WNT5A inhibition alters the malignant peripheral nerve sheath tumor microenvironment and enhances tumor growth.
ABSTRACT: Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft-tissue sarcomas that cause significant mortality in adults with neurofibromatosis type 1. We compared gene expression of growth factors in normal human nerves to MPNST and normal human Schwann cells to MPNST cell lines. We identified WNT5A as the most significantly upregulated ligand-coding gene and verified its protein expression in MPNST cell lines and tumors. In many contexts WNT5A acts as an oncogene. However, inhibiting WNT5A expression using shRNA did not alter MPNST cell proliferation, invasion, migration, or survival in vitro. Rather, shWNT5A-treated MPNST cells upregulated mRNAs associated with the remodeling of extracellular matrix and with immune cell communication. In addition, these cells secreted increased amounts of the proinflammatory cytokines CXCL1, CCL2, IL6, CXCL8, and ICAM1. Versus controls, shWNT5A-expressing MPNST cells formed larger tumors in vivo. Grafted tumors contained elevated macrophage/stromal cells, larger and more numerous blood vessels, and increased levels of Mmp9, Cxcl13, Lipocalin-1, and Ccl12. In some MPNST settings, these effects were mimicked by targeting the WNT5A receptor ROR2. These data suggest that the non-canonical Wnt ligand WNT5A inhibits MPNST tumor formation by modulating the MPNST microenvironment, so that blocking WNT5A accelerates tumor growth in vivo.
Project description: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.
Project description: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 beta-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.
Project description:We have previously shown that Wnt5A and ROR2, an orphan tyrosine kinase receptor, interact to mediate melanoma cell motility. In other cell types, this can occur through the interaction of ROR2 with the cytoskeletal protein filamin A. Here, we found that filamin A protein levels correlated with Wnt5A levels in melanoma cells. Small interfering RNA (siRNA) knockdown of WNT5A decreased filamin A expression. Knockdown of filamin A also corresponded to a decrease in melanoma cell motility. In metastatic cells, filamin A expression was predominant in the cytoplasm, which western analysis indicated was due to the cleavage of filamin A in these cells. Treatment of nonmetastatic melanoma cells with recombinant Wnt5A increased filamin A cleavage, and this could be prevented by the knockdown of ROR2 expression. Further, BAPTA-AM chelation of intracellular calcium also inhibited filamin A cleavage, leading to the hypothesis that Wnt5A/ROR2 signaling could cleave filamin A through activation of calcium-activated proteases, such as calpains. Indeed, WNT5A knockdown decreased calpain 1 expression, and by inhibiting calpain 1 either pharmacologically or using siRNA, it decreased cell motility. Our results indicate that Wnt5A activates calpain-1, leading to the cleavage of filamin A, which results in a remodeling of the cytoskeleton and an increase in melanoma cell motility.
Project description:Evolutionarily conserved receptor tyrosine kinase–like orphan receptor-1 and -2 (ROR1/2) are considered distinct receptors for Wnt5a and are implicated in noncanonical Wnt signaling in organogenesis and cancer metastasis. We found that Wnt5a enhanced proliferation and migration of chronic lymphocytic leukemia (CLL) cells and that these effects were blocked by the humanized anti-ROR1 mAb cirmtuzumab (UC-961). Treatment of CLL cells with Wnt5a induced ROR1 to oligomerize with ROR2 and recruit guanine exchange factors (GEFs), which activated Rac1 and RhoA; siRNA-mediated silencing of either ROR1 or ROR2 or treatment with UC-961 inhibited these effects. Using the ROR1-deficient CLL cell line MEC1, we demonstrated that ectopic ROR1 expression induced ROR1/ROR2 heterooligomers, which recruited GEFs, and enhanced proliferation, cytokine-directed migration, and engraftment potential of MEC1 cells in immune-deficient mice. Notably, treatment with UC-961 inhibited engraftment of ROR1+ leukemia cells in immune-competent ROR1-transgenic mice. Molecular analysis revealed that the extracellular Kringle domain is required for ROR1/ROR2 heterooligomerization and the cysteine-rich domain or intracellular proline-rich domain is required for Wnt5a-induced recruitment of GEFs to ROR1/ROR2. This study identifies an interaction between ROR1 and ROR2 that is required for Wnt5a signaling that promotes leukemia chemotaxis and proliferation.
Project description:Tyrosine kinase receptors represent targets of great interest for cancer therapy. Here we show, 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. As ROR2 has been shown to specifically interact with the non-canonical Wnt ligand, Wnt5A, this corroborates our earlier 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 show 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.
