Role of leukocyte cell-derived chemotaxin 2 as a biomarker in hepatocellular carcinoma

We sought to identify a secreted biomarker for β-catenin activation commonly seen in hepatocellular carcinoma (HCC). By examination of our previously published genearray of hepatocyte-specific β-catenin knockout (KO) livers, we identified secreted factors whose expression may be β-catenin-dependent. We verified expression and secretion of the leading factor in HCC cells transfected with mutated (Hep3BS33Y)-β- catenin. Serum levels of biomarker were next investigated in a mouse model of HCC with β-catenin gene (Ctnnb1) mutations and eventually in HCC patients. Leukocyte cell-derived chemotaxin-2 (LECT2) expression was decreased in KO livers. Hep3BS33Y expressed and secreted more LECT2 in media as compared to Hep3BWT. Mice developing HCC with Ctnnb1 mutations showed significantly higher serum LECT2 levels. However patients with CTNNB1 mutations showed LECT2 levels of 54.28±22.32 ng/mL (Mean ± SD; n = 8) that were insignificantly different from patients with non-neoplastic chronic liver disease (32.8±21.1 ng/mL; n = 15) or healthy volunteers (33.2±7.2 ng/mL; n = 11). Intriguingly, patients without β-catenin mutations showed significantly higher serum LECT2 levels (54.26 ± 22.25 ng/mL; n = 46). While β-catenin activation was evident in a subset of non-mutant β-catenin HCC group with high LECT2 expression, serum LECT2 was unequivocally similar between β-catenin-active and -normal group. Further analysis showed that LECT2 levels greater than 50 ng/ml diagnosed HCC in patients irrespective of β-catenin mutations with specificity of 96.1% and positive predictive value of 97.0%. Thus, LECT2 is regulated by β-catenin in HCC in both mice and men, but serum LECT2 reflects β-catenin activity only in mice. Serum LECT2 could be a potential biomarker of HCC in patients. © 2014 Okabe et al

β-Catenin Loss in Hepatocytes Promotes Hepatocellular Cancer after Diethylnitrosamine and Phenobarbital Administration to Mice

Hepatocellular Carcinoma (HCC) is the fifth most common cancer worldwide. β-Catenin, the central orchestrator of the canonical Wnt pathway and a known oncogene is paramount in HCC pathogenesis. Administration of phenobarbital (PB) containing water (0.05% w/v) as tumor promoter following initial injected intraperitoneal (IP) diethylnitrosamine (DEN) injection (5 µg/gm body weight) as a tumor inducer is commonly used model to study HCC in mice. Herein, nine fifteen-day male β-catenin knockout mice (KO) and fifteen wild-type littermate controls (WT) underwent DEN/PB treatment and were examined for hepatic tumorigenesis at eight months. Paradoxically, a significantly higher tumor burden was observed in KO (p<0.05). Tumors in KO were β-catenin and glutamine synthetase negative and HGF/Met, EGFR & IGFR signaling was unremarkable. A significant increase in PDGFRα and its ligand PDGF-CC leading to increased phosphotyrosine-720-PDGFRα was observed in tumor-bearing KO mice (p<0.05). Simultaneously, these livers displayed increased cell death, stellate cell activation, hepatic fibrosis and cell proliferation. Further, PDGF-CC significantly induced hepatoma cell proliferation especially following β-catenin suppression. Our studies also demonstrate that the utilized DEN/PB protocol in the WT C57BL/6 mice did not select for β-catenin gene mutations during hepatocarcinogenesis. Thus, DEN/PB enhanced HCC in mice lacking β-catenin in the liver may be due to their ineptness at regulating cell survival, leading to enhanced fibrosis and regeneration through PDGFRα activation. β-Catenin downregulation also made hepatoma cells more sensitive to receptor tyrosine kinases and thus may be exploited for therapeutics

Defective HNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis

Alcoholic hepatitis (AH) is a life-threatening condition characterized by profound hepatocellular dysfunction for which targeted treatments are urgently needed. Identification of molecular drivers is hampered by the lack of suitable animal models. By performing RNA sequencing in livers from patients with different phenotypes of alcohol-related liver disease (ALD), we show that development of AH is characterized by defective activity of liver-enriched transcription factors (LETFs). TGFβ1 is a key upstream transcriptome regulator in AH and induces the use of HNF4α P2 promoter in hepatocytes, which results in defective metabolic and synthetic functions. Gene polymorphisms in LETFs including HNF4α are not associated with the development of AH. In contrast, epigenetic studies show that AH livers have profound changes in DNA methylation state and chromatin remodeling, affecting HNF4α-dependent gene expression. We conclude that targeting TGFβ1 and epigenetic drivers that modulate HNF4α-dependent gene expression could be beneficial to improve hepatocellular function in patients with AH

siRNA-Mediated β-Catenin Knockdown in Human Hepatoma Cells Results in Decreased Growth and Survival1

