NOTCH-induced aldehyde dehydrogenase 1A1 deacetylation promotes breast cancer stem cells
Di Zhao, … , Kun-Liang Guan, Qun-Ying Lei
Di Zhao, … , Kun-Liang Guan, Qun-Ying Lei
Published November 10, 2014
Citation Information: J Clin Invest. 2014;124(12):5453-5465. https://doi.org/10.1172/JCI76611.
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Research Article Oncology

NOTCH-induced aldehyde dehydrogenase 1A1 deacetylation promotes breast cancer stem cells

Abstract

High aldehyde dehydrogenase (ALDH) activity is a marker commonly used to isolate stem cells, particularly breast cancer stem cells (CSCs). Here, we determined that ALDH1A1 activity is inhibited by acetylation of lysine 353 (K353) and that acetyltransferase P300/CBP–associated factor (PCAF) and deacetylase sirtuin 2 (SIRT2) are responsible for regulating the acetylation state of ALDH1A1 K353. Evaluation of breast carcinoma tissues from patients revealed that cells with high ALDH1 activity have low ALDH1A1 acetylation and are capable of self-renewal. Acetylation of ALDH1A1 inhibited both the stem cell population and self-renewal properties in breast cancer. Moreover, NOTCH signaling activated ALDH1A1 through the induction of SIRT2, leading to ALDH1A1 deacetylation and enzymatic activation to promote breast CSCs. In breast cancer xenograft models, replacement of endogenous ALDH1A1 with an acetylation mimetic mutant inhibited tumorigenesis and tumor growth. Together, the results from our study reveal a function and mechanism of ALDH1A1 acetylation in regulating breast CSCs.

Authors

Di Zhao, Yan Mo, Meng-Tian Li, Shao-Wu Zou, Zhou-Li Cheng, Yi-Ping Sun, Yue Xiong, Kun-Liang Guan, Qun-Ying Lei

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Figure 1

Acetylation at Lys-353 decreases ALDH1A1 enzyme activity.

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Acetylation at Lys-353 decreases ALDH1A1 enzyme activity. (A) ALDH1A1 wa...
(A) ALDH1A1 was acetylated, and FLAG-ALDH1A1 was transfected into 293T cells, followed by treatment with the deacetylase inhibitor TSA or NAM. ALDH1A1 acetylation was analyzed by Western blotting with pan–anti-acetyllysine antibody (α-Ac). (B) K353 mutation decreased ALDH1A1 acetylation. (C) Specificity of the antibody against K353-acetylated ALDH1A1 was determined. The nitrocellulose membrane was spotted with different amounts of acetylated K353 peptide or unmodified peptide, as indicated, and probed with anti-AcALDH1A1(K353) antibody (α-K353Ac). (D) The anti-AcALDH1A1(K353) antibody recognized WT but not K353R-mutant ALDH1A1. (E) Treatment with NAM increased endogenous ALDH1A1 acetylation at K353. HEPG2 cells were treated with NAM. Endogenous ALDH1A1 protein levels and K353 acetylation were determined. (F) Endogenous ALDH1A1 was acetylated at Lys-353. K353 acetylation of endogenous ALDH1A1 in human liver tissues was determined using the anti-AcALDH1A1(K353) antibody preincubated with or without acetylated K353 peptide. (G and H) Inhibition of SIRT family deacetylases reduced the enzyme activity of WT, but not K353R/Q-mutant, ALDH1A1. FLAG-tagged WT or mutant ALDH1A1 protein was expressed in 293T cells, and transfected cells were treated with or without NAM and then purified by IP using an anti-FLAG antibody. Enzymatic activity was measured and normalized to protein levels. (I) Acetylation at K353 inhibited ALDH1A1 enzyme activity. Recombinant WT and K353-acetylated ALDH1A1 protein were prepared by genetically encoding Nε-acetyllysine in E. coli. UnAc-ALDH1A1, unacetylated ALDH1A1. (G–I) Enzymatic activity was measured and normalized to protein levels. Relative enzyme activity data represent the mean ± SD of triplicate experiments.