Abstract

The cellular energy sensor AMP-activated protein kinase (AMPK) is a metabolic hub regulating various pathways involved in tumor metabolism. Here we report that vacuolar H+-ATPase (V-ATPase) inhibition differentially affects regulation of AMPK in tumor and nontumor cells and that this differential regulation contributes to the selectivity of V-ATPase inhibitors for tumor cells. In nonmalignant cells, the V-ATPase inhibitor archazolid increased phosphorylation and lysosomal localization of AMPK. We noted that AMPK localization has a prosurvival role, as AMPK silencing decreased cellular growth rates. In contrast, in cancer cells, we found that AMPK is constitutively active and that archazolid does not affect its phosphorylation and localization. Moreover, V-ATPase-independent AMPK induction in tumor cells protected them from archazolid-induced cytotoxicity, further underlining the role of AMPK as a prosurvival mediator. These observations indicate that AMPK regulation is uncoupled from V-ATPase activity in cancer cells and that this makes them more susceptible to cell death induction by V-ATPase inhibitors. In both tumor and healthy cells, V-ATPase inhibition induced a distinct metabolic regulatory cascade downstream of AMPK, affecting ATP and NADPH levels, glucose uptake, and reactive oxygen species production. We could attribute the prosurvival effects to AMPK's ability to maintain redox homeostasis by inhibiting reactive oxygen species production and maintaining NADPH levels. In summary, the results of our work indicate that V-ATPase inhibition has differential effects on AMPK-mediated metabolic regulation in cancer and healthy cells and explain the tumor-specific cytotoxicity of V-ATPase inhibition.

Highlights

  • The cellular energy sensor AMP-activated protein kinase (AMPK) is a metabolic hub regulating various pathways involved in tumor metabolism

  • We found that all tumor cells had constitutively activated AMPK and that archazolid had no effect on the activation level (Fig. 1A and Fig. S1A)

  • We found that tumor cells had an increased level of total AMPK and a slightly higher level of phosphorylated AMPK (Fig. 1D and Fig. S1D), indicating that AMPK activation is beneficial for cancer cells

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Summary

Results

To test whether inhibition of V-ATPase leads to AMPK activation, we treated different tumor (MDA-MB-231, MCF7, T24, and HUH7) and nontumor (HEK293, MCF10A, and HMLE) cells with archazolid and analyzed phosphorylation of AMPK on Thr-172. There was activation of AMPK in all tested cell lines and no difference between tumor and nontumor cells These results suggest that the differential effect seen on AMPK phosphorylation is not induced by a general stress response but specific for V-ATPase inhibition. To transfer of LKB1 to the lysosome in HEK293 cells, in contrast to tumor cells, where no difference between treated and nontreated cells could be observed, as shown by confocal microscopy (Fig. 2C) These results further suggest that there is a difference in AMPK activation by V-ATPase inhibition in tumor and nontumor cells. The tumor cell lines showed only a slight effect (MDAMB-231) or even a decrease (MCF7) in glucose uptake (Fig. 3, B and C), indicating distinct metabolic regulation of tumor and nontumor cells by V-ATPase inhibition

AMPK activation protects nontumor cells from archazolidinduced cytotoxicity
Discussion
Cell culture and compounds
Isolation of lysosomes
Apoptosis assay
Cytotoxicity assay
Glucose uptake
ROS measurement
Full Text
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