2.1 Chemistry

Compound syntheses and analyses were done by the company ValueTek Inc. (NJ, USA). All reagents and solvents were purchased from Sigma–Aldrich Chemical Co., Combi-Block Inc., Astatech Inc., Ark Pharma Inc., and Frontier Scientific Inc. and were used as received. HNMR spectra were recorded on a Bruker Fourier-400 spectrometer. Crude products were purified by silica gel chromatography. For all products, the purity was ascertained to be greater than 95% by the HPLC method using Shimadzu 2010HPLC-UV/MS system with a C-18 reverse phase column.

2.2 Mouse colony

Female BALB/c mice (6–8 week old) were purchased from Charles River. Studies using mice were performed in compliance with the Institutional Animal Care and Use Committee of Weill Medical College of Cornell University. All mice were housed in the facility of the Research Animal Resource Center of Weill Medical College of Cornell University.

2.3 Cell culture

Mouse 4T1 mammary tumor cells and human MDA-MB-231 breast tumor cells were obtained from ATCC. 4T1 cells were cultured in RPMI 1640 medium supplemented with 10% FBS. MDA-MB-231 cells were cultured in DMEM supplemented with 10% FBS.

2.4 Human fascin-1 expression and purification

Recombinant human fascin 1 was expressed as a GST fusion protein in BL21 Escherichia coli. 1-liter of 2YT medium with ampicillin was inoculated overnight with 3 mL of BL21/DE3 culture transformed with pGEX4T-fascin 1 plasmid and grown at 37 °C until attenuance at 600 nm (OD₆₀₀) reached about 0.5. The culture was then transferred to 17 °C and induced by the addition of 0.1 mM isopropyl β-d-thiogalactoside (IPTG) for 16 h. Bacteria were harvested by centrifugation at 5000 r.p.m. for 10 min. The pellets were suspended in 30 mL of Tris/HCl (20 mM Tris/HCl pH 8.0, 150 mM NaCl) supplemented with 0.2 mM PMSF, 1 mM DTT, 1% Triton X-100 and 1 mM EDTA. After sonication, the suspension was centrifuged at 15,000 r.p.m. for 30 min to remove the cell debris. The supernatant was then incubated for 2 h with 4 mL of glutathione beads (Sigma) at 4 °C. After extensive washing with Tris/HCl (20 mM Tris/HCl pH 8.0, 150 mM NaCl), the beads were resuspended in 10 mL of thrombin cleavage buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 2 mM CaCl₂, 1 mM DTT). Fascin was released from the beads by incubation overnight with 40–100 U of thrombin at 4 °C. After centrifugation, 0.2 mM PMSF was added to the supernatant to inactivate the remnant thrombin activity. The fascin protein was further concentrated with Centricon to about 50 mg/mL.

2.5 F-actin bundling assay

Actin-bundling activity was measured by low-speed centrifugation assay. Monomeric rabbit G-actin was induced to polymerize at room temperature in F-actin buffer (20 mM Tris-HCl at pH 8, 1 mM ATP, 1 mM DTT, 2 mM MgCl₂ and 100 mM KCl). Recombinant fascin proteins (wide-type or mutant) were subsequently incubated with F-actin for 30 min at room temperature and centrifuged for 15 min at 10,000 g in an Eppendorf 5415D tabletop centrifuge. Both supernatants and pellets were dissolved in an equivalent volume of SDS sample buffer, and the amount of fascin was determined by SDS-PAGE. We measured the intensities of fascin proteins in Coomassie-stained gels and then calculated the relative actin-bundling activity.

2.6 Boyden-chamber cell migration assay

MDA-MB-231 cells (5 × 10⁴) suspended in 100 μl starvation medium were added to the upper chamber of an insert (6.5 mm diameter, 8 μm pore size; Becton Dickson), and the insert was placed in a 24-well plate containing 700 μl starvation medium with or without 10% FBS. When used, inhibitors were added to both the upper and the lower chambers. Migration assays were performed for 6 h and cells were fixed with 3.7% formaldehyde. Cells were stained with crystal violet staining solution, and cells on the upper side of the insert were removed with a cotton swab. Three randomly selected fields (10 objectives) on the lower side of the insert were photographed, and the migrated cells were counted. Migration was expressed as average number of migrated cells in a field.

