Protein phosphatase 2A inhibition enhances radiation sensitivity and reduces tumor growth in chordoma

Protein phosphatase 2A inhibition enhances radiation sensitivity and reduces tumor growth in... Abstract Background Standard therapy for chordoma consists of surgical resection followed by high-dose irradiation. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase involved in signal transduction, cell cycle progression, cell differentiation, and DNA repair. LB100 is a small-molecule inhibitor of PP2A designed to sensitize cancer cells to DNA damage from irradiation and chemotherapy. A recently completed phase I trial of LB100 in solid tumors demonstrated its safety. Here, we show the therapeutic potential of LB100 in chordoma. Methods Three patient-derived chordoma cell lines were used: U-CH1, JHC7, and UM-Chor1. Cell proliferation was determined with LB100 alone and in combination with irradiation. Cell cycle progression was assessed by flow cytometry. Quantitative γ-H2AX immunofluorescence and immunoblot evaluated the effect of LB100 on radiation-induced DNA damage. Ultrastructural evidence for nuclear damage was investigated using Raman imaging and transmission electron microscopy. A xenograft model was established to determine potential clinical utility of adding LB100 to irradiation. Results PP2A inhibition in concert with irradiation demonstrated in vitro growth inhibition. The combination of LB100 and radiation also induced accumulation at the G2/M phase of the cell cycle, the stage most sensitive to radiation-induced damage. LB100 enhanced radiation-induced DNA double-strand breaks. Animals implanted with chordoma cells and treated with the combination of LB100 and radiation demonstrated tumor growth delay. Conclusions Combining LB100 and radiation enhanced DNA damage-induced cell death and delayed tumor growth in an animal model of chordoma. PP2A inhibition by LB100 treatment may improve the effectiveness of radiation therapy for chordoma. chordoma, LB100, PP2A, radiation therapy Chordomas are slow-growing yet locally aggressive primary bone tumors of the axial skeleton. They are believed to arise from the neoplastic transformation of notochordal remnant tissues accounting for the distribution of tumors along the midline axis.1 The most common sites of involvement are the sacrum, skull base, and the remaining mobile spine.2 For this reason, patients with chordomas are treated by a variety of different subspecialists depending on the location of the tumor.3–7 Because chordomas are locally invasive with metastatic potential, a complete en bloc surgical resection is clinically indicated to prevent or delay tumor recurrence. However, efforts to remove the entire tumor are often hampered by proximity to critical neurovascular structures of the skull base and the spine, leaving most patients with residual disease. Since effective medical therapy for chordoma is lacking, patients left with incomplete surgical resection require high-dose irradiation due to radioresistance of chordoma cells,2,8–11 increasing risk of injury to nearby normal tissue and contributing to morbidity.12 As such, there is a critical unmet need to identify strategies to improve the effectiveness of irradiation in patients with chordoma while minimizing treatment-associated toxicity.13 Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase with multifunctional roles in diverse physiologic processes and many pathological conditions, including cancer.14 PP2A mediates the DNA damage response,15 therefore the inhibition of PP2A catalytic activity sensitizes cancer cells to DNA damaging effects of ionizing radiation in part by altering homologous recombination repair capacity.16 Based on these previous studies of the PP2A role in DNA repair, and in concert with the observation that cancer cells exhibit altered DNA repair functions, we hypothesized that the combination of PP2A inhibition and irradiation may have complementary antitumor activity. LB100, a hydrophilic small-molecule derivative of cantharadin, is a potent inhibitor of PP2A. This compound was developed by Lixte Biotechnology through a cooperative research and development agreement based on our previous work.17–19 A recently completed phase I study confirmed the feasibility of safe administration of LB100 in solid tumors.20 Preclinical studies in a pancreatic cancer model suggest LB100 sensitizes cells to irradiation by interfering with DNA homologous recombination repair capacity.16 Chordomas are radioresistant in part due to extensive tissue hypoxia.21–24 Poor tissue oxygenation demands delivery of high-dose irradiation associated with increased toxicity, to achieve adequate antitumor response. Strategies that can enhance the effectiveness of radiation may offset the need to administer potentially toxic doses to sensitive tissues. In this study, we investigated the radiation sensitizing potential of LB100 in chordoma. Methods Chemicals, Cell Lines, and Radiation Treatment LB100 was provided by Lixte Biotechnology Holdings. The chemical structure of LB100 is shown in Fig. 1. LB100 was reconstituted in 0.1 N monosodium glutamate, pH 10.5 (pH was adjusted with NaOH), and stored at −80°C. The 3 patient-derived chordoma cell lines used in this study—U-CH1, UM-Chor1, and JHC7—were obtained from the Chordoma Foundation. Cells were maintained in Iscove’s modification of Dulbecco’s medium (IMDM) and Roswell Park Memorial Institute (RPMI) 1640 medium (4:1), supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and nonessential amino acids. Fig. 1 View largeDownload slide View largeDownload slide LB100 alters cell cycle state and augments antitumor effect of irradiation. Human chordoma cell lines, U-CH1, UM-Chor1, and JHC7, were used to evaluate the proliferative effect of LB100. Two human chordoma cell lines, U-CH1 and UM-Chor1, were used for cell cycle analysis. (A–C) As a single agent, LB100 has minimal effect on cell proliferation at clinically achievable concentrations (5 µM). Cell proliferation was quantified with XTT assays following 24, 48, or 72 h of treatment with increasing concentrations of LB100 and analyzed using a one-way ANOVA comparing the treatment groups with vehicle control. (D) Chemical structure of LB100. (E) LB100 strikingly enhances radiation-mediated cytotoxicity after 72 h of treatment. (F–I) Cell cycle analysis was performed 24 h following treatment with the indicated combinations of LB100 and radiation. The percentage of cells in the indicated cell cycle stage were quantified by flow cytometry and analyzed by a 2-way ANOVA. Cells were plated in triplicate. Fig. 1 View largeDownload slide View largeDownload slide LB100 alters cell cycle state and augments antitumor effect of irradiation. Human chordoma cell lines, U-CH1, UM-Chor1, and JHC7, were used to evaluate the proliferative effect of LB100. Two human chordoma cell lines, U-CH1 and UM-Chor1, were used for cell cycle analysis. (A–C) As a single agent, LB100 has minimal effect on cell proliferation at clinically achievable concentrations (5 µM). Cell proliferation was quantified with XTT assays following 24, 48, or 72 h of treatment with increasing concentrations of LB100 and analyzed using a one-way ANOVA comparing the treatment groups with vehicle control. (D) Chemical structure of LB100. (E) LB100 strikingly enhances radiation-mediated cytotoxicity after 72 h of treatment. (F–I) Cell cycle analysis was performed 24 h following treatment with the indicated combinations of LB100 and radiation. The percentage of cells in the indicated cell cycle stage were quantified by flow cytometry and analyzed by a 2-way ANOVA. Cells were plated in triplicate. Cell Proliferation Analysis Cell proliferation was determined with a 2,-3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay as indicated by the manufacturer’s instruction. U-CH1, UM-Chor1, and JHC7 cell lines were seeded in 96-well plates at 5 × 104 cells/well. After 24 h incubation, cells were treated with various doses (0–20 μM) of LB100 and incubated for 24, 48, and 72 hours. Activated-XTT solution was prepared by the manufacturer’s protocol and the cells incubated for 4 hours. Cell proliferation was calculated using the background-corrected absorbance as follows: Cell proliferation = [Aλ sample well / Aλ control well) / DMSO treated control]. Cell Cycle Analysis U-CH1 cells were seeded at 2 × 105 cells/well in a 6-well dish. The following day, cells were treated with 4 μM LB100 or vehicle control (DMSO) for 4 hours before receiving sham or 2 Gy irradiation. After overnight incubation, cells were pulsed for 4 hours with 10 μM 5-ethynyl-2′-deoxyuridine (EdU) and evaluated by flow cytometry. EdU uptake was detected with a Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Thermo Fisher Scientific, C10424) per the manufacturer’s protocol. DNA was stained with FxCycle violet stain (Thermo Fisher Scientific, F10347). Data were acquired on a BD LSRFortessa X50 flow cytometer and analyzed using FlowJo v9.9.4. Immunofluorescent Cytochemical Staining for γ-H2AX Cells were grown in chamber slides and exposed to LB100 (4 μM) for 4 hours prior to administration of 2 Gy or sham radiation. Cells were fixed with 2% paraformaldehyde, washed with phosphate buffered saline (PBS), permeabilized with 1% Triton X-100, washed again with PBS, and blocked with 1% bovine serum albumin (BSA). Mouse anti–γ-H2AX antibody (Millipore) was added at 1:500 and incubated overnight at 4°C. Cells were washed with 1% BSA, and goat anti-mouse‒fluorescein isothiocyanate antibody (Jackson ImmunoResearch) was added at 1:100 and incubated for 1 hour at room temperature. Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (Sigma). Coverslips were mounted with Vecta Shield anti-fade solution (Vector Labs) and slides examined on a Leica DMRXA fluorescent microscope (Leica Microsystems). γ-H2AX foci were quantitated in 50 cells per condition. Raman Imaging Microscopy Raman imaging microscopy was used to confirm DNA damage. Raman is a spectroscopic technique that measures the energy difference between the incident photons from a laser and the inelastic scattered photons collected at a detector. This difference is the energy required for different types of vibrations of the molecule. The vibration frequencies depend on the symmetry of the molecule, masses of atoms, distance between atoms, bond angle, and strength. The use of Raman via the Raman imaging microscope allows focusing of the laser through the microscope and the collection of Raman spectra for each pixel. Subsequent movement of the stage permits the acquisition of spectra in a new position. This approach provides a chemical and biochemical mapping of the entire cell in the culture medium. Due to its comprehensive chemical analysis, Raman spectra of DNA contain multiple peaks from allowed vibration frequencies that have been previously assigned to T, C G, A, single-strand DNA, double-strand DNA, backbone, or DNA attached to nanoparticles. Samples used for Raman were transferred into 35 mm sterile glass bottom dishes (Ibidi) and cultured for 24 hours. Raman spectra were acquired using a DXR 2xi Raman microscope (Thermo Fisher Scientific) with 24 mW of 780 nm laser through a 60x water immersed confocal objective, at 0.5 s exposure time for a 1 μm pixel size, between 50 and 3200 cm−1 spectral region. Spectra were collected and subsequently background corrected using the Raman silent region. Chemical maps were produced by the peak area function between 1085 and 1100 cm−1 using Thermo Fisher Scientific OMNIC software. Wound Healing Assay Seeded onto 12-well plates were 2 × 105 U-CH1 and UM-Chor1 cells. Once cells reached 100% confluence, “wounds” were created by scraping “#” lines with a 200 μL pipette tip, and then cells were washed 3 times in serum-free medium. Cells were exposed to medium, LB100 (4 μM), 2 Gy irradiation, or LB100 (4 μM) for 4 hours prior to administration of 2 Gy. The “wounds” were observed every 24 hours and photographed using an EVOS Cell Imaging System (EVOS XL Core Cell Imaging System, Thermo Fisher Scientific). Eight images were taken per well at each time point using a 10x objective. The distances between the 2 edges of the scratch (wound width) were measured at 3 sites for each image using ImageJ software. The migratory distances were calculated by subtracting the wound width at each time point from the wound width at the time zero point. To complete the experiment, cells were washed 2 times in PBS, fixed for 10 min with methanol, and stained using 0.2% crystal violet for 20 min. Invasion Assay Chordoma cell invasive capacity was assessed using Corning BioCoat Matrigel Invasion Chambers with BD Matrigel Matrix (Thermo Fisher Scientific) as previously described.22 Pre-coated membranes (8 μm pore size, Matrigel 100 μg/cm2) were rehydrated and seeded with 3 × 105 cells in 2 mL of IMDM/RPMI (4:1) with or without LB100 in triplicates into the upper part of each transwell. Cells were irradiated (2 Gy) 4 hours after treatment with LB100. The lower compartment was filled with 2.5 mL of IMDM/RPMI (4:1) supplemented with 0.1% BSA. After incubation for 96 hours at 37°C, non-invaded cells on the upper surface of the membrane were wiped with a cotton swab. Migrated cells on the lower surface of the filter were fixed with 70% ethanol for 2 minutes and stained with crystal violet (0.5%) for 15 minutes. Invasive activity was determined by counting cells in 4 microscopic fields per well, and the extent of invasion was expressed as an average number of cells per microscopic field. Western Blotting Immunoblotting was performed as previously described.25 Proteins were extracted by CellLytic M (Sigma-Aldrich) cell lysis buffer supplemented with protease inhibitor cocktail and phosphatase inhibitors. Protein quantification was measured by the Pierce BCA protein assay kit (Thermo Fisher Scientific). The protein bands were detected by conventional protocols for western blotting. Proteins were detected by using specific primary antibodies against γ-H2AX (Millipore) and glyceraldehyde 3-phosphate dehydrogenase (Santa Cruz Biotechnology), and subsequently with the appropriate horseradish peroxidase–conjugated secondary antibodies (Cell Signaling Technology). Subcutaneous Xenograft Model All animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Animals and approved by the Animal Care and Use Committee of the NIH. Six-week-old female NSG mice (NOD.Cg-Prkdcscid Il2rgtm1WjI/SzJ; Charles River) were used for the in vivo studies. U-CH1 cells (8 × 106 in 100 µL of Hanks’ Balanced Salt Solution) were subcutaneously injected into the right thigh. Xenografted tumors were measured twice a week from the first day of treatment and volumes were calculated using the formula: TV = (width)2 × (length)/2. When tumors reached ≈130 mm3, animals were randomized into 5 groups: untreated controls, LB100 (1 mg/kg), radiation (5 Gy), and combination LB100 and radiation (3 or 5 Gy). Each group was composed of 8 animals. LB100 was delivered by intraperitoneal injections at a dose of 1 mg/kg, twice a day for 3 days. Radiation treatments were given on 2 consecutive days. Endpoints were (i) the diameter of tumor reaching 2 cm and (ii) development of ulcerations. Survival analysis was performed by plotting the Kaplan–Meier curves. The log-rank test was used to compare the groups. Irradiation of Animals Animals were restrained in a custom jig that was developed and used extensively in the Radiation Oncology Branch and the Radiation Biology Branch of the NIH and placed in an orthovoltage radiotherapy unit maintained at the NIH. The mice were restrained in the custom leg jig for no more than 7 minutes. The rest of the mouse, except the leg, was covered with lead shielding. Previous studies have demonstrated this procedure to be of minimal stress to the animals, as the process is painless, odorless, and brief; therefore, no anesthetics were given. Irradiation was delivered using the Pantek radiation machine located in the NIH Animal Facility. Irradiation treatment was performed in an Xrad 320 irradiator (Precision X-ray) at a dose rate of 256.1 cGy/min. Delivered dose was verified by thermoluminescent dosimeter. Animals were observed during irradiation for any signs of discomfort. Animals that were unable to consume food or water or exhibited signs of illness were immediately euthanized. Statistical Analysis In vitro studies were subject to 3 independent experiments. Data are presented as mean ± SD. An ordinary one-way ANOVA test was used for comparison between more than 2 groups. P ≤ 0.05 was considered statistically significant. Values stated within text and figures represent mean ± SD. Statistics were performed on results from at least 2 independent replicates. Results LB100 Enhances Radiation-Mediated Attenuation of Chordoma Cell Proliferation To determine the sensitivity of chordoma cells to the PP2A inhibitor, LB100, three human chordoma cell lines were used: U-CH1, UM-Chor1, and JHC7. In vitro cell proliferation was assessed in response to LB100 treatment in dose- and time-dependent manners (Fig. 1A–C). LB100 as a single agent demonstrated a moderate and heterogeneous effect on cell proliferation at clinically achievable concentrations (up to 4–5 µM). U-CH1 and UM-Chor1 cells were more resistant than JHC7 and did not show significant decreases in proliferation until treatment at 10 µM (Fig. 1A–C). However, LB100 in combination with radiation treatment significantly reduced U-CH1 cell numbers compared with vehicle control or LB100 or radiation treatment alone (Fig. 1E). These data suggest that a clinical achievable dose of LB100 is effective when combined with radiation in the treatment of chordoma. LB100 Treatment Increases G2/M-Phase Arrest PP2A can influence multiple stages of the cell cycle, from G1/S transition to cytokinesis.26 To determine whether PP2A inhibition alters cell cycle in chordoma cells, EdU incorporation assays were performed in U-CH1 cells treated with LB100, radiation, or the combination. EdU uptake was assessed 24 hours following radiation to prevent cell loss due to treatment-related cytotoxicity. LB100, radiation, and the combination each led to a significant reduction of cells in S phase compared with vehicle control 24 hours following radiation treatment. Interestingly, cells treated with LB100 or LB100 with radiation had a significantly increased population of cells in the G2/M phase compared with control and radiation-only groups (Fig. 1F–H). The G0/G1 phase was not significantly altered by any treatment (Fig. 1I). Together, these data demonstrate that the PP2A inhibitor LB100 increases the proportion of cells in the radiation-sensitive G2/M phase of the cell cycle. LB100 Sensitizes Chordoma Cells to Radiation-Induced DNA Damage Through γ-H2AX Activation To evaluate for a potential radiation sensitizing effect of LB100, we screened U-CH1 and UM-Chor1 cells. To determine the effect of LB100 treatment on DNA damage and repair, γ-H2AX expression level was measured by immunofluorescence and immunoblot