C5aR deficiency attenuates the breast cancer development via the p38/p21 axis

Emerging evidence has shown activation of the complement component C5 to C5a in cancer tissues and C5aR expression in breast cancer cells relates to the tumor development and poor prognosis, suggesting the involvement of complement C5a/C5aR pathway in the breast cancer pathogenesis. In this study, we found that as compared to the non-tumoral tissues, both C5aR and MAPK/p38 showed an elevated expression, but p21/p-p21 showed lower expression, in the tumoral tissues of breast cancer patients. Mice deficient in C5aR or mice treated with the C5aR antagonist exhibited attenuation of breast cancer growth and reduction in the p38/p-p38 expression, but increase in p21/p-p21 expression, in the tumor tissues. Pre-treatment of the breast cancer cells with recombinant C5a resulted in reduced p21 expression, and MAPK/p38 inhibitors prevented C5a-induced reduction in p21 expression, suggesting the involvement of the MAPK/p38 signaling pathway in the C5a/C5aR-mediated suppression of p21/p-p21 expression. These results provide evidence that breast cancer development may rely on C5a/C5aR interaction, for which MAPK/p38 pathway participate in down-regulating the p21 expression. Inhibition of C5a/C5aR pathway is expected to be helpful for the treatment of patients with breast cancer.


INTRODUCTION
Breast cancer (BC) is reported to be the most common cancer among women worldwide [1,2]. Although there are numerous therapeutic strategies, including chemotherapy and radiotherapy as well as hormone and immune therapy, the mortality resulting from BC is the second leading cause of cancer deaths among women [3,4]. The underlying mechanism is complex. Recent evidence shows that the tumor microenvironment, AGING including inflammatory cells and molecules, plays a critical role in the tumor progression [5,6].
Complement system, a vital component of the innate immune system, plays an important role in the immunerelated inflammatory diseases [7]. Complement activation occurs in response to infection and a set of innate molecules. Once activated, the complement cascade generates a number of effector molecules, resulting in the cleavage of C3 and C5 to their activated forms, C3a and C5a, respectively and the terminal product C5b-9 [8]. C5a, acts as an inflammatory mediator when binding to its receptor, C5aR [9,10]. It has been previously reported that the complement system contributes to cancer progression. Higher C5a levels were found in the lung cancer cell lines than in the nonmalignant lung epithelial cells [11]. The expression of C5aR was upregulated in various human cancer tissues, and BC C5aR expression was related to tumor development and poor prognosis of the patients [12,13]. C5aR facilitated BC metastasis by altering T-Cell responses in the metastatic niche [14].
Controlling the cell cycle is a crucial process to regulate cell proliferation and apoptosis, especially in tumor cells [15]. P21 is a tumor suppressor that mediates cell cycle arrest at G1 or G2 phase. It was previously reported that the MAPK signal pathway regulates p21 transcription through transcription factors [16,17]. C5aR activates the PI3K/AKT and MAPK signaling pathways and potentiates cell growth [18]. C5a/C5aR signaling contributes to the pathogenesis of some malignant tumors, such as gastric, thyroid, and lung cancer [11,[19][20][21]. However, the underlying mechanisms of C5a/C5aR signaling-mediated BC development remains unclear. In this study, our data indicate that the C5a/C5aR pathway promotes BC development via activation of the MAPK/p38 pathway.

Upregulation of C5aR expression in the tumoral tissues of BC patients
To investigate the potential role of the C5a/C5aR pathway in BC, we analyzed C5aR expression in the tumoral tissues from BC patients at different clinical stages using IHC and western blotting. C5aR expression was significantly higher in the tumoral tissues than in the BC-adjacent non-tumoral tissues ( Figure 1A, 1B). However, the C5a and C5 serum levels of patients with BC (n=27) exhibited a remarkable reduction, compared to healthy volunteers (n=20) (C5a: P<0.001, Figure 1C; C5: P<0.001, Figure 1D). The drop in serum C5a and C5 levels detected in the advanced BC patients was possibly due to the excessive activation and consumption of the complement in the early stages.  Figure 1). C5aR deficiency caused a significant reduction in the tumor growth ( Figure 2A). Moreover, the expression of Ki67 and CD146, which are closely related to the cell proliferation and angiogenesis, was remarkably downregulated in C5aR deficient mice ( Figure 2B). Similar results were found when the mice were treated with the C5aRa (Figure 2C, 2D), a C5aR antagonist hexapeptide AcF (OP (D) ChaWR) [22]. In addition, the senescence of 4T-1 cells in vitro was significantly increased after C5aRa treatment (Supplementary Figure 2) while the invasion and metastasis of a non-adherent BC cell line MDA-MB-453 cells were accelerated after exposure to recombinant C5a (Supplementary Figure 3). All these results indicate the critical role of C5a/C5aR pathway in the pathogenesis of BC development.

