Polo-like kinase 1 inhibitor BI 6727 induces DNA damage and exerts strong antitumor activity in small cell lung cancer
Yuehong Wanga,1, Linying Wua,1, Yinan Yaoa, Guohua Lua, Liming Xub, Jianying Zhoua,∗
a Department of Respiratory Disease, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China
b Department of Pathology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China
A R T I C L E I N F O
Keywords:
PLK1 BI 6727
Small cell lung cancer DNA damage
A B S T R A C T
The prognosis of small cell lung cancer (SCLC) is poor despite its good initial response to chemotherapy. Polo- like kinase 1 (PLK1) is a crucial mitotic regulator that is overexpressed in many tumors, and its overexpression is associated with tumor aggressiveness and a poor prognosis. However, its role in SCLC is still poorly char- acterized. Based on immunohistochemistry findings, the PLK1 protein is expressed at higher levels in SCLC tumor samples than in normal lung tissue samples. The selective PLK1 inhibitor BI 6727 significantly induced the inhibition of proliferation and apoptosis in a dose-dependent manner in SCLC cell lines. FACS analysis showed an increase in the population of cells in the G2/M phase, followed by DNA damage and the consequent activation of the ataxia telangiectasia and Rad3-related (ATR)/ataxia telangiectasia mutated (ATM)-Chk1/Chk2 checkpoint pathway. In addition, BI 6727 treatment resulted in clearly attenuated growth and apoptosis in NCI-H446 Xe- nografts. The level of histone H2AX phosphorylation at serine-139 (γH2AX) was markedly increased both in vitro
and in vivo. Our findings indicate that BI 6727 has therapeutic potential for SCLC patients.
1. Introduction
Lung cancer is the leading cause of cancer-related deaths world- wide, and the American Cancer Society estimates that lung cancer was responsible for one-fourth of cancer-related deaths in the US in 2017 [1,2]. Small cell lung cancer (SCLC) accounts for 15–20% of all lung cancer cases, and approXimately two-thirds of SCLC patients have ex- trathoracic metastases at the time of diagnosis. Furthermore, the median survival of patients with SCLC without treatment is 2–4 months, and the five-year overall survival rate is only 5–7% [3–6]. SCLC is a malignancy that exhibits rapid tumor growth and the early develop- ment of widespread metastases, which makes it the most aggressive subtype of lung cancer [7,8].
Unfortunately, almost no significant advances have been made in the treatment of SCLC in the past 30 years. Although approXimately 80% of SCLC patients respond favorably to first-line chemotherapy with a platinum drug in combination with etoposide, 80–98% of patients die from recurrent disease within 2 years of diagnosis [4,9]. Although the second-line regimen of single-agent topotecan shows an improvement in overall survival (OS) compared with the best supportive care, it prolongs life by only a few weeks in relapsed patients [10–12].
Therefore, novel treatment strategies for SCLC are urgently needed.
Polo-like kinase 1 (PLK1) is a member of the polo-like kinase family that is composed of 5 highly conserved serine/threonine protein kinases (PLK 1-5). It was first described in 1988 in Drosophila, and it was shown to play an essential role in mitosis and meiosis; it has been extensively studied in recent years [13]. PLK1 regulates several mitotic events, including mitotic entrance, centrosome maturation, chromosome seg- regation, cytokinesis, and exit from mitosis. In addition, PLK1 partici- pates in DNA damage response and repair [14]. The overexpression of PLK1 has been found in numerous tumor types, including breast, col- orectal, endometrial, ovarian, pancreatic, and non-small cell lung cancer (NSCLC) [15]. Moreover, higher PLK1 expression correlates with tumor aggressiveness and a poor prognosis [16–19]. Because of its key role in the orchestration of the cell cycle and the progression of cancer, PLK1 could be an attractive therapeutic target for the treatment of tu- mors. Recently, several PLK1 inhibitors, such as BI 2536, BI 6727, and GSK 461364, have been investigated in various cancers [20,21]. BI 6727 (volasertib), a dihydropteridinone derivative, is an ATP-compe- titive kinase inhibitor with higher potency and selectivity than those of its predecessor, BI 2536 [22]. Several preclinical and clinical trials have shown that BI 6727 displays encouraging antitumor activity as a
∗ Corresponding author. Department of Respiratory Disease, The First Affiliated Hospital, College of Medicine, Zhejiang University, No.79, Qingchun Road, Hangzhou, 310003, China.
