Induction and analysis of the consequences of a lysosomal storage phenotype in a breast cancer cell line

Résumé

BACKGROUND – Lysosomal storage disorders (LSDs) are a group of genetic diseases characterized by the accumulation of material in lysosomes due to the deficiency of an acid hydrolase, membrane transporter or of the trafficking pathway that sorts acid hydrolases to lysosomes. Interestingly, it has been reported that the incidence of cancer is increased in LSD patients compared to control groups. However, the mechanism behind this higher risk of developing cancer has yet to be discovered. AIMS – Identification of treatments that cause a lysosomal storage phenotype in the breast cancer cell line MCF7 and characterization of the consequences on lysosome size, distribution and biogenesis, including an investigation of the putative activation of transcription factors that regulate lysosomal gene expression. A second aim is to analyze the consequences on parameters that support cancer cell aggressiveness, including acid hydrolase secretion and signs of Epithelial-to-Mesenchymal Transition (EMT). METHODS – Using immunofluorescence, RT-qPCR and biochemical methods (western blotting and enzyme assays), we characterized three models of induced lysosomal storage disorder: 1-incubation with sucrose, which accumulates in lysosomes after endocytosis; 2- incubation with a site 1-protease inhibitor (iS1P), thereby preventing the synthesis of Mannose 6-Phosphate sorting signals on lysosomal enzymes which are hypersecreted as a result (causing the accumulation of their substrates in lysosomes); 3- treatment with U18666A, which blocks cholesterol egress from lysosomes. RESULTS – We identified that all three treatments induce lysosomal expansion, accompanied by an increase of expression of some acid hydrolase and/or lysosomal membrane proteins, though not to the same extent with all treatments. Interestingly, we found a nuclear delocalization for the transcription factor EB (TFEB, a master regulator of lysosomal genes) in each storage condition, and detected SREBP activation after sucrose and U18666A treatments. This is another transcription factor that binds to the promoter of some lysosomal genes. Moreover, while it appears that the lysosomal storage phenotypes do not induce EMT under our experimental conditions, we also detected hypersecretion of cathepsin D, a lysosomal protease with reported pro-cancerous effects. CONCLUSION – Our results support that lysosomal storage defects can be induced in MCF7 breast cancer cells using either sucrose, iS1P or U18666A treatments. It appears that this results in the activation of transcription factors, TFEB and/or SREBP depending on the stored material. These activations could promote cancer cell growth, possibly via induction of lysosomal biogenesis, which would help to maintain sufficient amino acid levels to support growth. In addition, all conditions resulted in the hypersecretion of procathepsin D, which could promote cancer cell proliferation, migration and/or invasion. Taken together, our results highlight several mechanisms by which lysosomal storage phenotypes could affect the aggressivity of cancer cells.

la date de réponse	16 janv. 2023
langue originale	Anglais
L'institution diplômante	Universite de Namur
Superviseur	Marielle Boonen (Promoteur) & Michel Jadot (Copromoteur)