Apoptotic melanoma cells treated with Rb44L1 peptide were examined by TUNEL staining, using the in situ Cell Death Detection Kit according with the manufacture's instruction (Roche Applied Science, Madison, WI). B16F10-Nex2 melanoma cells (1 ×104 ) were seeded on 96-well clear-bottom black polystyrene microplate and incubated with 0, 130 and 260µM of Rb44L1 peptide for 18 h. After incubation, cells were fixed in formaldehyde 2% for 20 min at room temperature, washed in PBS, and incubated with Hoechst 33342 (Invitrogen, Eugene, OR), at 10µg/mL final concentration in the reaction buffer and TUNEL enzymatic substrate. Cells were washed and images were acquired and analyzed in a Cytell Cell image cytometer (GE Healthcare, Little Chalfont, UK).

#### Annexin V and Propidium Iodide Labeling

B16F10-Nex2 cells (5 ×105 ) were cultured in 6-well plates and further incubated with Rb44L1 at 0, 80 and 100µM for 18 h at 37◦C. After incubation, the Annexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich, St. Louis, MO) was used and cells labeled with propidium iodide (PI) and FITC annexin V (AV) were analyzed by flow cytometry (BD Bioscience FACSCanto II equipment, Franklin Lakes, NJ), using FlowJo software (TreeStar Inc., Ashland, OR).

### Cell Cycle Analysis

B16F10-Nex2 (5×105 ) cells were seeded in conical centrifugation tubes and incubated with 65µM Rb44L1 peptide for 16 h in suspension. After incubation, the cells were washed with PBS and fixed in ethanol 70% for 1 h at 4◦C. Cells were then washed again with PBS and stained with propidium iodide (PI) solution (50µg/ml PI, 0.1 mg/ml RNAse A) for 20 min at 4◦C in the dark. DNA fluorescence staining was acquired by FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). FlowJo software (Tree Star Inc., Ashland, OR) was used for post-acquisition analysis (20.000 events per sample). The microtubule depolymerizing CA4 (combretastatin A4, Sigma-Adrich, St. Louis, MO) was used at 75µM as positive control of G2/M cell cycle arrest.

#### Transmission Electron Microscopy

B16F10-Nex2 cells (1 ×106 ) were seeded in 6-well plates. Cells were then incubated with peptide Rb44L1 at 260µM for 18 h at 37◦C. Fixation, dehydration and staining of the samples were performed as previously described (23). Jeol 1200 EXII electron microscope (Tokyo, Japan) was used for image acquisition.

## Mitochondrial Membrane Potential (1ψm)

B16F10-Nex2 cells (1 ×104 ) were pre-incubated with the cationic lipophilic dye tetramethylrhodamine ethyl ester (TMRE) at 20 nM for 30 min, and then with peptide Rb44L1 at 0, 130, and 260µM for 6 h. After the incubation period, images of living cells were acquired and analyzed by Cytell Cell Imaging System (GE Healthcare, Little Chalfont, UK).

#### Superoxide Anion Measurement

Superoxide anion production was measured by dihydroethidium (DHE) assay. Briefly, 1 ×104 cells cultivated on 96-well clearbottom black plate were pre-incubated with DHE for 30 min at 37◦C. Rb44L1 was added at 130 and 260µM concentrations and fluorescence units were quantified after 16 h in a microplate reader (Molecular Devices M2, Sunnyvale, CA) adjusted for excitation at 370 nm and emission at 420 nm. As positive control, cells were treated with 5 mM of H2O2 at 37◦C for 20 min, and the negative control run with no peptide.

#### Cell Lysate Extracts and Western Blotting

B16F10-Nex2 cells (106 ) were incubated with 0 and 130µM of Rb44L1 peptide for different times (1, 3, 6, 8, and 24 h). After incubation, cells were washed in PBS and lysed with 300 µL of SDS sample buffer (62.5 mM Tris-HCl, pH 6.8 at 25◦C, 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue). Proteins from whole cell extracts were analyzed by Western blotting as previously described (20). The following primary, highly specific monoclonal antibodies, were used: rabbit anti-Bcl-2, -Bcl-xl, -Bax, -caspase-9 and cleaved caspase-9, caspase-3 and cleaved caspase-3, -Parp and cleaved Parp, and -GAPDH (for total protein loading control), with secondary anti-rabbit IgG conjugated with horseradish peroxidase (HRP). All antibodies were purchased from Cell Signaling Technology (Beverly, MA) except for anti-GAPDH, acquired from Sigma-Aldrich (St. Louis, MO). Immunoreaction was revealed using the LuminataTM Forte solution (Millipore, Billerica, MA) and images were acquired using Uvitec Cambridge (Cambridge, UK). The molecular mass of each protein was estimated based on a prestained protein standard (Spectra Multicolor, ThermoScientific, Waltham, MA). Full-length Western blotting membranes are displayed in **Figure S1**.

#### In vitro Angiogenesis Assay

The basement matrix GeltrexTM (Invitrogen, Eugene, OR) was added (30 µl/well) to coat a 96-well plate and allowed to polymerize for 40 min at 37◦C. HUVEC cells (5 ×103 ) suspended in RPMI medium supplemented with 0.2% of fetal calf serum were plated alone (control) or mixed with 5µM of Rb44L1 peptide. The cells were incubated at 37◦C for 6 h and images were captured with a microscope digital camera (Olympus, Tokyo, Japan). The numbers of pro-angiogenic structures (typically closed compartments or rings formed after endothelial cell sprouting) were counted from 3 different wells.

#### Ethics Statement

The present study is part of Project 2010/51423-0 granted by the São Paulo State Research Support Foundation (FAPESP), Brazil. The protocols used for animal experiments were carried out in accordance with the Ethics Committee of Federal University of São Paulo, Brazil and have been approved via document CEP 1234/2011.

#### Mice and Subcutaneous Melanoma Model

Eight-week-old male C57Bl/6 mice were acquired from the Center for Development of Experimental Models (CEDEME) at Federal University of São Paulo (UNIFESP), Brazil. The Ethics Committee for Animal Experimentation (UNIFESP) approved protocols of animal experiments. In the subcutaneous (s.c.) melanoma model, male C57Bl/6 mice (five per group) were subcutaneously grafted in the right flank with 1 ×105 syngeneic B16F10-Nex2 melanoma cells. Animals were subjected to 5 peritumoral daily doses of 300 µg (total 10 mg/kg) of Rb44L1. DMSO (1%) in PBS, was the vehicle control. Treatment started after the tumor size reached 80 mm3 as measured with a caliper. The tumor volume (V) was calculated by the formula V = 0.52 × d 2 × D, where d and D are short and long diameters of the tumor, respectively, measured every other day. Mice were euthanized at the end of experiments or when the tumor size reached the maximum allowed volume of 3,000 mm3 .

#### Live-Cell Imaging of Microtubule Dynamics

Real-time fluorescence microscopy of living B16F10-Nex2 melanoma cells previously modified by viral transduction for the expression of green fluorescent tubulin (CellLight R Reagents −2.0 BacMam, Life Technologies), was used to investigate the peptide interaction with microtubules. Viable green fluorescence protein (GFP) tubulin-expressing cells (1 ×104 ) were incubated with Rb44L1 and Rb29L1 at 260µM and fluorescent images were taken at 10-min intervals during 2 h using the time-lapse BioStation fluorescence microscope (Nikon Instruments, Inc, Melville, NY). For instance, humidity, temperature (37◦C) and CO2 (5%) were carefully controlled. Fluorescence analysis and quantification were performed with the ImageJ software and the video was processed with the NIS-Elements analysis software (Nikon, Tokyo) and Adobe After Effects software.

### Fluorescence Staining of F-Actin

B16F10-Nex2 cells (5 ×104 ) were seeded in 24-well microplates and incubated with different concentrations of Rb44L1 (0, 130 and 260µM) for 30 min and 3 h. After incubation, cells were fixed in 3.7% of formaldehyde for 20 min at 4◦C, blocked (1% BSA, 5% SFB, 0.1% Triton in 1X PBS) for 30 min at room temperature and stained with Hoechst 33342 (Invitrogen, Eugene, OR) and anti-phalloidin conjugated with FITC for 1 h at 37◦C. Images were acquired and analyzed by Cytell Cell Imaging System (GE healthcare, Little Chalfont, UK).

#### System Preparation and Molecular Dynamics

De novo peptide structure prediction was made by Pep-Fold3 webserver (30). We obtained the tubulin structure from PDB 4TV9 (31) (chains A and B). Protonation analysis was made by PROPKA3 (32). Energy minimization was carried out on GROMACS 5.1 (33) using CHARMM36 force field (34). Systems were built by CHARMM-GUI webserver (35, 36) with TIP3P water molecules (37) and counter ions, when charge balancing was required. Simulations consisted of 5,000 steps of steepest descent energy minimization, followed by 25 ps of NVT equilibration dynamics for L1-CDR peptides and 10 ns for tubulin. A NPT production molecular dynamics of 100 ns was carried out on GROMACS 5.1 using CHARMM36 force field for each system. Secondary structure assignment and hydrogen bonds (H-bonds) were analyzed by using VMD (38) plugins. Hbonds distance cut-off was set up at 3.0 Å with angle cut-off of 20◦ . All further MD analyses were made by GROMACS 5.1.

#### Normal Mode Calculations and Generation of Low-Energy Conformations

Normal mode analysis (NMA) was performed using CHARMM c41b1 (39) and CHARMM36 force filed using DIMB (40) module and excluding CMAP (41). A distance dependent dielectric constant was employed to treat the electrostatic shielding by the solvent as described by Philot et al. (42). We used the mode 08 (open/close of tubulin monomers) as directional constraint to generate low-energy conformers along the mode trajectory using the VMOD algorithm in CHARMM as depicted by Louet et al. (43). The restraints were applied only on Cα atoms and the energy was computed for all atoms. The structures were displaced from 0.0 Å to +6.0 Å (open direction) using steps of 1.0 Å, resulting in 7 intermediate low-energy structures along the mode.

#### Molecular Docking

In order to obtain different structures to perform molecular docking, we clustered the MD trajectory of each peptide. All MD frames were fitted to the reference structure and clustered with GROMOS method by using GROMACS 5.1, with a backbone RMSD cutoff of 2.0 Å for Rb29 and Rb44 and 5.0 Å for C36 (since the last is very flexible) resulting in 3, 11, and 8 different clusters, respectively. The center structure of each peptide cluster was then used in docking simulations, performed with Hex 8.0 (44). Hex depicts proteins as rigid bodies and makes a blind search through protein surface while it evaluates the interaction correlation by using the fast Fourier transformation algorithm. As described in Meissner et al. (45), solvation and desolvation effects were treated as surface phenomena, since the Hex algorithm models the interaction, excluding volume and complementarity of form. Approximately 350 solutions were found for each combination. We used BINANA 1.2 (46) as a rescore method to investigate the specific molecular basis guiding the interaction between tubulin and peptides.

#### Chemiluminescent Dot-Blotting

Peptide Rb44L1 binding to microtubule structures was determined by chemiluminescent (CL) dot-blotting as described elsewhere (26) with some modifications. Peptides C36L1 (positive control), Rb44L1, scrambled-Rb44L1 (Scr44) at 10 µg/10 µL each, or vehicle (1% DMSO in milli-Q water), were applied on nitrocellulose membranes. They were blocked with 5% BSA in 0.05% PBS-Tween 20. B16F10-Nex2 cell protein lysate (50µg/ml), prepared with non-denaturing protein extraction buffer according to the manufacturer's instructions (Cell Signaling, Beverly, MA), was applied onto the nitrocellulose membranes and incubated overnight at 4◦C. After washing, membranes were incubated with anti-alpha tubulin antibody (Sigma-Aldrich, St. Louis, MO) for 1 h at 37◦C followed by anti-rabbit IgG-HRP antibody for 1 h at 37◦C. Immunoreactivity was determined using the LuminataTM Forte solution (Millipore, Billerica, MA). Images were acquired by Uvitec Cambridge (Cambridge, UK) with 1-min membrane exposure time. No reactivity with the control peptide was observed. To investigate the influence of GTP and Mn2+ on the peptide binding with αtubulin, the membranes coated with 10 µg Rb44L1 or scrambled (Scr44) peptide were blotted with or without 1 mM GTP (Cytoskeleton, Denver, CO) and/or 1 mM Mn2SO. 4H2O (Sigma-Aldrich, St. Louis, MO) added to the cell lysate (50 µg/ml), for 2 h at 37◦C. Chemiluminescence was detected as described above but with short membrane exposure time (20 s).

#### Tubulin Polymerization Assay

Microtubule polymerization was evaluated using the Tubulin Polymerization Assay kit (Cytoskeleton, Inc., Denver, CO). Rb44L1 (130µM) or Scr44 (130µM); colchicine (50µM); Rb44L1 (130µM) + colchicine (50µM), diluted with 1% DMSO in distilled water were added to 50 µl of the tubulin reaction mix with optimized volumes for inhibitor detection containing 2 mg/ml or 1 mg/ml of tubulin in 80 mM PIPES (piperazine-N-N'-bis [2- ethane sulfonic acid] sodium salt), pH 6.9, 2 mM MgCl2, 0.5 mM EGTA (ethylene glycol-bis N,N,N',N'- tetra acetic acid), 60% v/v glycerol, 1 mM GTP, and 10µM of the fluorescent reporter. The black, flat bottom, half area 96-well plate, with the samples, was examined in a fluorescence microplate reader (SpectraMax-M2e, Molecular Devices, Sunnyvale, CA) every 1 min at 340 nm of excitation and 410 nm of emission for 40 or 180 min. To monitor the tubulin polymerization in the same condition as of the dot blotting assay, the reaction was prepared as described above with 2 mg/ml of purified tubulin in 0.1% of BSA in PBS and 3.4% of cell lysis buffer, without cell lysate.

#### Statistical Analysis

The software GraphPad Prism 5.0 (San Diego, CA) was utilized for all tests. Statistical differences between groups were compared by Student's t-test. Differences in survival time and rate were evaluated by the Kaplan-Meier survival curves. P-values are indicated as ∗p < 0.05, ∗∗p < 0.01 and, ∗∗∗p < 0.001.

### RESULTS

### L1-CDR Peptides Differ in Dynamic Features

Peptides Rb44L1 and Rb29L1 were studied in comparison with peptide (C36L1), which exerts cytotoxicity by depolymerization of microtubules and displays antitumor activities, as previously investigated (26).