in 2 chordoma cell lines at 24 hours (Fig. 2A–C). It was previously reported that in the absence of DNA damage, cells at G2/M phase can demonstrate elevated γ-H2AX levels, a phenomenon referred to as mitotic H2AX phosphorylation.27–29 Consistent with these reports, LB100 alone increased expression and foci of γ-H2AX as it drove cells to G2/M phase (Fig. 2A–C). Radiation alone also increased expression and foci of γ-H2AX (Fig. 2A–C; Supplementary Figure S1). Though we did not observe significant change in γ-H2AX expression levels between the LB100 alone group and the combination of LB100 plus radiation, cells treated with the combination of LB100 and radiation showed the highest γ-H2AX level for foci formation compared with the control group, the LB100 alone group, and the radiation alone group. Fig. 2 View largeDownload slide LB100 enhances radiation-induced DNA damage. (A) U-CH1 and UM-Chor1 cells were treated with 4 μM LB100 for 3 hours pre- and 24 hours post-radiation (2 Gy). At the end of drug exposure, cells were fixed and then subjected to immunofluorescence staining with 4′,6′-diamidino-2-phenylindole‒blue and fluorescein isothiocyanate‒green foci for γ-H2AX, a marker of DNA double-strand breaks. Representative images show increased γ-H2AX expression in the cells exposed to the combination of LB100 and irradiation. (B) Western blot analysis reveals increased protein level of γ-H2AX with LB100 treatment and combination treatment with LB100 and radiation in U-CH1 and UM-Chor1 cells. (C) Quantification of γ-H2AX volume intensity relative to glyceraldehyde 3-phosphate dehydrogenase volume intensity. (D) DNA backbone intensity and location in live chordoma cells as a function of different treatments. Optical image (top panel). Chemical image (lower panel). Image 1: untreated; image 2: treated with LB100; image 3: irradiated; image 4: irradiated and treated with LB100; image 5: overlay of Raman spectra indicating the peak at 1095 cm−1 corresponding to the DNA backbone. Scale bar = 10 μm. (E) Images obtained by transmission electron microscopy show nuclear fragmentation in cells treated with the combination of LB100 and irradiation. Fig. 2 View largeDownload slide LB100 enhances radiation-induced DNA damage. (A) U-CH1 and UM-Chor1 cells were treated with 4 μM LB100 for 3 hours pre- and 24 hours post-radiation (2 Gy). At the end of drug exposure, cells were fixed and then subjected to immunofluorescence staining with 4′,6′-diamidino-2-phenylindole‒blue and fluorescein isothiocyanate‒green foci for γ-H2AX, a marker of DNA double-strand breaks. Representative images show increased γ-H2AX expression in the cells exposed to the combination of LB100 and irradiation. (B) Western blot analysis reveals increased protein level of γ-H2AX with LB100 treatment and combination treatment with LB100 and radiation in U-CH1 and UM-Chor1 cells. (C) Quantification of γ-H2AX volume intensity relative to glyceraldehyde 3-phosphate dehydrogenase volume intensity. (D) DNA backbone intensity and location in live chordoma cells as a function of different treatments. Optical image (top panel). Chemical image (lower panel). Image 1: untreated; image 2: treated with LB100; image 3: irradiated; image 4: irradiated and treated with LB100; image 5: overlay of Raman spectra indicating the peak at 1095 cm−1 corresponding to the DNA backbone. Scale bar = 10 μm. (E) Images obtained by transmission electron microscopy show nuclear fragmentation in cells treated with the combination of LB100 and irradiation. To better understand these observations, we evaluated nuclear DNA damage using Raman imaging microscopy and transmission electron microscopy. Raman imaging microscopy was used to measure the intensity and location of the intact DNA backbone, which has a vibration at 1095 cm−1. Since the Raman signal is proportional to the concentration of the chemical species, by fragmenting the DNA backbone or interfering with its chemical structure, it is expected that the intensity of the intact peak at 1095 cm−1 will decrease or disappear. Raman imaging microscopy was used to demonstrate the relative effect of LB100 and radiation on the intensity and location of the DNA backbone in live chordoma cells. For simplicity, only the peak at 1095 cm−1 assigned to the O–P–O stretching of the DNA backbone was mapped.30–33 Irradiation of chordoma cells led to a decrease in the integrity of the DNA backbone (Fig. 2D, image 3) compared with control and LB100 alone (Fig. 2D, images 1 and 2), and this effect was further enhanced by the addition of LB100 (Fig. 2D, image 4). Chordoma cells from these 4 groups were further probed by transmission electron microscopy. The cells treated with the combination of LB100 and irradiation revealed significant nuclear fragmentation (Fig. 2E). Taken together, these results suggest that LB100 treatment enhances sensitivity of chordoma cells to radiation-induced damage. The Effect of LB100 on Chordoma Cell Migration and Invasion Tumor cell mobility plays a critical role in invasion and metastasis. Chordoma cells are highly invasive. Therefore, we investigated the effect of LB100 on cell motility. Serial images of the tumor cells were collected every 24 hours after inducing a wound injury. Representative images at 144 hours following treatment showed that exposure to LB100 attenuates cell motility (Fig. 3A and B). The time required for cells to migrate into the gap was significantly longer for cells treated with LB100 compared with radiation or vehicle control. Cell motility was further assessed with an in vitro invasion model. Cells exposed to LB100 with or without radiation exhibited decreased invasive capacity (Fig. 3C and D). Notably, in U-CH1 cells which are resistant to LB100 treatment, the combination of LB100 plus radiation decreased invasion compared with LB100 alone (Fig. 3D). Fig. 3 View largeDownload slide LB100 attenuates chordoma cell migration and invasion. (A) Representative images from the wound-healing assay for each cell line taken at 144 hours. (B) Migration distance was analyzed using ImageJ software (n = 3). Data are presented as mean ± SEM. A one-way ANOVA was used for statistical analyses (****P < 0.0001). (C) Representative images of a Matrigel invasion assay with U-CH1 and UM-Chor1 cell lines show a significant decrease in invasion in response to 96 hours of LB100 treatment (4 μM) and irradiation (2 Gy) (n = 3). Scale bar = 200 µm. (D) Invasion is quantified as number of cells per microscopic field (20x), and a one-way ANOVA was used to compare ± SEM. Fig. 3 View largeDownload slide LB100 attenuates chordoma cell migration and invasion. (A) Representative images from the wound-healing assay for each cell line taken at 144 hours. (B) Migration distance was analyzed using ImageJ software (n = 3). Data are presented as mean ± SEM. A one-way ANOVA was used for statistical analyses (****P < 0.0001). (C) Representative images of a Matrigel invasion assay with U-CH1 and UM-Chor1 cell lines show a significant decrease in invasion in response to 96 hours of LB100 treatment (4 μM) and irradiation (2 Gy) (n = 3). Scale bar = 200 µm. (D) Invasion is quantified as number of cells per microscopic field (20x), and a one-way ANOVA was used to compare ± SEM. LB100 in Combination with Irradiation Delays In Vivo Tumor Growth To determine the potential clinical utility of LB100, mice bearing U-CH1 subcutaneous xenograft tumors were randomized into 5 groups: (i) vehicle, (ii) LB100 (1 mg/kg), (iii) radiation (5 Gy), (iv) combination of LB100 (1 mg/kg) and irradiation (3 Gy), (v) combination of LB100 (1 mg/kg) and irradiation (5 Gy). Tumor volumes were significantly smaller in animals treated with the combination of LB100 and irradiation compared with other groups (control group P = 0.028, LB100 group P = 0.0014, 5 Gy radiation group P = 0.0273) (Fig. 4A–C). Tumors were further analyzed by immunohistochemistry for Ki-67 and assay by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) (Supplementary Figure S3). Ki-67 labeling was found to be decreased for the tumors exposed to LB100 and irradiation. TUNEL assay revealed increased staining in the combination arm, indicative of apoptosis. Interestingly, consistent with mitotic arrest, tumors treated with the combination of LB100 and irradiation demonstrated the presence of multinucleated cells. Fig. 4 View largeDownload slide LB100 in combination with irradiation significantly decreases tumor burden in vivo. To evaluate the benefit of combination treatment in vivo, mice bearing U-CH1 subcutaneous xenografts were randomized into 5 groups: vehicle control; LB100 (1 mg/kg); irradiation (5 Gy); combination LB100 (1 mg/kg) and irradiation (3 Gy); and combination LB100 (1 mg/kg) and irradiation (5 Gy). Treatment schedule was: intraperitoneal injections of LB100 given twice daily for 3 consecutive days. Radiation (3 or 5 Gy) was given on 2 consecutive days. (A and B) Xenograft tumor volumes of animals treated with the combination of LB100 and irradiation are significantly reduced compared with other groups (control group P = 0.028; LB100 group P = 0.0014; 5 Gy radiation group P = 0.0273). Data are presented as mean ± SEM. (C) Subcutaneously implanted tumors were surgically removed. Tumor sizes in the LB100 and irradiation combination groups are smaller. Fig. 4 View largeDownload slide LB100 in combination with irradiation significantly decreases tumor burden in vivo. To evaluate the benefit of combination treatment in vivo, mice bearing U-CH1 subcutaneous xenografts