Chemerin Promotes The Proliferation And Migration of Breast Cancer Cells Through JNK And ERK1/2 Signaling pathways

Breast cancer is the most common invasive malignancy. In 2020, the number of new cases of breast cancer worldwide has replaced lung cancer as the No.1 cancer in the global. Breast cancer is the leading cause of cancer death among women worldwide. Mammary tumorigenesis is severely linked to obesity, one potential connection is chemerin. Chemerin is a chemoattractant protein secreted by adipocytes, which contributes to the progression of breast cancer. Cell proliferation, migration, and invasion are cellular processes associated with various stages of metastasis. These processes are associated with mitogen-activated protein kinase (MAPK) signaling pathway. In this study, human breast cancer cell lines MCF-7 and MDA-MB-231were utilized to determine the effect of chemerin on cell proliferation, migration, and key proteins of MAPK signaling pathway. We found that chemerin promoted cell proliferation and migration in a concentration-dependent manner. Interestingly, these effects of chemerin were through promoting the proteins phosphorylation of ATF2 and ERK1/2 but not p38, in MAPK signaling pathway. Specic inhibitors of JNK and ERK1/2 pathway showed that the effect exerted by chemerin in cell proliferation and migration in breast cancer cells was dependent on these proteins. Our ndings suggest that chemerin promotes the development of mammary cancer cells through JNK and ERK signaling pathways. and cell migration rate with the increase of chemerin in a concentration-dependent after the treatment of MCF-7 MDA-MB-231 cancer cell lines with concentrations of chemerin. value and cell migration rate of the cancer cells were reduced. that ndings support groups: extracellular signal-regulated kinases (ERKs), p38 MAPKs, and c-Jun NH2-terminal kinases (JNKs). Activation of these MAPKs occurs through a cascade of upstream kinases. Our showed that cell migration was signicantly inhibited after MCF-7 & MDA-MB-231 human breast cancer cell lines treated by JNK and ERK pathway inhibitors, while cell migration was not signicantly affected by treatment with p38 pathway inhibitor. These results indicate that chemerin plays roles through JNK and ERK signaling pathways, not the P38 signaling pathway.


Introduction
Breast cancer is the most common malignancy in women worldwide, accounting for a quarter of cancer cases in women [1], and causing nearly 700,000 deaths globally each year [2]. According to the data released by the World Health Organization's International Agency for Research on Cancer (IARC) in 2020, the number of new cases of breast cancer worldwide has reached 2.26 million, which has replaced lung cancer as the No.1 cancer in the global [3]. Breast cancer is the leading cause of cancer death among women worldwide in 2020, accounting for 680,000 deaths [3]. There are many important risk factors for breast cancer, including obesity, estrogen, and so on [4]. Obesity, which is a worldwide epidemic [5], has been found to be associated with different kinds of cancers [6]. Post-menopausal obesity has especially given the increased risk of breast cancer [7]. It has been reported that post-menopausal obesity with an excess estrogen condition can cause a 63% increasing risk in breast cancer [6] through many biologically active factors including estrogen, leptin and chemerin, ect [8][9][10][11]. Chemerin, discovered adipokine recently, also known as tazarotene-induced gene 2(TIG2) or retinoic acid receptor responder 2(RARRES2), is a chemoattractant protein which was demonstrated to regulate adipogenesis, metabolism, immunity and tumorigenesis [12][13][14][15]. It has been closely associated with metabolic syndrome, obesity, in ammatory diseases and cancer [16][17][18][19][20][21][22][23][24][25]. At present, the relationship between chemerin and cancer is not clearly understood completely. The con icting reports, tumorigenic [25][26][27] and antitumor [12,28,29] effects, about the connection between chemerin and cancer, have caused this relation to be controversial. For breast cancer, some suspect that chemerin may be involved in a higher risk, albeit indirectly [30]. Another research [10] has showed that chemerin expression is positively relevant to patients' body weight, body mass index (BMI), tumor size, metastasis including lymph node metastasis and distant metastasis, and tumor grading, respectively, and is inversely relevant respectively to estrogen receptor (ER) and progesterone receptor (PR) expression in breast cancer tissues. Chemerin expression is a predictor and prognostic factor for breast cancer. While a cross sectional study [5] showed that chemerin concentration in serum could not be used in breast cancer stage by studying 117 patients with breast cancer.
Researchers found that chemerin concentrations in serum were no differences between patients with metastatic and non-metastatic cancer. These studies have an important limitation which was short of a control group without breast cancer. Moreover, some adipose-derived angiogenic factors have shown experimentally that they may promote breast cancer growth, though this has yet to be tested in the context of chemerin [31,32]. It has also been conjectured that chemerin may play a role in promoting metastasis through its angiogenic functions.
In this study, we evaluated the roles of chemerin in cell proliferation and migration in a cultured cell model of breast cancer, and its related mechanism. We utilized the human breast cancer cell lines MCF-7 and MDA-MB-231 and found that chemerin promoted cell proliferation and migration by up-regulating proteins phosphorylation of ATF2 and ERK1/2 of MAPK pathway but not p38. Using a combination of inhibitors for these proteins we found a decrease in cell proliferation and migration in breast cancer cells.
Here, we presented that chemerin contributed to cell proliferation and migration in breast cancer cell lines by the molecular mechanism of the signaling events which the chemerin system may be utilized or manipulated for clinical bene t.

