Antiproliferative and Antimigration Activities of Fluoro-Neplanocin A via Inhibition of Histone H3 Methylation in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is among the most aggressive and potentially metastatic malignancies. Most affected patients have poor clinical outcomes due to the lack of specific molecular targets on tumor cells. The upregulated expression of disruptor of telomeric silencing 1-like (DOT1L), a histone methyltransferase specific for the histone H3 lysine 79 residue (H3K79), is strongly correlated with TNBC cell aggressiveness. Therefore, DOT1L is considered a potential molecular target in TNBC. Fluoro-neplanocin A (F-NepA), an inhibitor of S-adenosylhomocysteine hydrolase, exhibited potent antiproliferative activity against various types of cancer cells, including breast cancers. However, the molecular mechanism underlying the anticancer activity of F-NepA in TNBC cells remains to be elucidated. We determined that F-NepA exhibited a higher growth-inhibitory activity against TNBC cells relative to non-TNBC breast cancer and normal breast epithelial cells. Moreover, F-NepA effectively downregulated the level of H3K79me2 in MDA-MB-231 TNBC cells by inhibiting DOT1L activity. F-NepA also significantly inhibited TNBC cell migration and invasion. These activities of F-NepA might be associated with the upregulation of E-cadherin and downregulation of N-cadherin and Vimentin in TNBC cells. Taken together, these data highlight F-NepA as a strong potential candidate for the targeted treatment of high-DOT1L-expressing TNBC.


Introduction
Breast cancer (BC) is the most commonly occurring cancer and second leading cause of cancer-related death in women worldwide [1,2]. Chemotherapy, radiation therapy, and surgical options such as mastectomy or lumpectomy have been identified as the most effective and common strategies for the treatment of early-stage BC. However, approximately 40% of patients with BC face a risk of cancer recurrence within a few years of treatment [3][4][5]. Recurrent BCs are not only highly metastatic but also tend to acquire resistance to previously used conventional therapies, including hormone therapy, chemotherapy, or targeted drugs. Consequently, 90% of deaths attributed to BC are associated with recurrent and/or metastatic disease [6,7].
It is reported that approximately 20% of BCs do not express hormonal receptors (estrogen receptor and progesterone receptor) and the human epidermal growth factor receptor 2 (HER2). Therefore, these BCs are classified as triple-negative BCs (TNBCs) [8,9]. On this line, the TNBCs do not respond to hormonal or targeted therapy and are also considered the most intractable and aggressive form of cancer.

Cell Proliferation Assay (SRB assay)
Cell proliferation was measured using a sulforhodamine B (SRB) assay [39]. Briefly, cells were seeded in 96-well plates and incubated for 30 min (for day 0 controls) or treated with test compounds for the indicated times. After incubation, the cells were fixed, dried, and stained with 0.4% SRB in 1% acetic acid solution. Unbound dye was removed by washing with 1% acetic acid, after which the stained cells were dissolved in 10 mM Tris (pH 10.0). The absorbance of the cell solution was measured at 515 nm, and this value was used to determine cell proliferation. IC 50 values were calculated by a nonlinear regression analysis with TableCurve 2D v5.01 software (Systant Software Inc., Richmond, CA, USA). All reagents used in the SRB assay were purchased from Sigma-Aldrich.

DOT1L Enzyme Activity Assay
DOT1L enzyme activity was measured using S-adenosyl methionine (SAM) as the methyl group donor and synthesized DOT1L as the substrate (Cat. No. 52202; BPS Bioscience, San Diego, CA, USA) according to the manufacturer's instructions.

Cell Viability (MTT Assay)
Cell viability was measured using an MTT assay. MDA-MB-231 cells were seeded in 12-well plates and incubated overnight. Next, the cells were treated with the indicated concentrations of F-NepA (0.2-3.2 µM) in growth media for the indicated times. Subsequently, the cells were gently washed Biomolecules 2020, 10, 530 4 of 16 twice with growth medium and incubated with 0.5 mg/mL MTT (Sigma-Aldrich) at 37 • C for 4 h. The formazan crystals formed by active mitochondria were dissolved in DMSO, and the absorbance in each well was measured at 570 nm and used to determine cell viability. The IC 50 values were calculated by a nonlinear regression analysis using TableCurve 2D v5.01 software (Systant Software Inc.). All reagents used in the MTT assay were purchased from Sigma-Aldrich.

