Long-Term Effects of Sirolimus on Human Skin TSC2-Null Fibroblast‒Like Cells

Tuberous sclerosis complex (TSC) is an autosomal-dominant disorder characterized by hamartomatous tumors of the skin, kidneys, brain, and lungs. TSC is caused by mutations in the TSC1 and TSC2 genes, which result in hyperactivation of the mTOR, leading to dysregulated cell growth and autophagy. Rapamycin (sirolimus) shrinks TSC tumors, but the clinical benefits of sirolimus are not sustained after its withdrawal. In this study, we studied the cellular processes critical for tumor formation and growth, including cell proliferation and cell size. TSC2^-/- and TSC2^+/- cells were isolated from TSC skin tumors and normal-appearing skin, respectively. Cells were incubated with sirolimus for 72 hours. Withdrawal of sirolimus from TSC2^-/- cells resulted in a highly proliferative phenotype and caused cells to enter the S phase of the cell cycle, with persistent phosphorylation of mTOR, p70 S6 kinase, ribosomal protein S6, and 4EB-P1; decreased cyclin D kinase inhibitors; and transient hyperactivation of protein kinase B. Sirolimus modulated the estrogen- and autophagy-dependent volume of TSC2^-/- cells. These results suggest that sirolimus may decrease the size of TSC tumors by reducing TSC2^-/- cell volume, altering the cell cycle, and reprogramming TSC2-null cells.