Cell line and culture
Human breast cancer cell lines MCF-7 & MDA-MB-231 were purchased from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China), and were cultured in RPMI-1640 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclon, USA) and 1000U/mL Penicillin & 100µg/mL streptomycin (Beijing Solaibao Technology Co. Ltd, China). Cells were incubated at 37℃ with 5% CO 2 under a humidi ed atmosphere. The following procedures were applied to all assays (i.e., cell proliferation assay, cell wound healing assay and western blot analyses). Breast cancer cell lines MCF-7 and MDA-MB-231 were cultured in serum-free medium for 18h. Then, along with the control (without any treatment), the following concentrations of chemerin (1, 10 and 100µg/L) were added to the respective wells. The cells were incubated with chemerin for 24 h to test cell proliferation. And concentrations of chemerin(100µg/L) or chemerin neutralizing antibody (100µg/L) were used to test cell proliferation & migration and protein expressions.

Cell proliferation assay
The MTT assay was used to evaluate the effect of chemerin on cell proliferation. MCF-7 & MDA-MB-231 cells were cultured in T-75 culture asks, up to 85% con uency. Cells were harvested and then seeded in 96-well plates overnight at a density of 5 × 10 3 cells per well contain RPMI-1640 medium 200µL. Afterwards, cells were grown in serum-free medium for 18h, chemerin (Peprotech, USA) with varying concentrations of 1, 10 and 100µg/L or chemerin neutralizing antibody (100µg/L) (Beijing Bosen Biology Co. LTD, China) was then added to the medium. The control group was treated with an equal volume of RPMI-1640 with 10% FBS. After 24 h, the cells were incubated with MTT (5mg/ml) 20µL for 4 h, then lysed in dimethylsulfoxide (DMSO) 150µL for 30min. Finally, the optical density (OD) at 490nm was measured with a microplate reader (Bio-Tek Instruments, Winooski, VT, USA). Cell proliferation assays were performed at least 3 times for each cell line (in replicates of 6 wells for each concentration of chemerin in each experiment). Data are presented as means ± SD for a representative experiment for each cell line.

Western blot analysis
Cells were cultured in 6-well plates in serum-free medium, then treated with chemerin (100µg/L) or chemerin neutralizing antibody (100µg/L) and incubated for 24h following the above-mentioned protocol. For extraction of total soluble proteins from the cultured cells, highly e cient RIPA tissue/cell lysates (Beijing Solaibao Technology Co. Ltd, China) were used. Cell lysates were sonicated and subsequently centrifuged at 12,000rpm for 20min at 4℃, and clear supernatants were collected. For the Western blot analysis, 10% SDS-PAGE was used and proteins were transferred to PVDF membranes (Immobilon-P, Millipore, Billerica, MA). Primary antibodies including phospho-speci c antibodies against the following proteins were used for probing: ATF-2, ERK1 & ERK2 and p38 (all antibodies were from Abcam, USA).
Mouse anti-actin antibody was from Beijing Zhongshan Jinqiao Biological Co. Ltd, China. Suitable secondary IgGs conjugated with horseradish peroxidase (HRP) were used as secondary antibody. Proteins were detected by Chemiluminescence Detection kit (Beijing Kangwei Biological Co. Ltd, China) and analyzed the gray values of the obtained bands by Gel imaging system (Bio-Rad, USA).

