AFAP1-AS1 Promotes Epithelial-Mesenchymal Transition and Tumorigenesis Through Wnt/β-Catenin Signaling Pathway in Triple-Negative Breast Cancer.

Long non-coding RNA (LncRNA) actin filament-associated protein1-antisense RNA 1 (AFAP1-AS1) is overexpressed in various types of cancers and plays an important role in tumor progression and prognosis. This study investigates the role of AFAP1-AS1 in tumor progression in triple-negative breast cancer (TNBC). We found that AFAP1-AS1 was overexpressed in TNBC tissues and cells. Overexpression of LncRNA AFAP1-AS1 was associated with poor prognosis in TNBC patients. Moreover, we demonstrated that upregulation of AFAP1-AS1 promoted cell proliferation and invasion, and inhibited cell apoptosis in vitro, while overexpression of AFAP1-AS1 promoted tumor growth in vivo. Our results also revealed that upregulation of AFAP1-AS1 activated Wnt/β-catenin pathway to promote tumorigenesis and cell invasion by increasing the expression of C-myc and epithelial-mesenchymal transition-related molecules in TNBC. Collectively, AFAP1-AS1 can be an independent prognostic marker and an effective therapeutic target of triple- negative breast cancer.

Background

Breast cancer is the most common cancer and causes the most deaths among all types of cancer in women worldwide (Torre et al., 2015). The 5-year survival rate of breast cancer is close to 90% because of the development of early screening and targeted therapeutic drugs for hormone or HER-2 (Cedolini et al., 2014). However, triple-negative breast cancer (TNBC), a special subtype of breast cancer, lacks hormone or HER-2 receptors, and therefore has no effective targeted therapy. TNBC has a higher risk of recurrence and metastasis than other subtypes of breast cancers (Foulkes et al., 2010; Perou, 2011). The overall prognosis of TNBC patients is significantly poorer than patients with other breast cancer subtypes (Kassam et al., 2009; Bianchini et al., 2016). In China, there is a large number of patients with TNBC, accounting for 15–20% of all breast cancer subtypes (Chen et al., 2016). In this circumstance, it is essential for us to explore the molecular pathogenesis of TNBC and find effective therapeutic targets in order to develop an effective treatment for these patients.

Long non-coding RNAs (LncRNAs) refer to a group of RNAs with more than 200 nucleotides in length, without protein coding function. LncRNAs often have low expression in cells but show more cell-specific expressions than mRNAs (Guttman and Rinn, 2012; Batista and Chang, 2013). LncRNAs have several functions, such as regulation of transcription, regulation of mRNA processing and regulation of mRNA post transcriptional control (Beermann et al., 2016). Actin filament-associated protein1-antisense RNA 1 (AFAP1-AS1) was originally discovered in esophageal adenocarcinoma in 2013 (Wu et al., 2013). In recent years, a large number of studies focused on AFAP1-AS1. It was found that AFAP1-AS1 is overexpressed in various types of cancers, including esophageal cancer (Wu et al., 2013), nasopharyngeal carcinoma (Bo et al., 2015), pancreatic ductal adenocarcinoma (Ye et al., 2015), lung cancer (Yu et al., 2015), ovarian cancer (Yang S.L. et al., 2016), hepatocellular carcinoma (Lu et al., 2016; Zhang et al., 2016), gastric cancer (Guo et al., 2017), biliary tract cancer (Lu et al., 2017), and colorectal cancer (Han et al., 2016; Li Q. et al., 2016; Wang et al., 2016). AFAP1-AS1 played an important role in tumor progression and prognosis of these patients. However, the role of AFAP1-AS1 in breast cancer, especially in TNBC, remains unclear.

The Wnt/β-catenin signaling pathway is an important signaling pathway in embryonic development and adult tissue homeostasis (Cha et al., 2016). In recent years, the aberrant expression of a Wnt/β-catenin signaling pathway has been reported to drive cancer initiation and progression (Xue et al., 2016; Tammela et al., 2017; Saponaro et al., 2018). Mutations in a gene-related Wnt/β-catenin signaling pathway has been discovered in many different types of cancers (Morin et al., 2016; Steinhart et al., 2017). A recent study showed that AFAP1-AS1 can promote tumor progression through a Wnt/β-catenin signaling pathway in tongue squamous cell carcinoma (Wang et al., 2018), but the underlying mechanisms are not fully understood. Furthermore, the expression of AFAP1-AS1 and its connection with the Wnt/β-catenin signaling pathway in breast cancer are still unknown.

