Activation of GPER suppresses migration and angiogenesis of triple negative breast cancer via inhibition of NF-κB/IL-6 signals

doi:10.1016/j.canlet.2016.11.003

Cancer Letters

Volume 386, 1 February 2017, Pages 12-23

https://doi.org/10.1016/j.canlet.2016.11.003 Get rights and content

Highlights

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GPER agonist G-1 inhibited IL-6 and VEGF-A in TNBC cells.
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G-1 or anti-IL-6 suppressed in vitro tube formation of HUVECs.
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Recombinant IL-6 reversed inhibitory effects of G-1 on angiogenesis and migration.
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G-1 inhibited the binding of NF-κB with IL-6 promoter.
•
The suppression of IL-6 by G-1 inhibited HIF-1α and STAT3 signals.

Abstract

Triple-negative breast cancer (TNBC) is characterized by high vascularity and frequent metastasis. Here, we found that activation of G protein-coupled estrogen receptor (GPER) by its specific agonist G-1 can significantly inhibit interleukin 6 (IL-6) and vascular endothelial growth factor A (VEGF-A). TNBC tissue microarrays from 100 TNBC patients revealed GPER is negatively associated with IL-6 levels and higher grade and stage. Activation of GPER or anti-IL-6 antibody can inhibit both in vitro tube formation of human umbilical vein endothelial cells (HUVECs) and migration of TNBC cells. While recombinant IL-6 supplementary can significantly reverse the inhibitory effects of G-1, suggesting the essential role of IL-6 in G-1 induced suppression of angiogenesis and invasiveness of TNBC cells. G-1 treatment decreased the phosphorylation, nuclear localization, transcriptional activities of NF-κB and suppressed its binding with IL-6 promoter. BAY11-7028, the inhibitor of NF-κB, can mimic the effect of G-1 to suppression of IL-6 and VEGF-A. While over expression of p65 can attenuate the inhibitory effects of G-1 on IL-6 and VEGF expression. The suppression of IL-6 by G-1 can further inhibit HIF-1α and STAT3 signals in TNBC cells by inhibition their expression, phosphorylation and/or nuclear localization. Moreover, G-1 also inhibited the in vivo NF-κB/IL-6 signals and angiogenesis and metastasis of MDA-MB-231 xenograft tumors. In conclusion, our study demonstrated that activation of GPER can suppress migration and angiogenesis of TNBC via inhibition of NF-κB/IL-6 signals, therefore it maybe act as an important target for TNBC treatment.

Introduction

Triple-negative breast cancer (TNBC) is defined by the absence of estrogen receptor (ER) and progesterone receptor (PGR) and low levels of human epidermal growth factor receptor 2 (HER-2). TNBC cells are characterized by the expression of basal or mesenchymal programs, which lead them more aggressive and easily to metastasis [1]. Furthermore, TNBC patients can't benefit from neither endocrine therapy nor targeted therapeutics due to the lack of ER and HER-2 [2]. Clinical data show that TNBC patients have the worst prognosis and highest mortality as compared with other subtypes of breast cancer [2]. Considering currently no targeted therapy drug has been approved by FDA (Food and Drug Administration) for TNBC treatment, there is an urgent need to illustrate the underlying molecular mechanisms responsible for the aggressive nature and progression of this subgroup.

Angiogenesis is an essential step for tumor development and initiated by chemoattractive and proliferative cytokines such as vascular endothelial growth factor (VEGF) [3]. Proangiogenic factors such as interleukin 6 (IL-6) and IL-8 can activate quiescent microvascular endothelial cells and induce them to form tube structures [4]. Angiogenesis plays an important role in the pathogenesis of TNBC [5]. TNBC patients have significant higher levels of VEGF and shorter survival times compared with non-TNBC [6]. Bevacizumab (Avastin; Genentech), which inhibits VEGF, can prolong progression-free survival in patients with TNBC [7], [8]. Proangiogenic factors including IL-6 and IL-8 are highly expressed and critical for growth of TNBCs but not ER-positive breast cancer cells [9]. Therefore, the identification of novel therapy directed towards angiogenesis may be an alternative way to improve outcome in this aggressive breast cancer subtype.

Estrogenic signals are critical for the progression of breast cancers in which ERα is considered as the main mediator. As to TNBC, recent study suggested that estrogenic signals can activate G protein-coupled estrogen receptor (GPER) [10] and its down downstream cascade signaling such as MAPK and PI3K/Akt signaling [11]. Recent data suggested that the expression of GPER was down-regulated during cancer tumorigenesis of ovarian [12] and TNBC [13] cancer patients. This is further confirmed by our studies that activation of GPER via its specific agonist G-1 can suppress the proliferation and epithelial mesenchymal transition (EMT) of TNBC cells [13], [14]. However, the specific status of non-genomic signaling mediated by GPER in cancer angiogenesis remains unclear.

