Jane J. Yu

Lymphangioleiomyomatosis (LAM) is a female-predominant lung disease that can lead to respiratory failure. LAM cells typically have inactivating tuberous sclerosis 2 (TSC2) mutations, leading to mTORC1 hyperactivation. The gender specificity of LAM suggests that female hormones contribute to disease progression. Clinical findings indicate that estradiol exacerbates LAM behaviors and symptoms. Although hormonal therapy with progesterone has been employed, the benefit in LAM improvement has not been achieved. We have previously found that estradiol promotes the survival and lung metastasis of cells lacking tuberin in a preclinical model of LAM. In this study, we hypothesize that progesterone alone or in combination with estradiol promotes metastatic behaviors of TSC2-deficient cells. In cell culture models of TSC2-deficient LAM patient-derived and rat uterine leiomyoma-derived cells, we found that progesterone treatment or progesterone plus estradiol resulted in increased phosphorylation of Protein Kinase B (Akt) and Extracellular signal-regulated kinases1/2 (ERK1/2), induced the proliferation, and enhanced the migration and invasiveness. In addition, treatment of progesterone plus estradiol synergistically decreased the levels of reactive oxygen species and enhanced cell survival under oxidative stress. In a murine model of LAM, treatment of progesterone plus estradiol promoted the growth of xenograft tumors; however, progesterone treatment did not affect the development of xenograft tumors of Tsc2-deficient cells. Importantly, treatment of progesterone plus estradiol resulted in alteration of lung morphology and significantly increased the number of lung micrometastases of Tsc2-deficient cells compared with estradiol treatment alone. Collectively, these data indicate that progesterone increases the metastatic potential of Tsc2-deficient LAM patient-derived cells in vitro and lung metastasis in vivo. Thus, targeting progesterone-mediated signaling events may have therapeutic benefit for LAM and possibly other hormonally dependent cancers.

Lymphangioleiomyomatosis (LAM) is a progressive neoplastic disorder that leads to lung destruction and respiratory failure primarily in women. LAM is typically caused by tuberous sclerosis complex 2 (TSC2) mutations resulting in mTORC1 activation in proliferative smooth muscle–like cells in the lung. The female predominance of LAM suggests that estradiol contributes to disease development. Metabolomic profiling identified an estradiol-enhanced prostaglandin biosynthesis signature in Tsc2-deficient (TSC⁻) cells, both in vitro and in vivo. Estradiol increased the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostaglandin biosynthesis, which was also increased at baseline in TSC-deficient cells and was not affected by rapamycin treatment. However, both Torin 1 treatment and Rictor knockdown led to reduced COX-2 expression and phospho-Akt-S473. Prostaglandin production was also increased in TSC-deficient cells. In preclinical models, both Celecoxib and aspirin reduced tumor development. LAM patients had significantly higher serum prostaglandin levels than healthy women. 15-epi-lipoxin-A₄ was identified in exhaled breath condensate from LAM subjects and was increased by aspirin treatment, indicative of functional COX-2 expression in the LAM airway. In vitro, 15-epi-lipoxin-A₄ reduced the proliferation of LAM patient–derived cells in a dose-dependent manner. Targeting COX-2 and prostaglandin pathways may have therapeutic value in LAM and TSC-related diseases, and possibly in other conditions associated with mTOR hyperactivation.

Lymphangioleiomyomatosis (LAM) is a destructive lung disease primarily affecting women. Genetic studies indicate that LAM cells carry inactivating tuberous sclerosis complex (TSC)–2 mutations, and metastasize to the lung. We previously discovered that estradiol increases the metastasis of TSC2-deficient cells in mice carrying xenograft tumors. Here, we investigate the molecular basis underlying the estradiol-induced lung metastasis of TSC2-deficient cells, and test the efficacy of Faslodex (an estrogen receptor antagonist) in a preclinical model of LAM. We used a xenograft tumor model in which estradiol induces the lung metastasis of TSC2-deficient cells. We analyzed the impact of Faslodex on tumor size, the extracellular matrix organization, the expression of matrix metalloproteinase (MMP)–2, and lung metastasis. We also examined the effects of estradiol and Faslodex on MMP2 expression and activity in tuberin-deficient cells in vitro. Estradiol resulted in a marked reduction of Type IV collagen deposition in xenograft tumors, associated with 2-fold greater MMP2 concentrations compared with placebo-treated mice. Faslodex normalized the Type IV collagen changes in xenograft tumors, enhanced the survival of the mice, and completely blocked lung metastases. In vitro, estradiol enhanced MMP2 transcripts, protein accumulation, and activity. These estradiol-induced changes in MMP2 were blocked by Faslodex. In TSC2-deficient cells, estradiol increased MMP2 concentrations in vitro and in vivo, and induced extracellular matrix remodeling. Faslodex inhibits the estradiol-induced lung metastasis of TSC2-deficient cells. Targeting estrogen receptors with Faslodex may be of efficacy in the treatment of LAM.

Tuberous sclerosis complex (TSC) is a tumor suppressor syndrome characterized by benign tumors in multiple organs, including the brain and kidney. TSC-associated tumors exhibit hyperactivation of mammalian target of rapamycin complex 1 (mTORC1), a direct inhibitor of autophagy. Autophagy can either promote or inhibit tumorigenesis, depending on the cellular context. The role of autophagy in the pathogenesis and treatment of the multisystem manifestations of TSC is unknown. We found that the combination of mTORC1 and autophagy inhibition was more effective than either treatment alone in inhibiting the survival of tuberin (TSC2)-null cells, growth of TSC2-null xenograft tumors, and development of spontaneous renal tumors in Tsc2 ^+/− mice. Down-regulation of Atg5 induced extensive central necrosis in TSC2-null xenograft tumors, and loss of one allele of Beclin1 almost completely blocked macroscopic renal tumor formation in Tsc2 ^+/− mice. Surprisingly, given the finding that lowering autophagy blocks TSC tumorigenesis, genetic down-regulation of p62/sequestosome 1 (SQSTM1), the autophagy substrate that accumulates in TSC tumors as a consequence of low autophagy levels, strongly inhibited the growth of TSC2-null xenograft tumors. These data demonstrate that autophagy is a critical component of TSC tumorigenesis, suggest that mTORC1 inhibitors may have autophagy-dependent prosurvival effects in TSC, and reveal two distinct therapeutic targets for TSC: autophagy and the autophagy target p62/SQSTM1.

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-β-estradiol (E ₂ ) 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 MEK-dependent pathways by E ₂ leads to pulmonary metastasis via enhanced survival of detached tuberin-null cells.