We sought to identify a secreted biomarker for -catenin activation commonly
We sought to identify a secreted biomarker for -catenin activation commonly seen in hepatocellular carcinoma (HCC). developing HCC with Ctnnb1 mutations showed significantly higher serum LECT2 levels. However patients with mutations showed LECT2 levels of 54.2822.32 ng/mL (Mean SD; n?=?8) that were insignificantly different from patients with non-neoplastic chronic liver disease (32.821.1 ng/mL; n?=?15) or healthy volunteers (33.27.2 ng/mL; n?=?11). Intriguingly, patients without -catenin mutations showed significantly higher serum LECT2 levels (54.26 22.25 ng/mL; n?=?46). While -catenin activation was evident in a subset of non-mutant -catenin HCC group with high expression, serum LECT2 was unequivocally comparable between -catenin-active and -normal group. Further analysis showed that LECT2 levels greater than 50 ng/ml diagnosed HCC in patients irrespective of -catenin mutations with specificity of 96.1% and positive predictive value of 97.0%. Thus, is usually regulated by -catenin in HCC in both mice and men, but serum LECT2 reflects -catenin activity only in mice. Serum LECT2 could be a potential biomarker of HCC in patients. Introduction Primary liver cancer, which is usually predominantly hepatocellular carcinoma (HCC), is the sixth most common cancer worldwide and the third most frequent cause of malignancy mortality . -Catenin gene (mutations are one of the major oncogenic gene alterations in HCC seen in 10C40%, while mutations affecting Axin1 are seen in around 10% of all HCCs . mutations are observed in exon-3 that contain phosphorylation sites essential for -catenin degradation leading to its stabilization and enhanced expression of target genes such as (and expression to be decreased in hepatocyte-specific -catenin knockout livers . Next, using an analysis in human HCC cells, we demonstrate that indeed expression and its protein levels reflect -catenin activity and hence hypothesize that it may be a good biomarker for HCC with -catenin activation. The power of LECT2 as a biomarker was validated first in a mouse liver tumor model where exon-3 mutation in -catenin gene and ensuing -catenin activation is usually implicated in HCC pathogenesis , . However, in HCC patients, serum LECT2 levels were not significantly different in ABT-888 manufacturer tumor with or without mutations when compared to patients with chronic liver disease or healthy volunteers. Furthermore, despite -catenin activation observed in an additional subset of non-mutated HCC, which showed high expression, serum LECT2 levels were not predictive for active -catenin signaling in the tumor. Interestingly though, irrespective of molecular aberrations, LECT2 levels were significantly higher in all HCC patients versus patients with cirrhosis or healthy controls. In fact, serum LECT2 50 ng/ml indicated HCC with high specificity and positive predictive value. Materials and Methods Cell lines and treatment Human HCC cell lines, Hep3B, HepG2, SNU449, SNU398, and HuH7, were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in Eagle’s SHC1 minimal essential medium (EMEM) or RPMI supplemented with 10% vol/vol FBS at 37C in a humidified 5% carbon dioxide atmosphere. For siRNA knockdown experiment, the cells were transfected using Lipofectamine 2000 (Life Technologies, Grand Island, NY) with -catenin (forward, and reverse, and reverse, was used as an internal ABT-888 manufacturer control. In human analyses, total ABT-888 manufacturer RNA was extracted from frozen tissues and qRT-PCR analysis performed as described previously . Enzyme-linked immunosorbent assay (ELISA) Serum LECT2 levels were measured by either human or mouse LECT2 ELISA kit (Medical & Biological Laboratories (MBL) Co, Ltd, Niigata, Japan) according to the manufacture’s protocol. Clinical tissue and serum samples All tissues and materials used in this study were obtained under an approved Institutional Review Board protocol at the University of Pittsburgh and Kumamoto University. Specifically, frozen tissues and serum samples were obtained from HCC patients in the Department of Surgery, University of Pittsburgh (Pittsburgh, PA; n?=?20) with a written informed consent approved by the University of Pittsburgh Institutional Review Board. Frozen tissues and serum samples from HCC patients were also collected by the Department of Gastroenterological Surgery, Kumamoto University (Kumamoto, Japan; n?=?45), with a written informed consent approved by the Institutional Review Board at ABT-888 manufacturer the Kumamoto University. Additional serum samples were obtained from patients with chronic liver disease (n?=?15) in the Department of Medicine that did not have any evidence.