ARPC2 promotes breast cancer proliferation and metastasis

Actin-related protein 2/3 complex (ARPC2) is an actin-binding component involved in the regulation of actin polymerization. It mediates the formation of branched actin networks and contacts the mother actin filament. Migration and invasion are key processes which enable tumor cells to infiltrate blood vessels or lymphatic vessels, and the actin pathway plays a very important role. Given that ARPC2 is critical to this progression, the present study focused on ARPC2 activity in breast cancer (BrCa) cell invasion and migration. Limited data are available on the expression and role of ARPC2 proteins in breast carcinomas. We screened the Oncomine database for messenger RNAs (mRNAs) that are upregulated in BrCa and found that ARPC2 was one of the most consistently involved mRNAs in BrCa. The analysis of immunohistochemical data revealed that ARPC2 expression was higher in breast cancerous tissues than in adjacent non-cancerous tissues. In addition, ARPC2 was highly associated with the tumor stage, nodal metastasis, and overall survival of patients with BrCa. We performed siRNA-ARPC2 transfection to investigate the effect of ARPC2 on the proliferation, migration, invasion and arrest of BrCa cells. It was revealed that ectopic ARPC2 expression significantly upregulated N-cadherin, vimentin, ZEB1, MMP-9 and MMP-3 expression and also activated the TGF-β pathway to contribute to epithelial-mesenchymal transition (EMT). These results collectively indicated that ARPC2 promoted the tumorigenesis of breast carcinoma and the initiation of EMT. Therefore, ARPC2 was revealed to be a potential therapeutic target in patients with BrCa.


Introduction
Breast cancer (BrCa) is the most prevalent malignancy among women. Approximately 252,710 new cases of invasive BrCa and 40,610 cancer-related deaths are expected to occur among American women in 2017 (1). BrCa is a heterogametic disease that can be categorized into luminal A, luminal B, human epithelial growth factor receptor-2 (HER-2) overexpressing and basal/triple negative (TN) molecular subtypes on the basis of gene expression profiling (2)(3)(4)(5). Given the high morbidity and poor prognosis of patients with BrCa, additional research is required to understand the potential molecular mechanism that mediates BrCa tumorigenesis. To date, a variety of biological factors have been identified to be involved in the development of this disease. Therefore, it is imperative to identify potential therapeutic targets such as progesterone receptor (PR) and estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2) among the various biological factors involved in BrCa development (6)(7)(8).
EMT is a specific physiological or pathophysiological phenomenon that is characterized by the loss of the epithelial phenotype and gain of mesenchymal characteristics by epithelial cells. These processes are evidenced by the decrease in E-cadherin expression and increase in vimentin/N-cadherin expression (18). The activation of EMT has been implicated in the metastasis of human tumors. In cancer, the frequent occurrence of EMT is associated with poor prognosis and distant metasis. TGF-β is a pathway involved in EMT regulation (19,20). The downstream factors of the TGF-β pathway include N-cadherin, vimentin, ZEB1, MMP-9 and MMP-3, and these factors have important roles in EMT regulation (21,22). However, whether the TGF-β/EMT pathway is involved in ARPC2-mediated carcinogenesis has never been reported.
The present study was aimed to determine whether ARPC2 is a BrCa oncogene and to further understand the mechanisms that underlie its in vitro and in vivo effect. By searching the Oncomine database and performing immunohistochemistry (IHC), we found that ARPC2 mRNA and protein expression levels were upregulated in BrCa. The results of the cell cycle, wound-healing, migration and invasion assays revealed that ARPC2 significantly promoted the proliferation, migration and invasion of BrCa cells. Clinical data, revealed that ARPC2 was associated with lymph node invasion and tumor stage. High ARPC2 expression levels were associated with a low overall survival (OS) rate. Lastly, we performed western blot analysis to further explore the potential mechanism of ARPC2 in BrCa. We found that ARPC2 increased the expression of EMT-inducing transcription factors (EMT-TFs), such as N-cadherin, vimentin, ZEB1, MMP-9 and MMP-3. Notably, ARPC2 may contribute to the malignant progression of BrCa.