In spite of the sequence similarity, the dynamics of L1- CDRs were very different from each other. Rb29L1 assumed a stable β-hairpin conformation, with residues 5 SLL and 13TYL forming the β-sheet (**Figures 1A,B**). In turn, Rb44L1 showed only an intermittent β-bridge between residues 5TI and 14YL (**Figures 1C,D**). C36L1, however, did not assume any ordered structure (**Figures 1E,F**). Root-mean-squared deviation (RMSD) of backbone heavy atoms and Cα rootmean-squared fluctuation (RMSF) calculations were performed to evaluate structure stability along the molecular dynamics (MD). Results confirmed the stability of Rb29L1, while C36L1 showed several conformational shifts (**Figure 1G**). Flexibility analysis confirmed this profile (**Figure 1H**). H-bonds formation during the dynamics could address these structural differences among the peptides. Rb29L1 showed more internal H-bonds than the other peptides, therefore it is more rigid. **Table 1** summarizes these interactions. The trajectories of each peptide MD were clustered, according to RMSD, onto representative conformations to perform docking simulations (**Figure S2**).

### In vitro Cytotoxicity of CDR Peptides

We investigated the anti-tumor potential of two L1-CDR-derived peptides: Rb44L1 from anti-Lewis B mAb and Rb29L1 from anti-A34 mAb. The IC50 values were determined for the Rb44L1 and Rb29L1 against different tumorigenic and non-tumorigenic cell lines (**Table 2**). Peptide Rb44L1 showed the lowest IC50 values as compared to Rb29L1. The concentrations of 130 µM (IC50) and 260µM (IC100), respectively, were therefore used in the subsequent experiments with B16F10-Nex2 melanoma cells. Rb44L1, was less active against non-tumorigenic cells, including murine and human fibroblasts, 3T3-NIH and GM637 cell lines. In the concentration range of 0 to 0.140 mM, no cytotoxicity was observed in these cells. Rb29L1 IC50 values were 3- to 10-fold higher than those of Rb44L1 in tumorigenic cell lines.

#### Rb44L1 Induces Apoptosis

Changes in the dynamics of the cytoskeleton have been implicated in the induction of apoptosis. Here, we show that Rb44L1 induced morphological alterations typical of apoptotic cell death such as cellular shrinkage, membrane blebs and cell rounding-up with pseudopodia retraction in B16F10- Nex2 melanoma cells when incubated with peptide at IC50 (130µM) and IC100 (260µM) for 18 h (**Figure 2A**). Chromatin condensation was observed in 95% and 98% of tumor cells treated with Rb44L1 at 130 and 260µM, respectively, for 18 h. DNA fragmentation was determined by green positive TUNEL staining in B16F10-Nex2 cells treated with 130 and 260µM of Rb44L1 (**Figures 2B,C**). Both DNA condensation and fragmentation were significantly higher in Rb44L1-treated cells as compared with the negative control (for chromatin condensation, ∗∗p < 0.01 at 130µM, ∗∗∗p < 0.001 at 260µM; and for DNA fragmentation, ∗∗∗p < 0.001 at both concentrations). Additionally, we observed that Rb44L1 could significantly enhance the translocation of phosphatidylserine (PS) to the outer leaflet of the plasma membrane, indicating early apoptosis. We observed a significant increase in the number of early apoptotic events in cells treated with Rb44L1 at 80 and 100µM, in comparison with untreated control cells (**Figure 2D**). Finally, Rb44L1 inhibited cell proliferation with cell cycle arrest, at 65µM (**Figure 2E**). The S-phase area decreased from 22.3 to 13.4%, with increase of the G2/M phase (from 21.8 to 33.5%). Microtubule depolymerizing combretastatin-A4 was used as positive control.

#### Morphological and Functional Alterations in Mitochondria and ROS Production

Transmission electron microscopy (TEM) of Rb44L1-treated B16F10-Nex2 cells, at 260µM for 18 h, showed condensed chromatin, nuclear membrane detachment, enlarged, and vacuolated mitochondria with damaged cristae surrounded by heavily injured cytoplasmic organelles compared to untreated cells (**Figure 3A**). The collapse of the mitochondria transmembrane potential (1ψm) was observed on early incubation with Rb44L1 (0, 130, and 260µM). After 6 h, reduction of TMRE fluorescence (53 and 94% reduction in cells treated with 130 and 260µM, respectively; ∗∗p < 0.01 and ∗∗∗p < 0.001 in relation to untreated cells) was observed indicating mitochondrial damage in these cells (**Figure 3B**).

FIGURE 1 | Secondary structure assignment during molecular dynamics and structural analysis of L1-CDRs. (A,B) Rb29L1 assumes a stable β-hairpin conformation during MD, showing a well established β-sheet between residues 5SLL and 13TYL; (C,D) Rb44L1 shows a recurrent β-bridge between residues 5TI and 14YL; (E,F) C36L1 presents the most flexible conformation, in its majority composed by turn and coil. Secondary structure color code: turn, in green; extended conformation (β-sheet), in yellow; isolated bridge, in gold; 3-10 helix, in blue; coil, in white; (G) root-mean-squared deviation of backbone atoms of Rb29L1, Rb44L1, and C36L1. Rb29L1 remains nearly at the same conformation during all MD, an effect also seen for Rb44L1, although with less intensity. C36L1, nonetheless, presented a great conformational variation; (H) root-mean-squared fluctuations of Cα atoms of Rb29L1, Rb44L1 and C36L1. Cα fluctuation, or flexibility, follows RMSD pattern. Rb29L1 presents a rigid structure while Rb44L1 and C36L1 are more flexible, the latter more pronounced.

Tumor cells were incubated with Rb44L1 at 130 and 260µM for 16 h and ROS levels were detected using DHE dye measured by fluorimetry. Hydrogen peroxide (H2O2) was used as positive control (Control +) at 5 mM. Accumulation of ROS (59% in relation to untreated cells; ∗∗∗p < 0.001) was observed in cells treated with Rb44L1 at both concentrations (**Figure 3C**).

#### Rb44L1 Elicited Caspase Activation

Different pro- and anti-apoptotic proteins in total cell lysates were evaluated by Western blotting in Rb44L1-treated B16F10- Nex2 cells at 130µM and different incubation periods. We observed that Rb44L1 induced early increase of pro-apoptotic Bax protein, followed by the cleavage of caspase-9, caspase-3 and TABLE 1 | Hydrogen bonds formation during molecular dynamics of L1-CDR peptides\*.

TABLE 2 | IC50 values of the bioactive peptide Rb44L1 and control Rb29L1 against tumorigenic and non-tumorigenic lineages after 16 h of incubation.

\*Non-tumorigenic cell lines.

PARP, together with downregulation of anti-apoptotic protein Bcl-2 (**Figure 3D**). GAPDH was used as loading control.

#### Rb44L1 Inhibited Angiogenesis in vitro

The cytotoxicity of Rb44L1 at different concentrations was assayed in the HUVEC lineage (**Figure 4A**). A non-cytotoxic concentration was used for the inhibition of endothelial cell (HUVEC) sprouting in GeltrexTM Matrix. Rb44L1 at 5µM for 6 h, significantly inhibited 90% of endothelial cell sprouting, with the number of compartments built by intercellular connections being compared to that of the control (∗∗p < 0.01; **Figures 4B,C**).

#### Antitumor Activity in vivo Against Subcutaneous Melanoma

The in vivo antitumor activity was also investigated in a subcutaneously grafted, syngeneic murine melanoma model. Peritumoral injections of Rb44L1 at 15 mg/Kg significantly delayed tumor volume progression (∗∗p < 0.01), and also prolonged mice survival (∗∗p < 0.01) (**Figures 4D,E**). Mice were euthanized at the scheduled end of experiments, or before, should the tumors ulcerate or reach the maximum allowed volume of 3,000 mm3 .

#### Rb44L1 Interacts With Microtubules and Induces Cytoskeleton Disruption in Melanoma Cells

Disruption of the microtubule integrity in B16F10-Nex2 cells was monitored during the incubation with Rb44L1 and Rb29L1. Microtubules were assessed by live-cell imaging using B16F10-Nex2 cells previously transduced with a genetic modified insect virus (baculovirus) containing a tubulingreen fluorescent fusion-protein construct (CellLight R , Life Technologies). The fluorescence of live murine melanoma cells was monitored and quantified for 2 h during incubation with 260µM of Rb44L1 and Rb29L1. The Rb44L1 peptide drastically reduced microtubule fluorescence compared to the negative control (**Figures 5A,B**), indicating that the microtubule network was depolymerized during the incubation with Rb44L1, whereas no depolymerization was seen in Rb29L1 treated cells. A representative video showing the kinetics of microtubule depolymerization in B16F10-Nex2 cells during the incubation with Rb44L1 and Rb29L1 is available in **Video S1**.

In addition to investigating whether Rb44L1 would also affect the integrity of F-actin, the reaction was assessed simultaneously using a phalloidin-FITC probe, as described in methods. We observed that F-actin integrity was completely lost after 3 h of incubation with Rb44L1 at 260 and 130µM (**Figure 5C**). Actin degradation occurred after the microtubule disruption process, as evidenced in the cytoskeleton integrity quantification analysis (**Figure 5D**), suggesting that actin filaments were disrupted as a consequence of microtubule depolymerization (∗p < 0.05 comparing microtubule and actin disruption). Less than 55 or 65% of cytotoxicity was seen when testing both concentrations of Rb44L1 at 260 and 130µM, respectively, in the first hours of incubation (**Figure S3**).

#### Normal Modes Expose Nonexchangeable Nucleotide and Colchicine Binding Sites

Normal mode analysis (NMA) was employed to investigate the opening motion of tubulin monomers. We hypothesized that this opening motion would be required to expose the nucleotide binding site located at α-tubulin (N-site) and dimer interface. Such exposition could favor the efficient docking of L1-CDR peptides and impair the tubulin dimer assembly, finally leading to microtubule dissociation. This motion was verified as the normal mode 8 (**Figures 6A,B**). Using the VMOD routine implemented on CHARMM, we performed a mass-weighted displacement of tubulin structure along mode 8, to produce energy-relaxed structures with gradually exposed nucleotide site. Tubulin residues originally in contact with GTP (contacts within 4.5 Å) showed a solvent-accessible surface area (SASA) of 511

Ų at the crystallographic structure (PDB 4TV9), while the same residues were more exposed after a displacement of 6 Å, presenting a SASA of 588 Ų (**Figures 6C,D**). The same occurred for the colchicine site, which presented a SASA of 225 Ų before the displacement and 236 Ų after mass-weighted displacement of 6 Å.

caspase-3,−9, cleaved PARP, Bax, Bcl-2, and Bcl-xl during Rb44-induced apoptosis are shown by Western blotting. GAPDH was used as loading control. A single cell-lysate sample was used in the same experiment and the Western blotting membranes were processed in parallel for antibody reactivity. Uncropped, full-length blottings are shown in Figure S1.

#### Docking Studies Reveal the Importance of Electrostatic Interactions

Docking calculations were performed using 7 tubulin structures generated from NMA displacement against the central structure of each L1-CDR peptide cluster (3 for Rb29L1, 11 for Rb44L1, and 8 for C36L1). In every docking round, an average of 350 different solutions was calculated. We then evaluated the best solution from Hex with BINANA to better understand the key binding characteristics governing the interaction.

Results indicated less favorable interactions for Rb29L1 than C36L1 and Rb44L1 in almost all displacements (as summarized in **Table 3**, detailed in **Table S1**, respectively), according to experimental results. At the best pose for Rb44L1 (docked with tubulin displaced by 2 Å) the 1R side-chain is buried in the cavity formed between tubulin monomers, participating in 3 of 6 H-bonds and 2 salt-bridges (**Figures 7A,C,E,G**). In fact, interactions involving 1R were observed in all displacements except at 3 Å and 4 Å. This indicates the putative importance of this residue to maintain the interaction with tubulin. When the 1R is replaced by alanine, the results showed a systematic worsening of energy values (as summarized in **Table 3**, detailed in **Table S1**). Biological assays confirmed this prediction since the R1A substitution in Rb44L1 was not cytotoxic to B16F10- Nex2 cells, in the 0 to 500µM range (data not shown). C36L1 pose analysis also indicated the involvement of a basic residue governing the interaction with tubulin. The 13K was present participating of H-bond, salt-bridge or cation-pi interactions in all tubulin displacements but at 5 Å. At the best pose–docked with tubulin displaced by 4 Å, 13K appeared in two H-bonds and in a salt-bridge (**Figures 7B,D,F,H**). Moreover, its side-chain was also buried in a cavity between tubulin monomers. On the other hand, although Rb29L1 had a greater number of H-bonds, the lack of

charged residues would contribute to predicted energies higher than the other peptides.

### Docking Studies Showed the L1-CDR Interactions Preferentially at the Nonexchangeable Nucleotide-Binding Site

We evaluated the best docking pose for both Rb44L1 and C36L1 in relation to the exposed nucleotide and colchicine binding sites. Rb44L1 interacted with three residues of the N-site (11Q, 69D, and 71E) and with one residue of the colchicine site (252K). The 1R participated in all interactions. C36L1, however, interacted with different residues of the N- site (71E, 11Q, 224Y, 206N, 177V) and one residue of colchicine site (179T). These interactions depended on 13K and 11N residues of the C36L1 peptide (**Figures 8A,B**). Rb29L1 showed interactions with tubulin similarly with those of C36L1 (177V, 179T, 206N, and 224Y). In contrast, there were interactions shared with Rb44L1 and C36L1 (11Q and 71E), which were absent in Rb29L1 (**Figure 8C**). Taken together, these results showed that tubulin-opening motion corresponded to a decrease of summed electrostatic energy values of the displaced structures (**Figure 8D**).

Both Rb44L1 and C36L1 interacted with the region of helices α2, α3 and α8 of α-tubulin subunit, and showed differences in relation to β-tubulin monomer. While Rb44L1 interacts with loops α1β1 and α7α8, C36L1 interacts with loop β9α11 and helix α11. The overlapping of C36L1 and Rb44L1 best poses showed residues 13K and 1R occupying the same region at the tubulin dimer interface, that is blocked by residues 70LEPT of α-tubulin and 243PGQL of β-tubulin in a minimized structure (**Figure 8E**). Rb44L1 interaction with α-tubulin subunit was further confirmed using a chemiluminescence dot-blotting assay. We observed that Rb44L1 significantly bound to α-tubulin present in B16F10-Nex2 cell extract, as compared to the negative

control and the scrambled peptide (Scr44), which was inactive. The C36L1 peptide was used as a positive control (**Figure 8F**). Different concentrations of the coated peptide Rb44L1 were tested and we found 10 µg/10 µl to give the best resolution in the dot-blotting (**Figure S4**). Interaction with β-actin was also evaluated and no reaction was seen (data not shown). As the docking studies revealed that the Rb44L1 interacted preferentially close to the N-site, we investigated the influence of additional GTP and Mn2+ on the peptide binding to α-tubulin in a dot-blotting assay with fixed peptide and melanoma cell lysate as a source of α-tubulin (monomeric, modified, dimeric). The peptide binding was enhanced in the presence of both GTP and Mn2+, but not with these agents added separately (**Figure S5**). Since the GTP N-site is nonexchangeable and noncatalytic, most likely the addition of GTP and Mn2+ triggered tubulin assembly by interacting on the E-site. Oligomeric tubulin bound to the peptide explains the increased reactivity with anti-α-tubulin antibody used to reveal the dot-blotting assay.

