Proapoptotic protein Bim attenuates estrogen-enhanced survival in lymphangioleiomyomatosis
Chenggang Li, Na Li, Xiaolei Liu, Erik Y. Zhang, Yang Sun, Kouhei Masuda, Jing Li, Julia Sun, Tasha Morrison, Xiangke Li, Yuanguang Chen, Jiang Wang, Nagla A. Karim, Yi Zhang, John Blenis, Mauricio J. Reginato, Elizabeth P. Henske, Jane J. Yu
Chenggang Li, Na Li, Xiaolei Liu, Erik Y. Zhang, Yang Sun, Kouhei Masuda, Jing Li, Julia Sun, Tasha Morrison, Xiangke Li, Yuanguang Chen, Jiang Wang, Nagla A. Karim, Yi Zhang, John Blenis, Mauricio J. Reginato, Elizabeth P. Henske, Jane J. Yu
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Research Article Cell biology Oncology

Proapoptotic protein Bim attenuates estrogen-enhanced survival in lymphangioleiomyomatosis

Abstract

Lymphangioleiomyomatosis (LAM) is a progressive lung disease that primarily affects young women. Genetic evidence suggests that LAM cells bearing TSC2 mutations migrate to the lungs, proliferate, and cause cystic remodeling. The female predominance indicates that estrogen plays a critical role in LAM pathogenesis, and we have proposed that estrogen promotes LAM cell metastasis by inhibition of anoikis. We report here that estrogen increased LAM patient–derived cells’ resistance to anoikis in vitro, accompanied by decreased accumulation of the proapoptotic protein Bim, an activator of anoikis. The resistance to anoikis was reversed by the proteasome inhibitor, bortezomib. Treatment of LAM patient–derived cells with estrogen plus bortezomib promoted anoikis compared with estrogen alone. Depletion of Bim by siRNA in TSC2-deficient cells resulted in anoikis resistance. Treatment of mice with bortezomib reduced estrogen-promoted lung colonization of TSC2-deficient cells. Importantly, molecular depletion of Bim by siRNA in Tsc2-deficient cells increased lung colonization in a mouse model. Collectively, these data indicate that Bim plays a key role in estrogen-enhanced survival of LAM patient–derived cells under detached conditions that occur with dissemination. Thus, targeting Bim may be a plausible future treatment strategy in patients with LAM.

Authors

Chenggang Li, Na Li, Xiaolei Liu, Erik Y. Zhang, Yang Sun, Kouhei Masuda, Jing Li, Julia Sun, Tasha Morrison, Xiangke Li, Yuanguang Chen, Jiang Wang, Nagla A. Karim, Yi Zhang, John Blenis, Mauricio J. Reginato, Elizabeth P. Henske, Jane J. Yu

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Figure 4

Erk1/2 and proteasome inhibition decreases estrogen-promoted cell survival and restores Bim levels in LAM cells.

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Erk1/2 and proteasome inhibition decreases estrogen-promoted cell surviv...
(A) ELT3 cells were seeded in poly-HEMA–coated plates, pretreated with or without 50 μM PD98059, a selective Erk1/2 inhibitor, or 100 nM proteasome inhibitor bortezomib (BTZ), or both PD98059 and BTZ for 15 minutes. The treatments were continued for an additional 24 hours in the presence of 10 nM estrogen (E2) or vehicle control. Cell death was analyzed by flow cytometry (n = 3). (B) The percentage of late apoptotic (annexin V+PI+) cells was determined (n = 3). (C) Immunoblotting analysis shows levels of phospho-Erk1/2, Erk1/2, Bim, and cell death markers cleaved caspase 3 and cleaved PARP in detached ELT3 cells treated with 10 nM E2 with or without 50 μM PD98059, or 100 nM BTZ, or both PD98059 and BTZ. Bim levels were quantified using densitometry, and normalized to β-actin. The lanes were run on the same gel, but were noncontiguous. (D) 621-101 cells were seeded in poly-HEMA–coated plates, pretreated with or without 50 μM PD98059, a selective Erk1/2 inhibitor, or 100 nM BTZ, or both PD98059 and BTZ for 15 minutes. The treatments were continued for an additional 24 hours in the presence of E2 (10 nM) or vehicle control. Cell death was analyzed by flow cytometry (n = 3). (E) The percentage of late apoptotic (annexin V+PI+) cells was determined (n = 3). (F) Immunoblotting analysis shows levels of phospho-Erk1/2, Erk1/2, Bim and cell death markers cleaved caspase 3 and cleaved PARP in detached 621-101 cells treated with 10 nM E2 with or without 50 μM PD98059, or 100 nM BTZ, or both PD98059 and BTZ. Bim levels were quantified using densitometry, and normalized to β-actin (obtained from replicate samples run in parallel). Results are representative of 3 experiments. Statistical analysis performed using a 1-way ANOVA test (Tukey’s multiple comparisons test multiple pair-wise comparisons between different groups). *P < 0.05, **P < 0.01, or ***P < 0.005 was considered significant.