Higher levels of TIMP-1 expression are associated with a poor prognosis in triple-negative breast cancer

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional protein that can directly regulate apoptosis and metastasis. In this study, we investigated the functional and molecular mechanisms by which TIMP-1 influences triple-negative breast cancer (TNBC). The expression level of TIMP-1 in breast cancer tissues was analyzed using the ONCOMINE microarray database. The overall survival of patients with distinct molecular subtypes of breast cancer stratified by TIMP-1 expression levels was evaluated using Kaplan–Meier analysis. Bisulfate sequencing PCR (BSP) was used to analyze the methylation status of the TIMP-1 promoter. Real-time-PCR (RT-PCR), Western blot and ELISA assays were used to evaluate gene and protein expression in cell lines and human tissue specimens. In addition, TIMP-1 function was analyzed using a series of in vitro and in vivo assays with cells in which TIMP-1 was inhibited using RNAi or neutralizing antibodies. We found that serum TIMP-1 levels were strongly enhanced in patients with TNBC and that elevated TIMP-1 levels were associated with a poor prognosis in TNBC. However, TIMP-1 levels were not significantly associated with overall survival in other subtypes of breast cancer or in the overall population of breast cancer patients. We also report the first evidence that the TIMP-1 promoter is hypomethylated in TNBC cell lines compared with non-TNBC cell lines, suggesting that aberrant TIMP-1 expression in TNBC results from reduced DNA methylation. RNAi-mediated silencing of TIMP-1 in TNBC cells induced cell cycle arrest at the G1 phase and reduced cyclin D1 expression. In addition, mechanistic analyses revealed that the p-Akt and p-NF-κB signaling pathways, but not the GSK-3β and MAPK1/2 pathways, are associated with TIMP-1 overexpression in TNBC cells. Moreover, neutralizing antibodies against TIMP-1 significantly decreased the rate of tumor growth in vivo. Our findings suggest that TIMP-1 is a biomarker indicative of a poor prognosis in TNBC patients and that targeting TIMP-1 may provide an attractive therapeutic intervention specifically for triple-negative breast cancer patients.


Background
Human breast cancer is a heterogeneous disease, and predicting treatment response and clinical outcomes is typically based on specific clinical and pathological features [1]. Breast cancer is molecularly classified into the luminal-A, luminal-B, HER2-overexpressing (HER2+) or triple-negative subtypes. Triple-negative breast cancer (TNBC) refers to a subtype of breast carcinoma characterized by the lack of expression of the 3 receptors most commonly targeted by standard breast cancer therapy: estrogen receptor alpha (ERα), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) [2]. In practice, TNBC is often used as a surrogate name for basal-like breast cancer [3]. There is currently no consensus on the optimal immunohistochemistry (IHC) panel to use to characterize basal-like tumors [4]. Although systematic therapeutic approaches have reduced cancerspecific mortality, TNBC is associated with relatively poor clinical outcomes compared with other subtypes of breast cancer [5,6]. In recent years, there has been a focus on further characterizing the various molecular markers and biomarkers associated with TNBC, including EGFR, VEGFR, c-Myc, C-kit, Poly (ADP-ribose) polymerase-1, HSP90, TOP-2A and spleen tyrosine kinase (SYK) [7,8]. These biomarkers might be valuable prognostic indicators and might represent potential therapeutic targets of TNBC treatment. Identifying novel biomarkers of TNBC might further contribute to the development of effective TNBC treatment approaches.
Tissue inhibitor of metalloproteinases-1 (TIMP-1), a member of the TIMP family of proteins comprising TIMP-1, 2, 3 and 4, was identified 2 decades ago and was initially characterized as an endogenous inhibitor of matrix metalloproteinases (MMPs) [9][10][11][12]. TIMP-1 has long been recognized for its role in extracellular matrix remodeling [13]. Emerging evidence indicates that TIMP-1 is frequently overexpressed in several types of human cancers, including prostate cancer [14], lung cancer [15], melanoma [16], glioblastoma [17] and breast cancer [18,19]. As a cytokine and a key regulator of ECM degradation, TIMP-1 has multiple functions associated with the tumor microenvironment and cancer progression [20]. In addition to its inhibitory activity against MMPs, TIMP-1 promotes cell proliferation in various cell types [21], including breast cancer cells [22,23], and it might also be associated with anti-apoptotic activity in breast cancer [24][25][26]. Although the anti-apoptotic activity of TIMP-1 in other cancers has been well demonstrated, some studies evaluating the role of TIMP-1 in breast cancer cell growth have reported conflicting results [23,27]. For example, in MDA-435 breast cancer cells, TIMP-1 was reported to promote cell growth by inhibiting MMPs [23]. In contrast, TIMP-1 was reported to inhibit cell growth in MCF-10A normal breast epithelial cells by decreasing cyclin D1 levels [27]. In TIMP-1-deficient mice, mammary epithelial cell proliferation is upregulated [28]. Thus, although several distinct signaling pathways and putative receptors have been implicated in TIMP-1 function [29][30][31][32], the mechanisms underlying the role of TIMP-1 in distinct subtypes of breast cancer remain unclear.
To gain new insights into the role of TIMP-1 during breast cancer progression, we examined TIMP-1 expression levels in serum derived from breast cancer patients and evaluated the prognostic value of TIMP-1 using a large publically available clinical microarray database of breast cancer specimens. Interestingly, we observed higher levels of TIMP-1 expression in patients with TNBC compared with control individuals, and this phenomenon was associated with a poor prognosis in TNBC patients. However, TIMP-1 expression levels were not associated with survival in other subtypes of breast cancer or in the overall population of breast cancer patients evaluated. Mechanistic analyses indicated that shRNAmediated knockdown of TIMP-1 in TNBC cells induced cell cycle arrest at the G1 phase and decreased cyclin D1 levels. Moreover, inhibiting TIMP-1 function prevented tumor growth in mice, suggesting that TIMP-1 inhibition might be a promising therapeutic strategy for treating TNBC.