were randomized into 5 groups: vehicle control; LB100 (1 mg/kg); irradiation (5 Gy); combination LB100 (1 mg/kg) and irradiation (3 Gy); and combination LB100 (1 mg/kg) and irradiation (5 Gy). Treatment schedule was: intraperitoneal injections of LB100 given twice daily for 3 consecutive days. Radiation (3 or 5 Gy) was given on 2 consecutive days. (A and B) Xenograft tumor volumes of animals treated with the combination of LB100 and irradiation are significantly reduced compared with other groups (control group P = 0.028; LB100 group P = 0.0014; 5 Gy radiation group P = 0.0273). Data are presented as mean ± SEM. (C) Subcutaneously implanted tumors were surgically removed. Tumor sizes in the LB100 and irradiation combination groups are smaller. Discussion Therapeutic options for patients with chordoma are limited. Since complete tumor resection is seldom achieved, external beam radiation therapy remains the standard of care, as effective medical therapies do not yet exist. However, because chordomas are radioresistant, high doses of ionizing radiation must be delivered to areas that border delicate neurovascular structures. Two main strategies are utilized to permit delivery of high-dose radiation to chordoma: (i) intensity-modulated radiation therapy and stereotactic radiosurgery, which better delineate borders of treatment to minimize toxicity to non-neoplastic tissues, and (ii) particle beam, such as proton, therapy, which concentrates intratumoral radiant energy. However, concurrent administration of a radiosensitizer would further enhance the effectiveness of these advanced technologies. As a single agent, LB100 had a modest anti-proliferative effect. However, when LB100 was given in combination with irradiation, we observed a marked inhibition of chordoma proliferation. To define a potential mechanism for the observed radiosensitizing property of LB100, we investigated the cell cycle state of chordoma cells and found that either as a single treatment or in combination with radiation, a greater proportion of chordoma cells were found to be in the G2/M phase of the cell cycle, a state most sensitive to the damaging effects of irradiation. Given this observation and published reports that PP2A participates in the DNA repair process, we searched for a candidate mechanism and found that the addition of LB100 to irradiation potentiated DNA double-strand breaks. This was evaluated by the presence of γ-H2AX, a product of rapid phosphorylation of histone H2AX at serine 139 which serves as a sensitive marker for DNA double-strand breaks induced by ionizing radiation or other genotoxic agents.34 Because cells in the G2/M phase of the cell cycle can demonstrate elevated expression of γ-H2AX in the absence of DNA damage, we used Raman spectroscopic imaging to confirm that cells treated with LB100 alone had similar concentrations of intact backbone DNA to control cells. This technique further corroborated the results of γ-H2AX experiments revealing that the combination of LB100 and irradiation contributed to the greatest amount of DNA damage. Ultrastructural analysis with electron microscopy revealed extensive nuclear fragmentation in cells exposed to the combination of LB100 and irradiation, further verifying DNA damaging effects of the combination treatment. These observations suggest that by interfering with the homologous recombination repair process, LB100 promotes persistent genomic damage. Although irradiation is delivered for antitumor effect, exposure of tumor cells to ionizing radiation may occasionally promote cancer growth and invasion.35–37 This may be particularly relevant for chordoma, since this is a disease characterized by infiltration of tumor cells into surrounding structures that often precludes complete surgical resection. However, we observed that chordoma cells treated with LB100 demonstrated attenuation of migration as well as invasion. Lastly, the antitumor activity of LB100 in combination with irradiation was confirmed in an in vivo xenograft model. The preclinical data presented here suggest that LB100 is capable of enhancing the therapeutic effects of irradiation in chordoma. Although LB100 as a single agent and in combination with cytotoxic chemotherapy was well tolerated in a recently completed phase I study, the safety profile in combination with irradiation has yet to be established.20 Preclinical work performed with a pancreatic cancer model indicates that LB100 may selectively target tumor cells. Exposure of normal intestinal cells to clinically achievable concentrations of LB100 failed to produce radiosensitization.16 This observation is consistent with our experimental data demonstrating LB100-induced radiosensitization is dependent on cell cycle state; nondividing cells may not be adversely affected. While additional studies to further characterize and confirm LB100-induced radiosensitization on dividing and nondividing cells is needed, this study offers a potential strategy to improve the antitumor effect of irradiation in chordoma. Supplementary Material Supplementary material is available at Neuro-Oncology online. Funding This work was supported by the Intramural Research Program, CCR, NCI, National Institutes of Health. Acknowledgments The authors thank Dr Christophe E. Redon (DNA Replication Group, Developmental Therapeutics Branch, Laboratory of Molecular Pharmacology, NCI) for technical assistance with in vitro irradiation experiments; Lixte Biotechnology for providing LB100; and the Chordoma Foundation for providing chordoma cell lines used in this study. Conflict of interest statement. No conflicts declared. References 1. Williams BJ , Raper DM , Godbout E , et al. Diagnosis and treatment of chordoma . J Natl Compr Canc Netw . 2013 ; 11 ( 6 ): 726 – 731 . Google Scholar CrossRef Search ADS PubMed 2. Stacchiotti S , Sommer J ; Chordoma Global Consensus Group . Building a global consensus approach to chordoma: a position paper from the medical and patient community . Lancet Oncol . 2015 ; 16 ( 2 ): e71 – e83 . Google Scholar CrossRef Search ADS PubMed 3. al-Mefty O , Borba LA . 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Google Scholar CrossRef Search ADS PubMed 11. Benk V , Liebsch NJ , Munzenrider JE , Efird J , McManus P , Suit H . Base of skull and cervical spine chordomas in children treated by high-dose irradiation . Int J Radiat Oncol Biol Phys . 1995 ; 31 ( 3 ): 577 – 581 . Google Scholar CrossRef Search ADS PubMed 12. Chen YL , Liebsch N , Kobayashi W , et al. Definitive high-dose photon/proton radiotherapy for unresected mobile spine and sacral chordomas . Spine (Phila Pa 1976) . 2013 ; 38 ( 15 ): E930 – E936 . Google Scholar CrossRef Search ADS PubMed 13. Park DM . Chordoma model . J Neurosurg . 2012 ; 116 ( 4 ): 799 – 800 ; discussion 800. Google Scholar CrossRef Search ADS PubMed 14. Perrotti D , Neviani P . Protein phosphatase 2A: a target for anticancer therapy . Lancet Oncol . 2013 ; 14 ( 6 ): e229 – e238 . Google Scholar CrossRef Search ADS PubMed 15. Lee DH , Chowdhury D . What goes on must come off: phosphatases gate-crash the DNA damage response . Trends Biochem Sci . 2011 ; 36 ( 11 ): 569 – 577 . Google Scholar CrossRef Search ADS PubMed 16. Wei D , Parsels LA , Karnak D , et al. Inhibition of protein phosphatase 2A radiosensitizes pancreatic cancers by modulating CDC25C/CDK1 and homologous recombination repair . Clin Cancer Res . 2013 ; 19 ( 16 ): 4422 – 4432 . Google Scholar CrossRef Search ADS PubMed 17. Park DM , Li J , Okamoto H , et al. N-CoR pathway targeting induces glioblastoma derived cancer stem cell differentiation . Cell Cycle . 2007 ; 6 ( 4 ): 467 – 470 . Google Scholar CrossRef Search ADS PubMed 18. Lu J , Zhuang Z , Song DK , et al. The effect of a PP2A inhibitor on the nuclear receptor corepressor pathway in glioma . J Neurosurg . 2010 ; 113 ( 2 ): 225 – 233 . Google Scholar CrossRef Search ADS PubMed 19. Lu J , Ksendzovsky A , Yang C , et al. CNTF receptor subunit α as a marker for glioma tumor-initiating cells and tumor grade: laboratory investigation . J Neurosurg . 2012 ; 117 ( 6 ): 1022 – 1031 . Google Scholar CrossRef Search ADS PubMed 20. Chung V , Mansfield AS , Braiteh F , et al. Safety, tolerability, and preliminary activity of LB-100, an inhibitor of protein phosphatase 2A, in patients with relapsed solid tumors: an open-label, dose escalation, first-in-human, phase I trial . Clin Cancer Res . 2017 ; 23 ( 13 ): 3277 – 3284 . Google Scholar CrossRef Search ADS PubMed 21. Cheney MD , Chen YL , Lim R , et al. [18F]-Fluoromisonidazole positron emission tomography/computed tomography visualization of tumor hypoxia in patients with chordoma of the mobile and sacrococcygeal spine . Int J Radiat Oncol Biol Phys . 2014 ; 90 ( 5 ): 1030 – 1036 . Google Scholar CrossRef Search ADS PubMed 22. Lee DH , Zhang Y , Kassam AB , et al. Combined PDGFR and HDAC inhibition overcomes PTEN disruption in chordoma . PLoS One . 2015 ; 10 ( 8 ): e0134426 . Google Scholar CrossRef Search ADS PubMed 23. Li X , Ji Z , Ma Y , Qiu X , Fan Q , Ma B . Expression of hypoxia-inducible factor-1α, vascular endothelial growth factor and matrix metalloproteinase-2 in sacral chordomas . Oncol Lett . 2012 ; 3 ( 6 ): 1268 – 1274 . Google Scholar CrossRef Search ADS PubMed 24. Mammar H , Kerrou K , Nataf V , et al. Positron emission tomography/computed tomography imaging of residual skull base chordoma before radiotherapy using fluoromisonidazole and fluorodeoxyglucose: potential consequences for dose painting . Int J Radiat Oncol Biol Phys . 