Wound Healing Assay
MDA-MB-231 and HCC1937 human TNBC cells were grown to 90% confluence in a six-well plate. Subsequently, each cell monolayer was artificially wounded using a Scratcher (SPL Life Sciences, Pocheon, Republic of Korea), and the detached cells were removed by washing with phosphate-buffered saline (PBS, Invitrogen Corp.). The wounded cultures were then incubated with medium containing 1% FBS and various concentrations of F-NepA for 24 h. The wounds were photographed at 0 and 24 h under an inverted microscope (Olympus, Tokyo, Japan). The wound areas were quantified using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA) and presented as the percent wound healing (%) relative to the wound area at 0 h [40].

Transwell Cell Invasion Assay
Twenty-four-well Transwell membrane inserts (diameter: 6.5 mm, pore size: 8 µm; Corning, Tewksbury, MA, USA) were each coated with 10 µl of type I collagen (0.5 mg/mL, BD Biosciences, San Diego, CA, USA) and 20 µl of a 1:20 mixture of Matrigel (BD Biosciences) in PBS. After treatment with the indicated compounds for 24 h, MDA-MB-231 and HCC1937 human TNBC cells were harvested, resuspended in serum-free medium, and plated (2 × 10 5 cells/chamber) in the upper chambers of the Matrigel-coated Transwell inserts. Medium containing 30% FBS was used as a chemoattractant in the lower chambers. After a 24 h incubation, the cells that had migrated to the outer surfaces of the lower chambers were fixed and stained using the Diff-Quik Staining Kit (Sysmex, Kobe, Japan) and imaged using a Vectra 3.0 Automated Quantitative Pathology Imaging System (Perkin Elmer, Waltham, MA, USA). Representative images from three separate experiments are shown, and the numbers of invaded cells were counted in five randomly selected microscopic fields (200× magnification) [41].

RNA Isolation and Real-Time Polymerase Chain Reaction (Real-Time PCR) Analysis
Total RNA was extracted from the cells using NucleoZOL reagent (Macherey-Nagel, Bethlehem, PA, USA). One microgram of total RNA per sample was reverse transcribed using a Reverse Transcription System (Cat. No. A3500; Promega, Madison, WI, USA) according to the manufacturer's instructions. Real-time PCR was conducted using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. The threshold cycle (C T ) was determined using Bio-Rad CFX manager 3.1 software. After normalizing the expression data to the level of the housekeeping gene ATCB (β-Actin mRNA), the comparative C T method was used to calculate the relative differences in mRNA expression between compound-treated cells and untreated controls [42]. The following primers were used for real-time PCR: CDH1, 5 -GTT ATT CCT CTC CCA TCA GCT G-3 and 5 -CTT GGC TGA GAG GAT GGT GTA A-3 ; CDH2, 5 -AGC CAA CCT TAA CTG AGG AGT-3 and 5 -GGC AAG TTG ATT GGA GGG ATG-3 ; VIM, 5 -AGA TGG CCC TTG ACA TTG AG-3 and 5 -TGG AAG AGG CAG AGA AAT CC; ATCB, 5 -AGC ACA ATG AAG ATC AAG AT-3 and 5 -TGT AAC GCA ACT AAG TCA TA-3 .

Statistical Analysis
The data are presented as mean values ± standard deviations (SDs) for the indicated numbers of independently performed experiments. All data are representative of the results of at least three independent experiments. Statistical significance was analyzed using Student's t-test or a one-way analysis of variance coupled with Dunnett's t-test. Differences were considered statistically significant at * p < 0.05, ** p < 0.01, *** p < 0.001.

