WMJ-S-001, a Novel Aliphatic Hydroxamate-Based Compound, Suppresses Lymphangiogenesis Through p38mapk-p53-survivin Signaling Cascade

Background and purpose: Angiogenesis and lymphangiogenesis are major routes for metastatic spread of tumor cells. It thus represent the rational targets for therapeutic intervention of cancer. Recently, we showed that a novel aliphatic hydroxamate-based compound, WMJ-S-001, exhibits anti-angiogenic, anti-inflammatory and anti-tumor properties. However, whether WMJ-S-001 is capable of suppressing lymphangiogenesis remains unclear. We are thus interested in exploring WMJ-S-001's anti-lymphangiogenic mechanisms in lymphatic endothelial cell (LECs). Experimental approach: WMJ-S-001's effects on LEC proliferation, migration and invasion, as well as signaling molecules activation were analyzed by immunoblotting, flow-cytometry, MTT, BrdU, migration and invasion assays. We performed tube formation assay to examine WMJ-S-001's ex vivo anti-lymphangiogenic effects. Key results: WMJ-S-001 inhibited serum-induced cell proliferation, migration, invasion in murine LECs (SV-LECs). WMJ-S-001 reduced the mRNA and protein levels of survivin. Survivin siRNA significantly suppressed serum-induced SV-LEC invasion. WMJ-S-001 induced p53 phosphorylation and increased its reporter activities. In addition, WMJ-S-001 increased p53 binding to the promoter region of survivin, while Sp1 binding to the region was decreased. WMJ-S-001 induced p38 mitogen-activated protein kinase (p38MAPK) activation. p38MPAK signaling blockade significantly inhibited p53 phosphorylation and restored survivin reduction in WMJ-S-001-stimulated SV-LCEs. Furthermore, WMJ-S-001 induced survivin reduction and inhibited cell proliferation, invasion and tube formation of primary human LECs. Conclusions and Implications: These observations indicate that WMJ-S-001 may suppress lymphatic endothelial remodeling and reduce lymphangiogenesis through p38MAPK-p53-survivin signaling. It also suggests that WMJ-S-001 is a potential lead compound in developing novel agents for the treatment of lymphangiogenesis-associated diseases and cancer.


