A Novel Synthetic Compound (E)-5-((4-oxo-4H-chromen-3-yl)methyleneamino)-1-phenyl-1H-pyrazole-4-carbonitrile Inhibits TNFα-Induced MMP9 Expression via EGR-1 Downregulation in MDA-MB-231 Human Breast Cancer Cells

Breast cancer is a common malignancy among women worldwide. Gelatinases such as matrix metallopeptidase 2 (MMP2) and MMP9 play crucial roles in cancer cell migration, invasion, and metastasis. To develop a novel platform compound, we synthesized a flavonoid derivative, (E)-5-((4-oxo-4H-chromen-3-yl)methyleneamino)-1-phenyl-1H-pyrazole-4-carbonitrile (named DK4023) and characterized its inhibitory effects on the motility and MMP2 and MMP9 expression of highly metastatic MDA-MB-231 breast cancer cells. We found that DK4023 inhibited tumor necrosis factor alpha (TNFα)-induced motility and F-actin formation of MDA-MB-231 cells. DK4023 also suppressed the TNFα-induced mRNA expression of MMP9 through the downregulation of the TNFα-extracellular signal-regulated kinase (ERK)/early growth response 1 (EGR-1) signaling axis. These results suggest that DK4023 could serve as a potential platform compound for the development of novel chemopreventive/chemotherapeutic agents against invasive breast cancer.


Introduction
Breast cancer is a common malignancy among women worldwide [1]. Metastasis, the spread of primary tumor cells to different organs through the blood or lymphatic vessels, is a typical hallmark of all malignant tumors and is responsible for high mortality among patients with cancer. Breast cancer commonly spreads to the regional lymph nodes, bone, liver, lungs, and brain [2]. Thus, the control over the process of metastasis at the early stages is imperative for the successful prevention and treatment of cancer, including breast cancer.
Tumor cell invasion involves their migration and penetration into neighboring tissues. Lymphovascular invasion is, in general, the first stage of carcinogenic events that initiate tumor metastasis [3]. The initial step of invasion is characterized by the breakdown of the basement membrane, a sheet-like structure of the extracellular matrix secreted by the epithelium [4]. Type IV collagen is a major component of the basement membrane, and gelatinases such as matrix metallopeptidase 9 (MMP9, also known as gelatinase B and 92 kDa type IV collagenase) and MMP2 (also known as gelatinase A and 72 kDa type IV collagenase) are responsible for the breakdown of type IV collagen [5]. MMP2 and MMP9 expression is known to be highly upregulated in almost every metastatic cancer cell type [5,6], and elevated MMP9 level is associated with the high potential of metastasis in several human carcinomas, including breast cancer [6][7][8]. MMP9 expression has been observed in invasive mammary carcinomas, but not in carcinomas in situ or hyperplastic mammary glands [9]. Experimental metastasis studies have also shown that the downregulation of MMP9 expression in cancer cells by ribozyme could reduce tumor foci in the lungs of mice [10], consistent with the results observed in Mmp2- [10] or Mmp9-deficient mice [11]. These observations suggest that a therapeutic strategy to manipulate MMP2 or MMP9 expression could be potentially advantageous for the development of anti-metastatic agents.

Effect of DK4023 on the TNFα-Induced Migration of MDA-MB-231 Cells
The tumor mass is composed of tissue-resident fibroblasts, peripherally recruited immune cells, and endothelial cells of surrounding blood vessels, as well as cancer cell populations. The local environment around the tumor mass includes various growth factors and cytokines, which are collectively referred to as a tumor microenvironment [21]. It has been well characterized that the inflammatory tumor microenvironment is closely associated with tumor development and progression [22,23]. TNFα is a major proinflammatory cytokine that is released from many cell types, including cancer cells, immune cells, and fibroblasts, in the tumor microenvironment [20]. It has been demonstrated that TNFα increases the expression of other cytokines and chemokines, including IL-1, IL-6, CCL2, CXCL8, and CXCL12 [24], induces epithelial-to-mesenchymal transition (EMT), through the activation of NF-κB and AP1, and facilitates the invasion and metastasis of breast cancer cells [21,25].
A crucial feature of invasive and metastatic breast cancer cells is the increase in their motility. To evaluate whether DK4023 could modulate the motility of metastatic MDA-MB-231 cells, we used an in vitro scratch-wound healing assay and measured the thickness of the scratched area. After scratching a confluent monolayer, cells were treated with TNFα (10 ng/mL) or TNFα (10 ng/mL) plus DK4023 (25 and 50 µM) (Figure 2a). At 12 h post-scratching, the scratched area decreased following TNFα treatment as compared that observed after vehicle treatment. In contrast, the TNFα-induced closure of the scratched area was significantly suppressed in the presence of DK4023 ( Figure 2b). As DK4023 did not exhibit cytotoxicity at concentrations around 50 µM (Figure 1), its inhibitory effect on the motility of MDA-MB-231 cells was not related to its cytotoxicity.
TNFα treatment as compared that observed after vehicle treatment. In contrast, the TNFα-induced   , which is known to build up higher-ordered structures such as stress fibers, lamellipodia, and 123 filopodia during cell movement [26]. As the dynamic rearrangement of the actin cytoskeleton plays 124 a crucial role in cell migration [27], we assessed whether DK4023 affects actin cytoskeletal 125 rearrangement. We used rhodamine-conjugated phalloidin to stain F-actin and found that TNFα