Project description:It has been shown that constitutively active Wnt5a-Ror2 signaling in osteosarcoma cell lines plays crucial roles in induced expression of matrix metalloproteinase-13 (MMP-13), required for their invasiveness; however, it remains largely unclear about the molecular basis of MMP-13 gene induction by Wnt5a-Ror2 signaling. Here we show by reporter assay that the activator protein 1 (AP1) (binding site in the promoter region of MMP-13 gene is primarily responsible for its transcriptional activation by Wnt5a-Ror2 signaling in osteosarcoma cell lines SaOS-2 and U2OS. Chromatin immunoprecipitation assays revealed that c-Jun and ATF2 are crucial transcription factors recruited to the AP1-binding site in the MMP-13 gene promoter during Wnt5a-Ror2 signaling in SaOS-2 cells. Using siRNA-mediated suppression or specific inhibitors, we also show that Dishevelled2 (Dvl2) and c-Jun N-terminal kinase are required for MMP-13 gene induction presumably via phosphorylation of c-Jun and ATF2 during Wnt5a-Ror2 signaling in SaOS-2 cells. Interestingly, Dvl2 and Rac1, but not Dvl3, are required for MMP-13 expression in SaOS-2 cells, whereas Dvl3, but not Dvl2 and Rac1, is required for its expression in U2OS cells, indicating the presence of distinct intracellular signaling machineries leading to expression of the same gene, in this case MMP-13 gene in different osteosarcoma cell lines. Moreover, we provide evidence suggesting that Wnt5a-Ror2 signaling might also be required for expression of MMP-13 gene during the development of the cartilaginous tissue.
Project description:Primary thyroid lymphoma (PTL) is a rare malignant thyroid tumor; its pathogenesis is closely related to chronic lymphocytic thyroiditis. The different pathological subtypes and stages of PTL have distinct clinical characteristics and prognosis, but the specific reasons are not clear. Wnt5a is a representative protein of non-canonical Wnt signaling. It plays an important role in many different types of tumors. This study is to explore the changes of Wnt5a and its receptor Ror2 in PTL development process and the clinical significance of their represent. We collected 22 PTL patient tumor specimens and clinical data. We observed the expression of Wnt5a and Ror2 in PTL tumor tissues by immunohistochemistry. Wnt5a was expressed positively in 12 (54.5 %) cases, and Ror2 was expressed positively in 18 (81.8 %) cases. The expression of Wnt5a had a significant difference in different pathological subtypes of PTL (P?<?0.05). Wnt5a and Ror2 expression were associated with local invasion and clinical stage, respectively (P?<?0.05), and had no significant correlation with age, gender, and tumor size. Although, no significant difference in overall survival was found between positive and negative groups of Wnt5a (P?=?0.416) or Ror2 (P?=?0.256), respectively. We still consider that Wnt5a and Ror2 play a complex and subtle role in the pathogenesis and progression of PTL and may become potential biomarkers and therapeutic targets of PTL.
Project description: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-? (IFN-?)-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-?-producing CD4(+) Th1 cells. In vitro experiments demonstrated that the Wnt5a-Ror2 signaling axis augmented the DCs priming effect of IFN-?, leading to enhanced lipopolysaccharide (LPS)-induced interleukin (IL)-12 expression. Taken together, these results suggest that Wnt5a promotes IFN-? signaling, leading to IL-12 expression in DCs, and thereby inducing Th1 differentiation in colitis.
Project description:Patients on peritoneal dialysis are at risk of developing peritoneal fibrosis and angiogenesis, which can lead to dysfunction of the peritoneal membrane. Recent evidence has identified cross-talk between transforming growth factor beta (TGFB) and the WNT/β-catenin pathway to induce fibrosis and angiogenesis. Limited evidence exists describing the role of non-canonical WNT signalling in peritoneal membrane injury. Non-canonical WNT5A is suggested to have different effects depending on the receptor environment. WNT5A has been implicated in antagonizing canonical WNT/β-catenin signalling in the presence of receptor tyrosine kinase-like orphan receptor (Ror2). We co-expressed TGFB and WNT5A using adenovirus and examined its role in the development of peritoneal fibrosis and angiogenesis. Treatment of mouse peritoneum with AdWNT5A decreased the submesothelial thickening and angiogenesis induced by AdTGFB. WNT5A appeared to block WNT/β-catenin signalling by inhibiting phosphorylation of glycogen synthase kinase 3 beta (GSK3B) and reducing levels of total β-catenin and target proteins. To examine the function of Ror2, we silenced Ror2 in a human mesothelial cell line. We treated cells with AdWNT5A and observed a significant increase in fibronectin compared with AdWNT5A alone. We also analysed fibronectin and vascular endothelial growth factor (VEGF) in a TGFB model of mesothelial cell injury. Both fibronectin and VEGF were significantly increased in response to Ror2 silencing when cells were exposed to TGFB. Our results suggest that WNT5A inhibits peritoneal injury and this is associated with a decrease in WNT/β-catenin signalling. In human mesothelial cells, Ror2 is involved in regulating levels of fibronectin and VEGF.
Project description:The present study investigated the prognostic significance of Wnt family member 5a (Wnt5a) and receptor tyrosine kinase-like orphan receptor 2 (Ror2) expression in laryngeal squamous cell carcinoma (LSCC). The protein expression levels of Wnt5a and Ror2 were analyzed in specimens from 137 patients with LSCC, using immunohistochemical staining of tissue microarrays and pairs of LSCC and adjacent tissue samples, and examined the associations between the two markers and various clinicopathological parameters. The Wnt5a and Ror2 expression levels were significantly higher in LSCC tissues than in normal tissue samples (Wnt5a, P=0.015; Ror2, P=0.039), and were significantly associated with high tumor stage (P<0.001), lymph node metastasis (Wnt5a, P=0.029; Ror2, P=0.018), and with each other (P=0.002). Patients with LSCC with high Wnt5a or Ror2 expression had poorer prognosis compared with those with low Wnt5a (P=0.022) or Ror2 (P=0.038) expression. Thus, Wnt5a and Ror2 may affect LSCC development, and are potential biomarkers in LSCC.