N-糖基化和经典Wnt信号通路促进Cthrc1的表达及其促进口腔癌细胞转移的功能
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摘要
背景和目的
Cthrc1首先在受伤的大鼠血管中发现,它是一种分泌型的糖基化蛋白,具有抑制胶原表达和促进细胞运动的功能。在TGF-β家族的成员(BMP4, BMP2, TGF-β)的影响下,Cthrc1在纤维母细胞和软骨细胞中的表达升高。Cthrc1也跟很多重要的信号通路有关系,比如Wnt和TGF-β。体内外实验模型都证明了Cthrc1抑制TGF-β信号通路的功能是通过减少Smad2和Smad3的磷酸化实现的。Cthrc1在肿瘤的发生和转移中的表达升高,但是它在其过程中的作用还不清楚。最近的研究证明Cthrc1可以激活Wnt/PCP信号通路,它是非经典Wnt途径的一种。Cthrc1的激活状态是一种锚在细胞膜表面的一种N末端糖基化的三聚体。虽然Cthrc1在肿瘤的发生发展中的表达升高,但是其过表达的机制和其在病理过程中的功能还不清楚。
Wnt/PCP信号通路在恶性肿瘤的发展中起着复杂的作用。在恶性肿瘤的早期阶段,Wnt/PCP通过抑制Wnt/β信号通路来抑制肿瘤的发展。随着肿瘤的成长,在肿瘤的晚期阶段,Wnt/PCP被激活并且可以促进肿瘤细胞的转移和侵袭,从而促进了肿瘤的转移。先前的研究证明了Wnt/β信号通路在口腔鳞状细胞癌中是激活的状态。并且我们过去的研究也报道了,在口腔鳞状细胞癌中E-cadherin的大量的N末端糖基化引起的细胞之间粘附的松解。E-cadherin的过度的糖基化又是由于DPAGT1的过表达引起的,DPAGT1是N末端糖基化的重要的调节基因。然而,我们近期的研究显示,DPAGT1基因又是经典Wnt信号通路的靶基因。经典Wnt信号通路的激活能够导致Wnt蛋白的N末端糖基化的增加,例如Wnt3a和LRP5/6,并且能够通过这个正反馈环引起DPAGT1的进一步高表达。这个反馈环的不适当的激活就能够促进口腔鳞状细胞癌的增值。
Cthrc1是分泌型的糖基化蛋白,所以我们可以推测,Cthrc1在口腔鳞状细胞癌中的过表达或许是DPAGT1和Wnt/β信号通路形成的这个反馈环作用的结果。并且,Cthrc1可能在口腔鳞状细胞癌中通过激活Wnt/PCP信号通路来促进肿瘤细胞的迁移和肿瘤的转移。
方法
在口腔鳞状细胞癌肿瘤标本和人舌鳞状细胞癌细胞Ca127中我们用western blot和免疫荧光的方法检测了Cthrc1和DPAGT1的蛋白水平的表达。对Cthrc1进行了去糖基实验。在ca127细胞中,我们沉默掉DPAGT1基因,然后检测了Cthrc1蛋白的表达水平。染色质免疫沉淀实验检测了在Cthrc1启动子上β-catenin和TCF的结合位点。在Ca127田胞中我们沉默掉Cthrc1基因,然后进行了细胞移动实验和刮伤愈合实验。
结果
在口腔鳞状细胞癌中Cthrc1表达升高。Cthrc1在很多人类实性肿瘤中表达升高,所以我们在肿瘤标本和肿瘤的临近正常上皮组织中用western blot和免疫荧光的方法检测了其蛋白表达水平。Cthrc1的表达在肿瘤组织中比正常组织中有非常显著的增高。在免疫荧光的结果中我们看到,在肿瘤上皮岛中表达升高,而在正常组织中几乎没有表达。过去的研究表明,Cthrc1的激活状态是一个锚在细胞膜上的N末端糖基化的三聚体,并且N末端的糖基化对其锚在细胞膜上起着重要的作用。所以我们在肿瘤标本及正常组织中用western blot和免疫荧光的办法检测了DPAGT1的表达,发现DPAGT1在肿瘤组织里的表达远远超过正常组织。并且在免疫荧光中我们看到,在正常上皮组织中DPAGT1仅仅在基底层表达,而在肿瘤组织中则是整个肿瘤上皮岛都有高表达。Cthrc1是糖基化蛋白,所以我们进行了去糖基实验,发现Cthrc1的激活状态确实有N末端糖基修饰。