Effect of DK4023 on the Actin Reorganization of MDA-MB-231 Cells
As shown in Figure 3, DK4023 reduced the TNFα-induced branched structures of the leader cells at the edge (arrow). Monomeric globular actin (G-actin) is polymerized into filamentous actin (F-actin), which is known to build up higher-ordered structures such as stress fibers, lamellipodia, and filopodia during cell movement [26]. As the dynamic rearrangement of the actin cytoskeleton plays a crucial role in cell migration [27], we assessed whether DK4023 affects actin cytoskeletal rearrangement. We used rhodamine-conjugated phalloidin to stain F-actin and found that TNFα treatment stimulated cytoskeletal rearrangement, as evident from the formation of F-actin-rich protrusions that appeared like lamellipodia (arrows) at the cell periphery ( Figure 3). After the treatment of cells with DK4023, the TNFα-induced F-actin-rich protrusions were substantially reduced. These data suggest that DK4023 prevents dynamic F-actin polymerization, resulting in the inhibition of cell motility.

Effect of DK4023 on the Expression of MMP9
As gelatinases play a critical role in cell migration, invasion, and metastasis, we investigated whether DK4023 modulates gelatinase activation. Gelatinase activity was determined using gelatin-gel zymography. DK4023 reduced the density of TNFα-induced gelatinolytic white band at 92 kDa MMP9, but not that of the band at 72 kDa MMP2 (Figure 4a). To investigate whether DK4023-induced suppression of MMP9 activity was associated with MMP9 downregulation, we determined the effect of DK4023 on the expression of MMP9 mRNA using conventional reverse-transcription polymerase chain reaction (RT-PCR). We found that DK4023 dose-dependently inhibited TNFα-induced mRNA expression of MMP9 but not MMP2, while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels remained unchanged ( Figure 4b). As TNFα induced MMP9 mRNA expression and proteolytic activity of MMP9 more efficiently than MMP2, we focused on the inhibitory effect of DK4023 on MMP9 expression in substantial experiments. To further evaluate the inhibitory effect of DK4023 on MMP9 expression at the transcriptional level, we measured MMP9 promoter activity using the pMMP9(−925/+13)-Luc luciferase-based promoter-reporter plasmid [28]. TNFα stimulated MMP9 promoter activity by 3.47 ± 0.351-fold, but this effect was significantly suppressed by 2.13 ± 0.351-and 1.47 ± 0.208-fold in the presence of 25 and 50 µM DK4023, respectively ( Figure 4c). Thus, DK4023 inhibits MMP9 mRNA expression.      through a ubiquitin-mediated proteolytic pathway [33]. We observed that TNFα enhanced the

Effect of DK4023 on the TNFα-Induced Activation of Nuclear Factor-Kappa B (NF-κB) Pathway
NF-κB is a well-known transcription factor involved in the regulation of TNFα-induced MMP9 expression [29][30][31][32]. In unstimulated cells, RelA/p65 and p50, which are the most abundant forms of NF-κB heterodimers, are inhibited by IκB in the cytoplasm. Upon exposure of cells to TNFα, IκB kinase (IKK) gets activated and phosphorylates IκB, subsequently resulting in the degradation of IκB through a ubiquitin-mediated proteolytic pathway [33]. We observed that TNFα enhanced the phosphorylation of IKK and RelA/p65 in a time-dependent manner, while IκB phosphorylation level increased within 10 min, then rapidly decreased, and slowly recovered thereafter in MDA-MB-231 cells (Figure 6a). To determine whether DK4023 modulates the NF-κB pathway, we pretreated MDA-MB-231 cells with DK4023 and measured the TNFα-induced phosphorylation status of three key proteins, IKK, IκB, and RelA/p65. DK4023 failed to significantly decrease the TNFα-induced phosphorylation of IKKα/β or RelA/p65 but could significantly increase the phosphorylation of IκB (Figure 6b). These data suggest that DK4023 may affect the NF-κB pathway through the stabilization of IκB, which could be necessary but not sufficient to downregulate MMP9 expression.