2012 ; 84 ( 3 ): 681 – 687 . Google Scholar CrossRef Search ADS PubMed 25. Soeda A , Park M , Lee D , et al. Hypoxia promotes expansion of the CD133-positive glioma stem cells through activation of HIF-1alpha . Oncogene . 2009 ; 28 ( 45 ): 3949 – 3959 . Google Scholar CrossRef Search ADS PubMed 26. Jiang Y . Regulation of the cell cycle by protein phosphatase 2A in Saccharomyces cerevisiae . Microbiol Mol Biol Rev . 2006 ; 70 ( 2 ): 440 – 449 . Google Scholar CrossRef Search ADS PubMed 27. McManus KJ , Hendzel MJ . ATM-dependent DNA damage-independent mitotic phosphorylation of H2AX in normally growing mammalian cells . Mol Biol Cell . 2005 ; 16 ( 10 ): 5013 – 5025 . Google Scholar CrossRef Search ADS PubMed 28. An J , Huang YC , Xu QZ , et al. DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression . BMC Mol Biol . 2010 ; 11 : 18 . Google Scholar CrossRef Search ADS PubMed 29. Tu WZ , Li B , Huang B , et al. γH2AX foci formation in the absence of DNA damage: mitotic H2AX phosphorylation is mediated by the DNA-PKcs/CHK2 pathway . FEBS Lett . 2013 ; 587 ( 21 ): 3437 – 3443 . Google Scholar CrossRef Search ADS PubMed 30. Barhoumi A , Zhang D , Tam F , Halas NJ . Surface-enhanced Raman spectroscopy of DNA . J Am Chem Soc . 2008 ; 130 ( 16 ): 5523 – 5529 . Google Scholar CrossRef Search ADS PubMed 31. Kulkarni A , Kim B , Dugasani SR , et al. A novel nanometric DNA thin film as a sensor for alpha radiation . Sci Rep . 2013 ; 3 : 2062 . Google Scholar CrossRef Search ADS PubMed 32. Draux F , Jeannesson P , Beljebbar A , et al. Raman spectral imaging of single living cancer cells: a preliminary study . Analyst . 2009 ; 134 ( 3 ): 542 – 548 . Google Scholar CrossRef Search ADS PubMed 33. Nawaz H , Bonnier F , Knief P , et al. Evaluation of the potential of Raman microspectroscopy for prediction of chemotherapeutic response to cisplatin in lung adenocarcinoma . Analyst . 2010 ; 135 ( 12 ): 3070 – 3076 . Google Scholar CrossRef Search ADS PubMed 34. Sak A , Stuschke M . Use of γH2AX and other biomarkers of double-strand breaks during radiotherapy . Semin Radiat Oncol . 2010 ; 20 ( 4 ): 223 – 231 . Google Scholar CrossRef Search ADS PubMed 35. Qian LW , Mizumoto K , Urashima T , et al. Radiation-induced increase in invasive potential of human pancreatic cancer cells and its blockade by a matrix metalloproteinase inhibitor, CGS27023 . Clin Cancer Res . 2002 ; 8 ( 4 ): 1223 – 1227 . Google Scholar PubMed 36. Park CM , Park MJ , Kwak HJ , et al. Ionizing radiation enhances matrix metalloproteinase-2 secretion and invasion of glioma cells through Src/epidermal growth factor receptor-mediated p38/Akt and phosphatidylinositol 3-kinase/Akt signaling pathways . Cancer Res . 2006 ; 66 ( 17 ): 8511 – 8519 . Google Scholar CrossRef Search ADS PubMed 37. Kaliski A , Maggiorella L , Cengel KA , et al. Angiogenesis and tumor growth inhibition by a matrix metalloproteinase inhibitor targeting radiation-induced invasion . Mol Cancer Ther . 2005 ; 4 ( 11 ): 1717 – 1728 . Google Scholar CrossRef Search ADS PubMed Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Neuro-Oncology Oxford University Press

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Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2017.
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Abstract

Abstract Background Standard therapy for chordoma consists of surgical resection followed by high-dose irradiation. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase involved in signal transduction, cell cycle progression, cell differentiation, and DNA repair. LB100 is a small-molecule inhibitor of PP2A designed to sensitize cancer cells to DNA damage from irradiation and chemotherapy. A recently completed phase I trial of LB100 in solid tumors demonstrated its safety. Here, we show the therapeutic potential of LB100 in chordoma. Methods Three patient-derived chordoma cell lines were used: U-CH1, JHC7, and UM-Chor1. Cell proliferation was determined with LB100 alone and in combination with irradiation. Cell cycle progression was assessed by flow cytometry. Quantitative γ-H2AX immunofluorescence and immunoblot evaluated the effect of LB100 on radiation-induced DNA damage. Ultrastructural evidence for nuclear damage was investigated using Raman imaging and transmission electron microscopy. A xenograft model was established to determine potential clinical utility of adding LB100 to irradiation. Results PP2A inhibition in concert with irradiation demonstrated in vitro growth inhibition. The combination of LB100 and radiation also induced accumulation at the G2/M phase of the cell cycle, the stage most sensitive to radiation-induced damage. LB100 enhanced radiation-induced DNA double-strand breaks. Animals implanted with chordoma cells and treated with the combination of LB100 and radiation demonstrated tumor growth delay. Conclusions Combining LB100 and radiation enhanced DNA damage-induced cell death and delayed tumor growth in an animal model of chordoma. PP2A inhibition by LB100 treatment may improve the effectiveness of radiation therapy for chordoma. chordoma, LB100, PP2A, radiation therapy Chordomas are slow-growing yet locally aggressive primary bone tumors of the axial skeleton. They are believed to arise from the neoplastic transformation of notochordal remnant tissues accounting for the distribution of tumors along the midline axis.1 The most common sites of involvement are the sacrum, skull base, and the remaining mobile spine.2 For this reason, patients with chordomas are treated by a variety of different subspecialists depending on the location of the tumor.3–7 Because chordomas are locally invasive with metastatic potential, a complete en bloc surgical resection is clinically indicated to prevent or delay tumor recurrence. However, efforts to remove the entire tumor are often hampered by proximity to critical neurovascular structures of the skull base and the spine, leaving most patients with residual disease. Since effective medical therapy for chordoma is lacking, patients left with incomplete surgical resection require high-dose irradiation due to radioresistance of chordoma cells,2,8–11 increasing risk of injury to nearby normal tissue and contributing to morbidity.12 As such, there is a critical unmet need to identify strategies to improve the effectiveness of irradiation in patients with chordoma while minimizing treatment-associated toxicity.13 Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase with multifunctional roles in diverse physiologic processes and many pathological conditions, including cancer.14 PP2A mediates the DNA damage response,15 therefore the inhibition of PP2A catalytic activity sensitizes cancer cells to DNA damaging effects of ionizing radiation in part by altering homologous recombination repair capacity.16 Based on these previous studies of the PP2A role in DNA repair, and in concert with the observation that cancer cells exhibit altered DNA repair functions, we hypothesized that the combination of PP2A inhibition and irradiation may have complementary antitumor activity. LB100, a hydrophilic small-molecule derivative of cantharadin, is a potent inhibitor of PP2A. This compound was developed by Lixte Biotechnology through a cooperative research and development agreement based on our previous work.17–19 A recently completed phase I study confirmed the feasibility of safe administration of LB100 in solid tumors.20 Preclinical studies in a pancreatic cancer model suggest LB100 sensitizes cells to irradiation by interfering with DNA homologous recombination repair capacity.16 Chordomas are radioresistant in part due to extensive tissue hypoxia.21–24 Poor tissue oxygenation demands delivery of high-dose irradiation associated with increased toxicity, to achieve adequate antitumor response. Strategies that can enhance the effectiveness of radiation may offset the need to administer potentially toxic doses to sensitive tissues. In this study, we investigated the radiation sensitizing potential of LB100 in chordoma. Methods Chemicals, Cell Lines, and Radiation Treatment LB100 was provided by Lixte Biotechnology Holdings. The chemical structure of LB100 is shown in Fig. 1. LB100 was reconstituted in 0.1 N monosodium glutamate, pH 10.5 (pH was adjusted with NaOH), and stored at −80°C. The 3 patient-derived chordoma cell lines used in this study—U-CH1, UM-Chor1, and JHC7—were obtained from the Chordoma Foundation. Cells were maintained in Iscove’s modification of Dulbecco’s medium (IMDM) and Roswell Park Memorial Institute (RPMI) 1640 medium (4:1), supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and nonessential amino acids. Fig. 1 View largeDownload slide View largeDownload slide LB100 alters cell cycle state and augments antitumor effect of irradiation. Human chordoma cell lines, U-CH1, UM-Chor1, and JHC7, were used to evaluate the proliferative effect of LB100. Two human chordoma cell lines, U-CH1 and UM-Chor1, were used for cell cycle analysis. (A–C) As a single agent, LB100 has minimal effect on cell proliferation at clinically achievable concentrations (5 µM). Cell proliferation was quantified with XTT assays following 24, 48, or 72 h of treatment with increasing concentrations of LB100 and analyzed using a one-way ANOVA comparing the treatment groups with vehicle control. (D) Chemical structure of LB100. (E) LB100 strikingly enhances radiation-mediated cytotoxicity after 72 h of treatment. (F–I) Cell cycle analysis was performed 24 h following treatment with the indicated