Dronedarone inhibits the proliferation of esophageal squamous cell carcinoma through the CDK4/CDK6-RB1 axis in vitro and in vivo
Bo LiJing ZhangYu YinYinhua LiYingying ChenXiaokun ZhaoAng LiLili ZhaoMingzhu LiZitong WangXuebo LuWenjie WuYueteng ZhangZigang DongKangdong LiuYanan Jiang
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Cyclin-dependent kinase 6
本研究においては,マウス精巣細胞へのクロラムフェニコールアセチルトランスフェラーゼ遺伝子のトランスフェクション効率をin vivoおよびin vitroで行ったリポフェクション法,エクレクトロポーレーション法ならびにパーティクルガン法にっいて比較した.併せて,in vivoエレクトロポーレーションによってマウスの生体精巣における大腸菌βガラクトシダーゼ遺伝子の三次元空間的な発現の獲得を試みた.4週齢のICR系統雄マウスを用いて全部で3回実験を行った.その結果,用いた3種類のトランスフェクション法のうちではin vivoエレクトロポーレーション法とin vivoパーティクルガン法が最も効率が良いことが判明した.in vivoエレクトロポーレーションを用いて大腸菌βガラクトシダーゼ遺伝子を導入したところ,マウス精巣において三次元空間的な明白な遺伝子発現がX-gal染色によって確認され,X-gal染色された細胞の中には精母細胞もしくは精子細胞に類似したものも含まれていた.最大導入可能DNA量,組織の損傷およびトランスフェクション効率を考慮に入れると, in vivoエレクトトポーレーションはマウスの生体精巣細胞への外来遺伝子導入に対して優れている方法であると結論された.
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MicroRNAs (miRNAs) are a large group of negative gene regulators that potentially play a critical role in tumorigenesis. Increasing evidences indicate that miR-145 acts a tumor suppressor in numerous human cancers. However, its role in oral carcinogenesis remains poorly defined. The aim of this study is to determine expression levels of miR-145 in oral squamous cell carcinomas (OSCCs) and normal mucosa tissues, and explore its biological functions in OSCCs.Reverse transcription quantitative real-time PCR (RT-qPCR) assay was used to evaluate expression levels of miR-145. The biological functions of miR-145 were determined by cell proliferation and colony formation, cell cycle and apoptosis, as well as cell invasion assay.MiR-145 was frequently down-regulated in OSCCs compared with normal mucosa tissues. Restoring miR-145 expression in OSCC cells dramatically suppressed cell proliferation and colony formation, and induced G1 phase arrest and cell apoptosis. Importantly, our data showed that miR-145 downregulated the expression of c-Myc and Cdk6, which have previously been identified as two direct targets of miR-145.Our data suggest that miR-145 exerts its tumor suppressor function by targeting c-Myc and Cdk6, leading to the inhibition of OSCC cell growth. MiR-145 rescue may thus be a rational for diagnostic and therapeutic applications in OSCC.
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皮膚血管は冷却刺激に応答して収縮し，体表面からの熱放散を制限する．この反応は，交感神経の興奮を介する全身性メカニズムと，局所的に皮膚血管の収縮反応性が増大する局所性メカニズムとが相乗的に機能して引き起こされる．局所性メカニズムの存在は，摘出血管を用いたin vitro実験によって証明されてきた．我々は，ラットやマウスを使ってin vivoで皮膚血流調節を解析する実験方法を確立した．皮膚循環は，様々な因子に起因する神経性の影響を強く受けるため，in vivoで皮膚血流を定量的に測定することは難しい．そこで，電位依存性Na+チャネル阻害薬であるテトロドトキシン（TTX）処置により神経伝導を遮断した条件下で皮膚血流を測定することを試みた．この条件下においても，ラットおよびマウスの後肢を局所冷却することにより，足底部の皮膚血流量が減少することを証明し，局所性メカニズムによる反応をin vivoで定量的に評価できることを示した．興味深いことに，マウスとラットでは，この反応の主要なメカニズムが異なっていた．すなわち，ラットでは，冷却刺激により血管あるいは周囲の細胞からATPが遊離され，これが交感神経終末のP2受容体に作用することでノルアドレナリン遊離を誘発し，平滑筋細胞の主にα1受容体の活性化を介して皮膚血管が収縮するのに対し，マウスでは，冷却刺激はRhoキナーゼの活性化を介して血管平滑筋のα2C受容体を介した収縮反応性を増大させることで皮膚血管を収縮させることが示唆された．In vivoでの解析は，レイノー病はもちろん，糖尿病やホルモン異常など末梢循環障害を来たす病態と皮膚循環調節との関係を解析する際に不可欠であり，この解析方法はこうした病態の治療薬や予防薬の開発にも役立つであろう．