ELISA
TIMP-1 levels in preoperative patient serum samples and cell-conditioned medium were detected using Quantikine Human TIMP-1 ELISA Kits (Cat. #DTM100, R&D Systems). ELISA assays were conducted according to the manufacturer's instructions. All the samples were analyzed in 3 wells in each experiment, and each experiment was repeated 3 times. The serum samples were collected from 81 patients prior to surgery. The use of the patient specimens was approved by the Institutional Ethics Committee of Shanghai Ninth People's Hospital affiliated with Shanghai JiaoTong University School of Medicine, and written consent was obtained from all participants.

Overall survival (OS) analysis
The Sorlie classification method used in the data set was used to assign patients to the different groups according to clinical breast cancer subtype. OS stratified by expression levels of the gene of interest was evaluated using Kaplan-Meier analysis, and comparisons between groups were evaluated using log-rank tests. The statistical analysis was performed according to the manufacturer's instructions [33] (http://kmplot.com/analysis/).

Cell cycle analysis
Cells cultured in 6-well plates were harvested, washed once in PBS and fixed in 70 % ethanol for 48 h at 4°C. The nuclei were stained with 50 μg/ml propidium iodide (PI) in 1 % Triton-X100/PBS containing 100 μg/ml DNase-free RNase, and the DNA content was analyzed using flow cytometry with the FACSCalibur platform (Becton Dickinson, San Jose, USA). The proportion of cells in each phase of the cell cycle was determined using the ModFit LT program (Verity Software House, USA).

Colony formation assay
For the colony formation assays, 1 × 10 3 cells were plated into 6-well plates and cultured for 10 days. At the end of the culture period, the cells were fixed with methanol for 30 min and stained with crystal violet for 30 min. The plates were washed several times with water, and the images of the optical density of the cells were captured using a digital camera.

Invasion assay
Cell invasion was examined using a reconstituted extracellular matrix membrane (Cat. #354480, BD Biosciences, San Jose, CA). Cells suspended in serum-free media at a concentration of 3 × 10 4 cells/0.5 ml were placed in the upper chambers, and complete media containing 10 % fetal bovine serum (FBS) and 1 % antibiotics (Invitrogen Corp., Carlsbad, CA) was added to the lower chambers. The chambers were incubated for 18-24 h at 37°C and 5 % CO 2 . After the incubation, the medium was completely removed from the upper and lower chambers, and the purple residue indicative of noninvasive cells was gently removed from the upper chamber using a cottontipped swab. Next, the chambers were fixed with methanol for 30 min and stained with crystal violet for an additional 30 min. The cells were counted in images of the membrane that had been captured using a microscope (Zeiss) with a 10x objective lens.

