Interferon-stimulated gene 15 modulates cell migration by interacting with Rac1 and contributes to lymph node metastasis of oral squamous cell carcinoma cells

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In an effort to understand the underlying mechanisms of lymph node metastasis in oral squamous cell carcinoma (OSCC), through in vivo selection, LN1-1 cells were previously established from OEC-M1 cells and showed enhanced lymphangiogenesis and lymphatic metastasis capabilities. In the current study, we use a stable isotope labeling with amino acids in cell culture (SILAC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic platform to compare LN1-1 to OEC-M1 cells. Interferon-stimulated gene 15 (ISG15) was found highly expressed in LN1-1 cells. Immunohistochemical analysis and meta-analysis of publicly available microarray datasets revealed that the ISG15 level was increased in human OSCC tissues and associated with poor disease outcome. Knockdown of ISG15 had minimal effects on tumor growth but did decrease tumor lymphangiogenesis and lymphatic metastasis of LN1-1 cells. Consistent with the in vivo assay, ISG15 knockdown did not impair cell growth but diminished cell migration, invasion, and transendothelial migration in vitro. ISG15-induced cell migration was independent of ISGylation and associated with membrane protrusion. Ectopic expression of ISG15 increased Rac1 activity and knockdown of Rac1 impaired ISG15-enhanced migration. Furthermore, Rac1 colocalized with ISG15 to a region of membrane protrusion and ISG15 coimmunoprecipitated with Rac1, especially with the Rac1-GDP form. Importantly, as shown by proximity ligation assays, ISG15 and Rac1 physically interacted with each other. Our results indicated that ISG15 affects cell migration by interacting with Rac1 and regulating Rac1 activity and contributes to lymphatic metastasis in OSCC.

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The authors would like to appreciate Drs. Lu-Hai Wang (China Medical University, Taichung, Taiwan) and Tze-Sing Huang (National Health Research Institutes, Miaoli, Taiwan) for providing technical supports in GTPase assay and PLA and Dr. Tsai-Kun Li (National Taiwan University, Taipei, Taiwan) for sharing the ISG15 and ISG15AA plasmids. The authors thank Dr. Szu-Heng Liu (National Health Research Institutes, Miaoli, Taiwan) and the protein core in Institute of Biological Chemistry and Institute of Molecular Biology, Academia Sinica (Taipei, Taiwan) for technical support in SILAC. The authors thank Dr. Yu-Shuen Wang (National Chiao Tung University, Hsinchu, Taiwan) for assistance on program implementation. The authors thank Dr. Shih-Sheng Jiang (National Health Research Institutes, Miaoli, Taiwan) and Taiwan Bioinformatics Institute Core Facility (National Core Facility Program for Biotechnology, MOST 105-2319-B-400-002) for assistances on using Oncomine and data analysis. The histological evaluation was assisted by the Pathology Core Laboratory, National Health Research Institutes, Taiwan. RNAi reagents were purchased from the National RNAi Core Facility at the Institute of Molecular Biology/Genomic Research Center, Academia Sinica, Taiwan.


This study was supported by grants NHRI CA-107-PP-04 and MOST 106-2314-B-400-026-MY3 from National Health Research Institutes and Ministry of Science and Technology, Taiwan.

Author information

Y-LC performed the work of molecular biology, biochemistry, and cell biology; W-LW performed the proteomic analysis; C-WJ contributed to manuscript preparation; Y-CY, S-HW, and F-YT contributed to the animal work and statistical analysis; Y-YS contributed to the histopathological examination of clinical samples; Y-LC and Y-WC contributed to experimental designs, data analysis, and manuscript writing; all authors contributed to edit the manuscript.

Correspondence to Ya-Wen Chen.

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