PATHOGENESIS AND PHARMACOLOGY OF ISOLATED HUMAN TSC2 SMOOTH MUSCLE CELLS. NOVEL THERAPEUTIC PERSPECTIVES

Cultured cells from human TSC2 angiomyolipomas can be a source of useful information for developing more appropriate pharmacological strategies aimed at blocking the life-threatening growth of smooth muscle cells in TSC and LAM. We have recently reported the novel finding that in vitro growth of smooth muscle-like cells derived from the renal angiomyolipoma (AML) of a TSC2 patient depends on the availability of EGF in the medium and functionality of EGFR receptor (EGFR). The sub-cloned smooth muscle cells (A+) are LOH with a germ line TSC2 exon 18 mutation consisting of a base pair change in amino acid 698 from a lysine to a stop codon (K698X) and do not express tuberin. Antibodies to EGF and IGF-1 receptors lead to the progressive loss of A+ cells, when added to the culture medium. The omission of EGF from the culture medium does not cause cell loss, and results in the induction of survivin that is also achieved by addition of IGF-I. This growth factor is auto-secreted by A+ cells at a constant rate (14.21 ng/1.5x10,000 cells) as long as they are kept in culture. Five weeks old Hsd:Athimic Nude-nu nu/nu mice were i.p. injected with 106 A+ cells previously labeled with PKH26-GL. Lungs, kidneys, uterus and neck, axial, intestinal, and renal lymph nodes were dissected out and properly processed after 30 days. Sections were cut and counterstained with DAPI, or a specific antibody against alfa-actin or HMB-45. Labeled A+ cells were abundantly detected in all the lymph nodes and were also positive for alfa-actin and HMB-45. Infiltration of A+ cells was also observed in lung and uterus of nude mice. Lungs presented emphysema-like alveolar destruction. All the controls injected with normal human aorta smooth muscle cells were negative. It has been shown that estradiol and tamoxifen stimulate growth of angiomyolipoma cells associated to LAM. Differently from the previous report, when A+ cells were incubated with 17-beta-estradiol, tamoxifen, and clomiphene no significant effects were observed. In conclusion A+ cells show all the characteristics of TSC and LAM cells and require EGF supplementation for proliferation. The killing activity of anti-EGF-R antibodies suggest new therapeutic perspectives for controlling smooth muscle cell growth in TSC complications and LAM.