2.7 Tumor metastasis in mice

Female BALB/c mice (6–8 week old) were purchased from Charles River. 4T1 tumor cells (1 × 10⁶) were injected subcutaneously into the abdominal mammary gland area of mice using 0.1 mL of a single-cell suspension in PBS on Day 0. Starting on Day 7, when the tumors averaged about ∼4–5 mm in diameter, G2 analogues or control solvent were given every day by intraperitoneal injection at 100 mg/kg per mouse until Day 24. On Day 25, the mice were sacrificed. This dosage regiment was well tolerated with no signs of overt toxicity. Every group included 5 mice. Numbers of metastatic 4T1 cells in lungs were determined by the clonogenic assay. In brief, lungs were removed from each mouse on Day 25, finely minced and digested in 5 mL of enzyme cocktail containing 1 × PBS and 1 mg/mL collagenase type IV for 2 h at 37 °C on a platform rocker. After incubation, samples were filtered through 70 μm nylon cell strainers and washed twice with PBS. Resulting cells were suspended, plated with a series of dilutions in 10 cm tissue culture dishes in RPMI 1640 medium containing 60 μM thioguanine for clonogenic growth. Since 4T1 tumor cells are resistant to 6-thioguanine, metastasized tumor cells formed foci after 7 days, at which time they were fixed with methanol and stained with 0.03% methylene blue for counting.

2.8 Fascin and F-actin binding assay

Actin-binding activity of fascin was measured by high-speed centrifugation assay. In high-speed centrifugation assay, monomeric rabbit G-actin (10 μM) was induced to polymerize at room temperature in F-actin buffer (20 mM Tris-HCl at pH8, 1 mM ATP, 1 mM DTT, 2 mM MgCl₂ and 100 mM KCl). Recombinant fascin proteins were subsequently incubated with F-actin for 60 min at room temperature and centrifuged for 30 min at 150,000 g. Both supernatants and pellets were dissolved in an equivalent volume of SDS sample buffer, and separated by SDS-PAGE.

2.9 Confocal fluorescence microscopy

Cells were seeded onto laminin-coated glass coverslips in starvation condition (no serum) for 2 h with or without inhibitors (50 μM). Cells were fixed with 3.7% formaldehyde in PBS for 10 min at room temperature, permeabilized with 0.1% Triton X-100 for 5 min, and then washed 3 times with PBS. Actin filaments were labeled with 2% Alexa 488-phalloidin. The coverslips were then mounted onto slides and imaged using Zeiss LSM510 confocal microscopy.

2.10 Fluorescence recovery after photobleaching (FRAP)

Cells were transfected using the Mirius LT1 Transfection Reagent (Mirius) following the manufacturer's instructions. 24 h after the transfection, the cells expressing GFP-FAK were seeded on fibronectin-coated (10 ug/mL in PBS, 1 h at RT) glass bottom dishes (MatTec). FRAP experiments were performed with a Zeiss 710 confocal microscope equipped with an environmental chamber, according to a protocol described before (Bordeleau et al., 2010). Photobleached regions consisted of a 2 μm wide circular region enclosing a selected focal adhesion. Fluorescence within the region was measured at low laser power before bleaching and then photobleached with 8 iterations using both the 405 and 488 nm laser lines at 100% laser power. Recovery was monitored at 488 nm at 0.5 s time intervals for 80 s. In some experiments, cells expressing GFP-FAK were pre-treated with 1 μM NP-G2-044. Fluorescence during recovery was normalized to the prebleach intensity and for the bleaching occurring during image acquisition, as before (Bordeleau et al., 2010). Relative recovery rates were computed with Matlab (MathWorks). The half-time for fluorescence recovery towards the asymptote was extracted from the plots after curve fitting of the data to a single exponential association algorithm.

2.11 Quantification of focal adhesion dynamics

A modified version of an adhesion dynamics assay was used to measure focal adhesion disassembly rate (Bordeleau et al., 2010). Briefly, time-lapse imaging on cells expressing GFP-FAK was performed on a Zeiss 710 confocal microscope equipped with an environmental chamber. The fluorescence of individual focal adhesion was measured as function of time in ImageJ. The assembly/disassembly dynamics of FAK-containing focal adhesions was reported in terms of normalized fluorescence intensity as function of time using Matlab. The assembly/disassembly rates were then computed from the numerical derivatives of each time dependent function. Each assembly and disassembly rate constant was calculated from measurements performed on at least 120 individual focal adhesions from 7 individual cells for each condition.