N末端糖基化能够增加口腔鳞状细胞癌中Cthrc1蛋白的稳定性。有研究证明N末端糖基化增加了Cthrc1的分泌,也是其锚在细胞膜上的重要因素。为了搞清楚N末端糖基化在Cthrc1过表达中起的作用,我们在cal27细胞中沉默掉了DPAGT1基因,从而使得N末端的糖基化减少。这样我们发现在沉默掉DPAGT1基因的cal27田胞中的Cthrc1的蛋白水平严重下降。随后我们用环己酰亚胺处理了经DPAGT1siRNA转染的ca127细胞及其对照组,我们发现N末端的糖基化使Cthrc1的半衰期增加,也就是增加了其稳定性。所以我们认为这可能是Cthrc1在口腔鳞状细胞癌中高表达的一个原因。
经典Wnt信号通路的激活也是口腔鳞状细胞癌中Cthrcl高表达的促进因素。我们过去的研究表明,在口腔鳞状细胞癌中DPAGT1与经典Wnt信号通路形成了一个正反馈的作用环。在我们的实验中,经过Wnt3a条件培养基培育过的ca127田胞的Cthrc1的表达水平增加。随后我们在染色质免疫沉淀实验中发现在Cthrc1的启动子上有β-catenin和TCF的结合位点。众所周知,β-catenin处在经典Wnt信号通路的最末端,TCF又是β-catenin的靶基因。所以经典Wnt信号通路的激活能够使Cthrc1勺表达升高。这也许是口腔鳞状细胞癌中Cthrc1蛋白表达升高的另一种机制。
Cthrc1促进了口腔鳞状细胞癌的迁移。我们大量的观察发现,利用DPAGT1cDNA上调Cthrc1表达之后,在刮伤愈合实验中伤口边缘的Cthrc1表达增多和伤口愈合加快,这都说明Cthrc1可能具有促进了细胞迁移的功能。因此我们在Ca127细胞中沉默掉Cthrc1,进行了细胞移动实验和刮伤愈合实验。在细胞移动实验中沉默组细胞移动率比对照组明显降低,在刮伤愈合实验中沉默组愈合速度比对照组明显减慢。这些结果表明在Ca127细胞中Cthrc1是具有促进细胞迁移的功能的。
DPAGT1转染的CAL27细胞中的Cthrc1在迁移的边缘位置表达升高的特点说明了Cthrc1的促进细胞迁移的功能是依赖于Wnt/RhoA信号通路的。确实如此,继western blot之后免疫沉淀实验表明Cthrc1与Wnt/PCP的配体Wnt5a,受体Fzd6有相互的结合作用。并且,Wnt5a在肿瘤中表达水平比正常组织中有明显的增高。所以,这些都说明在口腔鳞状细胞癌中,Cthrcl可能通过一个非经典Wnt信号通路Wnt/PCP信号通路来促进细胞的迁移。
结论
我们的结果证明了N末端糖基化和经典Wnt信号通路的激活能够引起Cthrcl的表达升高,也就是DPAGT1与Wnt/β信号通路形成的反馈环的异常激活能够导致Cthrc1在口腔鳞状细胞癌中的异常高表达。
结果也间接的说明,Cthrc1可能通过Wnt/PCP信号通路在口腔鳞状细胞癌的晚期促进癌细胞的迁移,从而导致肿瘤的转移。
Background and Objective
Collagen 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.
Methods
We 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.
Results
Cthrcl 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.
Conclusion
Our 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.
