Metformin induces autophagy level changes in heterozygous and CRISPR-edited TSC2 primary fibroblasts

Introduction: Tuberous Sclerosis Complex (TSC) is a rare genetic disorder caused by pathogenic variants in TSC1 or TSC2 genes, which lead to mTORC1 hyperactivation and autophagy suppression. Loss-of-heterozygosity (LOH) may worsen the dysregulation of these pathways and may trigger tumorigenesis. The standard treatment, rapamycin (mTORC1 inhibitor), presents limitations, suggesting that alternative therapeutic approaches must be investigated. Methods: Primary fibroblast cultures were established from skin biopsies of patients with and without the TSC2 pathogenic variant c.1008C>T (NM_000548). LOH was induced in mutant cells using CRISPR/Cas9 editing. Heterozygous, gene-edited and wild-type cells were treated for 12 hours with the autophagy-inducing compounds rapamycin and metformin or vehicle control (DMSO). Autophagy levels were assessed by flow cytometry using acridine orange staining and MAP1LC3 (LC3) immunofluorescence. mTORC1 activation was evaluated via immunofluorescence detection of phosphorylated S6K at the Ser434 residue. Results: Flow cytometry analysis indicated that autophagic cell proportion did not vary between treatments and DMSO in wild-type cells. Conversely, the treatments showed an increased number of autophagic cells compared to DMSO in heterozygous cells: rapamycin (16.2% vs 6.5%, p=0.006); metformin (17.6% vs 6.5%, p=0.002). In CRISPR-edited cells, both treatments induced increasing number of autophagic cells compared to heterozygous cells: rapamycin (27.1% vs 16.2%, p<0.001) and metformin (27.2% vs 17.6%, p<0.001). LC3 immunofluorescence revealed that heterozygous and gene-edited metformin-treated cells had a higher number and smaller autophagosomes in comparison to the control. Moreover, metformin-treated autophagosomes were smaller than rapamycin-treated. In addition, metformin-treated gene-edited cells exhibited lower activity of mTORC1, as indicated by reduced p-S6K immunofluorescence. Conclusions: Our study demonstrated that metformin induces autophagy in TSC cells, suggesting that this pathway may be one of the mechanisms by which metformin exerts its therapeutic effects in TSC.