2.12 Morphodynamics analysis

Image analysis was performed with Matlab according to a previously published method with some modifications (Johnson et al., 2015). Images were subjected to an adaptive Wiener filter (1 μm window) followed by a mean filter (3 μm window) to smoothen cell edges or a top-hat filter (1 μm ring) to extract fibrillary structures. The mean filter removes features smaller than the window size while the top-hat filter increases the contrast ratio of features smaller than the window size. The images were then automatically thresholded to obtain binary images. To obtain the lamellipodial structures from the mean filtered images, any holes inside the cell resulting from the thresholding were healed by filling them. This has the added advantage of removing any internal actin structures from the analysis. The resulting structures were then eroded to fit within the original size of the cell. To obtain the filopodia, the top-hat filtered images were further subjected to a median filter to correct for intensity variation while keeping the filopodial features. The final binary lamellipodial image was then subtracted from the filopodial image to remove any internal actin structure and retain only the outward extending filopodial components. Spatiotemporal maps were generated as described (Johnson et al., 2015). Briefly, the coordinate system of each image was transformed relative to the cell centroid along angular projections in 1° increment. To obtain the protrusion speed, the difference in overlap between each consecutive time step was calculated (yielding individual pixels values of −1 for retraction and 1 for protrusion events). The calculated pixel values along each angular direction were summed and then converted to velocity.

Overlapping spatiotemporal maps were generated by combining the filopodial structures and the mean filtered lamellipodial protrusion. Protruding structures were identified as the portion of the cell extending at a speed ≥1.25 μm/min for more than 1 min and spanning ≥8°. Filopodia were identified from the angular projection as objects having an elliptic eccentricity ≥0.7 and spanning ≤4°. This process excludes unwanted large membrane protrusions resulting from filtering errors. Since any identified filopodial components are the result of the lamellipodia mask subtraction, filopodial components do not overlap with detected protrusions. Therefore, overlapping components are defined as juxtaposed elements in the same, previous or subsequent video frame.

2.13 Statistical analysis

Data are expressed as mean±s.e.m. and analyzed by Student's t test with significance defined as p < 0.05.

3.1 Optimization of small-molecule fascin inhibitors

We had identified small-molecule inhibitors that decreased the actin-bundling activity of fascin from screening chemical libraries (Huang et al., 2015). One of the small-molecule inhibitors is N-(1-(4-(trifluoromethyl)benzyl)-1H-indazol-3-yl)furan-2-carboxamide (named G2) (Figure 1A). Compound G2 directly binds to fascin with a K_d value of 5–20 μM, and inhibited the actin-bundling activity of fascin [half-maximal inhibitory concentration (IC₅₀), 5–8 μM], but not L-plastin (another actin-bundling protein) (IC₅₀ > 100 μM) (Huang et al., 2015). Compound G2 blocked tumor cell migration and invasion (IC₅₀, 50–100 μM), and tumor metastasis in mouse models (decreased ∼95% at 100 mg/kg) (Huang et al., 2015). Hence Compound G2 is an attractive hit compound. To further explore and optimize the structure-activity-relationship of Compound G2 for greater potency to inhibit the actin-bundling activity of fascin, we designed, synthesized, and biologically evaluated G2 analogues and obtained improved fascin inhibitors (Figure 1 B–G). G2 analogues were individually tested for the ability to inhibit the actin-bundling activity of fascin (Some examples are shown in Figure 1 B–I). In this assay, purified fascin proteins were incubated with purified actin proteins in the absence or presence of different concentrations of analogues. Since one fascin molecule interacts with 4–5 actin molecules in the actin-fascin bundles, we used fascin (0.25 μM) and actin (1 μM) in a 1:4 ratio. After actin polymerization and bundling by fascin, the samples were centrifuged at low-speed to collect the actin bundles. Supernatants (free fascin, free actin and un-bundled F-actin polymers) and pellets (bundled actin-fascin filaments) were separated by SDS-PAGE. Gels were then stained with Coomassie blue to visualize the fascin and actin protein levels (some examples are shown in Figure 1C and F). The fractions of fascin proteins in the pellet (over total fascin proteins) were calculated and plotted against the concentrations of the analogues (Figure 1 D and G). Some of these modified analogues were more potent than G2. For example, NP-G2-044 had an IC₅₀ of ∼0.2 μM. We should note that, given our actin-bundling assay conditions, this IC₅₀ value is close to the lower sensitivity limit of our assay. Some of the modified compounds were inactive and these compounds could be used as negative controls. For example, Compounds NP-G2-112 and NP-G2-113 are structurally similar to Compounds NP-G2-044 and NP-G2-011, respectively, but were not active in inhibiting the activity of fascin (Figure 1H and I). These initial in vitro screenings of derivatives of Compound G2 will assist in the future development of compounds with improved potency, specificity, and pharmacological profiles for eventual clinical applications.

