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Serum amyloid P down-regulates CCL-1 expression, and inhibits Ras/MAPK signaling and development of breast cancer

Purpose: To investigate the role of serum amyloid component P (SAP) on Ras/MAPK pathway in the development of breast cancer (BC) via regulation of chemokine (CC motif) ligand 1 (CCL-1). Methods: Breast cancer (BC) and metastasis models were established using SAP-Tg transgenic mice and WT C57BL/6 mice. The effect of SAP on growth and metastasis was observed. Differentially expressed proteins in SAP-Tg and C57BL/6 serum were analyzed, and further determined by enzymelinked immunosorbent assay (ELISA) and quantitative polymerase chain reaction (qPCR). The effect of SAP on CCL1/Ras/MAPK signaling pathway was studied by immunoblotting. Results: Compared with WT control, SAP-Tg BC model showed a significant reduction in tumor volume and prolonged survival (p < 0.05). In the lung metastasis model, SAP-Tg mice showed a decreased number of nodules on the organ surface (p < 0.05). Protein microarray screening results showed that SAP inhibited CCL-1 expression (p < 0.05). CCL-1 mRNA level in SAP-Tg mice was significantly lower than WT control (p < 0.05). After stimulating RAW cells (mouse macrophage line) with SAP recombinant protein, ELISA results showed that CCL-1 secretion significantly decreased (p < 0.05). In both models, P38 and ERK1/2 activation in SAP-Tg mice were significantly lower than that in C57BL/6 mice. Conclusion: SAP inhibits the growth and metastasis of BC, possibly by reducing the secretion of CCL1 and inhibiting Ras/MAPK signaling pathway, thus suggesting a possible treatment strategy for breast cancer.


INTRODUCTION
Serum amyloid P (SAP), C-reactive protein (CRP) and PTX3 belong to pentraxins. SAP is expressed by liver cells and regulated by a range of cytokines such as IL-1, IL-6 and TNF-alpha. CCL-1 is the first discovered CC-class chemokine, which is mainly secreted by activated immune cells such as T cells, mast cells and macrophages. CCR8 is the only receptor of CCL-1 as currently known. Studies have shown that CCL-1 can regulate neurotransmitters and affect neuropathic pain, regulate the migration of nerve cells [1], promote the normal development of thymus, and play an important role in allergic reactions and asthma. CCL-1/CCR8 expression has been found in leukemia and lymphoma. They can protect against apoptosis of T lymphocytes and leukemia cells through an autocrine mechanism [2].
In gastric cancer, overexpression of CCL-1 may cause down-regulation of non-coding RNA GAS5, which in turn affects the ability of cells in vitro, and ultimately promotes proliferation and inhibits apoptosis of gastric cancer cells [3]. There are reports that CCR8 is highly expressed in colorectal cancer and that CCL-1 can promote the migration of SW480 cells, but has no effect on their proliferation [4]. The migration of UMUC3 and 5637 cells was significantly enhanced when overexpressing CCL-1 [5]. Recent studies have shown that malignant melanoma cells could overexpress CCR8, and their adjacent lymphatic vessels showed CCL-1 expression. The inhibition of CCR8 expression can suppress the migration of tumor cells, and recombinant CCL-1 factors can promote the migration of CCR8 + tumor cells [6]. CCL-1 is closely related to apoptosis of tumor cells. MAPK signal pathway is resistant to CCR8dependent CCL-1, which can promote the phosphorylation of ERK by overexpression of CC5-1 in BW5147 cells and promote apoptosis. CCL-1 may play a role through the MAPK signaling pathway [7].
MAPK is a highly conserved silk/threonine protein kinase, mostly expressed in most mammalian cytoplasm and nucleus [8]. Studies have shown that P38 MAPK signaling pathway is involved in the development of cell apoptosis through caspase [9], and caspase can activate JNK and P38 MAPK, causing some transcriptional protein expression (Fas, P53, etc), and ultimately induce cell apoptosis [10]. In order to reveal the effects of SAP on the growth and metastasis of mouse breast cancer (BC), we conducted the following study.