In this study, we analyzed the expression of AFAP1-AS1 in TNBC tissues and assessed the effect of AFAP1-AS1 on clinicopathological characteristics and prognosis. We then explored its function in TNBC by both in vitro and in vivo experiments. We found that the expression of AFAP1-AS1 is associated with tumorigenesis and prognosis in TNBC. Our results demonstrated that overexpression of AFAP1-AS1 can promote epithelial-mesenchymal transition (EMT) and tumorigenesis via the Wnt/β-catenin signaling pathway in TNBC.

Materials and Methods

Cell Culture

Cell lines were obtained from the American Type Culture Collection (Manassas, VA, United States). Two normal mammary epithelial cell lines (184A1 and MCF-10A) and eight human breast cancer cell lines (BT474, MCF-7, T47D, BT483, BT20, MDA-MB-468, BT549, and MDA-MB-231) were used. Each cell line was passaged in our laboratory for no more than 6 months. We maintained individual cell lines according to the supplier’s instructions. All cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (FBS; Gibco) in a humidified incubator with 5% CO2 at 37°C.

Clinical Sample

Our tissue samples included 31 TNBC tissues and 31 corresponding paired normal adjacent tissues. All tissues were prepared for quantitative real-time PCR (qRT-PCR) analysis. We also collected 238 matched human TNBC tissues between March 2005 and September 2009 from the Sun Yat-sen University Cancer Center. All these patients experienced modified radical mastectomy and postoperative chemotherapy (AC∗4-T∗4). The resected cancerous tissues and paired normal mammary tissues were immediately stored in RNA (Ambion). Our study was approved by the Ethics Committee of Sun Yat-sen University Cancer Center Health Authority (81372133). All procedures of collection and use of tissues followed the ethical standards formulated in the Helsinki Declaration.

SiRNA Transfection and qRT-PCR

The sequences of two siRNAs that targeted AFAP1-AS1 were siRNA1, 5′-CCTATCTGGTCAACACGTATT-3′ and siRNA2, 5′-GGGCTTCAATTTACAAGCATT-3′. The sequences of non-target negative controls (NC) were provided by Life Technologies. Cells were cultured overnight and transfected with either 50 nM siRNA1, siRNA2, or NC by Lipofectamine 3000 transfection reagent (Life Technologies, Carlsbad, CA, United States). RNA was extracted using TRIzol reagent according to the manufacturer’s instructions. SYBR Premix ExTaq II kit (Takara, Dalian, China) and CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, United States) were used to detect the expression of target gene, and the comparative CT method was used to evaluate the relative quantification of AFAP1-AS1. The sequence of primers were as follows: AFAP1-AS1,5′-CACACAGGGGAATGAAGAGG-3′ and 5′-AATGGTGGTAGGAGGGAGGA-3′; SLUG, 5′-CGAACTGGACACACATACAGTG-3′ and 5′-CTGAGGATCTCTGGTTGTGGT-3′; vimentin, 5′-TGCCAACCGGAACAACGAT-3′ and 5′-AATTCTCTTCCATTTCACGCATC-3′; fibronectin, 5′-AACAAATCTCCTGCCTGGGACTGA-3′ and 5′-TGAGTTGGCGGTGACATCAGAAGA-3′; ZEB1, 5′-GATGATGAATGCGAGTCAGATGC-3′ and 5′-CTGGTCCTCTTCAGGTGCC-3′; ZEB2, 5′-TTCTGCGACATAAATACG-3′ and 5′-GAGTGAAGCCTTGAGTGC-3′; E-cadherin, 5′-GAGAACGCATTGCCACATACAC-3′ and 5′-AAGAGCACCTTCCATGACAGAC-3′; β-actin, 5′-GTCACCGGAGTCCATCACGAT-3′and 5′-TCACCAACTGGGACGACATG-3′. β-actin was used as an endogenous control.