combinations of LB100 and radiation. The percentage of cells in the indicated cell cycle stage were quantified by flow cytometry and analyzed by a 2-way ANOVA. Cells were plated in triplicate. Fig. 1 View largeDownload slide View largeDownload slide LB100 alters cell cycle state and augments antitumor effect of irradiation. Human chordoma cell lines, U-CH1, UM-Chor1, and JHC7, were used to evaluate the proliferative effect of LB100. Two human chordoma cell lines, U-CH1 and UM-Chor1, were used for cell cycle analysis. (A–C) As a single agent, LB100 has minimal effect on cell proliferation at clinically achievable concentrations (5 µM). Cell proliferation was quantified with XTT assays following 24, 48, or 72 h of treatment with increasing concentrations of LB100 and analyzed using a one-way ANOVA comparing the treatment groups with vehicle control. (D) Chemical structure of LB100. (E) LB100 strikingly enhances radiation-mediated cytotoxicity after 72 h of treatment. (F–I) Cell cycle analysis was performed 24 h following treatment with the indicated combinations of LB100 and radiation. The percentage of cells in the indicated cell cycle stage were quantified by flow cytometry and analyzed by a 2-way ANOVA. Cells were plated in triplicate. Cell Proliferation Analysis Cell proliferation was determined with a 2,-3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay as indicated by the manufacturer’s instruction. U-CH1, UM-Chor1, and JHC7 cell lines were seeded in 96-well plates at 5 × 104 cells/well. After 24 h incubation, cells were treated with various doses (0–20 μM) of LB100 and incubated for 24, 48, and 72 hours. Activated-XTT solution was prepared by the manufacturer’s protocol and the cells incubated for 4 hours. Cell proliferation was calculated using the background-corrected absorbance as follows: Cell proliferation = [Aλ sample well / Aλ control well) / DMSO treated control]. Cell Cycle Analysis U-CH1 cells were seeded at 2 × 105 cells/well in a 6-well dish. The following day, cells were treated with 4 μM LB100 or vehicle control (DMSO) for 4 hours before receiving sham or 2 Gy irradiation. After overnight incubation, cells were pulsed for 4 hours with 10 μM 5-ethynyl-2′-deoxyuridine (EdU) and evaluated by flow cytometry. EdU uptake was detected with a Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Thermo Fisher Scientific, C10424) per the manufacturer’s protocol. DNA was stained with FxCycle violet stain (Thermo Fisher Scientific, F10347). Data were acquired on a BD LSRFortessa X50 flow cytometer and analyzed using FlowJo v9.9.4. Immunofluorescent Cytochemical Staining for γ-H2AX Cells were grown in chamber slides and exposed to LB100 (4 μM) for 4 hours prior to administration of 2 Gy or sham radiation. Cells were fixed with 2% paraformaldehyde, washed with phosphate buffered saline (PBS), permeabilized with 1% Triton X-100, washed again with PBS, and blocked with 1% bovine serum albumin (BSA). Mouse anti–γ-H2AX antibody (Millipore) was added at 1:500 and incubated overnight at 4°C. Cells were washed with 1% BSA, and goat anti-mouse‒fluorescein isothiocyanate antibody (Jackson ImmunoResearch) was added at 1:100 and incubated for 1 hour at room temperature. Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (Sigma). Coverslips were mounted with Vecta Shield anti-fade solution (Vector Labs) and slides examined on a Leica DMRXA fluorescent microscope (Leica Microsystems). γ-H2AX foci were quantitated in 50 cells per condition. Raman Imaging Microscopy Raman imaging microscopy was used to confirm DNA damage. Raman is a spectroscopic technique that measures the energy difference between the incident photons from a laser and the inelastic scattered photons collected at a detector. This difference is the energy required for different types of vibrations of the molecule. The vibration frequencies depend on the symmetry of the molecule, masses of atoms, distance between atoms, bond angle, and strength. The use of Raman via the Raman imaging microscope allows focusing of the laser through the microscope and the collection of Raman spectra for each pixel. Subsequent movement of the stage permits the acquisition of spectra in a new position. This approach provides a chemical and biochemical mapping of the entire cell in the culture medium. Due to its comprehensive chemical analysis, Raman spectra of DNA contain multiple peaks from allowed vibration frequencies that have been previously assigned to T, C G, A, single-strand DNA, double-strand DNA, backbone, or DNA attached to nanoparticles. Samples used for Raman were transferred into 35 mm sterile glass bottom dishes (Ibidi) and cultured for 24 hours. Raman spectra were acquired using a DXR 2xi Raman microscope (Thermo Fisher Scientific) with 24 mW of 780 nm laser through a 60x water immersed confocal objective, at 0.5 s exposure time for a 1 μm pixel size, between 50 and 3200 cm−1 spectral region. Spectra were collected and subsequently background corrected using the Raman silent region. Chemical maps were produced by the peak area function between 1085 and 1100 cm−1 using Thermo Fisher Scientific OMNIC software. Wound Healing Assay Seeded onto 12-well plates were 2 × 105 U-CH1 and UM-Chor1 cells. Once cells reached 100% confluence, “wounds” were created by scraping “#” lines with a 200 μL pipette tip, and then cells were washed 3 times in serum-free medium. Cells were exposed to medium, LB100 (4 μM), 2 Gy irradiation, or LB100 (4 μM) for 4 hours prior to administration of 2 Gy. The “wounds” were observed every 24 hours and photographed using an EVOS Cell Imaging System (EVOS XL Core Cell Imaging System, Thermo Fisher Scientific). Eight images were taken per well at each time point using a 10x objective. The distances between the 2 edges of the scratch (wound width) were measured at 3 sites for each image using ImageJ software. The migratory distances were calculated by subtracting the wound width at each time point from the wound width at the time zero point. To complete the experiment, cells were washed 2 times in PBS, fixed for 10 min with methanol, and stained using 0.2% crystal violet for 20 min. Invasion Assay Chordoma cell invasive capacity was assessed using Corning BioCoat Matrigel Invasion Chambers with BD Matrigel Matrix (Thermo Fisher Scientific) as previously described.22 Pre-coated membranes (8 μm pore size, Matrigel 100 μg/cm2) were rehydrated and seeded with 3 × 105 cells in 2 mL of IMDM/RPMI (4:1) with or without LB100 in triplicates into the upper part of each transwell. Cells were irradiated (2 Gy) 4 hours after treatment with LB100. The lower compartment was filled with 2.5 mL of IMDM/RPMI (4:1) supplemented with 0.1% BSA. After incubation for 96 hours at 37°C, non-invaded cells on the upper surface of the membrane were wiped with a cotton swab. Migrated cells on the lower surface of the filter were fixed with 70% ethanol for 2 minutes and stained with crystal violet (0.5%) for 15 minutes. Invasive activity was determined by counting cells in 4 microscopic fields per well, and the extent of invasion was expressed as an average number of cells per microscopic field. Western Blotting Immunoblotting was performed as previously described.25 Proteins were extracted by CellLytic M (Sigma-Aldrich) cell lysis buffer supplemented with protease inhibitor cocktail and phosphatase inhibitors. Protein quantification was measured by the Pierce BCA protein assay kit (Thermo Fisher Scientific). The protein bands were detected by conventional protocols for western blotting. Proteins were detected by using specific primary antibodies against γ-H2AX (Millipore) and glyceraldehyde 3-phosphate dehydrogenase (Santa Cruz Biotechnology), and subsequently with the appropriate horseradish peroxidase–conjugated secondary antibodies (Cell Signaling Technology). Subcutaneous Xenograft Model All animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Animals and approved by the Animal Care and Use Committee of the NIH. Six-week-old female NSG mice (NOD.Cg-Prkdcscid Il2rgtm1WjI/SzJ; Charles River) were used for the in vivo studies. U-CH1 cells (8 × 106 in 100 µL of Hanks’ Balanced Salt Solution) were subcutaneously injected into the right thigh. Xenografted tumors were measured twice a week from the first day of treatment and volumes were calculated using the formula: TV = (width)2 × (length)/2. When tumors reached ≈130 mm3, animals were randomized into 5 groups: untreated controls, LB100 (1 mg/kg), radiation (5 Gy), and combination LB100 and radiation (3 or 5 Gy). Each group was composed of 8 animals. LB100 was delivered by intraperitoneal injections at a dose of 1 mg/kg, twice a day for 3 days. Radiation treatments were given on 2 consecutive days. Endpoints were (i) the diameter of tumor reaching 2 cm and (ii) development of ulcerations. Survival analysis was performed by plotting the Kaplan–Meier curves. The log-rank test was used to compare the groups. Irradiation of Animals Animals were restrained in a custom jig that was developed and used extensively in the Radiation Oncology Branch and the Radiation Biology Branch of the NIH and placed in an orthovoltage radiotherapy unit maintained at the NIH. The mice were restrained in the custom leg jig for no more than 7 minutes. The rest of the mouse, except the leg, was covered with lead shielding. Previous studies have demonstrated this procedure to be of minimal stress to the animals, as the process is painless, odorless, and brief; therefore, no anesthetics were given. Irradiation was delivered