Materials and methods
Patients and tissue collection. We obtained 172 paraffinembedded surgical BrCa tissue specimens from the Department of Pathology at the First Affiliated Hospital of Anhui Medical University (Hefei, Anhui, China). We recruited 68 female patients with benign breast diseases from the same hospital. The 68 female patients were used as the control group. No patients had received preoperative adjuvant therapy. Two pathologists confirmed the diagnoses of breast tumors in all the samples used. All cases were followed-up for 60 months. Overall survival (OS) was calculated on the basis of data. Patient samples were categorized into two groups in accordance with median expression (high vs. low expression) (Table I) and assessed using a Kaplan-Meier survival plot. The protocol of the present study was approved by the Institutional Review Board of Anhui Medical University. Detailed information of patients including ER/PR expression, lymph node metastasis and age are listed in Table II. The agreement on the use of these tissue samples was approved by the Biomedical Ethics Committee of Anhui Medical University and the confidentiality of patient information was maintained. We obtained informed consent from all patients before enrolling participants in the present study.
Oncomine, The cancer genome atlas data and cBioPortal analysis. The Oncomine microarray database (http://www. oncomine.org) was used for analysis. We screened for relevant entries on ARPC2 expression in BrCa tissues and normal breast tissues. With 'Cancer vs. normal' analysis as the primary filter. Data sets were used in the present study in accordance with Ma et al (23), Curtis et al (24), Perou et al (25) and Zhao et al (26). We screened for differentially expressed genes (Pearson's correlation >0.4) in several important node locations of ARPC2 in BrCa (Pearson's correlation >0.4) by using BioPortal for Cancer Genomics (http://www.cbioportal.org). All searches were performed according to the cBioPortal's online instructions.
Cell culture. Human BrCa cell lines (MCF-10A, MCF-7, T47D, BT474 and MDA-MB-231) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured under ATCC-recommended conditions. Cells were routinely incubated at 37˚C and 5% CO 2 under a humidified atmosphere.
ARPC2 overexpression and downregulation. The full length of Homo sapiens ARPC2 was synthesized and subcloned into an entry vector by OriGene Technologies Technologies, Inc., (Beijing, China). siRNA-ARPC2 (siARPC2) was purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). MCF-7 and MDA-MB-231 cells were transfected using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in accordance to the manufacturer's instructions. Transfection efficiency was confirmed by performing reverse transcription-quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting.
Western blotting and RT-PCR. We used RT-PCR to evaluate the mRNA levels of ARPC2. Total RNA was isolated from cultured cells using TRIzol reagent. RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). qRT-PCR was performed using the SYBR Prime Script kit (Takara Bio, Inc., Otsu, Japan). ARPC: Forward primer, 5'-TCC GAC TCT ACC AGC TGA TGC-3' and reverse primer, 5'-AAG CTG GAC TCA TCC CAC AGC-3' . PCR was conducted at 95˚C for 15 min, followed by 40 cycles of 94˚C for 15 sec, 55˚C for 30 sec, and 64˚C for 30 sec. Experiment was repeated three times and the expression of ARPC2 was quantified using the 2 -ΔΔCq method (27). Cells were lysed via a lysis buffer which consisted of RIPA (Beyotime Institute of Biotechnology, Haimen, China). The protein concentration was determined using BCA kit. Equal amounts of total protein (10 µg) were separated by 10% SDS polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes, followed by blocking with 5% non-fat milk for 1 h. The membranes were incubated with primary antibodies overnight at 4˚C. Protein expression was quantified by densitometry (Quantity One software; Bio-Rad Laboratories, Hercules, CA, USA Colony formation assays. For the colony formation assay, transfected cells were grown in a fresh 6-well plate with RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum. For the cell colony formation assay, the transfected cells were seeded in 6-well plates (1x10 3 /well) and cultured for 14 days at 37˚C. After 14 days, all visible colonies per well were manually counted. Assays were performed in triplicate, and the results were calculated as the mean ± standard deviation (SD).

