Upregulation of the Wnt Co-Receptor LRP6 Promotes Hepatocarcinogenesis and Enhances Cell Invasion

Background: Activation of the Wnt/b-catenin signaling pathway plays a crucial role in hepatocellular carcinoma (HCC). Lowdensity lipoprotein (LDL) receptor-related protein-6 (LRP6) is one of the co-receptors of the Wnt/b-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 b-catenin. Moreover, overexpression of the full-length and constitutively active LRP6, respectively, activated the WNT/b-catenin signaling pathway, as shown by the TCF/b-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

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

<p>(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.</p

Clinicopathologic correlation of LRP6 transcript in HCC patients.

<p>IHC, immunohistochemical;</p>*<p>Nuclear stain or cytoplasmic overexpression.</p

Constitutively active LRP6 enhanced tumor cell growth <i>in vivo</i>.

<p>(A) <i>In vivo</i> 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.</p

Overexpression of LRP6 in HCC cell lines and human HCCs.

<p>(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.</p

Constitutively active LRP6 promoted both cell migration and invasion.

<p>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<0.001 for both). (B) Overexpression of myc-CA LRP6 promoted cell invasion in BEL-7402 cells. The numbers of invaded cells in CA LRP6 Clones #3 and #8 were significantly higher than the vector control cells (P<0.001 and  = 0.009, respectively).</p

Constitutively active LRP6 enhanced cell proliferation <i>in vitro.</i>

<p>(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, <0.001,  = 0.002 and 0.005, respectively).</p

Mitochondrial diseases in Hong Kong: prevalence, clinical characteristics and genetic landscape

Abstract Objective To determine the prevalence of mitochondrial diseases (MD) in Hong Kong (HK) and to evaluate the clinical characteristics and genetic landscape of MD patients in the region. Methods This study retrospectively reviewed the phenotypic and molecular characteristics of MD patients from participating public hospitals in HK between January 1985 to October 2020. Molecularly and/or enzymatically confirmed MD cases of any age were recruited via the Clinical Analysis and Reporting System (CDARS) using relevant keywords and/or International Classification of Disease (ICD) codes under the HK Hospital Authority or through the personal recollection of treating clinicians among the investigators. Results A total of 119 MD patients were recruited and analyzed in the study. The point prevalence of MD in HK was 1.02 in 100,000 people (95% confidence interval 0.81–1.28 in 100,000). 110 patients had molecularly proven MD and the other nine were diagnosed by OXPHOS enzymology analysis or mitochondrial DNA depletion analysis with unknown molecular basis. Pathogenic variants in the mitochondrial genome (72 patients) were more prevalent than those in the nuclear genome (38 patients) in our cohort. The most commonly involved organ system at disease onset was the neurological system, in which developmental delay, seizures or epilepsy, and stroke-like episodes were the most frequently reported presentations. The mortality rate in our cohort was 37%. Conclusion This study is a territory-wide overview of the clinical and genetic characteristics of MD patients in a Chinese population, providing the first available prevalence rate of MD in Hong Kong. The findings of this study aim to facilitate future in-depth evaluation of MD and lay the foundation to establish a local MD registry