Expression Levels of DOT1L and Antiproliferative Activities of NepA Analogs in Human BC Cells
We previously designed and synthesized analogs of NepA ( Figure 1A, 1) and reported the antiproliferative activities of these molecules against a panel of human cancer cell lines, including A549 (lung cancer cells), HCT-116 (colorectal cancer cells), SNU-638 (stomach cancer cells), MDA-MB-231 (BC cells), SK-HEP-1 (liver cancer cells), and PC-3 (prostate cancer cells). Of these analogs, F-NepA ( Figure 1A, 2) and N 6 -methyl-F-NepA ( Figure 1A, 3) exhibited potent growth-inhibitory activity against human cancer cells [37]. Our findings suggested that the inhibitory effect of NepA on the production of SAM via the inhibition of SAH might also affect the histone methylation status of human cancer cells. In BC, the upregulated methylation of H3K79 (H3K79me) is known to be correlated strongly with a poor clinical outcome [2]. Therefore, we focused on DOT1L expression and DOT1L-mediated methylation levels in BC cells. As shown in Figure 1B, the TNBC cell lines HCC38, HCC1937, and MDA-MB-231 exhibited higher DOT1L and H3K79me2 expression levels relative to those in normal breast epithelial (MCF10A) and non-TNBC cell lines (MCF-7, T-47D). In comparison, the expression level of E-cadherin, an epithelial marker, was found to be significantly low in TNBC cell lines. We further analyzed the clinical significance of DOT1L expression in TNBC patients with respect to relapse-free survival (RFS) according to the Kaplan−Meier method. We applied the auto-select best cutoff method to classify patients with BC. Patients were further classified according to their levels of DOT1L expression and probability of RFS. Among patients with TNBC, those with high DOT1L expression had a lower probability of RFS than did those with low DOT1L expression ( Figure 1C). We also found that NepA and its analogs exhibited more potent antiproliferative activity against TNBC cells relative to non-TNBC cells. In particular, F-NepA (2) showed a similar antiproliferative activity with NepA (1) against human BC cells, but F-NepA was less cytotoxic compared to NepA against normal breast epithelial cells (MCF10A). These findings indicate that the introduction of fluorine may reduce the cytotoxicity associated with NepA in normal cells (Table 1). In addition, the MDA-MB-231 and HCC1937 cells exhibited the relatively higher DOT1L expression levels and downregulated E-cadherin level among the tested human breast cancer cell lines. Therefore, these two cell lines were employed in subsequent experiments as representative of TNBC cells.

Effects of NepA Analogs on DOT1L Activity and H3K79me2 Expression
To further determine whether NepA analogs downregulated the methylation status of H3K79, we treated TNBC cells (MDA-MB-231 and HCC1937 cells) with NepA analogs for 48 h and analyzed the H3K79me2 levels by Western blotting. As expected, NepA (1) and F-NepA (2) exhibited remarkable abilities to suppress H3K79 dimethylation at the test concentration of 200 nM. In contrast, the N 6 -methyl derivative of F-NepA (3) exhibited negligible inhibitory activity against H3K79me2 (Figure 2A). In addition, a DOT1L enzymatic activity assay confirmed that NepA and F-NepA effectively inhibited DOT1L activity under cell-free conditions ( Figure 2B), suggesting that the suppressive effects of NepA analogs on the H3K79me2 levels in TNBC cells were associated with the inhibition of DOT1L enzymatic activity. Furthermore, the introduction of an N 6 -methyl group into F-NepA reduced the cancer cell antiproliferative activity of this analog and the DOT1L-mediated suppression of H3K79me2, indicating that the N 6 -amine moiety is a pivotal structural chemical component in the bioactivity of NepA analogs (Table 1, Figure 2). the N 6 -methyl derivative of F-NepA (3) exhibited negligible inhibitory activity against H3K79me2 (Figure 2A). In addition, a DOT1L enzymatic activity assay confirmed that NepA and F-NepA effectively inhibited DOT1L activity under cell-free conditions ( Figure 2B), suggesting that the suppressive effects of NepA analogs on the H3K79me2 levels in TNBC cells were associated with the inhibition of DOT1L enzymatic activity. Furthermore, the introduction of an N 6 -methyl group into F-NepA reduced the cancer cell antiproliferative activity of this analog and the DOT1L-mediated suppression of H3K79me2, indicating that the N 6 -amine moiety is a pivotal structural chemical component in the bioactivity of NepA analogs (Table 1, Figure 2). The relative intensities of the indicated protein levels were analyzed semi-quantitatively using NIH ImageJ software. (B) DOT1L (500 ng/well) enzyme activity levels were analyzed after incubation with NepA analogs (0.5-2 µM) for 2 h. All data are expressed as mean values ± SD (n = 3) and are representative of three separate experiments. **p < 0.01 indicates significant differences relative to the vehicle-treated control group.

