partially restored following dasatinib potentiation of ROR1 expression. Consequently, ROR1 silencing accentuates dasatinib killing of t(1;19) ALL cells.

NKX2-1/TITF1/TTF-1-Induced ROR1 is required to sustain EGFR survival signaling in lung adenocarcinoma.

We and others previously identified NKX2-1, also known as TITF1 and TTF-1, as a lineage-survival oncogene in lung adenocarcinomas. Here we show that NKX2-1 induces the expression of the receptor tyrosine kinase-like orphan receptor 1 (ROR1), which in turn sustains a favorable balance between prosurvival PI3K-AKT and pro-apoptotic p38 signaling, in part through ROR1 kinase-dependent c-Src activation, as well as kinase activity-independent sustainment of the EGFR-ERBB3 association, ERBB3 phosphorylation, and consequential PI3K activation. Notably, ROR1 knockdown effectively inhibited lung adenocarcinoma cell lines, irrespective of their EGFR status, including those with resistance to the EGFR tyrosine kinase inhibitor gefitinib. Our findings thus identify ROR1 as an "Achilles' heel" in lung adenocarcinoma, warranting future development of therapeutic strategies for this devastating cancer.

ROR1 expression is not a unique marker of CLL.

Recent studies have identified receptor tyrosine kinase-like orphan receptor 1 (ROR1) on the surface of chronic lymphoid leukaemia (CLL) cells. In order to determine whether ROR1 expression is a suitable surrogate marker for the diagnosis of CLL we analysed the mRNA level of ROR1 in different types of non-Hodgkin lymphomas (NHL), and detected elevated levels of ROR1 compared to control peripheral mononuclear cells in several entities (CLL ≥ mantle cell lymphoma (MCL) > marginal zone lymphoma (MZL) >> diffuse large B-cell lymphoma > follicular lymphoma). ROR1 protein was expressed intensely on the cell surface of lymphoma cells with leukaemic blood count detected by three colour immunofluorescence. Our results indicate that ROR1 expression is not limited to CLL cases, but it is more prevalent in NHLs, mainly in MCL where it is expressed intensely and MZL where it is expressed moderately, suggesting a general role of ROR1 in lymphoma genesis and/or maintenance. Copyright © 2010 John Wiley & Sons, Ltd.

Ror2 receptor requires tyrosine kinase activity to mediate Wnt5A signaling.

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 beta-catenin protein stabilization and entry into the nucleus, resulting in changes in target gene transcription. Emerging evidence suggests that Wnt5a can inhibit Wnt/beta-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 Axin2(LacZ), finding increased reporter activity in Ror2 null mice, demonstrating that Ror2 can also inhibit Wnt/beta-catenin signaling in the context of intact tissues.