{"title":"核糖体 S6 激酶(RSK)通过磷酸化组蛋白赖氨酸去甲基化酶 KDM4B,在 DNA 损伤反应中发挥着关键作用。","authors":"Wenwen Wu, Jing Zhu, Naoe Taira Nihira, Yukiko Togashi, Atsushi Goda, Junki Koike, Kiyoshi Yamaguchi, Yoichi Furukawa, Takuya Tomita, Yasushi Saeki, Yoshikazu Johmura, Makoto Nakanishi, Yasuo Miyoshi, Tomohiko Ohta","doi":"10.1186/s13058-024-01901-x","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Epigenetic dysregulation affecting oncogenic transcription and DNA damage response is a hallmark of cancer. The histone demethylase KDM4B, a factor regulating these processes, plays important roles in estrogen receptor-mediated transcription and DNA repair in breast cancer. However, how oncogenic phospho-signal transduction affects epigenetic regulation is not fully understood. Here we found that KDM4B phosphorylation by ribosomal S6 kinase (RSK), a downstream effector of the Ras/MAPK pathway, is critical for the function of KDM4B in response to DNA damage.</p><p><strong>Methods: </strong>KDM4B-knockout breast cancer cell lines were generated via CRISPR/Cas9-mediated gene editing. Re-expression of wild-type or phospho-site mutated KDM4B in knockout cells was performed by lentivirus-mediated gene transfer. Gene knockdown was achieved by RNA interference. DNA double-strand breaks (DSBs) were induced by ionizing radiation or laser-microirradiation. Protein accumulation at DSB sites was analyzed by immunofluorescence. KDM4B phosphorylation by RSK was assessed by in vitro and in vivo kinase assays. Gene and protein expression levels were analyzed by RT‒PCR and western blotting. The sensitivity of cells to ionizing radiation was examined by a clonogenic survival assay.</p><p><strong>Results: </strong>RSK phosphorylated KDM4B at Ser666, and inhibition of the phosphorylation by RSK depletion or RSK inhibitors abrogated KDM4B accumulation at the sites of DNA double-strand breaks (DSBs). DSB repair was significantly delayed in KDM4B-knockout cells or cells treated with RSK inhibitors. The replacement of endogenous KDM4B with the phosphomimetic mutant S666D restored KDM4B accumulation and DSB repair that had been inhibited by RSK inhibitors, suggesting a critical role for RSK at the specific serine residue of KDM4B in the effect of RSK inhibitors on DSB repair. As a consequence of these aberrant responses, inhibition of KDM4B phosphorylation increased the sensitivity of the cells to ionizing radiation.</p><p><strong>Conclusions: </strong>Overall, the present study uncovered a novel function of RSK on the DNA damage response, which provides an additional role of its inhibitor in cancer therapy.</p>","PeriodicalId":49227,"journal":{"name":"Breast Cancer Research","volume":null,"pages":null},"PeriodicalIF":7.4000,"publicationDate":"2024-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11492477/pdf/","citationCount":"0","resultStr":"{\"title\":\"Ribosomal S6 kinase (RSK) plays a critical role in DNA damage response via the phosphorylation of histone lysine demethylase KDM4B.\",\"authors\":\"Wenwen Wu, Jing Zhu, Naoe Taira Nihira, Yukiko Togashi, Atsushi Goda, Junki Koike, Kiyoshi Yamaguchi, Yoichi Furukawa, Takuya Tomita, Yasushi Saeki, Yoshikazu Johmura, Makoto Nakanishi, Yasuo Miyoshi, Tomohiko Ohta\",\"doi\":\"10.1186/s13058-024-01901-x\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>Epigenetic dysregulation affecting oncogenic transcription and DNA damage response is a hallmark of cancer. The histone demethylase KDM4B, a factor regulating these processes, plays important roles in estrogen receptor-mediated transcription and DNA repair in breast cancer. However, how oncogenic phospho-signal transduction affects epigenetic regulation is not fully understood. Here we found that KDM4B phosphorylation by ribosomal S6 kinase (RSK), a downstream effector of the Ras/MAPK pathway, is critical for the function of KDM4B in response to DNA damage.</p><p><strong>Methods: </strong>KDM4B-knockout breast cancer cell lines were generated via CRISPR/Cas9-mediated gene editing. Re-expression of wild-type or phospho-site mutated KDM4B in knockout cells was performed by lentivirus-mediated gene transfer. Gene knockdown was achieved by RNA interference. DNA double-strand breaks (DSBs) were induced by ionizing radiation or laser-microirradiation. Protein accumulation at DSB sites was analyzed by immunofluorescence. KDM4B phosphorylation by RSK was assessed by in vitro and in vivo kinase assays. Gene and protein expression levels were analyzed by RT‒PCR and western blotting. The sensitivity of cells to ionizing radiation was examined by a clonogenic survival assay.