Downregulated p21 expression in tumor tissues of BC patients
It was previously reported that the p21 expression was negatively correlated with tumor development in colorectal cancer and gastric carcinoma [23,24]. However, the role of p21 in the pathogenesis of BC is unclear. In the present study, we found high expression of p-p21 was significantly associated with less lymph node metastasis and early TNM stage (Table 1, p=0.016 and 0.042 respectively). Kaplan-Meier analysis was used to analyze the effect of p-p21 on the survival of BC patients. We showed that the patients with low p-p21 expression had a significantly (p<0.001) shorter disease-free survival (DFS) than those with higher p-p21 expression ( Figure 3A). Additionally, multivariate analysis indicated that p-p21 expression was an independent prognostic factor for DFS in the patients ( Table 2). Consistent with these results, lower p21 and p-p21 expression was found in the tumoral tissues than in the non-tumoral tissues in BC patients ( Figure 3B, 3C), suggesting that p21/p-p21 plays a protective role during the development of BC.     Figure 4). The expression of p21 and p-p21 were measured by western blotting, IHC and RT-PCR. The increased expression of the p21 and p-p21 was observed in tumor tissues of mice with C5aR-deficiency or treated with C5aRa ( Figure 4A-4D). This suggests that the C5a/C5aR signaling is critical for the reduction of p21 expression during the development of BC.

C5aR signaling suppresses p21 expression through activation of the MAPK/p38 pathway
Although the PI3K/AKT signaling pathway was closely related to the genesis and metastasis of some types of tumors, such as colorectal cancer and hepatocellular carcinoma [25,26], the levels of PI3K/AKT were comparable between the tumoral tissues and paracarcinoma tissue from BC patients with clinical stages ( Figure 5A). This is indicative of the non-association of the PI3K/AKT pathway and pathogenesis of BC. In contrast, compared with that in para-carcinoma tissue, the p-p38 levels in the tumoral tissues were significantly increased ( Figure 5B).
To explore the correlation of C5aR signaling with MAPK/p38 pathway activation during BC growth, we treated BC cell line MCF7 cells (which constitutively expressed p21 (Supplementary Figure 4) and C5aR (Supplementary Figure 5A) ) with the C5aRa for 24 h. It was found that p38 phosphorylation levels showed a time-dependent reduction ( Figure 6A) as well as cell proliferation was suppressed (Supplementary Figure  5B) and the cells underwent a p21-mediated G2/M phase arrest with a concentration-dependent manner (Supplementary Figure 5C). When mice were transplanted with the BC cell line 4T-1 cells, mice with the C5aR-deficiency or treated with C5aRa exhibited a remarkable decrease in p-p38 levels during the development of BC ( Figure 6B, 6C). Taken together, these in vitro and in vivo results demonstrate that the C5a/C5aR pathway participates in the MAPK/p38 pathway activation during BC development.
To further investigate whether MAPK/p38 is involved in the C5a/C5aR pathway-mediated downregulation of the cell cycle protein p21 in BC, we treated the BC cell line MCF-7 cells with the recombinant C5a and measured the expression of MAPK/p38 and p21. It was observed that the C5a stimulation caused an increased p38 phosphorylation ( Figure 7A), but a reduced p21 expression in the BC cells ( Figure 7B, 7C), and C5aRa treatment reversed these effects. Moreover, MAPK/p38 inhibitors attenuated C5a-mediated downregulation in p21 expression ( Figure 7D, 7E). All these results suggest that the MAPK/p38 pathway participates in the C5a/C5aR-mediated suppression of p21 expression during BC pathogenesis.

