The Methylation of the TSC2 Promoter Underlies the Abnormal Growth of TSC2 Angiomyolipoma-Derived Smooth Muscle Cells

Abstract

Tuberous sclerosis complex (TSC) is an autosomal-dominant disease that is caused by mutations in either the TSC1 or TSC2 gene. Smooth muscle-like cells (ASMs) were isolated from an angiomyolipoma of a patient with TSC. These cells lacked tuberin, were labeled by both HMB45 and CD44v6 antibodies, and had constitutive S6 phosphorylation. The cells bear a germline TSC2 intron 8-exon 9 junction mutation, but DNA analysis and polymerase chain reaction amplification failed to demonstrate loss of heterozygosity. Testing for an epigenetic alteration, we detected methylation of the TSC2 promoter. Its biological relevance was confirmed by tuberin expression and a reduction in HMB45 labeling and S6 constitutive phosphorylation after exposure to the chromatin-remodeling agents, trichostatin A and 5-azacytidine. These cells were named TSC2(-/meth) ASMs. Their proliferation required epidermal growth factor in the medium as previously described for TSC2(-/-) ASMs. Blockade of epidermal growth factor with monoclonal antibodies caused the death of TSC2(-/meth) ASMs. In addition, rapamycin effectively blocked the proliferation of these cells. Our data show for the first time that methylation of the TSC2 promoter might cause a complete loss of tuberin in TSC2 cells, and that the pathogenesis of angiomyolipomas might also originate from epigenetic defects in smooth muscle cells. Additionally, the effect of chromatin-remodeling agents in these cells suggests a further avenue for the treatment of TSC as well as lymphangioleiomyomatosis.

Loss of heterozygosity on tuberous sclerosis complex genes in multifocal micronodular pneumocyte hyperplasia

“…In fact, a recent study showed inactivation of TCS2 caused by methylation of the TSC2 gene promoter in angiomyolipoma-derived cells. 29 If so, each of them might be genetically distinct from another one with LOH, but we could not discern whether multifocal micronodular pneumocyte hyperplasia had those events on TSC genes in this study. Finally, we could not completely eliminate the possibility that tissue samples for PCR might be contaminated by normal cells even though our LOH analysis yielded reliable data.…”

Antibody αPEP13h Reacts With Lymphangioleiomyomatosis Cells in Lung Nodules

“…7 Expression of tuberin may be negatively regulated by DNA methylation. 8 Dysfunctional hamartin or tuberin leads to increased activity of the mammalian target of rapamycin (mTOR), resulting in increased cell size and proliferation. [9][10][11] As a result, mTOR has been targeted in clinical trials with rapamycin (sirolimus) or related rapalogs.…”

Chromatin Remodeling by Rosuvastatin Normalizes TSC2-/meth Cell Phenotype through the Expression of Tuberin

“…More recently, we isolated another type of a actin-positive TSC2 cell population from the AML of a male patient. These latter cells do not express tuberin for the methylation of the TSC2 promoter (TSC2 -/meth ASM cells); thus, epigenetic defects might also originate AML pathogenesis (Lesma et al, 2009). Aberrant DNA methylation of CpG islands in promoter regions of many genes has been observed in several types of cancer and is associated with tumor suppressor gene silencing (Jones and Baylin, 2002).…”

“…Growth and proliferation require specifically the presence of epidermal growth factor (EGF) in the growth medium. EGF cannot be replaced by insulin-like growth factor-1 (Lesma et al, , 2009Carelli et al, 2007). The EGF dependency ceases when these TSC2-deficient cells are made able to produce tuberin either by transfection of TSC2 gene in TSC2 2/2 ASM cells or treatment with chromatin remodeling agents in the case of TSC2 -/meth ASM cells.…”

“…The EGF dependency ceases when these TSC2-deficient cells are made able to produce tuberin either by transfection of TSC2 gene in TSC2 2/2 ASM cells or treatment with chromatin remodeling agents in the case of TSC2 -/meth ASM cells. In both cases, the restored function of the TSC2 gene normalizes rate of cell proliferation and extent of S6 phosphorylation (Lesma et al, 2008(Lesma et al, , 2009). The blockade of EGF or insulin-like growth factor-1 receptors by antibodies results in cell death.…”