HIF-2α expression and metabolic signaling require ACSS2 in clear cell renal cell carcinoma
Zachary A. Bacigalupa, Emily N. Arner, Logan M. Vlach, Melissa M. Wolf, Whitney A. Brown, Evan S. Krystofiak, Xiang Ye, Rachel A. Hongo, Madelyn Landis, Edith K. Amason, Kathryn E. Beckermann, W. Kimryn Rathmell, Jeffrey C. Rathmell
Zachary A. Bacigalupa, Emily N. Arner, Logan M. Vlach, Melissa M. Wolf, Whitney A. Brown, Evan S. Krystofiak, Xiang Ye, Rachel A. Hongo, Madelyn Landis, Edith K. Amason, Kathryn E. Beckermann, W. Kimryn Rathmell, Jeffrey C. Rathmell
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Research Article Cell biology Metabolism

HIF-2α expression and metabolic signaling require ACSS2 in clear cell renal cell carcinoma

Abstract

Clear cell renal cell carcinoma (ccRCC) is an aggressive cancer driven by VHL loss and aberrant HIF-2α signaling. Identifying means to regulate HIF-2α thus has potential therapeutic benefit. Acetyl-CoA synthetase 2 (ACSS2) converts acetate to acetyl-CoA and is associated with poor patient prognosis in ccRCC. Here we tested the effects of ACSS2 on HIF-2α and cancer cell metabolism and growth in ccRCC models and clinical samples. ACSS2 inhibition reduced HIF-2α levels and suppressed ccRCC cell line growth in vitro, in vivo, and in cultures of primary ccRCC patient tumors. This treatment reduced glycolytic signaling, cholesterol metabolism, and mitochondrial integrity, all of which are consistent with loss of HIF-2α. Mechanistically, ACSS2 inhibition decreased chromatin accessibility and HIF-2α expression and stability. While HIF-2α protein levels are widely regulated through pVHL-dependent proteolytic degradation, we identify a potential pVHL-independent pathway of degradation via the E3 ligase MUL1. We show that MUL1 can directly interact with HIF-2α and that overexpression of MUL1 decreased HIF-2α levels in a manner partially dependent on ACSS2. These findings identify multiple mechanisms to regulate HIF-2α stability and ACSS2 inhibition as a strategy to complement HIF-2α–targeted therapies and deplete pathogenically stabilized HIF-2α.

Authors

Zachary A. Bacigalupa, Emily N. Arner, Logan M. Vlach, Melissa M. Wolf, Whitney A. Brown, Evan S. Krystofiak, Xiang Ye, Rachel A. Hongo, Madelyn Landis, Edith K. Amason, Kathryn E. Beckermann, W. Kimryn Rathmell, Jeffrey C. Rathmell

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Figure 4

HIF-2α expression and degradation are regulated by ACSS2 activity.

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HIF-2α expression and degradation are regulated by ACSS2 activity. (A) H...
(A) Heatmap showing differential gene expression expressed as fold change (downregulated or upregulated at least 1.5-fold, adjusted P value ≤ 0.05) between control (blue; n = 3) and shACSS2 (red; n = 3) groups. Bar graph showing relative transcript counts from HIF-2α RCC signaling pathway comparing control (blue) and shACSS2 (red) groups. Statistical significance was determined using multiple unpaired, 2-tailed t tests (**P < 0.01; ***P < 0.001; ****P < 0.0001). (B) Representative Western blot analysis of HIF-2α (n = 3), ACSS2 (n = 3), and actin (n = 3) expression following 24-hour doxycycline induction of 786-O pTRIPZ control and pTRIPZ shACSS2 cells. (C) Representative Western blot analysis assessing expression of HIF-2α (n = 3), VEGFR2 (n = 2), Epo (n = 2), and actin (n = 3) in 786-O cells treated with DMSO or 1 μM, 5 μM, or 10 μM ACSS2i for 24 hours. (D) Representative Western blot analysis showing expression of ACSS2 (n = 6), HIF-2α (n = 4), VEGFR2 (n = 3), Epo (n = 3), EGLN3 (n = 3), and actin (n = 6) in 786-O cells transduced with pLX304 empty vector or pLX304 V5-ACSS2 overexpression vector. (E) Representative Western blot and bar chart quantification (n = 3) depicting HIF-2α expression in 786-O cells in the absence or presence of a 1-hour incubation with 10 μM MG132 prior to treatment with DMSO or 5 μM ACSS2i for 24 hours. (F) Western blot images analyzing LC3 A/B (n = 3), K48 polyubiquitination (n = 3), and MUL1 (n = 3) in 786-O cells treated with DMSO or 5 μM ACSS2i for 48 hours. Statistical significance was determined using 2-way ANOVA and Holm-Šidák test for multiple comparisons (*P < 0.05; **P < 0.005). Data are represented as mean ± SD. See also Supplemental Figure 2.