Glucose-6-phosphate dehydrogenase blockade potentiates tyrosine kinase inhibitor effect on breast cancer cells through autophagy perturbation

AbstractBackground: Glucose-6-phospate dehydrogenase (G6PD) is the limiting enzyme of the pentose phosphatepathway (PPP) correlated to cancer progression and drug resistance. We previously showed that G6PD inhibitionleads to Endoplasmic Reticulum (ER) stress often associated to autophagy deregulation. The latter can be inducedby target-based agents such as Lapatinib, an anti-HER2 tyrosine kinase inhibitor (TKI) largely used in breast cancertreatment.Methods: Here we investigate whether G6PD inhibition causes autophagy alteration, which can potentiateLapatinib effect on cancer cells. Immunofluorescence and flow cytometry for LC3B and lysosomes tracker wereused to study autophagy in cells treated with lapatinib and/or G6PD inhibitors (polydatin). Immunoblots for LC3Band p62 were performed to confirm autophagy flux analyses together with puncta and colocalization studies. Wegenerated a cell line overexpressing G6PD and performed synergism studies on cell growth inhibition induced byLapatinib and Polydatin using the median effect by Chou-Talay. Synergism studies were additionally validated withapoptosis analysis by annexin V/PI staining in the presence or absence of autophagy blockers.Results: We found that the inhibition of G6PD induced endoplasmic reticulum stress, which was responsible forthe deregulation of autophagy flux. Indeed, G6PD blockade caused a consistent increase of autophagosomesformation independently from mTOR status. Cells engineered to overexpress G6PD became resilient to autophagyand resistant to lapatinib. On the other hand, G6PD inhibition synergistically increased lapatinib-induced cytotoxiceffect on cancer cells, while autophagy blockade abolished this effect. Finally, in silico studies showed a significantcorrelation between G6PD expression and tumour relapse/resistance in patients.Conclusions: These results point out that autophagy and PPP are crucial players in TKI resistance, and highlight apeculiar vulnerability of breast cancer cells, where impairment of metabolic pathways and autophagy could beused to reinforce TKI efficacy in cancer treatment.Polydatin by Chou-Talay method. Synergism studies were further validated with apoptosisanalysis by annexin V/PI staining and performed as well in presence of autophagy blockers.Results: We show that the inhibition of G6PD induces endoplasmic reticulum stress, which isresponsible for the deregulation of autophagy flux. Indeed, G6PD blockade cause a consistentincrease of autophagosomes formation independently from mTOR status. Cells engineered tooverexpress G6PD become resilient to autophagy and resistant to lapatinib. On the otherhand, G6PD inhibition synergistically increases the lapatinib cytotoxic effect on cancer cells,while autophagy blockade at an early step abolishes this effect. Conclusion: These resultspoint out that autophagy and PPP are crucial players in TKI resistance, and highlight a peculiarvulnerability of breast cancer cells, where impairment of metabolic pathways and autophagycould be used to reinforce TKI efficacy in cancer treatment.