Molecular heterogeneity of TSC2-deficient cells

TSC is an autosomal-dominant genetic disorder resulting from the dysregulation of PI3K/Akt/mammalian target of rapamycin pathway. From specimens of three TSC2 patients we have isolated three populations of α-actin smooth muscle (ASM) cells. In angiomyolipoma TSC2-/- ASM cells the loss of tuberin is caused by Knudson’s double hit, while angiomyolipoma TSC2-/meth ASM and chylous LAM/TSC cells do not express tuberin for an epigenetic event. In these cells survival and proliferation are dependent from EGF that cannot be replaced by IGF-1. In TSC2-/- and TSC2-/meth ASM cells the extent of Akt phosphorylation was very low compared to LAM/TSC cells. IGF-1 increased phospho-Akt in TSC2-/- and TSC2-/meth ASM and slightly in LAM/TSC cells. Specific PI3K inhibitor, LY294002, reduced Akt and S6 phosphorylation in LAM/TSC cells, while did not have any effect in TSC2-/- and TSC2-/meth ASM cells. S6 phosphorylation was not increased by IGF-1 incubation in any cell type. Stable transfection of TSC2 and trichostatinA treatment caused tuberin expression in TSC2-/- and TSC2-/meth ASM cells, respectively, and restored the ability to regulate PI3K. Silencing of Akt in TSC2-/- ASM cells caused the reduction of S6 phosphorylation. In spite of the common lack of tuberin expression, the described TSC2-null cells showed differences in PI3K/Akt/mTOR pathway regulation and IGF-1 stimulation suggesting a molecular heterogeneity in TSC cells