#### Rb44L1 Inhibits Purified Tubulin Assembly

The microtubule destabilization effect of Rb44L1 was also evaluated using a fluorescence recombinant tubulin polymerization assay kit (Cystoskeleton, Inc., Denver, CO). In this setting and starting with 2 mg/ml tubulin, 0.2 mg/ml of Rb44L1 delayed tubulin assembly and reduced approximately 1/4 of the total assembly capacity compared to the control ( ∗∗∗p < 0.001) and the scrambled peptide, Scr44 (**Figures 9A,B**). This effect was significantly more evident at half the tubulin concentration (1 mg/ml) and 0.2 mg/ml of Rb44L1 (∗∗∗p < 0.001

compared to the control). Polymerization was inhibited in 3/4 followed by depolymerization, after approximately 150 min incubation (**Figures 9C,D**). Since colchicine is a well-known microtubule inhibitor and has a binding-site mostly on β-tubulin, we assayed the effect of simultaneous addition of colchicine and Rb44L1. Increased inhibition of tubulin assembly was observed with this combination, suggesting independent interaction sites of Rb44L1 and colchicine, ∗∗∗p < 0.001 compared to the colchicine alone (**Figure 9E**). It should be pointed out that single drugs such as the MT- depolymerizing colchicine and the MT-polymerizing paclitaxel when used in combination, the depolymerization effect has predominated (47).

#### DISCUSSION

The microtubules together with various stabilizing and destabilizing molecules display many important physiological functions. Due to their indispensability in the mitotic cell division, microtubules have been selected as preferred anticancer targets. Indeed, microtubule directed drugs are among the most commonly prescribed agents in cancer chemotherapy (2). Recently, anti-tumor peptides targeting microtubules (26) have been studied as tubulin interacting ligands that may evolve to be used in cancer therapy.

Novel anti-tumor peptides may have advantages over mAbs and tyrosine-kinase inhibitors, such as low cost, high specificity and potency due to their compatibility with targeted proteins, ability to penetrate the cell membrane, reduced immunogenicity, and improved safety (48). For example, the ADH-1 (Exherin), is an anticancer peptide distributed by Adhex Technologies R , which targets N-cadherin and induced partial and complete protective responses in patients with metastatic melanoma (49).

The microtubule destabilizing Ig VL CDR1 peptide (C36L1) triggered cytotoxic and cytostatic effects on melanoma cells in vitro (23). Besides C36L1, we found that another CDR-L1 derived peptide, from anti-Lewis B mAb, exhibited similar cytotoxic mechanisms, targeting microtubules (MT). In the present work, we studied the molecular structure and biological effects of different L1-CDR-derived peptides: C36L1, Rb44L1 and Rb29L1 on microtubules. We analyzed the structure of L1-CDRdestabilizing MT peptides C36L1 and Rb44L1, as compared to the inactive one, Rb29L1. The latter demonstrated the most stable and rigid structure, assuming a β-hairpin conformation with several high occupancy H-bonds. Rb44L1 showed less rigidity as compared to Rb29L1, with a stable β-bridge conformation, while C36L1 was the most flexible peptide among them.

The biological effects of the peptides were examined and Rb44L1 showed the highest cytotoxic activity, selectively in different cancer cell lines with no significant effects on nontumorigenic cell lines (**Table 2**).

Morphological and biochemical changes during tumor cells incubation with cytotoxic concentrations of Rb44L1 were

TABLE 3 | Key binding characteristics governing tubulin and L1-CDR interaction.

Best poses are highlighted in bold (energy values in kJ/mol).\*Predicted summed electrostatic energy by atom-type pair according to Gasteiger partial charges.

observed. Apoptosis was recognized by the remarkable shrinkage of the cytoplasm, roundup cells with pseudopodia retraction and shriveling without cell lysis, genomic DNA condensation and fragmentation, and exposure of phosphatidylserine at the surface of peptide-treated cells (50). The intrinsic pathway involves the functional deregulation of mitochondria, which may culminate in activation of caspases and the cascade of events that drives to cell death (51, 52). Early disruption of mitochondrial membrane potential, as evidenced by time-lapse fluorescence microscopy and TEM, together with later production of ROS, cleavage of caspase-9, caspase-3, the PARP, upregulation of Bax and downregulation of Bcl-2 were effects induced by Rb44L1, and they are all consistent with the intrinsic pathway of apoptosis (53, 54), strongly suggesting that this is the main in vitro cytotoxic mechanism of the peptide in melanoma cells.

P53 is activated in response to different stresses leading tumor cells to apoptosis and growth arrest (55). In this regard, accumulation of active p53 may also be attributed to disintegration of the cytoskeleton. Microtubule targeted-drugs are one of the main stimuli able to increase levels and activate p53 (56).

The main mechanism that seems to be involved in the intrinsic apoptosis by Rb44L1 peptide is the early disruption of the microtubules in melanoma cells. Rb44L1 destabilized labeled microtubules during early stages of incubation, as observed by fluorescence microscopy. In contrast, Rb29L1 did not affect the microtubule dynamics, under the same conditions.

The actin cytoskeleton integrity was also evaluated, as observed by fluorescence microscopy. Rb44L1 induced the degradation of actin filaments in melanoma cells to a maximum effect after 3 h of tumor cell treatment with this peptide. Alterations of actin dynamics are sufficient to induce apoptosis. They involve changes in F-actin levels, in the flux of actin through the filament pool, or both (57). In addition, F-actin depolymerization has been implicated in reduced MMP and elevated ROS production, together with shortening of cell lifespan (58), as observed in melanoma cells treated with Rb44L1. The peptide, however, did not directly interact with F-actin to induce depolymerization as suggested by a late kinetics, which follows microtubule depolymerization. In fact, the actin cytoskeleton integrity has been shown to be highly dependent on the microtubule dynamics (59, 60), which is crucial in tumor cells constantly entering the mitotic program as compared to non-tumorigenic cells (2). Cellular functions depend on the crosstalk between microtubules and actin filaments, in which specific proteins bind to microtubules and actin filaments simultaneously, promoting co-organization and coupled growth of both networks (61). Both cytoskeleton constituents are intrinsically related and rearranged during the progress of apoptosis. Important events are regulated by ROCK kinases that actively regulate the actomyosin contractile ring, a process facilitated by the early disruption of microtubules. Protrusions of the plasma membrane also called apoptotic bodies or blebs, are formed, with subsequent depolymerization of actin filaments (62).

Rb44L1 interaction with microtubules and induction of their depolymerization with subsequent degradation of actin filaments increased the number of tumor cells in the G2/M phase leading to a mitotic catastrophe. Such effects, coupled to inhibited angiogenesis as observed in vitro, are consistent with the described effects of other microtubule targeting drugs (2, 63). A schematic illustration of the effects induced by Rb44L1 on melanoma cells is detailed in the **Figure 10**.

Most importantly, this peptide showed a promising antitumor protective effect against subcutaneously grafted melanoma, with no systemic toxicity being observed.

Once proteins exist in equilibrium of multiple conformations in solution, we used a theoretical approach that mixed analyses of molecular dynamics and normal modes, to sample distinct structural states of α/β-tubulin dimer. This hybrid methodology allowed for the assignment of both local and collective motions of the system, that are essential dynamic features related to conformational selection and induced fit, respectively (64–66).

Microtubules are dynamic cellular structures that switch between growing and pruning cycles both in vivo and in vitro. Stabilization or destabilization of microtubule dynamics is promoted by a number of endogenous and exogenous compounds that regulate the process in different ways, either by competition with GTP (67), structural modification of the protein-protein interface between α and β monomers (8, 31, 68) or by allosteric mechanisms (69). One of the most frequently described mechanisms is the ligand binding at the colchicine site on β-tubulin, which is spatially next to an α-tubulin nucleotide binding site, with nonexchangeable, noncatalytic characteristics, known as N-site. Therefore, we explored the exposition of both binding sites as a molecular docking strategy, since their coupling might trigger the structural destabilization of tubulin dimer exerted by some L1-CDR peptides.

The tubulin heterodimer has two guanine binding sites: at the exchangeable, catalytic site (E-site) on the β chain, GTP is hydrolyzed to GDP during microtubule assembly; the nonexchangeable, noncatalytic site (N-site), on the α chain, is always occupied by GTP, suggesting that it may function as a structural cofactor of tubulin (70). Divalent cations have high affinity for both sites and their binding is associated to the structural stability of tubulin dimer (71). Mg2+ is a wellestablished ion required for microtubule assembly and stability, and contributes to strong GTP binding to the E-site (72). Q-band EPR and electron spin echo envelope modulation spectroscopy

showed that Mn2+ at both N and E-sites directly coordinated to the triphosphate of GTP, proving that the divalent cation at both sites directly interacts with GTP (73). Mn2+ slowly exchanged for Mg2+ at the N-site and other divalent and trivalent cations may also exchange at this site and play a role in the assembly of microtubules (74, 75). Chelation of divalent cations in general, inhibits the assembly of tubulin dimers.

L1-CDR peptides bound at the nucleotide/colchicine binding site at the dimer interface, but most of the interactions were made at the N-site. The best solution of Rb44L1 peptide was in an α/β-tubulin semi-open state. We observed that the 1R is a key residue for interaction with tubulin dimer. The mutation of this residue for alanine, weakened the interaction, increasing the free energy. This result was further corroborated by experimental assays. Interestingly, the 13K of C36L1 used the same tubulin cavity as that of 1R of Rb44L1, although C36L1 best docking pose was observed in an open conformation. This polar pocket may play an important role in tubulin depolymerization induced by L1-CDR peptides since the inactive Rb29L1 did not present a favorable interaction on this region.

The inactivity of Rb29L1 peptide is noteworthy, since its sequence is quite similar to Rb44L1 except between residues 4

and 7, which is TSLL in the former peptide and QTIT in the latter. Interestingly, the most favorable docking poses showed a different interaction pattern with tubulin, since Rb44L1 QTIT residues were less solvent exposed than Rb29L1 TSLL residues, which are 70 Å2 more exposed to solvent. This is a direct consequence of the observed R interaction pattern with buried tubulin residues (69D and 11Q) in the N-site, and could be related to the observed activity differences.

A dot-blotting assay showed that in the presence of both GTP and Mn2+, but not with these agents added separately, the Rb44L1 peptide bound with increased affinity to the tubulin α-chains of monomeric, modified or dimeric substrates from a tumor cell lysate. This may have occurred by the GTP-E site induced oligomerization of tubulin dimers present in the cell lysate during incubation, indicating that under the conditions used, the dot-blotting assay with fixed peptide did not impair tubulin assembly on the latter (**Figure S6**).

In contrast, what is the possible mechanism triggering Rb44L1 depolymerization of tubulin? We found that the surface overlapping of the docked conformation of the peptide and the closed α/β-tubulin revealed that the peptide represents a steric constraint to the protein in this conformation. The effect noticed in the overlapping regions of 1R, 3 S, and 12N residues, and the peptide size

of 1675.0 Å3 , which preclude the α/β-tubulin return to a closed conformation, is a source of structure destabilization (**Video S2**).

Taken together, we propose that Rb44L1 peptide is a novel candidate to be developed as a drug, acting on the microtubule network of tumor cells. Molecular docking on tubulin monomers in opening motion, and the possible mechanisms of action leading to microtubule depolymerization were explored in comparison with other Ig CDR-L1 derived peptides, all tested against in vitro models of melanoma cells.

#### DATA AVAILABILITY STATEMENT

Datasets are available on request. The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

#### ETHICS STATEMENT

This study was carried out in accordance with the recommendations of the Ethics Committee of Federal University of São Paulo, Brazil for animal manipulation and experimental procedures. Animals were provided by Centro de desenvolvimento de modelos experimentais para medicina e biologia (CEDEME), of Federal University of São Paulo. The protocol was approved by the Ethics Committee of Federal University of São Paulo, Brazil via document CEP 1234/2011.

### AUTHOR CONTRIBUTIONS

NG and CF performed the biological experiments, analyzed data, and designed the figures. MM carried out annexin V and propidium iodide labeling analysis and designed **Figure 2D**. RA carried out the cell cycle analysis and designed **Figure 2E**. PR-L and RC carried out all the chemical analysis. NG wrote the manuscript and LP and LT designed the research project. All authors reviewed and approved the manuscript.

### FUNDING

This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (Fapesp), grant 10/51423-0 and by the National Council for Scientific and Technological Development (CNPq).

#### ACKNOWLEDGMENTS

The authors acknowledge Carolina de Amat for the illustration of Rb44L1 effects.