INTRODUCTION
Lymphangiogenesis, the formation of new lymphatic vessels, occurs primarily in many physiological processes such as embryonic development, tissue repair and resolution of inflammatory reactions. It also contributes to a variety of pathological events including lymphedema, inflammatory diseases, and tumor metastasis (1). Metastatic spread of tumor cells is the major cause of morbidity and mortality and responsible for ∼90% of cancer-related deaths (2). A variety of mechanisms such as seeding of body cavities, local tissue invasion, invasion into lymphatics and hematogenous spread are involved in metastatic tumor spread. Although both blood and lymphatic systems contribute to tumor progression and metastasis, the dissemination of tumor cells via lymphatic vasculature is the most common route for most carcinomas (3,4). Increased number of tumor-associated lymphatic vessels has been shown closely correlated with metastasis and poor clinical outcome (5).
Lymphangiogenesis, similar to angiogenesis, is tightly regulated by lymphangiogenic factors and its cognate receptors (6). The member of the vascular endothelial growth factor (VEGF) family, VEGF-C, is currently the best-characterized lymphangiogenic factor. VEGF-C's lymphangiogenic effects is primarily mediated by VEGFR-3 (also known as flt-4). The expression of VEGFR-3 is largely restricted to the lymphatic endothelial cells (LECs) in normal adult tissues (7,8). It is reported in experimental xenograft models that VEGF-C stimulates tumor lymphangiogenesis, as well as lymph node metastasis (9,10). In addition, VEGF-C overexpression in tumor tissues significantly correlates with accelerated tumor progression and poor clinical outcome (11). Knocking down VEGF-C expression with siRNA significantly prevented lymphangiogenesis and enhanced chemo-sensitivity in breast cancer cells (12). Therefore, VEGF-C-associated lymphangiogenesis has emerged as a key prognostic marker and represents a promising therapeutic target for cancer intervention (13). To interfere with VEGF-C-VEGFR-3 signaling, a variety of strategies has been reported currently. These includes neutralizing antibodies or peptides that antagonize VEGFR-3 signaling, receptor traps or monoclonal antibodies targeting VEGF-C and small molecule receptor tyrosine kinase inhibitors of VEGFR-3 (14).
The smallest member of the inhibitor of apoptosis protein (IAP) family, survivin, is rarely detected in most terminally Abbreviations: IAP, inhibitor of apoptosis protein; LEC, lymphatic endothelial cell; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; p38MAPK, p38 mitogen-activated protein kinase; PI, propidium iodide; VEGF, vascular endothelial growth factor. differentiated adult tissues with notable exceptions of vascular endothelial or hematopoietic cells (15). However, a high survivin level is found in most common human cancers. Highly expressed survivin positively correlates with tumor progression and poor prognosis (16)(17)(18). Survivin not only suppresses cell death, but also participates in a variety of cellular events. These include cell migration, cell cycle progression (16), and angiogenesis, which may promote metastatic spread of tumor cells (17). Cai et al. (19) reported that survivin level is associated with VEGF-C level and the presence of lymphatic invasion in breast cancer. However, the association between endothelial survivin and lymphangiogenesis remains incompletely understood. It is likely that modulating survivin level may provide another means of regulating tumor lymphangiogenesis. Survivin expression is regulated primarily at the transcriptional level. Survivin is up-regulated by transcription factors such as signal transducer and activator of transcription 3 (STAT3) (20), specificity protein 1 (Sp1) (21) or hypoxiainducible factor-1α (HIF-1α). However, p53, a tumor suppressor, may cause survivin reduction (22,23). Pharmacological targeting of the p53-survivin cascade may be a potential therapeutic strategy in not only causing tumor cell death, but also suppressing lymphangiogenesis and tumor progression.
Hydroxamate, a key pharmacophore, has attracted considerable attention in drug development field due to its diverse pharmacological properties (24). Growing evidence demonstrates the potential use of hydroxamate derivatives as anti-tumor (20,25), anti-inflammatory (26), or anti-infectious (27) agents. Recently, we synthesized and showed that a novel aliphatic hydroxamate-based compound WMJ-S-001 exhibits anti-tumor (22), anti-inflammatory (28) and anti-angiogenic (20) properties. Given its potential as lead compound for drug discovery, we aimed to investigate WMJ-S-001's antilymphangiogenic effects and its underlying mechanisms in LECs.

MTT Assay
We used the colorimetric MTT assay to determine cell viability as described previously (28).

Cell Proliferation Assay (BrdU Incorporation Assay)
Human lymphatic endothelial cells (HLECs) (2 × 10 4 per well) seeded in 48-well tissue culture plates were starved in MV2 basal medium in the absence of growth supplements for 24 h. After starvation, cells were incubated in MV2 growth medium containing growth supplements with or without WMJ-S-001 at indicated concentrations for another 24 h. A Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche) based on the colorimetric detection of the incorporation of BrdU was used to determine cell proliferation following the manufacturer's instructions.

Western Analysis
After treatment, cells were harvested in lysis buffer [0.5% NP-40, 140 mM NaCl, 10 mM Tris (pH 7.0), 0.05 mM pepstatin A, 2 mM PMSF and 0.2 mM leupeptin]. Cell lysate with equal amounts of protein were subjected to SDS-PAGE and transferred onto a NC membrane (Pall Corporation, Washington, NY, U.S.A.). After transfer, membrane was incubated with 5% non-fat milk-containing blocking buffer for 1 h. Specific primary antibodies and horseradish peroxidaseconjugated secondary antibodies were used to recognize target proteins. Enhanced chemiluminescence was used to detect immunoreactivity according to manufacturer's instructions. To obtain the quantitative data, a computing densitometer with a scientific imaging system (Biospectrum AC System, UVP) was used.

Reporter Assay
After transfection, SV-LECs were treated with vehicle or WMJ-S-001 for another 24 h. A Dual-Glo luciferase assay system kit (Promega, Madison, WI, U.S.A.) was employed to determine the luciferase reporter activity per manufacturer's instructions. The reporter activity was normalized based on renilla-luciferase activity.

