The Prognostic Value of ADAMTS8 and Its Role as a Tumor Suppressor in Breast Cancer

Abstract A disintegrin-like and metalloprotease with therombospondin type1 motif 8 (ADAMTS8) plays an important role in many malignancies. However, the clinical and biological significance of ADAMTS8 in breast cancer remain unknown. In this study, the clinical data from 1066 breast cancer patients were analyzed by The Cancer Genome Atlas (TCGA) database, and were analyzed using the correlation between ADAMTS8 expression and the clinicopathological features and prognoses. The CCK-8 assay, clone formation assay, flow cytometry and Transwell assay were used to characterize the effects of ADAMTS8 on proliferation, migration and invasion of breast cancer cells. Gene set enrichment analysis (GSEA) and western blotting were used to identify the potential molecular mechanism on how ADAMTS8 exert its biological function. ADAMTS8 overexpression correlated longer overall survival (OS) and progression-free survival (PFS). ADAMTS8 was considered as an independent prognostic factor for OS. ADAMTS8 overexpression inhibited breast cancer cell proliferation, migration and invasion in vitro, and induced G2/M cell cycle arrest. ADAMTS8 was also involved in cell cycle regulation and was associated with the EGFR/Akt signaling pathway. ADAMTS8 knockdown showed the reverse effect. Together, the results showed that ADAMTS8 functioned as a tumor suppressor gene (TGS) and could be a prognostic biomarker for breast cancer.


Introduction
Breast cancer is a common malignancy in women, and the morbidity in many countries was ranked first in 2021 (1,2). It is a heterogeneous disease with distinct molecular subtypes, which are correlated with recurrence rate, overall survival, and therapy (3). Despite advances in the treatment of breast cancer, an effective therapy remains elusive (4). Therefore, identifying new biomarkers is crucial for the clinical diagnosis of breast cancer and for assessing the benefits for targeted therapy (5)(6)(7).
ADAMTS8 belongs to the Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) family and is a key antiangiogenic regulatory protein (8). ADAMTS8 inhibits epidermal growth factor receptor (EGFR) signaling and as well as vascular endothelial growth factor (VEGF)-mediated angiogenesis by endothelial cells, leading to decreased expression levels of p-MEK and p-ERK (9). A disintegrin-like and metalloprotease with therombospondin type1motif 8 (ADAMTS8) has structural similarity to a disintegrin-like and metalloprotease with therombospondin type1 motif 1 (ADAMTS1) is an important member of the anti-angiogenic family of the ADAMTS (10). The two molecules differ in that ADAMTS8 has only one C-terminal domain, which is crucial for the suppression of angiogenesis (9). Recently, ADAMTS family proteins have been found to be related to oncogenesis (9), and methylation of another family member, a disintegrin-like and metalloprotease with therombospondin type1 motif 9 (ADAMTS9), is essential for the occurrence and development of breast cancer (9). ADAMTS8 and ADAMTS9 are the two anti-angiogenic members of the ADAMTS family with aggrecanase properties (11)(12)(13), which is associated with tumor suppression (12). The ADAMTS8 gene is hypermethylated when associated with tumor growth and metastasis of gastric cancer (14).
ADAMTS8 could suppress cell proliferation in hepatocellular carcinoma and esophageal squamous cell carcinoma (15,16). However, the biological role and clinical significance of ADAMTS8 in breast cancer remain poorly defined. A comprehensive study of the published literature indicated that ADAMTS8 was downregulated in different breast cancer cell lines such as MDA-MB-231, BT549, MB468, MCF7, SK-BR-3, YCCB-1, and ZR-75-1, whereas the methylation of ADAMTS8 differed between the MDA-MB-231 and BT549 cell lines (9). Different cells (including HBL100, MDA-MB-231, YCC-B1, T47D, BT549, MCF7, SKBR3) were tested by real-time quanitative Polymerase Chain Reaction (PT-qPCR), and the result confirmed that MDA-MB-231 and BT549 cells were also downregulated in breast cancer cells (supplementary Figure 1). Therefore, we performed the functional experiments with MDA-MB-231 and BT549 cell lines to investigation the effect of breast cancer cell proliferation, migration and invasion. In this study, we explored the association between ADAMTS8 expression and DNA methylation. The clinicopathological features associated with ADAMTS8 expression were analyzed using The Cancer Genome Atlas (TCGA) database, and the prognostic significance of ADAMTS8 was first elaborated. Functional experiments verified that ADAMTS8 functioned as a tumor suppressor gene (TGS) in breast cancer. The potential mechanism and signaling pathways of ADAMTS8 in breast cancer were also characterized.

