2D-DIGE-MS Proteomics Approaches for Identification of Gelsolin and Peroxiredoxin 4 with Lymph Node Metastasis in Colorectal Cancer

Cheng-Yi Huang; Ko-Chao Lee; Shui-Yi Tung; Wen-Shin Huang; Chih-Chuan Teng; Kam-Fai Lee; Meng-Chiao Hsieh; Hsing-Chun Kuo

doi:10.3390/cancers14133189

2D-DIGE-MS Proteomics Approaches for Identification of Gelsolin and Peroxiredoxin 4 with Lymph Node Metastasis in Colorectal Cancer

Cancers (Basel). 2022 Jun 29;14(13):3189. doi: 10.3390/cancers14133189.

Authors

Cheng-Yi Huang¹, Ko-Chao Lee², Shui-Yi Tung^{3

4}, Wen-Shin Huang¹, Chih-Chuan Teng^{5

6}, Kam-Fai Lee⁷, Meng-Chiao Hsieh¹, Hsing-Chun Kuo^{5

6

8

9}

Affiliations

¹ Division of Colon and Rectal Surgery, Department of Surgery, Chang Gung Memorial Hospital, Chiayi 61363, Taiwan.
² Chang Gung Memorial Hospital-Kaohsiung Medical Center, Division of Colorectal Surgery, Department of Surgery, Chang Gung University College of Medicine, Kaohsiung 83301, Taiwan.
³ Department of Hepato-Gastroenterology, Chang Gung Memorial Hospital, Chiayi 61363, Taiwan.
⁴ College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan.
⁵ Division of Basic Medical Sciences, Department of Nursing, Chang Gung University of Science and Technology, Chiayi 61363, Taiwan.
⁶ Research Fellow, Chang Gung Memorial Hospital, Chiayi 61363, Taiwan.
⁷ Department of Pathology, Chang Gung Memorial Hospital, Chiayi 61363, Taiwan.
⁸ Research Center for Food and Cosmetic Safety, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan 33303, Taiwan.
⁹ Chronic Diseases and Health Promotion Research Center, Chang Gung University of Science and Technology, Chiayi 61363, Taiwan.

Abstract

Background/aims: A combination of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry approach was used to search for potential markers for prognosis and intervention of colorectal cancer (CRC) at different stages of lymph node metastasis (LMN). This quantitative proteomic survey aimed to investigate the LNM-associated proteins and evaluate the clinicopathological characteristics of these target proteins in CRC from stage I to stage IV.

Methods: Sixteen CRC cases were categorized into paired non-LNM and LNM groups, and two-dimensional difference gel electrophoresis and MS proteome analysis were performed. Differential protein expression between non-LNM and LNM CRC was further validated in a tissue microarray, including 40 paraffin-embedded samples by immunohistochemistry staining. Moreover, a Boyden chamber assay, flow cytometry, and shRNA were used to examine the epithelial-mesenchymal transition and mechanism invasiveness of the differentially expressed proteins in DLD-1 cells and in vivo xenograft mouse model.

Results: Eighteen differentially expressed proteins were found between non-LNM and LNM CRC tissues. Among them, protein levels of Gelsolin (GSN) and peroxiredoxin 4 (PRDX4) were abundant in node-positive CRC. Downregulation of GSN and PRDX4 markedly suppressed migration and invasiveness and also induced cell cycle G1/S arrest in DLD-1. Mechanistically, the EGFR/RhoA/PKCα/ERK pathways are critical for transcriptional activation of histone modification of H3 lysine 4 trimethylation (H3K4me3) of GSN and PRDX4 promoters, resulting in upregulation of GSN, PRDX4, Twist-1/2, cyclinD1, proliferating cell-nuclear antigen, β-catenin, N-cadherin, and matrix metalloprotein-9.

Conclusions: GSN and PRDX4 are novel regulators in CRC lymph node metastasis to potentially provide new insights into the mechanism of CRC progression and serve as a biomarker for CRC diagnosis at the metastatic stage.

Keywords: EGFR/RhoA/PKCα/ERK; colorectal cancer (CRC); gelsolin (GSN); lymph node metastasis; peroxiredoxin 4 (PRDX4).

Grants and funding

Funding for this study was provided in part by research grants BMRPD42, CLRPG8G0593, CMRPG6F0641, CMRPG6F0642, CMRPG6F0643, CMRPG6J0131, CMRPG6J0132, CMRPG6J0133, from Chang Gung Memorial Hospital, Chiayi, Taiwan, and by the Ministry of Science and Technology, Taiwan (MOST 110-2320-B-255-005-MY3).