Xenograft models
The mouse xenograft tumor assays were performed in the animal center of Shanghai Jiao Tong University School of Medicine after obtaining approval from the Shanghai Medical Experimental Animal Care Commission. Twenty 6-week old female mice were obtained from the Shanghai Medical Experimental Animal Care Commission. All the animal experiments were performed in a designated animal center. The mice were subcutaneously injected (2 injection sites per mouse) with 1 × 10 6 MDA-MB-468 cells and divided into 2 groups (N = 10 for each group). Five days later, the mice were injected with a neutralizing antibody against TIMP-1 (10 μg per 25 g of body weight) (Cat. #AF970, R&D Systems) or the IgG control, and the injections were repeated once a week for 4 weeks. Tumor volumes were measured regularly using the formula V = 0.5 × L × W 2 , where L was the longest diameter, and W was the shortest diameter, before the animals were sacrificed, and the tumors were isolated.

TIMP-1 expression was significantly elevated in breast cancer
To characterize the role of TIMP-1 in breast cancer, we analyzed TIMP-1 mRNA expression in breast cancer specimens from the publicly available cancer microarray database ONCOMINE (https://www.onc omine.org/). We found that TIMP-1 expression was significantly increased in invasive breast carcinoma (Fig. 1a) and ductal breast carcinoma (Fig. 1b) compared with normal breast tissues. We also evaluated the levels of TIMP-1 mRNA and protein in breast cancer cell lines and found that TIMP-1 expression was significantly elevated in the TNBC cell lines and HER2+ breast cancer cell lines (SK-BR-3) and the normal epithelial cell line (MCF-10A) at the mRNA and protein levels ( Fig. 1c and d).
TIMP-1 was initially identified in human serum derived from skin fibroblasts in 1975 [35]. Based on this finding, we assessed the association between serum levels of TIMP-1 breast cancer clinical parameters, including age (< or ≥50 years), T status, lymph node metastasis, and the ER, PR and HER2 status (Table 1). Serum TIMP-1 were significantly elevated in malignant tissues compared with benign tissues (p < 0.001) and in ER-negative breast cancer patients compared with ERpositive patients (p = 0.002). Stratifying TIMP-1 levels according to molecular subtype revealed that serum levels of TIMP-1 were significantly higher in patients with the luminal-A (p < 0.001), HER2+ (p < 0.001) and TNBC (p < 0.001) subtypes compared with patients with benign disease. However, there were no significant differences in TIMP-1 levels among the 3 malignant subtypes (p > 0.05, data not shown).
These results suggested that elevated TIMP-1 expression might play an important role in breast cancer development.

TIMP-1 predicts poor clinical outcomes in patients with TNBC
To further explore the relationship between TIMP-1 and clinical prognosis in patients with breast cancer, we evaluated the prognostic value of TIMP-1 in a large publically available clinical breast cancer microarray database [33] that includes data from 1027 patients (459 luminal-A, 308 luminal-B, 75 HER2+ and 185 TNBC). We found that higher levels of TIMP-1 expression were associated with poor overall survival (OS) in TNBC patients (p = 0.032, Fig. 2e) but not in the overall breast cancer population or in the other subtypes evaluated (p > 0.05, Fig. 2a-d).
Upregulation of TIMP-1 in TNBC is associated with promoter hypomethylation DNA methylation is a key epigenetic modification in the mammalian genome that regulates gene expression. To determine if DNA methylation is associated with the transcriptional silencing of TIMP-1 in different subtypes of breast cancer, we analyzed the promoter sequence of TIMP-1. We identified 1 CpG island located between bp −157 and −32 (Fig. 3a) and analyzed its methylation status in breast cancer cell lines and in clinical samples from breast cancer patients. As shown in Fig. 3b Fig. 3c, and the data are summarized in Fig. 3d. These data indicate that methylation of the TIMP-1 promoter is significantly greater in TNBC (p < 0.05). In addition, RT-PCR analysis of MDA-231 and BT474 cells treated with various concentrations of 5-Aza-2′-deoxycytidine (5-Aza) for 48 h demonstrated that TIMP-1 mRNA levels increased in BT474 cells but not in MDA-231 cells (Fig. 3e). Together, these findings indicate that high levels of TIMP-1 expression in TNBC might be associated with TIMP-1 promoter hypomethylation.

