Upregulation of the WNT co-receptor LRP6 in hepatocellular carcinoma

Abstract

Activation of WNT/β-catenin signaling pathway is closely related to cancer formation including hepatocellular carcinoma (HCC), which is one of the commonest fatal cancers worldwide1. Low-density lipoprotein (LDL) receptor-related protein-5/6 (LRP5/6) are co-receptors for WNT ligands2 that have been studied in embryonic development but rarely in cancers3. We hypothesize that LRP6 is a putative oncogene in the WNT signaling pathway where its overexpression may play an important role in hepatocarcinogenesis. In this study, with immunofluorescence staining of LRP6-EGFP-transfected BEL7402, we observed that LRP6 displayed subcellular localization in the membrane as well as cytoplasm. Theexpressions of Wnts, their receptors frizzled (Fz) and LDL receptorlike proteins (LRP) and other Wnt sigaling pathway-related genes in human HCC samples were screened by quantitative RT-PCR using Taqman Low Density Array (LDA). We observed frequent upregulation (50%) of LRP6 expression at mRNA and protein levels in human HCCs, as compared with their corresponding non-tumorous livers. LRP6 protein expression level also positively correlated with that of beta-catenin in human HCCs. LRP6-EGFP-overexpressing and shRNA LRP6 knockdown stable clones were established for functional assays. The effects of LRP6 on Wnt/β-catenin signaling and cell proliferation were assessed in HCC cell lines. An increased protein expression level of β-catenin was found in LRP6-overexpressing stable clones. Interestingly, we observed that degradation of LRP6 might involve the proteosome/ubiquitination pathway through its D-box motif. Treatment with proteosome inhibitor MG132 in BEL7402 HCC cells and 293T cells resulted in an increased LRP6 protein expression. In addition, in vivo ubiquitination assay showed that LRP-Ub conjugation was enhanced by MG132 treatment. The results suggest that LRP6 is degraded via the proteasome/ubiquitination pathway through its D-box motif. To conclude, LRP6 is overexpressed in HCC and correlates positively with beta-catenin levels. Its degradation likely involves proteosome/ubiquitination pathway. Our data suggest that LRP6 may be a putative oncogenic gene and may have important role in hepatocarcinogenesis.