Molecular characterization of growth and survival of human TSC2 gene-deficient smooth muscle-like cells

Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome resulting from the loss of function of the TSC1/2 complex. The TSC1 and TSC2 gene products, hamartin and tuberin, associate as a functional complex and inhibit the mammalian target of rapamycin (mTOR). The loss of tuberin function leads to constitutive S6K1 activation and phosphorylation of its ribosomal substrate S6. We have recently reported the isolation from a renal angiomyolipoma and characterization of smooth muscle-like cells (A+) showing loss of heterozigosity for the TSC2 gene. A+ cells require epidermal growth factor (EGF) supplementation to sustain proliferation, and exposure to antibodies to EGF receptor (EGFR) is lethal. In addition, A+ cells secrete insulin-like growth factor-1 (IGF-1). Incubation at plating time with rapamycin (1 and 5ng/ml) for 24 and 48 hours decreases phosphorylation of S6 in a dose- and time-dependent manner and normalizes A+ cell proliferation. Similar results were obtained after blockade of EGFR, while PD98059 (PD), an Erk inhibitor, had only a partial effect. On the other hand the effective rapamycin dose increases 100 fold when the drug was added 24 hours after A+ cell plating. EGF proliferative action cannot be substituted by IGF-1, although blockade of IGF-1 receptor with specific antibodies causes cell death. IGF-1 addition does not modify S6 phosphorylation but may play a role in survival since it promotes the expression of survivin, that is also induced by the lack of EGF in the growth medium. The expression of pro-apoptotic Smac/DIABLO is down-regulated by IGF-1 addition and unaffected by the omission of EGF. Survivin expression is also activated by exposure to anti-EGFR, PD and rapamycin. A+ cells are highly resistant to apoptotic stimuli, although caspase-3 is very abundant. In conclusion, the prevention within 48 hours of S6 phosphorylation and normalization of the proliferation rate by rapamycin and anti-EGFR support the direct involvement of EGF pathway in A+ cell TSC2 cellular phenotype, while the effects of autosecreted IGF-1 on survivin and Smac/DIABLO suggest its role on survival.