Cthrc1首先在受伤的大鼠血管中发现,它是一种分泌型的糖基化蛋白,具有抑制胶原表达和促进细胞运动的功能。在TGF-β家族的成员(BMP4, BMP2, TGF-β)的影响下,Cthrc1在纤维母细胞和软骨细胞中的表达升高。Cthrc1也跟很多重要的信号通路有关系,比如Wnt和TGF-β。体内外实验模型都证明了Cthrc1抑制TGF-β信号通路的功能是通过减少Smad2和Smad3的磷酸化实现的。Cthrc1在肿瘤的发生和转移中的表达升高,但是它在其过程中的作用还不清楚。最近的研究证明Cthrc1可以激活Wnt/PCP信号通路,它是非经典Wnt途径的一种。Cthrc1的激活状态是一种锚在细胞膜表面的一种N末端糖基化的三聚体。虽然Cthrc1在肿瘤的发生发展中的表达升高,但是其过表达的机制和其在病理过程中的功能还不清楚。
Wnt/PCP信号通路在恶性肿瘤的发展中起着复杂的作用。在恶性肿瘤的早期阶段,Wnt/PCP通过抑制Wnt/β信号通路来抑制肿瘤的发展。随着肿瘤的成长,在肿瘤的晚期阶段,Wnt/PCP被激活并且可以促进肿瘤细胞的转移和侵袭,从而促进了肿瘤的转移。先前的研究证明了Wnt/β信号通路在口腔鳞状细胞癌中是激活的状态。并且我们过去的研究也报道了,在口腔鳞状细胞癌中E-cadherin的大量的N末端糖基化引起的细胞之间粘附的松解。E-cadherin的过度的糖基化又是由于DPAGT1的过表达引起的,DPAGT1是N末端糖基化的重要的调节基因。然而,我们近期的研究显示,DPAGT1基因又是经典Wnt信号通路的靶基因。经典Wnt信号通路的激活能够导致Wnt蛋白的N末端糖基化的增加,例如Wnt3a和LRP5/6,并且能够通过这个正反馈环引起DPAGT1的进一步高表达。这个反馈环的不适当的激活就能够促进口腔鳞状细胞癌的增值。
Cthrc1是分泌型的糖基化蛋白,所以我们可以推测,Cthrc1在口腔鳞状细胞癌中的过表达或许是DPAGT1和Wnt/β信号通路形成的这个反馈环作用的结果。并且,Cthrc1可能在口腔鳞状细胞癌中通过激活Wnt/PCP信号通路来促进肿瘤细胞的迁移和肿瘤的转移。
方法
在口腔鳞状细胞癌肿瘤标本和人舌鳞状细胞癌细胞Ca127中我们用western blot和免疫荧光的方法检测了Cthrc1和DPAGT1的蛋白水平的表达。对Cthrc1进行了去糖基实验。在ca127细胞中,我们沉默掉DPAGT1基因,然后检测了Cthrc1蛋白的表达水平。染色质免疫沉淀实验检测了在Cthrc1启动子上β-catenin和TCF的结合位点。在Ca127田胞中我们沉默掉Cthrc1基因,然后进行了细胞移动实验和刮伤愈合实验。
结果
在口腔鳞状细胞癌中Cthrc1表达升高。Cthrc1在很多人类实性肿瘤中表达升高,所以我们在肿瘤标本和肿瘤的临近正常上皮组织中用western blot和免疫荧光的方法检测了其蛋白表达水平。Cthrc1的表达在肿瘤组织中比正常组织中有非常显著的增高。在免疫荧光的结果中我们看到,在肿瘤上皮岛中表达升高,而在正常组织中几乎没有表达。过去的研究表明,Cthrc1的激活状态是一个锚在细胞膜上的N末端糖基化的三聚体,并且N末端的糖基化对其锚在细胞膜上起着重要的作用。所以我们在肿瘤标本及正常组织中用western blot和免疫荧光的办法检测了DPAGT1的表达,发现DPAGT1在肿瘤组织里的表达远远超过正常组织。并且在免疫荧光中我们看到,在正常上皮组织中DPAGT1仅仅在基底层表达,而在肿瘤组织中则是整个肿瘤上皮岛都有高表达。Cthrc1是糖基化蛋白,所以我们进行了去糖基实验,发现Cthrc1的激活状态确实有N末端糖基修饰。
N末端糖基化能够增加口腔鳞状细胞癌中Cthrc1蛋白的稳定性。有研究证明N末端糖基化增加了Cthrc1的分泌,也是其锚在细胞膜上的重要因素。为了搞清楚N末端糖基化在Cthrc1过表达中起的作用,我们在cal27细胞中沉默掉了DPAGT1基因,从而使得N末端的糖基化减少。这样我们发现在沉默掉DPAGT1基因的cal27田胞中的Cthrc1的蛋白水平严重下降。随后我们用环己酰亚胺处理了经DPAGT1siRNA转染的ca127细胞及其对照组,我们发现N末端的糖基化使Cthrc1的半衰期增加,也就是增加了其稳定性。所以我们认为这可能是Cthrc1在口腔鳞状细胞癌中高表达的一个原因。