3.2 Mechanism of action of the fascin inhibitors

We investigated the biochemical mechanism of action by which fascin inhibitors block the actin-bundling function of fascin. To determine the possible binding region of Compounds NP-G2-011 and NP-G2-044 on fascin, we took advantage of mutant fascin proteins that we previously generated. We and others previously solved the X-ray crystal structure of fascin (Chen et al., 2010; Jansen et al., 2011; Sedeh et al., 2010). Based on the crystal structure, we generated 100 mutations of surface-exposed residues on fascin, and determined the actin-bundling and actin-binding activity of these 100 mutants (Yang et al., 2013). The residues that were critical for actin-bundling activity were clustered in three regions and may represent different actin-binding sites on fascin (Figure 2A). The X-ray crystal structures of fascin mutants from these three regions showed that these fascin mutants have the same structure (the inactive configuration) which is slightly different from that of the active configuration of wild-type fascin (Yang et al., 2013). Thus these mutations did not introduce major overall structural changes on fascin proteins. We have selected some of these fascin mutants and tested their sensitivity to Compounds NP-G2-011 and NP-G2-044. We found that fascin (E391A) (from the actin-binding site 1) retained the actin-bundling activity but was insensitive to the inhibition by Compounds NP-G2-011 and NP-G2-044 (up to 400 μM) (Figure 2 B and C). Together these data demonstrate that Compounds NP-G2-011 and NP-G2-044 may bind to the region around residue E391. However, we should note that our previous structural data showed that the actin-binding sites on fascin are conformationally interconnected. Mutations in any one of the actin-binding sites lead to structural changes in other actin-binding sites. Hence, we could not rule out the possibility of the binding of the fascin inhibitors to other actin-binding sites.

Based on studies on enzymes, there are four types of inhibitory mechanisms: competitive inhibition, uncompetitive inhibition, non-competitive inhibition, and mixed inhibition. Although both the fascin inhibitors and actin filaments could interact with fascin, given the different structures and sizes of the fascin inhibitors and F-actin filaments, it is unlikely the fascin inhibitors use a mechanism of competitive inhibition since competitive inhibitors are often similar in structure to the real substrate. It is also unlikely an uncompetitive inhibition (in which the inhibitor binds only to the substrate–enzyme complex) as the fascin inhibitors could directly bind to fascin in the absence of F-actin filaments (Huang et al., 2015). To distinguish the mechanisms of non-competitive inhibition (in which the binding of the inhibitor does not affect the binding of substrate) versus mixed inhibition (in which the binding of the inhibitor affects the binding of the substrate), we measured the binding of actin filaments to fascin (using high-speed centrifugation assay) in the presence of the small-molecule fascin inhibitors. As shown in Figure 2 D–I, Compounds G2, NP-G2-011 and NP-G2-044 all reduced the binding between fascin and actin filaments, with similar IC₅₀ values as their effects on the actin-bundling activity of fascin. With increased concentrations of inhibitors, the total amounts of actin filaments in the pellets were the same, but the amounts of bound fascin proteins were decreased (Figure 2 D, F, and H). Therefore, these data suggest these inhibitors act through mixed inhibition. These inhibitors likely change the conformation of fascin, reduce the binding of actin filaments, and thus lead to the inhibition of the bundling activity of fascin.