Cell Migration Assay
SV-LECs were seeded in the 12-well tissue culture plates. After growing to confluence, SV-LECS were starved with serum-free DMEM medium for 24 h. Pipette tips were used to create scratch wounds in monolayers of SV-LECs. Cells were washed with PBS, followed by the treatment with WMJ-S-001 at different concentrations in the presence or absence of 10% FBS for another 24 h. Cells were fixed with cold 4% paraformaldehyde and stained with 0.5% toluidine blue. After staining, an OLYMPUS Biological Microscope digital camera (Yuan Li Instrument Co., Taipei, Taiwan) was used to take photographs at 40× magnification. Cell migration rate was determined by calculating the migrated cells in the wound area.

Invasion Assay
We performed cell invasion assays as described previously (20). 0.2% gelatin solution was used to coat the lower face of the filter FIGURE 2 | compared with the control group; #p < 0.05, compared with the group treated with serum alone). (B) A total of 10 4 SV-LECs were seeded in the top gelatin-coated chamber and treated with vehicle or indicated concentrations of WMJ-S-001 using serum (10% FBS) as chemo-attractant. After 16 h, the SV-LECs that invaded through the gelatin-coated membrane were stained and quantified as described in the section Materials and Methods. Each column represents the mean ± S.E.M. of six independent experiments (*p < 0.05, compared with the control group; #p < 0.05, compared with the group treated with serum alone).
in the transwell plate (Corning, NY, U.S.A.). The lower chambers were filled with containing 10% FBS-containing DMEM medium (SV-LECs) or growth supplements-containing MV2 medium (HLECs). SV-LECs or HLECs (2 × 10 4 cells per chamber) were seeded in the upper chambers in the serum-free DMEM medium or MV2 basal medium with or without WMJ-S-001. After 18 h, the non-invaded cells in the upper chamber were removed by gently scraped with a cotton swab. The invaded cells in the lower face of the filter were fixed, stained with toluidine blue (0.5% in 4% paraformaldehyde) and photographed using an optical microscope (Nikon, Japan) at ×40. The number of stained cells that invaded through the filter were counted. We also quantified cell invasion by dissolving the stained cells in 33% acetic acid and measuring the absorbance at 570 nm.

Chromatin Immunoprecipitation (ChIP) Assay
After treatment as indicated, cells were cross-linked with formaldehyde (1%) for 10 min at 37 • C.

Tube Formation Assay
The basement membrane matrix, matrigel (Becton Dickinson, Mountain View, CA, USA) was used to perform the tube formation assay as previously described (32). Matrigel was polymerized for 30 min at 37 • C. HLECs were seeded onto the matrigel in MV2 basal medium with or without WMJ-S-001 at indicated concentrations. After 24 h, cells were photographed using an optical microscope (Nikon, Japan) at ×40.

Blinding and Randomization
We have different people analyzing data (analyst) and conducting experiments (operator) for blinding. The same cell in every single experiment was used to evaluate the WMJ-S-001's effects vs. the related control. Therefore, formal randomization was not employed.

Data and Statistical Analysis
In this study, the data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (33). Results represented as mean ± standard error of mean (S.E.M) (n ≥ 3), where 'n' refers to independent values, and not replicates. Normalization was performed to compare the differences after the treatment to control for unwanted sources of variation and to reveal relevant trends. unpaired Student's t-test for parametric analysis. Kruskal-Wallis test followed by Dunn's multiple comparison for nonparametric analysis or one-way analysis of variance (ANOVA) with Tukey's post-hoc test for parametric analysis was used to evaluate the statistical comparisons between more than two groups. A P-value smaller than 0.05 was defined as statistically significant.