ARPC2 expression levels in different BrCa cell lines.
We quantified the expression of ARPC2 in different types of human BrCa cell lines (MCF-7, T47D, BT474 and MDA-MB-231) and human breast epithelial cell line (MCF-10A) by RT-PCR and western blotting. As revealed in Fig. 1A and B, we found that ARPC2 was highly expressed in BrCa cell lines. However, changes in ARPC2 expression in four types of BrCa cell lines did not exhibit a consistent pattern. Since MCF-7 and MDA-MB-231 cell lines had a good tumorigenic effect, we selected MCF-7 and MB-MDA-231 cell lines as experimental models.
ARPC2 mRNA and protein expression in BrCa and normal tissues. We performed IHC to assess ARPC2 protein expression in different types of BrCa (n=172) and paired normal breast (n=68) tissues. As revealed in Fig. 1C, the stain for ARPC2 protein expression was predominantly located in the cytosol. Our results demonstrated that the expression level of ARPC2 protein in normal tissues was significantly lower than in the cancer tissues (Table I). The Kaplan-Meier curve and log-rank test analyses revealed that ARPC2 expression was associated with the OS of patients with BrCa. We evaluated the prognostic value of ARPC2 using the protein expression data and survival information of 172 patients with BrCa. We divided patient samples into two groups in accordance with the median expression (high vs. low expression). As revealed in Fig. 1D, high ARPC2 levels were associated with a low OS rate. Moreover, to illustrate the potential relationship between ARPC2 and BrCa progression, we used the Oncomine database (http://www. oncomine.org) for the analysis of ARPC2 mRNA expression. We found that ARPC2 mRNA expression was significantly upregulated in BrCa relative to that in normal breast tissues (Fig. 1E).

Relationship of ARPC2 with the clinicopathological characteristics and poor prognosis of patients with BrCa.
We analyzed the relationship between ARPC2 protein expression and the clinicopathological parameters of patients with BrCa. As presented in Table II, ARPC2 protein expression was significantly and positively associated with lymph node metastasis, tumor size and grade of BrCa (P<0.05). The protein expression of ARPC2, however, was not significantly associated with other clinical parameters, including age, HER-2 expression, and estrogen and progesterone receptor status of patients with BrCa.

ARPC2 silencing inhibits the migration and invasion capabilities of BrCa cell lines.
To delineate the role of ARPC2 in BrCa tumorigenesis, we knocked out ARPC2 in MDA-MB-231 cells and overexpressed ARPC2 expression in MCF-7 cell lines. We confirmed ARPC2 expression by performing western blotting and qRT-PCR ( Fig. 2A and B). To further confirm the role of ARPC2 in BrCa migration and invasion, we used a colony formation and scratch wound healing assays ( Fig. 2C and D). The colony forming ability of cells was decreased and the percentage of wound closure was increased by ARPC2 silencing in MDA-MB-231 cells. Similarly, the migration and the invasion of MDA-MB-231 was also inhibited by ARPC2 silencing as determined by Transwell assays. Thus, the indu-cive effect of ARPC2 in BrCa cell migration and invasion was revealed.

ARPC2 overexpression inhibits the growth of BrCa cells.
We assessed the effects of pcDNA3.1-ARPC2 transfection on cellular proliferation and growth through colony formation assays. Our results demonstrated that pcDNA3.1-ARPC2 displayed a relatively rapid proliferation rate compared to control cells when MCF-7 cells were transfected with pcDNA3.1-ARPC2. The colony forming ability (Fig. 2C) as well as the migration and invasion abilities (Fig. 2E) were enhanced as determined by colony formation and Transwell assays.
ARPC2 silencing inhibits BrCa cell proliferation and induces apoptosis. We downregulated ARPC2 expression in the MDA-MB-231 cell line through transfection with an ARPC2-specific siRNA or a non-specific siRNA control. As revealed in Fig. 3A, ARPC2 silencing altered the cell cycle of the MDA-MB-231 cell line. We found that a higher number of siARPC2 MDA-MB-231 cells were in the G1 phase compared with the control cells. In addition, the transfection of siARPC2 increased apoptosis of MDA-MB-231 cells, as determined by performing Annexin V-FITC/PI staining (Fig. 3B).
The results revealed that suppression of TGF-β signaling reduced Smad2 phosphorylation levels and downregulated vimentin expression (Fig. 4B). To investigate whether ARPC2 is required for TGF-β-induced EMT, we transfected MDA-MB-231 cells with siARPC2 or non-target control However, under the same conditions E-cadherin and vimentin expression levels were not altered in MB-MDA-231-siARPC2 cells (Fig. 4C). These experiments suggested that ARPC2 is involved in TGF-β-induced EMT.
ARPC2 regulates EMT-related protein expression. Cellular adhesive ability is associated with EMT. To determine whether ARPC2 expression triggers EMT in BrCa cells, we examined MMP-3, MMP-9 (cell migration marker), E-cadherin (epithelial cell marker), N-cadherin, vimentin (mesenchymal marker) and ZEB-1 protein expression after transfection with siARPC2 and pcDNA3.1-ARPC2 ( Fig. 4D and E).   (Fig. 5C). As revealed in Fig 5, a high ARPC2 level increased tumor growth and weight ( Fig. 5A and B), and generated lung or lymphonodus metastases in nude mice (Fig. 5D). We subsequently explored the effects of ARPC2 on tumor growth in vivo by IHC, the results of which revealed that tumour growth and tumor weight were significantly increased in the ARPC2-overexpression group compared with the negative control group and that the Ki-67 expression levels in the ARPC2-overexpression group were higher than those in the negative control group.

