Yonsei Med J.  2015 Sep;56(5):1296-1306. 10.3349/ymj.2015.56.5.1296.

Serum Dickkopf-1 as a Biomarker for the Diagnosis of Hepatocellular Carcinoma

• Kim SU
• Park JH
• Kim HS
• Lee JM
• Lee HG
• Kim H
• Choi SH
• Baek S
• Kim BK
• Park JY
• Kim DY
• Ahn SH
• Lee JD
• Han KH

• KMID: 2163621
• DOI: http://doi.org/10.3349/ymj.2015.56.5.1296

Abstract

PURPOSE
Dickkopf-1 (DKK-1) is a Wnt/beta-catenin signaling pathway inhibitor. We investigated whether DKK-1 is related to progression in hepatocellular carcinoma (HCC) cells and HCC patients.
MATERIALS AND METHODS
In vitro reverse-transcription polymerase chain reaction (RT-PCR), wound healing assays, invasion assays, and ELISAs of patient serum samples were employed. The diagnostic accuracy of the serum DKK-1 ELISA was assessed using receiver operating characteristic (ROC) curves and area under ROC (AUC) analyses.
RESULTS
RT-PCR showed high DKK-1 expression in Hep3B and low in 293 cells. Similarly, the secreted DKK-1 concentration in the culture media was high in Hep3B and low in 293 cells. Wound healing and invasion assays using 293, Huh7, and Hep3B cells showed that DKK-1 overexpression promoted cell migration and invasion, whereas DKK-1 knock-down inhibited them. When serum DKK-1 levels were assessed in 370 participants (217 with HCC and 153 without), it was significantly higher in HCC patients than in control groups (median 1.48 ng/mL vs. 0.90 ng/mL, p<0.001). The optimum DKK-1 cutoff level was 1.01 ng/mL (AUC=0.829; sensitivity 90.7%; specificity 62.0%). Although DKK-1 had a higher AUC than alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) (AUC=0.829 vs. 0.794 and 0.815, respectively), they were statistically similar (all p>0.05). When three biomarkers were combined (DKK-1 plus AFP plus DCP), they showed significantly higher AUC (AUC=0.952) than single marker, DKK-1 plus AFP, or DKK-1 plus DCP (all p<0.001).
CONCLUSION
DKK-1 might be a key regulator in HCC progression and a potential therapeutic target in HCC. Serum DKK-1 could complement the diagnostic accuracy of AFP and DCP.

Keyword

Dickkopf-1; hepatocellular carcinoma; biomarker

MeSH Terms

Area Under Curve
Biomarkers/blood/metabolism
Biomarkers, Tumor/blood
Carcinoma, Hepatocellular/blood/*diagnosis
Enzyme-Linked Immunosorbent Assay
Female
Humans
Intercellular Signaling Peptides and Proteins/*blood/*metabolism
Liver Neoplasms/blood/*diagnosis
Male
Middle Aged
Protein Precursors/blood/metabolism
Prothrombin/metabolism
ROC Curve
Reverse Transcriptase Polymerase Chain Reaction/*methods
Sensitivity and Specificity
alpha-Fetoproteins/analysis/metabolism
Biomarkers
Biomarkers, Tumor
Intercellular Signaling Peptides and Proteins
Protein Precursors
Prothrombin
alpha-Fetoproteins

Figure

• 

  Fig. 1 DKK-1 expression in Hep3B, Huh7, and 293 cells. (A) RT-PCR analysis showed high DKK-1 mRNA expression in Hep3B cells, low in 293 cells, and none in the negative control. (B) The DKK-1 concentration secreted in the culture media, determined by ELISA, was high in Hep3B cells, moderate in Huh7 cells, and low in 293 cells. (C) Following DKK-1 transfection, luciferase assays revealed that the β-catenin transcriptional activity was induced in 293 cells. *p<0.01. DKK-1, dickkopf-1.

• 

  Fig. 2 DKK-1 promotes cell migration and invasion. (A) DKK-1 overexpression promoted 293 cell migration in wound healing assays. (B) DKK-1 knock-down inhibited Huh7 cell migration in wound healing assays. (C) DKK-1 overexpression promoted Huh7 cell invasion, whereas DKK-1 knock-down inhibited it in invasion assays. *p<0.01. DKK-1, dickkopf-1.

• 

  Fig. 3 Knock-down of DKK-1 inhibits cell growth. (A) DKK-1 knock-down in Huh7 cells decreased the amount of secreted DKK-1 after 48 h, determined by ELISA. (B) DKK-1 knock-down inhibited Huh7 cell growth after 48 h, determined by MTT assays. *p<0.01. DKK-1, dickkopf-1.

• 

  Fig. 4 Serum DKK-1 concentration. Serum DKK-1 concentrations were significantly higher in HCC patients than those of all the control groups. DKK-1, dickkopf-1; HCC, hepatocellular carcinoma; TNM, Tumor-Node-Metastasis; BCLC, Barcelona Clinic Liver Cancer.

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