The PAD4 inhibitor GSK484 enhances the radiosensitivity of triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) accounts for approximately 10–20% of all breast cancers and is one of the leading causes of mortality among females. Radiotherapy is essential during the treatment of breast cancer. Growing evidence has indicated that peptidyl arginine deiminase-4 (PAD4) inhibitor can alleviate the development of multiple cancers, including breast cancer, through inhibiting cell proliferation. GSK484 is considered to be a highly potent PAD4-selective inhibitors. However, the potential role and mechanism of GSK484 in TNBC remain unclear. In this study, we intended to explore the effects of GSK484 on the radiosensitivity of TNBC cell lines (MDA-MB-231 and BT-549). We found that the pretreatment of GSK484 enhanced irradiation (IR)-induced inhibitory effects on cell proliferation, migration and invasion. Besides, our findings revealed that GSK484 facilitated TNBC cell apoptosis. IR treatment-induced increase of the protein level of ATG5 and ATG7, and decrease of p62 protein level were countervailed by GSK484. In addition, GSK484 enhanced DNA damage induced by IR. Moreover, in vivo experiments demonstrated that the combined treatment of IR and GSK484 showed an obvious decline of tumor growth in contrast to IR-alone or GSK484-alone treatment. Overall, GSK484 may serve as a radiosensitizer of TNBC through inhibiting IR-induced autophagy.

Introduction

Breast cancer is one of the most frequently diagnosed cancer and is a huge threat to female’s health worldwide. ^1,2 In 2005, triple-negative breast cancer (TNBC) was first defined as the tumor lacking estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor type 2 (HER2). ³ Moreover, it has been reported that TNBC accounts for about 10–20% of all breast cancer types, and is characterized by poor prognosis with rapid proliferation and high histological grade. ^4,5 Although radiotherapy is one of the primary treatments for TNBC and can increase overall survival in situ or infiltrating breast cancer, many patients experience radiotherapy failure due to the distant metastases and high recurrence. ^{6 –8} Therefore, identifying novel strategies is necessary to improve the effectiveness of radiosensitivity in TNBC.

Peptidylarginine deiminase 4 (PAD4) is a Ca²⁺-dependent enzyme that can convert the arginine side chains of histones to citrulline by deamination. ⁹ Mounting evidence has revealed that PAD4 plays important roles in various cancers, ¹⁰ such as breast cancer ¹¹ and non-small cell lung cancer. ¹² Moreover, recent studies have demonstrated that PAD4 inhibitor may provide a novel insight for the treatment of cancers. ^{13 –16} Compared with methotrexate-alone treatment, the combination of PAD inhibitor and methotrexate treatment increased the cytotoxic effect on prostate cancer cells. ¹⁷ As a newly discovered PAD4 inhibitor, GSK484 has been found to moderately alleviate cardiac dysfunction induced by myocardial infarction. ¹⁸ Also, it is reported that GSK484 abolished the suppressive influence of Kaempferol on tumor metastasis. ¹⁹ However, research on the role of GSK484 in cancers is quite limited and the potential role and mechanism of GSK484 in TNBC development remains undefined.

In the present study, we examined whether the PAD4 inhibitor GSK484 influences the sensitivity of TNBC cells to radiation both in vitro and in vivo. Our findings may lay the foundation for the exploration of underlying mechanism of GSK484-mediated radiosensitization, which may help to improve the efficiency of radiotherapy in TNBC treatment.

Materials and methods

Cell lines and cell culture

The human TNBC cell lines MDA-MB-231 and BT-549 were provided by American Type Culture Collection (USA). All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco, USA), and incubated in a humidified atmosphere with 5% CO₂ at 37%.

Drug and chemical

The PAD4 inhibitor GSK484 was obtained from Selleck Chemicals (USA) and dissolved in DMSO. The DMSO concentration of the solution used in this study was consistently lower than 0.1%. Cells were grown to 70%–80% confluence on plates and cultivated with a low dose of GSK484 for 1 h before irradiation.

Cell Counting Kit-8 (CCK-8) assay

Cells were detached using trypsinization, washed twice with PBS, and then seeded in 96-well plates (2 × 10³ cells/well). After 1, 2, 3, 4, 5 and 6 d incubation, the supernatant was removed, and cell growth was measured with a Cell Counting Kit-8 (CCK-8) assay (Dojindo, Japan) following the manufacturer’s instructions. The absorbance at 450 nm was tested using a microplate reader (168-1000 Model 680, Bio-Rad).

Colony formation assay

Exponential growth phase cells were irradiated with X-rays (0, 2, 4 and 6 Gy) at 3 Gy/min with a linear accelerator (Varian Medical Systems, USA) as previously described. ²⁰ Then, the cells were digested for counting. After 14 d incubation, the colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (Beyotime, Shanghai, China) for 30 min. Finally, the number of clones containing ≥ 50 cells was counted.

