N-glycosylation And Canonical Wnt Signaling Cooperate To Induce CTHRC1and Drive Oral Cancer Cell Migration

Background and ObjectiveCollagen Triple Helix Repeat Containingl (Cthrcl) was first identified in injured rat arteries, and it is a secreted glycoprotein that has the ability to inhibit collagen expression and promote cell migration. Expression of Cthrcl is increased in fibroblasts and chondrocytic cells in response to TGF-beta family members including BMP4, BMP2and TGF-beta. Cthrcl has been linked to major signaling pathways such as Wnt and TGF-beta. The ability of Cthrcl to inhibit TGF-beta signaling via a reduction in Smad2/Smad3phosphorylation has been demonstrated both in vivo and in vitro models. Cthrcl is also upregulated during tumorigenesis and metastasis; its role during this process is unknown. Recently studies suggested that Cthrcl maybe activate the Planar Cell Polarity (PCP) pathway of non-canonical Wnt signaling. And its active form is an N-glycosylated trimer anchored on the cell surface. N-glycosylation may contribute to the anchorage of Chtrcl on the cell surface. Although Cthrcl is upregulated during tumorigenesis and metastasis, the mechanism and the overexpression of Cthrc1and its function are not clear.Wnt/planar cell polarity(PCP) signaling pathway of non-canonical Wnt signaling pathway plays a complex role in cancer development. At early stages of cancer, Wnt/PCP inhibits cancer progression by antagonizing Wnt/p signaling. As tumor progress, Wnt/PCP gets activated and promotes tumor cell migration and invasion and supports angiogenesis, contributing to metastasis in late stages of cancer. Previous studies have already identified that Wnt/(3signaling is activated in oral cancer and we also reported that cellular discohesion in oral squamous cell carcinoma (OSCC) occurred, in part, due to inappropriate upregulation of DPAGT1, the first gene in the N-glycosylation pathway and its key regulator, which resulted in extensive N-glycosylation of E-cadherin. Recent work in our laboratory has shown that DPAGT1is a target of canonical Wnt signaling. Activation of canonical Wnt leads to increased N-glycosylation of Wnt proteins, Wnt3a and LRP5/6, leading to further upregulation of DPAGT1in a positive feedback loop. Inappropriate activation drives OSCC proliferation.Cthrcl is glycoprotein, so we hypotheses that its overexpression maybe involved in the feedback loop of DPAGT1and Wnt/β signaling pathway. And Cthrcl may be able to promote tumor spread at the later stages of cancer through activate the Wnt/PCP signaling pathway.MethodsWe examined Cthrcl protein, DPAGT1gene and some components of the Wnt/PCP signaling pathway in OSCC by western blot and immunofluorescence in cal27cells and tumor tissue specimens, and also Deglycosylation of Cthrcl was performedCthrc1protein level is detected after silencing DPAGT1gene in cal27cells. And Chip assays were performed to detect the binding sits of β-catenin and TCF on Cthrcl promoter. Cell migration experiments and scratch wound healing experiment were performed after silencing the Cthrcl in Ca127cells. ResultsCthrcl is overexpressed in OSCC, and it is modified with complex N-glycans. Cthrc1is upregulated in many solid human tumors, so we detected Cthrcl in adjacent epithelial and tumor samples lysates by western blot and immunofluoresence. Cthrc1is overexpressed in the OSCC. In OSCC, Cthrcl immunostaining got high signal throughout the tumor islands, but there was no signal in adjacent epithelial. Previous study suggested that the active form of Cthrc1is an N-glycosylated trimmer anchored on the cell membrane, and N-glycosylation may contribute to this anchorage of Cthrcl on the cell surface. So we examined the DPAGT1gene in OSCC and the adjacent epithelial lysates by western blot and immunofluorescence. DPAGT1expressed much more in tumor than in adjacent epithelial. In adjacent epithelia, DPAGT1staining was detected in the basal layer and diminished in the cytodifferentiated cells of the spinous cell layer. In OSCC, DPAGT1expression was extensive throughout the tumor islands. Cthrc1is a secreted glycoprotein, we examined its sensitivity to glycanases, EndoH and PNaseF. EndoH removes high mannose and hybrid N-glycans, whereas PNGaseF removes N-glycans at asparagine residues with the exception of complex N-glycans modified by fucose at the chitobiose core. Mobility shifts before and after PNGaseF treatments showed that Cthrc1was PNGaseF-sensitive, but not sensitive to EndoH, suggesting that Cthrcl is modified by complex N-glycans. N-glycosylation increases the stability of Cthrcl in OSCC. Previous study suggested that N-glycosylation may increase the secretion of Cthrc1, and may promote the anchorage of Cthrcl on cell surface. To identify the real role of N-glycosylation in Cthrcl overexpression, we reduced the DPAGT1gene expression with DPAGT1siRNA in ca127cells. The protein level of Cthrcl is dramatically decreased in silenced cal27cells, and the mRNA level of GPT is decreased by50%percent. Then we treated the NS and S ca127cells with cycloheximid, then got longer half-life of Cthrcl in NS than S. This suggested that N-glycosylation increased the stability of Cthrcl in OSCC. Maybe that is one of the mechanisms of Cthrc1overexpression in OSCC.Canonical Wnt signaling pathway contributes to the overexpression of Cthrcl in OSCC. Our previous study showed that the DPAGT1and canonical Wnt signaling pathway form a positive feedback loop in OSCC. In our experiment, the protein level of Cthrcl is increased in Wnt3a condition medium treated Ca127cells. Then we figured out that there are β-catenin and TCF binding sits on Cthrcl promoter by ChIP assay. We all know that β-catenin is the most downstream of the canonical Wnt pathway, and TCF is the target of the β-catenin. So, the activation of canonical Wnt signaling pathway may be able to upregulated the Cthrc1. That may be another mechanism of Cthrcl overexpression in OSCC.Cthrc1drives OSCC cell migration. Our collective observations that Cthrcl was upregulated by DPAGT1coincident with increased localization of Cthrcl to the wound edge and faster wound closure suggested that Cthrcl was involved in cell migration. Thus, we carried out trans-well migration assays with CAL27cells in which Cthrcl expression was inhibited by treatment with siRNA (S) and compared to cells treated with scrambled, non-silencing control RNA (NS). Results showed that inhibition of Cthrcl expression by85%reduced S cell migration to a10%level of NS cells. Furthermore, this extent of inhibition of Cthrcl reduced the motile behavior of S cells compared to NS cells in a scratch wound assay carried out with confluent monolayers of cells. These results showed that in cultured CAL27cells, Cthrcl was involved in cell migration.The observed increased expression and localization of Cthrc1to migrating edge cells in DPAGT1CAL27transfectants suggested that Cthrcl functioned by upregulating an RhoA-dependent actin-myosin rearrangements via a non-canonical Wnt pathway. Indeed, immunoprecipitation of Cthrcl followed by immunoblot demonstrated that Cthrcl interacted with a non-canonical Wnt ligand, Wnt5a, and receptor, Fzd6. Furthermore, levels of Wnt5a were more than5-fold higher in human OSCC specimens compared to AE. These results suggested that in OSCC, Cthrcl was likely to promote cell migration via a non-canonical Wnt pathway.ConclusionOur results here suggested that N-glycosylation and Wnt/β signaling pathway promote the overexpression of Cthrcl in oral cancer.And then perhaps Cthrc1promotes the role of PCP pathway in regulating the oral tumor’s invasion and migration.