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Human colorectal cancer-associated carbohydrate antigen on annexin A2 protein

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Abstract

Cancer-associated antigens are not only a good marker for monitoring cancer progression but are also useful for molecular target therapy. In this study, we aimed to generate a monoclonal antibody that preferentially reacts with colorectal cancer cells relative to noncancerous gland cells. We prepared antigens composed of HT-29 colorectal cancer cell lysates that were adsorbed by antibodies to sodium butyrate-induced enterocytically differentiated HT-29 cells. Subsequently, we generated a monoclonal antibody, designated 12G5A, which reacted with HT-29 colon cancer cells, but not with sodium butyrate-induced differentiated HT-29 cells. Immunohistochemical staining revealed 12G5A immunoreactivity in all 73 colon cancer tissue specimens examined at various degrees, but little or no immunoreactivity in noncancerous gland cells. Notably, high 12G5A immunoreactivity, which was determined as more than 50% of colon cancer cells intensively stained with 12G5A antibody, exhibited significantly higher association with a poor overall survival rate of patients with colorectal cancer (P = 0.0196) and unfavorable progression-free survival rate of patients with colorectal cancer (P = 0.0418). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, si-RNA silencing analysis, enzymatic deglycosylation, and tunicamycin treatment revealed that 12G5A recognized the glycosylated epitope on annexin A2 protein. Our findings indicate that 12G5A identified a cancer-associated glycosylation epitope on annexin A2, whose expression was related to unfavorable colorectal cancer behavior.

Key message

• 12G5A monoclonal antibody recognized a colorectal cancer-associated epitope.

• 12G5A antibody recognized the N-linked glycosylation epitope on annexin A2.

• 12G5A immunoreactivity was related to unfavorable colorectal cancer behavior.

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Availability of data and materials

The datasets used during the present study are available from the corresponding author upon reasonable request.

Funding

This study was supported by grants from the Ministry of Education of Japan (Grant nos. KAKEN 20 K07406).

Author information

Authors and Affiliations

  1. Department of Surgical Oncology, Gifu University, Gifu University Graduate School of Medicine, Gifu, Japan

    Hideharu Tanaka, Yoshinori Iwata, Itaru Yasufuku & Kazuhiro Yoshida

  2. Department of Pathology and Translational Research, Gifu University Graduate School of Medicine, Gifu, Japan

    Chiemi Saigo, Yusuke Kito & Tamotsu Takeuchi

Authors
  1. Hideharu Tanaka
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  2. Chiemi Saigo
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  3. Yoshinori Iwata
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  4. Itaru Yasufuku
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  5. Yusuke Kito
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  6. Kazuhiro Yoshida
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  7. Tamotsu Takeuchi
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Contributions

Kazuhiro Yoshida and Tamotsu Takeuchi participated in the design of the study, data interpretation, and manuscript drafting. Hideharu Tanaka, Chiemi Saigo, Yoshinori Iwata, Itaru Yasufuku, and Yusuke Kito performed the experiments. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work is appropriately investigated and resolved.

Corresponding author

Correspondence to Tamotsu Takeuchi.

Ethics declarations

Ethics approval

After receiving approval from the Institutional Review Board of the Gifu University Graduate School of Medicine (specific approval numbers: 2019–202 and 2019–0444) to carry out our retrospective study, we collected 55 specimens from surgically treated patients who were primarily diagnosed with colorectal cancer. Informed consent was obtained from all participants or their authorized representatives. This study was conducted in accordance with the ethical standards outlined in the Declaration of Helsinki, 1975.

The experimental protocol to obtain the antibodies was approved by the Animal Care Committee of Gifu University (specific approval number: H30–32).

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Not applicable.

Competing interests

The authors declare no competing interests.

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Supplementary information

Fig. S1

Immunofluorescence staining results demonstrating a greater reaction of 12G5A antibody with HT-29 colon cancer cells than with sodium butyrate-induced HT-29 cells. a and b HT-29 cells were cultured without (a) or with (b) 1-mM sodium butyrate. Cells were fixed with 4% (m/v) paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 10% goat serum. The cells were then incubated with 12G5A-cultured supernatant and then with Alexa Fluor 488-conjugated anti-mouse antibody. Images were acquired using a confocal laser scanning microscope (Leica TCS SP8, Germany). 12G5A immunoreactivity was visualized with green fluorescence, and the nucleus was stained using DAPI. Scale bar represents 20 μm. c Simultaneously, alkaline phosphatase activity was measured to monitor the enterocyte differentiation of HT-29 cells using p-nitrophenyl phosphate as substrate in triplicate. Alkaline phosphatase activity was enhanced by sodium butyrate. The mean and standard deviation of absorbance at 405 nm are shown. (PNG 152 kb)

High Resolution image (TIF 187 kb)

Fig. S2

Little or no 12G5A immunoreactivity was found in normal colon tissue microarray. Representative immunohistochemical staining of two human normal colorectal tissues using 12G5A antibody is shown. We obtained similar staining in 22 other normal microarray colorectal tissues. Scale bar represents 100 μm. (PNG 175 kb)

High Resolution image (TIF 764 kb)

Fig. S3

Immunoaffinity extraction of 12G5A antigen from SW480 cells. Approximately, 100-, 40-, and 30-kDa protein bands were observed after Coomassie brilliant blue staining (lane 12G5A). Significant protein bands were not obtained using the control murine IgM-binding M-270 epoxy magnetic beads (lane Mock). Each protein band was cut and analyzed by peptide mass fingerprinting using a MALDI-TOF assay. Interestingly, an approximately 40-kDa protein appeared to be human annexin A2 with a significant Mowse score, while the other two protein bands could not be identified with a sufficient Mowse score. Immunoblotting on the immunoprecipitation specimens indicated that a commercially available antibody to annexin A2 reacted with the 40-kDa protein, but not with the specimens of isotype control immunoprecipitants. (PNG 307 kb)

High Resolution image (TIF 2794 kb)

Fig. S4

siRNA-mediated silencing of annexin A2 gene in SW480 cells. Both the siRNAs, s1383 and s9548 (indicated as 2 and 3, respectively), successfully downregulated annexin A2 expression at the mRNA (a) and protein (b) levels compared to control siRNA-treated SW480 cells (indicated as 1). The relative expression rate based on that of the control siRNA-treated group was 0.107 ± 0.015 and 0.05 ± 0.01 in siRNA s1383 and s9548-treated SW480 cells, respectively (a). Note that there was little or no annexin A2 green protein band in siRNA-treated cells. Red bands indicate GAPDH protein loading in each lane (b) (PNG 103 kb)

High-resolution image (TIF 321 kb)

ESM 5

(PNG 1335 kb)

High Resolution image (TIF 3061 kb)

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Tanaka, H., Saigo, C., Iwata, Y. et al. Human colorectal cancer-associated carbohydrate antigen on annexin A2 protein. J Mol Med 99, 1115–1123 (2021). https://doi.org/10.1007/s00109-021-02077-z

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  • DOI: https://doi.org/10.1007/s00109-021-02077-z

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