Analyses using online databases
Prognostic data were obtained using the Kaplan-Meier Plotter plotter. The correlation study was performed using Pearson correlation analyze. The data of ADAMTS8 and DNA methylation were downloaded from this site (http://methhe.mbc. nctu.edu.tw/php/index.php). A 77 paired patient samples were analyzed for different expression in ADAMTS8 mRNA, and 99 paired patient samples were analyzed for different methylation expression in the ADAMTS8 promoter. The TCGA database (https://tcga-data.nci.nih.gov/ tcga/) was used to download Breast Cancer tissue gene expression data. A total of 1066 Breast Cancer patients with complete clinicopathological data were screened and sorted in terms of the median level of ADAMTS8 expression from high to low, with the first 50% as the high expression group and the last 50% as the low expression group.

Establishment of stable cell lines
Breast cancer cell lines (MDA-MB-231 and BT549) were obtained from the American Type Culture Collection. Both cell lines were cultured in the complete medium as described previously (11). The cells were transfected with pCDNA3.1 and ADAMTS8 plasmids using Lipofectamine 2000 (Invitrogen) for 14 days to construct stably expressing cell lines. Cells with empty vector pCDNA3.1 were used as a control. RNA and total proteins were prepared from transfected cells. Overexpression of ADAMTS8 was validated by q-PCR and western blotting. ADAMTS8 siRNA (RiboBio, Guangzhou, China) was transfected into MDA-MB-231 and BT549 cells by Lipofectamine 2000 (Invitrogen). Cells were cultured for 48 h. The siADAMTS8 expression was validated by western blotting.

RNA extraction and expression
RNA was extracted from cell lines with TRIzol reagent (Life Technologies, Carlsbad, CA) and treated with DNase I. The RNA was stored at À80 C, and its concentration was measured by NanoDrop2000 spectrophotometry. RT-PCR was performed on aliquots with 1 lg total RNA to generate 20 ll cDNA by using the Reverse Transcription system (Promega, Madison, WI). Semi-quantitative PCR was performed using SYBR Green PCR Master Mix (Thermo Fisher Scientific, Hong Kong, China) an ABI 7500 Real-Time PCR system (Applied Bio systems, Foster City, CA). Gene expression level was calculated by the 2 ÀDDCt method (9). GAPDH was acted as gene expression control. The primers were designed by premier 5 software. The related primer sequences were as follows:

Cell proliferation assay
Cell viability was measured using the CCK8 assay following a previous report (15). Breast cancer cell lines (MDA-MB-231 cells and BT549 cells) were seeded in 96-well plates (at a density of 2000 cells/well) using 100 mL complete medium per well. Cell viability was assessed by measuring OD values after 24, 48, and 72 h. Absorbance was measured at 490 nm using a microplate reader.

Colony formation assay
Cell proliferation was determined by a colony formation assay following a previous report (18). MDA-MB-231 cells were seeded in 6-well plates at a density of 1600 cells per well for 10 days. The BT549 cells were seeded in 6-well plates at a density of 800 cells per well for 2 weeks. Surviving clones (more than 50 cells per clone) were fixed with paraformaldehyde and stained with Gentian Violet.

Flow cytometry analysis
MDA-MB-231 and BT549 cells were cultured in 6-well plates and grown overnight. Cultures were then transfected with pCDNA3.1 and ADAMTS8 plasmids using Lipofectamine 2000, cells were transfected with pCDNA3.1 as a control. Cultured for 48 h, digested with trypsin, and fixed in ice-cold 70% ethanol at 4 C overnight. The cells were stained with 2.5 mL RNase 20 mg/mL, DNase free (Sigma-Aldrich, St, Louis, MO) and 50 mg/L propidium iodide for 30 min separately. Cell sorting was performed and data were analyzed by ModFit 3.0 software (Verity Software House, Topsham, ME).

