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. 2015;16(8):1220-30.
doi: 10.1080/15384047.2015.1056409. Epub 2015 Jun 29.

Activation of IL-8 via PI3K/Akt-dependent pathway is involved in leptin-mediated epithelial-mesenchymal transition in human breast cancer cells

Lin Wang  1 , Cuiping Tang, Hong Cao, Kuangfa Li, Xueli Pang, Liang Zhong, Weiqi Dang, Hao Tang, Yunxiu Huang, Lan Wei, Min Su, Tingmei Chen
Affiliations

Activation of IL-8 via PI3K/Akt-dependent pathway is involved in leptin-mediated epithelial-mesenchymal transition in human breast cancer cells

Lin Wang et al. Cancer Biol Ther. 2015.

Abstract

Background information: Previous studies have revealed that leptin may be involved in epithelial-mesenchymal transition (EMT), a crucial initiator of cancer progression to facilitate metastatic cascade, increase tumor recurrence, and ultimately cause poor prognosis. However, the underlying mechanism remains unclear. The aim of our present study was to investigate the effect of leptin on EMT of breast cancer cells and the underlying mechanism.

Results: Our data demonstrated that leptin significantly increased the phosphorylation of STAT3, Akt, and ERK1/2, elevated the expression of IL-8, and induced breast cancer cells to undergo EMT. The effect of leptin on IL-8 could visibly abolished by the inhibitor of PI3K LY294002. In addition, leptin-induced EMT of breast cancer cells was blocked by anti-IL-8 antibodies. Examination of the expression of ObR, leptin, IL-8 and EMT-related biomarkers in patient specimens demonstrated that malignant breast carcinoma with lymph node metastases (LNM), which represents poor prognosis, expressed higher levels of ObR, leptin, IL-8 than other types of breast cancer, and displayed more obvious EMT transversion. In vivo xenograft experiment revealed that leptin signally promoted tumor growth and metastasis and increased the expressions of IL-8 and EMT-related biomarkers.

Conclusions: Our results support that leptin-induced EMT in breast cancer cells requires IL-8 activation via the PI3K/Akt signal pathway.

Keywords: AKT, Protein Kinase B; COX-2, cyclooxygenase-2; EMT; EMT, epithelial-mesenchymal transition; ERK, extracellular signal-regulated kinase; IFN, interferon; IL-8; IL-8, Interleukin 8; JAK, Junas Kinase; LNM, lymph node metastases; MAPK, Mitogen-activated protein kinase; MMP, matrix metalloproteinase; NF-κB, Nuclear factor kappa B; Ob-R, Ob receptor; PI-3K, phosphatidylinositol-3 kinase; PI3K/Akt; STAT, signal transduction and activators of transcription; TGF, transforming growth factor; TNF, tumor necorsis factor; VEGF, vascular endothelial growth factor; breast cancer; leptin; mTOR, Mammalian Target Of Rapamycin; qRT-PCR, quantify reverse transcription-polymerase chain reaction.