WMJ-S-001 Inhibits SV-LEC Proliferation and Causes Apoptosis
It is still difficult to isolate and propagate LECs from different organs (7,34,35). This limits the studies on lymphangiogenesis or signaling mechanisms in LECs. In this study, we selected a SV40 large T-expressed immortalized murine LEC line (SV-LEC), which retain their "lymphatic" endothelial characteristics after repeated passages (30,36), to overcome these limitations. A MTT assay was used to examine whether WMJ-S-001, a novel aliphatic hydroxamate-based compound, affects SV-LEC viability. As shown in Figure 1A, WMJ-S-001 reduced SV-LEC viability in the concentration-and time-dependent manners. We next determined whether WMJ-S-001's inhibitory actions on the cell viability attributes to its anti-proliferative effects. After starvation with serum-free medium for 24 h, SV-LECs were stimulated with serum (10% FBS) for another 6 to 72 h in the presence or absence of WMJ-S-001 (10 µM). As shown in Figure 1B, WMJ-S-001 significantly reduced serum-induced SV-LEC proliferation. In addition, WMJ-S-001 concentration-dependently inhibited cell proliferation in SV-LECs after 48 h exposure to serum ( Figure 1C). We next used flow cytometry with propidium iodide (PI) staining to examine whether WMJ-S-001 alters cell cycle progression or induces apoptosis. Treatment of WMJ-S-001 for 24 h significantly reduced the percentage of PI-stained cells in the S region as compared with the control group ( Figure 1D). This effect was accompanied by a concomitant increase in the percentage of PI-stained cells in the G1 region after treatment of WMJ-S-001 ( Figure 1D). In addition, WMJ-S-001 at concentrations of 10 µM or lower did not significantly cause apoptosis (sub-G1, apoptotic region). However, WMJ-S-001 at 30 µM significantly induced cell apoptosis in SV-LECs ( Figure 1D). We next examined whether WMJ-S-001 activates caspase3, an apoptosis marker. WMJ-S-001 at concentrations higher than 10 µM (20 and 30 µM) markedly increased the cleaved (active) form of caspase 3 and its substrate, PARP ( Figure 1E). It suggests that inhibiting LEC proliferation and causing apoptosis may contribute to WMJ-S-001's antilymphangiogenic effects.

WMJ-S-001 Inhibits Serum-Induced LEC Migration and Invasion
We next examined whether WMJ-S-001 alters cell motility, a pivotal step in lymphangiogenesis, in SV-LECs after serum (10% FBS) exposure. WMJ-S-001 significantly inhibited seruminduced LEC migration as determined by wound-healing migration assay (Figure 2A). We also used transwell invasion assay to examine WMJ-S-001's effects on serum-induced cell invasion. As shown in Figure 2B, WMJ-S-001 at 10 and 30 µM significantly reduced the number of invading cell penetrating the gelatin-coated transwell filter barrier, using serum (10% FBS) as the chemoattractant. These observations indicate that WMJ-S-001 is capable of inhibiting LEC migration and invasion.

Surivin Reduction Contributes to WMJ-S-001's Inhibitory Effects on SV-LEC Invasion
Growing evidence has demonstrated that suvivin not only regulates mitosis and apoptosis, but also plays a critical role in angiogenesis (37,38). However, whether endothelial survivin participates in lymphangiogenesis remains unclear. We thus determined whether WMJ-S-001 alters survivin level in SV-LECs. Results from immunoblotting analysis demonstrated that survivin exhibits high expression levels in SV-LECs ( Figure 3A). However, treatment of cells with WMJ-S-001 for 18 or 24 h concentration-dependently caused survivin reduction ( Figure 3A). We also determined whether WMJ-S-001 affects survivin mRNA level in SV-LECs. As shown in Figure 3B, cell cycle arrest and apoptosis in SV-LECs ( Figure 3E). We examined whether survivin reduction affects SV-LEC motility. As shown in Figure 3F, survivin siRNA mimicked the WMJ-S-001's effects in reducing the number of invading cell penetrating the gelatin-coated transwell filter barrier, using serum as the chemoattractant. In contrast, negative control siRNA was without effects (Figure 3F). These observations suggest that survivin reduction may contribute to WMJ-S-001's inhibitory effects on SV-LEC motility and subsequent lymphangiogenesis.