Cell migration and invasion assays
Transwell chambers were used to detect cell migration and invasion ability (18,19). The cells were collected (2 Â 10 4 MDA-MB-231 cells and 4 Â 10 4 BT549 cells), washed in serum-free medium once and seeded onto the upper chamber. The upper chambers included 700 lL medium with 20% fetal bovine serum (FBS). The following steps were described previously (20).

Wound healing assay
Stably expressing cells were respectively cultured in different 6-well plates, and grown until confluent. Each scratch was made on the cell monolayer using 10 uL pipette tip. The cells were cultured in serum-free medium. The cells were taken respectively for 0, 24, and 48 h and were observed by Microscopic images (100Â magnification, Nikon, Japan). The results were assessed by the zero linear wound line. The data were calculated by ImaginJ software (National Institutes of Health, Bethesda, MD).

Western blot analysis
Total proteins were extracted and analyzed by western blotting as described previously (21,22). Gene set enrichment analysis (GSEA) and protein-protein interaction (PPI) network construction A total of 1066 Breast Cancer patients were screened from TCGA database and explored the enrichment of populations with high and low expression differences of ADAMTS8 in different pathways. GSEA was performed to analyze the associated between ADAMTS8 expression and biological processes (21). The value of the normalized enrichment score(NES)>1 and p < 0.05 were considered to be statistically significant difference. PPI network was predicted by STRING database (http://string-db.org) (Last accessed 17/10/2020).

Statistical analysis
All data are representative of three independent experiments. Statistical analyses were performed using GraphPad prism7.0 software (GraphPad, San Diego, CA) and ImageJ software. The experimental results were assessed by the X 2 test, Student's t-test and Fisher's exact test. p-Values of all results less than 0.05 were assumed to be statistically significant.

Results
Expression of ADAMTS8 in breast cancer tissue and clinicopathological features from TCGA database In this study, 77 paired patients' samples were analyzed for the different expressions of ADAMTS8 mRNA, and 99 paired patient samples were analyzed for different methylation in the ADAMTS8 promoter. ADAMTS8 expression was lower in breast tumor tissues than in the corresponding normal tissues (Figure 1(A)). ADAMTS8 DNA promoter methylation levels were higher in breast tumor tissues than in the corresponding normal tissues (Figure 1(B)). Moreover, TCGA database was used to examine the expression of ADAMTS8 mRNA in 1066 patients. ADAMTS8 expression was significantly correlated with age (p < 0.001), TNM stage (p < 0.001), tumor grade (p < 0.001) and distant metastasis (p ¼ 0.039) ( Table 1). IHC was performed in 29 pairs of breast tumor tissues and adjacent-normal tissues. The IHC staining intensity score was negative at the range of 100Â magnification in breast tumor tissues (Figure 1(C)). The IHC staining intensity score was negative at the range of 200Â magnification in breast tumor tissues (Figure 1(D)). The IHC staining intensity score was moderately positive at the range of 100Â magnification in the adjacent normal tissues (Figure 1(E)). The IHC staining intensity score was moderately positive at the range of 200Â magnification in the adjacent normal tissues (Figure 1(F)). Together, the results indicated that the ADAMTS8 staining score was significantly lower in breast tumor tissues than adjacent normal tissues.
High expression of ADAMTS8 predicts a better prognosis for breast cancer patients High ADAMTS8 expression was associated with long relapse-free survival (RFS) with low ADAMTS8 expression (p < 0.001) (Figure 2(A)). High ADAMTS8 expression was associated with long overall survival (OS) (p < 0.001) than patients with low ADAMTS8 expression ( Figure  2(B)). Multivariate analysis identified ADAMTS8 as an independent prognostic factor for OS (hazard ratio (HR) ¼ 0.647, 95% confidence interval (CI)[0.450-0.929], p ¼ 0.018) in breast cancer patients ( Table 2). The results indicated that ADAMTS8 could play an important anti-tumor role, and high ADAMTS8 expression was significantly correlated with good prognosis for breast cancer patients.  control group. The expression of ADAMTS8 was assessed by western blotting and q-PCR (Figure 3(A)). The cell proliferation ability was measured by the CCK8 assay. Overexpression of ADAMTS8 inhibited proliferation of MDA-MB-231 and BT549 cells (p < 0.01, Figure  3(B)). Clonogenic assays showed that colony formation ability was significantly lower in ADAMTS8-transfected cells than in vectortransfected cells (reduced to 30% of control for MDA-MB-231, p < 0.01; and 67% of control for BT549, p < 0.01, Figure 3(C)). These data suggested that ADAMTS8 inhibited the viability and growth of breast cancer cells.    vector-transfected cells and 20.30% in ADAMTS8-transfected cells, p < 0.01; BT549 cells: 13.71% in vector-transfected and 19.09% in ADAMTS8-transfected BT549 cells, p < 0.01, Figure 4(A)). The results demonstrate that