In the present study, we reported that activation of GPER by its agonist G-1 significantly inhibited expression of IL-6 and VEGF-A both in vitro and in vivo and then suppressed the angiogenesis and progression of TNBC. Furthermore, the down regulation of NF-κB was essential in G-1 decreased angiogenesis and IL-6 production. Our findings suggested that activation of GPER signaling might represent a novel therapeutic approach for TNBC treatment.

Section snippets

Cell culture and treatment

TNBC cells MDA-MB-231, BT-549, HS578T, and MDA-MB-468 were acquired from the American Type Culture Collection (Manassas, VA, USA), and cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 10 μg/ml streptomycin at 37 °C in a 5% CO₂ atmosphere. Cell were treated with 1 μM G-1, 10 ng/ml TGF-β, 40 ng/ml recombinant IL-6, 200 ng/ml anti-IL-6 antibody, 10 μM NF-κB inhibitor BAY11-7028 (BAY) unless other specified. For transfection studies, cells

Activation of GPER suppresses IL-6 and VEGF-A in TNBC cells

To identify the breast cancer-associated genes regulated by GPER activation, we performed PCR array analysis to check the variation of 84 genes commonly involved in regulation of signal transduction and biological pathways of breast carcinogenesis, progression and invasion of breast cancer cell lines (Breast Cancer PCR array PAHS-131Z, SABiosciences). The mRNA levels of a total of 24 genes were induced by at least 1.5-fold by one of the treatments by comparison with un-induced control cells (

Discussion

TNBC cells are characterized by high vascularity and frequent metastasis [2]. Metastasis is the major cause of tumor recurrence and patient death, whereas angiogenesis is essential for the in vivo growth of cancers. Most publications have focused on the regulatory function of GPER in endocrinology and cancer cell proliferation [10]. Here we demonstrated the suppression effects of GPER on angiogenesis and metastasis of the following evidences: in vitro studies showed GPER specific agonist can

Acknowledgements

This research was supported by the National Natural Science Foundation of China (Grant No. 81673454, No. 81672608, No. 81472470, No. 81302317, and No. 81572270), the Guangdong Natural Science Funds for Distinguished Young Scholar (No. 2014A030306025), the Pearl River S&T Nova Program of Guangzhou (No. 201506010039), the Opening Project Program of State Key Laboratory of Oncology in South China (No. HN2014-09), and the Science & Technology Planning Project of Guangdong Province (2013B060300005).

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¹: These authors contributed equally to this work.

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Original ArticleActivation of GPER suppresses migration and angiogenesis of triple negative breast cancer via inhibition of NF-κB/IL-6 signals

Highlights

Abstract

Introduction

Section snippets

Cell culture and treatment

Activation of GPER suppresses IL-6 and VEGF-A in TNBC cells

Discussion

Acknowledgements

Ann. Oncol. Off. J. Eur. Soc. Med. Oncol. ESMO

Cell

Ann. Oncol. Off. J. Eur. Soc. Med. Oncol. ESMO

Mol. Oncol.

Toxicol. Lett.

Biochim. Biophys. Acta

Crit. Rev. Oncol. Hematol.

Cancer Lett.

Mol. Cell

J. Neuroimmunol.

Cancer Cell

Cancer Cell

Cell

Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies

J. Clin. Invest

Molecular mechanisms and clinical applications of angiogenesis

Nature

Vascular proliferation is increased in basal-like breast cancer

Breast Cancer Res. Treat.

Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer

N. Engl. J. Med.

RIBBON-2: a randomized, double-blind, placebo-controlled, phase III trial evaluating the efficacy and safety of bevacizumab in combination with chemotherapy for second-line treatment of human epidermal growth factor receptor 2-negative metastatic breast cancer

J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol.

Growth of triple-negative breast Cancer cells relies upon coordinate autocrine expression of the proinflammatory cytokines IL-6 and IL-8

Cancer Res.

The G-protein-coupled estrogen receptor GPER in health and disease

Nat. Rev. Endocrinol.

G protein-coupled receptor 30 mediates estrogen-induced proliferation of primordial germ cells via EGFR/Akt/β-catenin signaling pathway

Endocrinology

GPER-1 acts as a tumor suppressor in ovarian cancer

J. Ovarian Res.

The activation of G protein-coupled receptor 30 (GPR30) inhibits proliferation of estrogen receptor-negative breast cancer cells in vitro and in vivo

Cell Death Dis.

Original Article
Activation of GPER suppresses migration and angiogenesis of triple negative breast cancer via inhibition of NF-κB/IL-6 signals