MTT Cell Viability Assay

An MTT assay was used to measure the viability of cells. Control samples and 1000 cells from each group were plated into each well of two 96-well plates. 20 μL of MTT substrate at a concentration of 2.5 mg/mL in PBS was added into each well. The plates were then maintained in a humidified incubator for an additional 4 h. Finally, the cells were solubilized in 150 μL of dimethylsulfoxide for colorimetric analysis (wavelength, 490 nm). One plate was analyzed immediately after the cells adhered, and the other plate was examined after 48 h. The percentage of cell viability was calculated by the following formula: cell viability = OD (treated)/OD (control) × 100.

Colony Formation Assay

1000 cells per milliliter were incubated in six-well plates covered with a layer of 0.6% agar containing 20% fetal bovine serum (FBS). Cells were prepared in 0.3% agar and seeded in triplicate. After the six-well plates were incubated at 37°C for 2 weeks, colonies were visible to the naked eye. These cells were then fixed with 4% formaldehyde and were stained with crystal violet (0.25%). Cell colonies were counted.

Cell Invasion and Migration Assays

Cell migration was examined by wound-healing assays. An artificial “wound” was created on a confluent cell monolayer. Scratches were treated with 10 μg/ml mitomycin C for 2 h, and photographs were taken using an inverted microscope (Olympus, Tokyo, Japan) after 24 h.

The cell invasion assay was conducted by seeding cells onto the basement membrane matrix present in the insert of a 24-well culture plate (EC matrix, Chemicon, Temecula, CA, United States). Fetal bovine serum was added to the lower chamber as a chemoattractant. After incubation for 48 h, the non-invading cells and EC matrix were gently removed with a cotton swab. Invasive cells located on the lower side of the chamber were stained with crystal violet, counted, and imaged.

Flow Cytometric Analysis of Apoptosis

Annexin V/propidium iodide (PI) staining and flow cytometry were used to analyze cell apoptosis. An Annexin V-fluorescein Isothiocyanate Apoptosis Detection Kit (KeyGen Biotech, Nanjing, China) was used following the manufacturer’s instructions. The apoptotic rate was detected with a flow cytometer, and the flow cytometry data was analyzed by Cell Quest Pro software.

Detection of Cell Apoptosis by JC-1 Staining

To measure cell apoptosis, JC-1 staining was used (Biyuntian Biochemistry Limited Company, China). Cells were cultured in 24-well plates for 48 h post-transfection, washed with PBS and incubated with a working solution of JC-1 for 20 min at 37°C. Cells were then rinsed twice with PBS, and stained with 1 mL of 10% DMEM medium containing 5 μmol/L JC-1. The stained cells were analyzed using a fluorescence microscope to determine color changes in the florescence from red to green.

Immunohistochemistry

Paraffin tissue sections were dewaxed, rehydrated, and placed in 10 mmol/L of citrate buffer (pH 6.0), then heated twice in a microwave oven for 5 min each. Sections were incubated with 3% H2O2 for 10 min, washed with PBS, blocked with 10% normal goat serum for 30 min, and then incubated with 4 mg/L purified anti-SLUG, anti-SNAIL, anti-β-catenin, and normal rabbit IgG as a control at 4°C overnight. After washing, the sections were stained with the catalyzed sign amplification system kit (DAKO code k5007) and visualized through photos taken.

Western Blot

RIPA lysis buffer with a proteinase inhibitor was used to extract protein from TNBC cells. The concentration of protein in the lysates was determined by the Protein BCA Assay Kit (Bio-Rad, United States). 20 μg of protein, mixed with 2 × SDS loading buffer, was loaded into each lane of 12% SDS-polyacrylamide gel. Electrophoresis was used to separate proteins in the lysates and the separated proteins were transferred into polyvinylidene difluoride membranes (Millipore, United States). The membranes were incubated at room temperature for 1 h with 5% skim milk powder to block the non-specific binding. Subsequently, the membranes were incubated with antibodies against β-catenin, C-myc, SLUG, SNAIL, vimentin, fibronectin, N-cadherin, E-cadherin, cleaved-caspase-3, cleaved-PARP, p-GSK3, GSK3, actin, and β-actin for 12 h at 4°C. The dilution of antibodies against β-catenin, C-myc, SLUG, SNAIL, vimentin, fibronectin, N-cadherin, E-cadherin, cleaved-caspase-3, cleaved-PARP, p-GSK3, and GSK3 was 1:1000. The dilution of anti-actin and anti-β-actin was 1:5000. Actin and β-actin were used as a protein loading control.