#### REFERENCES

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc. 2019.00025/full#supplementary-material

bridge between adaptive innate immunity. PLoS ONE (2009) 4:e8187. doi: 10.1371/journal.pone.0008187

by suppression of microtubule dynamics. Proc Natl Acad Sci USA. (2002) 99:10855–60. doi: 10.1073/pnas.132275599

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Girola, Resende-Lara, Figueiredo, Massaoka, Azevedo, Cunha, Polonelli and Travassos. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Celastrol Inhibits the Growth of Ovarian Cancer Cells in vitro and in vivo

Li-Na Xu1†, Na Zhao1†, Jin-Yan Chen2†, Piao-Piao Ye1 , Xing-Wei Nan1 , Hai-Hong Zhou1 , Qi-Wei Jiang3 , Yang Yang3 , Jia-Rong Huang3 , Meng-Ling Yuan3 , Zi-Hao Xing3 , Meng-Ning Wei 3 , Yao Li 3 , Zhi Shi 3 \* and Xiao-Jian Yan1,4 \*

*1 Department of Gynecology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China, 2 Department of Gynecology, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China, 3 Department of Cell Biology and Institute of Biomedicine, National Engineering Research Center of Genetic Medicine, Guangdong Provincial Key Laboratory of Bioengineering Medicine, College of Life Science and Technology, Jinan University, Guangzhou, China, 4 Center for Uterine Cancer Diagnosis & Therapy Research of Zhejiang Province, Women's Hospital and Institute of Translational Medicine, Zhejiang University School of Medicine, Zhejiang, China*

#### Edited by:

*Yan-Yan Yan, Shanxi Datong University, China*

#### Reviewed by:

*Changliang Shan, Nankai University, China Nana Zhang, Chinese Academy of Medical Sciences, China*

#### \*Correspondence:

*Zhi Shi<EMAIL_ADDRESS>Xiao-Jian Yan<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Cancer Molecular Targets and Therapeutics, a section of the journal Frontiers in Oncology*

Received: *06 December 2018* Accepted: *02 January 2019* Published: *28 January 2019*

#### Citation:

*Xu L-N, Zhao N, Chen J-Y, Ye P-P, Nan X-W, Zhou H-H, Jiang Q-W, Yang Y, Huang J-R, Yuan M-L, Xing Z-H, Wei M-N, Li Y, Shi Z and Yan X-J (2019) Celastrol Inhibits the Growth of Ovarian Cancer Cells in vitro and in vivo. Front. Oncol. 9:2. doi: 10.3389/fonc.2019.00002* Celastrol is a natural triterpene isolated from the Chinese plant Thunder God Vine with potent antitumor activity. However, the effect of celastrol on the growth of ovarian cancer cells *in vitro* and *in vivo* is still unclear. In this study, we found that celastrol induced cell growth inhibition, cell cycle arrest in G2/M phase and apoptosis with the increased intracellular reactive oxygen species (ROS) accumulation in ovarian cancer cells. Pretreatment with ROS scavenger N-acetyl-cysteine totally blocked the apoptosis induced by celastrol. Additionally, celastrol inhibited the growth of ovarian cancer xenografts in nude mice. Altogether, these findings suggest celastrol is a potential therapeutic agent for treating ovarian cancer.

Keywords: celastrol, reactive oxygen species, N-acetyl-cysteine, apoptosis, ovarian cancer

### INTRODUCTION

Ovarian cancer is the most lethal gynecologic cancer and the fifth leading cause of female cancer-related deaths in the United States in 2018 (1). Because of the late stage diagnoses, the prognosis of ovarian cancer remains poor, despite advances in aggressive surgery and combination chemotherapy (2–4). Current treatments for ovarian cancer are far from satisfactory, therefore it is of considerable interest to develop novel therapeutic agents to improve the outcomes of ovarian cancer.

Celastrol is a natural triterpene isolated from the Chinese plant Thunder God Vine (Tripterygium wilfordii),which has been reported with a wide range of bioactivities, such as antitumor (5), anti-inflammatory (6), antidiabetic activities (7) and antihypertensive (8). Celastrol has shown the potent antitumor activity in various cancers including prostate, breast, liver, colon, and lung (9–13). Although celastrol is able to induce apoptosis and inhibit proliferation, migration and invasion in ovarian cancer cells in vitro (14–16), the effect of celastrol on the growth of ovarian cancer cells in vivo is still unknown. Here, we have comprehensively investigated the antitumor activity of celastrol in ovarian cancer cells in vitro and in vivo.

### MATERIALS AND METHODS

#### Cells Lines and Reagents

The human ovarian cancer lines A2780 and SKOV3 were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 ng/ml) at 37◦C with 5% CO2 in a humidified incubator. Celastrol was purchased from Shanghai Tauto Biotechnology. N-acetyl-L-cysteine (NAC) and dihydroethidium (DHE) were purchased from Sigma-Aldrich. Methythiazolyldiphenyl-tetrazolium bromide (MTT), propidium iodide (PI) and other chemicals were purchased from Shanghai Sangon Biotech. Anti-p27 (610241), Anti-Cyclin B1 (554177), and Anti-Cyclin E (51-1459GR) antibodies were from BD Biosciences. Anti-RAF1 (SC-133) antibodies were from Santa Cruz Biotechnology. Anti-PARP (9542), Anti-AKT (4691), Anti-pAKT S473 (4060), Anti-ERK (4695), Anti-pERK T202/T204 (4370), Anti-JNK (9252), Anti-pJNK T183/Y185 (4668), Anti-p38 (9212), Anti-pp38 T180/Y182 (4511) antibodies were from Cell Signaling Technologies. Anti-GAPDH (LK9002T) antibodies were from Tianjin Sungene Biotech.

#### MTT Assay

Cells were seeded into a 96-well plate at a density of 0.5 ×104 cells/well. Then, different concentrations of celastrol (10 µL/well) were added to designated wells. After 72 h, 10 µL of MTT was added to each well at a final concentration of 0.5 mg/ml, and the plate was further incubated for 4 h, allowing viable cells to change the yellow MTT into dark-blue formazan crystals. Subsequently, the medium was discarded and 50 µL of dimethylsulfoxide (DMSO) was added to each well to dissolve the formazan crystals. The absorbance in individual well was determined at 570 nm by multidetection microplate reader 680 (BioRad, PA, USA). The concentrations required to inhibit growth by 50% (IC50) were calculated from survival curves using the Bliss method (17).

### Cell Cycle Analysis

Cells were harvested and washed twice with cold PBS and then fixed with 70% ice-cold ethanol at 4◦C for 30 min. After centrifugation at 200 × g for 10 min, cells were washed twice with PBS, resuspended with 0.5 mL PBS containing PI (50µg/mL), Triton X-100(0.1%, v/v), 0.1% sodium citrate, and DNase-free RNase (100µg/mL), and detected by flow cytometry (FCM) after 15 min incubation in the dark at room temperature. Fluorescence was measured at an excitation wave length of 480 nm through a FL-2 filter. Data were analyzed using ModFit LT 3.0 software (Becton Dickinson) (18, 19).

#### Apoptosis Analysis

Cell apoptosis was evaluated with FCM assay. Briefly, cells were harvested and washed twice with cold PBS, then stained with Annexin V-FITC and PI in the binding buffer, and detected by FACSCalibur FCM (BD, CA, USA) after 15 min incubation in

the dark at room temperature. Fluorescence was measured at an excitation wave length of 480 nm through FL-1 (530 nm) and FL-2 (585 nm) filters. The early apoptotic cells (Annexin V positive only) and late apoptotic cells (Annexin V and PI positive) were quantified (20).

### Western Blot Analysis

Cells were harvested and washed twice with cold PBS and then resuspended and lysed in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 ng/mL PMSF, 0.03% aprotinin, and 1µM sodium orthovanadate) at 4◦C for 30 min.

cell apoptosis was detected by FCM. The representative charts (A,C), quantified data (B,D), and Western blot results (E,F) of three independent experiments are shown. The same GAPDH image of Figure 2 has been used as loading control. \*\**P* < 0.01 vs. corresponding control.

Lysates were centrifuged at 14,000 × g for 10 min and supernatants were collected. Proteins were separated on 12% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% BSA and incubated with the indicated primary antibodies. Corresponding horseradish peroxidase-conjugated secondary antibodies were used against each primary antibody. Proteins were detected using the chemiluminescent detection reagents and films (21, 22).

#### Reactive Oxygen Species Assay

Cells were incubated with 10µM of DHE at 37◦C for 30 min, washed twice with PBS, and microphotographed under a conventional fluorescent microscope (Olympus, Japan) immediately. For each well, 5 fields were taken randomly. Then, cells were rapidly digested, harvested and washed twice with cold PBS, and detected by FCM. The DHE Fluorescence intensity was measured and quantified at an excitation wave length of 518 nm through PE filters (23, 24).

#### Nude Mice Xenograft Assay

Balb/c nude mice were obtained from the Guangdong Medical Laboratory Animal Center and maintained with sterilized food and water. This study was carried out in accordance with the recommendations of the Guidelines for the Care and Use of Laboratory Animals, and the protocol were approved by the Institutional Animal Care and Use Committee of Jinan University. Four female nude mice with 4–5 weeks old and 20–22 g weight were used for each group. Each mouse was injected subcutaneously with A2780 cells (4 × 106 in 100 µl of medium) under the left and right shoulders. Mice were randomized into two groups, when the subcutaneous tumors were approximately 0.3 × 0.3 cm2 (two perpendicular diameters) in size, and were injected intraperitoneally with vehicle alone (0.5% methylcellulose) and celastrol (2 mg/kg) every day. The body weights of mice and the two perpendicular diameters (A and B) of tumors were recorded every day. The tumor volume (V) was calculated as:

$$V = \pi/6 \left(1/2(A+B)\right)^3$$

The mice were anaesthetized after experiment, and tumor tissue was excised from the mice and weighted. The rate of inhibition (IR) was calculated according to the formula:

IR=1-Mean tumor weight of experimental group/Mean tumor weight of control group × 100% (25)

#### Statistical Analysis

A student's t-test was used to compare individual data points between two groups. A P-value of < 0.05 was set as the criterion for statistical significance.

#### RESULTS

#### Celastrol Inhibited the Growth of Ovarian Cancer Cells in vitro

To access the effect of celastrol on ovarian cancer cells, we treated two ovarian cancer cell lines A2780 and SKOV3 with the increasing concentrations of celastrol range from 0.1 to 10µM for 72 h. As shown in **Figures 1A,B**, the results of MTT assay revealed that the growth of two ovarian cancer cell lines was similarly inhibited by celastrol in a dose-dependent manner with the IC50 values were 2.11 and 2.29µM in A2780 and SKOV3 respectively. These data suggested that celastrol inhibits the growth of ovarian cancer cells.

### Celastrol Induced Cell Cycle Arrest in Ovarian Cancer Cells

To determine whether celastrol is able to induce cell cycle arrest, cell cycle distribution was examined after celastrol treatment. A2780 and SKOV3 cells were treated with 0.3, 1 and 3µM of celastrol for 48 h, then stained with PI and examined by FCM. As shown in **Figures 2A–D**, celastrol induced the accumulation in Sub G1 and G2/M phase and reduction in G0/G1 and S phase in two ovarian cancer cell lines. Next, the cell cycle related proteins were detected by Western Blot. As shown in **Figures 2E,F**, increased p27 and Cyclin B1 and decreased Cyclin E proteins were detected in celastrol-treated A2780 and SKOV3 cells. Together, these results indicated that celastrol induces cell cycle arrest in ovarian cancer cells.

### Celastrol Induced Apoptosis in Ovarian Cancer Cells

To determine whether celastrol could induce cell apoptosis, A2780 and SKOV3 cells were treated with indicated concentrations of celastrol for 48 h, apoptosis was assessed by FCM with Annexin V/PI staining. As shown in **Figures 3A–D**, celastrol dose-dependently induced early stage of apoptosis (Annexin V+/PI–) and late stage of apoptosis (Annexin V+/PI+) in both cells. Treatment of celastrol upregulated the protein expressions of cleaved-PARP, pp38 T180/Y182 and pJNK T183/Y185 but downregulated the protein expressions of pERK T202/Y204, pAKT S473 and RAF1 (**Figures 3E,F**). Consequently, these results suggest that celastrol induces cell apoptosis in ovarian cancer cells.

### ROS Generation Was Critical for Celastrol-Induced Apoptosis in Ovarian Cancer Cells

Numerous antitumor agents demonstrate antitumor activity via ROS-dependent activation of apoptotic cell death (26, 27). It has previously been reported that the elevated intracellular ROS mediated celastrol-induced apoptosis in several human cancer cells (28). Thus, we surmised that celastrol caused apoptosis in ovarian cancer cells was due to excessive ROS generation. Firstly, the cellular ROS was tagged by DHE fluorescence staining in celastrol-treated cells. As shown in **Figure 4**, celastrol enhanced the detectable red fluorescent signals of DHE in both A2780 and SKOV3 cells, suggesting the intracellular ROS levels were increased after celastrol treatment. Then we pre-treated A2780 and SKOV3 cells with NAC (a specific ROS scavenger), Celastrol-induced cell apoptosis were totally attenuated by NAC in both ovarian cancer cells

(**Figure 5**). Collectively, these results suggest that ROS generation was critical for celastrol-induced apoptosis in ovarian cancer cells.

quantified results (B,D) were shown. \*\**P* < 0.01 vs. corresponding control.

### Celastrol Inhibited the Tumor Growth of Ovarian Cancer in Nude Mice

To confirm the antitumor effects of celastrol in vivo, A2780 subcutaneous xenograft tumors were generated in the nude mice. As shown in **Figures 6A–E**, treatment of celastrol did inhibit the growth of A2780 xenograft tumors with the inhibition ratio of 28.60% by diminishing the tumor volumes and weights. Furthermore, mice body weight in celastrol group was close to that of control group, suggesting that celastrol at the indicated dose did not cause toxicity in mice (**Figure 6C**).

## DISCUSSION

Natural products attract more and more attention in the prevention and treatment of cancer in recent years. Products from the plant Tripterygium wilfordii, including celastrol and triptolide, are of special attention because of its superior antitumor activities against a variety of cancer types, and therefore are the traditional herb medicines considered to have the most potential in modern cancer therapy. For the treatment of ovarian cancer, triptolide has been shown to inhibit the proliferation, migration and invasion of ovarian cancer in multiple pathways (29–31) and demonstrated to exert efficacy in preclinical models (32). Celastrol has also been reported to induce apoptosis and inhibit proliferation, migration and invasion in ovarian cancer cells in vitro (14, 16), but the mechanism for its anti-tumor effect and the effect of celastrol on the growth of ovarian cancer cells in vivo are not fully understood. In our present study,

corresponding control.

we have demonstrated that celastrol mediated dose-dependent anti-growth effects on human ovarian cancer cell lines SKOV3 and A2780. The IC50 value after 72 h treatment with celastrol ranged from 2 to 3µM in these two human ovarian cancer cell lines, similarly to the IC50 value of celastrol of ovarian cancer in other articles (15, 16). We have also shown that celastrol induced both the early and late stage of apoptosis and cell cycle arrest in G2/M phase with obvious up-regulation of cleaved-PARP, pp38 T180/Y182, pJNK T183/Y185, p27 and Cyclin B1 and down-regulation of pERK T202/Y204, pAKT S473, RAF1 and Cyclin E in a dose-dependent manner. Similar with our results, celastrol can induce the activation of JNK and inactivation of AKT in multiple myeloma cells RPMI-8226 (33), activation of p38 in ovarian cancer cells OVCAR-8 and colorectal cancer

medium) under the left and right shoulders. When the subcutaneous tumors were approximately 0.3 × 0.3 cm in size, mice were randomized into two groups, and received intraperitoneal injection of vehicle alone (0.5% methylcellulose) or celastrol (2 mg/kg) every day. The body weight and tumor volume were recorded every day. After experiment, the mice were anesthetized, and tumor tissue was excised from the mice and weighted. The tumor volume (A), original tumors (B), body weight (C), tumor weight (D), and summary data (E) were shown. \**P* < 0.05 vs. corresponding control.

cells SW620 cells (34) and inactivation of ERK in hepatoma cells Hep3B (35). Furthermore, celastrol inhibited the growth of A2780 ovarian cancer subcutaneous xenograft tumors in nude mice by diminishing the tumor volumes and weights, and mice body weight in celastrol group was close to that of control group. These in vitro and in vivo data strongly indicate that celastrol may be a appropriate candidate for treating ovarian cancer.