p38MAPK Mediates WMJ-S-001-Induced p53 Activation and Survivin Reduction in SV-LECs
We investigated the underlying mechanisms of WMJ-S-001 in repressing survivin expression in SV-LECs. Transcription factor p53 participates in various cellular processes such as cell cycle arrest, apoptosis or senescence by regulating diverse target genes (39). It is reported that p53 may suppress survivin expression by counteracting Sp1 binding to the survivin promoter region (40). Therefore, we explored whether WMJ-S-001 induces p53 activation in SV-LECs. Post-translational modifications such as phosphorylation or acetylation play important roles in regulating p53's activity (41). We thus examined WMJ-S-001's effects on p53 phosphorylation and acetylation in SV-LECs. As shown in Figure 4A, 6 h exposure to WMJ-S-001 led to concentration-dependent increases in p53 phosphorylation and acetylation. Results from reporter assay showed that treatment of cells with WMJ-S-001 for 24 h significantly increased PG13luciferase (p53-luciferase) activity. WMJ-S-001 also increased the promoter-luciferase activity of p21, a p53 target gene, in SV-LECs ( Figure 4B). Moreover, we performed a ChIP analysis to examine whether Sp1 or p53 is recruited to the endogenous survivin promoter region (−236 to −26) containing putative Sp1 and p53 binding sites, in WMJ-S-001-stimulated SV-LECs. As shown in Figure 4C, 6 h WMJ-S-001 exposure increased p53 binding to the survivin promoter region (−236/−26). This effect was accompanied by a concomitant decrease in Sp1 binding to the region. p38-mediated signaling cascade has been shown previously to cause p53 phosphorylation and survivin reduction in cancer cells (23). We thus elucidated whether WMJ-S-001-induced p53 activation involves p38MAPK signaling in SV-LECs. As shown in Figure 4D, WMJ-S-001 time-dependently induced p38MAPK phosphorylation. A pharmacological p38MAPK inhibitor, p38 inhibitor III, significantly suppressed WMJ-S-001's effects in inducing p53 phosphorylation ( Figure 4E) and survivin reduction ( Figure 4F) in SV-LECs. It appears that WMJ-S-001 induces p38MAPK activation, resulting in p53 activation and survivin reduction in SV-LECs.

WMJ-S-001 Suppressed Cell Proliferation, Invasion and Tube Formation of Primary Human LECs
We next examined whether WMJ-S-001 reduces survivin expression and exhibits anti-lymphangiogenic activities in primary human LECs. As shown in Figure 5A, WMJ-S-001 at concentrations ranging from 10 to 30 µM significantly reduced survivin mRNA levels in human LECs (Figure 5A). WMJ-S-001's effects on cell proliferation in human LECs were examined using a BrdU incorporation assay. After starvation with MV2 basal medium for 24 h, human LECs were stimulated by MV2 growth medium in the absence or presence of WMJ-S-001 for another 24 h. The percentage of BrdU-labeled cells increased significantly after a 24 h treatment with MV2 growth medium. However, WMJ-S-001 reduced this increase in a concentration-dependent manner ( Figure 5B). The effects of WMJ-S-001 on human LEC invasion were also determined using transwell invasion assay. As shown in Figure 5C, WMJ-S-001 significantly reduced the number of invading cells penetrating the gelatin-coated transwell filter barrier using MV2 growth medium as the chemoattractant (Figure 5C). We also examined whether ribociclib, a potent proliferation inhibitor targeting cyclin-dependent kinases (CDKs) 4/6 (42), affects human LEC invasion. As shown in Figure 5D, ribociclib, similar to WMJ-S-001, suppressed human LEC invasion. It is likely that WMJ-S-001's inhibitory effects on LEC invasion may also attribute to its anti-proliferative properties. Another key step of lymphangiogenesis is the tubular formation of LECs. We thus examined WMJ-S-001's effect on this step. Human LECs were seeded on the matrigel surface in complete MV2 growth medium in the presence of vehicle as control or WMJ-S-001. As shown in Figure 5E, cells incubated with MV2 growth medium became elongated, and formed capillary-like structure within 16 h. WMJ-S-001, however, concentration-dependently reduced the formation of capillary-like network (Figure 5E). Similarly, WMJ-S-001 also significantly reduced VEGF-C-induced tubular formation of human LECs ( Figure 5E). Furthermore, WMJ-S-001 suppressed VEGF-C-induced ERK1/2 phosphorylation in human LECS (Figure 5F). Together these observations suggests that WMJ-S-001 exhibits anti-lymphangiogenic properties through suppressing cell proliferation, invasion and tubular formation of LECs. WMJ-S-001's actions in LECs may also attribute to survivin reduction.

