Estrogen promotes pulmonary metastases of tuberin-deficient cells, associated with extracellular matrix reorganization

B199 Lymphangioleiomyomatosis (LAM) is a rare disease that affects exclusively women. LAM is characterized by the proliferation of abnormal smooth muscle cells and cystic degeneration of lung parenchyma. Genetic studies suggest that LAM pathogenesis involves the metastasis of benign cells. The fact that LAM affects young women suggests that estrogen may promote metastasis. To determine whether estrogen promotes the metastasis of TSC2-deficient cells, rat uterine leiomyoma (ELT3) cells were injected subcutaneously into ovariectomized female or male CB17-scid mice implanted with estrogen or placebo pellets. The number of pulmonary metastases was scored in four 5-micron sections from both lungs.
 Estrogen significantly enhanced the frequency and the number of pulmonary metastases in both female and male mice. Estrogen-treated mice developed primary tumors that progressed more rapidly and attained a greater size than tumors in placebo-treated mice. The primary tumors and the metastases were strongly immunoreactive with anti-smooth muscle actin and anti-phospho-S6 antibody, as expected. Compared with placebo treated tumors, cells from the estrogen-treated tumors showed increased nuclear localization of phospho-p42/44 MAPK, suggesting a reactivation of signaling pathways that are impaired in tuberin-deficient ELT3 cells. Estrogen-treated tumors also expressed elevated levels of matrix metalloproteinase 2 (MMP2) compared with placebo-treated tumors.
 In both male and female mice, the estrogen-treated tumors exhibited an abnormal nuclear morphology and an altered ECM when compared with the placebo-treated tumors. The accumulation of type IV collagen, a major structural component of ECM, was greatly reduced in estrogen-treated tumors compared with placebo-treated tumors. These findings support a model in which estrogen reactivates MAPK in TSC2-deficient cells, leading to degradation of ECM and promoting pulmonary metastasis.
 To elucidate the signaling pathways responsible for estrogen-induced ELT3 cell metastasis, xenograft mice were treated with the estrogen receptor inhibitor Fulvestrant or the mTORC1 inhibitor RAD001. RAD001 completely blocked both the primary tumor development and the estrogen-induced metastasis. Fulvestrant did not inhibit the primary tumors, but completely blocked estrogen-promoted lung metastases.
 In vitro studies using cultured ELT3 cells supported the in vivo findings: estrogen rapidly stimulated phosphorylation and nuclear translocation of phospho-p42/44 MAPK, induced cellular MMP2 expression, and increased MMP2 activity in the conditioned media.
 In conclusion, we found that estrogen promotes the pulmonary metastases of tuberin-deficient ELT3 cells. This is associated with estrogen-stimulated MAPK activation and ECM alteration coupled with MMP2 expression and type IV collagen accumulation. RAD001 inhibited both primary tumor development and lung metastases. Fulvestrant inhibited the estrogen-induced lung metastases. We speculate that this animal model may have relevance to both LAM pathogenesis and to the development of estrogen-focused targeted therapeutic strategies for LAM.