Cancer Research Institute, Seoul National University, Seoul, Republic of Korea.
Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea.
Interdisciplinary Programs in Cancer Biology Major, Seoul National University Graduate School, Seoul, Republic of Korea.
Cancer Research Institute, Seoul National University, Seoul, Republic of Korea. kyunghunlee@snu.ac.kr.
Department of Internal Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea. kyunghunlee@snu.ac.kr.
Department of Internal Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea.
Department of Translational Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea.
Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea.
Cancer Research Institute, Seoul National University, Seoul, Republic of Korea. moisa@snu.ac.kr.
Interdisciplinary Programs in Cancer Biology Major, Seoul National University Graduate School, Seoul, Republic of Korea. moisa@snu.ac.kr.
Department of Internal Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea. moisa@snu.ac.kr.
Department of Translational Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea. moisa@snu.ac.kr.
Abstract
BACKGROUND Cyclin-dependent kinase 9 (CDK9) is a crucial regulator of transcriptional progression of RNA polymerase-II (RNAP2). RNA polymerases trapped in DNA can be a source of transcription-replication conflict (T-R conflict), which is a common source of replication stress. AZD4573, a highly selective CDK9 inhibitor, has been shown to induce apoptosis in leukemia cell lines, while its anti-tumor potential in breast cancer has yet to be elucidated.
METHODS To evaluate the cytotoxicity of AZD4573 in vitro, MTT assays were performed. The expression of signal transduction molecules was determined using Western blotting, immunoprecipitation, and immunofluorescence. Apoptotic cell death was verified by the annexin-V assay. DNA strand breaks and repair efficacy were evaluated through the alkaline comet assay. The siRNA knock-down system was used to confirm the action mechanism.
RESULTS AZD4573 induced T-R conflicts during S-phase, increasing replication stress and DNA strand breaks, resulting in apoptosis by induction of caspase-3. Furthermore, we identified Dead-box 25 (DDX25) helicase as a key mediator in resolving the T-R conflicts. Nuclear translocation of DDX25 correlated with reduced sensitivity to AZD4573 by the resolution of T-R conflicts.
CONCLUSIONS Inhibition of CDK9 by AZD4573 induces the accumulation of DNA damage through T-R conflicts. DDX25 helicases were identified as a key mediator in resolving T-R conflicts and the reduced sensitivity to AZD4573.
Breast cancerCyclin dependent kinase 9DNA damageTranscription-replication conflict
Figures
Fig. 1AZD4573 has a cytotoxic effect on breast cancer cells regardless of CDK9 or MCL-1 expression. A The cytotoxic effect of AZD4573 in breast cancer cells was measured using the MTT assay. Results were presented as a graph with standard error bars. B AZD4573 effectively inhibited CDK9 in breast cancer cell lines. Actin was used as a lading control. C Correlation between the IC50 value of AZD4573 and the mRNA expression of CDK9 is not statistically significant. The correlation was measured by the R value in a scatter plot of a linear regression line graph based on points. D CDK9 protein expressions were shown by Western blot. E Significant association between AZD4573 sensitivity and expression levels of MCL1 was not observedFig. 2AZD4573 induces T-R conflicts during replication. A T-R conflicts persisted in sensitive cells, whereas they were rapidly resolved in less-sensitive cells. Representative images from immunofluorescence staining of T-R conflicts in HCC70 cells, sensitive cells to AZD4573, and in T47D cells, less-sensitive cells to AZD4573. Microscopic images of stained cells were acquired at × 40 magnification. The number of S9.6 foci/cells is quantified using Sigmaplot and presented in a bar graph (means ± SE). Experiments were conducted in duplicate, and the results were repeated. At least 100 nuclei were analyzed for each experiment. B AZD4573 leads to DNA damage in replicating cells. γ-H2AX foci formation in S-phase was conducted via EdU-incorporation assay. Microscopic images of stained cells were acquired at × 40 magnification. The number of γ-H2AX foci was quantified by Sigmaplot and presented as a bar graph (means ± SE). Experiments were conducted in duplicate, and the results were repeated. At least 80 EdU-positive cells were analyzed for each experiment. C Decreased S-phase proportions were observed in sensitive cells. Cells were treated with indicated doses of AZD4573 for 24 h. Stacked bar graph represents the distribution of cells in G1, S, and G2/M phases, analyzed exclusively from the live cell population. Columns represent the average of three independent experiments. D Sensitive cells exhibited a reduced S-phase proportion following treatment with 25 nmol/L AZD4573 for 48 h, S-phase progression was examined by BrdU-labeling. BrdU positive cell population is quantified by Sigmaplot and presented in a bar graph (means ± SE). *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001. The scale bars represent 20 μmFig. 