</p><p><strong>Results: </strong>RSK phosphorylated KDM4B at Ser666, and inhibition of the phosphorylation by RSK depletion or RSK inhibitors abrogated KDM4B accumulation at the sites of DNA double-strand breaks (DSBs). DSB repair was significantly delayed in KDM4B-knockout cells or cells treated with RSK inhibitors. The replacement of endogenous KDM4B with the phosphomimetic mutant S666D restored KDM4B accumulation and DSB repair that had been inhibited by RSK inhibitors, suggesting a critical role for RSK at the specific serine residue of KDM4B in the effect of RSK inhibitors on DSB repair. As a consequence of these aberrant responses, inhibition of KDM4B phosphorylation increased the sensitivity of the cells to ionizing radiation.</p><p><strong>Conclusions: </strong>Overall, the present study uncovered a novel function of RSK on the DNA damage response, which provides an additional role of its inhibitor in cancer therapy.</p>\",\"PeriodicalId\":49227,\"journal\":{\"name\":\"Breast Cancer Research\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":7.4000,\"publicationDate\":\"2024-10-21\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11492477/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Breast Cancer Research\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1186/s13058-024-01901-x\",\"RegionNum\":1,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"Medicine\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Breast Cancer Research","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1186/s13058-024-01901-x","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"Medicine","Score":null,"Total":0}
引用次数: 0
摘要
背景:影响致癌转录和 DNA 损伤反应的表观遗传失调是癌症的一个特征。组蛋白去甲基化酶 KDM4B 是调节这些过程的因子,在雌激素受体介导的转录和乳腺癌 DNA 修复中发挥着重要作用。然而,致癌磷酸化信号转导如何影响表观遗传调控还不完全清楚。在这里,我们发现核糖体 S6 激酶(RSK)磷酸化 KDM4B,RSK 是 Ras/MAPK 通路的下游效应器,对于 KDM4B 应对 DNA 损伤的功能至关重要:方法:通过 CRISPR/Cas9 介导的基因编辑生成 KDM4B 基因敲除的乳腺癌细胞系。通过慢病毒介导的基因转移,在基因敲除细胞中重新表达野生型或磷酸化位点突变的 KDM4B。基因敲除是通过 RNA 干扰实现的。电离辐射或激光微照射诱导 DNA 双链断裂(DSB)。通过免疫荧光分析DSB位点的蛋白质积累。通过体外和体内激酶试验评估 RSK 磷酸化 KDM4B 的情况。基因和蛋白质表达水平通过 RT-PCR 和 Western 印迹进行分析。细胞对电离辐射的敏感性通过克隆存活试验进行了检测:结果:RSK 在 Ser666 处使 KDM4B 磷酸化,RSK 缺失或 RSK 抑制剂可抑制 KDM4B 在 DNA 双链断裂(DSB)部位的积累。在KDM4B基因敲除细胞或用RSK抑制剂处理的细胞中,DSB修复明显延迟。用磷酸拟态突变体 S666D 替代内源性 KDM4B 可恢复 KDM4B 的积累和被 RSK 抑制剂抑制的 DSB 修复,这表明 RSK 在 KDM4B 的特定丝氨酸残基上对 RSK 抑制剂对 DSB 修复的影响起着关键作用。由于这些异常反应,KDM4B磷酸化抑制增加了细胞对电离辐射的敏感性:总之,本研究发现了 RSK 在 DNA 损伤反应中的新功能,这为其抑制剂在癌症治疗中提供了新的作用。
Ribosomal S6 kinase (RSK) plays a critical role in DNA damage response via the phosphorylation of histone lysine demethylase KDM4B.
Background: Epigenetic dysregulation affecting oncogenic transcription and DNA damage response is a hallmark of cancer. The histone demethylase KDM4B, a factor regulating these processes, plays important roles in estrogen receptor-mediated transcription and DNA repair in breast cancer. However, how oncogenic phospho-signal transduction affects epigenetic regulation is not fully understood. Here we found that KDM4B phosphorylation by ribosomal S6 kinase (RSK), a downstream effector of the Ras/MAPK pathway, is critical for the function of KDM4B in response to DNA damage.
Methods: KDM4B-knockout breast cancer cell lines were generated via CRISPR/Cas9-mediated gene editing. Re-expression of wild-type or phospho-site mutated KDM4B in knockout cells was performed by lentivirus-mediated gene transfer. Gene knockdown was achieved by RNA interference. DNA double-strand breaks (DSBs) were induced by ionizing radiation or laser-microirradiation. Protein accumulation at DSB sites was analyzed by immunofluorescence. KDM4B phosphorylation by RSK was assessed by in vitro and in vivo kinase assays. Gene and protein expression levels were analyzed by RT‒PCR and western blotting. The sensitivity of cells to ionizing radiation was examined by a clonogenic survival assay.
Results: RSK phosphorylated KDM4B at Ser666, and inhibition of the phosphorylation by RSK depletion or RSK inhibitors abrogated KDM4B accumulation at the sites of DNA double-strand breaks (DSBs). DSB repair was significantly delayed in KDM4B-knockout cells or cells treated with RSK inhibitors. The replacement of endogenous KDM4B with the phosphomimetic mutant S666D restored KDM4B accumulation and DSB repair that had been inhibited by RSK inhibitors, suggesting a critical role for RSK at the specific serine residue of KDM4B in the effect of RSK inhibitors on DSB repair. As a consequence of these aberrant responses, inhibition of KDM4B phosphorylation increased the sensitivity of the cells to ionizing radiation.
Conclusions: Overall, the present study uncovered a novel function of RSK on the DNA damage response, which provides an additional role of its inhibitor in cancer therapy.
期刊介绍:
Breast Cancer Research, an international, peer-reviewed online journal, publishes original research, reviews, editorials, and reports. It features open-access research articles of exceptional interest across all areas of biology and medicine relevant to breast cancer. This includes normal mammary gland biology, with a special emphasis on the genetic, biochemical, and cellular basis of breast cancer. In addition to basic research, the journal covers preclinical, translational, and clinical studies with a biological basis, including Phase I and Phase II trials.