Pathogenesis and pharmacological evaluation of human TSC2 smooth muscle cells. Possible treatment options for TSC and lymphangioleiomyomatosis

Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM) are rare genetic diseases caused by mutations in TSC genes. TSC2-/- cells form hamartomas and invade lungs causing the fatal disease LAM. We have isolated from an angiomyolipoma a homogenous population of TSC2 smooth muscle-like (ASM) cells with LOH genetic lesion (TSC2-/-ASM cells). These cells grow and proliferate in EGF-dependent manner, while auto-secreted IGF-1 acts as a surviving factor. The constitutive phosphorylation of S6, and higher Akt and ERK phosphorylation are a paradigm of these cells. We validated our cells as a cellular model for studying TSC and LAM by TSC2 gene transfection, which promoted the biochemical normalization and eliminated EGF-dependency. The blockade of EGF-receptors with specific antibodies results in TSC2-/- ASM cells death and suggest a novel therapeutic avenue for TSC and LAM. We have recently found that suppression of TSC2 gene may also have an epigenetic origin, another pure ASM cell population was isolated in our lab. These cells showed the typical constitutive S6 phosphorylation, and lacked tuberin as the result of TSC2 promoter methylation as a second hit (TSC2-/methASM cells). Chromatin remodelling agents, such as trichostatin-A and 5-azacytidine de-methylated TSC2-/methASM cell promoter, that returned normal. TSC2-/methASM cells are pharmacologically comparable to TSC2-/-ASM cells. These data show for the first time that TSC2 pathogenesis might be originated also by epigenetic defects in ASM cells. Pulmonary proliferation of TSC2-/-ASM cells in LAM is characterized by cystic destruction of lung parenchyma. To develop a proper animal model for LAM and to validate our in vitro pharmacological observations we have administered by endonasal route TSC2-/-ASM cells to nude mice. TSC2-/-ASM cells massively penetrated into lungs, and from there reached the lymphatic vessels where they accumulated in the lymph nodes. Here these cells grow and proliferate. The progressive increase of TSC2-/-ASM cells in lung parenchyma correspond to the progressive invasion of lymphatic vessel and destruction of lung tissue. Anti-EGFR antibody treatment killed TSC2-/-ASM cells and reversed lung structural alterations, while rapamycin had a weaker effect. These data suggest a novel therapeutic approach for the control of the abnormal ASM cell growth in TSC and lymphangioleiomyomatosis