Effect of DK4023 on the TNFα-Induced Expression of Early Growth Response-1 (EGR-1)
EGR-1 is a Cys 2 His 2 -type zinc-finger transcription factor induced by mitogenic stimulation and DNA damage signals [34]. EGR-1 is known to directly bind to the EGR-1-binding element within the MMP9 gene promoter and transactivate the MMP9 promoter activity upon TNFα stimulation in HeLa cervical cancer cells [28]. In MDA-MB-231 cells, TNFα increased the level of EGR-1 protein in a time-dependent manner (Figure 7a). However, DK4023 decreased the TNFα-induced expression of EGR-1 in a dose-dependent manner (Figure 7b). To evaluate the role of EGR-1 in DK4023-induced MMP9 suppression, we used the wild-type MMP9 promoter construct and a site-directed mutant construct obtained by disrupting the EGR-1-binding site (EBS) at MMP9 promoter-reporter. TNFα stimulated MMP9 promoter activation in the wild-type construct, but its effect was lost following the disruption of EBS (Figure 7c). DK4023 treatment inhibited the TNFα-induced MMP9 promoter activation in the wild-type construct. These data suggest that EGR-1 is critical for mediating the TNFα-induced activation of MMP9 promoter and that EGR-1 downregulation by DK4023 is associated with the suppression of TNFα-induced MMP9 promoter activation.   (c) The effect of the mutation of the EGR-1-binding element in MMP9 promoter-reporter construct, pMMP9-Luc(−925/+13), on the TNFα-induced activation of MMP9 promoter was determined by luciferase reporter activity. WT, wild-type; mtEgr1, mutation of the EGR-1-binding sequence. Values were expressed as mean ± SD. *** p < 0.001, NS not significant (p > 0.05) by Dunnett's (a,b) or Sidak's multiple comparison test (c).

Cells and Chemicals
MDA-MB-231 human breast cancer cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in Dulbecco's modified Eagle's medium (Corning Cellgro, Manassas, VA, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Corning Cellgro). TNFα was purchased from Sigma-Aldrich (Saint Louis, MO, USA), and Pierce BCA Protein Assay Reagent was supplied by Thermo Scientific (Rockford, IL, USA). The Firefly and Renilla Dual-Glo ™ Luciferase Assay System was procured from Promega (Madison, WI, USA), and nitrocellulose membrane, from Bio-Rad Laboratories (Hercules, CA, USA).

Chemical Synthesis
Commercially available phenylhydrazine (I, 1.10 g, 10 mmol) and 2-(ethoxymethylidene) propanedinitrile (II, 10 mmol, 1.22 g) were dissolved in 20 mL of an ethanol-water (1:3) solution, and the resulting mixture was stirred at 25 • C for 20 h to obtain an orange color solid. The precipitate was filtered and washed with cold ethanol. The resulting solid of III, 5-amino-1-phenyl-1H-pyrazole-4-carbonitrile, was dried and used for the next reaction without any further purification. In brief, 5 mL of toluene solvent was mixed with equimolar amounts of 5-amino-1-phenyl-1H-pyrazole-4-carbonitrile (III, 60 mg, 0.3 mmol) and 4-oxo-4H-chromene-3-carbaldehyde (IV, 51 mg, 0.3 mmol), and the mixture was treated with a catalytic amount of sodium hydroxide (NaOH). The reaction mixture was stirred at 75 • C for 5 h. After reaction completion, the mixture was cooled to room temperature and the solvent was evaporated under the vacuum. The residues were purified with medium-pressure chromatography to obtain an analytically pure compound of V (named DK4023). The structure of DK4023 was verified using nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) as described previously [36]. Its spectroscopic data are summarized as follows: IR (cm −1 ) 3112.55 (N-H)

Cell Migration Assay
Cell motility was examined using an in vitro scratch-wound healing assay, as previously described [19]. In brief, MDA-MB-231 cells were cultured in six-well plates and allowed to reach confluency. A scratch wound was made with a pipette tip on the confluent cell layer, which was then pretreated with either vehicle (DMSO) or 25 µM DK4023. After 30 min, cells were treated with either vehicle (PBS) or 10 ng/mL TNFα. Gap closure was imaged at 12 h post-scratching using an EVOS FL Auto Cell Imaging System (Life Technologies, Carlsbad, CA, USA) and the area of the closed gap was quantified using ImageJ version 1.52a (http://imagej.nih.gov/ij/; Center for Information Technology, National Institute of Health, Bethesda, MA, USA).

