ARD1 stabilizes NRF2 through direct interaction and promotes colon cancer progression

Xizhu Fang; Yeon-Hwa Lee; Jeong-Hoon Jang; Su-Jung Kim; Seong Hoon Kim; Do-Hee Kim; Hye-Kyung Na; Kyung-Ok Kim; Jeong-Heum Baek; Young-Joon Surh

doi:10.1016/j.lfs.2022.121217

ARD1 stabilizes NRF2 through direct interaction and promotes colon cancer progression

Life Sci. 2023 Jan 15:313:121217. doi: 10.1016/j.lfs.2022.121217. Epub 2022 Nov 25.

Authors

Affiliations

¹ College of Pharmacy, Seoul National University, Seoul, South Korea.
² Department of Molecular Medicine and Biopharmaceutical Science, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, South Korea.
³ Department of Chemistry, College of Convergence and Integrated Science, Kyonggi University, Suwon, Gyeonggi-do, South Korea.
⁴ Department of Food Science and Biotechnology, College of Knowledge-Based Services Engineering, Sungshin Women's University, Seoul, South Korea.
⁵ Gachon Medical Research Institute, Gil Medical Center, Gachon University, Incheon, South Korea.
⁶ Division of Colon and Rectal Surgery, Department of Surgery, Gil Medical Center, Gachon University College of Medicine, Incheon, South Korea.
⁷ College of Pharmacy, Seoul National University, Seoul, South Korea; Cancer Research Institute, Seoul National University, Seoul, South Korea. Electronic address: surh@snu.ac.kr.

PMID: 36442525
DOI: 10.1016/j.lfs.2022.121217

Abstract

Aims: Aberrant overactivation/overexpression of NRF2 is implicated as a driving event in tumor progression, which has been attributed to its mutation or inactivation of the inhibitory protein, KEAP1. However, alternative mechanisms responsible for sustained activation of NRF2 are less understood.

Main methods: Human colon cancer cell lines and tissues obtained from colorectal cancer (CRC) patients were used. To examine the expression levels of ARD1 and NRF2, Western blot and immunofluorescence analyses were performed. To investigate the potential relevance of NRF2 and ARD1 to human CRC, NRF2 and ARD1 were individually silenced in human colon cancer cells (HCT-116) by transfection with their specific small interfering RNA (siRNA). To determine the functional role of ARD1 in NRF2 regulation, in situ proximate ligation, co-immunoprecipitation, nano-LC-ESI MS/MS, and in vitro acetylation assays were performed.

Key findings: ARD1 knockdown in human colon cancer cell lines significantly reduced the protein levels of NRF2 without affecting its mRNA expression; however, silencing of NRF2 did not alter ARD1 protein expression. In addition, these two proteins were co-localized and physically interacted with each other both in human colon cancer cells (HCT-116) and human colon tumor tissues. Mechanistically, ARD1 overexpression increased the acetylation levels of NRF2. Moreover, an in vitro acetylation assay and mass spectrometric analysis demonstrated that ARD1 could directly acetylate NRF2. Ectopic expression of mutant forms of ARD1 with defective acetyltransferase activity reduced the stability of NRF2.

Significance: In conclusion, ARD1 may potentiate the oncogenic function of NRF2 in human colon cancer by stabilizing this transcription factor.

Keywords: ARD1; Acetylation; Colorectal cancer; NRF2; Posttranslational modification.

MeSH terms

Cell Line
Colonic Neoplasms* / genetics
Humans
Kelch-Like ECH-Associated Protein 1 / genetics
NF-E2-Related Factor 2* / genetics
Tandem Mass Spectrometry

Substances

Kelch-Like ECH-Associated Protein 1
NF-E2-Related Factor 2
NAA10 protein, human