经典Wnt信号通路的激活也是口腔鳞状细胞癌中Cthrcl高表达的促进因素。我们过去的研究表明,在口腔鳞状细胞癌中DPAGT1与经典Wnt信号通路形成了一个正反馈的作用环。在我们的实验中,经过Wnt3a条件培养基培育过的ca127田胞的Cthrc1的表达水平增加。随后我们在染色质免疫沉淀实验中发现在Cthrc1的启动子上有β-catenin和TCF的结合位点。众所周知,β-catenin处在经典Wnt信号通路的最末端,TCF又是β-catenin的靶基因。所以经典Wnt信号通路的激活能够使Cthrc1勺表达升高。这也许是口腔鳞状细胞癌中Cthrc1蛋白表达升高的另一种机制。
Cthrc1促进了口腔鳞状细胞癌的迁移。我们大量的观察发现,利用DPAGT1cDNA上调Cthrc1表达之后,在刮伤愈合实验中伤口边缘的Cthrc1表达增多和伤口愈合加快,这都说明Cthrc1可能具有促进了细胞迁移的功能。因此我们在Ca127细胞中沉默掉Cthrc1,进行了细胞移动实验和刮伤愈合实验。在细胞移动实验中沉默组细胞移动率比对照组明显降低,在刮伤愈合实验中沉默组愈合速度比对照组明显减慢。这些结果表明在Ca127细胞中Cthrc1是具有促进细胞迁移的功能的。
DPAGT1转染的CAL27细胞中的Cthrc1在迁移的边缘位置表达升高的特点说明了Cthrc1的促进细胞迁移的功能是依赖于Wnt/RhoA信号通路的。确实如此,继western blot之后免疫沉淀实验表明Cthrc1与Wnt/PCP的配体Wnt5a,受体Fzd6有相互的结合作用。并且,Wnt5a在肿瘤中表达水平比正常组织中有明显的增高。所以,这些都说明在口腔鳞状细胞癌中,Cthrcl可能通过一个非经典Wnt信号通路Wnt/PCP信号通路来促进细胞的迁移。
结论
我们的结果证明了N末端糖基化和经典Wnt信号通路的激活能够引起Cthrcl的表达升高,也就是DPAGT1与Wnt/β信号通路形成的反馈环的异常激活能够导致Cthrc1在口腔鳞状细胞癌中的异常高表达。
结果也间接的说明,Cthrc1可能通过Wnt/PCP信号通路在口腔鳞状细胞癌的晚期促进癌细胞的迁移,从而导致肿瘤的转移。
Background and Objective
Collagen 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.
Methods
We 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.
Results
Cthrcl 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.
Conclusion
Our 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.
引文
1. Choi, S. and J.N. Myers, Molecular pathogenesis of oral squamous cell carcinoma: implications for therapy. J Dent Res,2008.87(1):p.14-32.
2. Rothenberg, S.M. and L.W. Ellisen, The molecular pathogenesis of head and neck squamous cell carcinoma. J Clin Invest.122(6):p.1951-7.
3. Perez-Ordonez, B., M. Beauchemin, and R.C. Jordan, Molecular biology of squamous cell carcinoma of the head and neck. J Clin Pathol,2006.59(5):p.445-53.
4. Stransky, N., et al., The mutational landscape of head and neck squamous cell carcinoma. Science.333(6046):p.1157-60.
5. Agrawal, N., et al., Exome sequencing of head and neck squamous cell carcinoma reveals inactivating mutations in NOTCH1. Science.333(6046):p.1154-7.
6. Perez-Sayans, M., et al., What real influence does the proto-oncogene c-myc have in OSCCbehavior? Oral Oncol.47(8):p.688-92.