Mouse Xenograft Model

The animal study was approved by the Ethics Committee of the Sun Yat-sen University Cancer Center (approval number 81372133). The procedures of the animal experiments followed the ethical standards. We washed MDA-MB-231 cells with PBS and then 2 × 106 cells were subcutaneously injected into the flank region of nude mice. The nude mice (5-week-old female nude mice) were divided into three groups, with 4 mice in each group. When the tumors grew to 50 mm3, 40 μl of PBS containing 1 μg of siRNA-ctr, siRNA#1, or siRNA#2 was injected into the tumors. The tumor growth was then measured by caliper once a week for 4 weeks. After 28 days, all nude mice were euthanized, and the tumors were collected to weigh and analyze the expression of AFAP1-AS1 and EMT markers.

Statistical Analysis

SPSS20.0 software and GraphPad Prism Software were used to perform the statistical analysis. Comparisons between groups were analyzed with t-test, one-way ANOVA, and Chi-square test. Kaplan-Meier method was used to perform the survival analysis. Differences were considered statistically significant if P < 0.05.

Results

AFAP1-AS1 Expression Up-Regulated in TNBC Cells and Tissues

In order to explore the role of AFAP1-AS1 in TNBC, we first investigated the mRNA expression of AFAP1-AS1 in 10 breast cell lines, including 2 normal mammary epithelial cell lines, 4 non-TNBC cell lines, and 4 TNBC cell lines, by qRT-PCR. We found that AFAP1-AS1 was overexpressed in 6 breast cancer cell lines, particularly in TNBC cell lines (including BT20, MDA-MB-468, BT549, and MDA-MB-231) (Figure 1A). We then detected the mRNA expression of AFAP1-AS1 in 31 TNBC tissues and corresponding paired normal tissues by qRT-PCR. We found that the mRNA expression of AFAP1-AS1 remarkably increased in TNBC tissues (P < 0.001; Figure 1B).

FIGURE 1

LncRNA AFAP1-AS1 is up-regulated in TNBC and up-regulation of LncRNA AFAP1-AS1 correlates with poor clinical outcomes. (A) The expression of LncRNA AFAP1-AS1 determined by qRT-PCR in 10 different mammary cell lines, including 2 HME cell line (MCF-10A and 184A1) and 8 breast cancer cell lines. AFAP1-AS1 expression was normalized by β-actin expression. (B) The expression of AFAP1-AS1 in 31 TNBC tissues and their matched normal adjacent tissues determined by qRT-PCR. (C) DFS curves for 238 TNBC patients with high or low AFAP1-AS1 expression. (D) OS curves for 238 TNBC patients with high or low AFAP1-AS1 expression. All data are shown as the mean ± SEM.

AFAP1-AS1 Overexpression Correlated With Poor Overall Survival in TNBC

To explore the correlation between AFAP1-AS1 expression and clinical characteristics of TNBC, we analyzed AFAP1-AS1 expression and clinicopathological variables in 238 TNBC cases. We used the mean level of AFAP1-AS1 as a cut-off point, and found that there were 106 patients with low expression and 132 patients with high expression of AFAP1-AS1. We then analyzed the correlation between AFAP1-AS1 expression and clinicopathological variables in TNBC by chi-square test. It was found that there was no significant correlation between the AFAP1-AS1 expression and clinicopathological features, including age, tumor status, lymph node status, distant metastasis, TNM stage, and histological grade (P = 0.569, 0.612, 0.849, 0.603, 0.696, and 0.747, respectively; Table 1). Furthermore, we analyzed the relationship between AFAP1-AS1 expression and prognosis of these 238 TNBC patients. The results indicated that higher expression of AFAP1-AS1 was associated with poorer disease-free survival (DFS) (P = 0.01; Figure 1C) and overall survival (OS) (P = 0.034; Figure 1D). These results indicated that AFAP1-AS1 could be an independent prognostic factor in TNBC.

Table 1

Association between LncRNA AFAP1-AS1 and clinicopathological characteristics in triple-negative breast cancer (Chi-square test).

