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. In vitro growth of TSC2-/- ASM cells depends on the functionality of EGF receptor. Indeed, incubation with anti-EGFR antibody leads to cell death in 12 days. Effects 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. 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. Our 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.
Anti-EGFR antibody efficiently and specifically inhibits human TSC2-/- smooth muscle cell proliferation. Possible treatment options for TSC and LAM / E. Lesma, V. Grande, S. Ancona, A.M. Di Giulio, A. Gorio. ((Intervento presentato al convegno TSC International Research Conference tenutosi a Brighton, UK nel 2008.
Anti-EGFR antibody efficiently and specifically inhibits human TSC2-/- smooth muscle cell proliferation. Possible treatment options for TSC and LAM
E. LesmaPrimo
;V. GrandeSecondo
;S. Ancona;A.M. Di GiulioPenultimo
;A. GorioUltimo
2008
Abstract
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. In vitro growth of TSC2-/- ASM cells depends on the functionality of EGF receptor. Indeed, incubation with anti-EGFR antibody leads to cell death in 12 days. Effects 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. 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. Our 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.Pubblicazioni consigliate
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