Suppression of GOLM1 by EGCG through HGF/HGFR/AKT/GSK-3β/β-catenin/c-Myc signaling pathway inhibits cell migration of MDA-MB-231

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Abstract

Golgi Membrane Protein 1 (GOLM1) has been identified as a prime target for cancer therapy because it overexpresses in many solid tumors, increases tumor growth and metastasis and leads to unfavorable survival. Though various approaches including siRNA interference and antibody targeting have been attempted, GOLM1 has remained an un-targetable molecule because of its mainly intracellular location and the lack of domains that could possibly be interfered with by small molecules. Numerous natural anti-tumoral plant substances have been identified, while their possible function on GOLM1 has never been revealed.

This is the first report to study the relationship between GOLM1 downregulation and natural anti-tumoral plant substances and the possible mechanism. Among three tested possible migration-inhibiting natural substances (Epigallocatechin gallate (EGCG), Betulinic acid (BA) and Lupeol), EGCG showed the most potent inhibition effect on GOLM1 expression and MDA-MB-231 cell migration. Knocking down GOLM1 expression further increased the EGCG treatment effect. Molecular docking prediction and following experiments suggested that EGCG may inhibit GOLM1 expression and MDA-MB-231 cells migration through HGF/HGFR/AKT/GSK-3/β-catenin/c-Myc signaling pathway.

In all, EGCG is the first identified GOLM1 downregulation natural product. Silencing GOLM1 may be a novel mechanism of potentiated anti-cancer migration effects and cytotoxic effect of EGCG. In addition, this study shed a new way for cancer therapy by combination of GOLM1 silencing and EGCG treatment in the future.

Introduction

Breast cancer is the most common cancer in women (Torre et al., 2017; Anastasiadi et al., 2017). The latest statistic published by the American Cancer Society in 2018 shows that breast cancer has the highest morbidity and mortality among women (Bray et al., 2018). A large number of clinical treatments for breast cancer depends on the expression of biomarkers, such as estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor-2 (Her2) (Roodi et al., 1995; Cuzick et al., 2011; Badve et al., 2010). Due to the lack of common targeted receptors in triple negative breast cancer (TNBC), the current therapies of TNBC focus on the chemotherapy by combining chemical compounds which are also toxic to normal cells, such as paclitaxel and anthracycline (Crown et al., 2002). Among different breast cancer subtypes, TNBC has the strongest drug resistance, the highest relapse rate and most importantly, the poorest overall survival because of aggressive metastasis. And epithelial to mesenchyme transition (EMT) plays a vital role in tumor metastasis (Brabletz et al., 2018). Therefore, novel low-toxic therapeutic strategies with anti-tumor and anti-EMT effect to overcome TNBC are urgently needed.

Golgi Membrane Protein 1 (GOLM1, also called GP73 or GOLPH2), a type Ⅱ transmembrane protein in Golgi apparatus, has attracted wide attention because it is highly expressed in various tumors including liver, lung, prostate and colorectal cancer (Kladney et al., 2000; Yang et al., 2019; Wang and WanYvonne, 2020) and positively relevant to EMT and cell migration of them (Chen and Yang, 2019; Ding et al., 2019; Song et al., 2018; Kojima et al., 2014; Zhang et al., 2019; Thiery et al., 2009; ArunaLi and M., 2018). In hepatocellular carcinoma cells, up-regulation of GOLM1 promotes cancer progression and metastasis by modulating EGFR/RTK signaling pathway (Ye et al., 2016), regulating TGFβ/Smad2 signal pathway (Yang et al., 2018), reducing N-cadherin and E-cadherin expression (Liu et al., 2016), or inducing cAMP Responsive Element Binding protein (CREB) and MMP-13 expression (Jin et al., 2015). Knockdown of GOLM1 reduces proliferation, migration and invasion of glioblastoma (GBM) cells, as well as xenograft tumor growth by impacting the nuclear translocation of β-catenin and inhibiting the Wnt/β-catenin pathway (Ding et al., 2019). Down-regulation of GOLM1 suppresses proliferation and aggressiveness of non-small-cell carcinoma cells through inhibiting PI3K-AKT pathway (Chen and Yang, 2019). Zhang et al. first reported the correlation between GOLM1 and breast cancer and found that GOLM1 promoted cell growth and metastasis of breast cancer by upregulation MMP13 expression in 2019 (Zhang et al., 2019). The large number of GOLM1 positive malignant tumors indicates that GOLM1 may be a potential target for cancer therapy. Various approaches to exploit the properties of GOLM1 for therapy have been attempted. Ye et al. found that GOLM1 level determined chemotherapeutic resistance in human hepatocellular carcinoma cells (Ye et al., 2018). To create the antibody targeting of GOLM1 shed by circulating soluble sGOLM1, Liewen et al. developed two candidate antibodies (G2-1 and G2-2) that targeted the part of GOLM1 remaining at the cell surface after proteolytic cleavage and verified the durable response with GOLM1-directed antibody-toxic drug-conjugate in patient-derived colorectal and lung cancer xenograft (PDX)-models (Liewen et al., 2019). Though siRNA interference and antibody neutralization has shown some success in preclinical models, until now GOLM1 has remained an undruggable target.