EXPERIMENTAL
Animal studies and mouse model establishment The animals were raised at Department of Animal Experiment Center, Second Hospital of Shandong University. All the animal procedure were approved by Emory Institutional Animal Care and Use Committee and the approval reference number is KYLL-2016(GJ)A-0033. A high standard of pre-and post-operative care was applied in accordance with the appropriate international guidelines for animal studies [11]. The SAP-Tg transgenic mice (SAP overexpressed) was established by cloning cDNA of mSAP into the vector. The detailed procedure are as followed: First, the plasmid was linearized and then injected into fertilized eggs and give birth to chimeric (SAP-Tg +/-) mice. Chimeric (SAP-Tg +/-) mice were screened and the positive rate was about half. Secondly, by chimeric mice (SAP-Tg +/-) match, we obtained the SAP-Tg(+/+) mice. And then the SAP-Tg(+/+) mice backcrossed with C57BL/6 mice at least 8 bands. Finally, the obtained C57BL/6 mice were identified several times to obtain a SAP-Tg(+/+) C57BL/6. BC model was established with freshly prepared methyl nitrosourea solution (MNU, 5 mg/ml, prepared with acetic acid, pH6.0). Mice were i.p. injected with MNU (50 mg/kg) and observed for 5 weeks. The mouse mammary gland was felt every day after the injection which confirmed that a period of 5 weeks was enough for tumor formation.
For the metastasis model, MCF-7 cell suspension (5 × 10 6 cells) was inoculated through the tail vein. Lung metastasis was established in 18-21 days. The lungs were collected after sacrificing the animals.

Immunohistochemistry
After the tissue was removed, it was fixed in formalin overnight, dehydrated, embedded, placed in 4 o C for cooling, and sectioned in paraffin into 4-μm sections. The slices were baked for 1 h at 65 o C, and underwent dewaxing according to standard protocols. After washing 3 times with PBS buffer (once 5 min), sections were boiled with sodium citrate buffer solution (pH = 6.0), and then washed three times with buffer with PBS (once 5 min). Sections were treated with 0.3 % hydrogen peroxide at room temperature for 30 min, washed 3 times with PBS, and incubated with the primary antibody (anti-Ki67, CST, USA) overnight. The next day, sections were washed at room temperature and secondary antibody (1: 100, with 1 % BSA) incubated for 40 min, and washed 3 times. The sections were DAB stained washed again and stained with hematoxylin for 3 min. For negative control, PBS was applied instead of the primary antibody.

H & E staining
The procedure was as follow: Dewaxing (xylene I, II, each 20 min), anhydrous ethanol (15 min), anhydrous ethanol (15 min, 95 % alcohol (10 min), 95 % Alcohol (10 min), 80 % alcohol (5 min), 70 % alcohol (5 min), water (3 min). Sections were immersed into the hematoxylin dye for 5 min, and then washed with water for 5 min, and then placed in the eosin dye for 20 s and washed. After dehydration, neutral gum was added to the sections and covered with coverslips for observation.

Real-time qPCR
Reverse transcription kit was purchased from TaKaRa company. Trizol was added to the cells and centrifuged (12000 r/min) for 10 min at 4 o C. The supernatant was transferred to a new EP tube and placed on ice for 5 min. The supernatant was added to one-fifth volume of chloroform (compared to Trizol), rapidly vortex shocked for 15 s, and placed on ice for 3 min. The samples were then centrifuged (12000 rpm) for 15 min at 4 o C. The upper layer was transfered to a new EP tube and added to equal volume of isopropyl alcohol. Again, the mixture was centrifuged (12000 rpm) for 15 min at 4 o C, and the supernatant was discarded. Seventy five percent ethanol (in DEPC water) was added and mixed. After centrifugation at 4 ℃ (12000 rpm) for 5 min, the supernatant was discarded. The liquid RNA was slightly dried and added 20 μL DEPC water. Real-time qPCR was performed as follows. The primer sequence is: The reaction procedure was as follows: predenaturation 95ºC, 10min; denaturation at 95 ºC, 30 s; annealing 55 ºC, extension at 72 ºC for 30 s, 40 cycles, followed by melting curve analysis.