ADAMTS8 inhibits cell migration and invasion in breast cancer cells
To assess metastasis-related abilities, cell migration and invasion assays were performed. Migration and invasion abilities were lower in ADAMTS8-transfected cells than in vector-transfected lines (p < 0.01; Figure 4(B)). The results demonstrated that ADAMTS8 inhibited the migration and invasion of breast cancer cells.

Discussion
ADAMTS8, located in chromosome 11q25, is an important member of the angio-inhibitory clade of the ADAMTS family (8). ADAMTS8 regulates tumor suppressor genes (TSGs) by epigenetic silencing, which is associated with growth, metastasis and poor prognosis in several malignant tumors (9). ADAMTS9 and ADAMTS18, as the same member of the ADAMTS family as ADAMTS8, were downregulated in breast cancer tissues compared with non-tumor tissues (11,22). In this study, we also found that ADAMTS8 downregulated in breast cancer tissues compared with non-tumor tissues. We also found that the methylation level of ADAMTS8 was significantly higher in breast cancer tissue than in non-tumor tissues. These data suggested that decreased ADAMTS8 mRNA expression was correlated with higher levels of ADAMTS8 methylation. TCGA database analysis indicated that ADAMTS8 expression was significantly associated with age, TNM, and distant metastasis in breast cancer patients (Table 1). These results indicated that ADAMTS8 may play a key role in the clinical diagnosis of breast cancer, and it could be a potential molecular target for anticancer therapy. ADAMTS8 overexpression predicted a better prognosis regarding PFS and OS, suggesting that ADAMTS8 may be considered a prognostic predictor in breast cancer. Multivariate analysis confirmed that ADAMTS8 was an independent prognostic factor for OS in breast cancer patients (Table 2).
However, the ADAMTS8 underlying biological mechanism remains unclear. Previous studies showed that ADAMTS8 inhibited cell viability, colony formation, cell cycle, invasion and migration in hepatocellular carcinomas, nasopharyngeal carcinomas, and colorectal cancer, as well as inducing apoptosis (9,15,20). Consistent with these studies, the present results showed that overexpression of ADAMTS8 inhibited proliferation, cell cycle progression, migration and invasion in breast cancer cell lines, with no significant effect on cell apoptosis. Specifically, overexpression of ADAMTS8 blocked cell division at the G2/M phase and suppressed migration and invasion in vitro. Moreover, ADAMTS8 knockdown displayed the reverse functions in proliferation and migration. The data suggested that ADAMTS8 functioned as a potential TSG in breast cancer.
The GSEA result validated significant negative relevance between ADAMTS8 expression and the cell cycle. We further found that ADAMTS8 was considered as a TSG through its involvement in cell cycle regulation, and possibly correlated with EGFR/Akt signaling pathway. A number of studies have shown that breast cancer progression and metastasis are closely related to abnormal protein activation, such as EGFR and EMT-associated proteins (23)(24)(25). EGFR has been reported in more than 50% of breast cancer with the activation of EGFR regulating downstream signal transduction (26). Moreover, ADAMTS8 displayed antitumor properties through antiagonizing EGFR-MEK-ERK signaling (9). EGFR, an upstream regulator of the Akt signaling pathway (26,27), is affected by ADAMTS1 (28). ADAMTS8 is structurally similar to ADAMTS1, Akt pathway is influenced by ADAMTS8 (28,29). Akt signaling pathway alterations are common in human malignancies and the pathway is hyperactive in breast cancer, promoting tumor progression by activating downstream molecules including p-Akt (28), p21, p27, MDM2, and NF-kB (25). Moreover, the Akt pathway promotes cell proliferation by phosphorylating CDK inhibitors, such as p21 and p27 (30). In our study, ectopic