ALDH2(E487K) mutation increases protein turnover and promotes murine hepatocarcinogenesis

Abstract

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) in the liver removes toxic aldehydes including acetaldehyde, an intermediate of ethanol metabolism. Nearly 40% of East Asians inherit an inactive ALDH2*2 variant, which has a lysine-for-glutamate substitution at position 487 (E487K), and show a characteristic alcohol flush reaction after drinking and a higher risk for gastrointestinal cancers. Here we report the characterization of knockin mice in which the ALDH2(E487K) mutation is inserted into the endogenous murine Aldh2 locus. These mutants recapitulate essentially all human phenotypes including impaired clearance of acetaldehyde, increased sensitivity to acute or chronic alcohol-induced toxicity, and reduced ALDH2 expression due to a dominant-negative effect of the mutation. When treated with a chemical carcinogen, these mutants exhibit increased DNA damage response in hepatocytes, pronounced liver injury, and accelerated development of hepatocellular carcinoma (HCC). Importantly, ALDH2 protein levels are also significantly lower in patient HCC than in peritumor or normal liver tissues. Our results reveal that ALDH2 functions as a tumor suppressor by maintaining genomic stability in the liver, and the common human ALDH2 variant would present a significant risk factor for hepatocarcinogenesis. Our study suggests that the ALDH2*2 allele-alcohol interaction may be an even greater human public health hazard than previously appreciated.

Keywords: ALDH2*2 polymorphism; Asian flush; alcohol metabolism; liver cancer; mouse model.

Fig. 1.

Generation of Aldh2^E487K KI mice. (A) Schematic drawings of the murine Aldh2 allele and targeting construct, Aldh2^E487K KI allele generated by homologous recombination in ES cells, and final Aldh2^E487K KI allele after Cre-mediated removal of the neomycin-resistance cassette. The dotted lines indicate regions of homology and asterisks indicate the altered exon. (B) PCR genotyping of WT, Aldh2^E487K/+, and Aldh2^E487K/E487K mice (“M” indicates molecular weight marker). (C) Confirmation of the E487K mutation by direct sequencing of PCR-amplified genomic DNA; *indicates the position of the nucleotide alteration producing the E487K mutation.

Fig. 2.

Significantly reduced ALDH2 enzymatic activity in Aldh2^E487K KI mouse hepatocytes. Primary hepatocytes from WT and Aldh2^E487K KI mice were analyzed for ALDH2 activity in vitro (mean ± SEM; **P < 0.01; n = 6) (A) and for ACE clearance over a short (B) or long (C) time course (mean ± SEM; n = 3).

Fig. 3.

Defective alcohol metabolism in Aldh2^E487K KI mice after acute ethanol challenge. Serum ACE concentrations from WT and Aldh2^E487K KI mice acutely challenged with ethanol at 2 g/kg (A), 4 g/kg (B), or 8 g/kg (C) over 12 h (mean ± SEM; n = 3). Behavioral scores of mice subjected to 2 g/kg (D), 4 g/kg (E), or 8 g/kg (F) ethanol over the same time course (mean ± SEM; n = 6).

Fig. S1.

Survival rates after acute ethanol challenge. WT and Aldh2^E487K KI mice were acutely challenged with ethanol at 2, 4, or 8 g/kg (mean ± SEM; n = 6 each).

Fig. 4.

Increased sensitivity of Aldh2^E487K KI mice to damage induced by chronic ethanol challenge. (A and B) Survival (A) and body weight (B) of WT and Aldh2^E487K KI mice treated with 4 g/kg ethanol for 6 wk (mean ± SEM; n = 9). (C) White blood cell counts from whole-blood smears of chronically treated mice (mean ± SEM; *P < 0.05, **P < 0.01; n = 3). (D) Histology and cleaved caspase 3 staining of treated mouse liver sections (400×).

Fig. 5.