ELISA assay
CCL-1 ELISA Kit was purchased from Guangzhou Ruibo Biotechnology Co., Ltd. One hundred microliters of sample and standard were added per well. They were incubated at 37 o C for 90 min, and then 100 μL Biotin-labeled antibody were added and incubated for 60-min at 37 o C. The plate was washed 3 times with 0.01M TBS for and 100 μL ABC was added per well and incubated at 37 o C for 30 min. It was washed again 5 timeswith 0.01M TBS before TMB dark reaction at 37 o C for 30 min. The reaction was stopped with TMB solution.

Western blot
Protein was extracted with the protein extraction kit from Beyotime Co. The protein lysate was added to 20 times the volume of the tissue. The amount of protein was quantified using the BCA Kit from Thermo Fisher. The BCA solution was prepared at a ratio of A: B = 50: 1, then 2 μL of protein extract, 18 μl PBS, and 200 μl of AB mixture were added to each well. For electrophoresis, 20 μL protein was loaded. The electric voltage was: 80 V in the stacking gel and 120 V in the separation gel. The protein was transferred to PVDF membrane under a constant current (250 mA) at 4 o C. The membranes were blocked with 5 % TBST-skim milk for 1 h at 37 o C, and then incubated overnight at 4 °C with primary antibodies. The blots were then incubated with the secondary antibodies. After washing (3 times), the blots were immersed in the luminous fluid. Signals were captured after 30s exposure.

Protein microarray
Orbital blood was collected and centrifuged. The supernatant was stored at -70 o C. Protein microarray kit (Angfei Co., USA) was reheated for 1h at RT. The protein concentration was determined using the BCA method, and the total protein concentration was adjusted to 500 μg/mL. Two arrays were placed in a capsule, and an equal volume of 200 μL samples were added and incubated for 2 h in the dark. After washing (3 times each 5min), the primary antibodies (1: 100) were added and incubated at 4 o C overnight. Then the arrays were washed (3 times each 5min) and the secondary antibodies (1: 100) were added1 h later, the film was washed and incubated with the developer (Thermo Fisher Co., Ltd. USA) for 2 min. The blots were captured for analysis.

Statistical analysis
Data were analyzed by SPSS. Student's t-test was used to identify significant differences. In all cases, p < 0.05 was considered to be statistically significant. *P < 0.05, **p < 0.01, ***p < 0.001. Image Pro Plus was applied to analyze the grey level of immunohistochemistry results.

Effect of SAP on the growth of mouse breast cancer
In order to study the effect of SAP on the growth of BC in mice, we established a mouse BC model, and analyzed the tumor volume and the survival rate of different mice. SAP-Tg mice showed a significantly lower volume compared with the C57BL/6 mice ( Figure 1A). The tumor weight of SAP-Tg mice was significantly lower than that of the C57BL/6 mice (Fig. 1B, p <  0.05).Also, transgenic mice had a significantly longer overall survival than C57BL/6 ( Figure 1C, p < 0.01). To further determine proliferation level of the tumors in the different groups, BrdU staining was performed. The SAP-Tg mice showed a lower level of BrdU+ cell number than the C57BL/6 mice ( Figure 1D, p < 0.01). The above results suggested a strong anti-tumor effect of SAP in BC development.

Effect of SAP on metastasis of mouse breast cancer
To further investigate the effect of SAP on lung metastasis, we established a lung metastasis model using tail vein injection. After inoculation, we counted the number of tumor nodules on the surface of all lung lobes. Consistent with the tumor growth results, SAP-Tg mice exhibited fewer metastatic nodules in their lungs than C57BL/6 ( Fig. 2A, B, P<0.01). Using H&E staining, the total number of metastatic nodules inside of lungs were counted, which was also lower in SAP-Tg mice compared with the C57BL/6 group ( Figure 2C, p < 0.01).