Immunofluorescence

TNBC cells were cultured with GSK484 at concentrations of 100 and 200 nmol/L for 1 h, and then irradiated with 8 Gy X-rays as previously described. ²⁰ After 2 h, cells were fixed with 4% paraformaldehyde. Each sample was analyzed at the same parameters using a laser scanning confocal microscope. To calculate the average number of foci per nucleus, the number of γH2A.X foci in the nuclei of at least 50 cells was counted in a double-blinded manner.

Nude mice xenografts

All mice experiments were handled in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (8th Edition, 2011, National Research Council). The experiment was conducted as previously described. ²⁰ Specifically, a total of 5 × 10⁶ MDA-MB-231 cells were injected subcutaneously into the 4th mammary fat pads of lactiferous ducts the in 20 female BALB/c mice (6-week-old; SLAC Laboratory Animal, Shanghai, China). Tumor volume was calculated using the following formula: tumor volume (mm³) = a × b² × 0.5 (a means the longest diameter of the tumor, b represents the shortest diameter of the tumor, and 0.5 is an empirical constant). When tumors reached about 0.5 cm in diameter, mice were randomly divided into four groups (Vehicle, 15 Gy alone, GSK484 4 mg/kg, and 15 Gy + GSK484 4 mg/kg). GSK484 was administered at a dose of 4 mg/kg ip in 2-hydroxypropyl-β-cyclodextrin (Sigma-Aldrich, USA) at 1 hour before irradiation. The tumor areas were irradiated with 6 MV X-rays (15 Gy in one fraction) in 15 Gy alone and 15 Gy + GSK484 4 mg/kg groups.

Transwell assay

GSK484-alone, IR-alone or IR+GSK484 treated cells (1 × 10⁵ cells/well) were planted on upper chambers coated with Matrigel and contained serum-free DMEM (Gibco, USA). DMEM containing 10% FBS (Gibco, USA) was added to the lower chamber. Then, cells were cultured for 48 hours at 37% with 5% CO₂. Next, non-invaded cells were cleared, the invaded cells were fixed with methanol and stained with crystal violet. Cell migration was performed as above steps except the upper chambers without Matrigel.

Flow cytometric analysis of cell apoptosis

TNBC cells following treatment were collected and washed with PBS. After adjusting the cell density to 1 × 10⁵ cells/mL, cells were stained by Annexin V-FITC and PI according to the manufacturer’s introductions. Cell apoptosis was assessed using flow cytometry (FACScan; BD Biosciences, USA).

Western blot analysis

Western blot analysis was carried out to measure the level of different proteins in TNBC cells. Total proteins were extracted using radioimmunoprecipitation assay lysis buffer (Beyotime, Haimen, China) supplemented with phenylmethylsulfonyl fluoride. The bicinchoninic acid protein assay kit (Beyotime, Haimen, China) was utilized to detect the protein concentration. Subsequently, proteins were separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were transferred to the polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Then, the membranes were blocked with 5% non-fat milk for 2 h at room temperature and incubated with primary antibodies at 4% overnight, followed by further incubation with secondary antibodies for 2 h. The primary antibodies were provided by Abcam (Shanghai, China): MMP2 (ab92536), MMP9 (ab76003), Cleaved caspase-3 (ab2302), Bax (ab53154), Bcl-2 (ab182858), PAD4 (ab214810), LC3 (ab51520), p62 (ab109012), ATG5 (ab108327), ATG7 (ab133528) and β-actin (ab8227). The bands were visualized by Enhanced Chemiluminescence Substrate kit (Millipore, USA) and Bio-Rad image analysis system (Bio-Rad Laboratories, Inc., USA).

Statistical analysis

Data were displayed as the mean ± standard deviation (SD) or the mean ± standard error (SEM). Statistical analyses were analyzed using SPSS 22.0 software (SPSS, USA) and GraphPad Prism 6.0 (GraphPad Software Inc, USA). Comparisons between different groups were performed by the Student’s t test or ANOVA. Survival fraction curve fitting was performed with the linear-quadratic model via the equation y = exp [– (a × x + b × x²)] as previously described. ²¹ P < 0.05 was considered significant.