Statistical analysis
Results are presented as means ±standard error of the mean (SEM) for a representative experiment for each cell line. Data were statistically analyzed using GraphPad Prism version 5 software (GraphPad Software, Inc., CA, USA). Analysis of more than two groups was performed using one-way ANOVA and the pairwise comparisons using t test. Difference at p<0.05 was considered signi cant.

Chemerin promotes cell proliferation in a concentrationdependent manner
Breast cancer cells MCF-7 and MDA-MB-231 were treated with different concentrations of chemerin (1, 10, 100µg/L). After 24h, the cell proliferation was detected by MTT method. The results showed that OD value of each concentration of chemerin treatment group was higher than that of the control group (p< 0.05, Fig. 1). The higher the concentration of chemerin, the bigger OD value is. The promotion effect of 100µg/L chemerin treatment group was the most obvious.

Chemerin promotes cell migration in a concentrationdependent manner
MCF-7 and MDA-MB-231 breast cancer cells were treated with different concentrations of chemerin (1, 10, 100µg/L), and cell migration ability was detected by scratch assay. The results showed that after 48 h of scratching, the cell migration rate of each concentration of chemerin treatment group was higher than that of the control group (p<0.05 or p<0.01, Fig. 2). The higher the concentration of chemerin, the stronger the promotion effect of cell migration is. The promotion effect of 100µg/L chemerin treatment group was the most obvious.

Chemerin neutralizing antibody inhibits cell proliferation
Breast cancer cells MCF-7 and MDA-MB-231 were treated with chemerin (100µg/L) and chemerin neutralizing antibody (100µg/L), respectively. The cell proliferation was detected by MTT assay. The results showed that compared with the control group, the OD value of MCF-7 and MDA-MB-231 breast cancer cells was signi cantly increased after chemerin was treated for 24h, while the OD value of the chemerin neutralizing antibody group was lower than that of the control group. This indicated that chemerin neutralizing antibody could signi cantly reduce the proliferation ability of breast cancer cells (p<0.05 or p<0.01, Fig. 3).

Chemerin neutralizing antibody inhibits cell migration
Breast cancer cells MCF-7 and MDA-MB-231 were treated with chemerin (100µg/L) and chemerin neutralizing antibody (100µg/L), respectively. Cell migration was detected by scratch assay. The results showed that 48h after scratching, the cell migration rate of the control group was signi cantly lower than that of the chemerin treatment group, while the cell migration rate of the chemerin neutralizing antibody group was signi cantly lower than that of the control group (p <0.05, Fig. 4).
2.5 Chemerin enhances protein ATF2 and ERK1/2 phosphorylated expressions but no change for p38 MCF-7 and MDA-MB-231 breast cancer cell lines were cultured and respectively treated with chemerin (100µg/L) and chemerin neutralizing antibody (100µg/L), the total protein was extracted after 24 h. ATF2, ERK1/2 and p38 proteins of JNK, ERK1/2 and p38 signaling pathway was detected by Western blot. The results showed that in MCF-7 cells, the protein expression levels of total ATF2, ERK1/2 and p38 had no signi cant differences compared with the control group. The phosphorylation of ATF2 and ERK1/2 proteins was signi cantly increased after chemerin treatment, but signi cantly decreased in the chemerin neutralizing antibody group (p <0.05). The expression of phosphorylated p38 protein was not signi cantly changed either being treated by chemerin or chemerin neutralizing antibody (see Fig. 5A

Effects of signaling pathway inhibitors on the ability of chemerin to promote the migration of breast cancer cells
Two breast cancer cell lines, MCF-7 and MDA-MB-231, were cultured and seeded into 6-well plates. After the cells were lled, the scratch experiment was performed. Before treated with signaling pathway inhibitors, each well was added with chemerin (100µg/L). Then, equal amounts of DMSO, JNK signaling pathway inhibitor SP600125, ERK1/2 signaling pathway inhibitor FR180204 and p38 signaling pathway inhibitor SB203580 were respectively added into different well. Samples were taken at 0, 24 and 48 h under an inverted microscope. Three sites were taken from each group for recording and cell migration rate was calculated. The results showed that the cell migration rate in groups of JNK and ERK1/2 signaling pathway inhibitor group were signi cantly smaller than that in the DMSO group (p<0.001). The cell migration rate was smaller in the p38 signaling pathway inhibitor group, but there was no statistically different (Fig. 6).