3T-R conflicts result in the accumulation of DNA damage leading to caspase-3-dependent apoptosis. A Expression levels of DNA damage response molecules in breast cancer cells were increased in sensitive cells following the increasing doses (0, 15, 25 nmol/L) of AZD4573 treatment for 48 h. Actin was used as a loading control. B Accumulation of DNA damage was observed in breast cancer cells via the comet assay. Cells were incubated with 25 nmol/L of AZD4573 for 48 h. C sub-G1 population was increased in sensitive cells. Population of sub-G1 is quantified by Sigmaplot and presented in a bar graph (means ± SE). D Cells that are undergoing apoptosis were increased in sensitive cells to AZD4573. Representative scatter plots of PI (y-axis) vs. Annexin-V (x-axis). The population of apoptotic cells was presented as the mean ± SD of triplicate experiments. E Increased expression levels of cleaved PARP and cleaved caspase-3 were observed in sensitive cells by Western blot. Cells were treated with increasing doses (0, 15, 25 nmol/L) of AZD4573 for 48 h. F Complete activation of caspase-3 and the increased expression of cleaved PARP were not observed in sensitive cells treated with AZD4573 and Z -VAD FMK Cells were treated 25 nmol/L AZD4573 alone or with 50 μmol/L of Z-VAD, a pan-caspase inhibitor, for 48 hFig. 4Knock-down of CDK9 results in cell growth inhibition, the accumulation of DNA damage, and increased T-R conflicts. A siRNA-mediated reduction of CDK9 expression and increased DNA damage was confirmed in HCC1937 cells. Actin was used as a loading control. B Viability of CDK9 down-regulated HCC1937 cells was measured by MTT assay. Results were presented as means with standard error. C T-R conflicts were induced in the siCDK9 transfected HCC1937 cells. Representative images from immunofluorescence staining of T-R conflicts in siCDK9 transfected HCC1937 cells. The number of S9.6 foci obtained in triplicate is quantified using Sigmaplot and presented in a bar graph (means ± SE). At least 100 nuclei were analyzed for each experiment. *p-value < 0.05Fig. 5DDX25 helicase is involved in T-R conflicts resolution through nuclear translocation. A Significant differences in the expression levels of DDX25 was observed according to the drug sensitivity. B Expression levels of DDX helicases remained unchanged in breast cancer cells. Actin was used as a loading control. C The amount of DDX25 helicase within the nuclear subfraction was elevated in less-sensitive cells to AZD4573. Cells were treated 25 nmol/L of AZD4573 for 3 h. A-tubulin and lamin B1 were used as cytoplasmic and nuclear markers. D DDX25 nuclear translocation was significantly increased in less-sensitive cells to AZD4573. DDX25 helicase foci formation in the nucleus was conducted via immunofluorescence assay. The number of DDX25 helicase foci was quantified by Sigmaplot and presented as a bar graph (means ± SE). Experiments were conducted in duplicate, and results were repeated. At least 30 nuclei were analyzed for each experiment. E DDX25 directly interacts with T-R conflicts. Breast cancer whole extracts were immunoprecipitated with DNA-RNA hybrids (S9.6) antibody or IgG antibody. F siRNA-mediated reduction of DDX25 expression was confirmed by Western blot. G Persistent T-R conflicts were observed in siDDX25-transfected T47D and HCC1428 cells. S9.6 foci formation in nucleus was conducted via immunofluorescence assay. The number of S9.6 foci was quantified by Sigmaplot and presented as a bar graph (means ± SE). *p-value < 0.05, **p-value < 0.05Fig. 6AZD4573 induces T-R conflicts and inhibits tumor growth in xenograft mouse model. Tumors were removed from the mice after the vehicle control or drug treatment ended. A IHC stain for T-R conflicts using S9.6 antibody showed an increase in the AZD4573-treated tumor. Microscopic images of stained cells were acquired at × 40 magnification. B Significant expression of DDX25 was not observed in the AZD4573-treated and control tumors. C Lower proliferation index measured by Ki-67 immunostaining was noted in AZD4573-treated tumors. The scale bars represent 50 μm. Analysis were conducted in duplicate and results were repeated. **p-value < 0.05
그림 출처: PubMed Central 공개 논문 (PMC12297744)
Conflict of interest statement
Declarations. Ethics approval and consent to participate: The animal study was reviewed and approved by the Committee on Institutional Animal Care and Use Committee (IACUC) of the Institute of Biomedical Science, Seoul National University, under the approval number 23–0379-S1A0, and were performed according to relevant guidelines and regulations. The maximal volume of tumor was permitted by their ethics committee was 2000 mm3. All the tumor in this study were below 2000 mm3. Consent for publication: Not applicable. Competing interests: Seock-Ah Im is a recipient of research funds from AstraZeneca Inc., Boryung Pharm, Daiichi Sankyo Co., Ltd, Daewoong Pharm, Pfizer, Roche and has consultant and advisory role for AstraZeneca, Daiichi Sankyo, Eisai, Hanmi Corp, MSD, Novartis, Pfizer, and Roche. Kyung-Hun Lee reports honoraria from AstraZeneca, Boryung Pharm, Daiichi Sankyo, Eli Lilly, Novartis, and Pfizer. Jiwon Koh has advisory role for AstraZeneca outside of this work. The other authors declared that they have no conflict of interest.
