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 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.背景 结节性硬化症（Tuberous sclerosis complex, TSC）是一种由TSC1或TSC2基因突变引发的肿瘤综合征，其特征为错构瘤（hamartoma）的形成。本团队此前从一名TSC2患者的血管平滑肌脂肪瘤（angiomyolipoma）中分离出一群均一的平滑肌样细胞（TSC2−/− ASM细胞），该细胞携带TSC2基因突变，同时存在TSC2杂合性缺失（loss of heterozygosity, LOH），因此无法产生TSC2基因产物马铃薯球蛋白（tuberin）。TSC2−/− ASM细胞的增殖依赖表皮生长因子（epidermal growth factor, EGF）。 方法与结果 本研究检测了EGF对TSC2−/− ASM细胞以及转染TSC2基因的TSC2−/− ASM细胞增殖的影响。与TSC2−/− ASM细胞不同，转染TSC2基因的细胞生长不再依赖EGF。此外，Akt、PTEN、Erk与S6的磷酸化水平显著降低。EGF是TSC2−/− ASM细胞的增殖因子。将TSC2−/− ASM细胞暴露于抗EGFR抗体后，其增殖受到显著抑制，恢复了对HMB45抗体的反应性——HMB45抗体为TSC2−/−细胞表型的标志物——并抑制了S6与ERK的组成型磷酸化。用雷帕霉素（rapamycin）处理TSC2−/− ASM细胞可降低其增殖速率，但仅在细胞接种时添加才有效。尽管雷帕霉素可有效抑制S6磷酸化，但在恢复HMB45反应性与阻断ERK磷酸化方面，其效果不及抗EGFR抗体。在TSC2−/− ASM细胞中，特异性PI3K抑制剂（如LY294002、渥曼青霉素（wortmannin））以及Akt1小干扰RNA（siRNA）对S6与ERK磷酸化的影响极弱。在转染TSC2基因后，细胞对Akt抑制剂产生了敏感性。 结论 本研究结果显示，对于TSC−/− ASM细胞的生长与存活而言，不依赖EGF的通路较胰岛素样生长因子I（insulin-like growth factor-I, IGF-I）介导的通路更为关键，且这种EGF依赖性正是由于缺乏马铃薯球蛋白（tuberin）所导致的。