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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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Serum LECT2 levels in mice with β-catenin gene mutated HCC.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. (* p<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. Glutamine Synthetase (Glul) and Lect2 expression in three tumor lesions (T1-T3) were assessed by qRT-PCR. Gene expression of background liver tissues surrounding tumor are shown as N.
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pone-0098817-g002: Serum LECT2 levels in mice with β-catenin gene mutated HCC.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. (* p<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. Glutamine Synthetase (Glul) and Lect2 expression in three tumor lesions (T1-T3) were assessed by qRT-PCR. Gene expression of background liver tissues surrounding tumor are shown as N.

Mentions: To investigate if Lect2 could be a serum biomarker in mice, we used a murine HCC model, which utilizes β-catenin signaling as a major mechanism of carcinogenesis. Tumor induction by a single injection of diethylnitrosamine followed by exposure to PB as described in methods and elsewhere has been shown to select for exon-3 mutations in β-catenin gene to give rise to HCC at 6-8 months [14], [15]. Indeed, HCC observed in mice with this protocol were strongly GS-positive and had nuclear β-catenin accumulation as observed by immunohistochemical staining (Figure 2A). Genetic alteration in β-catenin gene contributing to β-catenin activation was confirmed by direct sequencing (Figure 2B). We could recognize liver tumor formation in 9 mice out of 13 that were subjected to DEN/PB protocol. Strikingly, the 9 mice with evidence of histological tumor burden showed significantly (p<0.01) higher serum Lect2 levels (55.9±19.9 ng/mL) as compared to the 4 non-tumor bearing mice (24.9±5.5 ng/mL) (Figure 2C). To confirm mutations in β-catenin gene due to existing tumor heterogeneity and also verify corresponding Lect2 expression, we extracted both genomic DNA and total RNA from same nodules (Figure 2D). Direct sequencing showed a common mutation (S33Y) in T1, T2 and T3 nodules (Figure 2E). We next examined Lect2 mRNA expression as well as Glul mRNA (encoding GS protein) expression in these tumor nodules. All 3-tumor nodules had high Lect2 and Glul expressions as compared to the background liver (Figure 2F). Thus, in mice Lect2 expression and eventually its secretion is upregulated by β-catenin mutations in HCC, which can be detected in serum and hence may be a useful biomarker in this mouse model.


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)

Serum LECT2 levels in mice with β-catenin gene mutated HCC.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. (* p<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. Glutamine Synthetase (Glul) and Lect2 expression in three tumor lesions (T1-T3) were assessed by qRT-PCR. Gene expression of background liver tissues surrounding tumor are shown as N.
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pone-0098817-g002: Serum LECT2 levels in mice with β-catenin gene mutated HCC.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. (* p<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. Glutamine Synthetase (Glul) and Lect2 expression in three tumor lesions (T1-T3) were assessed by qRT-PCR. Gene expression of background liver tissues surrounding tumor are shown as N.
Mentions: To investigate if Lect2 could be a serum biomarker in mice, we used a murine HCC model, which utilizes β-catenin signaling as a major mechanism of carcinogenesis. Tumor induction by a single injection of diethylnitrosamine followed by exposure to PB as described in methods and elsewhere has been shown to select for exon-3 mutations in β-catenin gene to give rise to HCC at 6-8 months [14], [15]. Indeed, HCC observed in mice with this protocol were strongly GS-positive and had nuclear β-catenin accumulation as observed by immunohistochemical staining (Figure 2A). Genetic alteration in β-catenin gene contributing to β-catenin activation was confirmed by direct sequencing (Figure 2B). We could recognize liver tumor formation in 9 mice out of 13 that were subjected to DEN/PB protocol. Strikingly, the 9 mice with evidence of histological tumor burden showed significantly (p<0.01) higher serum Lect2 levels (55.9±19.9 ng/mL) as compared to the 4 non-tumor bearing mice (24.9±5.5 ng/mL) (Figure 2C). To confirm mutations in β-catenin gene due to existing tumor heterogeneity and also verify corresponding Lect2 expression, we extracted both genomic DNA and total RNA from same nodules (Figure 2D). Direct sequencing showed a common mutation (S33Y) in T1, T2 and T3 nodules (Figure 2E). We next examined Lect2 mRNA expression as well as Glul mRNA (encoding GS protein) expression in these tumor nodules. All 3-tumor nodules had high Lect2 and Glul expressions as compared to the background liver (Figure 2F). Thus, in mice Lect2 expression and eventually its secretion is upregulated by β-catenin mutations in HCC, which can be detected in serum and hence may be a useful biomarker in this mouse model.

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