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Figures

Figure 1.
Figure 1.
Leptin induces epithelial-mesenchymal transition of breast cancer cells. (A) Proteins were extracted from MCF-7, SK-BR-3 and MD-MB-231 cells. Western blotting analysis using specific antibodies demonstrated that Ob-Rb (125 kDa) and Ob-Rt (100 kDa) were expressed in the breast cancer cells. (B) Immunofluorescence assay with specific antibodies demonstrated leptin receptors were presented on the membrane of breast cancer cells. Original magnification: × 400 (C) The pictures of microscope showed EMT-related morphological changes. The presence of cell-cell junction dissolution, loss of apical-basolateral cell polarity emerged in MCF-7 and SK-BR-3 cells treated with leptin after 16 h serum starvation, while variations of morphological features were not observed in MD-MB-231 cells. Original magnification: ×200 (D) Westren blot analysisdemostrated that leptin downregulated E-cadherin, while upregulated Vimentin and Fibronectin of MCF-7 and SK-BR-3 cells. The EMT-related proteins in MD-MB-231 cells were not investigated.
Figure 2.
Figure 2.
Leptin-Leptin ObR axis was required for activation of IL-8, STAT3, ERK1/2, AKT in breast cancer cells. Serum-starved MCF-7 and SK-BR-3 cells were treated with different concentrations of leptin from 20 ng/ml to 200 ng/ml for 24 h or treated with 100 ng/ml leptin for different time from 0 h to 48 h. Q-PCR and Western blotting analysisdemonstrated leptin facilitated IL8 mRNA or protein of MCF-7 and SK-BR-3 cells with dose (A, C) and time-dependent (B, D) manner. The effect reached to peak level after MCF-7 and SK-BR-3 cells were treated with 100 ng/ml leptin for 24 h.(E) Serum-starved MCF-7 and SK-BR-3 cells were treated with 100 ng/ml leptin for various time intervals from 0h to 24 h. Westren blotting analysis was performed to detect the levels of phosphorylared and total STAT3, ERK1/2, AKT. (F) Antibodies against ObR (4 μg/ml) addition to MCF-7 and SK-BR-3 cells prior to leptin exposure absolutely abolished the expression of phosphorylation STAT3 and AKT (P < 0.05), while no changes was observed in phosphotylation ERK1/2. The specific antibody also down- regulated secretion of IL-8 from breast cancer cells that were treated with leptin (P < 0.05).
Figure 2.
Figure 2.
(Continued)
Figure 3.
Figure 3.
Leptin-induced epithelial-mesenchymal transition of breast cancer cells were abolished in the presence of AKT inhibitor and monoclonal Ab against IL-8. Serum-starved MCF-7 and SK-BR-3 cells were pretreated with 50 μM AG490 or 20 μM LY294002 for 1 h followed by 100 ng/ml leptin treatment for 24 h, DMSO were added as control. For IL-8 blocking experiments, specific antibodies to IL-8 or control IgG were added in serum-starved MCF-7 and SK-BR-3 cells cultures together with leptin and then incubated for 48 h. (A) QPCR and (B) Western blotting analysis indicated that LY294002 could abolish leptin-induced activation of IL-8 of MCF-7, SK-BR-3 cells (P < 0.05). (B) Western blotting analysis indicated that LY294002 could also restore the changes of Vimentin, Fibronectin and E-cadherin achieved by leptin treatment. (C)Western blotting analysis indicated that Akt depletion using Akt-siRNA could also restore the changes of IL-8, Vimentin and E-cadherin achieved by leptin treatment compared with nontargeting control siRNA. (D)Western blotting and (E) Immunofluorescence assay with Fluorescent confocal microscopy showed that comparing with control IgG, IL-8 specific antibody could completely abolish the hallmarks of leptin-induced EMT of MCF-7 and SK-BR-3 cells (P < 0.05).(F)Cell scratch assay and (G)Transwell chamber showed that leptin obviously advanced the migration and invasion capability of MCF-7 and SK-BR-3 cells after 36 h incubation, respectively. While IL-8 antibody could reduce the effects compared with control Ab (P < 0.05). Original magnification: ×200.
Figure 3.
Figure 3.
(Continued)
Figure 3.
Figure 3.
(Continued)
Figure 4.
Figure 4.
Expression of ObR, Leptin, IL-8, E-cadherin and Vimentin in breast cancer tissues. Clinical pathological specimens of breast tissues were collected and subjected to immunohistochemistry analysis using ObR, Leptin, IL-8, E-cadherin and Vimentin antibodies. A, B and C represented benign hyperplasia breast tissues, invasive breast carcinoma without metastasis and carcinoma with its lymph node metastases (LNM). The staining intensity of ObR, Leptin, IL-8, and Vimentin in invasive breast carcinoma without axillary lymph node metastases was lower than in LNM and stronger compared to that in benign breast tissue, with contrary staining intensity of E-cadherin expression.
Figure 5.
Figure 5.
In vivo evidence for Leptin-mediated alterations of EMT markers and IL-8 expression levels. Groups of female nude mice (n = 5) were injected with 1×107 MCF-7 tumor cells in the mammary fat, and 15 d later, intratumor injection of PBS or leptin at 0.1 μg/g was performed biweekly until the 30th day, respectively. The mice were sacrificed. (A, B) Tumor volume and (B) primary weight of mice treated with PBS were significantly smaller than that of leptin injection (P < 0.001). (D) Survival time curve revealed that leptin reduced survival rate of tumor-bearing mice compared with group PBS (P < 0.001). (E) H&E staining demonstrated leptin promoted lung and liver metastasis of breast cancer xenografts. (F) Leptin increased expression of IL-8, Ki67 and Vimentin while decreased expression of E-cadherin evaluated by IHC.
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