In-Gel Gelatinase Activity Assay
Gelatin zymography was carried out using the Novex Zymogram gel system (Novex, San Diego, CA), as previously described [39]. In brief, MDA-MB-231 cells (3 × 10 5 ) were cultured in a serum-free medium in 24-well plates for 24 h and then pretreated with either vehicle (DMSO) or DK4023 (25 or 50 µM). After 30 min, cells were treated with either vehicle (PBS) or TNFα (10 ng/mL). After 24 h, the culture medium was collected following the removal of cell debris and mixed with a non-reducing sample buffer (62.5 mM Tris-HCl [pH 6.8], 4% SDS, 25% glycerol, and 0.01% bromophenol blue). Equal amounts of each sample were electrophoresed using Novex pre-cast gels (10% acrylamide-0.1% gelatin). After electrophoresis, gels were equilibrated by incubation first in Novex Zymogram renaturation buffer for 30 min and then overnight in Zymogram developing buffer at 37 • C. Destaining was performed in methanol/acetic acid/distilled water (25:7:68, by volume) until gelatinolytic white bands appeared on the blue background. Relative gelatinolytic activities of MMP2 and MMP9 were determined by quantifying the white band intensities using ImageJ version 1.52a.

3-D Spheroid Culture and Invasion Assay
The 3-D invasion assay was carried out using the Cultrex 3-D Spheroid Cell Invasion Assay kit (Trevigen, Inc., Gaithersburg, MD, USA), as previously described [39]. Briefly, after forming spheroids of MDA-MB-231 cells, spheroids were embedded in matrigel-based extracellular matrix components and treated with or without 10 ng/mL TNFα in the presence or absence of 25 µM DK4023 for 7 days. Invasive protrusions into the surrounding extracellular matrix were visualized with an EVOS ® FL Auto Cell Imaging System (Life Technologies).

Immunoblot Analysis
Immunoblotting was performed as previously described [28]. In brief, MDA-MB-231 cells were lysed in a buffer comprising 20 mM HEPES (pH 7.2), 1% Triton X-100, 10% glycerol, 150 mM sodium hydroxide (NaCl), 10 µg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride and electrophoresed on 10% SDS polyacrylamide gel electrophoresis (PAGE) gels. The separated protein bands were transferred onto nitrocellulose membranes, which were incubated with appropriate primary and horseradish peroxidase-conjugated secondary antibodies. The blots were then developed using an Amersham ECL Western Blotting Detection Kit (GE Healthcare Life Science, Chicago, IL, USA).

Statistical Analysis
Statistical significance was analyzed using GraphPad PRISM software version 8.2.1 (GraphPad Software Inc., La Jolla, CA, USA). A value of p < 0.05 was considered significant.

Conclusions
The present study identified a novel synthetic isoflavone derivative, DK4023, as an anti-invasive agent against metastatic breast cancer cells. Natural isoflavones, such as genistein and daidzein, are known to inhibit TNFα-induced migration and invasion of human breast cancer cells by preventing the inhibition of NF-κB [17]. This study demonstrated that DK4023 inhibited TNFα-induced migration and lamellipodium formation of highly metastatic MDA-MB-231 cells. Our findings also show that DK4023 suppressed TNFα-induced expression of MMP9 mRNA through the downregulation of the ERK1/2-mediated expression of EGR-1, independently of NF-κB. More importantly, DK4023 substantially attenuated the invasive capability of MDA-MB-231 breast cancer cells. Although additional in vivo studies are warranted to validate the clinical efficacy of DK4023 in a metastasis animal model, we propose that DK4023 can serve as a promising agent to target the TNFα-ERK1/2 MAPK-EGR-1-MMP9 signaling pathway for the development of a chemopreventive or chemotherapeutic adjuvant, which can be used in combination with conventional chemotherapy, radiotherapy, or immunotherapy against metastatic cancers, particularly breast cancer.