Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome resulting from the loss of function of the hamartin/tuberin complex. Two genes are implicated in TSC, TSC1 and TSC2, respectively encoding hamartin and tuberin. The complex inhibits target of rapamycin (TOR)-mediated signaling to S6 kinase and participates in the control of cellular growth. From a surgically removed angiomyolipoma of a TSC2 patient we have isolated alpha-actin-positive smooth muscle cells, named A+, bearing mutation on exon 18, with loss of heterozigosity for TSC2 gene. S6K1 is constitutively activated, while IGF-1 is abundantly released and involved in cell survival rather than proliferation. EGF is necessary to promote proliferation of TSC2-/- A+ cells. Following retroviral transduction of TSC2 gene in A+ cells, tuberin was successfully expressed and EGF-growth dependency was lost, thus it appears that EGF requirement for A+ cell growth is due to lack of tuberin. TSC2-/- A+ cells were positively labeled by HMB45 antibody but, following TSC2 transduction, A+ cells were negative. In TSC2-transfected cells phosphorylation of Akt and S6 was reduced, with the expression of Akt and S6 unaltered. Also PTEN phosphorylation decreased drastically. Exposure for 2 hours with IGF-1 (50ng/ml) slightly activated PI3K in TSC2-/- A+ cells; this was not affected by inhibitors such as LY294002 (LY) and wortmannin. After reintroduction of TSC2 gene the sensitivity to these inhibitors was restored and LY (20M) addition reduced basal and IGF-1-induced Akt phosphorylation, and inhibited activation of S6K1 and PTEN. The phenotype of TSC2-/- A+ cells was also assessed after antiEGFR and rapamycin incubation. When A+ cells were exposed at plating time to antiEGFR (5g/ml) labelling with HMB45 was down-regulated within 5 days. A smaller effect was observed with rapamycin (5ng/ml). The proliferation rate of TSC2-/- A+ cell was reduced by rapamycin when added at plating time, but a delay of 3 hours caused the loss of rapamycin effect. AntiEGFR was effective when added in both conditions. In conclusion our study shows that lack of tuberin affects greatly the EGF dependency for growth and IGF-1 related metabolism. The insensitivity to IGF-1 pathway inhibitors is reversed by the reintroduction of TSC2 gene. The superior effects of antiEGFR versus rapamycin suggest a novel therapeutic strategy for TSC and LAM.
Phenotype reversion of human TSC2-/- smooth muscle cells / E. Lesma, V. Grande, E. Isaia, A.M. Di Giulio, A. Gorio. ((Intervento presentato al convegno LAM International Research Symphosium tenutosi a Cincinnati nel 2007.
|Titolo:||Phenotype reversion of human TSC2-/- smooth muscle cells|
LESMA, ELENA ANNA (Primo)
GRANDE, VERA (Secondo)
DI GIULIO, ANNA MARIA (Penultimo)
GORIO, ALFREDO (Ultimo)
|Data di pubblicazione:||20-apr-2007|
|Parole Chiave:||TSC ; LAM ; anti-EGFR|
|Settore Scientifico Disciplinare:||Settore BIO/14 - Farmacologia|
|Citazione:||Phenotype reversion of human TSC2-/- smooth muscle cells / E. Lesma, V. Grande, E. Isaia, A.M. Di Giulio, A. Gorio. ((Intervento presentato al convegno LAM International Research Symphosium tenutosi a Cincinnati nel 2007.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|