Results

Effects of GSK484 on TNBC cell progression and radiosensitivity

First, we observed that the IC₅₀ values of GSK484 in MDA-MB-231 and BT-549 cells were 49 nM and 51 nM (Figure 1A). Accordingly, we chose GSK484 at concentrations of 25 nM to treat MDA-MB-231 and BT-549 cells. Then, following the methods of previous research, ²⁰ we detected the survival fraction of MDA-MB-231 and BT-549 cells after IR (0, 1, 2, 3, 4, 5, 6, 7 and 8 Gy) and found that GSK484 treatment decreased the survival fraction of MDA-MB-231 and BT-549 cells after IR (Figure 1B). Next, result from colony formation assay indicated that the proliferation of TNBC cells treated with IR alone was gradually weakened, and these effects were enhanced by the combination of GSK484 and IR (Figure 1C). Similar results were obtained via transwell assays. It is evident from Figure 1D that the combination of GSK484 and IR conspicuously enhanced the inhibitory effects of IR on TNBC cell migration and invasion. Besides, we observed that the protein levels of matrix metalloproteinases (MMP2 and MMP9) were notably decreased after GSK484 or IR treatment, and these effects could be strengthened with the combination of GSK484 and IR in TNBC cells (Figure 1E). Taken together, these findings demonstrated that compared with IR treatment alone, the combination of GSK484 and IR markedly increased the radiosensitivity of TNBC cells in vitro.

Figure 1.

Effects of GSK484 on TNBC cell proliferation and radiosensitivity. (A) The IC₅₀s of GSK484 in MDA-MB-231 and BT-549 cells (B) Survival fraction curves of MDA-MB-231 and BT-549 cells after treatment of GSK484 or IR. (C) Numbers of clones at 14 d after IR in TNBC cells treated with and without GSK484. (D) Cell migration and invasion were detected by transwell assays. (E) The protein levels of MMP2 and MMP9 were examined by western blot analysis. *P < 0.05.

GSK484 facilitates cell apoptosis and promotes the radiosensitivity of TNBC

Subsequently, flow cytometry was performed to measure TNBC cell apoptosis. As presented in Figure 2A, the combined treatment of IR and GSK484 conspicuously increased cell apoptosis compared to those treated with IR or GSK484 separately in MDA-MB-231 and BT-549 cells. Moreover, the treatment of IR or GSK484 significantly increased the protein levels of cleaved caspase-3 and Bax, while decreased the protein levels of Bcl-2 in MDA-MB-231 and BT-549 cells. These effects were enhanced by the combination treatment of IR and GSK484 (Figure 2B). Furthermore, western blot analysis elucidated that IR treatment-triggered the increased protein levels of ATG5, ATG7, as well as the decreased protein level of p62 were recovered by GSK484 treatment. Meanwhile, GSK484 counteracted the transformation of LC3-I to LC3-II induced by IR treatment (Figure 2C). Collectively, GSK484 accelerated cell apoptosis and enhanced the radiosensitivity in TNBC.

Figure 2.

GSK484 facilitates cell apoptosis in TNBC. (A) MDA-MB-231 and BT549 cell apoptosis was analyzed by flow cytometry under the treatment of IR and GSK484. (B) Western blot analysis was carried out to evaluate the protein levels of cleaved caspase-3, Bax and Bcl-2 in TNBC cells treated with IR or GSK484. (C) Western blot analysis was performed to assess the protein levels of LC3, p62, ATG 5 and ATG 7. *P < 0.05.

GSK484 enhances IR-triggered DNA damage

To further verify whether GSK484 plays a role during DNA damage repair, immunofluorescence assay was carried out to detect γH2A.X. We noted a remarkable increase in γH2A.X after IR treatment with GSK484 (Figure 3A). For calculating the average number of foci per nuclei and the percentages of nuclei with more than 10 foci, at least 50 MDA-MB-231 and BT-549 cells were used to count the number of γH2A.X foci in the nuclei. The results demonstrated that compared with the control group, TNBC cells received IR or IR + GSK484 treatment exhibited memorably elevated percentage of nuclei with more than 10 foci. In the meantime, compared with TNBC cells treated with IR alone, the percentage of nuclei with more than 10 foci was obviously increased by the combined treatment of IR and GSK484 (Figure 3B). Moreover, Since GSK484 is a highly potent PAD4-selective inhibitors, and PAD4 has been reported to affect DNA damage, ²² thus we evaluated the influence of GSK484 on the protein level of PAD4. Western blot analysis demonstrated that compared with control group, the protein level of PAD4 in TNBC cells was decreased by GSK484 but increased by IR; the introduction of GSK484 abolished IR-induced elevation of PAD4 protein level (Figure 3C). To sum up, GSK484 enhanced IR-induced DNA damage in the progression of TNBC.

Figure 3.

GSK484 enhances IR-triggered DNA damage. (A) Immunofluorescence assay showed the number of γH2A.X foci in treated MDA-MB-231 and BT-549 cells. (B) Compared with the IR-alone group, the IR + GSK484 group exhibited significantly increased percentage of more than 10 foci in MDA-MB-231 and BT-549 cells. (C) The protein level of PAD4 in TNBC cells was measured by western blot. *P < 0.05.