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Abstract CDK4 and CDK6 kinases are master regulators of cell cycle progression, and they have thus been attractive targets for cancer therapy. CDK4/6 inhibitors (CDK4/6i) showed significant clinical effectiveness in ER+ breast cancers, and three CDK4/6i are now FDA-approved for this indication. However, in several tumor types, CDK4/6i have only been modestly effective, but the underlying mechanism(s) accounting for the discrepancy have remained elusive. To identify the basis of resistance to CDK4/6i, we employed a variety of biochemical, genetic and proteomic approaches, we developed a potent and selective CDK4/6-directed degrader (PROTAC) to investigate in-cell binding of compounds and validated our findings in large tumor and clinical-based data. Our studies revealed that tumor response to CDK4/6i is critically determined by the expression of CDK6. Tumors with low CDK6 expression (CDK6-low) depend on CDK4, and are uniformly sensitive to CDK4/6i. CDK6-low tumors include both entire cancer types (e.g. Ewing Sarcomas, MCL), as well as subgroups of large tumor types, such as portion of non-small cell lung adenocarcinomas (NSCLC). We further validated this finding in NSCLC patients previously treated with CDK4/6i in a clinical trial. In contrast, tumors that express both CDK4 and CDK6 universally depend on CDK6, which is expressed as either CDK4/6i-sensitive CDK6 (CDK6-S) or CDK4/6-resistant CDK6 (CDK6-R) in different cells. Using a CDK4/6-directed PROTAC, we further found that CDK4/6i binds strongly CDK6-S but weakly CDK6-R, indicating different conformations adopted by CDK6 in the two states. An unbiased proteomic analysis identified binding of components of the HSP90/CDC37 complex associated with the state of CDK6: in tumors in which CDK6 is expressed as a thermo-unstable, strong HSP90/CDC37-client, CDK4/6i (and consequently CDK4/6 PROTACs) bind and potently inhibit CDK6 and downstream Rb/E2F signaling. In contrast, tumors resistant to CDK4/6i express CDK6 as a thermostable, weak HSP90/CDC37-client that binds weakly to CDK4/6i. Our data uncover the expression state of CDK6 and its dependence on the HSP90/CDC37 complex as a critical determinant of tumor response to CDK4/6i. We further identify low CDK6 expression as a molecular predictor of tumor sensitivity to current CDK4/6i, indicating a potential biomarker for selection of patients that are more likely to benefit from these drugs. Finally, our findings underline the need for novel inhibitors targeting the thermostable CDK6-R to be used for the treatment of the large number of patients with of Rb-proficient solid tumors that are resistant to current clinical CDK4/6i. Thus, this unexpected dualism in the dependence of CDK6 kinase on HSP90 explains resistance of a large portion of tumors to current CDK4/6i, and provides a roadmap for developing more effective CDK4/6-directed pharmacologic strategies for cancer therapy. Citation Format: Xuewei Wu, Xiaobao Yang, Yan Xiong, Ruitong Li, Takahiro Ito, Tamer Ahmed, Zoi Karoulia, Christos Adamopoulos, Hong Wang, Li Wang, Ling Xie, Beatrix Ueberheide, Stuart Aaronson, Xian Chen, William Sellers, Sean Buchanan, Jian Jin, Poulikos I. Poulikakos. Tumor resistance to CDK4/6 inhibitors and degraders determined by the expression state of CDK6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 41.
