Abstract
Simple SummaryMetformin 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.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.
Highlights
The emerging importance of energy metabolism in cancer has led to the investigation of metabolic drugs as potential anti-cancer therapies [1,2]
To quantify the effect of metformin and 2DG on mitochondrial mass, MDA-MB231 cells were stained with nonyl acridine orange (NAO), a potential-independent dye that binds cardiolipin (Figure 1A)
Orange, which accumulates in the polarized mitochondria, revealed a 125% increase in mitochondrial mass in 4.8DG and 150% in Met + 0.6DG treated cells (Figure 1B)
Summary
The emerging importance of energy metabolism in cancer has led to the investigation of metabolic drugs as potential anti-cancer therapies [1,2]. Metformin acts systemically by inhibiting gluconeogenesis in the liver and improving glycemic control, but can act directly on cancer cells by inhibiting complex I of the respiratory electron transport chain (ETC) [6], which leads to AMPK activation [7]. Metformin can act independently of AMPK activation, for example, by inhibiting mTOR signaling and inducing cell cycle arrest [8,9], and by its direct effects on cancer cells metabolism, suppressing biosynthetic reactions that rely on reduced cofactors and reduced nucleotide levels [9–11]. The induction of mitochondrial biogenesis could importantly influence the response of cancer cells to metabolic drugs such as metformin, as additional respiratory capacity could render cells more resilient to energy stress, as has been demonstrated for breast cancer’s resistance to metformin in vivo [15]. Few studies to date have evaluated the effect of metformin on mitochondrial biogenesis in cancer cells
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