Clinical characteristics	Cases	AFAP1-AS1				P-value
	Total (N = 238)	High	No. %	Low	No. %
Age (years)						P = 0.569
≤50	144	82	56.9%	62	43.1%
>50	94	50	53.2%	44	46.8%
Tumor status						P = 0.612
T1	66	33	50.0%	33	50.0%
T2	151	88	58.3%	63	41.7%
T3	11	5	45.5%	6	54.5%
T4	10	6	60.0%	4	40.0%
Lymph node status						P = 0.849
N0	121	65	53.7%	56	46.3%
N1	69	39	56.5%	30	43.5%
N2	26	14	53.8%	12	46.2%
N3	22	14	63.6%	8	36.4%
Distant metastasis						P = 0.603
Yes	4	2	50.0%	2	50.0%
No	234	130	55.6%	104	44.4%
TNM stage						P = 0.696
I	46	22	47.8%	24	52.2%
II	138	79	57.2%	59	42.8%
III	50	29	58.0%	21	42.0%
IV	4	2	50.0%	2	50.0%
Histological grade						P = 0.747
G1	3	2	66.7%	1	33.3%
G2	140	75	53.6%	65	46.4%
G3	95	55	57.9%	40	42.1%

AFAP1-AS1 Knockdown Suppressed TNBC Cell Proliferation in vitro

To investigate the biological role of AFAP1-AS1 in TNBC cell proliferation, we knocked down LncRNA AFAP1-AS1 with small interfering RNA (siRNA) in MDA-MB-231 and BT549 cells, and detected that the expression of AFAP1-AS1 was significantly decreased by siRNA (Figure 2A). We then performed a MTT cell viability assay and a colony formation assay. Compared with the control groups, we found that AFAP1-AS1 knockdown markedly inhibited cell proliferation and colony formation in MDA-MB-231 and BT549 cells (Figures 2B,C).

FIGURE 2

AFAP1-AS1 knockdown suppressed cell proliferation and colony formation in TNBC cells. (A) siRNA knockdown efficiency of AFAP1-AS1 expression in MDA-MB-231 and BT-549 cells by qRT-PCR (∗∗P < 0.01). (B) MTT assay on the cell viability of MDA-MB-231 and BT-549 cells infected with siRNA-AFAP1-AS1, or siRNA-ctr (∗P < 0.05 and ∗∗P < 0.01). (C) Colony formation assays on MDA-MB-231 and BT-549 cells transfected with siRNA-ctr, siRNA-#1, or siRNA-#2 (∗∗P < 0.01). The representative data from three independent experiments are shown.

AFAP1-AS1 Knockdown Enhanced TNBC Cell Apoptosis in vitro

To understand the role of AFAP1-AS1 in cell apoptosis, we knocked down AFAP1-AS1 in TNBC cells and performed an Annexin V-FITC/PI staining-based FACS analysis and JC-1 staining. The results showed that apoptosis of MDA-MB 231 and BT-549 cells were significantly increased in AFAP1-AS1 knockdown groups compared with the control groups (Figures 3A,B). Furthermore, we used Western blotting to examine the change of expression of cleaved-caspase-3 and cleaved-PARP after knocking down AFAP1-AS1. The results showed that, compared with the control groups, the expression of cleaved-caspase-3 and cleaved-PARP were significantly increased in AFAP1-AS1 knockdown groups (Figure 3C). These results suggested that AFAP1-AS1 might mediate the signaling pathway of cell apoptosis.

FIGURE 3

AFAP1-AS1 knockdown enhanced apoptosis. (A) Annexin V-FITC/PI-staining on cell apoptosis in MDA-MB-231 and BT-549 cells evaluated by flow cytometry 24 h after siRNA transfection (∗∗P < 0.01). (B) JC-1 staining (Biyuntian Biochemistry Limited Company, China) was used to measure cell apoptosis. Downregulated AFAP1-AS1 could significantly increase the apoptosis rate of MDA-MB 231 and BT-549 cells. (C) The expressions of cleaved-caspase-3 and cleaved-PARP in MDA-MB 231 and BT-549 cells detected by Western blot. Actin was used as the loading control (∗∗P < 0.01). The representative data from three independent experiments are shown.

AFAP1-AS1 Knockdown Inhibited Cell Invasion and Migration in vitro

To detect the effect of AFAP1-AS1 on cell invasion and migration, we performed wound-healing assays and a transwell invasion assay in TNBC cells. The result of wound-healing assays showed that cell migration of TNBC cell was significantly inhibited in AFAP1-AS1 knockdown groups compared with control groups (Figure 4A). The result of the transwell invasion assay showed that cell invasion of TNBC cells was significantly decreased in AFAP1-AS1 knockdown groups compared with control groups (Figure 4B). These results suggested that AFAP1-AS1 could promote cell invasion and migration in vitro.