β-Catenin, the chief oncogenic component of the canonical Wnt pathway, is known to be involved in a variety of cancers, including hepatocellular carcinoma (HCC). Although the mechanism of β-catenin activation in HCC is multifactorial, it is indisputably implicated at various stages of hepatocarcinogenesis, making it an attractive therapeutic target. Here we investigate the effect of small interfering RNA-mediated β-catenin knockdown on the growth and survival of human hepatoma cell lines with (HepG2) and without (Hep3B) β-catenin mutations. Transfection of HepG2 and Hep3B cells with human β-catenin (CTNNB1) small interfering RNA resulted in a significant β-catenin decrease, as confirmed by Western blot analyses and immunofluorescence, also leading to decreased expression of known target genes such as cyclin D1 and glutamine synthetase. The decrease in β-catenin activity was confirmed by TOPflash reporter luciferase assay. The functional impact of diminished β-catenin was exhibited as temporal decrease in tumor cell viability by the MTT assay. A concomitant decrease in tumor cell proliferation was also evident with [3H]thymidine incorporation and verified with soft agar assays. Thus, β-catenin is essential for the survival and growth of hepatoma cells independent of mutations in the β-catenin gene and provide a proof of principle for the significance of the therapeutic inhibition of β-catenin in HCC

A Novel Transgenic Mouse Model Implicates <i>Sirt2</i> as a Promoter of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths globally. Incidence rates are steadily increasing, creating an unmet need for new therapeutic options. Recently, the inhibition of sirtuin-2 (Sirt2) was proposed as a potential treatment for HCC, despite contradictory findings of its role as both a tumor promoter and suppressor in vitro. Sirt2 functions as a lysine deacetylase enzyme. However, little is known about its biological influence, despite its implication in several age-related diseases. This study evaluated Sirt2’s role in HCC in vivo using an inducible c-MYC transgene in Sirt2+/+ and Sirt2−/− mice. Sirt2−/− HCC mice had smaller, less proliferative, and more differentiated liver tumors, suggesting that Sirt2 functions as a tumor promoter in this context. Furthermore, Sirt2−/− HCCs had significantly less c-MYC oncoprotein and reduction in c-MYC nuclear localization. The RNA-seq showed that only three genes were significantly dysregulated due to loss of Sirt2, suggesting the underlying mechanism is due to Sirt2-mediated changes in the acetylome, and that the therapeutic inhibition of Sirt2 would not perturb the oncogenic transcriptome. The findings of this study suggest that Sirt2 inhibition could be a promising molecular target for slowing HCC growth

Aberrant Wnt/β-Catenin Signaling in Pancreatic Adenocarcinoma

Wnt/β-catenin signaling plays an important role in normal development. However, its aberrant activation is associated with several cancers. The aim of this study is to examine the Wnt/β-catenin pathway in patients with advanced pancreatic adenocarcinoma (n = 31). Paraffin sections from tumors (n = 16) and normal pancreata (n = 3) were used to determine the localization of β-catenin. An additional 15 frozen tumors, adjacent normal pancreata (n = 5), or normal pancreata (n = 4) were utilized for protein isolation. Tumors were also examined for mutations in exon 3 of the CTNNB1 gene. More than 65% of the tumors showed an increase in total β-catenin, consistent with its enhanced membranous, cytoplasmic, and nuclear localization, but only two showed mutations in CTNNB1. The majority of the remaining tumors demonstrated concurrent increases in Wnt-1 and frizzled-2 (positive regulators) and a decrease in Ser45/Thr41-phospho-β-catenin. Electrophoretic mobility shift assay demonstrated β-catenin-T-cell factor binding in tumors only. Adenomatous polyposis coli and axin, which are both negative regulators, remained unchanged. Unexpectedly, total glycogen synthase kinase-3β protein was elevated in these tumors. Elevated levels of E-cadherin were also observed, although E-cadherin-β-catenin association in tumors remained unaffected. Thus, Wnt/β-catenin activation was observed in 65% of pancreatic adenocarcinomas, independently of β-catenin gene mutations in most tumors

Tumor-bearing KO mice display active PDGFRα signaling when compared to WT.

<p>A. Representative western blots from 8 KO and 9 WT show a dramatic increase in total levels of PDGFRα and modest increase in PDGFRβ in the KO. Actin loading verifies equal loading. B. Average integrated optical density (IOD) obtained from scanned autoradiographs shown in Fig. 5A revealed significantly higher PDGFRα levels in KO (p = 0.002). C. Western blot from representative samples shows a dramatic increase in PDGF-CC, a ligand for PDGFRα in KO whereas PDGF-AA and BB remained unremarkable between the two groups. D. Bar graph depicts a significant increase in Tyr720-PDGFRα in KO as compared to WT (p<0.05). E. Insignificant differences were evident in Tyr-849-PDGFRα between the WT and KO.</p

β-Catenin suppression engenders mitogenicity to PDGF-CC in hepatoma cell culture.

<p>PDGF-CC (10 ng/ml) treatment does not increase DNA synthesis as compared to HCl treatment of Hep3B cells. β-Catenin knockdown led to significant decrease in thymidine incorporation as compared to control siRNA (p<0.0005). However PDGF-CC treatment led to a significant increase in thymidine incorporation in β-catenin-suppressed as compared to control siRNA-transfected cells (p<0.005).</p