Rewiring of Endolysosomal Signaling with Nutrient Depletion in Cancer Cells

Purpose: Limited nutrient availability in the tumor microenvironment can cause rewiring of signaling and metabolic networks to confer cancer cells with survival advantages. We aimed to identify the signaling pathways that can enable cancer cells to survive under nutrient depletion and cope with cytotoxic effects of chemotherapy agents. Methods: Nutrient depletion was mediated by the depletion of glucose, L-glutamine and serum from the culture medium. Cell viability was determined with MTT and colony formation assays, tumor forming capacity with a Chorioallantoic membrane (CAM) assay and activation of nutrient sensing and endolysosomal trafficking pathways was determined with Western blot. Lysosomal abundance, localization, and trapping of chemotherapy drugs were determined with immunocytochemistry, immunoelectron microscopy and Lysotracker assay. Results: Nutrient depletion resulted in the survival of a population of cancer cells with high viability and capacity to form tumors. These cells also displayed an increase in the abundance and size of lysosomes. Moreover, lysosomes were mainly perinuclear in nutrient depleted (ND) cells; this localization was mediated by a rapid post-transcriptional increase in the endolysosomal trafficking protein Rab7a. The acidic lysosomes in ND cells could trap weakly basic drugs such as doxorubicin, mediating resistance, which could be partially reversed with bafilomycinA1. An in vivo CAM assay indicated a remarkable decrease in Ki-67 positivity when ND cells were treated with 5-FU and bafilomycinA1 compared to ND cells treated with either agent alone. Conclusion: Nutrient depletion activates lysosomal signaling that activates multiple survival pathways in cancer.