Serum LECT2 levels in mice with β-catenin gene mutated HCC.

<p>A. Representative picture of β-catenin (left panel) and GS (right panel) immunohistochemistry of liver of a tumor bearing mouse at 8 months after DEN/PB treatment. Magnification, 100×. B. Using frozen tissue from a representative tumor, β-catenin gene exon-3 mutation affecting codon 33 (red box) was confirmed by direct sequencing. C. Serum LECT2 levels were significantly (*) increased in tumor bearing versus non-tumor bearing DEN/PB treated mice as analyzed by ELISA. (* <i>p</i><0.01). D. Representative pictures of frozen sections from which tumors (T1-T3) were scraped for direct sequencing. E. Sequence analysis from three tumor lesions (T1-T3) show S33Y-β-catenin gene mutations in codon 33 (red boxes) by direct sequencing. F. <i>Glutamine Synthetase (Glul)</i> and <i>Lect2</i> expression in three tumor lesions (T1-T3) were assessed by qRT-PCR. Gene expression of background liver tissues surrounding tumor are shown as N.</p

Clinical characteristics of control patients with cirrhosis but no HCC.

†<p>HCV, hepatitis type C virus; ETOH, alcoholic; NASH, non-alcoholic steatohepatitis; AIH, autoimmune hepatitis.</p><p>*serum LECT2 level value was very high (103.9 ng/mL).</p

Candidate genes encoding secreted protein.

<p>Candidate genes encoding secreted protein.</p

Regulation of LECT2 expression by β-catenin.

<p>A. Strategy to identify biomarker for β-catenin activation. Microarray analysis was performed using liver tissue from hepatocyte-specific β-catenin knockout (KO) and wild-type (WT) mice, which identified 14 secreted targets. <i>Lect</i>2 expression was 117-fold lower in KO livers. B. β-Catenin expression in Hep3B cells and stable cell lines established with wild-type β-catenin (Hep3B WT)- or mutated β-catenin (Hep3B S33Y)-transfected cells. C. Representative Western blot shows increased LECT2 protein levels in Hep3B S33Y cells as compared to Hep3B WT. D. Hep3B S33Y cells transfected with either <i>β-catenin</i> or <i>control</i> siRNA showed decreased β-catenin and LECT2 protein levels in a representative Western blot. E. Increased LECT2 protein levels were observed in culture media collected from Hep3B S33Y cells as compared to Hep3B WT as analyzed by ELISA. Basal media was used as a negative control. F. Occupation of <i>Lect2</i> promoter by TCF4 especially in Hep3B S33Y cells was as assessed by ChIP. Albumin promoter is not regulated by β-catenin but by HNF1α, which is used as quality control for chromatin.</p

Clinical characteristics of patients with HCC.

<p>Clinical characteristics of patients with HCC.</p

The role of serum LECT2 level as a diagnostic biomarker in HCC.

<p>A. Serum LECT2 levels in all HCC patients as compared to patients with chronic liver fibrosis (CH/LC), and healthy volunteer (HV) as assessed by ELISA. (*<i>p</i><0.01). B. ROC analysis for the utility of LECT2 as a diagnostic marker of HCC with AUC = 0.82. C. Fisher's Exact test shows that based on the cut-off value of serum LECT2 level at 50 ng/mL, sensitivity, specificity, positive predictive value, negative predictive value for the diagnosis of HCC were 59.3%, 96.1%, 97.0%, and 53.2%, respectively.</p

No correlation of β-catenin mutations or β-catenin activation to serum LECT2 levels in patients.

<p>A. Serum LECT2 levels in patients with HCC with <i>CTNNB1</i> mutations, absent <i>CTNNB1</i> mutations, patients with chronic liver fibrosis (CH/LC), and healthy volunteer (HV) as assessed by ELISA. (*<i>p</i><0.05). B. No correlation observed between <i>LECT2</i> expression in tumor and serum levels of LECT2 in HCC patients (n = 28). C. No correlation observed between <i>LECT2</i> expression in β-catenin mutated tumors and serum levels of LECT2 in these HCC patients (n = 4). D. No correlation observed between <i>LECT2</i> expression in non-β-catenin mutated tumors and serum levels of LECT2 in these HCC patients (n = 24). E. Heat map shows expression of β-catenin target genes in β-catenin-mutated (MT) and non-mutated wild-type (WT) HCC patients (n = 28). Genes assessed included <i>AXIN2</i>, <i>REGUCALCIN</i>, <i>LECT2</i>, and <i>GLUL</i>. (+) indicates β-catenin activity as seen by increased expression of at least 2 target genes whereas (−) indicates absent β-catenin activation reflected by lack of target gene expression. F. Serum LECT2 levels showed insignificant difference in HCC patients that lacked β-catenin gene mutations but showed high expression of β-catenin target genes versus patients who have neither β-catenin gene mutations nor any increase in β-catenin target gene expression. G. β-Catenin target gene expression shown by qRT-PCR. Steel-Dwass test was performed to compare the values among three groups. *, <i>p</i><0.05. (+) indicates β-catenin activity as seen by increased expression of at least 2 target genes whereas (−) indicates lack of β-catenin activity due lack of target gene expression.</p

EZH2 Is Associated with Malignant Behavior in Pancreatic IPMN via p27^Kip1 Downregulation

<div><p>Background</p><p>The epigenetic mechanism of tumorigenesis in pancreatic intraductal papillary mucinous neoplasm (IPMN) remains largely unknown. The aim of this study is to examine the role of enhancer of zeste homologue 2 (EZH2) alteration in pancreatic IPMN progression.</p><p>Methods</p><p>Fifty-four surgically resected pancreatic IPMN specimens, including a total of 181 lesions (normal duct in 48, adenoma in 50, borderline atypia in 53, carcinoma in situ (CIS) in 19, and invasive carcinoma in 11) were analyzed by immunohistochemical staining (EZH2, Ki-67, p27^Kip1). Using paraffin embedded sections, total RNA was successfully extracted from 20 IPMN lesions (borderline IPMN in 9, CIS in 6, invasive carcinoma in 5) and 7 pancreatic normal ducts, and then levels of <i>EZH2</i> and <i>p27^Kip1</i> mRNA were analyzed by real time PCR.</p><p>Results</p><p>In immunohistochemical analysis, cell proliferative activity revealed by Ki-67 positive nuclei was increased during IPMN progression (normal ductp<0.0001). EZH2-positive cells in malignant IPMN did not express p27^Kip1. <i>EZH2</i> mRNA expressions in malignant lesions were significantly higher than those in benign lesions (<i>p</i><0.0001). In contrast, <i>p27^Kip1</i> mRNA in malignant lesions was significantly decreased compared to those in benign lesion (<i>p</i><0.05), and there was an inverse correlation between <i>EZH2</i> and <i>p27^Kip1</i> mRNA levels (<i>p</i> = 0.0109).</p><p>Conclusion</p><p>EZH2 is associated with the accelerated cell proliferation and malignant step in pancreatic IPMN via the downregulation of p27^Kip1.</p></div

H&E staining of IPMNs.

<p>IPMNs consist of different degrees of dysplasia characterized by multifocality.</p

The expression patterns of p27^Kip1, p21^WAF1, and PTEN in normal duct and IPMN lesions.

<p>a) Immunohistochemical staining of p27^Kip1 in IPMN lesions (×40). CIS, carcinoma in situ. b) The percentage of p27^Kip1-positive nuclear cells in IPMN lesions. ****, <i>p</i><0.05. c) The percentage of p21^WAF1-positive nuclear cells in IPMN lesions. d) The percentage of PTEN-positive cells in IPMN lesions.</p