7. Priyadarsini, R.V., et al., The flavonoid quercetin modulates the hallmark capabilities of hamster buccal pouch tumors. Nutr Cancer.63(2):p.218-26.
8. Jamal, B., et al., Aberrant amplification of the crosstalk between canonical Wnt signaling and N-glycosylation gene DPAGT1 promotes oral cancer. Oral Oncol.48(6):p.523-9.
9. MacDonald, B.T., K. Tamai, and X. He, Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell,2009.17(1):p.9-26.
10. Logan, C.Y. and R. Nusse, The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol,2004.20:p.781-810.
11. Wend, P., et al., Wnt signaling in stem and cancer stem cells. Semin Cell Dev Biol.21(8): p.855-63.
12. Song, J., et al., Characterization of side populations in HNSCC:highly invasive, chemoresistant and abnormal Wnt signaling. PLoS One.5(7):p. e11456.
13. Gordon, M.D. and R. Nusse, Wnt signaling:multiple pathways, multiple receptors, and multiple transcription factors. J Biol Chem,2006.281(32):p.22429-33.
14. van Amerongen, R. and R. Nusse, Towards an integrated view of Wnt signaling in development. Development, 2009.136(19):p.3205-14.
15. Sethi, J.K. and A. Vidal-Puig, Wnt signalling and the control of cellular metabolism. Biochem J.427(1):p.1-17.
16. Heuberger, J. and W. Birchmeier, Interplay of cadherin-mediated cell adhesion and canonical Wnt signaling. Cold Spring Harb Perspect Biol.2(2):p. a002915.
17. Sengupta, P.K., M.P. Bouchie, and M.A. Kukuruzinska, N-glycosylation gene DPAGT1 is a target of the Wnt/beta-catenin signaling pathway. J Biol Chem.285(41):p.31164-73.
18. Helenius, A. and M. Aebi, Intracellular functions of N-linked glycans. Science,2001. 291(5512):p.2364-9.
19. te Heesen, S., et al., Yeast Wbplp and Swplp form a protein complex essential for oligosaccharyl transferase activity. EMBO J,1993.12(1):p.279-84.
20. Sengupta, P.K., et al., Coordinate regulation of N-glycosylation gene DPACT1, canonical Wnt signaling and E-cadherin adhesion. J Cell Sci.
21. Marek, K.W., I.K. Vijay, and J.D. Marth, A recessive deletion in the GlcNAc-1-phosphotransferase gene results in peri-implantation embryonic lethality. Glycobiology, 1999.9(11):p.1263-71.
22. Haegel, H., et al., Lack of beta-catenin affects mouse development at gastrulation. Development, 1995.121(11):p.3529-37.
23. Dennis, J.W., M. Granovsky, and C.E. Warren, Glycoprotein glycosylation and cancer progression. Biochim Biophys Acta,1999.1473(1):p.21-34.
24. Nita-Lazar, M., et al., Overexpression of DPAGT1 leads to aberrant N-glycosylation of E-cadherin and cellular discohesion in oral cancer. Cancer Res,2009.69(14):p.5673-80.
25. Pyagay, P., et al., Collagen triple helix repeat containing 1, a novel secreted protein in injured and diseased arteries, inhibits collagen expression and promotes cell migration. Circ Res,2005.96(2):p.261-8.
26. Yamamoto, S., et al., Cthrd selectively activates the planar cell polarity pathway of Wnt signaling by stabilizing the Wnt-receptor complex. Dev Cell,2008.15(1):p.23-36.
27. Turashvili, G., et al., Novel markers for differentiation of lobular and ductal invasive breast carcinomas by laser microdissection and microarray analysis. BMC Cancer,2007. 7:p.55.
28. Tang, L, et al., Aberrant expression of collagen triple helix repeat containing 1 in human solid cancers. Clin Cancer Res,2006.12(12):p.3716-22.
29. Park, E.H., et al., Collagen triple helix repeat containing-1 promotes pancreatic cancer progression by regulating migration and adhesion of tumor cells. Carcinogenesis.34(3): p.694-702.
30. Goto, M., et al., Rapl stabilizes beta-catenin and enhances beta-catenin-dependent transcription and invasion in squamous cell carcinoma of the head and neck. Clin Cancer Res.16(1):p.65-76.