DISCUSSION
The C5a/C5aR pathway has been increasingly recognized as a crucial contributor to cancer progression. Blockade of the C5a/C5aR signaling resulted in an impaired tumor growth in mice [27]. A higher mRNA level of C5aR showed a negative relationship with the survival of ovarian cancer patients and C5a silencing reduced the tumor growth in ovarian cancer cells [28]. In BC, C5aR-positive patients had a lower survival rate than C5aR-negative patients, reiterating that C5aR is a predictor of poor prognosis in BC [13]. Consistently, our data showed that the tumors volume remarkably reduced in C5aR-deficient mice or mice treated with C5aR antagonists, indicating that the C5a/C5aR pathway plays a critical role in the pathogenesis of BC. It was previously showed in a mouse model of BC, that C5a/C5aR system facilitated metastasis in the lungs by creating a microenvironment favourable for cancer cells (by suppressing effector CD8 + and CD4 + T cell responses) [14]. The reduced tumor growth in mice with pharmacologic blockade of C5aR or its genetic ablation in C5aR was caused by AGING  AGING the increase in antitumor immune response, or synergistically inhibiting cancer cell growth by both cancer microenvironment and C5a-C5aR interaction in cancer cells? This needs to be clarified in the future.
The PI3K/AKT pathway participates in a broad range of cell processes, including proliferation, apoptosis, metabolism regulation, and cell cycle progression.
Hyper-activation of the PI3K pathway is involved in  were pre-incubated with C5aRa (10 nM) for 60 min before exposure to recombinant murine C5a (480 ng/mL) for 30 min or 24 h. The cell lysates were assessed by western blotting using a polyclonal antibody against p-p38, p21 and the mRNA levels of p21 were measured by RT-PCR. (D, E) MCF-7 cells were pre-incubated with p38 inhibitor SB203580 (50 mM) for 60 min before exposure to recombinant murine C5a (480 ng/mL) for 24 h and then p21 expression levels were detected by western blotting with polyclonal antibody against p21 and the mRNA levels of p21 were measured by RT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001. GAPDH was used as the loading control. All in vitro measurements were performed in independent quadruplicates with matching results.
AGING tumor development [29]. Blocking the PI3K/AKT pathway was beneficial for the clinical treatment of lung cancer [30]. However, our data showed that the MAPK/p38 pathway, and not the PI3K/AKT pathway, was activated, in BC progression. Similar results were observed in other labs. Zheng et al. found that downregulating the phosphorylation of p38/MAPK signaling pathway led to a reduction in the migration of cervical cancer cells [31]. The results of Davidson et al. showed that p38 was expressed in 54/55 (98%) specimens, and its phosphorylated form was found in 51/55 (92%) in effusions from patients diagnosed with serious ovarian carcinoma, suggesting that p38/MAPK signaling was closely related to the incidence of ovarian cancer [32]. The discrepancy is probably contributed to the difference in cancer types. It is reported that p38 is more likely to be activated in women once they develop a tumor since women generally produce high levels of estrogen [33].
P21 is negatively associated with the cancer occurrence [34,35]. Consistent with these results, we observed that the p21 expression was reduced in the tumor tissues and its low expression was closely associated with poor survival rates in BC patients. In contrast, the data from Yu. et al. showed that the increased expression of p21 caused chemo-resistance in BC cells with HER2 overexpression [36]. P21 overexpression is also associated with the poor prognosis in BC survival [37]. The contradictory functions of the p21 during cancer development may be related to its sub-cellular localization [38]. It was demonstrated that the cytoplasmic localization of p21 was strongly associated with poor prognosis [39][40][41]. P21 expression in the nucleus promotes tumor cell apoptosis, if expressed in the cytoplasm inhibits its apoptosis. However, the underlying mechanism of p21 expression in BC development needs further exploration in the future.
Collectively, these results reveal that the C5a/C5aR pathway is critical for BC development, for which MAPK/p38 pathway participates in down-regulating p21 expression. In order to investigate the role of C5a in PI3K/AKT activation, the MCF-7 cells were stimulated with 480 ng/mL C5a (Biovision, USA) for 30 min. For some assays, the MCF-7 cells were pre-treated with 10 nM C5aRa (GL Biochem, China) for 60 min before stimulation with C5a. To investigate the changes in the p21 expression induced by the C5a stimulation, the MCF-7 cells were incubated for 24 h in the media alone or in the media supplemented with C5a, with or without C5aRa pre-treatment. To determine whether the C5amediated impairment of p21 expression is MAPK/p38dependent, the MCF-7 cells were pre-treated with 50 μM SB203580 (a p38 inhibitor) for 60 min and then AGING stimulated with C5a for 30 min with or without C5aRa pre-treatment. Cells were collected in the indicated time points for western blotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay.

Cell cycle
About 5 × 10 6 human MCF-7 BC cells were harvested at 25 °C after pre-treatment with various concentrations of C5a for 24 h. The supernatant was removed, and the cells were fixed in 70% ice-cold ethanol overnight. Ethanol-fixed cells were re-suspended in 500 μL PBS (PH=7.4) containing 0.1 mg/mL RNase and 40 μg/mL propidium iodide (PI) and were incubated at 37 °C for 30 min. The cell cycle distribution was analyzed using a flow cytometer (BD FACS Canto II, USA).

Mice studies
Wild Mice developed tumors on day 15 after the 4T-1 injection and were sacrificed by retro-orbital bleeding. The tumors tissues were collected immediately and prepared for analysis by immunohistochemistry (IHC), western blotting and qRT-PCR.

Western blotting
In brief, equal amounts of total protein extracts from the cultured cells or tissues were separated using 8-12%

Enzyme-linked immunosorbent assay
The concentrations of C5 and C5a in the serum samples of the BC patients and healthy volunteers were measured using ELISA kits (USCN, China) targeting various factors.

In vitro breast cancer cells migration and invasion assay
For transwell migration assays, 2.5 ~ 5 × 10 4 nonadherent BC cell line MDA-MB-453 cells were plated in the top chamber with the non-coated membrane (24well insert; pore size, 8 μm; BD Biosciences). For invasion assays, 1.25 × 10 5 cells were plated in the top chamber with Matrigel-coated membrane (24-well insert; pore size, 8 μm; BD Biosciences). In both assays, cells were plated in medium without serum, and medium supplemented with 480 ng/mL recombinant C5a were used as a chemoattractant in the lower chamber. Cells were incubated for 12 h and cells that did not migrate or invade through the pores were removed by a cotton swab. Cells on the lower surface of the membrane were stained with the crystal violet dye and counted. The results were observed under the microscope. Magnification: 200 ×.

Statistical analysis
Differences between groups were evaluated by t test, as specified in the figure legends, by using GraphPad prism Software (La Jolla, CA, USA). Significance was assumed at a p value of 0.05 or less.

Availability of data and materials
The data are fully available without restriction.