www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 65), pp: 109632-109645 Research Paper LRP6 promotes invasion and metastasis of colorectal cancer through cytoskeleton dynamics Qian Yao1,*, Yu An1,*, Wei Hou1, Ya-Nan Cao1, Meng-Fei Yao1, Ning-Ning Ma1, Lin Hou1, Hong Zhang2, Hai-Jing Liu1 and Bo Zhang1 1Department of Pathology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China 2Department of Pathology, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, China *These authors have contributed equally to this work Correspondence to: Hai-Jing Liu, email: [email protected] Bo Zhang, email: [email protected] Keywords: colorectal cancer; Wnt signaling; LRP6; metastasis; cytoskeleton Received: May 09, 2017 Accepted: October 28, 2017 Published: November 30, 2017 Copyright: Yao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Low density lipoprotein (LDL) receptor-related protein-6 (LRP6) is an important co-receptor of Wnt pathway, which plays a predominant role in development and progression of colorectal cancer. Recently, dysregulation of LRP6 has proved to be involved in the progression of cancers, but its biological role and clinical significance in colorectal cancer remain unclear. In present study, we revealed that phosphorylation of LRP6 was aberrantly upregulated in colorectal carcinoma correlating with TNM or Dukes staging and worse prognosis. In addition, phosphorylated LRP6 was positively correlated with nuclear accumulation of β-catenin. Overexpression or activation of LRP6 could activate Wnt signaling and promote tumor cell migration in vitro. The activation of LRP6 could induce microtubule dynamics and actin remodeling, probably through regulation of microtubule-associated protein 1B (MAP1B), microtubule actin cross-linking factor 1 (MACF1) and Rho GTPase--RhoA and Rac1. The investigation suggests that LRP6 may be a potential prognostic marker and therapeutic target in the progression of colorectal cancers. INTRODUCTION and progression of colorectal cancers in past decades. Especially, nuclear translocation of β-catenin, a hallmark Colorectal cancer is the third most common of Wnt signaling activation, has proved to be a critical cancer and fourth leading cause of cancer-related deaths role in many aspects of cellular biological activities, such worldwide [1, 2] and there is potential for distant as growth, differentiation, EMT, as well as cytoskeleton metastasis following surgical excision of the primary remodeling [6, 7]. Nevertheless, Wnt signaling could be lesion [3]. Despite the introduction of new treatments, further enhanced by its secreted ligands even in APC- the 5-year survival rate for metastatic colorectal cancer mutated situation, suggesting that that hyperactivation of remains below 10% [4]. Hence, the underlying mechanism Wnt pathway may be contributed by other alterations in of metastasis, predictive biomarkers and therapeutic targets colorectal cancers [8-11]. for colorectal cancer are under extensive exploration. Loss Low-density lipoprotein (LDL) receptor-related of function mutation in APC (adenomatous polyposis coli) protein-6 (LRP6) is one of the co-receptors of Wnt tumor suppressor gene has been detected in about 85% pathway, which form a signalosome with Wnt ligand of sporadic colorectal cancers, which leads to activation and Frizzled receptor to activate downstream cascade. of Wnt signaling pathway [5]. In fact, activation of Wnt The phosphorylation of PPSPXS motifs of LRP6 could signaling has been clarified to be involved in initiation recruit other Wnt signaling components Disheveled (Dvl), www.impactjournals.com/oncotarget 109632 Oncotarget Axin and GSK3β to signalosome to amplify and stabilize Positive p-LRP6 was found in 92 of 115 cases (80.0%) of its phosphorylation, which can directly inhibit GSK3β advanced carcinoma (T3 and T4), while early carcinomas activity leading to multiple downstream cascades [12- (T1 and T2) showed positive staining in just 35.3% (24/68) 14]. Therefore, LRP6 represents an important regulatory (p<0.001, χ2=36.801). 