Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor

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Abstract

Cellular amino acid uptake is critical for mTOR complex 1 (mTORC1) activation and cell proliferation. However, the regulation of amino acid uptake is not well-understood. Here we describe a role for asparagine as an amino acid exchange factor: intracellular asparagine exchanges with extracellular amino acids. Through asparagine synthetase knockdown and altering of media asparagine concentrations, we show that intracellular asparagine levels regulate uptake of amino acids, especially serine, arginine and histidine. Through its exchange factor role, asparagine regulates mTORC1 activity and protein synthesis. In addition, we show that asparagine regulation of serine uptake influences serine metabolism and nucleotide synthesis, suggesting that asparagine is involved in coordinating protein and nucleotide synthesis. Finally, we show that maintenance of intracellular asparagine levels is critical for cancer cell growth. Collectively, our results indicate that asparagine is an important regulator of cancer cell amino acid homeostasis, anabolic metabolism and proliferation.

Figures

  • Figure 1 | Resistance to glutamine withdrawal or glutaminase inhibition causes cellular asparagine dependence. (a–d) Proliferation curves of LPS2 parental, LPS2 glutamine (Q)-independent, SUM159PT parental and SUM159PT CB-839-resistant cells in the presence or absence of 0.1mM asparagine (N) in the medium. (e) Percentages of intracellular 13C-labelled asparagine in LPS2 parental and glutamine-independent, as well as SUM159PT parental and CB-839-resistant cells labelled with U-13C-asparagine in the medium for 24 h, as determined by LC-MS. (f) The per cent change in medium asparagine levels as determined by LC-MS after 24-h incubation time for the indicated cells or for medium in an empty tissue culture plate (blank). Error bars denote s.d. of the mean (n¼ 3). P values were calculated by the Student’s t-test: *Po0.05; **Po0.01; ***Po0.001; NS, not significant.
  • Figure 2 | Asparagine levels regulate cell proliferation. (a) Relative growth rates of cancer cell lines stably expressing scrambled shRNA (Scr) or ASNS shRNA. Growth rates are normalized to the scrambled shRNA control for each cell line. (b) Immunoblot showing ASNS protein levels on stable expression of scrambled (Scr) or ASNS shRNA in cell lines shown in a. (c) Relative growth rates of HeLa cells stably expressing scrambled shRNA (Scr) or ASNS
  • Figure 3 | Asparagine is an amino acid exchange factor. Relative glutamine (a) and asparagine (b) levels in the medium from LPS2 cells, as measured by LC-MS, before and after amino acid (AA) stimulation following pre-loading of the cells with glutamine (Q) and/or asparagine (N). Serum- and amino acid-starved LPS2 cells were pre-loaded with 2mM glutamine, 2mM asparagine or 2mM glutamine and 2mM asparagine for 60min prior to stimulation for 30min with an amino acid mixture (AA medium) lacking glutamine and asparagine. ‘Blank’ indicates measurements from plates lacking cells. ‘No Pre’ indicates measurements from plates of LPS2 cells not pre-loaded with glutamine or asparagine. Relative intracellular glutamine (c) and asparagine
  • Figure 4 | Asparagine levels regulate serine uptake and serine metabolism gene expression. (a) Asparagine levels in the media, measured by LC-MS, from serum- and amino acid-starved LPS2 cells pre-loaded with asparagine (Pre N) and unstimulated (No AA) or stimulated with different amino acid sub-categories: non-polar, basic or Ser/Thr. Non-polar amino acids include leucine, isoleucine, methionine, tryptophan and phenylalanine; basic amino acids include lysine, arginine and histidine; Ser/Thr includes serine and threonine. (b) Relative intracellular asparagine levels in amino acid-starved glutamineindependent LPS2 cells as measured by LC-MS following 1-, 5- or 120-min stimulation with complete glutamine-free medium. Prior to stimulation, cells were either pre-loaded with asparagine (Pre N) or starve medium (No pre). Declining intracellular asparagine levels over time indicates exchange with
  • Figure 5 | Asparagine regulates mTORC1 activation and autophagy. (a) Immunoblotting of lysates from serum- and amino acid-starved LPS2 parental cells pre-loaded with starve medium (No pre), glutamine (Pre Q) or asparagine (Pre N), followed by amino acid stimulation for 0, 5 or 15min. Lysates were probed with a phospho-specific antibody towards the mTOR target S6K at T389 and with anti-tubulin. (b) Immunoblot comparing ASNS levels in lysates from LPS2, HeLa and A431 cells under non-starved conditions. (c) Immunoblot showing phosphorylation of downstream mTOR effector S6 ribosomal protein (S235/236) following starvation of LPS2 parental cells of glutamine ( Q) or asparagine ( N) for the indicated times. ‘þ ’ indicates no starvation. (d) Immunoblot showing S6 ribosomal protein phosphorylation (S235/236) following glutamine and asparagine starvation of LPS2 parental cells for
  • Figure 6 | Asparagine levels regulate mRNA translation. (a) Immunoblot of HeLa lysates with (shASNSþDox) and without (shASNS Dox) a 48-h induction of ASNS shRNA expression and HeLa lysates 48 h after doxycycline induction of scrambled shRNA (Scr) or ASNS shRNA (shASNS). Lysates were immunoblotted for ASNS, phospho-4E-BP1 (S65), total 4E-BP1, phospho-S6K (T389), total S6K, phospho-S6 (S235/236), total S6 and tubulin. Green arrow indicates phosphorylated form of 4E-BP1; red arrow indicates unphosphorylated form. (b) Immunoblot showing levels of eIF4E and 4E-BP1
  • Figure 7 | Asparagine levels regulate nucleotide synthesis. (a) Immunoblot of HeLa lysates 48 h post doxycycline-induced expression of scrambled shRNA or ASNS shRNA in asparagine-free DMEM ( Asn) or DMEM supplemented with 0.1mM asparagine (þAsn). Lysates were immunoblotted for mTOR activity marker pS6K, phospho-CAD (S1859), total CAD and tubulin. (b) Immunoblot of HeLa lysates with (shASNSþDox) and without
  • Figure 8 | Asparagine coordinates protein and nucleotide synthesis.

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APA

Krall, A. S., Xu, S., Graeber, T. G., Braas, D., & Christofk, H. R. (2016). Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor. Nature Communications, 7. https://doi.org/10.1038/ncomms11457

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