Discussion
Breast cancer is the most common malignant tumor in women in the world at present. Many bioactive substances, for example leptin, which produced by adipose tissue, are closely related to the incidence of breast cancer. Chemerin is a protein produced by adipose tissue and is encoded by the RARRES2 gene, which translates into a protein containing 163 amino acids with a molecular weight of 18.618KDa [34,35]. This protein is synthesized by adipose tissue triggered by TNF-α[36] and consists of a 20-amino acid hydrophobic N-terminal signal peptide, an intervening 137-amino acid cysteine folded domain and a 6amino acid pre-C-terminal fragment, with low activity [37]. Chemerin is the endogenous ligand of chemokine-like receptor 1 (CMKLR1) or ChemR23 (also known as DEZ), which is one of G-protein-coupled receptor, and plays a biological role by binding to these receptors. A number of studies have shown that chemerin plays a role in a various of pathophysiological processes such as obesity [18,38], in ammatory [39,40], metabolic syndrome [41], psoriasis [42], dilated cardiomyopathy [43], non-alcoholic steatohepatitis [44], and polycystic ovary syndrome [45]. At the same time, chemerin is also one of the key molecules involved in the process of oncogenesis and immune surveillance. The expression level of chemerin will also change during the occurrence and development of different cancers. Speci cally, chemerin can promote or inhibit tumor occurrence and development [10,16,20,[46][47][48]. Studies have con rmed [49][50][51][52] that the major binding receptor of chemerin, CMKLR1, is expressed on the surface of immune cells and various malignant tumor cells. Second atypical receptor of chemerin, chemokine CCmotif receptor-like 2(CCRL2), is mainly expressed in endothelial cells, macrophages, dendritic cells, and so on. This Helps focus chemerin on activated endothelial cells and sites of in ammation. Though chemerin has not been deeply studied in breast cancer, there are several preliminary pieces of evidence showing that chemerin may have a tumor-promoting effect. Chemerin is known to be associated with obesity and obesity and obesity-related parameters, such as blood pressure and BMI, and postmenopausal obesity is associated with an increased risk of breast cancer, so some researchers suspect that chemerin may be correlated to a higher risk of breast cancer, albeit with indirect evidence [30]. It has also been speculated that chemerin may play a role in instigating metastasis through its angiogenic functions. However, in breast adenocarcinoma, which is one of the most common types of breast cancer, the expression of chemerin is signi cantly down-regulated [12], suggesting that chemerin may have an antitumor effect in breast cancer. This anti-tumor role was also veri ed by Kim [53]. Our study showed that OD value and cell migration rate increased with the increase of chemerin concentration in a concentration-dependent manner after the treatment of MCF-7 & MDA-MB-231 human breast cancer cell lines with different concentrations of chemerin. Moreover, after treatment with chemerin neutralizing antibody, the OD value and cell migration rate of the cancer cells were reduced. These results suggest that chemerin can promote the proliferation and migration of breast cancer cells. Our ndings support the tumor-promoting effect of chemerin.
Chemerin is a chemotactic protein that acts as a ligand for both CMKLR1, one of the G-protein-coupled receptors, and GPR1 and CCRL2. Only the biological actions of chemerin/CMKLR1 axis have been clearly described at present [39,[54][55][56]. CMKLR1 is known to be expressed on the surface of a variety of malignant tumor cells [49][50][51][52]. The stimulation of chemerin can induce cell invasion of human mesenchymal stromal cells [19], gastric cancer cells [20], and esophageal squamous cancer cell [26]. In patients with advanced stage of non-small cell lung cancer (NSCLC) receiving platinum-based chemotherapy, the SNP rs1878022, located within an intron in the CMKLR1 gene on chromosome12q23.3, was positively statistical associated with decreased overall survival rate [57]. This suggests that CMKLR1 could have a role in NSCLC, and in vitro studies are in agreement. Study has shown that chemerin activates key mitogen-activated protein kinase (MAPK) and Akt signaling pathways and stimulates gelatinolytic activity of matrix metalloproteinase (MMP) to induce cell migration and angiogenesis [58]. The MAPK signaling pathway is one of the original signaling systems which exists in all eukaryotes, and controls fundamental cellular processes such as proliferation, differentiation, survival and apoptosis. Based on the structure and function, mammalian MAPKs can be divided into three groups: extracellular signal-regulated kinases (ERKs), p38 MAPKs, and c-Jun NH2-terminal kinases (JNKs). Activation of these MAPKs occurs through a cascade of upstream kinases. Our study showed that cell migration was signi cantly inhibited after MCF-7 & MDA-MB-231 human breast cancer cell lines treated by JNK and ERK pathway inhibitors, while cell migration was not signi cantly affected by treatment with p38 pathway inhibitor. These results indicate that chemerin plays roles through JNK and ERK signaling pathways, not the P38 signaling pathway.
Although our study has showed that chemerin promotes breast cancer growth through JNK and ERK signaling pathways, it is still unclear whether chemerin can be used as therapeutic target or biomarker for breast cancer and it needs in-depth study by a large number of researchers.