DISCUSSION
Most cancer-related deaths are caused by tumor metastasis (2). Targeting the major routes for metastatic spread of tumor cells, angiogenesis and lymphangiogenesis, thus represents a promising therapeutic strategy for cancer intervention. To date, the European Medicines Agency (EMEA) or the U.S. Food and Drug Administration (FDA) has already approved several agents targeting angiogenesis including monoclonal antibodies and small molecule inhibitors for the treatment of certain types of cancer (43)(44)(45). In addition, some lymphangiogenesis inhibitors have been shown effective in reducing tumor progression and metastasis in solid tumors (46,47). These observations led to increased efforts in discovering and developing novel agents targeting angiogenesis or lymphangiogenesis. Growing evidence shows beneficial effects of hydroxamate-based compounds in the treatment of cancer (24,25,31). Recently, we synthesized and identified a novel aliphatic hydroxamate-based compound, WMJ-S-001, that exhibits anti-tumor (22) and anti-angiogenesis (20) properties in in vitro and in vivo models. In this study, we further demonstrated that WMJ-S-001 also suppressed lymphangiogenesis using LECs as a cell model. We also demonstrated that p38MAPK-p53-survivin signaling might participate in WMJ-S-001's anti-lymphangiogenic actions. Beyond its anti-apoptotic effects, survivin regulates a variety of cellular events such as cell cycle progression, cell migration and angiogenesis, which may enhance tumor metastasis and progression (16,17,37,48). However, the association between endothelial survivin and lymphangiogenesis, remains to be fully defined. In the present study, we showed that endothelial survivin reduction significantly suppressed LEC invasion, a key step in lymphangiogenesis. Similar to previous studies (22,40), we noted that WMJ-S-001-induced survivin reduction attributes to the activation of p53 in LECs. WMJ-S-001-activated p53 also led to cell cycle regulator p21 transcriptional activation, which blocks cell cycle machinery. It appears that WMJ-S-001's anti-proliferative effects in LECs may involve additional cell cycle regulatory proteins. In addition to suppressing cell cycle progression, WMJ-S-001 at concentrations higher than 10 µM also caused caspase 3 activation and apoptosis. It is likely that WMJ-S-001's anti-lymphangiogenic effects may also attribute to its apoptotic mechanisms when its concentrations is higher than 10 µM.
We showed that p38MAPK mediates p53 phosphorylation and survivin reduction in LECs after WMJ-S-001 exposure. The precise mechanisms underlying WMJ-S-001-induced p38MAPK activation in LECs remains to be established. Chen et al. (49) reported that activating Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-1 (SHP-1)-PP2A-p38MAPK signaling cascade leads to p53 activation and vascular smooth muscle cell death. We have established that SHP-1 activation is involved in WMJ-S-001's anti-angiogenic effects in HUVECs (20). We also noted that SHP-1 signaling blockade by NSC-87877 reduces WMJ-S-001's effects on survivin and p21 levels in both HUVECs and HCT116 colorectal cancer cells (unpublished data). It raises the possibility that WMJ-S-001-induced p53 activation and subsequent cellular events may also involve SHP-1 in LECs.
Several lines of evidence demonstrated that angiogenesis inhibitors targeting the VEGF-VEGF-R signaling might enhance the evasive and adaptive resistance to the therapies in tumor cells. The underlying mechanisms by which the cancer cells develop resistance remain incompletely understood. The bevacizumab (a clinical used VEGF monoclonal antibody)adapted tumor cells may switch their dependence to alternative proangiogenic signaling and enhancement of lymphaticmediated metastasis (55). Sunitinib, a multi-targeted tyrosine kinase inhibitor, also induced evasive adaption in cancer cells through an alternative neurophilin 1(NRP-1)-mediated signaling (56). Whereas, sunitinib-adapted tumor exhibits less lymphatic metastasis, as it targets both lymphangiogenesis and angiogenesis (57). It is likely that single-target agents may be inadequate as therapeutics. We have shown in this study that p38MAPK-p53-mediated survivin reduction, at least in part, contributes to WMJ-S-001's anti-lymphangiogenic actions in LECs. Moreover, WMJ-S-001 has additional properties such as anti-tumor (22), anti-angiogenic (20) and anti-inflammatory (28) activities. The precise mechanisms underlying these activities remain to be fully define. Together these findings, however, support WMJ-S-001's potential as a promising lead compound in developing novel agents for oncologic therapy.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author.