31. Nita-Lazar, M., et al., Hypoglycosylated E-cadherin promotes the assembly of tight junctions through the recruitment of PP2A to adherens junctions. Exp Cell Res.316(11):p. 1871-84.
32. Parri, M. and P. Chiarugi, Rac and Rho GTPases in cancer cell motility control. Cell Commun Signal.8:p.23.
33. Polakis, P., Wnt signaling and cancer. Genes Dev,2000.14(15):p.1837-51.
34. Lau, K.S., et al., Complex N-glycan number and degree of branching cooperate to regulate cell proliferation and differentiation. Cell,2007.129(1):p.123-34.
35. Mendelsohn, R., et al., Complex N-glycan and metabolic control in tumor cells. Cancer Res,2007.67(20):p.9771-80.
36. Katoh, M., WNT/PCP signaling pathway and human cancer (review). Oncol Rep,2005. 14(6):p.1583-8.
37. Luga, V., et al., Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration. Cell.151(7):p.1542-56.
38. Dennis, J.W., I.R. Nabi, and M. Demetriou, Metabolism, cell surface organization, and disease. Cell,2009.139(7):p.1229-41.
39. Wang, P., et al., CTHRC1 is upregulated by promoter demethylation and transforming growth factor-betal and may be associated with metastasis in human gastric cancer. Cancer Sci.103(7):p.1327-33.
40. Watanabe, S., et al., A novel glycosylation signal regulates transforming growth factor beta receptors as evidenced by endo-beta-galactosidase C expression in rodent cells. Glycobiology.21(4):p.482-92.
41. Guturi, K.K., et al., Mechanism of beta-catenin-mediated transcriptional regulation of epidermal growth factor receptor expression in glycogen synthase kinase 3 beta-inactivated prostate cancer cells. J Biol Chem.287(22):p.18287-96.
42. Contessa, J.N., et al., Inhibition ofN-linked glycosylation disrupts receptor tyrosine kinase signaling in tumor cells. Cancer Res,2008.68(10):p.3803-9.
43. Schlessinger, J., Cell signaling by receptor tyrosine kinases. Cell,2000.103(2):p.211-25.
44. Masiakowski, P. and R.D. Carroll, A novel family of cell surface receptors with tyrosine kinase-like domain. J Biol Chem,1992.267(36):p.26181-90.
45. Oldridge, M., et al., Dominant mutations in ROR2, encoding an orphan receptor tyrosine kinase, cause brachydactyly type B. Nat Genet,2000.24(3):p.275-8.
46. Patton, M.A. and A.R. Afzal, Robinowsyndrome. J Med Genet,2002.39(5):p.305-10.
47. Nusse, R. and H.E. Varmus, Wnt genes. Cell,1992.69(7):p.1073-87.
48. Oishi, I., et al., The receptor tyrosine kinase Ror2 is involved in non-canonical Wnt5a/JNK signalling pathway. Genes Cells,2003.8(7):p.645-54.
49. Katoh, M., Comparative genomics on ROR1 and ROR2 orthologs. Oncol Rep,2005.14(5): p.1381-4.
50. Nishita, M., et al., Filopodia formation mediated by receptor tyrosine kinase Ror2 is required for Wnt5a-induced cell migration. J Cell Biol,2006.175(4):p.555-62.
51. Huang, C.L., et al., Wnt5a expression is associated with the tumor proliferation and the stromal vascular endothelial growth factor--an expression in non-small-cell lung cancer. J Clin Oncol,2005.23(34):p.8765-73.
52. Kurayoshi, M., et al., Expression of Wnt-5α is correlated with aggressiveness of gastric cancer by stimulating cell migration and invasion. Cancer Res, 2006.66(21):p.10439-48.
2. Rothenberg, S.M. and L.W. Ellisen, The molecular pathogenesis of head and neck squamous cell carcinoma. J Clin Invest.122(6):p.1951-7.
3. Perez-Ordonez, B., M. Beauchemin, and R.C. Jordan, Molecular biology of squamous cell carcinoma of the head and neck. J Clin Pathol,2006.59(5):p.445-53.