3.3 Effects on actin cytoskeletal reorganization by improved fascin-specific inhibitors

Next we explored the cellular effects of the improved fascin inhibitors on actin cytoskeletal reorganization which is critical for cell migration. Fascin is the main actin-bundling protein in filopodia. To critically evaluate that the fascin inhibitors indeed engage fascin in cells, it would be best to have a fascin mutant that does not bind to a fascin inhibitor but retains its actin-bundling activity. This mutant fascin should confer resistance to the fascin inhibitor in cells. Our above data show that fascin(E391A) is such a fascin mutant. Therefore, to investigate the specificity of these fascin inhibitors NP-G2-011 and NP-G2-044 in cells, we examined the filopodial formation in 4T1 breast tumor cells expressing wild-type fascin or fascin(E391A) mutant. While NP-G2-011 and NP-G2-044 abolished bradykinin-induced filopodial formation in 4T1 cells with wild-type fascin (Figure 3 A and C), NP-G2-011 and NP-G2-044 did not affect the filopodial formation in 4T1 cells re-expressing fascin(E391A) mutant (Figure 3 B and C). These data demonstrate that NP-G2-011 and NP-G2-044 engage the target fascin and block filopodial formation in cells, and confirm a critical role for the actin-bundling activity of fascin in filopodial formation.

Then we investigated the effect on lamellipodial formation. PDGF (platelet-derived growth factor) treatment could induce the formation of lamellipodia and membrane ruffles in a Rac-dependent manner (Jaffe and Hall, 2005). As shown in Figure 4 A and C, while PDGF induced lamellipodial formation in cells, this induction was blocked by the fascin inhibitors NP-G2-011 and NP-G2-044. To ascertain these inhibitors acted via fascin to block lamellipodial formation, we used 4T1 cells expressing fascin(E391A). PDGF still induced lamellipodial formation in these cells, but the induction was insensitive to NP-G2-044 (Figure 4, B and C). We further explored the mechanism by which fascin contributes to lamellipodial formation. Given the well-established role of fascin in filopodial formation, we tested the hypothesis that filopodia serve as templates for the formation of new lamellipodia (Johnson et al., 2015). To visualize the formation of new lamellipodia, we expressed actin-GFP in MDA-MB-231 cells. High-resolution time-lapse videos of randomly migrating cells were acquired and analyzed (Figure 4 D–H). As shown in Figure 4D, a filopodium (marked by the arrows at 0 and 30 s time points) extended and formed a lamellipodium (fan-shaped membrane protrusion at 60–150 s time points). Spatiotemporal maps of the protrusion and retraction speed of cell membrane extensions were generated (Figure 4E, left panel). These angular projection maps showed that lamellipodia coincide with protrusion over filopodia (Figure 4E, right panel). While NP-G2-044 treatment did not affect the overall speed at which the lamellipodia were extending (Figure 4F), fascin inhibition lowered the overall angular portion of the cell contour covered by lamellipodia (Figure 4G). Furthermore, in the absence of NP-G2-044, 78% of the lamellipodia overlapped with pre-existing filopodia (Figure 4H). NP-G2-044 significantly decreased the overlap of lamellipodia and filopodia (to 57%) (Figure 4H). These data demonstrate that filopodia could act as initiation hubs for new lamellipodia, and suggest that NP-G2-044 decreases filopodial formation resulting in decreased lamellipodial formation.

