Estrogen promotes the survival and pulmonary metastasis of tuberin-null cells

Abstract

Lymphangioleiomyomatosis (LAM) is an often fatal disease primarily affecting young women in which tuberin (TSC2)-null cells metastasize to the lungs. The mechanisms underlying the striking female predominance of LAM are unknown. We report here that 17-␤-estradiol (E2) causes a 3-to 5-fold increase in pulmonary metastases in male and female mice, respectively, and a striking increase in circulating tumor cells in mice bearing tuberin-null xenograft tumors. E 2-induced metastasis is associated with activation of p42/44 MAPK and is completely inhibited by treatment with the MEK1/2 inhibitor, CI-1040. In vitro, E 2 inhibits anoikis of tuberin-null cells. Finally, using a bioluminescence approach, we found that E 2 enhances the survival and lung colonization of intravenously injected tuberin-null cells by 3-fold, which is blocked by treatment with CI-1040. Taken together these results reveal a new model for LAM pathogenesis in which activation of MEKdependent pathways by E 2 leads to pulmonary metastasis via enhanced survival of detached tuberin-null cells.L AM, the pulmonary manifestation of tuberous sclerosis complex (TSC), affects women almost exclusively (1). LAM affects 30Ϫ40% of women with TSC (2, 3). In a Mayo Clinic series, LAM was the third most frequent cause of TSC-related death, after renal disease and brain tumors (4). LAM can also occur in women who do not have germline mutations in TSC1 or TSC2 (sporadic LAM). LAM cells from both TSC-LAM and sporadic LAM carry inactivating mutations in both alleles of the TSC1 or TSC2 genes (5). The protein products of TSC1 and TSC2, hamartin and tuberin, respectively, form heterodimers (6, 7) that inhibit the small GTPase Ras homologue enriched in brain (Rheb), via tuberin's highly conserved GTPase activating domain. In its active form, Rheb activates the mammalian target of rapamycin (mTOR) complex 1 (TORC1), which is a key regulator of protein translation, cell size, and cell proliferation (8). Evidence of TORC1 activation, including hyperphosphorylation of ribosomal protein S6, has been observed in tumor specimens from TSC patients and LAM patients (9-11). Independent of its activation of mTOR, Rheb inhibits the activity of B-Raf and C-Raf/Raf-1 kinase, resulting in reduced phosphorylation of p42/44 MAPK (12-14), but the impact of the Raf/MEK/ MAPK pathway on disease pathogenesis is undefined.LAM is characterized pathologically by widespread proliferation of abnormal smooth muscle cells and by cystic changes within the lung parenchyma (1). About 60% of women with the sporadic form of LAM also have renal angiomyolipomas. The presence of TSC2 mutations in LAM cells and renal angiomyolipoma cells from women with sporadic LAM, but not in normal tissues, has led to the hypothesis that LAM cells spread to the lungs via a metastatic mechanism, despite the fact that LAM cells have a histologically benign appearance (15,16). Genetic and fluorescent in situ hybridization analyses of recurrent LAM after lung transplantation support this benign metastatic model (16).The female...

Integration of mTOR and estrogen–ERK2 signaling in lymphangioleiomyomatosis pathogenesis

“…E 2 , upon binding to its estrogen receptor (ER), has been reported to regulate transcriptiondependent and -independent signaling events (14). Thus, in addition to its ability to promote changes in gene expression (10,(15)(16)(17)(18), E 2 can induce the activation of signaling proteins such as Src, Akt, and ERK-MAP kinase (14). The importance of ERK-MAP kinase in LAM was suggested by a recent report showing that E 2 promoted the MEK-dependent invasion of cells derived from Eker rat uterine leiomyoma (ELT3 cells) into the lungs of ovariectomized mice (18).…”

“…Thus, in addition to its ability to promote changes in gene expression (10,(15)(16)(17)(18), E 2 can induce the activation of signaling proteins such as Src, Akt, and ERK-MAP kinase (14). The importance of ERK-MAP kinase in LAM was suggested by a recent report showing that E 2 promoted the MEK-dependent invasion of cells derived from Eker rat uterine leiomyoma (ELT3 cells) into the lungs of ovariectomized mice (18). However, the molecular basis for E 2 -dependent ERK contribution to the enhanced invasive phenotype in the presence of constitutively activated mTORC1 was not defined, and whether E 2 promoted a similar response in patient-derived cells remained to be determined.…”

mTOR Signaling in Lymphangioleiomyomatosis

“…[136][137][138][139] Separate studies suggested that prolactin stimulates mTOR, and modifies STAT1 activity in mTOR-dependent fashion. 140,141 Further studies are required to better understand how mTORCs interact with androgen or estrogen signaling pathways.…”

mTOR Activation, Lymphangiogenesis, and Estrogen-Mediated Cell Survival: The “Perfect Storm” of Pro-Metastatic Factors in LAM Pathogenesis

“…Estrogen also enhances the number of circulating tumor cells and the survival of intravenously injected ELT3 -cells. 38 In vitro, estrogen-treated ELT3 cells exhibit induced resistance to anoikis and decreased levels of caspase-3 cleavage. Using the Xenogen bioluminescent imaging (Caliper Life Sciences, Hopkinton, MA), estrogen treatment causes a twofold increase in lung colonization of intravenously injected ELT3-luciferase cells at 3 h, and a five-fold increase at 24 h (Fig.…”