Department of Urology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
Department of Urology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
Urol Oncol. 2023 May;41(5):253.e11-253.e20. doi: 10.1016/j.urolonc.2023.01.010. Epub 2023 Feb 21.
CDK4/6 inhibitors have proven their potency for the treatment of cancer but only in combination with hormone or targeted therapies. The aim of this study was the identification of molecules that are involved in response mechanisms to CDK4/6 inhibitors and the development of novel combination therapies with corresponding inhibitors in bladder cancer. Genes of response to therapy and genes that confer resistance to the CDK4/6 inhibitor palbociclib were identified by performing an analysis of published literature and own published data using a CRISPR-dCas9 genome wide gain of function screen. Genes that were down-regulated upon treatment were compared with genes that confer resistance when up-regulated. Two of the top 5 genes were validated by quantitative PCR and western blotting upon treatment with palbociclib in the bladder cancer cell lines T24, RT112 and UMUC3. As inhibitors for combination therapy, we used ciprofloxacin, paprotrain, ispinesib and SR31527. Analysis of synergy was done using the "zero interaction potency" model. Cell growth was examined using sulforhodamine B staining. A list of genes that met the requirements for inclusion in the study was generated from 7 publications. Of the 5 most relevant genes, MCM6 and KIFC1 were chosen and their down-regulation upon treatment with palbociclib was confirmed by qPCR and immunoblotting. The combination of inhibitors against both, KIFC1 and MCM6 with PD resulted in a synergistic inhibition of cell growth. We have identified 2 molecular targets whose inhibition has promising potential for effective combination therapies with the CDK4/6 inhibitor palbociclib.
CDK4/6 抑制剂已被证明在癌症治疗方面具有强大的功效，但仅与激素或靶向治疗联合使用。本研究旨在鉴定参与 CDK4/6 抑制剂反应机制的分子，并开发针对膀胱癌的新型联合治疗方法和相应的抑制剂。通过对已发表文献和使用 CRISPR-dCas9 全基因组功能获得筛选的自有发表数据进行分析，确定了对治疗有反应的基因和对 CDK4/6 抑制剂 palbociclib 产生耐药性的基因。将治疗后下调的基因与上调时产生耐药性的基因进行比较。在膀胱癌细胞系 T24、RT112 和 UMUC3 中用 palbociclib 处理后，通过定量 PCR 和 Western blot 验证了前 5 个基因中的 2 个。作为联合治疗的抑制剂，我们使用环丙沙星、paprotrain、ispinesib 和 SR31527。使用“零相互作用效价”模型分析协同作用。通过磺酰罗丹明 B 染色法检测细胞生长。从 7 篇出版物中生成了一份符合纳入研究要求的基因列表。在 5 个最相关的基因中，选择了 MCM6 和 KIFC1，并通过 qPCR 和免疫印迹证实了它们在用 palbociclib 处理后下调。针对 KIFC1 和 MCM6 的抑制剂与 PD 的联合使用导致细胞生长的协同抑制。我们已经确定了 2 个分子靶标，它们的抑制对 CDK4/6 抑制剂 palbociclib 的有效联合治疗具有很大的潜力。