Finally we investigated any possible effect on stress fiber formation and focal adhesion turnover. Stress fibers are contractile actin bundles and provide force for cell adhesion and migration. LPA (lysophosphatidic acid) could induce actin stress fiber formation through a RhoA-dependent pathway (Jaffe and Hall, 2005). While we observed the induction of actin stress fiber formation by LPA, this induction was reduced by NP-G2-011 and NP-G2-044 (Figure 5 A and C). On the other hand, LPA could still induce actin stress fiber formation in cells expressing fascin(E391A) even in the presence of NP-G2-044 (Figure 5 B and C). These data demonstrate a role for fascin in actin stress fiber formation. Stress fibers are linked to the extracellular matrix via focal adhesions which are dynamic complexes. Focal adhesions allow the cell to communicate with and respond to its environment. We investigated the role of fascin in focal adhesion dynamics using our new pharmacological inhibitors of fascin. Since focal adhesion turnover has previously been shown to be related to FAK (a non-receptor tyrosine kinase) residency within focal adhesions (Bordeleau et al., 2010; Hamadi et al., 2005), we performed FRAP (fluorescence recovery after photobleaching) experiments on FAK-GFP expressing MDA-MB-231 cells. Confocal live-cell images of MDA-MB-231 cells expressing GFP-FAK were taken before photobleaching (Figure 5D). The recovery after photobleaching was recorded by time-lapse imaging (Figure 5E). The kinetics of recovery of GFP-FAK after photobleaching of focal adhesions from cells pre-treated with vehicle or NP-G2-044 were illustrated in Figure 5F. Upon NP-G2-044 treatment, the recovery half-life computed from the fluorescence recovery experiments decreased from 6.2 s to 3.4 s (Figure 5G), indicative of lower FAK residency and suggesting a more stable focal adhesion (Bordeleau et al., 2010; Hamadi et al., 2005). Furthermore, measurements of the focal adhesion turnover during a 40-min period using GFP-FAK expressing MDA-MB-231 cells and time-lapse confocal live-cell imaging revealed a corresponding 20–25% reduction in both the assembly and disassembly rates after fascin inhibition (Figure 5 H–L). Together, our data demonstrate a role for the actin-bundling activity of fascin in various actin cytoskeletal reorganization processes.

3.4 Inhibition of tumor cell migration

To test the effect of these G2 analogues on tumor cell migration, we studied the migration of tumor cells in the absence or presence of the analogues. We used the quantitative Boyden chamber assay. MDA-MB-231 human metastatic breast tumor cells were loaded onto the top of the Boyden chamber. After about six hours, cells migrated into the bottom of the chamber filter were counted. Among the analogues, four of them had improved potency in blocking tumor cell migration comparing to Compound G2 (Figure 6 A–F). While Compound G2 inhibited the migration of MDA-MB-231 tumor cells with an IC₅₀ of 50–100 μM, three of these analogues (NP-G2-036, NP-G2-044 and NP-G2-050) blocked tumor cell migration with IC₅₀ values of ∼10 μM (Figure 6 D–F). Compound NP-G2-112, which did not inhibit the actin-bundling activity of fascin, did not block the migration of MDA-MB-231 cells (Figure 6G). Hence some of the analogues of Compound G2 are with improved potency in cellular studies.

3.5 Inhibition of tumor metastasis in mouse models

Finally we tested the effect of Compounds NP-G2-011 and NP-G2-044 on tumor metastasis in animal models. In the orthotropic spontaneous tumor metastasis mouse model, 4T1 breast tumor cells were injected into the orthotropic site (the mammary gland) and then the metastasis to the lung was monitored (Chen et al., 2010; Shan et al., 2005; Yang et al., 2009) (Figure 7A). The 4T1 mouse tumor closely mimics human breast cancer in its anatomical site, immunogenicity, growth characteristics, and metastatic properties (Pulaski and Ostrand-Rosenberg, 1998). From the mammary gland, the 4T1 tumor spontaneously metastasizes to a variety of target organs including lung, bone, brain, and liver. Seven days after implantation of 4T1 tumor cells, we injected the mice with control solvent or Compound NP-G2-011 or NP-G2-044 (intraperitoneally and daily at 100 mg/kg). After 18 days, the mice were sacrificed and examined for metastasis in the lungs (Chen et al., 2010; Shan et al., 2005; Yang et al., 2009). Whereas mice injected with control solvent exhibited large numbers of metastasized 4T1 cells in the lungs, the number of metastasized 4T1 cells in the lungs of mice treated with Compounds NP-G2-011 or NP-G2-044 was reduced by ∼95% (Figure 7A). We observed that Compound NP-G2-011 or NP-G2-044 treatment had no effect on the body weight of the mice (Figure 7B). These compounds had a minor inhibitory effect on the growth of primary tumors at later stages as measure by the volume of the primary tumors in mice (Figure 7C) and the weight of the dissected primary tumors (Figure 7D).