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Science 338 (6110): 1069-1072

Copyright © 2012 by the American Association for the Advancement of Science

SAICAR Stimulates Pyruvate Kinase Isoform M2 and Promotes Cancer Cell Survival in Glucose-Limited Conditions

Kirstie E. Keller1, Irene S. Tan2, and Young-Sam Lee1,*

1 Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
2 National Institutes of Health–Johns Hopkins University Graduate Partnerships Program, Baltimore, MD 21218, USA.


Figure 1
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Fig. 1. Identification of SAICAR as a regulator of PKM2. (A) LC-MS total ion current chromatographs of PKM2-copurified metabolites from glucose-rich (top) and glucose-free cells (bottom). A metabolite further characterized (fig. S1) is noted with an asterisk (*). A549 cells were incubated in fresh Dulbecco’s modified Eagle’s medium (10% dialyzed fetal bovine serum) containing either 25 mM or no glucose for 30 min, and used for metabolite extraction as described in materials and methods. Extracted metabolites were mixed with recombinant human PKM2 for 30 min at 37°C, cooled on ice, and subjected to gel-filtration chromatography (exclusion limit: 10 kD) at 4°C. Metabolites that copurified with PKM2 were subjected to LC-MS analysis (C18, electrospray ionization, positive mode). (B) Structure of SAICAR. (C) Effect of enzymatically synthesized SAICAR (fig. S2) on PK activities of (bullet) PKM2 and (o) PKM1. Data are means ± SD (n = 3 independent experiments).

 

Figure 2
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Fig. 2. SAICAR binds to PKM2 in glucose-depleted cancer cells. (A) Top: Cellular concentration of SAICAR in HeLa cells in response to glucose depletion. HeLa cells were incubated in glucose-free media, and the time-course changes in SAICAR concentration were measured by LC-MS as described in materials and methods. Bottom: Pyruvate and lactate concentration changes are also noted. (B) Effect of extracellular glucose concentration on cellular SAICAR amount. Cellular concentration of SAICAR was measured in (o) HeLa and in ({nabla}) NIH 3T3 cells 30 min after incubation in media with various glucose concentrations. (C) Effect of cell type. HeLa, H1299, HuMEC, and HuALF were incubated in 25 mM (filled bars) or in no glucose media (gray bars) for 30 min and used for the analysis of SAICAR level. Data are means ± SD (n = 3). Other cancer cell lines tested (A549 and U87) also showed glucose-depletion–dependent increase of cellular SAICAR level. (D) LC-MS peak area of SAICAR copurified with PKM1, PKM2, or StrepII tag from H1299 cells incubated for 30 min in fresh 25 mM or no glucose media. (E) Amount of FBP that copurified with the protein of interest analyzed as in (D). n.d., not detectable.

 

Figure 3
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Fig. 3. SAICAR level affects glucose metabolism, cancer cell survival, and proliferation. (A) A schematic diagram showing the synthesis and metabolism of SAICAR. (B) Cellular levels of SAICAR in HeLa control-kd (black bars), adsl-kd (open bars), and paics-kd (gray bars) before and after 30 min of incubation in glucose-free media. Data are the means ± SD (n = 3). (C and D) Effect of SAICAR on (C) glucose uptake and (D) lactate fermentation. The amount of glucose and lactate in the media was measured by enzymatic assays and LC-MS analysis, respectively (normalized with total cellular protein concentration). Bars denote means ± SD, respectively. (E) Survivals of HeLa control-kd (control; cells transfected with random shRNA vector), adsl-kd, paics-kd, and paics-kd adsl-kd cells in glucose-free media were measured by a Trypan blue exclusion method. Data are means ± SD (n = 3, where >50 cells were counted to calculate the percentage of live cells in each measurement).

 

Figure 4
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Fig. 4. SAICAR-PKM2 interaction is responsible for the promotion of cancer cell survival. (A and B) Identification of a PKM2 mutant insensitive to SAICAR. Effect of (A) SAICAR and (B) FBP on pyruvate kinase activities of PKM2 Q393K (Gln393->Lys) (o) and PKM2 K433E (Lys433->Glu) (bullet) mutants are shown. Data are means ± SD (n = 3). (C and D) Cell viability in glucose-depletion conditions was measured as in Fig. 3E in HeLa cells expressing (C) PKM2* and (D) PKM2* Q393K mutant while endogenous PKM2 expression is knocked down.Data are means ± SD (n = 3, >50 cells were counted for each measurement). Control: cells transfected with scrambled shRNA vectors.

 


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