using the Pantek radiation machine located in the NIH Animal Facility. Irradiation treatment was performed in an Xrad 320 irradiator (Precision X-ray) at a dose rate of 256.1 cGy/min. Delivered dose was verified by thermoluminescent dosimeter. Animals were observed during irradiation for any signs of discomfort. Animals that were unable to consume food or water or exhibited signs of illness were immediately euthanized. Statistical Analysis In vitro studies were subject to 3 independent experiments. Data are presented as mean ± SD. An ordinary one-way ANOVA test was used for comparison between more than 2 groups. P ≤ 0.05 was considered statistically significant. Values stated within text and figures represent mean ± SD. Statistics were performed on results from at least 2 independent replicates. Results LB100 Enhances Radiation-Mediated Attenuation of Chordoma Cell Proliferation To determine the sensitivity of chordoma cells to the PP2A inhibitor, LB100, three human chordoma cell lines were used: U-CH1, UM-Chor1, and JHC7. In vitro cell proliferation was assessed in response to LB100 treatment in dose- and time-dependent manners (Fig. 1A–C). LB100 as a single agent demonstrated a moderate and heterogeneous effect on cell proliferation at clinically achievable concentrations (up to 4–5 µM). U-CH1 and UM-Chor1 cells were more resistant than JHC7 and did not show significant decreases in proliferation until treatment at 10 µM (Fig. 1A–C). However, LB100 in combination with radiation treatment significantly reduced U-CH1 cell numbers compared with vehicle control or LB100 or radiation treatment alone (Fig. 1E). These data suggest that a clinical achievable dose of LB100 is effective when combined with radiation in the treatment of chordoma. LB100 Treatment Increases G2/M-Phase Arrest PP2A can influence multiple stages of the cell cycle, from G1/S transition to cytokinesis.26 To determine whether PP2A inhibition alters cell cycle in chordoma cells, EdU incorporation assays were performed in U-CH1 cells treated with LB100, radiation, or the combination. EdU uptake was assessed 24 hours following radiation to prevent cell loss due to treatment-related cytotoxicity. LB100, radiation, and the combination each led to a significant reduction of cells in S phase compared with vehicle control 24 hours following radiation treatment. Interestingly, cells treated with LB100 or LB100 with radiation had a significantly increased population of cells in the G2/M phase compared with control and radiation-only groups (Fig. 1F–H). The G0/G1 phase was not significantly altered by any treatment (Fig. 1I). Together, these data demonstrate that the PP2A inhibitor LB100 increases the proportion of cells in the radiation-sensitive G2/M phase of the cell cycle. LB100 Sensitizes Chordoma Cells to Radiation-Induced DNA Damage Through γ-H2AX Activation To evaluate for a potential radiation sensitizing effect of LB100, we screened U-CH1 and UM-Chor1 cells. To determine the effect of LB100 treatment on DNA damage and repair, γ-H2AX expression level was measured by immunofluorescence and immunoblot in 2 chordoma cell lines at 24 hours (Fig. 2A–C). It was previously reported that in the absence of DNA damage, cells at G2/M phase can demonstrate elevated γ-H2AX levels, a phenomenon referred to as mitotic H2AX phosphorylation.27–29 Consistent with these reports, LB100 alone increased expression and foci of γ-H2AX as it drove cells to G2/M phase (Fig. 2A–C). Radiation alone also increased expression and foci of γ-H2AX (Fig. 2A–C; Supplementary Figure S1). Though we did not observe significant change in γ-H2AX expression levels between the LB100 alone group and the combination of LB100 plus radiation, cells treated with the combination of LB100 and radiation showed the highest γ-H2AX level for foci formation compared with the control group, the LB100 alone group, and the radiation alone group. Fig. 2 View largeDownload slide LB100 enhances radiation-induced DNA damage. (A) U-CH1 and UM-Chor1 cells were treated with 4 μM LB100 for 3 hours pre- and 24 hours post-radiation (2 Gy). At the end of drug exposure, cells were fixed and then subjected to immunofluorescence staining with 4′,6′-diamidino-2-phenylindole‒blue and fluorescein isothiocyanate‒green foci for γ-H2AX, a marker of DNA double-strand breaks. Representative images show increased γ-H2AX expression in the cells exposed to the combination of LB100 and irradiation. (B) Western blot analysis reveals increased protein level of γ-H2AX with LB100 treatment and combination treatment with LB100 and radiation in U-CH1 and UM-Chor1 cells. (C) Quantification of γ-H2AX volume intensity relative to glyceraldehyde 3-phosphate dehydrogenase volume intensity. (D) DNA backbone intensity and location in live chordoma cells as a function of different treatments. Optical image (top panel). Chemical image (lower panel). Image 1: untreated; image 2: treated with LB100; image 3: irradiated; image 4: irradiated and treated with LB100; image 5: overlay of Raman spectra indicating the peak at 1095 cm−1 corresponding to the DNA backbone. Scale bar = 10 μm. (E) Images obtained by transmission electron microscopy show nuclear fragmentation in cells treated with the combination of LB100 and irradiation. Fig. 2 View largeDownload slide LB100 enhances radiation-induced DNA damage. (A) U-CH1 and UM-Chor1 cells were treated with 4 μM LB100 for 3 hours pre- and 24 hours post-radiation (2 Gy). At the end of drug exposure, cells were fixed and then subjected to immunofluorescence staining with 4′,6′-diamidino-2-phenylindole‒blue and fluorescein isothiocyanate‒green foci for γ-H2AX, a marker of DNA double-strand breaks. Representative images show increased γ-H2AX expression in the cells exposed to the combination of LB100 and irradiation. (B) Western blot analysis reveals increased protein level of γ-H2AX with LB100 treatment and combination treatment with LB100 and radiation in U-CH1 and UM-Chor1 cells. (C) Quantification of γ-H2AX volume intensity relative to glyceraldehyde 3-phosphate dehydrogenase volume intensity. (D) DNA backbone intensity and location in live chordoma cells as a function of different treatments. Optical image (top panel). Chemical image (lower panel). Image 1: untreated; image 2: treated with LB100; image 3: irradiated; image 4: irradiated and treated with LB100; image 5: overlay of Raman spectra indicating the peak at 1095 cm−1 corresponding to the DNA backbone. Scale bar = 10 μm. (E) Images obtained by transmission electron microscopy show nuclear fragmentation in cells treated with the combination of LB100 and irradiation. To better understand these observations, we evaluated nuclear DNA damage using Raman imaging microscopy and transmission electron microscopy. Raman imaging microscopy was used to measure the intensity and location of the intact DNA backbone, which has a vibration at 1095 cm−1. Since the Raman signal is proportional to the concentration of the chemical species, by fragmenting the DNA backbone or interfering with its chemical structure, it is expected that the intensity of the intact peak at 1095 cm−1 will decrease or disappear. Raman imaging microscopy was used to demonstrate the relative effect of LB100 and radiation on the intensity and location of the DNA backbone in live chordoma cells. For simplicity, only the peak at 1095 cm−1 assigned to the O–P–O stretching of the DNA backbone was mapped.30–33 Irradiation of chordoma cells led to a decrease in the integrity of the DNA backbone (Fig. 2D, image 3) compared with control and LB100 alone (Fig. 2D, images 1 and 2), and this effect was further enhanced by the addition of LB100 (Fig. 2D, image 4). Chordoma cells from these 4 groups were further probed by transmission electron microscopy. The cells treated with the combination of LB100 and irradiation revealed significant nuclear fragmentation (Fig. 2E). Taken together, these results suggest that LB100 treatment enhances sensitivity of chordoma cells to radiation-induced damage. The Effect of LB100 on Chordoma Cell Migration and Invasion Tumor cell mobility plays a critical role in invasion and metastasis. Chordoma cells are highly invasive. Therefore, we investigated the effect of LB100 on cell motility. Serial images of the tumor cells were collected every 24 hours after inducing a wound injury. Representative images at 144 hours following treatment showed that exposure to LB100 attenuates cell motility (Fig. 3A and B). The time required for cells to migrate into the gap was significantly longer for cells treated with LB100 compared with radiation or vehicle control. Cell motility was further assessed with an in vitro invasion model. Cells exposed to LB100 with or without radiation exhibited decreased invasive capacity (Fig. 3C and D). Notably, in U-CH1 cells which are resistant to LB100 treatment, the combination of LB100 plus radiation decreased invasion compared with LB100 alone (Fig. 3D). Fig. 3 View largeDownload slide LB100 attenuates chordoma cell migration and invasion. (A) Representative images from the wound-healing assay for each cell line taken at 144 hours. (B) Migration distance was analyzed using ImageJ software (n = 3). Data are presented as mean ± SEM. A one-way ANOVA was used for statistical analyses (****P < 0.0001). (C) Representative images of a Matrigel invasion assay with U-CH1 and UM-Chor1 cell lines show a significant decrease in invasion in response to 96 hours of LB100 treatment (4 μM) and irradiation (2 Gy) (n = 3). Scale bar = 