TIMP-1 silencing induces cell cycle arrest in the G1 phase in TNBC cells
Based on the observation that TIMP-1 is highly expressed in TNBC cells, we evaluated the role of TIMP-1 in TNBC cells by transfecting MDA-MB-468 and MDA-MB-231 cells with a vector expressing a short hairpin RNA (shRNA) targeting TIMP-1. Three shRNAs, referred to as shTIMP1-1#, −2#, and −3#, were used in these experiments. The shRNA knockdown efficiency of TIMP-1 expression was confirmed using real-time PCR and ELISA assays in both cell lines.
To determine the role of TIMP-1 in TNBC cell proliferation, we examined cell cycle distribution using flow cytometry. TIMP-1 knockdown increased the proportion of cells in the G1 phase (81.61 % of shTIMP1-1# cells and 74.92 % of shTIMP1-3# cells vs. 59.66 % of control cells, Fig. 4c), and decreased the proportion of cells in the G2 and M phases compared with the control (Fig. 4c). Similar results were observed in MDA-231 cells (Fig. 4d). In addition, TIMP-1 knockdown significantly reduced colony formation in MDA-MB-468 and MDA-MB-231 cells compared with the control (Fig. 4e and f). CCK-8 assays showed that cell growth was restrained in TIMP-1 knockdown MDA-468 cells (Fig. 4g). However, no differences in cell invasion were observed between TIMP-1 knockdown TNBC cells and control TNBC cells (Fig. 4h).
Together, these results demonstrate that the loss of TIMP-1 expression can induce cell cycle arrest in the G1 phase and reduce colony formation in TNBC cells.

The Akt signaling pathway is associated with TIMP-1regulated cyclin D1 expression in TNBC cells
To further investigate the molecular mechanism by which TIMP-1 regulates TNBC cell cycle distribution, we examined the levels of cyclin proteins in TIMP-1 knockdown and control MDA-MB-468 cells using Western blot. Cyclin D1, a protein encoded by the CCND1 gene, is required for cell cycle progression from the G1 to the M phase [36]. As shown in Fig. 5a-c, cyclin D1 levels decreased in TIMP-1 knockdown cells. In contrast, TIMP-1 overexpression enhanced cyclin D1 expression in MCF-10A cells (Fig. 5d). The results indicated that TIMP-1 induced cell cycle arrest by upregulating cyclin D1 expression at the mRNA and protein levels.

Blocking TIMP-1 activity with neutralizing antibodies inhibits tumor growth
To determine if TIMP-1 is involved in tumor growth in vivo, we used a neutralizing antibody to block TIMP-1 activity in TNBC cells. We used this approach rather than engineering TIMP-1 knockdown cells as TIMP-1 is a secreted protein. Ultimately, 13 tumors derived from the cancer cell injections were identified in each group and used for further analysis. A significantly lower rate of tumor growth was observed in mice injected with neutralizing antibodies against TIMP-1 compared with mice injected with the control IgG. The 26 tumors 5 weeks after the tumor cell injections are shown in Fig. 6a. We observed a strong reduction in tumor volume and total tumor burden in mice injected with the neutralizing antibody compared with control mice (Fig. 6b and c). Together, these data suggest that blocking TIMP-1 activity might be an effective approach for treating triple-negative breast cancer.

