Upregulation of the Wnt co-receptor LRP6 promotes hepatocarcinogenesis and enhances cell invasion

Abstract

Background: Activation of the Wnt/β-catenin signaling pathway plays a crucial role in hepatocellular carcinoma (HCC). Low-density lipoprotein (LDL) receptor-related protein-6 (LRP6) is one of the co-receptors of the Wnt/β-catenin pathway and forms a signaling complex with Wnt ligand and Frizzled receptor to activate downstream signaling. However, the role of LRP6 in hepatocarcinogenesis is unclear. In this study, we examined its expression and roles in human HCC.

Methodology/principal findings: Using real-time quantitative RT-PCR, we found that LRP6 was frequently (45%) overexpressed in human HCCs (P = 0.003). In vitro studies showed that ectopic expression of LRP6 increased the protein level of β-catenin. Moreover, overexpression of the full-length and constitutively active LRP6, respectively, activated the WNT/β-catenin signaling pathway, as shown by the TCF/β-catenin reporter assay. With regard to the effects of LRP6 overexpression in HCC cells, stable overexpression of the constitutively active LRP6 in BEL-7402 HCC cells enhanced cell proliferation, cell migration, and invasion in vitro as well as tumorigenicity in nude mice.

Conclusions/significance: Our findings indicate that overexpression of LRP6 contributes to the hyperactivation of the Wnt/β-catenin signaling pathway in human HCCs and suggest it may play a role in hepatocarcinogenesis.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Overexpression of LRP6 in HCC cell lines and human HCCs.

(A) Quantitative real-time PCR and Western blotting. Both transcripts and protein of LRP6 were expressed in all seven HCC cell lines. (B) In human HCCs, the transcript level of LRP6 was frequently (45%) up-regulated as compared with their corresponding non-tumorous livers (P = 0.003). (C) Western blot analysis. LRP6 protein level was determined in 28 HCC pairs and was found to be overexpressed in 9 (32%) cases. The 9 cases with LRP6 protein overexpression in the tumors are shown. (D) Immunohistochemical analysis. LRP6 protein was found to be overexpressed in the HCC as compared with the corresponding non-tumorous liver. High power magnification of the tumor showed strong positive staining of LRP6 protein in the cytoplasm (white arrows) and also the membranes (black short arrows) of the tumor cells.

Figure 2. Ectopic expression of LRP6 activated the Wnt/β-catenin pathway.

(A) Schematic diagram showing the structural domains of Myc-tagged full length form of LRP6 (myc-FL LRP6) and the constitutively active form (myc-CA LRP6). (B) Western blotting. Myc-FL LRP6 and myc-CA LRP6 were transiently overexpressed in BEL-7402 HCC cell line and human embryonic kidney cell HEK293T cells. The protein level of β-catenin was increased in both BEL-7402 HCC cell line and HEK293T cells. (C) TOP/FOP luciferase reporter assay. Expression of myc-FL LRP6 led to an activation of TCF/β-catenin reporter up to ∼40-fold (without Wnt3a treatment) and ∼120-fold (with Wnt3a treatment), respectively, as compared with the vector control. The fold of induction in myc-CA LRP6 cells reached ∼150-fold even without Wnt3a treatment. (D) Similar results were also observed in HEK293T cells.

Figure 3. Constitutively active LRP6 enhanced cell proliferation in vitro.

(A) LRP6 expressing BEL-7402 stable cells were established using myc-CA LRP6 construct. The protein level of β-catenin was upregulated as compared with the parental BEL-7402 cells. (B) Cell proliferation assay. The numbers of cells of CA LRP6 Clones #1, #3, #8 and #11 on Day 7 were significantly higher than the vector control BEL-7402 cells (P = 0.032,

Figure 4. Constitutively active LRP6 promoted both cell migration and invasion.

Cell migration and invasion assays were performed using LRP6-stably expressing BEL-7402 cells. (A) Overexpression of myc-CA LRP6 enhanced cell migration in BEL-7402 cells. The numbers of migrated cells in CA LRP6 Clones #3 and #8 were significantly higher than the vector control (P

Figure 5. Constitutively active LRP6 enhanced tumor cell growth in vivo.

(A) In vivo nude mice injection assay was performed by injecting myc-CA LRP6 stably expressing and vector control BEL-7402 cells subcutaneously into the flank of the nude mice. (B) The tumor sizes of two myc-CA LRP6 stably expressing tumors were significantly higher as compared with the tumor of vector control (P<0.001). (C) The tumor weight of Clone #3 was higher in myc-CA LRP6 than the control vector (P = 0.002). Another clone (Clone #8) showed a trend of higher tumor weight but the difference did not reach statistical significance (P = 0.165). Error bar = SEM.