E-mail address: [email protected] (J. Zhou).
1 These authors have contributed equally to this work.
https://doi.org/10.1016/j.canlet.2018.08.007
Received 30 May 2018; Received in revised form 14 July 2018; Accepted 9 August 2018
0304-3835/©2018PublishedbyElsevierB.V.
monotherapy or as part of PLK1 inhibitor-based combination treat- ments in various tumors, including NSCLC [23–30]. A combination therapy with BI 6727 and low-dose cytarabine (LDAC) had a promising result with significantly prolonged median event-free survival and OS in patients with acute myeloid leukemia (AML) who were ineligible for intensive induction therapy [31]. As a result, the Food and Drug Ad- ministration (FDA) has recently awarded volasertib breakthrough therapy status after a significant improvement was observed in patients with AML, and a phase III trial (NCT01721876) has been conducted to assess the efficacy and safety of the combination of volasertib with LDAC.
However, the efficacy of PLK1 inhibition in SCLC is still unclear. Cao et al. [32] showed that downregulation of miR-886-3p is closely correlated with shorter survival in patients with SCLC. MiR-886-3p was shown to potently repress the proliferation, migration, and invasion of NCI-H446 cells in cell culture via suppression of the expression of its
target genes, PLK1 and TGF-β1, at the posttranscriptional level. Re- cently, a study found that PLK1 specifically binds to the F-boX and WD
repeat domain-containing 7 (Fbw7) ubiquitin ligase and promotes its autopolyubiquitinylation and proteasomal degradation, counteracting the Fbw7-mediated degradation of N-myc, which reinforces myc-regu- lated oncogenic programs. Inhibitors of PLK1 preferentially induce the apoptosis of MYCN-amplified tumor cells in SCLC and synergistically potentiate the therapeutic efficacies of Bcl2 antagonists [33]. Further- more, the screening of oncology drugs and investigational agents showed that approXimately half of the 63 SCLC cell lines were sensitive to polo-like kinase inhibitors, including BI 6727 [34].
These findings reveal that PLK1 inhibition may be an effective therapeutic strategy for the treatment of SCLC. In this study, we showed that PLK1 is overexpressed in SCLC tumor tissue when compared with normal lung tissues, determined the efficacy of PLK1 inhibition with BI 6727 in several SCLC cell lines and a tumor-bearing mouse model, and explored the potential mechanisms of action of BI 6727.
2. Materials and methods
2.1. Chemicals and cell culture
BI 6727was obtained from Selleck Chemicals. NCI- H446, NCI-H69, NCI-H209, and DMS 153 cells were was obtained from the American Type Culture Collection (Manassas, VA, USA). HBE cells were pur- chased from Chinese Academy of Sciences Cell Bank (Shanghai, China). All cells were grown in their recommended medium at 37 °C with 5% CO2 in a humidified incubator.
2.2. Cell viability assays
SCLC cells and HBE cells plated in triplicate were incubated with 0.1% dimethyl sulfoXide (vehicle control) or BI 6727 for 72 h at 9 dif- ferent concentrations, and the maximum dose of 1.2 μM was chosen because this dose is the peak drug concentration in humans (Cmax) [27].
Cell viability was measured using the Cell Counting Kit-8 assay (Do- jindo Molecular Technologies, Tokyo, Japan) according to the manu- facturer’s instructions. The IC50 of BI 6727 in SCLC cells was calculated using CalcuSyn software version 2.1(Biosoft, Cambridge, UK). At least 3 replicates were tested for each cell line on different days.
2.3. Colony formation assays
For adherent cells, NCI-H446 cells were plated in a 6-well plate at a density of 600 cells/well, treated with 0.1% dimethyl sulfoXide or BI 6727 for 24 h and then incubated in drug-free medium for 14 days with regular medium changes every 48–72 h. NCI-H209 cells grown in sus- pension were diluted to a density of 2 × 104 cells/ml in 0.35% low melting-point agarose (LMP) in RPMI medium supplemented with 10% FBS. The cell suspension was added to each well of a 6-well plate with
an underlayer of 0.7% LMP and then cultured at 37 °C for 14–20 days. The plates were stained with crystal violet, and the total number of colonies per well was counted using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA). The median number from 3 replicates was used for statistical analysis.
