Selective elimination of neuroblastoma cells by synergistic effect of Akt kinase inhibitor and tetrathiomolybdate

Abstract

Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis‐activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti‐cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down‐regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells.

Neuroprotective effects of ammonium tetrathiomolybdate, a slow-release sulfide donor, in a rodent model of regional stroke

“…We previously described the unique sulfide-releasing properties of ATTM [23], a treatment used over many decades as an oral copper chelator to treat Wilson's disease and, more recently, investigated as an anti-cancer agent [42]. We could demonstrate organ protection and outcome benefit in reperfusion models of myocardial infarction, global brain injury, and resuscitated hemorrhage [23], and were thus keen to extend these results to a relevant preclinical stroke model.…”

CRISPR/Cas9 nickase mediated targeting of urokinase receptor gene inhibits neuroblastoma cell proliferation

“…Akt pathway is considered to be a clinically relevant and promising target for neuroblastoma treatment [ 4 , 67 , 68 ]. Akt is a serine/threonine kinase that is often hyperactivated in aggressive neuroblastomas, primary and metastatic tumors and cell lines [ 4 , 67 – 69 ]. Phosphorylation of Akt at Thr308 (catalytical domain) is necessary and sufficient for Akt activation, whereas phosphorylation at Ser473 (C-terminal regulatory domain) is not sufficient but is required for optimal Akt activation [ 70 ].…”

“…Akt Thr308 up-regulates mammalian target of rapamycin (mTORC1) and p70S6K, which enhance protein synthesis. Phosphorylation of Akt at Ser473 by mTORC2 promotes anti-apoptotic and cell survival pathways [ 4 , 68 , 69 ]. In vitro activation of PI3K/Akt pathway is associated with enhanced survival and proliferation of SH-SY5Y, SK-N-BE(2) and Neuro 2A neuroblastoma cell lines [ 69 , 71 ], while in vivo PI3K/Akt inhibition reduces tumor growth and MYCN protein expression [ 72 ].…”

“…Phosphorylation of Akt at Ser473 by mTORC2 promotes anti-apoptotic and cell survival pathways [ 4 , 68 , 69 ]. In vitro activation of PI3K/Akt pathway is associated with enhanced survival and proliferation of SH-SY5Y, SK-N-BE(2) and Neuro 2A neuroblastoma cell lines [ 69 , 71 ], while in vivo PI3K/Akt inhibition reduces tumor growth and MYCN protein expression [ 72 ]. In the current study, the decrease in cell proliferation in uPAR deficient clones was accompanied by a significant (3.3-fold) reduction of Akt phosphorylation at Thr308 residue and a less pronounced (1.8-fold) decrease in Akt phosphorylation at Ser473 (Figure 9A and 9D ); the total Akt content remained unchanged.…”

Developing preclinical models of neuroblastoma: driving therapeutic testing

“…This work highlighted how spheroids may assist in identifying therapies that may be more successful clinically, either for killing macroscopic tumors [157]. Spheroids have also been used to evaluate specific pathway inhibitors such as multikinase inhibitors and oxidative phosphorylation inhibitors [158,159].…”