SLC4A11 mediates ammonia import and promotes cancer stemness in hepatocellular carcinoma

Abstract

End-stage liver disease is marked by portal hypertension, systemic elevations in ammonia, and development of hepatocellular carcinoma (HCC). While these clinical consequences of cirrhosis are well described, it remains poorly understood whether hepatic insufficiency and the accompanying elevations in ammonia contribute to HCC carcinogenesis. Using preclinical models, we discovered that ammonia entered the cell through the transporter SLC4A11 and served as a nitrogen source for amino acid and nucleotide biosynthesis. Elevated ammonia promoted cancer stem cell properties in vitro and tumor initiation in vivo. Enhancing ammonia clearance reduced HCC stemness and tumor growth. In patients, elevations in serum ammonia were associated with an increased incidence of HCC. Taken together, this study forms the foundation for clinical investigations using ammonia-lowering agents as potential therapies to mitigate HCC incidence and aggressiveness.

Keywords: Amino acid metabolism; Liver cancer; Metabolism; Oncogenes; Oncology.

Figure 1. Elevated ammonia is associated with an increased incidence of and poor prognosis in HCC.

(A) CONSORT diagram of patient population. (B) Scatter plot depicting correlation between mean ammonia concentration and HCC incidence fit using linear regression of 95% of the 48,476-patient cohort with mean ammonia concentrations ≤ 100 μM/L. (C) Two-year landmark analysis of HCC incidence in patients with high and low ammonia using propensity score matching. (D) OS of patients with high and low ammonia using propensity score matching. Hazard ratio log-rank test, P values, and 95% confidence intervals indicated.

Figure 2. Ammonia promotes the acquisition of cancer stem cell properties in vitro.

(A–C) Representative bright-field micrographs, number of hepatospheres, and diameter of hepatospheres formed with and without ammonium chloride (10 mM) in HepG2 (A), HUH7 (B), and mHCC (C) cells. Data are shown as mean ± SD (n = 7–9). Scale bar: 1,000 μm. (D and E) CD44 mRNA expression in HepG2 (D) and HUH7 (E) cells with and without ammonium chloride (10 mM). Data are shown as mean ± SD (n = 3). (F) CD44 surface expression by flow cytometry in control and ammonium chloride–treated (10 mM) mHCC hepatospheres. Mean fluorescence intensity (MFI) fold change ± SD (n = 3). (G–I) ALDH activity in HepG2 (G), HUH7 (H), and mHCC (I) hepatospheres with and without ammonium chloride (10 mM) (n = 3). *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005 by 2-tailed t test.

Figure 3. Ammonia contributes to tumor initiation in vivo.

(A and B) Control and ammonium chloride–treated (10 mM) hepatospheres derived from HepG2 (A) and mHCC (B) cells were dissociated into single cells and implanted into NSG mice at the indicated cell numbers (n = 12 tumors per arm). Tumor initiation and TIC frequency was quantified using extreme limiting dilution analysis. (C and D) HepG2 tumor volume and tumor weight was quantified in the 500-cell titration group. (E and F) mHCC tumor volume and tumor weight was quantified in the 500-cell titration group. Data are represented as means ± SD. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005 by 2-tailed t test.

Figure 4. SLC4A11 functions as an ammonia importer in hepatocellular carcinoma stem cells.

(A) SLC4A11 mRNA expression was quantified in 2D versus 3D culture of HepG2 (left) and HUH7 (right) cells. Data are shown as mean ± SD (n = 3). (B) SLC4A11 mRNA expression was quantified in HepG2 (left) and HUH7 (right) hepatospheres with and without ammonium chloride (10 mM). Data are shown as mean ± SD (n = 3). (C and D) SLC4A11 was depleted in HepG2 (C) and mHCC (D) cells by Crispr/Cas9 using 2–3 independent gRNAs, and ammonia concentration in control and SLC4A11-KO hepatospheres with and without ammonium chloride (10 mM) was quantified. Data are shown as mean ± SD (n = 3). *P ≤ 0.05, **P ≤ 0.005 by 2-tailed t test (A–D). (E) tdTomato-tagged SLC4A11 was overexpressed in mHCC cells and ammonia concentration in control, and SLC4A11 overexpression hepatospheres with and without ammonium chloride (10 mM) was quantified. Data are shown as mean ± SD (n = 3). ***P ≤ 0.0005 by 1-way ANOVA for the entire group with multiple comparisons using Tukey’s test.

Figure 5. SLC4A11-mediated ammonia transport sustains a cancer stem cell phenotype.

(A and B) Hepatosphere number in control and SLC4A11 KO mHCC (A) and HepG2 (B) cells with and without ammonium chloride (10 mM). Data are shown as mean ± SD (n = 3). *P ≤ 0.05, **P ≤ 0.005 by 2-tailed t test (A and B). (C) Hepatosphere number in control and SLC4A11 overexpressing mHCC cells with and without ammonium chloride (10 mM). Data are shown as mean ± SD (n = 3). (D) CD44 mRNA expression and (E) ALDH activity in control and SLC4A11 KO mHCC hepatospheres in the presence of ammonium chloride (10 mM). Data are shown as mean ± SD (n = 3). *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005 by 1-way ANOVA with multiple comparisons using Tukey’s test (C–E).

Figure 6. Ammonia augments amino acid and nucleotide biosynthesis in a SLC4A1-dependent manner.

(A) Schematic depicting LC-MS–based nitrogen tracing in control and SLC4A11-KO HepG2 hepatospheres with and without 8 hours of ammonium chloride (10 mM) (created with BioRender.com). (B) Isotopologue enrichment of top 30 metabolites in control and SLC4A11-KO HepG2 hepatospheres (background subtracted from ¹⁵NH₄Cl-treated samples, n = 3). (C) KEGG gene set enrichment analysis for 15 pathways significantly enriched by hypergeometric testing (n = 3). (D and E) Normalized isotopologue enrichment of representative amino acids and nucleotides in control and SLC4A11-KO HepG2 hepatospheres. *P ≤ 0.05 by 2-tailed t test.

Figure 7. Ammonia clearance reduces tumor burden and cancer stem cell markers in vivo.

(A) Ammonia concentration in livers of control and HCC tumor–bearing C57BL/6J mice following hydrodynamic transfection of Myc, gp53/Cas9, and sleeping beauty transposase. Data are shown as mean ± SD (n = 9 per arm). (B–F) Representative photo and liver weight (B), ammonia concentration (C), CD44 mRNA expression (D), SLC4A11 mRNA expression (E), and ALDH activity (F) of tumor-bearing C57BL/6J mice generated by hydrodynamic tail vein injection with and without ornithine treatment. Data are shown as mean ± SD (n = 8 per arm). *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005 by 2-tailed t test (A–F). (G and H) Representative photo and tumor weight (G), and ammonia concentration (H) of mHCC tumors established in NSG mice with the indicated conditions. Data are shown as mean ± SD (n = 10 per arm). *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005 by 1-way ANOVA with multiple comparisons using Tukey’s test.