Biological roles of ROS were intricate and important in cancer cells (36). The intracellular ROS plays a significant role in regulating multifarious cell physiological process such as growth, differentiation, death and so on (37). ROS changes the cellular redox condition, induces DNA damage and influences the activities of tumor suppressor or oncogene, thereby involving in the initiation and progression of cancer (38, 39). Lots of studies have shown that cancer cells normally produce more ROS than normal cells (40). Interestingly, accumulating evidence suggests that cancer cells are more vulnerable to ROSinduced death because they are under the increased oxidative stress (41). A variety of agents like YM155, dinaciclib and triptolide may be selectively toxic to tumor cells because they enhanced intracellular oxidant stress and push these already stressed cells beyond their limitation (24, 38, 42, 43). In addition, previous studies have demonstrated that ROS plays a pivotal role in celastrol-induced apoptosis in multiple cancers, such as colon cancer, liver cancer, osteosarcoma, etc. (9, 28, 44). In this study, we have found that the intracellular ROS levels were increased after celastrol treatment, and pretreated with ROS scavenger NAC totally attenuated celastrolinduced cell apoptosis in ovarian cancer cells. It has been reported that celastrol enhanced the intracellular ROS to induce apoptosis by inhibiting mitochondrial respiratory chain

complex I activity in lung cancer H1299 cells (45). Whether celastrol induces ROS accumulation to trigger apoptosis in the same way in ovarian cancer cells need to be further investigated.

In summary, our data have shown that celastrol induced cell growth inhibition, cell cycle arrest in G2/M phase and apoptosis with the increased intracellular ROS accumulation in ovarian cancer cells in vitro and in vivo. Pretreatment with NAC totally blocked the apoptosis induced by celastrol. Altogether, these findings suggest celastrol is a potential therapeutic agent for treating ovarian cancer.

#### AUTHOR CONTRIBUTIONS

L-NX, NZ, J-YC, ZS, and X-JY designed the experiments, performed the experiments, analyzed the data, and wrote the paper. X-WN, H-HZ, P-PY, Q-WJ, YY, J-RH, M-LY, Z-HX,

#### REFERENCES

M-NW, and YL performed the experiments. All authors read and approved the final manuscript.

#### FUNDING

This work was supported by funds from the National Key Research and Development Program of China (No. 2017YFA0505104 to ZS), the National Natural Science Foundation of China (No. 81772540 to ZS and No. 81503293 to X-JY), the Guangdong Natural Science Funds for Distinguished Young Scholar (No. 2014A030306001 to ZS), the Guangdong Special Support Program for Young Talent (No. 2015TQ01R350 to ZS), the Science and Technology Program of Guangdong (No. 2016A050502027 to ZS) and the Science and Technology Program of Guangzhou (No.<PHONE_NUMBER>58 to ZS), the Zhejiang major disease diagnosis and treatment technology research center (No. JBZX-201803 to X-JY).

in human ovarian cancer cells. Oxid Med Cell Longev. (2014) 2014:906804. doi: 10.1155/2014/906804

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Xu, Zhao, Chen, Ye, Nan, Zhou, Jiang, Yang, Huang, Yuan, Xing, Wei, Li, Shi and Yan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Inhibition of FAK Signaling Elicits Lamin A/C-Associated Nuclear Deformity and Cellular Senescence

Hsiang-Hao Chuang1 , Pei-Hui Wang1 , Sheng-Wen Niu2 , Yen-Yi Zhen2 , Ming-Shyan Huang3 , Michael Hsiao4,5 and Chih-Jen Yang1,6,7 \*

*1 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan, 2 Division of Nephrology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan, 3 Department of Internal Medicine, E-Da Cancer Hospital, School of Medicine, I-Shou University, Kaohsiung, Taiwan, 4 Genomics Research Center, Academia Sinica, Taipei, Taiwan, 5 Department of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, 6 Department of Internal Medicine, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan, 7 Department of Respiratory Therapy, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan*

Focal adhesion kinase (FAK) is a non-receptor kinase that facilitates tumor aggressiveness. The effects of FAK inhibition include arresting proliferation, limiting metastasis, and inhibiting angiogenesis. PF-573228 is an ATP-competitive inhibitor of FAK. Treating lung cancer cells with PF-573228 resulted in FAK inactivation and changes in the expressions of lamin A/C and nuclear deformity. Since lamin A/C downregulation or deficiency was associated with cellular senescence, the senescence-associated β-galactosidase (SA-β-gal) assay was used to investigate whether PF-573228 treatment drove cellular senescence, which showed more SA-β-gal-positive cells in culture. p53 is known to play a pivotal role in mediating the progression of cellular senescence, and the PF-573228-treated lung cancer cells resulted in a higher p53 expression level. Subsequently, the FAK depletion in lung cancer cells was employed to confirm the role of FAK inhibition on cellular senescence. FAK depletion and pharmacological inhibition of lung cancer cells elicited similar patterns of cellular senescence, lamin A/C downregulation, and p53 upregulation, implying that FAK signaling is associated with the expression of p53 and the maintenance of lamin A/C levels to shape regular nuclear morphology and manage anti-senescence. Conversely, FAK inactivation led to p53 upregulation, disorganization of the nuclear matrix, and consequently cellular senescence. Our data suggest a new FAK signaling pathway, in that abolishing FAK signaling can activate the senescence program in cells. Triggering cellular senescence could be a new therapeutic approach to limit tumor growth.

Keywords: non-small cell lung cancer, senescence, focal adhesion kinase, nuclear deformity, lamin A/C

### INTRODUCTION

Focal adhesion kinase (FAK) is a multifunctional non-receptor tyrosine kinase that participates in a variety of signaling axes (1–5). In response to extracellular stimuli, FAK translocates to the focal adhesion complex, and mediates molecular signaling for cellular events (6–9). In focal adhesion, FAK cascades signal the focal adhesion complex to promote cell proliferation and migration

#### Edited by:

*Zhe-Sheng Chen, St. John's University, United States*

#### Reviewed by:

*Shiv K. Gupta, Mayo Clinic, United States Ji-Ye Yin, Central South University, China*

> \*Correspondence: *Chih-Jen Yang<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Cancer Molecular Targets and Therapeutics, a section of the journal Frontiers in Oncology*

Received: *24 October 2018* Accepted: *08 January 2019* Published: *30 January 2019*

#### Citation:

*Chuang H-H, Wang P-H, Niu S-W, Zhen Y-Y, Huang M-S, Hsiao M and Yang C-J (2019) Inhibition of FAK Signaling Elicits Lamin A/C-Associated Nuclear Deformity and Cellular Senescence. Front. Oncol. 9:22. doi: 10.3389/fonc.2019.00022*

**207**

(4, 9, 10). In addition to the focal adhesion complex and cytoplasm, FAK is also present in the cell nucleus (3, 11). Nuclear FAK acts as a cotranscriptional factor in gene transcription and is involved in p53 degradation, in contrast to its enzymatic function in protein phosphorylation (3, 11, 12). Whereas, FAK in the focal adhesion complex affects the expressions of cyclin B1 and cyclin D1 to program tumor cell proliferation (6, 13, 14), nuclear FAK elicits p53 degradation to drive cell cycle progression (11, 12).

The biological roles of FAK in cell migration and proliferation have also been implicated in pathological progression and in the development of cancer. There are several lines of evidence suggesting that FAK activity can manipulate tumor phenotypes, leading to uncontrolled proliferation, neovascularization, and metastasis (5, 15, 16), and the FAK signaling to tumor cell propagation represents tumorigenic capacity (5, 11). Given the crucial roles of FAK in these malignant processes, FAK is regarded to be a potential target for anti-cancer therapy (17, 18). Experiments have shown that FAK depletion results in silencing of cancer-promoting gene expressions in human hepatocellular carcinoma (HCC) xenotransplants in nude and severe combined immunodeficiency (SCID) mice (19). Moreover, the enzymatic function of FAK involves in proliferation and metastasis (9, 15, 20). Suppressing the catalytic activity of FAK or sequestering FAK in the cytoplasm has been reported to potentially perturb FAK signaling, which implies that chemical inhibitors of the enzymatic activity of FAK may be a pharmacological strategy to limit cancer growth and metastasis (6, 7, 21, 22).

Triggering cell apoptosis and arresting cell cycle progression with pharmacological regimens are common strategies to limit tumor cell growth. FAK inhibition represents an anti-cancer therapeutic strategy, as FAK inhibitors have effects on antiangiogenesis, anti-proliferation, and anti-invasion effects (5, 15, 23). Besides, inducing cellular senescence in tumor cells could be a new therapeutic approach to limit tumor cell growth (24). Although therapy-induced senescence (TIS) in cancer cells may result from deficient apoptosis (25), driving cancer cells to cellular senescence could be a way to limit tumor propagation (26). In general, chemotherapy-induced DNA damage, telomere shortening, and oncogenic stress are the three main pathological causes of senescence (24, 27–32), and the induction of cellular senescence with these drug regimens is a side effect. Cases of βgal-positive lung cancer biopsies in response to chemotherapy have been reported (24, 29, 33). Inducing cellular senescence could be a new approach to limit cancer growth based on phenotypic aging without DNA damage or genomic instability (31, 34). Chromatin or nuclear skeleton disorganization could be a cause of cellular senescence instead of oncogenic stress and replicative failure (8, 26).

Recent pharmacological advances in cancer therapy have led to an increased focus on developing chemical compounds that are able to target specific molecules in tumor cells to both improve efficacy and lower toxicity (4, 35). PF-573228, which competes with ATP binding to abolish the catalytic function of FAK, can inhibit the phosphorylation of FAK at tyrosine 576/577 and FAK kinase function (36, 37). Consequently, PF-573228 efficiently suppresses both the growth and metastasis of epithelial carcinoma (4, 36). The pharmacological effects of PF-573228 have been characterized based on the inhibition of FAK catalytic activity (36). In this study, we hypothesized that inhibiting the enzymatic function of FAK would stop lung cancer cell growth and invasion. Interestingly, the enzymatic inactivation of FAK resulted in nuclear deformity. When we investigated the cause and effect of nuclear deformity by PF-573228, we observed that p53 upregulation, lamin A/C downregulation, and cellular senescence in the lung cancer cells exposed to PF-573228. Strikingly, perturbation of FAK signaling led to downregulation of lamin A/C and cellular senescence rather than proliferative arrest, and halted migration of the lung cancer cells. These results showed that treatment with a FAK inhibitor could be a therapeutic approach to abrogate tumor growth. In addition, these findings revealed the crucial role of FAK signaling in antisenescence, and that inhibition of FAK resulted in the progression of senescence.

### MATERIALS AND METHODS

#### Materials

Detailed information on the materials is listed in **Supplementary Table S1**.

### Cell Culture and Drug Treatment

A549 cells, H1299 cells, and H460 cells were purchased from ATCC. The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37◦C in a humidified atmosphere at 5% CO2, and treated with PF-573228 (TOCRIS, Bristol, UK) at concentrations of 0, 0.1, 1, or 10µM.

#### Western Blot Analysis

The cells were harvested and lysed in 1x RIPA buffer (Merck, Darmstadt, Germany) containing protease and phosphatase inhibitors. The protein concentration was determined using a Bio-Rad DC protein assay kit (Bio-Rad, California, USA). For Western blot analysis, 30 µg of total protein was applied to SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked in 5% skim milk for 2 h in TBST buffer (20 mM Tris-Cl, 150 mM NaCl, 0.1% Tween 20, pH 7.4). After blocking, the membranes were probed with the primary antibody overnight. Antibodies against FAK, p-FAK, cyclin B1, p53, and lamin A/C were used in immunoblotting. The given protein bands were identified by horseradish peroxidase-conjugated secondary antibodies and developed with an enhanced chemiluminescence solution.

## Flow Cytometry Cell Cycle Analysis

The cells were harvested and washed with PBS buffer, and then fixed in 70% (v/v) ethanol. The fixed cells were stained with propidium iodide solution and injected into an Attune NxT Flow Cytometer (Life Tech, California, USA) to analyze the cell cycle profile.

#### Immunofluorescent Staining and Immunofluorescent Microscopic Imaging

A Leica DMi8S epifluorescence microscope (Wetzlars, Germany) equipped with an X-Cite XCT10A (Lumen Dynamics, Wiesbaden, Germany) light source, filters and objectives (10x, 20x, 40x, and 63x) was used to observe fluorescent signals in the cells. In addition to epifluorescence, confocal images were captured using an OLYMPUS FV1000 confocal laser scanning microscope equipped with a light source, filters and objectives (10x, 20x, 40x, 63x, and 100x). Cells were seeded on 12-mm coverslips in a 24-well culture plate. The cells were harvested and fixed in 4% paraformaldehyde in PBS for 10 min, and permeabilized in 0.5% Triton in PBS for 5 min. After fixation, the cells were subjected to immunofluorescent staining with antibodies recognizing FAK and emerin. Phalloidin-TRITC was used as an additional reagent. Cell nuclei were stained with 0.2µg/mL 4′ , 6-diamidino-2-phenylindole (DAPI).

### Senescence-Associated β-Galactosidase Staining

The cells were fixed with 4% paraformaldehyde for 15 min. After fixation, acidic β-galactosidase (SA-β-gal) was assayed in senescence assay buffer (1 mg/mL 5-bromo-4-chloro-3-indolyl β D-galactopyranoside (X-gal), 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, 150 mM NaCl, 40 mM citric acid, and 40 mM Na2HPO4 at pH 6.0) in the dark at 37◦C for 16 h. SA-β-gal activity was detected based on SA-β-gal-hydrolyzed X-gal, which produces a blue color. All chemical reagents were purchased from Sigma-Aldrich.

### Cell Growth Assay

The cells were trypsinized, resuspended in 1xPBS, and stained with trypan blue (Sigma-Aldrich). The number of cells was counted with a hemocytometer.

#### Lentiviral Production and Infection

Lentivirus-associated plasmids encoding luciferase, and FAK short hairpin RNA (shRNA) were obtained from the National RNAi Core Facility of Academia Sinica, Taiwan. The production and infection of lentiviruses were performed according to the guidelines of the National RNAi Core Facility.

#### Statistical Analysis

The experimental data were digitized and analyzed. Data are presented as the mean ± the standard error of the mean (SEM). One-way ANOVA was used to compare digitized data and measurements from independent experiments in two or more groups, and the Student's T-test was used to compare two independent samples. A p < 0.05 was considered to indicate a statistically significant difference.