76.6% (59/77) of cases with lymph node in transducing Wnt stimulation. Recent reports reveal node metastasis was positive p-LRP6, but only 48.8% that LRP6 contributed to tumor progression in colorectal (39/80) (p<0.001, χ2=12.995) without metastasis showed cancer and some other cancers [15-18]. However, the p-LRP6 expression. In addition, there was a positive mechanism about LRP6 in colorectal cancers is seldom linear correlation between p-LRP6 expression and TNM investigated. or Dukes staging (p<0.001). Kaplan-Meier single-factor In this study, we demonstrated that phosphorylated analysis and log-rank test demonstrated a statistically LRP6 was markedly up-regulate in colorectal carcinoma significant decrease in disease-free survival (DFS) in and indicated poor prognosis. The activation of LRP6 patients with positive staining of p-LRP6 (p=0.003) could enhance Wnt signaling and promote cell migration. (Figure 1C). However, evaluation of patients’ overall LRP6 governed the cytoskeleton stability and remodeling survival (OS) displayed a borderline significance between through microtubule-related protein: microtubule- positive and negative expression of p-LRP6 (p=0.058) associated protein 1B (MAP1B) and microtubule actin (Figure 1D). cross-linking factor 1 (MACF1) as well as Rho GTPases. Taken together, these data proved that the p-LRP6 was involved in Wnt signaling activation and correlated RESULTS with a more aggressive phenotype with higher metastatic potential and worse prognosis. Expression of Phosphorylated LRP6 is correlated with aggressive clinical behavior in Activation of LRP6 increases Wnt/β-catenin colorectal carcinoma signaling in colorectal cancer cells Immuno-staining of both LRP6 and phosphorylated To evaluate the role of LRP6 in activation of Wnt LRP6 (p-LRP6) were performed in colorectal carcinoma signaling, the plasmids LRP6-WT (wild-type LRP6), tissue. The expression of LRP6 was negative in most cases LRP6-DA (dominant active, reserving the intracellular (91.2%, 62/68) with few positive (8.8%, 6/68). However, domain (ICD) of LRP6, residues 1126-1613) and LRP6- the immunostaining of p-LRP6 was in a diffuse pattern DN (dominant negative, reserving the extracellular domain in cytoplasm with no apparent membranous expression. of LRP6, residues 1-1114) were constructed (Figure 2A). Comparing to non-neoplastic glandular cells (4.8%, With transfection of Lovo and HCT116 cells with above 1/21) and adenoma tissue (5.3%, 2/38), the expression plasmids, β-catenin immunofluorescence staining and of p-LRP6 was much more intense in carcinoma with luciferase assay were carried out. The results showed that higher positive level (62.3%, 114/183), which displayed nuclear staining of β-catenin was obviously enhanced the significant statistical difference (p<0.001) (Figure 1A, in LRP6-WT and LRP6-DA group with attenuated 1B). It was noteworthy that positive p-LRP6 was usually membrane expression comparing to control or LRP6-DN observed at the invasive front tumor zone in infiltrating (p<0.001) (Figure 2B, 2C). Expectedly, overexpression of nests of cancers (Figure 1A). LRP6-WT and LRP6-DA led to an activation of TCF/β- Wnt signaling plays a pivotal role in colorectal catenin reporter up to about 2-fold, as compared to the carcinogenesis and the nuclear accumulation of β-catenin vector control or LRP6-DN (p<0.001) (Figure 2D). The is widely accepted as the hallmark of Wnt signaling data demonstrated that activation of LRP6 could increase activation [19, 20]. Accordingly, the expressions of Wnt signaling activity in colorectal cancer cells. β-catenin was immunohistochemically evaluated. Positive nuclear expression of β-catenin was 74.3% (136/183) in Activation of LRP6 promotes migration of carcinoma, which was higher than adenoma tissue (55.3%, colorectal cancer cells 21/38) (p<0.05) (Supplementary Figure 1). At the same time, the cytoplasm expression of β-catenin was also found To determine whether up-regulation of LRP6 would more frequently in carcinoma cells (79.8%, 146/183) than influence cancer cell migration and invasion, Transwell adenoma (57.9%, 22/38) (p<0.05) (Supplementary Figure assays with or without Matrigel were performed. In 1A, 1B). Besides, a positive correlation between p-LRP6 migration assay, Lovo and HCT116 cells transfected and nuclear expression of β-catenin was established with LRP6-WT or LRP6-DA plasmids demonstrated (Supplementary Table 1). much higher motility potential than vector control group The correlations between p-LRP6 expression and or LRP6-DN group (p<0.001) (Figure 2E, 2F). As for clinicopathologic parameters such as age, gender, tumor invasion assay, we failed to found the statistical difference size, depth of invasion, lymph node metastasis, TNM and between each group, however, the results showed the Dukes staging were analyzed (Supplementary Table 2). tendency of promoted invasive potential in LRP6-DA and www.impactjournals.com/oncotarget 109633 Oncotarget LRP6-WT transfected cells (p=0.087) (Supplementary invasion, which arise the question about how LRP6 Figure 2). manage to control such aggressive behavior. It was widely Above evidence suggested the possible role of LRP6 agreed that migratory cancer cells undergo