Numerous natural substances with low toxicity and anti-tumor activity have been identified. Apigenin could inhibit cell invasion via PI3K/Akt pathway in MDA-MB-231 cells (Lee et al., 2008). Betulinic acid (BA) represses tumor metastasis through endoplasmic reticulum stress apoptotic pathway (Cai et al., 2018). Epigallocatechin gallate (EGCG) inhibited invasion of TNBC Cells (MDA-MB-231) by downregulating MMP-9 expression through FAK/ERK/NF-κB and Ap-1 or reducing TNF-α-mediated VCAM-1 expression (Sen and Chatterjee, 2011; Ko et al., 2015). The combination treatment of cisplatin and resveratrol induced apoptosis and autophagy in malignant mesothelioma cells (Lee et al., 2016).

Knockdown of GOLM1 reduced proliferation, migration and invasion of numerous tumors. However, it has never reported any natural substances suppressing cell proliferation and metastasis through blocking of GOLM1. To find GOLM1 down-regulating natural anti-tumor products, cell lines with GOLM1 high express should be considered. Our previous studies found that GOLM1 was highly expressed in TNBC cell lines (MDA-MB-231 cells) and knocking down GOLM1 significantly inhibited the migration and invasion of MDA-MB-231 (date not published). In this study, the function of selected natural anti-tumor substances (EGCG, BA, Lupeol) on GOLM1 were tested and EGCG was found to have the most potent down-regulation effect on GOLM1 expression. Further negative relationship between EGCG and GOLM1 were verified and the possible mechanisms how EGCG affected GOLM1 expression and cell-migration in MDA-MB-231 cell lines were studied.

Section snippets

Cell culture

Breast cancer cells MDA-MB-231 were obtained from the Cell Bank Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), and cultured in DMEM high glucose medium supplemented with 1% penicillin, 1% streptomycin (KeyGEN, Jiangsu, China) and 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Inc., USA) in a humidified incubator with 5% CO2 at 37 °C.

Cell viability

Cell viability was detected by MTT assay. In brief, MDA-MB-231 cells were inoculated in 96-well plates at a density of

Identification of GOLM1 expression down-regulating natural products in MDA-MB-231 cells

Knockdown of GOLM1 reduced proliferation, migration and invasion of numerous tumors. Numerous natural products capable of reducing tumor cell proliferation, migration and invasion have been identified. However, their possible function of natural products on GOLM1 expression has never been revealed. The prime purpose of this study was to find potential GOLM1 expression down-regulating natural products. We first determined whether the candidate plant extracts (EGCG, BA, Lupeol) could inhibit the

Discussion

Due to its specific spatial organization of several signal transduction pathways, the Golgi is increasingly being viewed as a hub for different signaling pathways (Millarte and Farhan, 2012). GOLM1, a Golgi phosphoprotein, has been indicated to play a crucial role in tumor metastasis (Kladney et al., 2000; Chen and Yang, 2019). Our previous study has revealed that GOLM1 is overexpressed in TNBC MDA-MB-231 cells, and knockdown of GOLM1 reduces cell migration and invasion. Ding et al. detected

Funding

This work was supported by the National Key R&D Program of China [grant number 2019YFD0901805], the 111 Project (B18022), the Fundamental Research Funds for the Central Universities [grant number 22221818014], and the Open Project Funding of the State Key Laboratory of Bioreactor Engineering, ECUST (ZDXM2019).

Submission declaration and verification

I, the writer of the thesis “Epigallocatechin gallate suppresses the migration of MDA-MB-231 breast cancer cells by downregulating GOLM1”, agree to authorize this thesis to be enrolled in Food and Chemical Toxicology. I also agree to authorize the Food and Chemical Toxicology Press exclusive right to edit, publish and distribute the above thesis, and insuring that this authorization will not invade other's right or benefit. Any infringement dispute or loss caused by the use of the above right

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Liming Zhao reports was provided by the National Key R&D Program of China. Liming Zhao reports was provided by the 111 Project. Liming Zhao reports was provided by the Fundamental Research Funds for the Central Universities. Liming Zhao reports was provided by the Open Project Funding of the State Key Laboratory of Bioreactor Engineering.

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