4. Stransky, N., et al., The mutational landscape of head and neck squamous cell carcinoma. Science.333(6046):p.1157-60.
5. Agrawal, N., et al., Exome sequencing of head and neck squamous cell carcinoma reveals inactivating mutations in NOTCH1. Science.333(6046):p.1154-7.
6. Perez-Sayans, M., et al., What real influence does the proto-oncogene c-myc have in OSCCbehavior? Oral Oncol.47(8):p.688-92.
7. Priyadarsini, R.V., et al., The flavonoid quercetin modulates the hallmark capabilities of hamster buccal pouch tumors. Nutr Cancer.63(2):p.218-26.
8. Jamal, B., et al., Aberrant amplification of the crosstalk between canonical Wnt signaling and N-glycosylation gene DPAGT1 promotes oral cancer. Oral Oncol.48(6):p.523-9.
9. MacDonald, B.T., K. Tamai, and X. He, Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell,2009.17(1):p.9-26.
10. Logan, C.Y. and R. Nusse, The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol,2004.20:p.781-810.
11. Wend, P., et al., Wnt signaling in stem and cancer stem cells. Semin Cell Dev Biol.21(8): p.855-63.
12. Song, J., et al., Characterization of side populations in HNSCC:highly invasive, chemoresistant and abnormal Wnt signaling. PLoS One.5(7):p. e11456.
13. Gordon, M.D. and R. Nusse, Wnt signaling:multiple pathways, multiple receptors, and multiple transcription factors. J Biol Chem,2006.281(32):p.22429-33.
14. van Amerongen, R. and R. Nusse, Towards an integrated view of Wnt signaling in development. Development, 2009.136(19):p.3205-14.
15. Sethi, J.K. and A. Vidal-Puig, Wnt signalling and the control of cellular metabolism. Biochem J.427(1):p.1-17.
16. Heuberger, J. and W. Birchmeier, Interplay of cadherin-mediated cell adhesion and canonical Wnt signaling. Cold Spring Harb Perspect Biol.2(2):p. a002915.
17. Sengupta, P.K., M.P. Bouchie, and M.A. Kukuruzinska, N-glycosylation gene DPAGT1 is a target of the Wnt/beta-catenin signaling pathway. J Biol Chem.285(41):p.31164-73.
18. Helenius, A. and M. Aebi, Intracellular functions of N-linked glycans. Science,2001. 291(5512):p.2364-9.
19. te Heesen, S., et al., Yeast Wbplp and Swplp form a protein complex essential for oligosaccharyl transferase activity. EMBO J,1993.12(1):p.279-84.
20. Sengupta, P.K., et al., Coordinate regulation of N-glycosylation gene DPACT1, canonical Wnt signaling and E-cadherin adhesion. J Cell Sci.
21. Marek, K.W., I.K. Vijay, and J.D. Marth, A recessive deletion in the GlcNAc-1-phosphotransferase gene results in peri-implantation embryonic lethality. Glycobiology, 1999.9(11):p.1263-71.
22. Haegel, H., et al., Lack of beta-catenin affects mouse development at gastrulation. Development, 1995.121(11):p.3529-37.
23. Dennis, J.W., M. Granovsky, and C.E. Warren, Glycoprotein glycosylation and cancer progression. Biochim Biophys Acta,1999.1473(1):p.21-34.
24. Nita-Lazar, M., et al., Overexpression of DPAGT1 leads to aberrant N-glycosylation of E-cadherin and cellular discohesion in oral cancer. Cancer Res,2009.69(14):p.5673-80.
25. Pyagay, P., et al., Collagen triple helix repeat containing 1, a novel secreted protein in injured and diseased arteries, inhibits collagen expression and promotes cell migration. Circ Res,2005.96(2):p.261-8.
26. Yamamoto, S., et al., Cthrd selectively activates the planar cell polarity pathway of Wnt signaling by stabilizing the Wnt-receptor complex. Dev Cell,2008.15(1):p.23-36.
27. Turashvili, G., et al., Novel markers for differentiation of lobular and ductal invasive breast carcinomas by laser microdissection and microarray analysis. BMC Cancer,2007. 7:p.55.
28. Tang, L, et al., Aberrant expression of collagen triple helix repeat containing 1 in human solid cancers. Clin Cancer Res,2006.12(12):p.3716-22.