FIGURE 4

AFAP1-AS1 knockdown inhibited cell invasion and migration. (A) Wound-healing assay was used to measure cell migration. These pictures show that cell migration of TNBC cells was significantly inhibited in AFAP1-AS1 knockdown groups. (B) Transwell invasion assay was used to measure cell invasion of MDA-MB-231 cells transfected with siRNA-ctr, siRNA#1, or siRNA#2 (∗∗P < 0.01). The representative data from three independent experiments are shown.

Silencing AFAP1-AS1 in TNBC Cells Inhibited Tumor Growth in vivo

Based on the above in vitro results and prognostic analysis, we performed an animal experiment to further explore the role of AFAP1-AS1 in TNBC. We adopted the breast cancer tumor xenografts model in nude mice. The mice (5-week-old female) were divided into three groups with 4 mice in each group. We injected 40 μl of PBS containing 1 μg of siRNA-ctr, siRNA#1, or siRNA#2 into the tumors. After 28 days, all mice were euthanized, and the tumors were collected and weighed. It was found that the mean weight of tumors from the AFAP1-AS1 knockdown groups were significantly lower than that in the control group (Figure 5A). We also confirmed that compared with the control group, the expression of AFAP1-AS1 was remarkably decreased in AFAP1-AS1 knockdown groups (Figure 5B). The results showed that AFAP1-AS1 in TNBC cells could promote tumor growth in vivo.

FIGURE 5

Silencing AFAP1-AS1 inhibited tumorigenesis in vivo. (A) A representative picture of the morphology of tumor xenografts after 28 days of treatment (left). The tumor weights of the xenografts are summarized in the right panel. All results are expressed as the mean ± SD of three independent experiments (∗P < 0.05). (B) siRNA knockdown efficiency of AFAP1-AS1 expression in tumors by qRT-PCR (∗∗P < 0.01). (C) AFAP1-AS1 cellular localization indicating AFAP1-AS1 mainly localized in the cytoplasm of cells.

AFAP1-AS1 Knockdown Reduced the Expression of C-myc and EMT-Related Molecules Through Wnt/β-Catenin Signaling Pathway

Because AFAP1-AS1 knockdown inhibited TNBC cell proliferation and invasion, we investigated the possible molecular mechanisms that regulate these processes in TNBC cells. We found that the expression of AFAP1-AS1 was localized in the cytoplasm of TNBC cells (Figure 5C). Since the Wnt/β-catenin signaling pathway, C-myc, and EMT play important roles in tumorigenesis and cell invasion, we performed immunohistochemical staining to examine the expression of SLUG, SNAIL, and β-catenin in AFAP1-AS1 knockdown TNBC tissues. It was found that SLUG, SNAIL, and β-catenin expressions were negative in AFAP1-AS1 knockdown tumor xenografts (Figure 6). Furthermore, we investigated the expression of the Wnt/β-catenin signaling pathway, C-myc and EMT-related molecules by Western blotting. It was found that AFAP1-AS1 knockdown significantly decreased the expression of Wnt/β-catenin signaling pathway-related molecules (β-catenin, p-GSK3), while the expression of GSK3 had no remarkable difference compared with the control groups. In addition, AFAP1-AS1 knockdown also decreased the expression of C-myc and EMT-related molecules (SLUG, SNAIL, vimentin, fibronectin, and N-cadherin), but increased the expression of E-cadherin in both MDA-MB-231 and BT549 cells (Figure 7). These results demonstrated that AFAP1-AS1 might activate Wnt/β-catenin signaling pathway to promote EMT in TNBC.

FIGURE 6

Immunohistochemistry assay of β-catenin, SLUG, and SNAIL expressions in TNBC tissues. β-catenin, SLUG, and SNAIL expressions were positive in TNBC tissues without AFAP1-AS1 downregulation, while β-catenin, SLUG, and SNAIL expressions were negative in AFAP1-AS1 knockdown TNBC tissues.

