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. 2016 Oct 21;35(1):166.
doi: 10.1186/s13046-016-0446-4.

Leptin Promotes Epithelial-Mesenchymal Transition of Breast Cancer via the Upregulation of Pyruvate Kinase M2

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Free PMC article

Leptin Promotes Epithelial-Mesenchymal Transition of Breast Cancer via the Upregulation of Pyruvate Kinase M2

Lan Wei et al. J Exp Clin Cancer Res. .
Free PMC article

Abstract

Background: Accumulating researches have shown that epithelial-mesenchymal transition (EMT) contributes to tumor metastasis. Leptin, a key adipokine secreted from adipocytes, shapes the tumor microenvironment, potentiates the migration of breast cancer cells and angiogenesis, and is also involved in EMT. However, the potential mechanism remains unknown. This study aims to explore the effect of leptin on EMT in breast cancer cells and the underlying mechanism.

Methods: With the assessment of EMT-associated marker expression in MCF-7, SK-BR-3, and MDA-MB-468 cells, the effect of leptin on breast cancer cells was analyzed. Besides, an array of pathway inhibitors as well as RNA interference targeting pyruvate kinase M2 (PKM2) were used to clarify the underlying mechanism of leptin-mediated EMT in vitro and in vivo.

Results: The results demonstrated that leptin promoted breast cancer cells EMT, visibly activated the PI3K/AKT signaling pathway, and upregulated PKM2 expression. An antibody against the leptin receptor (anti-ObR) and the PI3K/AKT signaling pathway inhibitor LY294002 significantly abolished leptin-induced PKM2 expression and EMT-associated marker expression. SiRNA targeting PKM2 partially abolished leptin-induced migration, invasion, and EMT-associated marker expression. In vivo xenograft experiments indicated that RNA interference against PKM2 suppressed breast cancer growth and metastasis.

Conclusions: Our data suggest that leptin promotes EMT in breast cancer cells via the upregulation of PKM2 expression as well as activation of PI3K/AKT signaling pathway, and PKM2 might be one of the key points and potential targets for breast cancer therapy.

Keywords: Breast cancer; Epithelial-mesenchymal transition; Leptin; Pyruvate kinase M2.

Figures

Fig. 1
Fig. 1
Leptin promoted epithelial-mesenchymal transition in breast cancer cell lines. a Western blotting analysis with specific antibodies demonstrated that Ob-Rb and Ob-Rt were expressed in MCF-7, SK-BR-3, and MDA-MB-468 cells. b An immunofluorescence assay showed leptin receptor expression on the membrane of breast cancer cells. Original magnification: ×400 c Morphological changes of breast cancer cells treated with leptin. The image showed cell-cell junction dissolution and loss of apical-basolateral cell polarity in MCF-7, SK-BR-3, and MDA-MB-468 cells after treatment with 200 ng/mL leptin. Original magnification: ×200 d Western blotting analysis demonstrated that leptin treatment upregulated vimentin and fibronectin expression, but downregulated E-cadherin expression in MCF-7, SK-BR-3, and MDA-MB-468 cells. All images were representative examples from three independent experiments
Fig. 2
Fig. 2
Leptin increased PKM2 expression. (af) Serum-starved MCF-7 and SK-BR-3 cells were exposed to concentrations of leptin ranged from 50 ng/mL to 200 ng/mL or 200 ng/mL leptin for 0 h to 24 h. Q-PCR and Western blotting analysis demonstrated that leptin promoted PKM2 mRNA and protein expression in a dose- and time-dependent manner in MCF-7 and SK-BR-3 cells
Fig. 3
Fig. 3
PKM2-siRNA abolished leptin-induced epithelial-mesenchymal transition in breast cancer cells. PKM2-siRNA treatment of MCF-7 and SK-BR-3 cells prior to 200 ng/mL leptin exposure decreased leptin-induced migration (a) and invasion (bd) (P < 0.05). (e) PKM2-siRNA abolished leptin-induced downregulation of E-cadherin and upregulation of vimentin, fibronectin and Twist in MCF-7 and SK-BR-3 cells (P < 0.05)
Fig. 4
Fig. 4
Leptin increased PKM2 expression and induced EMT in breast cancer cells via the activation of PI3K/AKT. a Serum-starved MCF-7 and SK-BR-3 cells were treated with PD98059 (MAPK inhibitor, 1.34 μmol/mL), AG490 (STAT3 inhibitor, 5 μmol/mL) and LY294002 (PI3K inhibitor, 10 μmol/mL) for 1 h, followed by 200 ng/mL leptin treatment for 24 h. DMSO was used as a control. Western blotting analysis demonstrated that the PI3K inhibitor LY294002 abolished leptin-mediated increase in PKM2 expression in MCF-7 and SK-BR-3 cells (P < 0.05). b Antibodies against ObR (4 μg/mL) abolished the leptin-mediated increase in p-AKT, p-PKM2 and PKM2 expression in MCF-7 and SK-BR-3 cells (P < 0.05). c The PI3K inhibitor LY294002 abolished leptin-mediated downregulation of E-cadherin expression and the upregulation of vimentin, fibronectin, p-PKM2 and PKM2 expression in MCF-7 and SK-BR-3 cells
Fig. 5
Fig. 5
RNA interference against PKM2 inhibited tumor progression in nude mice. SKBR-3, SKBR-3-LV, and SKBR-3-shPKM2 tumor cells (5 × 106 cells/per inoculation) were injected into mammary fat pads of 5-week-old female nude mice (n = 9/group). Tumor volumes were measured using calipers and defined as ab2/2, where a and b corresponded to length and width, respectively. A cohort of mice was sacrificed 5 weeks after injection. (a, b, c) Tumor volume and primary weights of mice in the SKBR-3-shPKM2 group were significantly smaller than that of mice in the SKBR-3 and SKBR-3-LV groups (P < 0.01). (d) A survival curve revealed that RNA interference against PKM2 increased the survival of tumor-bearing mice, compared with mice in the SKBR-3 and SKBR-3-LV groups (P < 0.01). (ef) H&E staining demonstrated that RNA interference against PKM2 inhibited lung metastasis of breast cancer xenografts. Immunohistochemistry staining demonstrated that RNA interference against PKM2 resulted in the increased expression of E-cadherin and decreased expression of vimentin, fibronectin and Twist

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