GSK484 enhances the radiosensitivity of TNBC in vivo

To figure out the synergistic sensitization effects of GSK484 in vivo, we established the xenograft model through inoculating MDA-MB-231 cells into the mammary fat pads of Balb/c mice with or without GSK484 treatment. As shown in Figure 4A, compared with the groups treated with IR alone or GSK484 alone, the tumor size of the xenografts was significantly reduced in the group treated with GSK484 combined with IR. Likewise, an obvious decline of both tumor volume and body weight was found in mice with a combination treatment in contrast to the treatment of IR or GSK484 alone (Figure 4B–C). In conclusion, combined treatment of IR and GSK484 enhanced the radiosensitivity of TNBC in vivo.

Figure 4.

GSK484 enhances the radiosensitivity of TNBC in vivo. (A) The photos of xenograft tumors obtained from sacrificed mice were shown. (B–C) Tumor volume and body weight were measured based on xenografted tumor model. *P < 0.05.

Discussion

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with approximately 0.2 million cases diagnosed worldwide per year. ^5,23 Compared with other breast cancer types, TNBC is more likely to recur locally or metastasize to the lung and brain during 3–5 years after diagnosis. ^7,24 Although the treatment of TNBC, especially radiotherapy, has made great progress over the past few decades, the effectiveness is still limited. ⁸ Thus, developing novel therapeutic strategies is necessary for improving the prognoses of TNBC patients.

Increasing reports have revealed that PAD4 participates in the development of several cancers, such as non-small cell lung cancer and lung metastasis of breast cancer. ^11,12 Moreover, PAD4 inhibitors have been considered as potential targets for cancer treatment. ^{13 –17} A recent study demonstrates that PAD4-specific inhibitor GSK484 could alleviate myocardial infarction triggered cardiac dysfunction. ¹⁸ In our research, we found that GSK484 sensitized MDA-MB-231 and BT-549 cells to ionizing radiation by enhancing the inhibition of post-irradiation cell proliferation, migration and invasion. Meanwhile, combined treatment of IR and GSK484 significantly enhanced the radiosensitivity of TNBC in vivo.

As we all know, radiation can induce DNA damage through the direct ionization, excitation or production of free radicals. ^25,26 Recent studies have indicated that DNA damage inhibits cell cycle process, and initiates DNA repair mechanisms at the same time. Radiation facilitates tumor cell death mainly by producing unrepaired DNA damage. ^{27 –29} In another word, the capacity of cancer cells to repair DNA damage is of vital significance in determining their radiosensitivity.

Hence, targeting DNA damage repair may become an effective mean to enhance radiosensitivity and improve therapeutic activity. Increasing evidence has indicated that the inhibition of DNA repair is greatly beneficial to achieve radiosensitivity in cancer cells. For example, MK-8776 decreases the DNA repair capacity and increases the radiosensitivity of TNBC cells. ²⁰ Targeting DNA repair improves radiosensitivity of head and neck squamous cell carcinoma. ³⁰ Likewise, our findings revealed that GSK484 strengthened the radiosensitivity of TNBC cells possibly by facilitating cell apoptosis and strengthening DNA damage.

Autophagy is a protein degradation mechanism that transports damaged organelles and long-lived proteins in autophagosomes to lysosomes for degradation and recycle. ³¹ Cells usually induce autophagy to maintain their longevity when exposed to unfavorable environment. ³² Growing evidence has manifested that autophagy is closely correlated with the progression of cancers. For instance, suppression of autophagy strengthens the inhibitory effect of phenethyl isothiocyanate on lung cancer metastasis. ³³ Licarin A regulates the development of non-small cell lung cancer by accelerating autophagy dependent cell apoptosis. ³⁴ MK-8776 serves as a radiosensitizer in TNBC by inhibiting autophagy. ²⁰ In recent years, targeting autophagy to enhance current treatments in multiple cancers, including breast cancer, has achieved promising results. ^{35 –38} Furthermore, a previous study indicated that PAD4 inhibitors might have an impact on cancer progression by regulating autophagy. ³⁹ Therefore, we aimed to investigate the correlation between GSK484 and autophagy in TNBC. Our data revealed that GSK484 reversed IR-induced the transformation of LC3-I to LC3-II, suggesting that GSK484 increases the radiosensitivity of TNBC via the inhibition of autophagy.

In conclusion, our findings demonstrate that the PAD4 inhibitor GSK484 significantly strengthens the radiosensitivity of TNBC cells through inhibiting cell proliferation, migration, invasion, autophagy, and enhancing cell apoptosis. GSK484 may play the role of radiosensitizer in TNBC treatment. However, further experiments are needed to figure out the association between GSK484 and cell autophagy in the future.