Dual Effect of Combined Metformin and 2-Deoxy-D-Glucose Treatment on Mitochondrial Biogenesis and PD-L1 Expression in Triple-Negative Breast Cancer Cells

SIMPLE SUMMARY: Metformin and 2-deoxy-D-glucose are metabolic drugs with multiple and incompletely understood anti-cancer effects. Their combination can cause breast cancer cell detachment from the growth surface. Mitochondria are important for detached cell survival and metastasis, but how metformin and 2DG affect cancer mitochondria is largely unknown. We found that metformin and 2-deoxy-D-glucose together increased mitochondrial mass in triple-negative breast cancer cells due to the enlargement of mitochondria, and did not decrease their degradation. Both the reduction in protein-attached sugars and reduced ATP production seemed to be involved in triggering the process. Metformin and 2-deoxy-D-glucose can reduce immune checkpoint PD-L1 levels, responsible for immune escape. We found that the reduction in protein-attached sugars caused by metformin and 2DG also reduced PD-L1 levels on breast cancer cells and its partner receptor PD-1 on activated T cells. While the activation of T cells was reduced, they mostly maintained their effector functions. Metformin and 2-deoxy-D-glucose could therefore potentially improve anti-cancer immunity. ABSTRACT: Metformin and 2-deoxy-D-glucose (2DG) exhibit multiple metabolic and immunomodulatory anti-cancer effects, such as suppressed proliferation or PD-L1 expression. Their combination or 2DG alone induce triple-negative breast cancer (TNBC) cell detachment, but their effects on mitochondria, crucial for anchorage-independent growth and metastasis formation, have not yet been evaluated. In the present study, we explored the effects of metformin, 2DG and their combination (metformin + 2DG) on TNBC cell mitochondria in vitro. Metformin + 2DG increased mitochondrial mass in TNBC cells. This was associated with an increased size but not number of morphologically normal mitochondria and driven by the induction of mitochondrial biogenesis rather than suppressed mitophagy. 2DG and metformin + 2DG strongly induced the unfolded protein response by inhibiting protein N-glycosylation. Together with adequate energy stress, this was one of the possible triggers of mitochondrial enlargement. Suppressed N-glycosylation by 2DG or metformin + 2DG also caused PD-L1 deglycosylation and reduced surface expression in MDA-MB-231 cells. PD-L1 was increased in low glucose and normalized by both drugs. 2DG and metformin + 2DG reduced PD-1 expression in Jurkat cells beyond the effects on activation, while cytokine secretion was mostly preserved. Despite increasing mitochondrial mass in TNBC cells, metformin and 2DG could therefore potentially be used as an adjunct therapy to improve anti-tumor immunity in TNBC.