Anti-EGFR antibody efficiently and specifically inhibits human TSC2-/- smooth muscle cell proliferation. Possible treatment options for TSC and LAM

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 Findings

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. 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. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, wortmannin) and Akt1 siRNA had little effect on S6 and ERK phosphorylation. Following TSC2-gene transfection, Akt inhibitor sensitivity was observed.

Conclusion

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.