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-beta-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.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Estrogen promotes the lung metastasis of tuberin-deficient ELT3 cells in female and male mice. (A) The proliferation of ELT3 cells in response to E2 was measured by 3H-thymidine incorporation after 5 days of growth. (B–J) ELT3 cells were injected s.c. into the flanks of female ovariectomized and male SCID mice implanted with E2 (n = 9) or placebo (n = 10) pellets. (B) Lung metastases were scored from E2 (n = 9) or placebo-treated (n = 10) mice. (C) The number of lung metastases in male mice was scored from placebo (n = 10) and E2-treated (n = 9) mice. (D–I) Consecutive lung sections containing metastases (arrows) from an E2-treated female mouse were stained with H&E (D), anti-smooth muscle actin (E), and anti-phospho-S6 (F). (Scale bar, 50 μM.) (G) Anti-phospho-S6 immunostain of the primary xenograft tumor of an estrogen-treated female mouse. (H and I) Phospho-S6 immunoreactivity of a metastasis (H) and xenograft tumor (I) of an estrogen-treated male mouse. (Scale bar, 20 μM.) *, P < 0.05, Student's t test.

Fig. 2.

Estrogen increases circulating tumor cells in mice bearing xenograft tumors and enhances the survival and lung seeding of intravenously injected Tsc2-null cells. (A) DNA prepared from the blood of placebo (n = 3) and E2-treated (n = 3) mice bearing xenograft tumors of similar size (≈1,000 mm3) was analyzed by real-time PCR using rat-specific primers to quantitate circulating tumor cells. (B) Levels of circulating tumor cell DNA 6 h after i.v. injection of ELT3 cells into placebo (n = 3) and E2-treated (n = 3) mice. (C) Levels of tumor cell DNA in the lungs 24 h after i.v. injection of ELT3 cells into placebo (n = 3) and E2-treated (n = 3) mice. *, P < 0.05, Student's t test.

Fig. 3.

Estrogen promotes the lung colonization of Tsc2-null ELT3 cells. (A) ELT3-luciferase cells were injected intravenously into ovariectomized female placebo (n = 3) and E2-treated (n = 3) mice. Lung colonization was measured using bioluminescence at 1, 3, and 24 h after injection. Representative images are shown. (B) Total photon flux/second present in the chest regions in placebo (n = 3) and E2-treated (n = 3) animals. *, P < 0.05, Student's t test. (C) Lungs were dissected 24 h postcell injection and bioluminescence was imaged in Petri dishes.

Fig. 4.

Estrogen activates p42/44 MAPK in ELT3 cells in vitro and in vivo. (A) ELT3 cells were grown in phenol red-free and serum-free media for 24 h and then stimulated with 10 nM E2 for 0, 5, 15, or 120 min. Levels of phosphorylated p42/44 MAPK and total MAPK were determined by immunoblot analysis. Pretreatment with PD98059 blocked E2-induced MAPK activation. β-Actin immunoblotting was included as a loading control. (B) Levels of phosphorylated C-Raf/Raf-1 and total Raf-1 after E2 stimulation. (C) Levels of phosphorylated S6 after E2 stimulation. (D) The nuclear and cytoplasmic fractions were separated, and levels of phospho-p42/44 MAPK were examined by immunoblot analysis. Anti-ELK1 and anti-α-tubulin were included as loading controls for the nuclear and cytosolic fractions, respectively. (E and F) Pulmonary metastases from an E2-treated mouse showed hyperphosphorylation of p42/44 MAPK. (Scale bar, 50 μM and 125 μM.) (G and H) Phospho-p42/44 MAPK (T202/Y204) immunostaining of primary tumor sections from placebo-treated (G) and E2-treated (H) mice. (Scale bar, 20 μM.) (I) Percentage of cells with nuclear immunoreactivity of phospho-p42/44 MAPK was scored from 4 random fields per section. *, P < 0.05, Student's t test.

Fig. 5.

Estrogen increases the resistance of ELT3 cells to anoikis. ELT3 cells were grown in phenol red-free and serum-free media for 24 h and then treated with 10 nM E2 for 24 h before culturing on PolyHEMA plates. The MEK1/2 inhibitor PD98059 was begun 15 min before detachment. (A) The level of cleaved caspase-3 was determined by immunoblot analysis. α-Tubulin is included as a loading control. (B) DNA fragmentation was assessed by ELISA. (C) Cell growth was measured by 3H-thymidine incorporation after 24 h of growth on PolyHEMA plates in the presence or absence of E2, followed by 24 h of growth on adherent plates in the absence of E2. (D) Levels of phospho-p42/44 MAPK, MAPK, Bim, cleaved caspase-3, phospho-S6K, and phospho-S6 were determined by immunoblot analysis. α-Tubulin is included as a loading control. *, P < 0.05, Student's t test.

Fig. 6.

The MEK1/2 inhibitor CI-1040 blocks the estrogen-driven metastasis of ELT3 cells in vivo. ELT3 cells were injected into female ovariectomized nude mice implanted with estrogen or placebo pellets. Animals were treated with CI-1040 (150 mg/kg/day by gavage, twice a day) starting 1 day post-ELT3 cell inoculation for the xenograft experiments (A–E), or 2 days before cell inoculation for i.v. injection (F). (A) Tumor development was recorded as the percentage of tumor-free animals post-cell inoculation. (B) The primary tumor area was calculated at 7 weeks post-cell inoculation. (C) The level of circulating ELT3 cells was measured from blood samples of xenograft animals using rat-specific qPCR amplification. (D) The percentage of mice with lung metastases in the placebo and estrogen-treated groups was compared. (E) The number of lung metastases was scored. (F) ELT3-luciferase cells were injected intravenously into ovariectomized female E2-treated (n = 5) and CI-1040 plus E2-treated (n = 5) mice. Lung colonization was measured using bioluminescence 2 and 5 h after injection. Total photon flux/second present in the chest regions were quantified and compared between E2 (n = 5) and CI-1040 plus E2-treated (n = 5) animals. Lungs were dissected and imaged 60 h post-cell injection. Total photon flux/second present in ex vivo lungs were quantified and compared between E2 (n = 5) and CI-1040 plus E2-treated (n = 5) animals. *, P < 0.05, Student's t test.

Fig. 7.

The mTOR inhibitor RAD001 blocks primary tumor development and estrogen-driven metastasis of ELT3 cells in vivo. ELT3 cells were injected into female ovariectomized nude mice implanted with estrogen or placebo pellets. Animals were treated with RAD001 (4 mg/kg/day by gavage) starting 1 day post-ELT3 cell inoculation. (A) The primary tumor area was calculated at 8 weeks post-cell inoculation. (B) The number of lung metastases was scored at 8 weeks post-cell inoculation. *, P < 0.05, Student's t test.