Tuberous sclerosis complex (TSC) is a tumor suppressor gene disorder characterized by mutations in the TSC1 or TSC2 genes. These mutations lead to the development of benign tumors involving smooth muscle cells, causing life-threatening lymphangioleiomyomatosis. We isolated and characterized two types of cells bearing a mutation in TSC2 exon 18 from a renal angiomyolipoma of a TSC patient: one population of alpha-actin-positive smooth muscle-like cells with loss of heterozygosity for the TSC2 gene (A(+) cells) and another of nonloss of heterozygosity keratin 8/18-positive epithelial-like cells (R(+) cells). Unlike control aortic vascular smooth muscle cells, A(+) cells required epidermal growth factor (EGF) to grow and substituting EGF with insulin-like growth factor (IGF)-1 failed to increase the cell number; however, omission of EGF did not cause cell loss. The A(+) cells constantly released IGF-1 into the culture medium and constitutively showed a high degree of S6K phosphorylation even when grown in serum-free medium. Exposure to antibodies against EGF and IGF-1 receptors caused a rapid loss of A(+) cells: 50% by 5 days and 100% by 12 days. Signal transduction mediated by EGF and IGF-I receptors is therefore involved in A(+) cell survival. These results may offer a novel therapeutic perspective for the treatment of TSC complications and lymphangioleiomyomatosis.

Tuberous sclerosis complex (TSC) is caused by mutations in TSC1 or TSC2 genes. Lymphangioleiomyomatosis (LAM) can be sporadic or associated with TSC and is characterized by widespread pulmonary proliferation of abnormal α-smooth muscle (ASM)-like cells. We investigated the features of ASM cells isolated from chylous thorax of a patient affected by LAM associated with TSC, named LAM/TSC cells, bearing a germline TSC2 mutation and an epigenetic defect causing the absence of tuberin. Proliferation of LAM/TSC cells is epidermal growth factor (EGF)-dependent and blockade of EGF receptor causes cell death as we previously showed in cells lacking tuberin. LAM/TSC cells spontaneously detach probably for the inactivation of the focal adhesion kinase (FAK)/Akt/mTOR pathway and display the ability to survive independently from adhesion. Non-adherent LAM/TSC cells show an extremely low proliferation rate consistent with tumour stem-cell characteristics. Moreover, LAM/TSC cells bear characteristics of stemness and secrete high amount of interleukin (IL)-6 and IL-8. Anti-EGF receptor antibodies and rapamycin affect proliferation and viability of non-adherent cells. In conclusion, the understanding of LAM/TSC cell features is important in the assessment of cell invasiveness in LAM and TSC and should provide a useful model to test therapeutic approaches aimed at controlling their migratory ability.

Background Tuberous sclerosis complex (TSC), a tumor syndrome caused by mutations in TSC1 or TSC2 genes, is characterized by the development of hamartomas. We previously isolated, from an angiomyolipoma of a TSC2 patient, a homogenous population of smooth muscle-like cells (TSC2−/− ASM cells) that have a mutation in the TSC2 gene as well as TSC2 loss of heterozygosity (LOH) and consequently, do not produce the TSC2 gene product, tuberin. TSC2−/− ASM cell proliferation is EGF-dependent.Methods and FindingsEffects of EGF on proliferation of TSC2−/− ASM cells and TSC2−/− ASM cells transfected with TSC2 gene were determined. In contrast to TSC2−/− ASM cells, growth of TSC2-transfected cells was not dependent on EGF. Moreover, phosphorylation of Akt, PTEN, Erk and S6 was significantly decreased. EGF is a proliferative factor of TSC2−/− ASM cells. Exposure of TSC2−/− ASM cells to anti-EGFR antibodies significantly inhibited their proliferation, reverted reactivity to HMB45 antibody, a marker of TSC2−/− cell phenotype, and inhibited constitutive phosphorylation of S6 and ERK. Exposure of TSC2−/− ASM cells to rapamycin reduced the proliferation rate, but only when added at plating time. Although rapamycin efficiently inhibited S6 phosphorylation, it was less efficient than anti-EGFR antibody in reverting HMB45 reactivity and blocking ERK phosphorylation. In TSC2−/− ASM cells specific PI3K inhibitors (e.g. LY294002, wortmannin) and Akt1 siRNA had little effect on S6 and ERK phosphorylation. Following TSC2-gene transfection, Akt inhibitor sensitivity was observed.ConclusionOur results show that an EGF independent pathway is more important than that involving IGF-I for growth and survival of TSC−/− ASM cells, and such EGF-dependency is the result of the lack of tuberin.

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