## RESULTS

### PF-573228 Causes Cessation of the Propagation of Lung Cancer Cells

Focal adhesion signaling is involved in cell proliferation, and FAK plays a key role in the focal adhesion complex that relays focal adhesion signals to the cell proliferation program (9, 15). Given the role of FAK signaling in tumor growth and metastasis, we hypothesized that inhibiting the catalytic activity of FAK may disrupt FAK signaling and blunt tumor cell proliferation. Therefore, we treated three distinct nonsmall cell lung cancer cell lines (A549 lung adenocarcinoma cells and H460 and H1299 large cell carcinoma cells) with PF-573228, an enzymatic inhibitor of FAK. PF-573228 was administered to the lung cancer cells for 4 days at three doses: 0.1, 1, or 10µM. The growth curves showed that 10µM PF-573228 effectively induced cessation of cell growth (**Figures 1A–C**).

We then examined the expression level of the cell cycle regulator cyclin B1, which has been reported to be a downstream effector of FAK signaling. Western blot analysis showed that cyclin B1 expression levels were much lower after the cells were exposed to 10µM PF-573228 (**Figures 1D–F**). To further characterize the effect of PF-573228 treatment on cell cycle progression, we analyzed the cell cycle distribution using flow cytometry analysis. The results showed that a low PF-573228 concentration had little influence on cell cycle progression (**Figures 1G–I**), whereas a high PF-573228 concentration (10µM) halted cell cycle progression at the G2/M transition (**Figures 1J,K**). This showed that PF-573228 treatment effectively suppressed multiplication of lung cancer cells.

### PF-573228 Administration Inactivates FAK

Since phosphorylation of FAK at Tyr-576 and Tyr-577 (p-FAK) represents enzymatic activation of FAK (37), an antibody against p-FAK was used to confirm the kinase activity of FAK and verify the effect of PF-573228 on FAK inactivation. FAK activity was practically blocked by 10µM PF-573228 in A549 cells (**Figures 2A,C**). To further confirm the inactivation of FAK by PF-573228 treatment, we also examined the phosphorylation of tyrosine 397 in FAK (pTyr-397). The results showed that the intensity of pTyr-397 was decreased after PF-573228 treatment (**Figure S1**). FAK is a key regulator of integrin signaling for focal adhesion assembly (38, 39). In addition to FAK inhibition, PF-573228 has been shown to perturb integrin-based signaling for focal adhesion maturation (36).

Treatment of the lung cancer cells with PF-573228 resulted in failure of FAK activation, and translocation to focal adhesion was observed in immunofluorescent imaging (**Figure 2B**). When the cells were cultured in PF-573228-free medium, more FAK translocated to focal adhesions, which appeared as plaque-like patterns in the cell periphery that formed at the tips of stress fibers, as visualized in cells stained with phalloidin-labeled Factin and an antibody against FAK (**Figure 2B**). By contrast, in the A549 cells treated with PF-573228, only a few FAK molecules translocated to focal adhesions, and tiny FAK-based focal adhesions formed at the tips of F-actin bundles, indicating failure of focal adhesion maturation (**Figure 2B**). The sizes of focal adhesions were measured and the areas of FAK at the tips of the actin stress fibers were digitized using Image Pro software. The sizes of focal adhesions ranged from 12 to 28 µm2 in the cells without PF-573228 treatment, whereas the extent of FAK-based focal adhesion was approximately 3–12 µm2 after PF-573228 treatment (**Figure 2D**).

FIGURE 1 | PF-573228 inhibited lung cancer cell growth. Three different types of lung cancer cells, (A) A549 lung adenocarcinoma and (B) H460, and (C) H1299 large cell carcinoma, were selected for the PF-573228 administration regimen. Cell growth curves of the three lung cancer cell lines treated with various doses of PF-573228 for 4 days were established. The administration of PF-573228 at 10µM to the lung cancer cells effectively suppressed cell growth *in vitro*, as proliferative activity totally ceased in the cells exposed to 10µM PF-573228. (D) On the third day, PF-573228-treated cells were harvested and subjected to Western blot analysis for cyclin B1. Cyclin B1 levels were much higher in A549 cells with 1µM PF-573228 or without PF-573228 treatment than in the cells treated with higher concentrations of PF-573228. (E) After 10µM PF-573228 treatment, cyclin B1 levels declined markedly in H460 cells. (F) PF-573228 administration slightly reduced cyclin B1 levels in H1299 cells. A549 cells were harvested and subjected to flow cytometry analysis for cell cycle profiling after PF-573228 treatment for 3 days. The concentrations of PF-573228 were 0µM (G), 0.1µM (H), 1µM (I) and 10µM (J), respectively, (K) After 10µM PF-573228 treatment, the G2/M ratio was significantly extended. The apoptotic ratio was also increased in A549 cells with 10µM PF-573228 treatment.

PF-573228 were reduced to less than half and one tenth compared with the cells without PF-573228 treatment, respectively. (D) The sizes of FAK-based focal

adhesions were 19 µm2 on average in the cells without PF-573228 treatment and 6.4 µm2 on average in the cells without PF-573228 treatment.

### Aberrant Nuclear Appearance and Lamin A/C Downregulation Occur Concurrently in the Lung Cancer Cells Exposed to PF-573228

In the absence of PF-573228, most cells contained oval or round nuclei, as visualized by DAPI staining (**Figure 2B**). Interestingly, a distorted nuclear morphology was observed in the A549 cells treated with PF-573228 (**Figure 2B**). As DAPI staining was insufficient to clearly visualize the nuclear appearance in detail, an antibody against emerin (40), a nuclear inner membrane protein, was used to visualize the nuclear shape in the PF-573228 treated A549 cells. Emerin staining revealed that the cells without PF-573228 treatment harbored oval-like nuclei. By contrast, most nuclei were larger and had irregular shapes with invagination in the cells upon exposure to 10µM PF-573228 (**Figure 3A**).

Lamin A/C is the nuclear skeleton responsible for maintaining and stabilizing the nuclear architecture (41–44). Because interrupting FAK signaling resulted in nuclear deformity (**Figure 2B**), changes in lamin A/C expression levels were assessed. The effects of FAK signaling on the expressions of lamin A/C and other nuclear skeletal proteins inferred that the nuclear deformity caused by PF-573228 was attributable to changes in lamin A/C expression.

To investigate changes in lamin A/C expressions in cells exposed to PF-573228, PF-573228-treated cells were harvested and subjected to Western blot analysis. Inhibition of FAK activity led to lower p-FAK levels (**Figures 3B,C**) and deformed nuclei in the lung cancer cells (**Figure 3A**). To quantify the expressions of lamin A and C, the intensities of their protein bands were normalized to β-actin (**Figure 3B**). The expressions of lamin A and C were much lower in the A549, H460, and H1299 cells treated with PF-573228 compared to those without PF-573228 treatment (**Figure 3B** and **Figures S2A,B**). The lamin A and C band intensities were quantified and plotted in bar charts. The ratios of lamin A/β-actin and lamin C/β-actin in A549 cells exposed to PF-573228 were reduced by one third and one half, respectively, compared to A549 cells without PF-573228

treatment (**Figures 3D,E**). Similar trends of downregulation of lamin A and lamin C by PF-573228 treatment were also detected in the two other lung cancer cell lines (**Figures S2A,B**).

### Lung Cancer Cells Are Destined to Senescence After Inhibition of FAK Enzymatic Function

Mutant LMNA, mutations that affect lamin A/C expression, and lamin A/C depletion in cells have been associated with premature aging and cellular senescence (8, 30, 32, 42, 45). Based on the concurrent lamin A/C downregulation and nuclear deformity observed in lung cancer cells exposed to PF-573228 (**Figures 3A,B**), we examined the development of cellular senescence in lung cancer cells treated with PF-573228. The SA-β-gal activity in cells was assayed by in situ staining using the chromogenic substrate X-gal, which colored SA-β-galpositive cells blue. As noted in **Figure 4A**, blue cells were clearly visible in the cells treated with PF-573228 (**Figure 4A**), whereas a sporadic distribution of blue-colored cells was observed in the cells without PF-573228 treatment (**Figure 4A**). The bar chart in **Figure 4B** shows that nearly 90% of the cells exposed to a higher dose of PF-573228 were positive for SAβ-gal, compared to ∼20% of the cells exposed to a lower dose of PF-573228, and ∼1% of the cells without PF-573228 treatment.

in the 1µM PF-573228-treated A549 cell population, and more than 80% in the 10µM PF-573228-treated A549 cell population. (C) A549 cells were treated with 0, 1, or 10µM PF-573228 for 4 days. p53 was not obviously increased in 1µM PF-573228 treated-A549 cells and was significantly elevated in 10µM PF-573228-treated A549 cells. (D) p53 levels approximately tripled in A549 cells exposed to 10µM PF-573228 compared to cells with or without 1µM PF-573228 treatment.

### Upregulation of p53 in Cells Exposed to PF-573228

Disruption of FAK signaling by PF-573228 caused cellular senescence. However, the mechanisms by which inhibition of FAK signaling affects senescence programming remain unclear. Cellular senescence in chemotherapy-affected cancer cells has been observed in several studies (24, 29, 46). In addition, clinical studies have reported that p53 plays a role in the development of cellular senescence in chemotherapy-affected cancer cells (46, 47). p53 is known to be a transcription factor in programed senescence and cell cycle arrest (48), and it may play a similar role in the cellular senescence program in lung cancer cells exposed to PF-573228 as in cells in which FAK signaling is interrupted.

To investigate whether or not p53 plays a role in PF-573228 induced cellular senescence, p53 expression levels were examined in PF-573228-treated cells. Western blot analysis showed that p53 expression levels increased significantly by more than 3 fold compared to cells without PF-573228 treatment and cells treated with a low concentration of PF-573228 (**Figures 4C,D**, and **Figure S3**).

#### Engagement of FAK Signaling With Nuclear Integrity and p53 Expression

FAK is not the only molecule targeted by PF-573228 (36). Although FAK enzymatic activity was blocked by PF-573228 administration, off-target effects could also have turned off other kinases, for example, cyclin-dependent kinases. Therefore, signaling perturbations of other kinases may have caused the pathogenic senescence in the lung cancer cells.

If FAK has an anti-senescence effect, FAK depletion would cause anti-senescence to fail and escalate senescence programing. To clarify the role of loss of FAK signaling in the development of cellular senescence and nuclear deformity with changes in lamin A and lamin C expressions, we used an shRNA targeting FAK to deplete the expression of FAK in lung cancer cells. After introducing shFAK into lung cancer cells, cells harboring shFAK were selected. To assess FAK knockdown, two shFAK clones were selected for Western blot analysis and senescence assays, which showed that shFAK successfully caused FAK depletion in A549, H460, and H1299 cells (**Figure 5A** and **Figure S4A**). In addition, the impact of FAK depletion on the downregulation of lamin C and cyclin B1 and upregulation of p53 was validated (**Figure 5A** and **Figure S4A**). We also assessed nuclear appearance using emerin staining, which revealed that A549 cells without FAK depletion harbored oval-like nuclei. By contrast, the ratio of nuclei harboring slightly larger and irregular shapes was increased in the cells with FAK depletion (**Figure 5B**). Similar results were obtained in the H460 and H1299 cells (**Figures S4B,C**). In addition, lung cancer cells harboring shLuc or shFAK were subjected to senescence assays, which revealed

more SA-β-gal-positive A549 cells harboring shFAK (**Figure 5C**). By contrast, few SA-β-gal-positive cells were visible in those harboring shLuc. The SA-β-gal-positive cells represented ∼0.7% of the shLuc A549 population. In the two shFAK cell clones, SA-β-gal-positive cells represented ∼11 and 15%, respectively (**Figure 5D**). Similar results were observed in H460 and H1299 cells (**Figures S4D–F**). However, H460 cells grew in single and multiple layers (**Figure S4D**), and it was difficult to measure the ratio of SA-β-gal-positive cells.

### Senescent Cells Reactivate Their Proliferative Activity After PF-573228 Withdrawal From Cell Culture

Aging and cellular senescence are often present in replicative failure, oncogenic induction, and telomere shortening (8, 34, 49). In clinical cases, chemotherapy or radiation has been shown to induce cellular senescence, and DNA damage or genomic instability is thought to be the pathological cause. Therapyinduced senescence can be classified as replicative senescence (24, 27, 33, 48), and replicative senescence is able to cease tumor growth (47, 50). However, this cellular senescence is reversible (31, 51, 52). Senescent cells expressing low levels of p16 have been shown to reversibly exit senescence when p53 expression levels fall (52).

In this study, FAK signaling downregulated the expression of p53, and inhibition of FAK signaling upregulated the expression of p53 in A549 cells (**Figures 4C,D**, **5A**). The A549 cells that entered senescence due to PF-573228 administration exhibited regrowth and return to a non-senescent state after withdrawal of PF-573228 from the culture (**Figures 6A–C**). The proliferative activity of A549 cells was based on cyclin B1 expression after the cells were incubated in PF-573228-free medium (**Figure 6D**).

### Restoration of Lamin a and Lamin C Expressions in Senescent Lung Cancer Cells After PF-573228 Withdrawal

We then tested whether senescence in lung cancer cells with FAK inhibition was reversible or irreversible. The three lung

cancer cells lines were cultured in medium containing 10µM PF-573228 to induce cell senescence in a 5-day induction course, after which the expressions of lamin A and lamin C decreased (**Figure 7A**). After the induction of senescence, PF-573228 withdrawal was scheduled over 6 days. Lamin A and lamin C expressions in A549 cells and H1299 cells gradually recovered after PF-573228 withdrawal (**Figures 7A,B**). By contrast, lamin A and lamin C levels in H460 cells remained lower when senescent H460 cells were incubated in PF-573228-free medium (**Figures 7A,B**). After PF-573228 withdrawal, senescent A549 cells escaped from senescence, as the SA-β-gal-positive A549 cell population declined to nearly half by the fifth day of PF-73228 withdrawal (**Figure 8A**).