SAP regulates CCL-1 secretion by macrophages
We collected the orbital blood and obtained the serum by centrifugation. The cytokine CCL-1 in the serum was determined. The SAP-Tg mice showed a significantly lower levelofCCL-1 compared with C57BL/6 mice, as demonstrated by the RayBiotech protein microarray ( Figure  3A). For further confirmation, quantitative PCR was performed and the results showed that the CCL-1 mRNA level in the peripheral blood of SAP-Tg mice was significantly lower (Fig. 3B,  P<0.05). The protein expression of CCL-1 was further determined using the mouse CCL-1 ELISA kit. For normal individuals, two strains had similar CCL-1 expression in the serum. However, in tumor-bearing groups, the expression of CCL-1 in SAP-Tg mice was significantly lower than that in C57BL/6 mice (p < 0.01, Figure 3C).
In the lung metastasis models, SAP-Tg mice also showed lessCCL-1 expression in the serum ( Figure 3D, p < 0.01). Given CCL-1 was mainly secreted by macrophages, we further examined SAP generation through the secretion from macrophages.
First, we recovered the macrophage cell line RAW264.7, followed by SAP administration (50 ng/mL) or the same amount used for treatment. After 24 h, the supernatant was collected for CCL-1 ELISA assay. Compared with DMSO (the vehicle), CCL-1 secretion from the SAP-treated cells was significantly decreased ( Figure 3E, p < 0.05).

SAP affects the Ras/MAPK signaling pathway in breast cancer
To further investigate whether SAP affects the activation of P38, ERK1/2 and K-ras through CCL-1, we examined these signaling by Western blotting. As shown in Figure 4A, p-P38 and p-ERK level were significantly lower in the SAP-Tgmice established BC model compared with the WT (P < 0.01). Similarly, in the metastasis model ( Figure 4B), SAP-Tg mice exhibited not only decreased expression o fp-P38, p-ERK but also Kras (p < 0.01).

DISCUSSION
SAP is a serum protein expressed by liver cells. It is upregulated after acute inflammation in mice, but not in the human body. To date, little is yet known about the roles of SAP on growth and metastasis of BC tumor. Through our screening, we found a lower CCL-1 expression in the SAP-Tg mice compared with the WT control. Chemokines are a class of small molecules, which promote leukocyte migration toward themselves [12]. CCL-1 is among the first discovered CC class of chemokines, which are mainly secreted by activated T cells, mast cells and macrophages [13][14][15][16][17]. Recently, it has been shown that autocrine CCL-1 produced from leukocellular lymphoma and adult cell leukemia has an anti-apoptotic effect [18]. Around the lymph node margins, endothelial cells secret CCL-1 and cause a high expression of CCR8, thereby affecting the growth of breast cancer and lymphatic metastasis. This study was to investigate the effect of SAP on in vivo growth and metastasis of BC. We used protein Microarray technology to probe the mechanism under lying the inhibition of SAP on growth and metastasis, and observed a significant difference in CCL-1 expression between SAP-Tg mice and WT control. This difference was validated by Real-time PCR and ELISA assay. We demonstrated the role of SAP on CCL-1 secreted by macrophages. Given CCL-1 could also be secreted by T cells and mast cells, we cannot exclude the involvement of T cells and mast cells so far. This is a limitation of the present work and is to be addressed in a further study.
CCL-1 may promote the proliferation and migration of tumor cells through MAPK signaling pathway [19]. It has been widely reported that p38 and JNK inhibitors can suppress the proliferation of tumor cells by inhibiting gastrin expression [20]. Gastrin can promote the proliferation of Colo320 cells by inhibiting p38 activation [21]. ERK MAPK signal pathway has a key function for tumor cell invasion, drug resistance and so on. In our subcutaneously implanted BC model and the metastasis model, activation (phosphorylation) of P38 and ERK in the SAPTg mice were significantly lower than the WT control; and K-ras was even more reduced in the metastasis model. These findings were consistent with the known tumor promoting role of Ras/MAPK signaling pathway.

CONCLUSION
The findings of this study indicate that SAP significantly inhibits BC growth and metastasis in mice. SAP inhibits macrophage-secreted CCL-1, thereby attenuating the activation of CCL-1-Ras/MAPK axis. SAP can be used as a novel factor for understanding the mechanism of BC development and a new target for BC treatment.

Open Access
This is an Open Access article that uses a funding model which does not charge readers or their institutions for access and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/ 4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/rea d), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.