Copper Chaperone for Superoxide Dismutase Promotes Breast Cancer Cell Proliferation and Migration via ROS-Mediated MAPK/ERK Signaling

Copper chaperone for superoxide dismutase (CCS) is a critical component of oxidation–reduction system and functions as a potential tumor promoter in several cancers. However, the function and clinical significance of CCS in breast cancer remain unclear. Here, we found CCS was highly expressed in breast cancer, where it promoted breast cancer cell proliferation and migration. Suppression of CCS expression was sufficient to attenuate the phosphorylation level of ERK1/2 and increase the accumulation of reactive oxygen species (ROS). Mechanistically, we found that knockdown of CCS decreases the activity of ERK1/2 mediated by the accumulation of ROS, which leads to the inhibition of cell proliferation and migration. In summary, these results indicated that CCS promotes the growth and migration of breast cancer cells via regulating the ERK1/2 activity mediated by ROS.


INTRODUCTION
Breast cancer is the leading cause of cancer-related deaths in women worldwide (Christofori, 2006;Bray et al., 2018). Breast cancer patients with metastases have an extremely poor prognosis (Gupta et al., 2005;Bacac and Stamenkovic, 2008;Thiery, 2009;Chaffer and Weinberg, 2011;Valastyan and Weinberg, 2011). Thus, exploring new targets for breast cancer treatment is important.
Copper, a redox-active transition metal essential for most living organisms, serves as a catalytic cofactor for enzymes that function in antioxidant defense, iron homeostasis, cellular respiration, and a variety of biochemical processes (Mandinov et al., 2003;Lowndes and Harris, 2005;Ashino et al., 2010;Xu et al., 2016;Sciegienka et al., 2017). The uncontrolled accumulation of copper could lead to increased oxidative stress and inappropriate binding to macromolecules. Copper chaperone for superoxide dismutase (CCS) delivers copper to specific cellular destinations and to superoxide dismutase (SOD1) (Kawamata and Manfredi, 2008;Ulloa, 2009). Mounting evidences suggest that CCS plays a crucial role in oxidative metabolism (Kawamata and Manfredi, 2008;Leitch et al., 2009;Suzuki et al., 2013a;Wang et al., 2015). Blockade of the copper-trafficking chaperone CCS contributes to the increased cellular reactive oxygen species (ROS) level due to the overall accumulation of copper inside the cells and the decreased SOD1 activity (Ulloa, 2009). Wang et al reported that inhibiting CCS blocks lung cancer and leukemia cell growth (Wang et al., 2015). In addition, they show that blocking copper trafficking induces cellular oxidative stress and reduces cellular ATP levels. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. However, the mechanisms underlying the relationship between CCS and tumorigenesis are still largely unknown, although the positive correlation between CCS and redox homeostasis has been revealed (Wang et al., 2015). Therefore, this study aimed to explore the critical role and molecular mechanism of CCS in migration and proliferation of breast cancer.
In aforementioned study by Wang et al, a CCS inhibitor was developed and shown to have the same effect as knocking down CCS in cancer cells (Wang et al., 2015). However, the precise role of CCS in migration and proliferation of breast cancer cells is unknown. In the present study, we report that CCS is highly expressed in breast cancer tissues and invasive breast cancer cells and promotes cell proliferation and migration. Furthermore, we found that inhibition of CCS by shRNA or an inhibitor blocks breast cancer proliferation and migration by triggering ROS mediated ERK activity. These results suggest that metastasis-prone breast cancer cells reprogram oxidative metabolism to promote cell proliferation and migration. Targeting CCS may represent a promising approach for selectively causing cell proliferation and migration in breast cancer cells.

Small Interference RNA Transfection
MDA-MB-231, MCF-7, and BEAS-2B cells (2 × 10 5 ) were seeded into 6-well plates and cultured in a humidified incubator at 37°C and 5% CO 2 for 24 h. Cells were transfected with a negative control siRNA (NC-siRNA) and siRNA targeting CCS by Lipofectamine RNA iMAX (Invitrogen corporation). Transfected cells were cultured for 48°C before being used for further experiments. The sequences of targeted CCS siRNA were 5′-GUCUUGGUACACACCACUCUA-3′. The sequences of negative control siRNA were 5′-UUCUCCGAACGUGUCACGUTT-3′(forward). All siRNA sequences were purchased from the Invitrogen Ribobio corporation of Guangzhou.