2.4. Cell cycle and apoptosis analysis
SCLC cell lines were treated with different concentrations of BI 6727 for 48 h and then apoptosis was analyzed with the FITC Annexin V Apoptosis Detection Kit (BD Pharmingen, NJ, USA). Cells were fiXed, permeabilized and stained with propidium iodide after 48 h incubation with BI 6727 using the Cell Cycle Phase Determination Kit (Cayman Chemical, Ann Arbor, Michigan USA) to analyze the cell cycle. Flow cytometry analyses were performed using the BD FACSVerse system (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo soft- ware.
2.5. Comet assay
The OXiSelect™ Comet Assay Kit (Cell Biolabs, San Diego, CA, USA) was used to evaluate DNA damage in SCLC cells. Individual cells at 1× 105 cells/ml were suspended in cold phosphate-buffered saline (PBS) and miXed with comet agarose at a 1:10 ratio (v/v), and then 75 μl/well was pipetted onto the -precoated slides. After being allowed to
solidify for 15 min at 4 °C, the slides were treated with lysis buffer for
1 h and an alkaline solution for 30 min in the dark at 4 °C to denature the DNA. Then, the slides were subjected to alkaline electrophoresis at 1 V/cm, 300 mA, for 30 min. Finally, the slides were dried and stained with Vista Green DNA Dye solution for 15 min and viewed by epi- fluorescence microscopy using an FITC filter. The analysis of DNA da- mage was conducted using CASP software (available at http://www. casp.of.pl), and 100 randomly selected comets were evaluated for each sample.
2.6. Western blot analysis
Cells without or with the indicated concentrations of cisplatin, etoposide, or volasertib were lysed in cell lysis buffer containing pro- tease and phosphatase inhibitors. The protein concentrations of the cell lysates were determined using the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Then, equivalent quantities (50 μg) of protein were separated with 10–15% sodium dodecyl sulfate-
polyvinylidene gel electrophoresis and transferred to polyvinylidene
difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany), which were then probed with antibodies against cyclin B, PLK1, cleaved
-poly-ADP ribose polymerase (PARP), cleaved caspase-3, γH2AX
(Ser139), phospho (p)-ataxia telangiectasia mutated (ATM) (Ser1981)/ total (t)-ATM, p-ATR (Ser428)/t-ATR, p-Chk1(Ser345)/t-Chk1, p- Chk2(Thr68)/t-Chk2 or GAPDH from Cell Signaling Technology (Danvers, MA, USA). After an incubation with goat anti-rabbit anti- bodies, the proteins were detected using enhanced chemiluminescence (ECL) reagent.
2.7. In vivo model
NCI-H446 cells (2 × 106 cells per mouse) were injected sub- cutaneously into four-week-old female BALB/c-nude mice (Shanghai EXperimental Animal Center, Chinese Academy of Science). Tumors were measured with a caliper twice weekly, and tumor volumes were calculated using the following formula: volume=(length × width2)/2. When the tumors reached a size of 100–150 mm3, the mice were ran- domized to either the group receiving the 30 mg/kg BI 6727 treatment (BI 6727 was suspended in 0.1 N HCl and then diluted in saline) or the group receiving only the vehicle weekly via the tail vein (6 mice per group). The animals were sacrificed after 3 weeks of treatment, and the
Fig. 1. Levels of PLK1 protein in SCLC and normal lung tissues, as assessed by immunohistochemistry. (A) The expression of PLK1 in normal lung tissue was barely detectable while SCLC tumor tissues showed high PLK1 expression. Magnification: 200 × . (B) PLK1 scores were used to evaluate PLK1 IHC staining and were calculated for every patient. Data are presented as the mean ± SD, ***P < 0.001 compared with normal lung tissue.
tumors were collected and photographed. All animal procedures were by IHC staining of samples from 48 SCLC patients and 15 patients with
conducted in accordance with the guidelines of the Animal Ethics Committee of Zhejiang University.
