Selective Inhibition of Growth of Tuberous Sclerosis Complex 2–Null Cells by Atorvastatin Is Associated with Impaired Rheb and Rho GTPase Function and Reduced mTOR/S6 Kinase Activity

Abstract

Inactivating mutations in the tuberous sclerosis complex 2 (TSC2) gene, which encodes tuberin, result in the development of TSC and lymphangioleiomyomatosis (LAM). The tumor suppressor effect of tuberin lies in its GTPase-activating protein activity toward Ras homologue enriched in brain (Rheb), a Ras GTPase superfamily member. The statins, 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, have pleiotropic effects which may involve interference with the isoprenylation of Ras and Rho GTPases. We show that atorvastatin selectively inhibits the proliferation of Tsc2 À/À mouse embryo fibroblasts and ELT-3 smooth muscle cells in response to serum and estrogen, and under serum-free conditions. The isoprenoids farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) significantly reverse atorvastatin-induced inhibition of Tsc2 À/À cell growth, suggesting that atorvastatin dually targets a farnesylated protein, such as Rheb, and a geranylgeranylated protein, such as Rho, both of which have elevated activity in Tsc2 À/À cells.Atorvastatin reduced Rheb isoprenylation, GTP loading, and membrane localization. Atorvastatin also inhibited the constitutive phosphorylation of mammalian target of rapamycin, S6 kinase, and S6 found in Tsc2 À/À cells in an FPP-reversible manner and attenuated the high levels of phosphorylated S6 in Tsc2-heterozygous mice. Atorvastatin, but not rapamycin, attenuated the increased levels of activated RhoA in Tsc2 À/À cells, and this was reversed by GGPP. These results suggest that atorvastatin may inhibit both rapamycin-sensitive and rapamycin-insensitive mechanisms of tuberin-null cell growth, likely via Rheb and Rho inhibition, respectively. Atorvastatin may have potential therapeutic benefit in TSC syndromes, including LAM. [Cancer Res 2007;67(20):9878-86]

Rheb activates AMPK and reduces p27Kip1 levels in Tsc2-null cells via mTORC1-independent mechanisms: implications for cell proliferation and tumorigenesis

“…Consistent with this possibility, we showed that Rheb depletion reduced the tumorigenic activity of Tsc2-null cells, and correlated with an in vivo upregulation of p27 levels. Our results agree with previous findings indicating that farnesyl transferase inhibitors and atorvastatin, which block Rheb activity, inhibit the growth of Tsc2-deficient cells (Gau et al, 2005;Finlay et al, 2007).…”

“…As Rheb is constitutively activated in Tsc2-deficient cells (Garami et al, 2003;Finlay et al, 2007), we depleted Rheb using RNA interference to investigate its role in regulating AMPK activity. Rheb small interfering RNA (siRNA) decreased Rheb mRNA and protein levels ( Figure 1a).…”

“…As Rheb is constitutively activated in Tsc2-null cells (Garami et al, 2003;Finlay et al, 2007), we sought to determine whether it regulates AMPK activity. Here, we show that Rheb but not mTORC1 activates AMPK.…”

Autophagy in hypoxia-ischemia induced brain injury: Evidences and speculations

“…1). Interestingly, and in keeping with our results, statins can impair Rheb and Rho GTPase function and reduce mTOR/S6 kinase activity 27 and this may explain their efficacy as prophylactic treatment in neonatal HI. Increased Rheb-TOR signaling, on the other hand, enhances sensitivity to oxidative stress 28 which seems to play a pivotal role in the progression of brain damage in the neonate.…”

The changing face of a rare disease: lymphangioleiomyomatosis

“…Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme and thereby block geranylgeranylation of Rho GTPases and farnesylation of Ras and Rheb [137]. Simvastatin was shown to inhibit RhoA GTPase activity and proliferation of TSC-null cells and TSC2-null tumour growth in mice, and to promote apoptosis [49].…”