Cell Proliferation Assay
Cell proliferation assays were performed by seeding 5 × 10 4 cells in 6-well plates and culturing the cells at 37°C. Relative cell proliferation was determined by cell numbers recorded at 4 days after being seeded and normalized to that of each of the cell lines at the starting time (t = 0 h).
SDS-PAGE and transferred onto PVDF membranes (Millipore). The membranes were blocked with 5% non-fat milk for 2 h and then incubated overnight at 4°C with the primary antibody and 1 h at room temperature with secondary antibody. Signals were detected using luminol substrate solution.

Transwell Migration Assay
For the Transwell (24-well insert, 8 mm pore size with polycarbonate membrane; Corning Costar, Lowell, MA, USA) migration assays, 600-mL media supplemented with 10% FBS was added to the lower chamber, and the cells resuspended in serum-free media were added to the upper insert after transfection. Transwell membranes were fixed and stained using crystal violet after specified time. The cells adhering to the lower surface of the membrane were counted under a light microscope (Olympus, Tokyo, Japan) at a magnification of 200.

Wound Healing Assay
To determine cell motility, cells were seeded into 6-well plates and grown to 90% confluence. A monolayer of the cells was then scratched with a sterile micropipette tip, followed by washing with PBS to remove cellular debris. The cell migration was observed and counted under a light microscope (Olympus, Tokyo, Japan) at a magnification of 200. The cells that migrated across the black lines were counted in three randomly chosen fields from each triplicate treatment.

Intracellular Reactive Oxygen Species (ROS) Production
The amount of intracellular ROS was measured by detecting dichlorodihydrofluorescein, which is the cleavage product of carboxy-H 2 DCFDA (Invitrogen) by ROS. A total of 200,000 cells were seeded in 6-well plate. Twenty-four hours after seeding, cells were washed with PBS and loaded with 12.5 μM carboxy-H 2 DCFDA for 60 min. The cells were harvested, resuspended in PBS, and analyzed using a FACS (BD Biosciences; excitation and emission at 490 and 530 nm, respectively).

Bioinformatics Analysis
The public Gene Expression Omnibus datasets (GSE9574 and GSE21422) and the TCGA (The Cancer Genome Atlas) dataset were used for bioinformatics analysis.

Statistical Analysis
The concentration of DC_AC50 required to reduce cell proliferation by 50% (IC50) was determined graphically using the Dose-response-Stimulation function in GraphPad Prism7 (San Diego, CA, United States). Statistical analyses of the significance of differences between groups were performed using Student's t-test with GraphPad Prism7. All data were obtained from three independent experiments performed in triplicate and were presented as the mean ± standard error. p < 0.05 was considered to indicate a statistically significant difference.

Higher CCS Gene Expression in Breast Cancer Patients
Bioinformatics analysis has been used to discover previously unknown function of genes associated with cancer. To determine the role of CCS in human breast cancer, we first examined the expression of CCS utilizing Gene Expression Omnibus (GEO) profiles; we found that the expression of CCS was higher in breast cancer tissue than in noncancerous tissue ( Figure 1A, GSE9574). We also confirmed these finding using The Cancer Genome Atlas (TCGA) dataset. CCS expression was also significantly higher in breast cancer tissue than in noncancerous tissue in the Cancer Genome Atlas (TCGA) ( Figure 1B). In addition, we also found that the expression of CCS was higher in invasive ductal carcinoma (IDC) than in ductal carcinoma (DCIS) (Figure 1A, GSE21422). To validate these findings, we checked CCS expression in various breast cancer cells lines. CCS was differentially expressed in several breast cancer cell lines, including MCF-7, T47D, MDA-MB-231, and SUM159. Of note, the expression of CCS was higher in T47D and MDA-MB-231 cell lines compared to MCF-7 and SUM159 cells ( Figure 1D). All these findings indicate the potential role of CCS in tumor formation and progression.

CCS Promotes Breast Cancer Cell Proliferation in vitro
We found that the expression of CCS was higher in breast cancer tissue than in noncancerous tissue, suggesting the potential role of CCS in breast cancer cell proliferation. To test our hypothesis, we generated stable cell lines in which CCS was knocked down in MDA-MB-231 cells (Figure 2A lower) and exogenously expressed in MCF-7 and SUM159 cells (Figures 2B,C lower). Cell number counting assays showed that knockdown of CCS reduced the proliferation of MDA-MB-231 cells (Figure 2A upper), while exogenous expression of CCS demonstrated the opposite effect (Figures 2B,C upper). To validate these findings, we knocked down the expression of CCS in MDA-MB-231, MCF-7, and BEAS-2B cells using siRNA. Cell number counting assays showed that knockdown of CCS significantly inhibited the proliferation of metastasisprone breast cancer cell lines MDA-MB-231 but did not have any effect on the proliferation of breast cancer MCF-7 cells or normal BEAS-2B cells (Figures 2D-F). Real-time PCR was used to determine the knockdown efficiency of CCS by siRNA ( Figure 2G). Next, we sought to explore the role DC_AC50, a potent and selective CCS inhibitor, in breast cancer. DC_AC50 has been shown to inhibit the proliferation of acute leukemia cells (Wang et al., 2015). We treated cells with DC_AC50 and found that MCF-7 cells exhibited significantly higher resistance to DC_AC50 than MDA-MB-231 cells (Figure 2H). Meanwhile, DC_AC50 treatment resulted in decreased cell proliferation of MDA-MB-231 cells in a time and dose-dependent manner (Figure 2I). These results imply that CCS plays an important role in breast cancer cell proliferation and suggests that CCS is a promising anti-cancer target.

