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 2

Bim is the key mediator of anoikis.

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Bim is the key mediator of anoikis. (A) ELT3 cells were transfected with...
(A) ELT3 cells were transfected with 2 independent rat Bim siRNAs or control siRNA for 48 hours. Bim transcript levels were measured using real-time RT-PCR (n = 3). (B) ELT3 cells were transfected with 2 independent rat Bim siRNAs or control siRNA for 48 hours, followed by 24-hour culture in poly-HEMA–coated plates. Levels of Bim, cleaved caspase 3, and cleaved PARP (rat) were analyzed by immunoblotting; Bim levels were quantified using densitometry, and normalized to β-actin. (C) ELT3 cells transfected with 2 independent rat Bim siRNAs were stained with a BD Annexin V: FITC Apoptosis Detection Kit I. Anoikis was determined by flow cytometry (BD FACSCanto II). Annexin V+PI+ indicated cells undergoing anoikis (n = 3). (D) 621-101 cells were transfected with 2 independent human Bim siRNAs or control siRNA for 48 hours. Bim transcript levels were measured using real-time RT-PCR (n = 3). (E) 621-101 cells were transfected with 2 independent human Bim siRNAs or control siRNA for 48 hours, followed by a 24-hour culture in poly-HEMA–coated plates. Levels of Bim, cleaved caspase 3, and cleaved PARP (human) were analyzed by immunoblotting; Bim levels were quantified using densitometry, and normalized to β-actin. (F) 621-101 cells transfected with 2 independent human Bim siRNAs were stained with a BD Annexin V: FITC Apoptosis Detection Kit I. Anoikis was determined by flow cytometry (BD FACSCanto II). Annexin V+PI+ indicated cells undergoing anoikis (n = 3). Results are representative of 3 experiments. Statistical analysis performed using a 1-way ANOVA test (Dunnett’s multiple comparisons test comparing Bim siRNA-1, Bim siRNA-2 with siControl). *P < 0.05, **P < 0.01, or ***P < 0.005 was considered significant.