Ammonium regulates mTOR signalling

mTORC1 and mTORC2 are two distinct mammalian TOR (target of rapamycin) complexes that regulate cell growth and metabolism. In cancer, genetic alterations lead to activation of mTOR signalling impacting tumour metabolism. Upregulated glutaminolysis is part of the metabolic reaction occurring in cancer that liberates high levels of ammonium, a toxic waste product. Although the importance of glutamine as a tumour nutrient is recognized, little is known about the potential effects of ammonium produced by glutaminolysis in tumours. In their new study,  Merhi et al., 2017 identify ammonium as a dose-dependent regulator of mTORC2, mTORC1 and of proliferation in cancer cells.

To study potential signalling pathways responding to ammonium, the authors performed a kinase array with lysates of MCF-7 breast cancer cells and detected an increase in the phosphorylation of the kinases AKT-S473 and ERK1/2 upon ammonium addition in a time and dose dependent manner. This was confirmed by western blot. Similarly, the authors observed AKT phosphorylation in other cancer and fibroblast cell lines. The upstream pathway leading to the induction of AKT phosphorylation was investigated. Pre-treatment of MCF-7 cells with a PI3K inhibitor impaired AKT phosphorylation and prevented its induction by ammonium supplementation. mTORC2 has been shown to promote cell proliferation and survival by phosphorylation and activation of the AKT and SGK and the phosphorylation of AKT-S473 is a good readout for mTORC2 activity (Oh et al., 2011). Merhi et al. show that siRNA-mediated knockdown of RICTOR (a subunit of mTORC2), leads to reduction of basal and ammonium-induced AKT-S473 phosphorylation. In addition, ammonium also induced NDRG1-T346 phosphorylation, another readout of mTORC2 activity. Knockdown of YES1 kinase and pharmacological inhibition of the focal adhesion kinase (FAK) decreased ammonium-induced AKT-S473 and NDRG1-T346 phosphorylation. The authors also addressed if integrins, known regulators of FAK signalling, was involved in the ammonium-induced AKT-S473 phosphorylation. Results showed that knockdown of ITGβ1 decreased the basal and the ammonium-induced AKT-S473 phosphorylation. Collectively the data indicates that ammonium-induced activation of mTORC2 involves ITGβ1, FAK, YES1, and PI3K signalling.

Ammonium treatment has been shown to induce a transient increase in calcium in cultured astrocytes (Rose et al., 2005). To explore this, the authors assessed the role of calcium in ammonium-induced mTORC2 activation. Pre-treatment of cells with a calcium chelator decreased basal and ammonium-induced AKT-S473 and NDRG1-T346 phosphorylation. In addition, an increase in cytoplasmic calcium concentration induced a rapid increase in AKT-S473 phosphorylation in a mTORC2- dependent way, suggesting that ammonium-induced mTORC2-dependent AKT phosphorylation is modulated by intracellular calcium mobilization.

The impact of ammonium on the activity of mTORC1 remains unclear. The kinase array revealed a decreased phosphorylation of p70S6K-T389, an mTORC1 readout, after treatment with ammonium, suggesting ammonium potentially inhibits mTORC1. Western blot analysis confirmed this mild decrease in phosphorylation. However, this weak dephosphorylation appeared transient, suggesting that the inhibition of mTORC1 was quickly compensated by a reactivation. AKT activation has been shown to promote mTORC1 by inhibiting the TSC complex. In addition, AKT-mediated phosphorylation of another negative regulator of mTORC1, PRAS40, prevents its inhibitory role (Dibble et al., 2015). Merhi et al. found that ammonium induced the rapid phosphorylation of TSC2-T1462, PRAS40-T246 and of 4EBP1 – another readout of mTORC1 activity – suggesting consequent rapid mTORC1 activation. This shows an additional regulatory process upon ammonium addition that transiently counteracts the mTORC1-mediated stimulation of p70S6K-T389 phosphorylation. Treatment with an inhibitor of AKT, not only prevented the ammonium-induced phosphorylation of AKT, but also reduced the phosphorylation of TSC2, PRAS40 and 4EBP1, consistent with ammonium-induced activation of mTORC1 being AKT-dependent.

The impact of ammonium on the proliferation of MCF-7 cells was also assessed. Adding high concentrations of ammonium (~15 mM) resulted in significant cell growth inhibition while low concentrations stimulated growth both in the presence or absence of glutamine supplementation.

In summary, the authors show that ammonium triggers mTORC2-dependent phosphorylation of AKT-S473 in cancer cells. The mTORC2 activation occurs via the PI3K pathway and relies on YES1 and FAK kinases, on integrin ITGβ1 and on intracellular calcium stores mobilization. In addition, ammonium also leads to an AKT-dependent stimulation of mTORC1 signalling and to a dose-dependent stimulation of proliferation. This study identifies that ammonium, a waste product of cancer cells, impacts both mTORC2 and mTORC1 signalling and brings insights into the molecular mechanism of the ammonium-mediated regulation and tumour growth.

FLCN, the gene responsible for BHD Syndrome, has been shown to regulate mTOR signalling. Our interactive folliculin signalling diagram illustrates the relationship between Folliculin (FLCN) and several proteins and signalling pathways including mTORC1 and mTORC2.

  • Merhi A, Delrée P, & Marini AM (2017). The metabolic waste ammonium regulates mTORC2 and mTORC1 signaling. Scientific reports, 7 PMID: 28303961

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