### DISCUSSION

FAK is a signaling mediator of integrin-based signaling and is associated with epidermal growth factor receptor (EGFR) signaling (23, 53). FAK-associated cross-talk between EGFR and integrin pathways have been shown to lead to tumor growth and metastasis in lung cancer (39, 53–55). Phosphorylation at tyrosine 576/577 (p-FAK) has been reported to result in catalytic activity and to be involved in tumor cell proliferation and metastasis (2, 12, 15, 16). Therefore, inhibition of the enzymatic function of FAK has been proposed to be a therapeutic strategy to limit tumor growth, angiogenesis, and metastasis (5, 7, 16, 23). In the present study, we tested the pharmacological effect of PF-573228 on inhibiting FAK activity and limiting lung cancer cell growth. When lung cancer cells were treated with PF-573228, an abnormal nuclear shape was observed. A similar cytological phenomenon has been reported in previous studies (56), however the molecular mechanism has not been clearly elucidated. In addition, nuclear lobulation and distorted nuclear morphology have been reported in cells with the LNMA mutation or lamin A/C downregulation (41–43). The LNMA mutation or lamin A/C downregulation has been shown to result in nuclear distortion

with a pathogenic tendency to develop aging and senescence (8, 30, 42, 57). The nuclear deformity in PF-573228-treated lung cancer cells (**Figures 2B**, **3A–C**) supports a pathophysiological impact from the inactivation of FAK signaling to downregulate lamin A/C.

In this study, we examined the expressions of lamin A/C and assayed SA-β-gal activity in lung cancer cells exposed to PF-573228. Our experimental results demonstrated that FAK inhibition and FAK depletion elicited similar downregulation of lamin A/C, upregulation of p53, and cellular senescence (**Figures 4A,C,D**, **5A,C**). These results imply that FAK signaling regulates the expression of lamin A/C to maintain a regular nuclear shape and activate anti-senescence programs (**Figure 8B**). The finding that FAK signaling affects lamin A/C expressions and influences the cellular context in which lamin A/C organizes the nuclear architecture is an important biological theme. The degradation of lamin A/C has recently been reported to be regulated by Akt1 or cdk5 signaling (30, 58). Akt signaling was shown to slightly alter the amount of lamin A/C in cells, but this small change in lamin A/C expressions did not seem to have a notable effect on nuclear shape. On the other hand, nuclear FAK has also been shown to act as a transactivator to regulate gene expressions and stem cell differentiation rather than stem cell renewal (59). In addition, nuclear FAK and Oct-4 have been shown to coordinate gene expression programming with the expression of Oct-4 in stem cell renewal. However, the role of nuclear FAK in gene expression programming does not seem to be associated with changes in lamin A/C expressions. Furthermore, we found that PF-573228 treatment does not dramatically affect nuclear translocation of FAK in A549 cells (**Figure S5**). This implied that FAKmediated signaling to maintain lamin A/C expression may not be through transcriptional regulation. By contrast, inactivation of FAK signaling or nestin silencing (30) has been shown to significantly downregulate lamin A/C and cause round or oval nuclei to become lobulated or irregular in shape. Our results indicated that FAK-mediated signaling is crucial to maintain nuclear shape and, potentially, for chromatin reorganization.

In addition to the downregulation of lamin A/C, this study showed that FAK inhibition-mediated p53 upregulation also played a crucial role in cellular senescence, and that p53 was increased during FAK inhibition either by a small compound or shRNA-mediated downregulation. Lim et al. demonstrated that nuclear FAK could promote p53 downregulation via enhanced Mdm2-dependent p53 ubiquitination in a kinase-independent manner (11). However, in our study, both the amount of

FAK protein and its enzymatic function affected the expression level of p53. PF-573228 treatment suppressed the enzymatic activity of FAK but did not significantly affect its abundance. However, an obvious effect on cell senescence was observed in the inhibitor treatment group. This result implies an important role of FAK enzymatic function in suppressing senescence. Downstream signaling such as the PI3K/Akt axis may play a critical role in modulating Mdm2 function and p53 regulation, and p53 activation may suppress cell proliferation and further trigger senescence. However, FAK inhibition also repressed the proliferation of p53 null cancer cells such as H1299 cells and induced senescence. FAK inhibition also reduced lamin A/C expressions in H1299 cells, with changes in chromatin integrity followed by the induction of senescence. These observations indicate that the induction of cellular senescence by perturbations in lamin A/C-mediated chromatin alterations is independent of p53 (30, 34, 60).

Downregulation or degradation of lamin A/C and upregulation of p53 by PF-573228 treatment or FAK depletion are the main causes of cellular senescence. Baell et al. reported that inhibition of histone acetyltransferase could induce cellular senescence (26), and that the pharmacological effects of VM-8014 and VM-1119 on chromatin remodeling caused cellular senescence (26). The induction of senescence by PF-573228, VM-8014, and VM-1119 may also be due to defective chromatin remolding. Because FAK and lamin A/C are also involved in chromatin remodeling (43, 55, 61), this cellular senescence is likely to be reversible (**Figures 6A–C**, **8A**). Therefore, when the enzymatic activity of FAK is restored, lamin A/C and cyclin B1 expression levels recover (**Figures 7A,B**, **6D**).

Inhibition of FAK signaling may have a therapeutic role in limiting cancer cell growth. In the present study, we demonstrated that disruption of the FAK signaling pathway led to cellular senescence in lung cancer cells. We also tested the sensitivity of human normal lung epithelial cells, BEAS-2B, to PF-573228 treatment. It appeared that a high concentration of PF-573228 could attenuate the propagation of BEAS-2B cells. However, the BEAS-2B cells cultured in medium containing serum still underwent cell cycle progression with a low proliferative rate (**Figure S6A**). This implies that oncogene addiction occurs in lung cancer cells for FAK signaling (54). We also evaluated whether FAK inhibition causes cellular senescence in BEAS-2B cells. The results showed that a high dose of PF-573228 treatment promoted cellular senescence in BEAS-2B cells (**Figures S6B,C**). However, the ratio of SA-β-gal positive cells was <3% (**Figure S6C**). This implies that normal cells are more insensitive to high concentrations of PF-573228 than lung cancer cells and FAK inhibitors have a therapeutic potential for cancer treatment. However, there was no evidence showing that FAK signaling can result in anti-senescence and convert senescent cells to non-senescent cells upon FAK inhibitor withdrawal in A549 cells (**Figures 8A,B**). We calculated the ratios of SA-βgal-positive and SA-β-gal-negative A549 cells after PF-573228 withdrawal and plotted curves with the timing of PF-573228 withdrawal. The slope of the curve for SA-β-gal-negative cells over 5 days indicated a reduction of 5,402 cells per day in the linear variation of SA-β-gal-negative cell numbers (**Figure 8A**). SA-β-gal-negative cells increased exponentially after PF-573228 withdrawal in A549 cells, and the curves of the SA-β-gal-negative cell growth were convergent with the growth curve of total A549 cells (**Figure 8A**). These results may be due to reversion of some of the senescent cells to non-senescent cells, as described in the schematic representation of FAK signaling in anti-senescence and PF-573228 treatment signaling cellular senescence (**Figure 8B**).

Previously, therapeutic outcomes were measured in terms of anti-angiogenesis, anti-proliferation, and anti-invasion (16, 39, 62). In this study, FAK inhibition limited lung cancer cell propagation by inducing cellular senescence (**Figure 8B**). Driving cell senescence programing is a new trend for the treatment of tumor diseases (26), as this therapeutic approach does not chemically elicit genomic evolution in cancer cells and does not severely damage non-cancer cells (63). Although cellular senescence does not kill tumor cells, limiting cancer growth could eliminate cancer cell malignancy. However, cellular senescence is an inducer of autophagy (64) and increases susceptibility to cell-mediated cytotoxicity by activated killer cells (65). Furthermore, FAK inhibition also increases immune surveillance (66). Consequently, FAK appears to be an attractive target for pharmacological strategies for cancer therapy. Our data reveal a signaling pathway for senescence and support a therapeutic strategy for cancer.

#### AUTHOR CONTRIBUTIONS

H-HC, Y-YZ, and C-JY conceptualized and designed this study. P-HW devised the methodology. H-HC, P-HW, and Y-YZ performed the experiments. S-WN and Y-YZ performed formal analysis. M-SH and MH provided the resources. H-HC and Y-YZ

#### REFERENCES

wrote the draft. M-SH, MH, and C-JY reviewed and edited the manuscript. All authors reviewed the manuscript.

#### FUNDING

This work was supported by the Ministry of Science and Technology [MOST 102-2314-B-037-028-MY3] to C-JY; Kaohsiung Municipal Ta-Tung Hospital [grant number KMTTH-106-006]. This research was also supported by Academia Sinica and Ministry of Science and Technology [MOST 106-0210-01-15-02, MOST 107-0210-01-19-01] to MH.

#### ACKNOWLEDGMENTS

We thank Dr. Lee, Che-Hsin for technical assistance for the flow cytometry analysis.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc. 2019.00022/full#supplementary-material

nuclear architecture in Hutchinson-Gilford progeria syndrome. Proc Natl Acad Sci USA (2004)101:8963–8. doi: 10.1073/pnas.0402943101

beta-galactosidase with fibroblastic aging. Exp Gerontol. (2003) 38:887–95. doi: 10.1016/S0531-5565(03)00132-3

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Chuang, Wang, Niu, Zhen, Huang, Hsiao and Yang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Molecular Targeted Therapy in the Treatment of Chordoma: A Systematic Review

Tong Meng1,2,3†, Jiali Jin4†, Cong Jiang5 , Runzhi Huang1 , Huabin Yin2,3 \*, Dianwen Song2,3 \* and Liming Cheng1,6 \*

#### Edited by:

*Jian-ye Zhang, Guangzhou Medical University, China*

#### Reviewed by:

*Claudia Palena, National Cancer Institute (NCI), United States Victor C. Kok, Asia University, Taiwan*

#### \*Correspondence:

*Huabin Yin<EMAIL_ADDRESS>Dianwen Song<EMAIL_ADDRESS>Liming Cheng<EMAIL_ADDRESS>
*†These authors have contributed equally to this work and share first authorship*

#### Specialty section:

*This article was submitted to Cancer Molecular Targets and Therapeutics, a section of the journal Frontiers in Oncology*

Received: *05 November 2018* Accepted: *10 January 2019* Published: *01 February 2019*

#### Citation:

*Meng T, Jin J, Jiang C, Huang R, Yin H, Song D and Cheng L (2019) Molecular Targeted Therapy in the Treatment of Chordoma: A Systematic Review. Front. Oncol. 9:30. doi: 10.3389/fonc.2019.00030* *1 Division of Spine, Department of Orthopedics, Tongji Hospital Affiliated to Tongji University School of Medicine, Shanghai, China, 2 Shanghai Bone Tumor Institution, Shanghai, China, 3 Department of Orthopedics, Shanghai General Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China, 4 Department of Central Laboratory, Shanghai Tenth People's Hospital of Tongji University, School of Medicine, School of Life Sciences and Technology, Tongji University, Shanghai, China, 5 Beth Israel Deaconess Medical Center, BIDMC Cancer Center, Harvard Medical School, Cancer Research Institute, Boston, MA, United States, 6 Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration, Ministry of Education, Tongji University, Shanghai, China*

Objectives: Chordoma is a rare bone malignancy that affects the spine and skull base. Treatment dilemma leads to a high rate of local relapse and distant metastases. Molecular targeted therapy (MTT) is an option for advanced chordoma, but its therapeutic efficacy and safety have not been investigated systematically. Therefore, a systematic review was conducted on studies reporting MTT regimens for chordoma.

Methods: Clinical trials, case series and case reports on chordoma MTT were identified using MEDLINE, Cochrane library and EMBASE, and systematically reviewed. Data on clinical outcomes, such as median overall survival, progression-free survival, response rate and adverse events (AEs) were extracted and analyzed.

Results: Thirty-three eligible studies were selected for the systematic review, which indicated that imatinib and erlotinib were the most frequently used molecular targeted inhibitors (MTIs) for chordoma. For PDGFR-positive and/or EGFR-positive chordoma, clinical benefits were achieved with acceptable AEs. Monotherapy is preferred as the first-line of treatment, and combined drug therapy as the second-line treatment. In addition, the brachyury vaccine has shown promising results.

Conclusions: The selection of MTIs for patients with advanced or relapsed chordoma should be based on gene mutation screening and immunohistochemistry (IHC). Monotherapy of TKIs is recommended as the first-line management, and combination therapy (two TKIs or TKI plus mTOR inhibitor) may be the choice for drug-resistant chordoma. Brachyury vaccine is a promising therapeutic strategy and requires more clinical trials to evaluate its safety and efficacy.

Keywords: molecular targeted therapy, bone tumor, chordoma, systematic review, imatinib, erlotinib

## INTRODUCTION

Chordoma is a relatively rare malignant bone tumor with an incidence of 0.08 per 100,000 (1). It accounts for 1–4% of all bone malignancies, and ∼20% of primary spine tumors (2). Although it can occur at any segment of the spine, the predominant site of chordoma are fused segments like clivus and sacrococcyx (3). It is an indolent malignancy that progresses slowly, but exhibits strong local aggressiveness and often grows into huge masses that compress vital nerves and blood vessels (4). In addition, since chordoma is usually unresponsive to the conventional radiotherapy and cytotoxic chemotherapy, surgery is the primary therapeutic option (1, 5). Large case series including our previous one have shown that a total resection of the tumor, with the goal of negative microscopic margins, is crucial for long-term positive outcomes (6). However, the complex anatomy of the spine and the relatively large tumor volume make a clean resection technically challenging, leading to a high rate of local relapse and distant metastases (7). Regarding to this advanced setting, conventional therapeutic methods were shown to be not highly effective (1). Therefore, novel therapeutic strategies are needed to prolong patients' survival and improve the quality of life.

Pathologically, chordoma arises from residual notochord cells within the vertebral body (8), as verified on the basis of genetic and immuno-phenotypic biomarkers (9). New insights into the molecular mechanism underlying chordoma have also identified novel therapeutic targets (5). Molecular targeted therapy (MTT) in chordoma includes (1) imatinib and dasatinib against platelet-derived growth factor receptors (PDGFR) and stem cell factor receptor (KIT) (10, 11); (2) erlotinib, lapatinib, gefitinib, and cetuximab against epidermal growth factor receptor (EGFR) and erbB-2/human epidermal growth factor receptor 2 (HER2/neu) (12, 13); (3) sorafenib, pazopanib, and sunitinib that target angiogenic factors like vascular endothelial growth factor receptor (VEGFR) (14–16); and (4) temsirolimus and sirolimus that target the phosphoinositide 3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway (17).

The indications for MTTs in chordoma patients are largely based on a few prospective clinical trials, small retrospective studies, and even case reports (10–17). However, the efficacy and safety of MTT regimens in chordoma patients, as well as the underlying molecular mechanisms, lack systematic investigation. Therefore, we conducted a systematic review on MTT regimens in chordoma patients to determine the clinical outcomes and underlying molecular mechanisms.