200 µm. (D) Invasion is quantified as number of cells per microscopic field (20x), and a one-way ANOVA was used to compare ± SEM. Fig. 3 View largeDownload slide LB100 attenuates chordoma cell migration and invasion. (A) Representative images from the wound-healing assay for each cell line taken at 144 hours. (B) Migration distance was analyzed using ImageJ software (n = 3). Data are presented as mean ± SEM. A one-way ANOVA was used for statistical analyses (****P < 0.0001). (C) Representative images of a Matrigel invasion assay with U-CH1 and UM-Chor1 cell lines show a significant decrease in invasion in response to 96 hours of LB100 treatment (4 μM) and irradiation (2 Gy) (n = 3). Scale bar = 200 µm. (D) Invasion is quantified as number of cells per microscopic field (20x), and a one-way ANOVA was used to compare ± SEM. LB100 in Combination with Irradiation Delays In Vivo Tumor Growth To determine the potential clinical utility of LB100, mice bearing U-CH1 subcutaneous xenograft tumors were randomized into 5 groups: (i) vehicle, (ii) LB100 (1 mg/kg), (iii) radiation (5 Gy), (iv) combination of LB100 (1 mg/kg) and irradiation (3 Gy), (v) combination of LB100 (1 mg/kg) and irradiation (5 Gy). Tumor volumes were significantly smaller in animals treated with the combination of LB100 and irradiation compared with other groups (control group P = 0.028, LB100 group P = 0.0014, 5 Gy radiation group P = 0.0273) (Fig. 4A–C). Tumors were further analyzed by immunohistochemistry for Ki-67 and assay by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) (Supplementary Figure S3). Ki-67 labeling was found to be decreased for the tumors exposed to LB100 and irradiation. TUNEL assay revealed increased staining in the combination arm, indicative of apoptosis. Interestingly, consistent with mitotic arrest, tumors treated with the combination of LB100 and irradiation demonstrated the presence of multinucleated cells. Fig. 4 View largeDownload slide LB100 in combination with irradiation significantly decreases tumor burden in vivo. To evaluate the benefit of combination treatment in vivo, mice bearing U-CH1 subcutaneous xenografts were randomized into 5 groups: vehicle control; LB100 (1 mg/kg); irradiation (5 Gy); combination LB100 (1 mg/kg) and irradiation (3 Gy); and combination LB100 (1 mg/kg) and irradiation (5 Gy). Treatment schedule was: intraperitoneal injections of LB100 given twice daily for 3 consecutive days. Radiation (3 or 5 Gy) was given on 2 consecutive days. (A and B) Xenograft tumor volumes of animals treated with the combination of LB100 and irradiation are significantly reduced compared with other groups (control group P = 0.028; LB100 group P = 0.0014; 5 Gy radiation group P = 0.0273). Data are presented as mean ± SEM. (C) Subcutaneously implanted tumors were surgically removed. Tumor sizes in the LB100 and irradiation combination groups are smaller. Fig. 4 View largeDownload slide LB100 in combination with irradiation significantly decreases tumor burden in vivo. To evaluate the benefit of combination treatment in vivo, mice bearing U-CH1 subcutaneous xenografts were randomized into 5 groups: vehicle control; LB100 (1 mg/kg); irradiation (5 Gy); combination LB100 (1 mg/kg) and irradiation (3 Gy); and combination LB100 (1 mg/kg) and irradiation (5 Gy). Treatment schedule was: intraperitoneal injections of LB100 given twice daily for 3 consecutive days. Radiation (3 or 5 Gy) was given on 2 consecutive days. (A and B) Xenograft tumor volumes of animals treated with the combination of LB100 and irradiation are significantly reduced compared with other groups (control group P = 0.028; LB100 group P = 0.0014; 5 Gy radiation group P = 0.0273). Data are presented as mean ± SEM. (C) Subcutaneously implanted tumors were surgically removed. Tumor sizes in the LB100 and irradiation combination groups are smaller. Discussion Therapeutic options for patients with chordoma are limited. Since complete tumor resection is seldom achieved, external beam radiation therapy remains the standard of care, as effective medical therapies do not yet exist. However, because chordomas are radioresistant, high doses of ionizing radiation must be delivered to areas that border delicate neurovascular structures. Two main strategies are utilized to permit delivery of high-dose radiation to chordoma: (i) intensity-modulated radiation therapy and stereotactic radiosurgery, which better delineate borders of treatment to minimize toxicity to non-neoplastic tissues, and (ii) particle beam, such as proton, therapy, which concentrates intratumoral radiant energy. However, concurrent administration of a radiosensitizer would further enhance the effectiveness of these advanced technologies. As a single agent, LB100 had a modest anti-proliferative effect. However, when LB100 was given in combination with irradiation, we observed a marked inhibition of chordoma proliferation. To define a potential mechanism for the observed radiosensitizing property of LB100, we investigated the cell cycle state of chordoma cells and found that either as a single treatment or in combination with radiation, a greater proportion of chordoma cells were found to be in the G2/M phase of the cell cycle, a state most sensitive to the damaging effects of irradiation. Given this observation and published reports that PP2A participates in the DNA repair process, we searched for a candidate mechanism and found that the addition of LB100 to irradiation potentiated DNA double-strand breaks. This was evaluated by the presence of γ-H2AX, a product of rapid phosphorylation of histone H2AX at serine 139 which serves as a sensitive marker for DNA double-strand breaks induced by ionizing radiation or other genotoxic agents.34 Because cells in the G2/M phase of the cell cycle can demonstrate elevated expression of γ-H2AX in the absence of DNA damage, we used Raman spectroscopic imaging to confirm that cells treated with LB100 alone had similar concentrations of intact backbone DNA to control cells. This technique further corroborated the results of γ-H2AX experiments revealing that the combination of LB100 and irradiation contributed to the greatest amount of DNA damage. Ultrastructural analysis with electron microscopy revealed extensive nuclear fragmentation in cells exposed to the combination of LB100 and irradiation, further verifying DNA damaging effects of the combination treatment. These observations suggest that by interfering with the homologous recombination repair process, LB100 promotes persistent genomic damage. Although irradiation is delivered for antitumor effect, exposure of tumor cells to ionizing radiation may occasionally promote cancer growth and invasion.35–37 This may be particularly relevant for chordoma, since this is a disease characterized by infiltration of tumor cells into surrounding structures that often precludes complete surgical resection. However, we observed that chordoma cells treated with LB100 demonstrated attenuation of migration as well as invasion. Lastly, the antitumor activity of LB100 in combination with irradiation was confirmed in an in vivo xenograft model. The preclinical data presented here suggest that LB100 is capable of enhancing the therapeutic effects of irradiation in chordoma. Although LB100 as a single agent and in combination with cytotoxic chemotherapy was well tolerated in a recently completed phase I study, the safety profile in combination with irradiation has yet to be established.20 Preclinical work performed with a pancreatic cancer model indicates that LB100 may selectively target tumor cells. Exposure of normal intestinal cells to clinically achievable concentrations of LB100 failed to produce radiosensitization.16 This observation is consistent with our experimental data demonstrating LB100-induced radiosensitization is dependent on cell cycle state; nondividing cells may not be adversely affected. While additional studies to further characterize and confirm LB100-induced radiosensitization on dividing and nondividing cells is needed, this study offers a potential strategy to improve the antitumor effect of irradiation in chordoma. Supplementary Material Supplementary material is available at Neuro-Oncology online. Funding This work was supported by the Intramural Research Program, CCR, NCI, National Institutes of Health. Acknowledgments The authors thank Dr Christophe E. Redon (DNA Replication Group, Developmental Therapeutics Branch, Laboratory of Molecular Pharmacology, NCI) for technical assistance with in vitro irradiation experiments; Lixte Biotechnology for providing LB100; and the Chordoma Foundation for providing chordoma cell lines used in this study. Conflict of interest statement. No conflicts declared. References 1. Williams BJ , Raper DM , Godbout E , et al. Diagnosis and treatment of chordoma . J Natl Compr Canc Netw . 2013 ; 11 ( 6 ): 726 – 731 . Google Scholar CrossRef Search ADS PubMed 2. Stacchiotti S , Sommer J ; Chordoma Global Consensus Group . Building a global consensus approach to chordoma: a position paper from the medical and patient community . Lancet Oncol . 2015 ; 16 ( 2 ): e71 – e83 . Google Scholar CrossRef Search ADS PubMed 3. al-Mefty O , Borba LA . 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This work is written by (a) US Government employee(s) and is in the public domain in the US.

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Neuro-OncologyOxford University Press

Published: Dec 23, 2017

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