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Background: The cell cycle kinase Cdk6 is a major regulator of cell cycle progression from G1 to S phase. Cdk4/6 kinase inhibitors are approved for the treatment of breast cancer and show promising results in pre-clinical studies of other cancers, including hematological malignancies. Cdk6 was also shown to exhibit kinase-independent functions including transcriptional regulation of gene expression. Aims: A systematic understanding of the kinase independent functions of Cdk6 should allow for a better comprehension of the mechanism of action of Cdk4/6 inhibitors. Methods: To model kinase-independent functions of Cdk6 in Acute Lymphoblastic Leukemia (ALL), mouse pre/pro B-cell lines were generated. Whole bone marrow from mice carrying a kinase-dead mutant of Cdk6 (Cdk6-K43M), Cdk6 knock-out (Cdk6-KO) or wildtype Cdk6 (Cdk6-WT) were retrovirally transduced with the BCR-ABL1p185 fusion gene. The resulting cell lines were analyzed in-vivo in transplant settings as well as on the molecular level where RNA-seq and ChIP-seq were performed. Results: Tail vain injection of Cdk6-K43M, Cdk6-KO and Cdk6-WT pre/pro B-cell lines into NSG mice led to the development of lymphoid malignancies albeit with different latencies depending on genotype. Mice injected with Cdk6-KO lines showed a significantly longer survival than mice injected with Cdk6-WT lines. The injection of Cdk6-K43M cells led to an intermediate phenotype with significantly shorter survival than Cdk6-KO, but significantly longer survival than Cdk6-WT. Subcutaneous injection of the cell lines also allowed for the formation of tumors, where tumor weight was largest in mice injected with Cdk6-WT cells, intermediate in the case of Cdk6-K43M cells and lowest in the case of Cdk6-KO cells. Therefore, while kinase-dead Cdk6 had reduced tumorigenic potential compared to Cdk6-WT, loss of kinase function alone exhibited stronger tumorigenic potential than the complete Cdk6 KO. Differential gene expression analysis between cell lines of the three genotypes revealed common alterations associated with Cdk6-K43M and Cdk6-KO compared to Cdk6-WT. Some of these changes could directly be attributed to the loss of kinase function, including for example the downregulation of E2F target genes. Additionally, changes specific for either Cdk6-K43M or Cdk6-KO were observed. As Cdk6-K43M retains kinase-independent functionality, which includes transcriptional regulation, we investigated the DNA binding profile of transcriptional complexes containing Cdk6-K43M. ChIP-seq analysis revealed that such complexes bind the DNA at largely similar sites as complexes containing Cdk6-WT. The majority of genes differentially expressed between Cdk6-WT and Cdk6-K43M were associated with transcriptional complexes harboring Cdk6. Summary/Conclusion: Pre/pro B-cell lines carrying kinase-dead Cdk6 (Cdk6-K43M) showed intermediate tumorigenic potential between lines with Cdk6-WT and Cdk6-KO. While Cdk6-K43M is associated with the DNA at similar sites as Cdk6-WT, there are distinct transcriptional changes associated with this mutation. The further elucidation of these alterations is expected to provide a better understanding of both, kinase independent functions of Cdk6 as well as the effects of its pharmacological inhibition.
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Cdk4 and cdk6 have long been considered functional homologues, despite the fact that tissue-specific differences are detected in the single cdk4 or cdk6 knockout animals. To explore the role of cdk4 and cdk6 in the same model system, we overexpressed variants of cdk4 and cdk6 in Mv1Lu cells to determine their effect on cell cycle progression. We found that cells that overexpressed cdk4 were able to reenter the cell cycle from a contact arrested quiescent state in a kinase-dependent fashion, consistent with the role of this kinase in G0-G1 phase exit. However, we also found that expression of catalytically inactive variants of cdk6 accelerated G0 release, enabled the cell to overcome TGF-β-mediated growth arrest, proliferate in the absence of cyclin D-associated kinase activity and maintain cdk2 activity, while cells expressing catalytically inactive cdk4 were not. This suggested that cdk6 expression was able to affect cell cycle progression in a kinase-independent manner that was distinct from the actions of cdk4. Cdk4 appears unable to associate with cyclin D in the absence of a required assembly factor, such as p27Kip1, but, by gel filtration chromatography, we detected the presence of a previously unidentified, catalytically inactive cyclin D-cdk6 dimer, which might contribute to the kinase-independent, pro-proliferative activity of cdk6.
Cyclin-dependent kinase 6
Cyclin-dependent kinase 4
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Cyclin D
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