GPI/AMF inhibition blocks the development of the metastatic phenotype of mature multi-cellular tumor spheroids

Epithelial–mesenchymal transition (EMT) and cellular invasiveness are two pivotal processes for the development of metastatic tumor phenotypes. The metastatic profile of non-metastatic MCF-7 cells growing as multi-cellular tumor microspheroids (MCTSs) was analyzed by determining the contents of the EMT, invasive and migratory proteins, as well as their migration and invasiveness potential and capacity to secrete active cytokines such as the glucose phosphate isomerase/AMF (GPI/AMF). As for the control, the same analysis was also performed in MCF-7 and MDA-MB-231 (highly metastatic, MDA) monolayer cells, and in stage IIIB and IV human metastatic breast biopsies. The proliferative cell layers (PRL) of mature MCF-7 MCTSs, MDA monolayer cells and metastatic biopsies exhibited increased cellular contents (2–15 times) of EMT (β-catenin, SNAIL), migratory (vimentin, cytokeratin, and fibronectin) and invasive (MMP-1, VEGF) proteins versus MCF-7 monolayer cells, quiescent cell layers of mature MCF-7 MCTS and non-metastatic breast biopsies. The increase in metastatic proteins correlated with substantially elevated cellular abilities for migration (18-times) and invasiveness (13-times) and with the higher level (6-times) of the cytokine GPI/AMF in the extracellular medium of PRL, as compared to MCF-7 monolayer cells. Interestingly, the addition of the GPI/AMF inhibitors erythrose-4-phosphate or 6-phosphogluconate at micromolar doses significantly decreased its extracellular activity (> 80%), with a concomitant diminution in the metastatic protein content and migratory tumor cell capacity, and with no inhibitory effect on tumor lactate production or toxicity on 3T3 mouse fibroblasts. The present findings provide new insights into the discovery of metabolic inhibitors to be used as complementary therapy against metastatic and aggressive tumors.