2.8. TUNEL assay
EXperimental
benign nodules who underwent surgery. The samples were used with the approval of the Ethics Committee of the First Affiliated Hospital of Zhejiang University. The tissue samples were prepared as 4 μm-thick sections; the sections were deparaffinized and rehydrated, and the an-
tigens were unmasked by heat-induced epitope retrieval for 1 h. The
Apoptosis was detected in tissue sections using the In Situ Cell Death Detection Kit, Fluorescein (Roche, Basel, Switzerland), according to the instructions provided with the kit. Briefly, the sections were dewaxed,
rehydrated, and permeabilized with 20 μg/ml proteinase K solution for 30 min at room temperature. After rinses with PBS, sections were in-
cubated with the TUNEL reaction miXture (Enzyme Solution was miXed with Label Solution at a 1:9 volumetric ratio) in a humidified atmo- sphere for 1 h at 37 °C in the dark. The nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole). After rinses with PBS, sections were mounted with antifade mounting medium and observed under a fluorescence microscope.
2.9. Immunofluorescence assay
Tumor samples were fiXed in 10% (v/v) formalin for at least 24 h at room temperature, embedded in paraffin and prepared on slides (4 μm in thickness). Then, the sections were deparaffinized in xylene, rehy-
drated by graded ethanol, and stained with hematoXylin-eosin (H&E) to observe pathological changes. The levels of phospho-H2A.X (pSer139) were detected using immunofluorescence staining. Tumor tissues were boiled in sodium citrate buffer and preincubated with 0.5% Trion X-100 and 5% bovine serum albumin in PBS for 2 h at room temperature. After an incubation with specific antibodies against phospho-H2A.X over- night at 4 °C, the sections were incubated with fluorescent secondary antibodies for another 2 h at room temperature. The sections were mounted with antifade mounting medium prior to observation using a confocal fluorescence microscope.
2.10. Immunohistochemistry staining and evaluation
The expression level of the PLK1 protein in lung tissues was detected
samples were incubated with anti-human PLK1 monoclonal antibody (1:30 dilution; Cell Signal Technology) at 37 °C for 1 h. After being washed in PBS, the sections were stained with DAB and counterstained with hematoXylin. The IHC images were captured and evaluated ac- cording to the distribution and immunoreactivity of PLK1. The results were evaluated by 2 independent observers. The percentage of PLK1- positive cells was recorded as 0 if 0–1% of the cells were stained, 1 if 1–5% of the cells were stained, 2 if 6–29% of the cells were stained, 3 if 30–59% of the cells were stained and 4 if more than 60% of the cells were stained. The staining intensity was recorded as 0 if no staining was observed, 1 for weak staining, 2 for moderate staining and 3 for strong staining. Then, these values were multiplied to provide a single PLK1 score for each sample [35].
2.11. Statistical analysis
The data are presented as the mean values ± SD from 3 or more independent repetitions. Two-tailed unpaired Student's t tests were performed to determine significance, and P < 0.05 was defined as statistical significance.
3. Results
3.1. The expression of PLK1 protein was up-regulated in SCLC patients
Overexpression of PLK1 has been reported in several types of tumors [15–19] [36], but the expression of PLK1 in patients with SCLC had not been determined. The expression of the PLK1 protein was determined by immunohistochemistry in tumor samples obtained from 23 patients with limited-stage SCLC (LS-SCLC) and 25 patients with extensive stage SCLC (ES-SCLC) as well as in lung tissue samples from 15 patients with
benign nodules who underwent surgery. PLK1 staining was barely de- tectable in most normal lung tissues, while SCLC tumor tissues ex- hibited moderate to intense diffuse staining of PLK1. The staining in- tensity in normal lung tissues was generally 0-1, while it was usually 2- 3 in SCLC tissues (Fig. 1A). Although PLK1 overexpression has been shown to be closely related to the metastasis and TNM stage of tumors [35], no significant difference in the expression of PLK1 was observed between LS-SCLC (4.109 ± 0.306) and ES-SCLC (5.036 ± 0.409,
P = 0.0723) tumor tissues (Fig. 1B).