FIGURE 7

AFAP1-AS1 knockdown reduced the expression of C-myc and EMT-related molecules through Wnt/β-catenin signaling pathway. The expressions of β-catenin, GSK3, p-GSK3, C-myc, SLUG, SNAIL, vimentin, fibronectin, N-cadherin, and E-cadherin in MDA-MB 231 and BT-549 cells detected by Western blot. β-actin was used as the loading control. AFAP1-AS1 knockdown significantly decreased the expression of β-catenin, p-GSK3, C-myc, and EMT-related molecules (SLUG, SNAIL, vimentin, fibronectin, and N-cadherin), but increased the expression of E-cadherin in both MDA-MB-231 and BT549 cells (∗∗P < 0.01).

Discussion

Triple-negative breast cancer is one of the breast cancer subtypes. Patients suffering from TNBC do not have sensitivity to hormone therapy or targeted therapy. These patients often have poor OS and DFS (Kalimutho et al., 2015; Torre et al., 2015; Chen et al., 2016). It is critical to find useful biomarkers and targeted therapies for TNBC. Accumulating evidence indicate that non-coding RNAs, a group of RNAs with no protein coding function, such as circular RNAs (circRNAs) and LncRNAs, play important roles in tumor initiation and progression in various types of cancers (Huarte, 2015; Liu et al., 2015; Beermann et al., 2016; Zheng et al., 2016; He et al., 2017). AFAP1-AS1 is one of the LncRNAs and its expression has been suggested to be related to tumor growth and prognoses in many different types of cancers (Zhang et al., 2018), such as retinoblastoma (Hao et al., 2018), esophageal cancer (Wu et al., 2013), nasopharyngeal carcinoma (Bo et al., 2015), pancreatic ductal adenocarcinoma (Ye et al., 2015), lung cancer (Yu et al., 2015), ovarian cancer (Yang S.L. et al., 2016), hepatocellular carcinoma (Lu et al., 2016; Zhang et al., 2016), gastric cancer (Guo et al., 2017), biliary tract cancer (Lu et al., 2017), and colorectal cancer (Han et al., 2016; Li Q. et al., 2016; Wang et al., 2016). However, a limited number of studies focused on LncRNA AFAP1-AS1 in TNBC. Yang F. et al. (2016) described that AFAP1-AS1 was up-regulated in TNBC and promoted cancer proliferation and invasion. However, the number of samples in their study was small, and the mechanism of AFAP1-AS1 in TNBC was not explored. In this study, we confirmed that AFAP1-AS1 was overexpressed in TNBC tissues. Our result was consistent with what Yang et al. reported. By studying 238 TNBC patients who experienced modified radical mastectomy and chemotherapy, we found that upregulated AFAP1-AS1 was correlated with poor disease-free survival and poor overall survival but had no significant association with clinicopathological variables of TNBC patients. The reason why AFAP1-AS1 had no association with clinicopathological characteristics might be that the majority of patients in the present study were diagnosed with early stage cancer, and the metastasis related with AFAP1-AS1 was difficult to detect at an early stage. A larger sample size might be required to further explore this relationship. Nevertheless, these results indicated that TNBC patients with upregulated AFAP1-AS1 might be more likely to have circulating tumor cells in the peripheral blood at early stage, and therefore, these patients had poorer prognosis. AFAP1-AS1 could be recognized as an independent prognostic factor of TNBC.

We investigated the roles of AFAP1-AS1 on TNBC cells in vitro. Yang F. et al. (2016) reported that up-regulation of AFAP1-AS1 could promote proliferation and invasion of TNBC cells in vitro. Through our studies, we found that knockdown of AFAP1-AS1 inhibited the tumorigenesis and invasion of TNBC cells and enhanced TNBC cells apoptosis. We also found that upregulated AFAP1-AS1 could promote tumor growth in nude mice. These results suggested that LncRNA AFAP1-AS1 could be a potential therapeutic target in TNBC.

