Figure 1. Co-occurrence of ADHFE1 and MYC amplifications in human breast tumors and accelerated tumor growth of ADHFE1-overexpressing MCF7 cells.

(A) Accumulation of D- and L-2-hydroxyglutarate and their ratio (D to L ratio above bars) in 15 human breast tumors and 4 adjacent noncancerous breast tissues (N). (B) Co-occurrence of ADHFE1 and MYC amplifications in human breast tumors (P < 0.001). Shown are 473 TCGA breast tumors with amplification (dark red) or overexpression (light red) of 1 of the 5 listed genes previously associated with D-2HG. Blue bars: deletion or reduced expression. IDH, isocitrate dehydrogenase; PHGDH, phosphoglycerate dehydrogenase. (C) High ADHFE1 protein expression by immunohistochemistry (IHC) is associated with decreased survival of patients with ER-negative breast cancer. P = 0.012 by the log-rank test. HR, hazard ratio. (D) Western blots. Top left panel: MYC upregulates ADHFE1. MYC signaling was induced (MYC-ER fusion in HMEC-MYC) or suppressed (inducible shRNA in SUM159T) with 4-hydroxytamoxifen (+) or doxycycline (+), respectively. HMEC, human mammary epithelial cell. Lower panel: ADHFE1 transgene expression in MCF10A and MCF12A cells increases MYC. Right panel: Induction of aldo-keto reductase AKR7A2, but not AKR1A1, in HMEC-MYC cells after 4-hydroxytamoxifen–stimulated MYC signaling (+TAM). (E) Increased tumor growth of MCF7 cells over-expressing either ADHFE1, MYC, or both ADHFE1 and MYC (ADHFE1-MYC). Solid lines show median for each group. Tumor growth was measured 16 weeks after injection of MCF7 cells into mammary fat pads. Two-sided t test for comparisons with control group (n = 10 per group). P = 0.05 for MYC versus ADHFE1-MYC tumors. (F) Increased 2-hydroxyglutarate (2HG) and 4-hydroxybutyrate (4HB) levels in MCF7 tumors overexpressing ADHFE1 and MYC (n = 10 per group). *P < 0.05, **P < 0.01, versus control group using 2-sided t test. Shown is the mean ± SD. ANOVA test for differences between groups was used in E and F: P < 0.001.