Abstract 5091: Lymphovascular tumor emboli exhibit a quiescent state refractory to chemotherapy and targeted therapies because of endogenously low metabolism

Abstract

Abstract Previous studies with a human xenograft model of inflammatory breast cancer, MARY-X, demonstrated that overexpressed E-cadherin moderated the formation of the lymphovascular embolus in vivo and tumoral spheroids of super-high density in vitro. In vitro, MARY-X cells grow very slowly as spheroids and the consumption of nutrients is markedly decreased, suggesting that the metabolism in the MARY-X spheroids is also very low. This low metabolism may also be exhibited in vivo within the lymphovascular embolus and may be responsible for its resistance to chemotherapy. This study investigated the mechanisms which lead to a low level of metabolism by initially analyzing AMPK, a metabolic checkpoint coordinating cell growth with energy status and the mTOR pathway, a pathway that is a highly conserved nutrient-responsive regulator of cell growth found in all eukaryotes. The levels of phosphorylated AMPK proteins (AMPKα and β subunits) decrease gradually with the formation of MARY-X spheroids in vitro. Since it had been reported that E-cadherin inhibits the activity of LKB1, a protein upstream of AMPK, which activates both AMPK and several AMPK-related kinases, the decrease in AMPK activity in the MARY-X spheroids could be the result of the homophilic E-cadherin interactions mediating the tight spheroid aggregates. mTOR activity, on the other hand, increases within the MARY-X spheroids. Because AMPK inversely regulates mTOR activity, it is easy to understand why mTOR activity increases. Using rapamycin on the spheroids led to the down-regulation in the activity of p70 S6 kinase but less effects on the activity of 4E-PB1. Most importantly, the treated spheroids did not dissociate nor undergo apoptosis. Use of Ly294002, a PI3K/Akt inhibitor, completely abolished the activities of both p70 S6 kinase and 4E-BP1, the substrates of mTOR. The treated spheroids dissociated and the cells underwent apoptosis. Compound C (AMPK inhibitor) blocked the activity of AMPK, which resulted in a marked upregulation of the mTOR pathway with great increases in both p70 S6 kinase and 4E-BP1 and an increase in resulting metabolism. However the treated spheroids, just like with Ly294002, dissociated and underwent apoptosis. Increasing the cellular metabolism leads to cell death even in enriched medium. Growing MARY-X in serum free media results in the activation of AMPK, when intracellular ATP levels decline and intracellular AMP increases, as occurs during nutrient deprivation or hypoxia. AMPK activation normally inhibits the mTOR pathway but in MARY-X spheroids grown in serum-free medium, the mTOR pathway is not inhibited, cellular metabolism remains low and the spheroids do not dissociate or undergo apoptosis. If these observations can be confirmed in in vivo studies, we may need to rethink our targeting strategies in the face of a quiescent tumor embolus exhibiting low metabolism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5091.