To explore the mechanism of AFAP1-AS1 in TNBC, we first detected the localization of AFAP1-AS1 in TNBC cells. We found that AFAP1-AS1 mainly localized in the cytoplasm of cells, indicating that AFAP1-AS1 might perform its function in the cytoplasm. It was reported that β-catenin is an important driver of cancer initiation and progression (Cui et al., 2018). The Wnt/β-catenin signaling pathway has been proved to play critical roles in tumorigenesis and progression in many cancers (Nusse and Clevers, 2017; Tammela et al., 2017). Previous studies also indicated that the Wnt/β-catenin signaling pathway could enhance breast cancer invasion and metastasis (Dey et al., 2013; Li Y. et al., 2016; Tang et al., 2018). We assumed AFAP1-AS1 could promote tumorigenesis of TNBC through the Wnt/β-catenin signaling pathway. There are several molecules that belong to the Wnt/β-catenin signaling pathway. We detected the expression of these molecules, including β-catenin. Our results showed that AFAP1-AS1 knockdown remarkably reduced the expression of β-catenin in TNBC cells. It suggested that AFAP1-AS1 could increase the expression of β-catenin to promote the Wnt/β-catenin signaling pathway. Nusse and Clevers (2017) suggested that expression of β-catenin is controlled by the destruction complex consisting of adenomatous polyposis coli (APC), casein kinase 1 (CK1), glycogen synthase kinase 3 (GSK3), β-transducin repeat-containing protein (β-TrCP), and scaffolding protein Axin. We assumed that AFAP1-AS1 might suppress the function of β-catenin destruction complex. To confirm this assumption, we examined the expression of AFAP1-AS1 on GSK3 and p-GSK3. We found that AFAP1-AS1 up-regulated the phosphorylation of GSK3 to reduce the function of GSK3. This result indicated that AFAP1-AS1 could promote a Wnt/β-catenin signaling pathway in TNBC. We further explored how AFAP1-AS1 promoted proliferation and invasion through the Wnt/β-catenin signaling pathway in TNBC. Previous studies have found that the Wnt/β-catenin signaling pathway was related to C-myc and EMT (McCrea and Gottardi, 2016; Nusse and Clevers, 2017). It is known that C-myc is an important gene regulating cell cycle progression, and EMT plays an important role in tumor migration and metastasis (De Craene and Berx, 2013; Nakaya and Sheng, 2013; Nieto et al., 2016). EMT-related molecules include SLUG, SNAIL, vimentin, fibronectin, E-cadherin, and N-cadherin. When cancer progression and metastasis happen, the expression of SLUG, SNAIL, fibronectin, vimentin, and N-cadherin increased while the expression of E-cadherin decreased (Lamouille et al., 2014; Du and Shim, 2016). Our results showed that AFAP1-AS1 knockdown remarkably reduced the expression of c-myc. It is known that down-regulation of c-myc could slow down cell cycle. Our results suggested that AFAP1-AS1 could promote proliferation of TNBC by activating the proto-oncogene C-myc through Wnt/β-catenin signaling pathway. Our results also showed that AFAP1-AS1 knockdown decreased the expression of SLUG, SNAIL, vimentin, fibronectin, and N-cadherin, but increased the expression of E-cadherin in TNBC cells. These results indicated that AFAP1-AS1 might promote TNBC progression and metastasis by activating EMT through the Wnt/β-catenin signaling pathway.

There are some limits in this study. First, our clinical samples mostly come from patients with early stage TNBC, so we did not observe the correlation between AFAP1-AS1 and clinicopathological characteristics. Second, the details of molecular mechanisms between AFAP1-AS1 and the Wnt/β-catenin signaling pathway are still unclear and further studies are needed.

Conclusion

In conclusion, our study indicated that AFAP1-AS1 is overexpressed in TNBC tumor tissues and cells. Overexpression of AFAP1-AS1 is associated with poor prognosis in TNBC patients. Studies in vitro confirmed that overexpression of AFAP1-AS1 promoted cell proliferation and invasion, and inhibited cell apoptosis in TNBC. Study in vivo showed upregulated AFAP1-AS1 could promote tumor growth in nude mice. Upregulation of AFAP1-AS1 might activate the Wnt/β-catenin signaling pathway and increase the expression of C-myc and EMT-related molecules to promote tumorigenesis and invasion (Figure 8). Collectively, AFAP1-AS1 could be an independent prognostic marker and a potential therapeutic target for TNBC.

FIGURE 8

Diagrammatic sketch of molecular mechanism.

Author Contributions

XSX, XQ, and HT designed the experiments. PL, YK, and CS performed the experiments. KZ analyzed the data and wrote the paper. XMX was the group leader and offered financial support. HT and XSX revised the paper. All authors reviewed the manuscript.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.