Reduced levels and stability of ALDH2 protein in liver of ALDH2*2 humans and Aldh2^E487K KI mice. (A and B) Representative Western blot (A) and quantification (B) relative to β-actin of human liver ALDH2 with the indicated genotypes. (C) Measurement of ALDH2 mRNA by real-time quantitative PCR in these human samples. (D and E) Representative Western blot (D) and quantification (E) normalized to β-actin of ALDH2 in heterozygous and homozygous Aldh2^E487K KI mouse liver. (F) Measurement of ALDH2 mRNA by real-time quantitative PCR in these mouse samples. (G and H) Western blot (G) and quantification (H) of ALDH2 in primary hepatocytes from Aldh2^E487K KI mice after cycloheximide treatment to determine the half-life of ALDH2 proteins (mean ± SEM; *P < 0.01; **P < 0.001; n = 3).

Fig. S2.

Chronic ethanol challenge in Aldh2^E487K knockin mice. (A) Representative images of white blood cells stained from a whole-blood smear. (B) H&E staining of esophagus and stomach in Aldh2^E487K knockin mice (n = 3).

Fig. S3.

PCR genotyping of the ALDH2 polymorphism in human liver tissues. (A) PCR fragments spanning the polymorphism amplified from genomic DNA of 20 human liver tissues. (B) Digested PCR fragments from A to identify the presence of the mutation. C1, C2, and C3 are positive controls for ALDH2*1/2*1, ALDH2*1/2*2, and ALDH2*2/2*2 genotypes, respectively. Patients 1, 4, 5, 9, 12, 15, 17, and 20 are heterozygotes.

Fig. S4.

PCR genotyping of the ALDH2 polymorphism in human liver tumors. (A) PCR fragments spanning the polymorphism amplified from genomic DNA of 31 human HCC samples. (B) Digested PCR fragments from A to identify the presence of the mutation. C1, C2, and C3 are positive controls for ALDH2*1/2*1, ALDH2*1/2*2, and ALDH2*2/2*2 genotypes, respectively.

Fig. 6.

Reduced ALDH2 expression in human liver tumor samples independent of the ALDH2 polymorphism. (A and B) Western blot of ALDH2 in liver tumor (T) and peritumor (PT) tissues from (A) ALDH2*1/2*1 patients or (B) ALDH2*2/2*1 patients. N, normal WT liver. (C) Quantification of ALDH2 levels normalized to β-actin (mean ± SEM; *P < 0.05, **P < 0.01).

Fig. S5.

Immunohistochemical staining for ALDH2 in hepatoma sections from chemical (DEN)-induced and DDB1^{F/F;Alb-Cre+/−} genetic liver cancer models. Dashed lines outline tumor (T) and nontumor (NT) areas; 40×, 100×, and 200× amplified sections are shown. Three DEN mice are shown.

Fig. 7.

Accelerated liver tumor development in DEN-induced Aldh2^E487K KI mice. (A) Outline of the experimental protocol. (B) Representative photographs of livers dissected from WT or KI mice 8 mo after DEN treatment. (C and D) Total number (C) and maximal volumes (D) of liver tumors (>0.5 mm) from WT and KI mice. (E) Serum ALT activity to estimate liver injury. (F) Histology and phospho-H2AX staining (400×) of liver tissue sections (mean ± SEM; *P < 0.05, **P < 0.01; n = 4).

Fig. S6.

Skin hyperpigmentation in Aldh2^E487K KI mice receiving chronic ethanol feeding. (A) Photographs of the enhanced pigmentation of paw skin of Aldh2^E487K KI mice treated with ethanol (4 g/kg) for 6 wk. (B) H&E staining of corresponding paw skin.

Fig. S7.

Proteasome-independent mechanism for the instability of ALDH2(E487K) mutant protein. (A) Western blot (WB) analysis of ALDH2 levels in primary hepatocytes isolated from WT or KI mice after treatment with the proteasome inhibitor Mg132. p21 levels were monitored as a control for Mg132 efficiency. “NC” indicates vehicle-only controls. (B) Ubiquitination assays for ALDH2 WT and E487K mutants expressed in HEK293 cells. Flag-ALDH2 was pulled down and probed for HA-UB. Flag-CLC2, positive control. IP, immunoprecipitation; M, E487K mutant; W, WT.