A metabolic shift toward glycolysis enables cancer cells to maintain survival upon concomitant glutamine deprivation and V-ATPase inhibition.

It is widely known that most cancer cells display an increased reliance on glutaminolysis to sustain proliferation and survival. Combining glutamine deprivation with additional anti-cancer therapies is an intensively investigated approach to increase therapeutic effectiveness. In this study, we examined a combination of glutamine deprivation by starvation or pharmacological tools, with the anti-cancer agent archazolid, an inhibitor of the lysosomal V-ATPase. We show that glutamine deprivation leads to lysosomal acidification and induction of pro-survival autophagy, which could be prevented by archazolid. Surprisingly, a combination of glutamine deprivation with archazolid did not lead to synergistic induction of cell death or reduction in proliferation. Investigating the underlying mechanisms revealed elevated expression and activity of amino acid transporters SLC1A5, SLC38A1 upon starvation, whereas archazolid had no additional effect. Furthermore, we found that the export of lysosomal glutamine derived from exogenous sources plays no role in the phenotype as knock-down of SLC38A7, the lysosomal glutamine exporter, could not increase V-ATPase inhibition-induced cell death or reduce proliferation. Analysis of the cellular metabolic phenotype revealed that glutamine deprivation led to a significant increase in glycolytic activity, indicated by an elevated glycolytic capacity and reserve, when V-ATPase function was inhibited concomitantly. This was confirmed by increased glutamine uptake, augmented lactate production, and an increase in hexokinase activity. Our study, therefore, provides evidence, that glutamine deprivation induces autophagy, which can be prevented by simultaneous inhibition of V-ATPase function. However, this does not lead to a therapeutic benefit, as cells are able to circumvent cell death and growth inhibition by a metabolic shift toward glycolysis.

Tropomyosin concentration but not formin nucleators mDia1 and mDia3 determines the level of tropomyosin incorporation into actin filaments.

The majority of actin filaments in human cells exist as a co-polymer with tropomyosin, which determines the functionality of actin filaments in an isoform dependent manner. Tropomyosin isoforms are sorted to different actin filament populations and in yeast this process is determined by formins, however it remains unclear what process determines tropomyosin isoform sorting in mammalian cells. We have tested the roles of two major formin nucleators, mDia1 and mDia3, in the recruitment of specific tropomyosin isoforms in mammals. Despite observing poorer cell-cell attachments in mDia1 and mDia3 KD cells and an actin bundle organisation defect with mDia1 knock down; depletion of mDia1 and mDia3 individually and concurrently did not result in any significant impact on tropomyosin recruitment to actin filaments, as observed via immunofluorescence and measured via biochemical assays. Conversely, in the presence of excess Tpm3.1, the absolute amount of Tpm3.1-containing actin filaments is not fixed by actin filament nucleators but rather depends on the cell concentration of Tpm3.1. We conclude that mDia1 and mDia3 are not essential for tropomyosin recruitment and that tropomyosin incorporation into actin filaments is concentration dependent.