3.2. Inhibition of PLK1 significantly suppressed proliferation and viability of SCLC cell lines
To explore the efficacy of BI 6727 in SCLC cells, we detected the cell viability of NCI-H446, DMS153, NCI-H69, and NCI-H209 cells treated with BI 6727 for 72 h. Cell growth was significantly reduced in a dose- dependent manner. BI 6727 effectively inhibited SCLC cell growth, and the IC50 in SCLC cells was in the nanomolar range (< 100 nM). We treated human bronchial epithelial (HBE) cells, a line isolated from the normal bronchial epithelium, with BI 6727 to investigate whether BI 6727 inhibits the proliferation of normal lung cells. HBE cells were relatively unaffected by BI 6727 at concentrations that severely reduced the viability of SCLC cells, suggesting that at concentrations that exert distinct effects on SCLC cells, BI 6727 is not toXic to normal lung cells (Fig. 2A). Consistently, low levels of colony formation were observed in NCI-H446 and NCI-H209 cells treated with BI 6727 (P < 0.01, Fig. 2B–C).
3.3. PLK1 inhibition led to G2/M arrest and apoptosis in SCLC cell lines
We treated SCLC cells with BI 6727 at different concentrations for 48 h and observed a significant dose-dependent accumulation of 4 N DNA content in NCI-H446 and DMS153 cells, indicating G2/M arrest (Fig. 3A and B). PLK1 plays an important role in the regulation of mi- totic entry and exit. PLK1 regulates mitotic exit through phosphor- ylating the anaphase-promoting complex (APC), which is responsible for the degradation of cyclin B1 [37]. Inhibition of the activation of PLK1 resulted in the accumulation of cyclin B1, blocking cell cycle progression (Fig. 3C). Induction of apoptosis was confirmed by flow cytometry analysis of Annexin V-positive cells; the percentage of apoptotic cells was markedly increased in NCI-H446, NCI-H69, NCI- H209 and DMS153 cells after treatment with BI 6727 (Fig. 4A and B). We also observed a dose-dependent increase in the levels of cleaved PARP in cells after 48 h of treatment with BI 6727. Western blots using an antibody against cleaved caspase-3 were performed to determine the involvement of caspase-3 in the cells undergoing apoptosis. Levels of the active p17 and p12 fragments were increased in cells treated with BI 6727 (Fig. 4C). Based on these results, the BI 6727 treatment induced caspase-3-dependent apoptosis in SCLC cells.
3.4. BI 6727 treatment induced DNA damage in SCLC cell lines
PLK1 is a target of the DNA damage checkpoint [38], and PLK1 inhibition can cause DNA damage. To investigate whether BI 6727 treatment induced DNA damage in SCLC cells, we exposed the cells to 50 nM BI 6727 for 48 h and then performed a comet assay, which measured DNA single-strand breaks and double-strand breaks (DSBs). The presence of a comet tail indicated the presence of denatured DNA,
Fig. 2. PLK1 inhibition diminished proliferation in SCLC cell lines. (A) Dose-response curves showing the effect of BI 6727 on the viability of NCI-H446, DMS153, NCI-H69 and NCI-H209 cells. Cells were plated in triplicate and treated with 0.1%DMSO as a control or with the indicated doses of BI 6727 for 72 h; then, cell viability was masured using the CCK8 assay. The IC50 of BI 6727 in each cell lines was calculated using CalcuSyn software. (B) Results from colony formation assays of NCI-H446 and NCI-H209 cells treated with increasing concentrations of BI 6727 or 0.1% DMSO. Cells were seeded onto a siX-well plate and were treated with DMSO or BI 6727 after a 24 h incubation. Cell colonies were stained with crystal violet and photographed after 14 days. (C) The numbers of NCI-H446 and NCI-H209 colonies were calculated. Data are presented as mean values and standard deviations (SD). **P < 0.01 compared with the control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3. G2/M cell cycle arrest in SCLC cells upon BI 6727 treatment. (A and B) Cell cycle analyses of NCI-H446 and DMS153 cells were conducted after treatment with various concentrations of BI 6727 for 48 h. Percentages of cells in each cell cycle stage are reported as the averages of triplicate experiments. (C) Western blot analysis of PLK1 and cyclin B1 in SCLC cells treated with BI 6727 treatment. Lysates from SCLC cells were probed with antibodies against PLK1, cyclin B1 and GAPDH after treatment with BI 6727 or 0.1% DMSO for 48 h, as proteasomal degradation of cyclin B1 depends on PLK1.