F-NepA Selectively Suppresses DOT1L-Mediated H3K79 Methylation in Human TNBC Cells
Despite the potent antiproliferative activity exhibited by NepA against numerous types of human cancer cells, the high toxicity and reversible SAH inhibition associated with this agent has limited its clinical development and use [30,43]. Compared to NepA, F-NepA irreversibly inhibits SAH and thus exhibits great potential as a therapeutic agent that targets TNBCs with upregulated DOT1L expression. The methylation of each H3 residue is governed by a specific enzyme (e.g., SET1A/B for H3K4, G9a for H3K9, or EZH1/2 for H3K27) [44,45]. After confirming the potency of F-NepA against DOT1L, we further evaluated whether F-NepA would affect the methylation of specific H3 lysine residues in human TNBC cells. Although treatment with F-NepA for 48 h effectively suppressed dimethylation at H3K79 in a concentration-dependent manner, no significant effects were observed on other H3 residues ( Figure 3A). These findings suggest that F-NepA selectively suppresses DOT1L, as indicated by the downregulation of H3K79me2, without altering the activities of other histone methyltransferases. Moreover, as shown in Figure 3B, F-NepA exhibited cytotoxicity against MDA-MB-231 in a timeand concentration-dependent manner. This analog also evoked significant morphological changes in treated cells, which exhibited either shrunken or pointed shapes relative to the appearances of vehicle-treated control cells when analyzed under an inverted phase-contrast microscope ( Figure 3C). These morphological changes might be partly concomitant with mesenchymal-epithelial transition induced by treatment of F-NepA.

F-NepA Modulates EMT-Associated Gene Expressions in Human TNBC Cells
TNBCs exhibiting aberrant DOT1L expression eventually undergo EMT. This process is strongly associated with cancer cell metastasis to other organs [46], which is a main cause of lethality in all BC cases [47]. Since cancer metastasis is a multistep process including cell migration and invasion, we determined whether F-NepA is able to regulate the metastatic potential of human TNBC cells via the inhibition of these two processes which might be coincident with DOT1L expression. Notably, F-NepA treatment of MDA-MB-231 and HCC1937 cells for 24 h effectively inhibited wound closure in a wound healing assay (cell migration, Figure 4A) and cell invasion in a Transwell-invasion assay ( Figure 4B) in a concentration-dependent manner. To further elucidate the molecular mechanism underlying the inhibition of cancer cell migration and invasion by F-NepA, we evaluated the effects of F-NepA on the expression levels of EMT-associated genes using real-time PCR and Western blotting. Treatment with F-NepA for 24 h induced the upregulation of CDH1, which encodes the epithelial biomarker E-cadherin, and downregulated the expression of the genes CDH2 and Vim, which respectively encode the typical mesenchymal biomarkers N-cadherin and Vimentin, in human TNBC cells ( Figure 5A). In the same cell lines, treatment with F-NepA for 48 h similarly regulated the protein levels of EMT markers, including E-cadherin, N-cadherin, and Vimentin, in a concentration-dependent manner ( Figure 5B). These results suggest that the antimigration and anti-invasion activities of F-NepA might be associated with the suppression of the DOT1L-dependent EMT signaling pathway in TNBC cells.

F-NepA Reverses TGF-β Induced EMT Biomarker Expressions
EMT is a complex and reversible process that is triggered by signals, such as hypoxic condition, TGF-β, and TNF-α [19]. Since the upregulation of the EMT signaling pathway is known to be a main cause of tumor metastasis, we further designed and performed the experiments for evaluating the effect of F-NepA in TGF-β-induced EMT models. Preferentially, to confirm the effect of TGF-β on induction of EMT signaling molecules, MCF10A, a normal breast epithelial cell line, was treated with TGF-β (5 ng/mL) for indicated times (days). As a result, TGF-β treatment effectively downregulated the expression of an epithelial marker (E-cadherin), and upregulated the expression of mesenchymal markers (N-cadherin and Vimentin) in a time-dependent manner ( Figure 6A). The downregulated E-cadherin expression in TGF-β-induced MCF10A cells was slightly recovered by F-NepA treatment for 48 h ( Figure 6B). Similarly, F-NepA effectively upregulated E-cadherin expression and downregulated N-cadherin and Vimentin expressions in two TGF-β-induced highly metastatic human TNBC cells ( Figure 6C). Taken together, these data suggest that the TGF-β-induced EMT process could be effectively reversed by F-NepA treatment in breast epithelial cells as well as in highly metastatic TNBC cells.   cadherin expression in TGF-β-induced MCF10A cells was slightly recovered by F-NepA treatment for 48 h ( Figure 6B). Similarly, F-NepA effectively upregulated E-cadherin expression and downregulated N-cadherin and Vimentin expressions in two TGF-β-induced highly metastatic human TNBC cells ( Figure 6C). Taken together, these data suggest that the TGF-β-induced EMT process could be effectively reversed by F-NepA treatment in breast epithelial cells as well as in highly metastatic TNBC cells.