Discussion
TIMP-1 is a small secretory glycoprotein with multiple functions, including anti-apoptotic activity and inhibiting matrix metalloproteinases [13,26]. Numerous studies have demonstrated that TIMP-1 levels are elevated in several types of human cancer, including breast cancer [19]. Breast cancer is a heterogeneous disease composed of distinct molecular subtypes with different phenotypes. Triple-negative breast cancer, which is defined by the absence of ER, PR and HER-2 expression, represents 15 % of breast cancer cases [37]. Among the different subtypes of breast cancers, TNBC is associated with the poorest clinical prognosis, and no effective targeted therapies are currently available [38]. Actually, little is known about the function and molecular mechanism of TIMP-1 in TNBC [39].
In this study, we found that TIMP-1 expression was elevated in TNBC cell lines and TNBC patients compared with non-TNBC cells and non-TNBC breast cancer patients and that increased TIMP-1 expression was associated with a poor prognosis in TNBC patients. Our epigenetic analysis provided the first evidence that elevated TIMP-1 expression in TNBC is associated with a reduction in TIMP-1 promoter methylation. These findings indicate that TIMP-1 expression might be linked to more aggressive subtypes of breast cancer and are consistent with previous studies reporting that TIMP-1 expression is associated with a poor prognosis in breast cancer [40], colorectal cancer [41], laryngeal squamous cell carcinoma [42] and hepatocellular carcinoma [43]. An increase in TIMP-1 mRNA levels induced by 5-Aza treatment has also been observed in melanoma [44] and gestational tissues [45], indicating that promoter methylation mediates the expression of TIMP-1 in various cell types. As a member of the TIMP family of proteins, TIMP-1 was initially characterized as an endogenous inhibitor of MMPs and A Disintegrin and metalloproteinase domaincontaining protein 10 (ADAM10) [46]. However, in recent years, several reports have focused on the cytokine-like functions of TIMP-1 in multiple biological processes [20,47]. In this study, TIMP-1 down-regulation significantly decreased cyclin D1 expression at both the mRNA and protein levels and disrupted Akt and NF-κB signaling, suggesting that Akt/NF-κB signaling might mediate the effects TIMP-1 exerts on cell cycle regulation in TNBC. Despite previous reports that GSK3β signaling pathway plays a critical role in cyclin D1 degradation [48] and that TIMP-1 activates human breast epithelial cells via the PI3K and MAPK signaling pathways [29], we found that the GSK-3β and MAPK1/2 pathways were unaffected in TIMP-1 knockdown TNBC cells or TNBC cells treated with exogenous TIMP-1. In a recent study, TIMP-1 was reported to phosphorylate Akt at Thr308 in human  [47]. Other studies have also reported that TIMP-1 can bind to CD63 or the pro-MMP9/CD44 complex, thereby activating survival pathways in some cells [32,49]. The physiologic receptor of TIMP-1 remains unclear; therefore, further investigation into TIMP-1 receptors and the intracellular processes mediated by TIMP-1 might provide novel insights into the molecular mechanisms of TIMP-1 in breast cancer cells or other types of cancer cells.
In this study; however, we did not observe defects in cell migration in TIMP-1 knockdown cells. A potential explanation for this finding is that knocking down a single factor is not sufficient to discernably disrupt cell migration in the highly aggressive TNBC cell lines we evaluated.
As the role of TIMP-1 in promoting proliferation in various cell types has been well established, efforts have been put forth to evaluate the effect of blocking TIMP-1 signaling in inflammation-associated diseases by targeting CD63 [31]. As TIMP-1 is secreted in the tumor microenvironment, we used a TIMP-1 neutralizing antibody to block TIMP-1 activity rather than use TIMP-1 knockdown cell lines. We found that the inhibition of TIMP-1 activity markedly suppressed tumor growth in mice, consistent with observations in mouse models of prostate cancer [50]. Targeting TIMP-1 or its receptor is widely used in the treatment of immune disease. In this study we investigated the potential use of TIMP-1 antibody in cancer therapy.

Conclusions
TIMP-1 was highly expressed in TNBC patients and was associated with a poor prognosis. The TIMP-1 promoter was hypomethylated in TNBC cells, resulting in an increase in proliferation and cyclin D1 levels via the p-Akt and p-NF-κB pathways. Treatment with a neutralizing antibody against TIMP-1 significantly decreased tumor growth in vivo. In summary, our results suggest that TIMP-1 might serve as a prognostic biomarker indicative of poor outcomes and be an effective therapeutic target of TNBC treatment. Disclosure of potential conflicts of interest.

IgG
Anti Tumor growth was significantly inhibited after 4 weeks in mice injected with a neutralizing antibody against TIMP-1 compared with the control mice (p = 0.0098). The data are presented as the mean ± SD of triplicate measurements. c Tumor weight was measured at the time the mice were sacrificed. Quantification of total tumor burden demonstrated that tumor growth was suppressed by TIMP-1 inhibition. The data are presented as the mean ± SD; n = 13; p = 0.0278