Leptin promotes migration and invasion of breast cancer cells by stimulating IL-8 production in M2 macrophages

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

doi:10.18632/oncotarget.11761

Abstract

This study aims to investigate the mechanisms underlying leptin-mediated crosstalk between tumor-associated macrophages (M2 macrophages) and breast cancer cells. THP1 human leukemic monocytes were induced to differentiate into M2 macrophages by PMA (100 nM) and IL-4 (20 ng/mL). Quantitative RT-PCR and Western blot revealed that leptin (100 nM) significantly increased the expression of leptin receptor (ObR) in the M2 macrophages (P < 0.01) and stimulated interleukin (IL)-8 expression in the M2 macrophages, mouse macrophage cells RAW264.7, and primary mouse peritoneal macrophages in a dose- and time-dependent manner. Leptin-induced IL-8 production was sensitive to the ERK inhibitor PD980590 (10 μmol/L), p38 MAPK inhibitor SB203580 (20 μmol/L), and anti-ObR neutralizing antibody (4 μg/mL). Leptin (100 ng/mL) substantially increased the phosphorylation of p38 and ERK1/2. Thus, leptin may induce IL-8 production in M2 macrophages by interacting with ObR to activate the p38 and ERK signaling pathways. Scratch and transwell chamber assay showed that both recombinant IL-8 and leptin-induced M2 macrophage-derived IL-8 promoted the migration and invasion of human breast cancer cells MCF7 and MDA-MB-231 (All P < 0.01). In a nude mice xenograft model of breast cancer (n = 5 per group), injection of leptin (0.1 μg/g) dramatically increased tumor volume and mass, reduced survival, exacerbated pulmonary metastasis, and elevated IL-8 and Ki67 expression in the tumor tissue (All P < 0.05) compared with PBS injection. Depletion of mouse macrophage by Clophosome®-clodronate liposome and injection of anti-mouse IL-8 neutralizing antibodies in the xenograft tumor significantly attenuated those leptin-mediated stimulations (All P < 0.05). These findings indicate that leptin may promote tumor growth and metastasis by stimulating IL-8 production in tumor-associated macrophage.

Highlights

Obesity and high adipose tissue mass, which increase the risk of aggressive tumor phenotype and worsen survival of breast cancer [1,2,3,4,5], can cause excessive secretion of leptin and other pro-inflammatory factors [6,7,8]
Flow cytometry revealed that M2 macrophages, which were induced from THP1 cells by Phorbol esters (PMA) (100 nM) and IL-4 (20 ng/mL), expressed lower levels of IL-12 (1.38%) and higher levels of Transforming growth factor (TGF)-β (70.97%) and IL-10 (19.63%) compared with THP1 cells and THP1 macrophages (Figure 1B)
The expression of the mannose receptor CD206, a M2 macrophage surface marker, was dramatically increased in M2 macrophages (Figure 1B), suggesting that exposure to PMA and IL-4 appears to successfully induce THP1 cells to differentiate into M2 macrophages

Summary

Introduction

Obesity and high adipose tissue mass, which increase the risk of aggressive tumor phenotype and worsen survival of breast cancer [1,2,3,4,5], can cause excessive secretion of leptin and other pro-inflammatory factors [6,7,8]. High leptin levels have been found to increase breast cancer risk [9,10,11,12,13,14]. Leptin levels and the expression of leptin receptor (ObR) are substantially higher in breast cancer cells and tissues than in normal mammary epithelial cells and tissues [9,10,11]. Women with increased levels of serum leptin often have a higher incidence of breast cancer than women with normal leptin levels [12,13,14]. High levels of serum leptin may lead to aberrant intracellular signaling, which results in cancer development [15, 18]

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