CCS Promotes Breast Cancer Cells Migration
We found that the expression of CCS was higher in invasive ductal carcinoma than in ductal carcinoma (Figure 1C), suggesting the potential role of CCS in promoting breast cancer migration. Next, we explore the role of CCS in the motility of the breast cancer cells. We performed a transwell migration assay that showed knockdown of CCS significantly inhibited breast cell migratory abilities in MDA-MB-231 (Figure 3A), while exogenous express CCS exhibited the opposite effects in MCF-7 and SUM159 cells (Figures 3B,C). To validate these finding, we treated MDA-MB-231 with CCS inhibitor, DC_AC50, and performed a transwell migration assay. We found that DC_AC50 blocked MDA-MB-231 cell migration in a dose-dependent manner ( Figure 3D). In addition, we also assessed migration of MDA-MB-231 in a wound healing assay. We found that knockdown or inhibition of CCS dramatically suppressed MDA-MB-231 cell migratory abilities (Figures 3E,F). To consolidate our findings, we overexpressed FLAG tagged CCS in MCF-7 cells. As expected, overexpression of CCS accelerated breast cancer cell migration in a wound healing assay (Figure 3G). Taken together, our results suggest that CCS plays an important role in promoting breast cancer cells migration. (G) Cell migration in CCS overexpressing and control MCF-7 cells was determined by the wound healing assay. The modified migration assay was evaluated by calculating the ratio of the cell numbers through the chamber or wound closure after the wound healing assay. All results performed above are presented as mean ± SD from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001, ns: not significant.

CCS Promotes Breast Cancer Migration via MAPK/ERK Signaling
Activation of survival signaling has been shown to play an essential role in tumor development (Baud and Karin, 2001).
Several studies have demonstrated that the MAPK/ERK signaling pathway is activated in cancer cells to promote cancer cell proliferation, migration, and invasion (Rajalingam et al., 2005;El Touny et al., 2014). Therefore, we examined whether (E) Cell migration in CCS overexpressing and control MCF-7 cells treated with U0126 was determined by wound healing assay. The modified migration assay was evaluated by calculating the ratio of the cell numbers through the chamber or wound closure after the wound healing assay. (F) Cell proliferation was determined by cell number counting assays in CCS stable knockdown MDA-MB-231 cells with overexpression of exogenous HA-tagged MEK. All results performed above are presented as mean ± SD from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001, ns not significant.
MAPK/ERK signaling is involved in CCS mediated cell proliferation and migration. To test this hypothesis, we examined the ERK1/2 and MEK1/2 activity in CCS knockdown MDA-MB-231 cells. Western blotting shows that the activity of ERK1/2 was drastically decreased in CCS knockdown MDA-MB-231 cells ( Figure 4A). Additionally, overexpression of FLAG tagged CCS increased the activity of ERK1/2 in MCF-7 cells (Figure 4B), but the increased activity of ERK1/2 was blocked in MCF-7 with ERK inhibitor U0126 ( Figure 4C). To validate the role of MAPK signaling in the process of CCS-induced migration and proliferation in breast cancer cells, we first reactivated ERK by transfecting exogenous HA tagged MEK into MDA-MB-CCS-KD cells.
As expect, the replenishment of MEK in MDA-MB-231-CCS-KD cells could partially rescue the capability of migration in MDA-MB-231-CCS-KD cells due to the reactivation of ERK1/2 ( Figure 4D). Secondly, we demonstrated that inhibition of MEK with U0126 treatment inhibited CCS-induced cell migration ( Figure 4E). Thirdly, overexpression of MEK in MDA-MB-231-CCS-KD cells partially rescues the decreased cell proliferation in CCS knockdown MDA-MB-231 cells ( Figure 4F). These results suggest that activation of the MAPK/ ERK pathway is essential for the CCS-promoted migration abilities and cell proliferation of breast cancer cells.