expression of ADAMTS8 decreased Akt signaling and affected the expression of its downstream genes. ADAMTS8 increases the activity of the CDK inhibitor p27 and suppresses the activity of the cycle-related proteins, cyclin B1 and Cdc2 p34, which regulate the G2/M phase (31,32). EGFR, a critical regulator of signaling pathway, is inhibited by Versican G3, a product of ADAMTS degradation. Versican G3 contains an EGF-like domain that mediates interaction with EGFR and ADAMTS8 (33,34). Versican G3 activates the EGFR pathway to enhance growth and metastasis in breast cancers (35). ADAMTS8 play a important role in tumorigenesis, invasion and metastasis, including EMT, regulation of growth factor activities integrin functions, and angiogenesis (36,37). In our study, ADAMTS8 overexpression was associated with decreased EGFR expression, which affected the expression of Akt and p-Akt and downstream cell-cycleassociated proteins (p27, cyclin B1, and Cdc2 p34), thereby affecting the expression of downstream EMT-associated proteins (SNAI1, SLUG, MMP1, and MMP1). These results indicated that ectopic ADAMTS8 induced G2/M phase arrest by regulating the EGFR/Akt signaling pathway. GSEA analysis confirmed that ectopic ADAMTS8 was involved in the negative regulation of the cell cycle. Moreover, ectopic ADAMTS8 inhibited EMT related-proteins, providing insight into the mechanism underlying the function of ADAMTS8.
PPI network analysis identified the downstream associated proteins with ADAMTS8, including ACAN, ADAMTS3, ADAMTS19, ADAMTS10, THBS1, POFUT2, ADAMTS17, ADAMTS14, ADAMTSL2, and ADAMTS13. ADAMTS8 was closely associated with the regulation of ADAMTS family members. ADAMTS8 is involved in the regulation of the expression of multiple members of the ADAMTS family. ADAMTS family member play an important role in tumor angiogenesis and affect angiogenesis through a variety of different pathways, Further studies could continue to explore the function and mechanism of ADAMTS8 from the aspect of tumor angiogenesis. Moreover, ACAN is an important component of the extracellular matrix of cartilage tissue and plays an important role in the development of cartilage, which has been reported in Hodgkin's lymphoma (38) and adrenocortical adenoma (39). THBS1 is a secreted protein expressed in embryos and healthy adult, which is involved in the regulation of cell-matrix interactions and has antiangiogenic functions. POFUT2 is a protein-coding gene, associated with the regulation of EMT, and it is necessary for the secretion of ADAMTS family members, such as ADAMSL1, ADAMTS9, and ADAMTS13 (40). PPI network analysis of functional interactions of ADAMTS8 could provide important information to understand the mechanisms underlying the occurrence and development of breast cancer.
However, some limitations were unavoidable due to the lack of experimental resources. First, the study of the mechanism was incomplete, so further studies are needed to elucidate the detailed mechanisms of how ADAMTS8 modulates associated-signaling pathways. Second, the experimental results in vivo still needed to be confirmed.

Conclusion
This study demonstrated that ADAMTS8 was downregulated in breast cancer and could be considered as a new TSG. It inhibited cell proliferation, migration and invasion, and blocked cycle division at the G2/M phase. The underlying mechanism could involve the EGFR/Akt signaling pathway. ADAMTS8 could function as a prognostic biomarker, and the present data supported its potential for prognostic evaluation in breast cancer.

Ethical approval
Informed consent was obtained from each participant included in the study. This research was approved by the Institutional Ethics Committees of the First Affiliated Hospital of Chongqing Medical University (Approval notice: #2017-61) and was performed in accordance with the tenets of the Declaration of Helsinki.