Mitochondrial glutamine metabolism via GOT2 supports pancreatic cancer growth through senescence inhibition

Abstract

Cellular senescence, which leads to a cell cycle arrest of damaged or dysfunctional cells, is an important mechanism to restrain the malignant progression of cancer cells. Because metabolic changes underlie many cell-fate decisions, it has been suggested that cell metabolism might play key roles in senescence pathways. Here, we show that mitochondrial glutamine metabolism regulates senescence in human pancreatic ductal adenocarcinoma (PDAC) cells. Glutamine deprivation or inhibition of mitochondrial aspartate transaminase (GOT2) results in a profound induction of senescence and a suppression of PDAC growth. Glutamine carbon flow through GOT2 is required to create NADPH and to maintain the cellular redox state. We found that elevated reactive oxygen species levels by GOT2 knockdown lead to the cyclin-dependent kinase inhibitor p27-mediated senescence. Importantly, PDAC cells exhibit distinct dependence on this pathway, whereas knockdown of GOT2 did not induce senescence in non-transformed cells. The essentiality of GOT2 in senescence regulation of PDAC, which is dispensable in their normal counterparts, may have profound implications for the development of strategies to treat these refractory cancers.

Fig. 1. The Inhibition of mitochondrial glutamine metabolism induces senescence in PDAC cells

a, 8988T cells were plated in complete media which was replaced the following day with Gln-free medium. Percentages of SA-β-gal positive cells are shown. b, Representative images of SA β-gal staining for 8988T cells in the presence or absence of Gln. c, Percentages of SA-β-gal positive cells in 8988T cells treated with increasing concentrations of DON. d, Percentages of SA-β-gal positive cells in control (shGFP) and GLS knockdown (shGLS) 8988T cells (left). Western blot confirmed knockdown of GLS expression (right). β-actin serves as a loading control. All error bars ± SEM. **p < 0.01 and ***p < 0.001

Fig. 2. Mitochondrial transaminase GOT2 is responsible for the regulation of PDAC senescence

a, Senescence induction of 8988T cells treated with EGCG (50 μM) or AOA (0.5 mM). b and c, Senescence induction of 8988T cells expressing a control shRNA (shGFP) or two independent shRNAs targeting GLUD1 b, or GOT2 c. d, Relative proliferation of 8988T cells expressing a control shRNA or shRNAs to GOT2. e, Percentage of cells in G0/G1, S and G2/M phases in 8988T cells expressing a control shRNA or a shRNA to GOT2. f, Percentages of SA-β-gal positive cells in GOT2 overexpressed (GOT2-OE) 8988T cells expressing a control shRNA or shRNAs to GOT2. g, Numbers of promyelocytic leukemia nuclear bodies (PML-NBs; left) and immunofluorescent staining using nuclear (DAPI; blue) and PML (green) in 8988T cells expressing a control shRNA or shRNAs to GOT2 (right).All error bars ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 3. GOT2 regulates senescence in PDAC cells but not in normal cells

a and b, Senescence induction a, and relative proliferation b, of PDAC cell lines (Panc1, PL45 and Tu8902) expressing a control shRNA (shGFP) or two independent shRNAs to GOT2. c, Percentages of SA-β-gal positive cells in HPDE, WI38 and HEK293T cells expressing a control shRNA (shGFP) or two independent shRNAs to GOT2. All error bars ± SEM. **p < 0.01 and ***p < 0.001

Fig. 4. GOT2 suppresses PDAC senescence by regulating cellular ROS

a, Relative ROS levels in 8988T cells expressing a control shRNA (shGFP) or a shRNA to GOT2 with or without NAC (10 mM). b, Senescence induction of 8988T cells expressing a control shRNA or two independent shRNAs to GOT2 supplemented with or without NAC. c, Senescence induction in control or AOA treated 8988T cells cultured with or without NAC. d, NADPH/NADP⁺ ratio in 8988T cells expressing a control shRNA or shRNAs to GOT2 supplemented with or without OAA (4 mM). e and f, Senescence induction e, and relative proliferation f, of 8988T cells expressing a control shRNA or two independent shRNAs to GOT2 supplemented with or without OAA (4 mM, or Asp (4 mM. All error bars ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 5. p27 is responsible for GOT2 knockdown-mediated senescence induction in PDAC cells

a, Percentages of SA-β-gal positives cells in 8988T cells expressing a control shRNA or two independent shRNAs to GOT2 supplemented with or without PFT-α. b, Relative mRNA levels of indicated genes in 8988T cells expressing a control shRNA or a shRNA to GOT2. c, p21 and p53 protein levels in whole-cell lysates from 8988T cells expressing a control shRNA or shRNAs to GOT2. β-actin serves as a loading control. d, The effect of GOT2 knockdown on p27 protein levels in 8988T cells expressing a control shRNA or shRNAs to GOT2. e and f, p27 protein levels in 8988T cells expressing a control shRNA or a shRNA to GOT2 supplemented with or without NAC e, OAA or Asp f. g, Percentages of SA-β-gal positive cells in control and GOT2 knockdown 8988T cells transfected with p27 or control siRNA as indicated. h, A proposed model depicting the regulation of PDAC senescence by GOT2. All error bars ± SEM. **p < 0.01 and ***p < 0.001