Anti-LRP6 monoclonal antibody, clone 2D20

The 22 gamma-Protocadherin (gamma-Pcdh) adhesion molecules encoded by the Pcdhg gene cluster play critical roles in nervous system development, including regulation of dendrite arborisation, neuronal survival, and synaptogenesis. Recently, they have been implicated in suppression of tumour cell growth by inhibition of canonical Wnt signalling, though the mechanisms through which this occurs remain unknown. Here, we show differential regulation of Wnt signalling by individual gamma-Pcdhs: The C3 isoform uniquely inhibits the pathway, whilst 13 other isoforms upregulate signalling. Focusing on the C3 isoform, we show that its unique variable cytoplasmic domain (VCD) is the critical one for Wnt pathway inhibition. gamma-Pcdh-C3, but not other isoforms, physically interacts with Axin1, a key component of the canonical Wnt pathway. The C3 VCD competes with Dishevelled for binding to the DIX domain of Axin1, which stabilizes Axin1 at the membrane and leads to reduced phosphorylation of Wnt co-receptor Lrp6. Finally, we present evidence that Wnt pathway activity can be modulated up (by gamma-Pcdh-A1) or down (by gamma-Pcdh-C3) in the cerebral cortex in vivo, using conditional transgenic alleles. Together, these data delineate opposing roles for gamma-Pcdh isoforms in regulating Wnt signalling and identify Axin1 as a novel protein interactor of the widely-expressed gamma-Pcdh-C3 isoform.

The signaling pathway involving the R-spondins and its cognate receptor, GPR48/LGR4, is crucial in development and carcinogenesis. However, the functional implications of the R-spondin-GPR48/LGR4 pathway in thyroid remain to be identified. The aim of this study was to investigate the role of R-spondin-GPR48/LGR4 signaling in papillary thyroid carcinomas. We retrospectively reviewed a total of 214 patients who underwent total thyroidectomy and cervical lymph node dissection for papillary thyroid carcinoma. The role of GPR48/LGR4 in proliferation and migration was examined in thyroid cancer cell lines. R-spondin 2, and GPR48/LGR4 were expressed at significantly higher levels in thyroid cancer than in normal controls. Elevated GPR48/LGR4 expression was significantly associated with tumor size (P=0.049), lymph node metastasis (P=0.004), recurrence (P=0.037), and the BRAFV600E mutation (P=0.003). Moreover, high GPR48/LGR4 expression was an independent risk factor for lymph node metastasis (P=0.027) and the BRAFV600E mutation (P=0.009). in vitro assays demonstrated that elevated expression of GPR48/LGR4 promoted proliferation and migration of thyroid cancer cells, whereas downregulation of GPR48/LGR4 decreased proliferation and migration by inhibition of the beta-catenin pathway. Moreover, treatment of thyroid cancer cells with exogenous R-spondin 2 induced activation of the beta-catenin pathway through GPR48/LGR4. The R-spondin 2-GPR48/LGR4 signaling axis also induced the phosphorylation of ERK, as well as phosphorylation of LRP6 and serine 9 of GSK3 beta. Our findings demonstrate that upregulation of the R-spondin 2-GPR48/LGR4 pathway contributes to tumor aggressiveness in papillary thyroid carcinoma by promoting ERK phosphorylation, suggesting that this pathway represents a novel therapeutic target for treatment of differentiated thyroid cancer.