Availability of data and materials
All data generated or analysed during this study are included in this published article.

Ethics approval and consent to participate
This article does not contain any studies with human participants or animals performed by any of the authors, therefore no ethic approval or consent is required. No administrative permission and/or licenses is acquired by this study to access the original data used in this research.   Cell proliferation assay after 24 h chemerin or chemerin neutralizing antibody treatment. The OD values of both MCF-7 and MDA-MB-231 breast cancer cells after being treated with chemerin (100μg/L) were higher than that of the control group p<0.01 while the OD values of the chemerin neutralizing antibody group were lower than that of the control group p<0.05 . *p<0.05, **p<0.01 Effect of chemerin neutralizing antibody on migration of breast cancer cells. The migration of breast cancer cells MCF-7 and MDA-MB-231 after treatment with chemerin (100μg/L) and chemerin neutralizing antibody (100μg/L) was detected at 0, 24 and 48 h. The cell migration rate of chemerin treatment group in MCF-7 was signi cantly higher than that of the control group (p<0.001), and the cell migration rate of chemerin treatment group in MDA-MB-231 was also higher than that of the control group (p<0.05). The cell migration rate of the chemerin neutralizing antibody group was lower than that of the control group both in breast cancer cells MCF-7 and MDA-MB-231 (p<0.05). *p<0.05, ***p<0.001 Protein expression of ATF2, ERK1/2 and p38 in breast cancer cells treated with chemerin (100μg/L) and chemerin neutralizing antibody (100μg/L) . The protein expression levels of total ATF2, ERK1/2 and p38 in both MCF-7 and MDA-MB-231 cells were no signi cantly different among the three groups. The phosphorylation of ATF2 and ERK1/2 proteins was signi cantly increased after chemerin treatment, but signi cantly decreased in the chemerin neutralizing antibody group both in MCF-7 and MDA-MB-231 cells (A, B, C) . There was not statistically signi cant in the expression of phosphorylated p38 protein among the three groups both in MCF-7 and MDA-MB-231 cells(A, B, C). *p<0.05 Effects of JNK, ERK1/2 and p38 signaling pathway inhibitors on the ability of chemerin to promote the migration of breast cancer cells. MCF-7 and MDA-MB-231 cells were treated with DMSO, JNK signaling pathway inhibitor SP600125, ERK1/2 signaling pathway inhibitor FR180204 and P38 signaling pathway inhibitor SB203580, respectively, and the cell migration rates were observed at 0, 24 and 48 h. The migration rate in groups of JNK and ERK1/2 signaling pathway inhibitor group in MCF-7 and MDA-MB-231 cells were signi cantly smaller than that in the DMSO group (p<0.001), but there was no statistical signi cance between the p38 signaling pathway inhibitor group and the DMSO group. ***p<0.001

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