#### MATERIALS AND METHODS

#### Search Strategy

For this systematic review, we used standard procedures from PRISMA guidelines (18). A comprehensive, systematic search was performed using MEDLINE (via PubMed), Cochrane Library and EMBASE. To find appropriate studies in MEDLINE, we used a combination of terms related to the MeSH terms "Chordoma/drug therapy" OR the free-text searching "Chordoma" AND ("targeted therapy OR inhibitor OR inhibit OR inhibition"). This search was further modified as appropriate for Cochrane Library and EMBASE. Initial search was performed on January 17, 2018 and repeated on July 1, 2018.

### Eligibility Criteria

Studies were deemed eligible for the assessment of MTTs in patients with chordoma, irrespective of previous and subsequent other treatment. Only English language publications were included. For clinical trials, case series and case reports published exclusively in abstract or news form, only those containing new data were analyzed. For literature reviews, new personal unpublished data is also included. Reference lists of selected studies and previous reviews associated with similar topics were screened manually. New clinical trials for chordoma were found from Chordoma Foundation, ClinicalTrials.gov, EU Clinical Trials Register and WHO International Clinical Trials Registry Platform. Although gray literature (such as unpublished reports, conference abstracts and dissertations) might provide some negative results and decrease the publication bias, we did not access them, because they were usually not peer reviewed and might be later published in peer-reviewed journals.

## Data Extraction and Synthesis

After removal of duplicates, titles and abstracts of all identified publications were systematically screened by two independent reviewers (MT and YHB). Discrepancies between reviewers were resolved by discussion. When eligibility criteria seemed to be met, the two reviewers (MT and YHB) independently assessed retrieved full texts and extracted information. If disagreements were still remained, the third reviewer (SDW) helped to reach an agreement. We contacted with the Chordoma Foundation in order to get helpful information. Additionally, we corresponded with researchers clarify study eligibility if the published study was unclear, although responses were poor. Extracted data were study characteristics (study design, first author, year of publication), patient characteristics (total number, history of treatment) and tumor characteristics (gene mutation and immunohistochemistry), MTT information (type of agents, dosage, course of treatment and adverse events), evaluation criteria (Choi's criteria, the response evaluation criteria in solid tumor (RECIST), clinical and radiological or metabolic response), and survival (duration of follow-up, progression-free survival and overall survival).

### RESULTS

### Search Results

The flow-chart for the selection and exclusion of relevant publications is shown in **Figure 1**. We identified 293 studies in the initial screening, and after removing duplicates and papers based on their titles and abstracts, selected 64 publications for full-text assessment. Twenty-seven studies met our inclusion criteria, and six more were included—three from manually searching the reference list of the selected articles, two from repeated search and one with the help of the Chordoma

Foundation. Finally, 33 studies were included in this systematic review.

## Study Characteristics

Among 33 studies, nine studies were clinical trials (10– 12, 14, 15, 19–22), with eight retrospective case series (16, 17, 23–28), and 16 case reports (13, 29–43). Imatinib was assessed in 18 studies with a total of 221 patients (10, 16, 17, 19, 23–28, 32, 34–36, 38, 39, 41, 42), erlotinib in 10 studies with 16 patients (13, 17, 22, 33, 35, 38, 40–42), cetuximab in five studies (seven patients) (13, 30, 31, 33, 41), sorafenib in four studies (65 patients) (15, 17, 21, 37), pazopanib in four studies with seven patients (16, 28, 41, 43) and sunitinib in three studies with 11 patients (14, 17, 28). Sirolimus, thalidomide, bevacizumab, gefitinib, linsitinib, and everolimus were accessed in two studies each (13, 22, 25, 28–31, 33, 34, 40– 42), whereas dasatinib (32 patients) (11), lapatinib (18 patients) (12), rapamycin (one patients) (34), temosirolimus (one patients) (17) and yeast-brachyury (GI-6301) vaccine (11 patients) (20) were only analyzed in one study each (**Figures 2** and **3**). Monotherapy of MTTs was reported in 24 studies (10–12, 14–17, 20, 21, 23, 24, 26–28, 32, 34–39, 41, 43, 44) with combination therapy in 13 studies (13, 19, 22, 25, 28–31, 33, 39– 42).

RECIST evaluation criteria was used in 19 studies (10–15, 17, 19, 20, 22, 25–28, 32, 40, 42, 43, 45) and Choi's criteria is applied in three studies (11, 12, 25). Twenty-one studies were evaluated by clinical/radiological or metabolic responses (16, 23–27, 29– 43). Adverse events (AEs) were reported in 25 studies, including hematological anomalies like anemia, thrombocytopenia, as well as non-hematological AEs like fatigue, fever, anorexia, QTc prolongation, abnormal liver function, nausea, and vomiting (10–15, 19, 22, 23, 26–38, 40, 43, 45).

#### Efficacy and Safety of MTT Regimens in Chordoma Patients PDGFR Inhibitors (Table 1)

Imatinib mesylate (IM), a specific tyrosine kinase inhibitor (TKI) targeting PDGFR and KIT (10, 46), was the most frequentlyused MTT in chordoma patients. Eighteen studies investigated the therapeutic efficacy of IM on 221 patients (10, 16, 17, 19, 23– 28, 32, 34–36, 38, 39, 41, 42), including three clinical trials (10, 19, molecular targeted inhibitor.

28), seven retrospective case series (16, 17, 23–27), and eight case reports (32, 34–36, 38, 39, 41, 42). Fourteen studies (204 patients) analyzed the efficacy of imatinib as monotherapy (10, 16, 17, 23, 24, 26–28, 32, 34–36, 39, 41), of which four studies (181 patients) used RECIST and 3 were focused on PDGFRβ-expressing chordoma. In these four studies, four patients achieved partial response (PR) (2.2%), 133 cases sustained stable disease (SD) (73.5%) and 44 cases experienced progressive disease (PD) (24.3%) (10, 17, 26, 27, 32). Clinical/radiological or metabolic responses were evaluated in 13 studies (85 patients), with 33 patients achieving PR (38.8%), 23 patients sustaining SD (27.1%) and 29 patients experiencing PD (34.1%) (16, 23, 24, 26–28, 32, 34–36, 38, 39, 41). Five of the above studies (73 patients) focused on PDGFRβ-expressing chordoma, with 45.2% PR, 31.5% SD, and 23.3% PD cases (23, 24, 26, 27, 36), and eight studies included 12 patients that experienced PD within a short period of time.

Progression-free survival (PFS) and overall survival (OS) are important indices of clinical outcome, and they were reported in two large case-studies (10, 27). Stacchiotti et al. conducted a phase II trial in 56 patients with chordoma, and the median PFS and OS were 9 and 35 months, respectively (10). A retrospective study on 46 chordoma patients reported a median PFS of 9.9 months (27).

AEs were reported in eight studies (10, 23, 27, 32, 34, 36), with skin rash being the most common, followed by oedema, chronic anemia, fatigue and fluid retention (10, 26). Subacute intraventricular hemorrhage was seen in one case of clivus chordoma treated with imatinib (36).

Dasatinib, an inhibitor of PDGFR and Src, was evaluated in a phase II study (NCT00464620) (11) on 32 patients. The median PFS and 6 months PFS rate were 6.3 months and 54%, respectively. The 2- and 5-years OS rate were 43 and 18%, respectively. Six patients had an objective response (OR) according to Choi criteria and one for RECIST. Fatigue, fever, anorexia, nausea, and vomiting occurred in more than 5% of the patients.

#### EGFR Inhibitors (Table 2)

Erlotinib was the most commonly used anti-EGFR agent and was analyzed in 10 studies (16 patients) for the treatment of chordoma (13, 17, 22, 32, 33, 35, 38, 40–42), including one clinical trials (22), one retrospective case study (17) and eight case reports (13, 17, 22, 32, 33, 35, 38, 40–42). Monotherapy with erlotinib was used in five studies (nine patients) (17, 32, 35, 38, 42), three (seven patients) of which were evaluated by RECIST (17, 32, 42), reporting PR in two patients and SD in five patients. Three case reports were evaluated by clinical/radiological or metabolic responses (32, 35, 38). All achieved PR and significant tumor bulk reduction was seen in two patients (70 and 46%, respectively). Skin rashes were commonly seen in the erlotinib-treated patients.

Lapatinib monotherapy was evaluated in a phase II clinical trial on 18 patients with EGFR-positive chordoma (12). Six patients achieved PR and seven sustained SD, with the median PFS of 6 months according to the Choi criteria. In contrast, all patients had SD by RECIST criteria with the median PFS of 8 months. Most patients experienced G ≥ 2 AEs.

Combined therapy with EGFR inhibitors was used in seven studies (eight patients) (13, 22, 30, 31, 33, 40, 41). Erlotinib was also the most common agents used in the combined MTT regimens (five studies, seven patients) (13, 22, 33, 40, 41).

Linsitinib, an inhibitor of IGF-1R/insulin receptor (INSR), was evaluated in a phase I study in combination with erlotinib (NCT00739453) (22). One patient with chordoma achieved PR for 18 months according to RECIST, with a PFS of 5 years. AEs included QTc prolongation, abnormal liver function, hyperglycemia and anorexia (22, 40).

The anti-EGFR monoclonal antibody (mAb) cetuximab was applied in combination with erlotinib in one patient with EGFRpositive chordoma, and he had a SD for 6 months (41). However, four patients with EGFR-negative chordoma experienced PD after receiving the same regimen. The treatment failure prompted a switch to bevacizumab, an anti-VEGF mAb (13, 33). Following this change, two patients achieved PR and another two presented SD. Treatment-related fatigue was observed in one patient (13, 33). Combined regimen of cetuximab and gefitinib was also effective in two cases of EGFR-positive chordoma (30, 31), where

#### Frontiers in Oncology | www.frontiersin.org

one achieved a PR for 9 months and the other had a 44% reduction in tumor bulk. Pronounced AEs, such as rash, acne, diarrhea, and skin defects, were reported in both cases (30, 31).

#### VEGFR Inhibitors (Table 3)

Sorafenib, a TKI against VEGFR and PDGFR, was assessed in four studies (15, 17, 21, 37). A phase II trial was conducted on 27 patients with chordomas (NCT00874874) (15), and OR was observed in one patient as per RECIST. The 12 months PFS and OS rates were 73.0 and 86.5%, respectively. In a study on 11 patients treated with sorafenib, PR was obtained in one patient, with SD in nine patients and PD in one patient according to RECIST (17). Another study assessing sorafenib reported a PFS of 12 months (37). However, sorafenib was limited by severe AEs like thrombocytopenia and diarrhea, and the rates of grade 3 and 4 toxicity were 77.8 and 14.8%, respectively.

Sunitinib, a multi-targeting TKI against VEGFR and PDGFR, was assessed in three studies (14, 17, 28). A phase II trial on sunitinib was conducted on nine patients (14), four of which achieved SD according to RECIST, concurrent to a qualitative decrease in tumor density, along with a median PFS of 12 months (14). Two patients treated with sunitinib had at least SD according to RECIST (17, 28), and one achieved a PR after a 27 months SD (28). The major toxicities were of grade 1 or 2 (14).

Pazopanib, another VEGFR inhibitor, was analyzed in seven patients (16, 28, 41, 43), of which four sustained SD with the median PFS of 15 months and the remaining three experienced PD. Thalidomide, an inhibitor of VEGF, was used as a second-line treatment for chordoma after failure of imatinib, rapamycin and other chemotherapy (29, 34). While one patient achieved a 50% tumor reduction, another experienced a PD (29, 34). In addition, severe toxicities of grade 3 and 4 were reported in both cases.

#### Other Molecular Targeted Inhibitors (MTIs) (Table 4)

Monotherapy with the mTOR inhibitors rapamycin and everolimus were ineffective in chordoma patients (34, 41). The combined MTT regimen of everolimus and imatinib resulted in sustained SD in one patient, with a PFS of 16 months (42). In addition, IM plus sirolimus was used in 10 patients with IMrefractory chordoma and activated mTOR (25). Nine patients were assessed, of which one achieved PR, seven sustained SD and one experienced PD according to RECIST. According to Choi criteria, seven patients achieved PR, and one sustained SD and one experienced PD. The same MTT regimen was also used against IM- and sunitinib-refractory chordoma but was not effective due to short of the mTOR expression (28). A phase I trial evaluated the effect of IM plus metronomic cyclophosphamide (MC)-based chemotherapy on 7 IM- and sunitinib-refractory chordoma patients (19). The median PFS was 10.2 months, and the 12 months PFS and OS rates were 42.9 and 85.7%, respectively according to RECIST. No dose-limiting toxicity and drug pharmacokinetic interactions were observed.

#### Brachyury Vaccine (Table 4)

A phase I dose-escalation trial using a recombinant Saccharomyces cerevisiae (yeast) vaccine encoding brachyury (GI-6301) was conducted on 11 patients (20), and 10 evaluable

patients showed a median PFS of 8.3 months. One patient achieved PR, with eight sustaining SD and one experiencing PD at 3 months according to RECIST. Seven patients had no evidence of PD, giving a clinical benefit rate of 70% at 5 months. The most common AEs were injection site reactions.

Ongoing and planned clinical trials on chordoma MTT are listed in **Table 5** .

#### DISCUSSION

Novel therapeutic strategies against chordoma are urgently needed to prolong the overall survival and relieve symptoms. Elucidation of the underlying molecular mechanisms of chordoma have helped identify numerous potential therapeutic targets (47 , 48), and several anti-chordoma agents are currently being tested in animal models and clinical trials. This systematic review is focused on the pharmacological management of chordoma patients and the clinical outcomes. Furthermore, the molecular mechanisms of MTT action have also been assessed.

#### Molecular Targets

Chordoma is a genetically heterogeneous tumor with frequent imbalances of large chromosomal regions. Somatic duplications of the notochordal transcription factor brachyury (47 , 48), chromosomal copy loss of phosphatase and tensin homolog (PTEN) (49), tuberous sclerosis complex (TSC) (50), cyclindependent kinase inhibitor 2A and 2B (CDKN2A and CDKN2B) (51), SMARCB1 (49), and PIK3CA (9) mutations are key aspects of chordoma pathogenesis, and therefore potential targets.

RTKs are the key players in the development and progression of chordoma, and their mutated forms can activate signaling cascades resulting in dysregulation of many essential proteins. Therefore, mutational analyses and IHC can greatly assist oncologists to determine the optimal inhibitors (52 –56). It needs to be emphasized that mutations in the molecular targets are clinically more relevant than their immunoreactivity, since target overexpression is not always driven by the activation of the corresponding signaling pathway. For example, high levels of EGFR in the chordoma cell line JHC7 was not accompanied by activated EGFR signaling (57).

#### Indications and Evaluation Criteria for MTTs

MTTs are not the first treatment options for chordoma, and only recommended for advanced or recurrent chordoma that are unresponsive to either surgical resection or radiotherapy.