J Breast Cancer.  2018 Mar;21(1):87-90. 10.4048/jbc.2018.21.1.87.

Identification of the Thioredoxin-Like 2 Autoantibody as a Specific Biomarker for Triple-Negative Breast Cancer

• Chung JM
• Jung Y
• Kim YP
• Song J
• Kim S
• Kim JY
• Kwon M
• Yoon JH
• Kim MD
• Lee JK
• Chung DY
• Lee SY
• Kang J
• Kang HC

• KMID: 2441872
• DOI: http://doi.org/10.4048/jbc.2018.21.1.87

Abstract

Triple-negative breast cancer (TNBC) has a higher risk of death within 5 years of being diagnosed than the other forms of breast cancer. It is the second leading cause of death due to cancer among women. Currently, however, no diagnostic blood-based biomarker exists to identify the early stages of TNBC. To address this point, we utilized a human protein microarray system to identify serum autoantibodies that showed different expression patterns between TNBC and normal serum samples, and identified five autoantibodies showing TNBC-specific expression. Among them, we selected the thioredoxin-like 2 (TXNL2) autoantibody and evaluated its diagnostic relevance by dot blot analysis with the recombinant TXNL2 protein. We demonstrated that the TXNL2 autoantibody showed 2- to 6-fold higher expression in TNBC samples than in normal samples suggesting that serum TXNL2 autoantibodies are potential biomarkers for TNBC.

Keyword

Autoantibodies; Biomarkers; Breast neoplasms; Protein array analysis

MeSH Terms

Autoantibodies
Biomarkers
Breast Neoplasms
Cause of Death
Female
Humans
Protein Array Analysis
Triple Negative Breast Neoplasms*
Autoantibodies
Biomarkers

Figure

• 

  Figure 1 Identification of five autoantibodies for triple-negative breast cancer (TNBC) biomarkers using a 17K human protein microarray system. (A) To identify novel TNBC-specific biomarkers, normal or TNBC patient sera containing autoantibodies were applied on 17K protein microarray. TNBC-specific autoantibodies were detected by anti-human Alexa Flour 546 (Invitrogen) and scanned by GenePix 4000B (Molecular Devices). Signal intensity for each spot was obtained as the ratio of foreground to background signals and was normalized with the glutathione s-transferase signal intensity. The mean signal intensity of each protein on the chip was calculated. (B) Five autoantibodies, thioredoxin-like 2 (TXNL2), ADP-ribosylhydrolase like 1 (ADPRHL1), glycyl-TRNA synthetase (GARS), spinocerebellar ataxia type 3 protein (ATXN3), and solute carrier family 16 member 4 (SLC16A4), were determined by the GenePix analysis. The differential expression of these five autoantibody biomarkers were calculated by statistical analysis. (C) High-power image of 17K protein microarray. Yellow arrow indicated TXNL2 autoantibody and white arrow was shown IgG as a positive control. TXNL2 autoantibody specifically and strongly interacted with the TXNL2 antigen.

• 

  Figure 2 Thioredoxin-like 2 (TXNL2) autoantibody is a potential biomarker for diagnosing triple-negative breast cancer (TNBC). (A) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the recombinant glutathione s-transferase (GST)-tagged TXNL2 protein. Total cell lysate and each subcellular fractions of the isopropyl β-D-1-thiogalactopyranoside-induced Escherichia coli BL21 (DE3) expressing GST-tagged TXNL2 protein was loaded in SDS-PAGE. The soluble fraction containing GST-tagged TXNL2 protein was applied in a Glutathione Sepharose 4B resin (GE Healthcare). Subsequently resin conjugated TXNL2 was detached by elution buffer containing reduced glutathione. (B) Superdex 75 (GE Healthcare) gel-filtration fractionation profile for the further purification of the target protein. (C) SDS-PAGE profile of GST-tagged TXNL2 protein eluted from gel filtration. (D) A high-performance liquid chromatography chromatogram of recombinant GST-tagged TXNL2 protein. (E) Dot blot analysis of TXNL2 for detection of TXNL2 autoantibody in TNBC serum sample. Purified GST or GST tagged TXNL2 protein was spotted on nitrocellulose membrane and applied with normal or TNBC patient sera. GST was used as a negative control. Autoantibody against TXNL2 was detected by anti-human horseradish peroxidase conjugated antibody and developed using an enhanced chemiluminescent substrate. (F) Graph shows that amount of TXNL2 autoantibody derived from human sera. TXNL2 autoantibody was expressed 2- to 6-fold higher in TNBC serum than in normal. Densitometric analysis was performed using the ImageJ program (https://imagej.net/).

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