CCS Activates MAPK/ERK Signaling via ROS
The inhibition of CCS leads to increased ROS levels. Thus, we hypothesized that CCS regulates the activity of ERK1/2 through ROS. To test this hypothesis, we examined ROS levels in MDA-MB-231 cells treated with CCS shRNA or DC_AC50. Indeed, we found that knockdown or inhibition of CCS significantly increases the cellular ROS levels (Figures 5A,B), while the increased ROS was blocked by treating cells with antioxidant N-Acetyl-L-cysteine (NAC; Figure 5A). In addition, we also observed that NAC abrogates the decreased activity of ERK1/2 in CCS knockdown MDA-MB-231 cells ( Figure 5C). Consistently, we also found that H 2 O 2 impaired phosphorylation of ERK1/2 in a dose-dependent manner but did not affect the total expression level of ERK1/2 ( Figure 5D). Finally, we found that NAC could rescue the decreased cell proliferation and migration of MDA-MB-231 CCS knockdown cells (Figures 5E,F). These results further support the idea that inhibition of CCS induces a ROS overload, which impairs MAPK/ERK signaling to attenuate cancer cell proliferation.
The combined results presented here also establish CCS as a viable anticancer target and copper trafficking as a new pathway for future therapeutic development.

DISCUSSION
Rapid cellular growth and migratory abilities play a crucial role in tumorigenesis and metastasis, which have been recognized to be associated with ROS levels (Aykin-Burns et al., 2009;Doskey et al., 2016). Those cells that survive oxidative stress stand a good chance to have acquired adaptive mechanisms to counteract the potential toxic effects of elevated ROS and to promote cell-survival pathways (Irmak et al., 2003). CCS, a co-enzyme of SOD1, is a critical component of the oxidationreduction system in cancer, and its differential expression in different types of breast cancer suggests a relationship between CCS and cancer cell growth and migration (Figure 6). However, the link between CCS-activated ROS and the occurrence and development of tumors is still in its infancy.
FIGURE 6 | Proposed working model. Schematic model shows that the role and mechanism of CCS in promoting breast cancer cell proliferation and migration.
In this study, we utilized MDA-MB-231 cells (triple-negative breast cancer) and MCF-7 cells (estrogen receptor positive breast cancer) as human cell line models and identified a novel function and mechanism of CCS in facilitating breast cancer cell proliferation and migration. This novel mechanism provides a link between oxidative metabolism and survival signaling. Wang et al. reported that inhibition of CCS leads to a selective suppression of cancer cell proliferation (Wang et al., 2015). Consistent with this, we found that knockdown of CCS significantly reduced cell proliferation in MDA-MB-231 cells but not MCF-7. Interestingly, we revealed a novel function of CCS in regulating migration of breast cancer cells by transwell and wound healing assays. In all, we show that CCS not only plays a vital role in cell proliferation, but it also drives breast cancer migration.
Previous evidence has shown that CCS serves as a co-enzyme of SOD1 to activate its catalytic activity, which is a critical component of oxidation-reduction system (Suzuki et al., 2013b). Since ROS associated oxidative stress has been proven to play important roles in several cancer types and served as promising target for therapy (Perše, 2013;Sosa et al., 2013), we hypothesized that dysregulated ROS levels provide a second signal for CCS-induced proliferation and migration in breast cancer cells. In our study, we showed that knockdown or inhibition of CCS led to increased total ROS levels in MDA-MB-231. ROS overload blocks the activation of the MAPK/ERK pathway, which plays a critical role in tumor formation and progression (Berger et al., 2017;Mayo et al., 2017). By mimicking oxidative stress with H 2 O 2 treatment, we were able to suppress the phosphorylation level of ERK1/2, which could be reversed upon treatment with antioxidant NAC. Furthermore, we found that the activation of MAPK/ERK pathways was essential for CCS-induced cell proliferation and migration. Treatment of MCF-7 with U0126-EtOH, a highly selective ERK kinase inhibitor, diminished CCS-induced migration. Conversely, overexpression of MEK enhanced the phosphorylation level of ERK1/2 and partially rescued migration in CCS knockdown MDA-MB-231 cells.
In summary, CCS-mediated ROS decreases the activation of ERK1/2, resulting in attenuation of cell proliferation and migration. Thus, CCS may be a therapeutic strategy to suppress tumor growth and metastasis.

AUTHOR CONTRIBUTIONS
RL and YL performed and analyze all the experiments. XZ and JW drafted the work an provided intellectual content. LL and CS edited the language and figures. CS, SL and SZ designed the study and wrote the manuscript.