Discussion
Recurrent and metastatic BrCa gravely threatens the health of women worldwide. Thus, intensive studies on the potential candidate molecules involved in the metastasis of BrCa are urgently required for the development of BrCa treatment. Previous studies on microarray profiling have revealed that ARPC2 is aberrantly expressed during BrCa progression. In the present study, it was revealed that ARPC2 expression was increased in BrCa cell lines (MCF-7, BT474, MDA-MB-231 and T47D). Furthermore, ARPC2 expression was uniquely linked with the invasion, apoptosis and proliferation of mammary carcinoma cells. This positive association was further supported by evidence obtained through in vitro studies and a nude mouse model. The analysis of clinical specimens, IHC results, and clinicopathological and prognostic information indicated that ARPC2 was significantly associated with tumor size, lymph node metastasis, and tumor grade. Moreover, Kaplan-Meier analyses revealed that ARPC2 expression was linked with the poor OS of patients with BrCa. Therefore, we proposed that ARPC2 has regulatory functions in BrCa development and that it can be treated as an independent prognostic marker for BrCa. The evolutionarily conserved Arp2/3 complex consists of five subunits (ARPC1-5) (28,29), and two actin-related proteins ARP2 and ARP3. ARPC2 can promote actin assembly in lamellipodia and participate in lamellipodial protrusions, which promote cell shape change and locomotion. ARPC2, along with some co-expressed genes, plays a crucial role in invasion and metastasis (30)(31)(32). To explore the regulatory network of ARPC2, we found that ARPC2 may be involved in EMT, a key early event in breast tumor invasion and metastasis. A growing body of evidence has revealed that EMT, a specific physiological or pathophysiological phenomenon, is characterized by the conversion of epithelial cells into mesenchymal-like cells through the loss of polarity and disruption of cell-cell connections (33,34). Although the significance of EMT-induced carcinogenesis has been defined, its molecular mechanism remains unclear.
EMT involves the dissolution of epithelial tight junctions, the modulation of adherent junctions, the remodeling of the cytoskeleton, and the loss of apical-basal polarity. Matrix metalloproteinases (MMPs) are EMT-related proteins (35) and ZEB1 is crucial for the transcriptional regulation of EMT (14). N-cadherin and vimentin are present in the microfilaments (actins) and microtubules (tubulins) (36,37). Arp2/3 subunits exhibit a tissue-specific expression pattern characterized by the increased expression of mesenchymal proteins. To directly test this hypothesis, we quantified the expression levels of ARPC2, E-cadherin, vimentin, ZEB1, MMP-9 and MMP-3 proteins. We found that in vitro ARPC2 increased breast tumor cell migration and invasion, as well as vimentin, ZEB1, MMP-9 and MMP-3 proteins. The results demonstrated that ARPC2 is an oncogene or has oncogenic effects on BrCa cell growth and metastasis by stimulating EMT. Recently, a study revealed that a TGF-β/Smad-dependent gene regulated fibronectin and smooth muscle actin (38) in human lung mesenchymal cells. In the present study, we found that TGF-β signaling was activated in ARPC2-induced EMT. In addition, TGF-β signaling induced ARPC2 expression in breast epithelial cells, and the knockdown of ARPC2 blocked TGF-β-induced EMT. We proposed that a feedback loop exists between ARPC2 and TGF-β expression. This feedback loop could regulate EMT and BrCa progression.
To the best of our knowledge, we are the first to report that ARPC2 plays a critical role in BrCa in vitro and in vivo. ARPC2 overexpression promoted BrCa cell proliferation, invasion, and metastasis through EMT. Our results indicated that ARPC2 is a potential biomarker or therapeutic target for BrCa.