Drug testing in-vitro has traditionally been performed in hyperglycemic cell culture concentrations, yet many tumours and their microenvironments are hypoglycemic. Here, we tested whether reduced glucose levels can influence drug resistance and investigated underlying mechanisms. PIK3CA mutant (AGS, HGC27), and wildtype (MKN45, NUGC4) gastric cancer (GC) cells were cultured in high glucose (HG, 25mM) or low glucose (LG, 5mM) media and tested for sensitivity to two cytotoxic compounds, 5-Fluorouracil and carboplatin, the PI3K/mTOR inhibitor, PI103, and the mTOR inhibitor, Ku-0063794. All cells had increased resistance to 5-Fluorouracil and carboplatin when cultured in LG compared to HG conditions despite having similar growth and cell cycle characteristics. For PI103 and Ku-0063794, only the PIK3CA mutant cells displayed increased resistance in LG conditions. The PIK3CA mutant LG cells had selectively increased p-mTOR, p-S6, p-4EBP1, GLUT1, and lactate production, and reduced reactive oxygen species, consistent with increased glycolysis. Combination analysis indicated PI103 and Ku-0063794 were synergistic in PIK3CA mutant LG cells only. Synergism was accompanied by reduced mTOR signalling and increased autophagy. Hypoglycaemia can increase drug resistance, especially in cells with a high dependence on glycolysis. Dual inhibition of the PI3K/mTOR pathway may be able to attenuate hypoglycemia-associated resistance.
Figure 6. Model of “synthetic lethal addiction” to rationalize selective synergy of PI103 and Ku-0063794 in PIK3CA mutant cells cultured in LG conditions. In PIK3CA wildtype cells, PI3K activates AKT, mTOR, S6, 4EBP1, protein translation and glycolysis (black arrows). In PIK3CA mutant cells, additional activation of AKT, mTOR, S6, 4EBP1, protein translation and glycolysis occurs (red arrows). Under glucose deprivation and in cells with enhanced glycolysis, GLUT1 levels are increased, leading to activation of mTOR signalling, protein translation and glycolysis (blue arrows). Synergy between PI103 and Ku-0063794 is observed only in PIK3CA mutant cells grown in LG conditions as these cells are dependent on hyperactivation of the PI3K/mTOR pathway. PIK3CA mutant cells cultured in HG conditions or PIK3CA wildtype cells in LG or HG conditions are less dependent on PI3K/mTOR activation, and hence the dual blockade of the PI3K/mTOR pathway is less detrimental. Autophagy occurs in cells with synergy due to the inhibition of mTOR.
B Bhattacharya1, S H H Low1, C Soh2, N Kamal Mustapa2, M Beloueche-Babari3, K X Koh1, J Loh1, R Soong1, 4*
1 Cancer Science Institute of Singapore, National University of Singapore, Singapore
2 Department of Pharmacy, National University of Singapore, Singapore
3 Division of Radiotherapy and Imaging, The Institute of Cancer Research & Royal Marsden NHS Foundation Trust Sutton, Surrey, United Kingdom
4 Department of Pathology, National University Health System, Singapore