Discussion
Nucleosides, which are endogenous to all cells of the body, are essential to the maintenance and regulation of numerous biological functions, including signaling pathways, energy metabolism, and heredity control [48,49]. Since the first descriptions of the chemical synthesis of adenosine and guanosine in the 1940s, modified nucleosides have become valuable therapeutic agents in an enormous number of situations because of their relative safety and significant activity levels [25,50]. Nucleoside analogs comprise a main class of small molecule-based antiviral, antitumor, and antibacterial agents [28,51,52]. Previously, we reported the synthesis and biological activity of halo-analogs of NepA, among which we identified F-NepA as a significantly more potent inhibitor of SAH relative to the parent compound, whereas chloro-and bromo-substituted analogs were relatively less active [33]. In another recent report [37], we described the profound antiproliferative activity of F-NepA against a panel of cancer cells. Therefore, in this study, we aimed to determine whether a decrease in SAM via the F-NepA-mediated inhibition would also affect the methylation status of H3 in human TNBC cells.
The regulation of aberrantly activated H3 methyltransferases is considered an attractive anticancer strategy [53,54]. In this regard, DOT1L plays a major role in regulating gene expression and chromosome structure during development and gene transcription by targeting H3K79 [55]. However, recent findings suggest that the dysregulation of DOT1L expression and activity is closely associated with aggressive behavior and metastatic potential in human TNBC cells [19]. Accordingly, we analyzed the relationships between the clinical outcomes of TNBCs and the expression of DOT1L. Notably, we observed an inverse correlation between DOT1L expression and the RFS rate in patients with TNBC, suggesting that DOT1L may be a therapeutic biomarker of TNBC. Interestingly, F-NepA effectively inhibited the proliferation of human TNBC cells, which constitutively overexpress DOT1L, via the inhibition of both DOT1L activity and the DOT1L-mediated selective suppression of H3K79 dimethylation. Furthermore, a structure-activity relationship study of NepA analogs revealed that the free N 6 -amine group of NepA was necessary for the cytotoxic and inhibitory effects of this agent against DOT1L. We further observed that the introduction of fluorine at the 6 -position of NepA reduced the toxicity associated with the parent compound against a normal breast epithelial cell line. These findings support the possibility that further design and development efforts may yield more bioactive compounds as therapeutic agents against TNBC.
Accumulating evidence suggests that the inhibition of DOT1L-mediated processes may also suppress the metastasis of human cancer cells [56,57]. Despite this possibility, early DOT1L inhibitors, such as EPZ-5676 (developed by Epizyme and Celgene), did not noticeably suppress the growth and metastasis of solid tumors, including BCs [20]. Compared with previously identified DOT1L inhibitors, F-NepA significantly inhibited the migration and invasion of cultured human TNBC cells in vitro. F-NepA treatment also downregulated the protein levels of mesenchymal markers (N-cadherin and Vimentin) while upregulating the level of an epithelial maker (E-cadherin), suggesting that F-NepA can suppress the migration and invasion potential of TNBCs expressing high levels of DOT1L. Moreover, we found that F-NepA is able to effectively regulate the TGF-β-induced EMT processes in both a normal breast epithelial cell and human TNBC cells.

Conclusion
In summary, our findings highlight DOT1L inhibition as a compelling potential strategy for the targeted therapy of human TNBCs. F-NepA effectively inhibits the DOT1L-mediated methylation of H3K79 and thus suppresses the migration and invasion of TNBC cells. Although an evaluation of the antitumor and antimetastatic activity of F-NepA in animal models is needed, F-NepA appears to be a promising lead compound in the discovery and development of novel therapeutic agents for the treatment of aggressive TNBC.

Patents
Jeong, L.S. Novel fluoro-homoneplanocin A and nucleoside derivatives, method for the synthesis thereof, and the pharmaceutical compositions for treating cancers containing the same as an active ingredient. KR 20130127883, 2013; US 20130310403, 2013.