29. Park, E.H., et al., Collagen triple helix repeat containing-1 promotes pancreatic cancer progression by regulating migration and adhesion of tumor cells. Carcinogenesis.34(3): p.694-702.
30. Goto, M., et al., Rapl stabilizes beta-catenin and enhances beta-catenin-dependent transcription and invasion in squamous cell carcinoma of the head and neck. Clin Cancer Res.16(1):p.65-76.
31. Nita-Lazar, M., et al., Hypoglycosylated E-cadherin promotes the assembly of tight junctions through the recruitment of PP2A to adherens junctions. Exp Cell Res.316(11):p. 1871-84.
32. Parri, M. and P. Chiarugi, Rac and Rho GTPases in cancer cell motility control. Cell Commun Signal.8:p.23.
33. Polakis, P., Wnt signaling and cancer. Genes Dev,2000.14(15):p.1837-51.
34. Lau, K.S., et al., Complex N-glycan number and degree of branching cooperate to regulate cell proliferation and differentiation. Cell,2007.129(1):p.123-34.
35. Mendelsohn, R., et al., Complex N-glycan and metabolic control in tumor cells. Cancer Res,2007.67(20):p.9771-80.
36. Katoh, M., WNT/PCP signaling pathway and human cancer (review). Oncol Rep,2005. 14(6):p.1583-8.
37. Luga, V., et al., Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration. Cell.151(7):p.1542-56.
38. Dennis, J.W., I.R. Nabi, and M. Demetriou, Metabolism, cell surface organization, and disease. Cell,2009.139(7):p.1229-41.
39. Wang, P., et al., CTHRC1 is upregulated by promoter demethylation and transforming growth factor-betal and may be associated with metastasis in human gastric cancer. Cancer Sci.103(7):p.1327-33.
40. Watanabe, S., et al., A novel glycosylation signal regulates transforming growth factor beta receptors as evidenced by endo-beta-galactosidase C expression in rodent cells. Glycobiology.21(4):p.482-92.
41. Guturi, K.K., et al., Mechanism of beta-catenin-mediated transcriptional regulation of epidermal growth factor receptor expression in glycogen synthase kinase 3 beta-inactivated prostate cancer cells. J Biol Chem.287(22):p.18287-96.
42. Contessa, J.N., et al., Inhibition ofN-linked glycosylation disrupts receptor tyrosine kinase signaling in tumor cells. Cancer Res,2008.68(10):p.3803-9.
43. Schlessinger, J., Cell signaling by receptor tyrosine kinases. Cell,2000.103(2):p.211-25.
44. Masiakowski, P. and R.D. Carroll, A novel family of cell surface receptors with tyrosine kinase-like domain. J Biol Chem,1992.267(36):p.26181-90.
45. Oldridge, M., et al., Dominant mutations in ROR2, encoding an orphan receptor tyrosine kinase, cause brachydactyly type B. Nat Genet,2000.24(3):p.275-8.
46. Patton, M.A. and A.R. Afzal, Robinowsyndrome. J Med Genet,2002.39(5):p.305-10.
47. Nusse, R. and H.E. Varmus, Wnt genes. Cell,1992.69(7):p.1073-87.
48. Oishi, I., et al., The receptor tyrosine kinase Ror2 is involved in non-canonical Wnt5a/JNK signalling pathway. Genes Cells,2003.8(7):p.645-54.
49. Katoh, M., Comparative genomics on ROR1 and ROR2 orthologs. Oncol Rep,2005.14(5): p.1381-4.
50. Nishita, M., et al., Filopodia formation mediated by receptor tyrosine kinase Ror2 is required for Wnt5a-induced cell migration. J Cell Biol,2006.175(4):p.555-62.
51. Huang, C.L., et al., Wnt5a expression is associated with the tumor proliferation and the stromal vascular endothelial growth factor--an expression in non-small-cell lung cancer. J Clin Oncol,2005.23(34):p.8765-73.
52. Kurayoshi, M., et al., Expression of Wnt-5α is correlated with aggressiveness of gastric cancer by stimulating cell migration and invasion. Cancer Res, 2006.66(21):p.10439-48.
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