and the tail moment was used to quantify the degree of DNA damage. The tail moment of BI 6727-treated cells was remarkably increased compared to that of vehicle-treated cells (Fig. 5A and B). Histone H2AX
phosphorylation at serine-139 (γH2AX) is a sensitive marker of un- repaired DSB; DNA damage results in the rapid phosphorylation of
H2AX by the PIKK family, including ATM and ATR [39]. Western blotting showed that the level of γH2AX was increased in NCI-H446 and DMS153 cells in a time-dependent manner after treatment with BI 6727
(Fig. 5C). The phosphorylation of ATM and ATR is generally observed with DNA damage [40]. Indeed, accumulated phosphorylation of ATM and ATR at the Ser1981 and Ser428 sites was observed in BI 6727- treated cells. Chk1 and Chk2 are the kinases downstream of ATR and ATM, and they were shown to be phosphorylated at the Ser317 and Thr68 sites, respectively (Fig. 5C).
3.5. BI 6727 treatment attenuated the growth of SCLC xenograft tumors and induced apoptosis and DNA damage in vivo
We then examined the activity of BI 6727 in an NCI-H446 Xenograft model. Compared with the control treatment, we observed a significant trend of decreased tumor growth in the BI 6727 treatment group (1849 ± 123.1 mm3 vs. 887.0 ± 57.27 mm3, N = 6, P < 0.0001;
Fig. 6A and B), and the treatment was well tolerated, with no apparent changes in body weight (Fig. S1). We also observed trends of more necrotic cells in NCI-H446 Xenografts treated with BI6727 than control on H&E staining (Fig. S2). The detection of apoptosis in the xenograft tumors using the TUNEL assay determined that the number of apoptotic cells was higher in the BI 6727 treatment group than in the control group. These results suggested that BI 6727 suppressed the progression
of SCLC in vivo (Fig. 6C and D). To elucidate whether BI 6727 was able to promote DNA damage in vivo, we assessed the expression of γH2AX in the xenograft tumor model by immunohistochemistry. Then, we
observed the effects of BI 6727 treatment on the xenograft tumor model and found that the expression of γH2AX was higher in the BI 6727 treatment group than in the control group. The formation of extensive γH2AX foci indicated a large number of DNA breaks (Fig. 6E).
4. Discussion
Here, we provided preclinical evidence of the potential for BI 6727 to act as a therapeutic agent in the treatment of SCLC. The expression of PLK1 in SCLC was markedly increased compared to that in the non- tumor lung tissues. Treatment of SCLC cell lines with BI 6727 resulted in G2/M cell cycle arrest and apoptosis. Simultaneously, we observed significantly attenuated tumor growth and increased apoptosis in the BI 6727-treated SCLC Xenograft model. Moreover, treatment with BI 6727 led to DNA damage in SCLC cell lines and the xenograft model.
The overexpression of PLK1 is associated with poor prognosis in many types of cancer. Here, we detected elevated protein levels of PLK1 in most of the clinical SCLC tumor samples. In recent studies, the ex- pression level of PLK1 was confirmed to be correlated with tumor stage and the OS of patients, and several investigations demonstrated that PLK1 was an independent prognostic factor in gastric cancer, esopha- geal carcinoma, hepatocellular carcinoma and NSCLC [17,35,41,42]. However, in our study, no difference was found in the expression of PLK1 between LS-SCLC and ES-SCLC.
SCLC patients face a very poor prognosis due to the aggressiveness and recurrence of this type of cancer. SCLC is a neuroendocrine tumor with a rapid doubling time [8], which means that drugs that arrest the cell cycle could slow down the proliferation of SCLC cells. PLK1 plays a vital role in the regulation of cell cycle progression; the expression of PLK1 increases in the S phase, peaks during the late G2 to M phase and declines during mitotic exit [43]. In addition, a previous study showed that the sensitivity to the inhibition of PLK1 might be dictated by a
Fig. 4. PLK1 inhibition induced apoptosis in SCLC cell lines. (A) SCLC cells were treated with 0.1% DMSO or different concentrations of BI 6727 for 48 h. Then, cells were subjected to flow cytometry analysis after FITC-Annexin V and propidium iodide (PI) staining. (B) Percentages of apoptotic cells are presented as the mean values ± SD. *P < 0.05; **P < 0.01. (C) SCLC cells were treated with 0.1% DMSO or the indicated concentrations of BI 6727 for 48 h, and protein extracts were subjected to SDS-PAGE and immunoblotted with antibodies against cleaved PARP and cleaved caspase-3. GAPDH was detected as a loading control for all whole cell extracts.
characteristically high proliferative index and a high degree of ded- ifferentiation in tumors [44]. Furthermore, PLK1 was confirmed to be necessary for the survival of only cancer cells and not of normal cells; the depletion of PLK1 led to mitotic catastrophe in HeLa cells. In striking contrast, normal HTERT- RPE1 and MCF10A cells showed no apparent defects in cell proliferation or cell cycle arrest after the de- pletion of PLK1 [45]. These findings suggested that inhibition of PLK1 activity or suppression of its expression could be a treatment strategy for SCLC. PLK1 depletion leads to G2/M cell cycle arrest in various tumors, such as NSCLC, melanoma, and thyroid carcinoma [20,22–24,44]. Simultaneously, various studies indicated that DNA damage triggered the activation of checkpoints to induce cell cycle arrest or even apoptosis if the damage overwhelmed the DNA repair capacity. Consistent with these findings, our results indicated that the inhibition of PLK1 induced the arrest of SCLC cells in the G2/M phase and then led to widespread apoptosis. Similar to the in vitro findings, BI 6727 notably slowed the growth of the SCLC Xenograft tumors and induced DNA damage.
PLK1 is a target of the DNA damage checkpoint and seems to be required for mitotic reentry after DNA damage checkpoint recovery [46]. The present study demonstrates that the overexpression of PLK1 phosphorylates and inactivates the ATM/ATR-Chk1/Chk2 pathway, which overrides the checkpoint barrier and facilitates the occurrence of
G2/M transition with damaged DNA, eventually leading to a much greater risk of tumorigenesis [47,48]. Further, RNA-seq analysis de- monstrated that the overexpression of PLK1 clearly affected the ex- pression of DNA damage repair genes in mouse embryo fibroblasts [49]. We demonstrated that BI 6727 induced DNA damage in SCLC both in vitro and in vivo, as expected. These were confirmed by the detection of
a strikingly increased level of expression of γH2AX and the formation of
comet tails in individual cells as revealed by the comet assay. After a double-stranded DNA break, alteration of the chromatin structure promotes the phosphorylation of H2AX by ATM, ATR and DNA-PK at the Ser139 site. Thus, we further detected the activity of the ATR/ATM- Chk1/Chk2 kinases and determined that these proteins were activated after 2 h of treatment with BI 6727. These results suggested that BI 6727 acts by causing DNA damage in SCLC cells. One previous study showed that the inhibition of PLK1 by BI 6727 combined with erlotinib significantly enhanced DNA damage via the activation of ATR/Chk1 in erlotinib-resistant NSCLC, suggesting that DNA damage potentiated the lethality of the inhibition of PLK1 and EGFR [24]. In addition, the knockdown of PLK1 in HT-29 and HCT116 cells resulted in the acti- vation of pChk1 and pChk2, followed by increased expression of γH2AX; however, PLK1 knockdown caused post-mitotic DNA damage
[50]. PLK1 depletion induces DNA damage not only in the G2/M phase
but also in the S phase. Another report showed that PLK1 depletion
Fig. 5. PLK1 inhibition induced DNA damage in SCLC cell lines. (A) SCLC cells were incubated with 0.1% DMSO or 50 nM BI 6727 for 48 h and then subjected to a comet assay, which measures both single-strand and double-strand DNA breaks. Subsequently, images of cells on slides were captured with an epifluorescence microscop. (B) For the evaluation of the comet patterns, at least 100 randomly selected nuclei from each sample were analyzed using CASP software, and tail moments in individual cells were calculated. *P < 0.05; ***P < 0.001. (C) BI 6727 activated the DNA damage sensing ATM and ATR pathway. NCI-H446 and DMS153 cells were treated with 100 nM BI 6727 for the indicated times (0, 2, 4, or 8 h). The cells were harvested, and Western blot analysis was performed with specific antibodies, as indicated.
leads to disruption of the formation of the DNA prereplicative complex, reduced DNA synthesis and DNA damage in the early S phase, prior to caspase activation. The proposed mechanism was that the depletion of PLK1 allowed accumulation of Emi1 and inactivation of APC, which prevented the destruction of geminin, leading to the disruption of chromatin and, subsequently, DNA damage [51].
In a recent phase II study, BI 2536 failed to demonstrate significant antitumor activity in 23 patients with relapsed SCLC; there were no objective responses nor any prolongation of PFS seen among these patients. The short terminal half-life of BI 2536 in patients and its low intratumoral accumulation results in the insufficient treatment of many advanced solid tumors [52]. In addition, dose-limiting toXicities such as neutropenia may prevent BI 2536 from reaching effective antitumor levels. BI 6727 showed a favorable pharmacokinetic profile, a high volume of distribution, prolonged terminal half-life, and manageable toXicities compared to BI 2536. The first-in-man study showed that BI 6727 has encouraging antitumor activity in advanced solid tumors, and 2 patients acquired prolonged treatment benefit, with PFS greater than 1 year [27]. Moreover, a recent phase II clinical trial in patients with
platinum-resistant or refractory ovarian cancer showed that 6 patients (11%) who received BI 6727 monotherapy achieved PFS for more than 1 year, while none of the patients receiving single-agent chemotherapy achieved PFS greater than 1 year; this result suggested that BI 6727 might achieve a durable response. Studies should be performed to in- vestigate biomarkers that can aid in the selection of patients who are more likely to respond to BI 6727 [30]. In a study of BI 2536 in AML patients, bone marrow biopsies after treatment showed mitotic arrest and the apoptosis of malignant cells, indicating a possible on-target effect [53]. In preclinical studies, BI 6727 was similarly highly effica- cious in lung cancer and melanoma cells resistant to taxanes or vinca alkaloids and showed promising activity in a taxane-resistant Xenograft model of colorectal cancer [22]. The studies by Fulda et al. identified the synthetic lethality of BI 6727 and microtubule-destabilizing drugs such as vincristine, vinblastine and vinorelbine in preclinical rhabdo- myosarcoma models and Ewing sarcoma cells through triggered mitotic arrest [54,55]. These findings demonstrated that BI 6727 might pro- duce synergistic effects with these antineoplastic drugs or be effective in patients who have become resistant to conventional
Fig. 6. Efficacy of PLK1 inhibition in NCI-H446 Xenograft models. Nude mice bearing established NCI-H446 SCLC tumors with an average size of 100–150 mm3 were treated i. v. once a week with either vehicle or 30 mg/kg doses of BI 6727 for 3 cycles. (A)Tumor volumes were determined twice weekly after the onset of treatment. Tumor volume are presented as the mean ± SD, ***P < 0.001. (B) Mice were sacrificed after 3 weeks of treatment, and tumors were carefully dissected and photographed. (C) TUNEL analysis of paraffin-embedded tumor tissue showed the change in the apoptosis of NCI-H446 Xenografts upon treatment with the control and BI 6727. Magnification: 400 × . (D) The percentage of nuclei stained with TUNEL. Data are presented showed as the mean ± SD, **P < 0.01. (E) Immunofluorescence images showed an increase in γH2AX expression in tumors collected from mice treated with BI 6727. Magnification: 400 × .
chemotherapeutic drugs. An ongoing phase III trial is being carried out to investigate the efficacy of BI 6727 combined with LDAC in AML patients who were ineligible for intensive remission induction therapy. These preclinical and clinical findings suggest that the use of BI 6727 as a second-line therapy or in combination with traditional chemotherapy is a promising avenue for future studies.
Altogether, these data strongly suggest that targeting PLK1 attenu- ates the growth of SCLC and induces DNA damage in vitro and in vivo. Our findings established PLK1 as a very promising target in SCLC. However, the validation of biomarkers for PLK1 inhibitors to help identify appropriate patients for treatment will be necessary to support the clinical utility of BI 6727. Meanwhile, combining BI 6727 with chemotherapy might be a promising therapeutic regimen.
Acknowledgments
This work was supported by a grant from the National Natural Science Foundation of China [No.81670017].
Appendix A. Supplementary data
Supplementary data related to this article can be found at https://
doi.org/10.1016/j.canlet.2018.08.007.
Conflicts of interest
The authors have no competing interests to declare.
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