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

Okabe H, Delgado E, Lee JM, Yang J, Kinoshita H, Hayashi H, Tsung A, Behari J, Beppu T, Baba H, Monga SP - PLoS ONE (2014)

Bottom Line: Leukocyte cell-derived chemotaxin-2 (LECT2) expression was decreased in KO livers.Hep3BS33Y expressed and secreted more LECT2 in media as compared to Hep3BWT.Intriguingly, patients without β-catenin mutations showed significantly higher serum LECT2 levels (54.26 ± 22.25 ng/mL; n = 46).

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America; Department of Gastroenterological Surgery, Graduate School of Life Sciences, Kumamoto University, Kumamoto, Japan.

ABSTRACT
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.

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Regulation of LECT2 expression by β-catenin.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. Lect2 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 β-catenin or control 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 Lect2 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.
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pone-0098817-g001: Regulation of LECT2 expression by β-catenin.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. Lect2 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 β-catenin or control 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 Lect2 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.

Mentions: To identify specific biomarkers for β-catenin upregulation, we utilized our previous dataset of microarray analysis comparing gene expression of liver tissue of hepatocyte-specific β-catenin knockout (KO) with that of wild type (WT) [13]. From a list of 2963 upregulated genes in WT as compared to KO (with fold change > 5), we identified 14 genes that encode for secreted proteins (Table 1). The leading candidate lect2 showed a 117-fold lower expression in KO as compared to WT (Figure 1A). To validate if LECT2 expression could be induced by β-catenin activation, we used two previously generated stable human cell lines, Hep3B cells expressing wild type β-catenin (Hep3BWT) and Hep3B cells expressing S33Y-mutated β-catenin (Hep3BS33Y), the latter showing highest β-catenin levels (Figure 1B) [17]. We compared LECT2 expression between these cell lines using qRT-PCR analysis and detected a notable increase in its expression in Hep3BS33Y cells (data not shown). Whole cell lysates also showed increased LECT2 protein levels in Hep3BS33Y as compared to Hep3BWT cells (Figure 1C). To see if LECT2 upregulation is caused by active β-catenin, Hep3BS33Y were transiently transfected with β-catenin or control siRNA. β-Catenin knockdown led to a notable decrease in LECT2 protein levels (Figure 1D).


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

Okabe H, Delgado E, Lee JM, Yang J, Kinoshita H, Hayashi H, Tsung A, Behari J, Beppu T, Baba H, Monga SP - PLoS ONE (2014)

Regulation of LECT2 expression by β-catenin.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. Lect2 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 β-catenin or control 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 Lect2 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.
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getmorefigures.php?uid=PMC4043833&req=5

pone-0098817-g001: Regulation of LECT2 expression by β-catenin.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. Lect2 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 β-catenin or control 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 Lect2 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.
Mentions: To identify specific biomarkers for β-catenin upregulation, we utilized our previous dataset of microarray analysis comparing gene expression of liver tissue of hepatocyte-specific β-catenin knockout (KO) with that of wild type (WT) [13]. From a list of 2963 upregulated genes in WT as compared to KO (with fold change > 5), we identified 14 genes that encode for secreted proteins (Table 1). The leading candidate lect2 showed a 117-fold lower expression in KO as compared to WT (Figure 1A). To validate if LECT2 expression could be induced by β-catenin activation, we used two previously generated stable human cell lines, Hep3B cells expressing wild type β-catenin (Hep3BWT) and Hep3B cells expressing S33Y-mutated β-catenin (Hep3BS33Y), the latter showing highest β-catenin levels (Figure 1B) [17]. We compared LECT2 expression between these cell lines using qRT-PCR analysis and detected a notable increase in its expression in Hep3BS33Y cells (data not shown). Whole cell lysates also showed increased LECT2 protein levels in Hep3BS33Y as compared to Hep3BWT cells (Figure 1C). To see if LECT2 upregulation is caused by active β-catenin, Hep3BS33Y were transiently transfected with β-catenin or control siRNA. β-Catenin knockdown led to a notable decrease in LECT2 protein levels (Figure 1D).

Bottom Line: Leukocyte cell-derived chemotaxin-2 (LECT2) expression was decreased in KO livers.Hep3BS33Y expressed and secreted more LECT2 in media as compared to Hep3BWT.Intriguingly, patients without β-catenin mutations showed significantly higher serum LECT2 levels (54.26 ± 22.25 ng/mL; n = 46).

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America; Department of Gastroenterological Surgery, Graduate School of Life Sciences, Kumamoto University, Kumamoto, Japan.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus