Up to 20% of patients with biliary tract cancer (BTC) have alterations in DNA damage response (DDR) genes, including homologous recombination (HR) genes. Therefore, the DDR pathway could be a promising target for new drug development in BTC. We aim to investigate the anti-tumor effects using poly(ADP-ribose) polymerase (PARP) and WEE1 inhibitors in BTC.
We used 10 BTC cell lines to evaluate an anti-tumor effect of olaparib (a PARP inhibitor) and AZD1775 (a WEE1 inhibitor) in
In this study, we observed a modest anti-proliferative effect of olaparib. DNA double-strand break (DSB) and apoptosis were increased by olaparib in BTC cells. However, olaparib-induced DNA DSB was repaired through the HR pathway, and G2 arrest was induced to secure the time for repair. As AZD1775 typically regulates the G2/M checkpoint, we combined olaparib with AZD1775 to abrogate G2 arrest. We observed that AZD1775 downregulated p-CDK1, a G2/M cell cycle checkpoint protein, and induced early mitotic entry. AZD1775 also decreased CtIP and RAD51 expression and disrupted HR repair. In xenograft model, olaparib plus AZD1775 treatment reduced tumor growth more potently than did monotherapy with either drug.
This is the first study to suggest that olaparib combined with AZD1775 can induce synergistic anti-tumor effects against BTC. Combination therapy that blocks dual PARP and WEE1 has the potential to be further clinically developed for BTC patients.
The DNA damage response (DDR) pathway is an essential target for cancer treatment, and the effectiveness of DDR-targeted agents has been evaluated in a variety of cancers [
Olaparib disrupts single-strand break (SSB) repair by trapping PARP in DNA; this disruption of SSB repair at the replication fork provokes the conversion of unrepaired SSB to a more genotoxic single-ended double-strand break (seDSB) [
Furthermore, olaparib induces G2 arrest, which allows time to repair DNA damage [
The WEE1 checkpoint kinase has been shown to control both S-phase and G2/M progression by regulating CDK1/2 [
In this study, we aimed to investigate the combination of olaparib and AZD1775 as a new DDR-targeted therapy for BTC. We added AZD1775 to olaparib to regulate CDK1, the key molecule that causes G2 arrest in BTC, and focused on the effects of AZD1775 abrogating the G2 arrest and blocking the HR pathway to enhance the efficacy of olaparib.
Ten human BTC cell lines were used in this study. SNU245, SNU308, SNU478, SNU869, SNU1079, and SNU1196 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). HuCCT-1 and TFK-1 cells were obtained from the RIKEN BioResource Center (Ibaraki, Japan). SNU2670 and SNU2773 cell lines were established from patient-derived cancer cells, as previously described [
Cell viability was measured using MTT assay. First, cells were plated in 96-well plates and incubated overnight at 37°C before the addition of olaparib and AZD1775 alone or in combination. Five days later, 50 μL of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) solution (Sigma-Aldrich, St. Louis, MO) was added to each well and incubated for 4 hours at 37°C. The MTT solution and medium were removed, and 150 μL DMSO was added to each well. The absorbance was measured at 540 nm using a VersaMax Microplate Reader (Molecular Devices, San Jose, CA), and the cell viability was calculated.
Cells were collected after treatment with olaparib, AZD1775, or both for 24 hours and 72 hours. The cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in RIPA buffer containing protease inhibitors for 30 minutes on ice. Proteins were obtained by centrifugation at 13,000 rpm for 20 minutes, and equal amounts were used for western blotting. Primary antibodies against the following proteins were purchased from Cell Signaling Technology (Beverley, MA): PARP (#9532), caspase-3 (#9662), caspase-7 (#9492), WEE1 (#4936), p-cdc2-Tyr15 (#9111), CtIP (#9201), NBS1 (#3002), p-ATM-Ser1981 (#5883), p-Chk2-Thr68 (#2661), ATR (#2790), p-CHK1-Ser345 (#2341), p-CDC25C-Ser216 (#9528), p53 (#9282), p-p53-Ser15 (#9284), p21 (#2947), and cyclin B1 (#4138). GAPDH (#sc-25778) and RAD51 (#sc-398587) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The p-CDK2-Tyr15(#ab-76146) antibody was purchased from Abcam (Cambridge, UK). Anti–γ-H2AX antibody (#05-636) was obtained from Millipore (Billerica, MA). p-ATR-Thr1989 (#GTX-128145) was purchased from GeneTex (Irvine, CA), and secondary antibodies were purchased from Thermo Fisher Scientific (Waltham, MA).
Treated cells were trypsinized, harvested, and fixed with 70% ethanol at 20°C for more than 2 days. Ethanol was removed, and the cells were resuspended in fluorescent activated cell sorting (FACS) buffer and incubated with 7 μL of RNase A (Invitrogen) (20 mg/mL) for 10 minutes at 37°C. Samples were stained with 13 μL of propidium iodide (PI; Sigma-Aldrich) and analyzed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).
Cells were seeded on a 6-well plate and treated with olaparib, AZD1775, or both. Cells were cultured at 37°C in a 5% CO2 incubator for 10 days. The colonies were rinsed with PBS and stained with Coomassie blue for 3 hours. The stained colonies were counted using the CellCounter plugins.
Cells were seeded in 60-mm dishes and treated with olaparib, AZD1775, or both. Treated cells were harvested with trypsin and resuspended in binding buffer (#556547, BD Biosciences). The apoptosis assay was performed by double staining with 1 μL Annexin V-FITC and 2 μL PI (#556547, BD Biosciences). and incubated for 15 minutes at RT in the dark. The cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences). Cells in early and late apoptosis were defined as Annexin V-FITC-positive/PI-negative and Annexin V-FITC-positive/PI-positive cells.
Treated cells were harvested with trypsin and fixed with 70% ethanol for at least 4 hours at 20°C. After washing with staining buffer (#420201, BioLegend, San Diego, CA), 20 μL of p-HH3 antibody (#558217, BD Bioscience), and 80 μL of staining buffer were added to each sample for 20 minutes at room temperature. Cells were washed with staining buffer and incubated with 200 μL of staining buffer and 2 μL of RNase A for 10 minutes at 37°C. Next, 5 μL of PI was added, and the cells were analyzed using a FACSCalibur flow cytometer. Each experiment was repeated three times.
For RAD51 and γ-H2AX foci analysis, cells were seeded on a confocal dish, incubated for 48 hours, and then treated for 24 hours. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. Cells were incubated with a rabbit monoclonal RAD51 (#sc-398587) antibody and mouse monoclonal γ-H2AX (#05-636) antibodies overnight at 4°C. Samples were stained with fluorochrome-conjugated secondary antibody, Alexa Fluor 488 anti-rabbit (#2147635) and Alexa Fluor 594 anti-mouse (#2179228) (Invitrogen, Carlsbad, CA), and counterstained with DAPI (Sigma-Aldrich). Immunofluorescence was visualized using a Zeiss LSM 800 laser scanning microscope at 40× magnification.
Treated cells were harvested, resuspended at 5×105 cells/mL in ice-cold PBS, and combined with molten LMAgarose at a ratio of 1:10. Next, the samples were moved onto comet slides and incubated at 4°C in the dark for 40 minutes. After dipping the slides in pre-cooled lysis solution at 4°C for 40 minutes, the slides were gently moved in freshly prepared alkaline unwinding solution (200 mM NaOH, 1 mM EDTA, pH > 13) for 30 minutes at room temperature in the dark. Electrophoresis was conducted for 30 minutes, and the samples were dried at room temperature overnight. Next, 100 μL of diluted SYBR Green staining solution was dropped onto each circle of agarose, and the samples were covered with a coverslip. The tail moment and intensity were measured using the Comet Assay IV program (Andor Technology, Belfast, UK). Three independent experiments were performed for each condition.
After seeding cells in 100-mm dishes and incubated at 37°C with normal medium. SiRNA specific for
Four-week-old female athymic nude mice were purchased from Orient Bio Inc. (Seongnam, Korea). SNU869 cells (1×107 cells) were subcutaneously injected into each mouse. When the tumor volume reached 250 mm3, the mice were randomly divided into four groups of five mice each. Olaparib (50 mg/kg) and AZD1775 (25 mg/kg) were administered orally once a day for 4 weeks (5 days on/2 days off), and the control group was treated with vehicle (0.5% methyl cellulose, Sigma-Aldrich) via oral gavage. Body weight and tumor size were measured every other day. The tumor volume was calculated using the following formula: tumor volume=[(width)2×height]/2.
The isolated xenograft tumor tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The paraffin blocks were sectioned on glass slides, and the slides were deparaffinized and dehydrated. The samples were detected by immunohistochemical staining with p-CDK1-Y15 (AP0016) antibody, and proliferation was evaluated using an anti-Ki67 antibody (GeneTex Inc.). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay-based ApopTag Plus Peroxidase
Statistical analyses were conducted using SigmaPlot ver. 10.0 (Systat Software Inc., San Jose, CA). Experimental data are presented as the mean±standard error. All statistical tests were two-sided. Differences were considered statistically significant at p < 0.05. The half-maximal inhibitory concentration (IC50) of the agents was also determined using the SigmaPlot software. The effects of combined drug were analyzed by calculating the combination index (CI) using CalcuSyn software (Biosoft, Cambridge, UK). CIs of < 1, 1, and > 1 to indicate synergistic, additive, and antagonistic effects, respectively.
To assess the anti-proliferative effects of olaparib in BTC cell lines, the MTT assay was performed after treatment with olaparib for 120 hours. The anti-proliferative effect of olaparib in 10 BTC cells was modest. The IC50 in four cell lines (SNU869, HuCCT-1, SNU2773, and SNU478) was between 2.96–6.49 μM, while that of the other six was higher than 10 μM (
Two cell lines were selected for further experiments based on the IC50 of olaparib in the MTT assay. SNU869 showed the lowest IC50, and SNU308 showed an IC50 higher than 10 μM. We observed that olaparib-induced DNA damage and apoptosis by western blotting. The expression of γ-H2AX, cleavage of PARP, cleavage of caspase-7, and cleavage of caspase-3 were induced in both cell lines (
We also observed an increase in G2/M phases in two BTC cell lines in a dose-dependent manner (
We observed that olaparib-induced G2 arrest, allowing cancer cells the time to repair DNA damage (
In the annexin V and cell cycle analyses, the annexin V-positive cells and the sub-G1 proportions were higher in the combination therapy than in either monotherapy (
As olaparib-treated cells induced G2 arrest, we hypothesized that AZD1775 might increase the sensitivity of olaparib by inducing early mitotic entry of cancer cells. We therefore performed a phospho-histone H3 staining assay to detect the increase in mitotic cells for 6 hours and 24 hours. The proportion of p-HH3-positive cells decreased at 6 hours after treated with olaparib in G2/M phase, but, when co-treated with AZD1775, cells undergo mitosis (
Combination treatment with olaparib and AZD1775 increased p-ATR, p-CHK1, p-p53, and p21, but WEE1, p-CDK1, p-CDK2, and p-CDC25C expression decreased due to AZD1775 (
These data showed that the inhibition of WEE1 overrode olaparib-induced G2 arrest and forced olaparib-treated cells to enter mitosis.
An immunofluorescence assay was performed to investigate the HR-disrupting effects of AZD1775. The number of γ-H2AX foci formation was more significantly increased by combination treatment with olaparib and AZD1775 than by monotherapy with either drug. On the other hand, olaparib-induced RAD51 foci formation was decreased by the combination treatment with AZD1775 (
Additionally, we were transfected the cells with siWEE1 to test whether WEE1 inhibition could also have the same effect of AZD1775. Transfection with siWEE1 showed effective knockdown of WEE1 expression, and reduced the expression of p-CDK1, RAD51, and CtIP proteins (
To determine whether olaparib plus AZD1775 could exert an anti-tumor effect
In this study, we’ve found that olaparib combined with AZD1775 can induce synergistic anti-tumor effects against BTC. Olaparib monotherapy had a modest anti-proliferative effect in 10 BTC cell lines. After olaparib treatment, DNA damage and apoptosis were increased; however, G2 arrest was simultaneously induced, enabling for DNA damage repair. Previous studies have shown that olaparib induces G2 arrest through the p53-p21-CDK1 axis [
It is known that in response to DNA damage, the G2 checkpoint is regulated by two main mechanisms: p53-p21-CDK1 or CHK1/2-CDC25C-CDK1 [
Olaparib induces DNA DSB; however, damaged DNA was mostly repaired by the HR pathway [
Additionally, CtIP initiates HR repair by interacting with the MRE11-RAD50-NBS1 complex and by promoting DNA end resection [
Even though BTCs harbor DDR alterations in significant amount of cases, DDR-targeted strategies for new drug development have not been much explored so far in BTC. We’ve pre-clinically tested the ATR inhibitor monotherapy, ATR inhibitor in combination with cisplatin, ATR inhibitor in combination with WEE1 inhibitor, PARP inhibitor in combination with ATR inhibitor, etc. in BTC [
This study is the first to give preclinical evidence that dual-inhibition of PARP and WEE1 has a synergistic anti-tumor effect in BTC. This combination strategy of PARP inhibitor with WEE1 inhibitor could have the potential to be further developed clinically in patients with advanced BTC.
Animal tests were performed at the Institute for Experimental Animals, College of Medicine, Seoul National University (Seoul, Korea) according to institutional guidelines, with prior approval from the Institutional Animal Care and Use Committee.
Conceived and designed the analysis: Seo HR, Oh DY.
Collected the data: Seo HR.
Contributed data or analysis tools: Seo HR, Nam AR, Bang JH, Oh KS, Kim JM.
Performed the analysis: Seo HR, Nam AR, Bang JH, Oh KS, Kim JM, Yoon JS, Kim TY, Oh DY.
Wrote the paper: Seo HR, Oh DY.
Oh DY received a research grant from AstraZeneca and served as a consultant/advisor to AstraZeneca. No conflicts of interest are disclosed by the other authors.
This research was supported by the Research Fund from the SNUH (grant No. 03-2019-0220), Institute of Smart Healthcare Innovative Medical Sciences, a Brain Korea 21 four program, Seoul National University, and the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2021R1A2C2007430). We would like to thank Editage for English language editing.
Olaparib-induced DNA damage and simultaneously activated G2/M checkpoint. (A) MTT assay was performed in 10 biliary tract cancer cell lines for 5 days after treatment with olaparib (0, 0.01, 0.1, 1, and 10 μM). The half-maximal inhibitory concentration (IC50) of olaparib was calculated using SigmaPlot. Error bars represent mean±standar deviation (SD). (B) The proteins that indicated the DNA damage and apoptosis were analyzed by western blotting after treatment with olaparib (0, 1, and 10 μM) for 72 hours. (C) Cell cycle stages were determined by flow cytometry after 72-hour treatment with olaparib (0, 1, and 10 μM). Error bars represent mean±SD. *p < 0.05, **p < 0.01, ***p < 0.001. (D) The levels of cell cycle proteins and homologous recombination proteins were assessed by western blotting of cells treated with 0, 1, and 10 μM olaparib for 24 hours.
Combination therapy with olaparib and AZD1775 induced more potent DNA damage and apoptosis. (A) Combination effect was investigated by MTT for 5 days before treatment with olaparib (0, 0.01, 0.1, 1, and 10 μM) and AZD1775 (0, 0.001, 0.01, 0.1, and 1 μM), dose ratio was 10:1. CI value was analyzed by CalcuSyn software. If the value is lower than 1 is synergistic, and the cell lines that higher than 1 exhibit an antagonistic effect. (B) Colony-forming analysis was performed with olaparib (0, 1, 5 μM), AZD1775 (0, 0.01, 0.05, 0.1 μM) or both for 10 days. Error bars represent mean±standard deviation (SD) (n=3). (C) Comet analysis was conducted after treatment with olaparib (5 μM), AZD1775 (0.2 μM) or both for 72 hours. The tail intensity and tail moment were analyzed using the Comet assay IV program. The experiment was repeated three times. Data are expressed as mean±standrad error of mean (n=100). (D) Apoptosis analysis was performed using annexin V/propidium iodide (PI) double staining after 24-hour and 72-hour treatment with olaparib (5 μM), AZD1775 (0.1 μM) or both. Error bars represent mean±SD (n=4). (E) Cell cycle analysis were performed with PI-stained BTC cells using flow cytometry after 72-hour treatment with olaparib (5 μM), AZD1775 (0.2 μM) or both. Error bars represent mean±SD (n=3). (F) Western blotting of two cell lines was performed after 72-hour incubation with olaparib, AZD1775, or both. *p < 0.05, **p < 0.01, ***p < 0.001.
AZD1775 abrogated G2 phase arrest and induced early mitotic entry. (A) The phospho-histone H3 (p-HH3)-positive population was analyzed by flow cytometry after 6-hour and 24-hour treatment with olaparib (5 μM), AZD1775 (0.1 μM), or both. The experiment was repeated four times. #p < 0.05, ##p < 0.01, in G2/M phase. *p < 0.05, in all cell cycle phases. (B) Cell cycle assay was performed after 6-hour and 24-hour treatment with olaparib (5 μM), AZD1775 (0.1 μM), or both (n=3). Error bars represent mean±standard deviation. *p < 0.05. (C) Western blotting of cell cycle arrest-related signaling proteins was performed in biliary tract cancer cells treated with olaparib (5 μM), AZD1775 (0.2 μM), or both for 24 hours.
AZD1775 disrupted homologous recombination (HR) repair and enhanced the effects of olaparib. (A) Immunofluorescence staining of γ-H2AX (red) and RAD51 (green) in SNU869 cells treated with olaparib (5 μM), AZD1775 (0.2 μM), or a combination of olaparib and AZD1775 for 24 hours. Cells with more than five foci were counted; 100 cells were analyzed, with experiments repeated 3 times. A confocal microscope at ×400 magnification was used. Error bars represent mean±standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001. (B) Western blotting was performed after 24-hour treatment to show decrease in HR-related proteins. (C) Cells were transfected with WEE1-specific siRNAs (50 nM) for 6 hours and cells were used for western blotting. (D) After transfected with siWEE1, cells were treated with olaparib 24 hours, and western blotting was conducted. (E) After transfected with siWEE1, we treated olaparib for 72 hours, and then performed western blotting.
Combination treatment of olaparib with AZD1775 showed Anti-tumor growth effects in a xenograft mouse model. (A) Vehicle, olaparib (50 mg/kg), AZD1775 (25 mg/kg), or both drugs were administered orally once a day for 4 weeks (5 days on/2 days off) to all groups of SNU869 xenograft mice (n=5 per group). Tumor volumes were measured three times weekly. Data are expressed as mean±standard error of mean (SEM). *p < 0.05. (B) Mouse weights (n=5 per group) were measured three times weekly. Data are expressed as mean±SEM. (C) Western blotting was performed for tumor-specific proteins in each group after tumor isolation. (D) SNU869-xenografted tumors were stained with H&E (×200), Ki67 (×200), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL; ×200), and p-CDK1 (×400) for immunohistochemical analysis. Scale bars=200 μm (H&E, Ki67, and TUNEL), 60 μm (p-CDK1). Immunohistochemical staining was conducted with identical blocks per group.