Sathe Anuja, Koshy Nicole, Schmid Sebastian C, Thalgott Mark, Schwarzenböck Sarah M, Krause Bernd J, Holm Per S, Gschwend Juergen E, Retz Margitta, Nawroth Roman
Department of Urology, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; Department of Nuclear Medicine, Rostock University Medical Centre (SMS, BJK), Rostock, Germany.
Department of Urology, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; Department of Nuclear Medicine, Rostock University Medical Centre (SMS, BJK), Rostock, Germany.
J Urol. 2016 Mar;195(3):771-9. doi: 10.1016/j.juro.2015.08.082. Epub 2015 Aug 28.
The retinoblastoma signaling network is frequently altered in advanced bladder cancer. We investigated the potential of CDK4/6 as a therapeutic target and determined biomarkers for patient stratification.
Genetic alterations were analyzed using public databases, including TCGA (The Cancer Genome Atlas), COSMIC (Catalogue of Somatic Mutations in Cancer) and CCLE (Cancer Cell Line Encyclopedia). Effects of the CDK4/6-inhibitor PD-0332991 or LY2835219 were examined in 10 bladder cancer cell lines by immunoblot, cell viability, apoptosis and cell cycle progression. Efficacy of the PD-0332991 and cisplatin combination was analyzed using the combination index. Gene expression level was determined by quantitative polymerase chain reaction. Cytomegalovirus promoter regulated recombinant retinoblastoma was used for reconstitution. Three-dimensional xenografts were grown on chicken chorioallantoic membrane and analyzed by measuring tumor weight and immunohistochemical expression of total retinoblastoma and Ki-67.
PD-0332991 treatment decreased the proliferation of retinoblastoma positive bladder cancer cell lines and was synergistic in combination with cisplatin. PD-0332991 or LY2835219 treatment decreased the phosphorylation, total protein and transcript level of retinoblastoma. Treatment resulted in a decrease in E2F target gene expression (CCNA2 and CCNE2) and cell cycle progression from G0/G1 to the S-phase but did not affect apoptosis. In retinoblastoma negative cells reconstituted with recombinant retinoblastoma PD-0332991 affected only phosphorylation and not the total retinoblastoma level. These cells remained resistant to treatment. In 3-dimensional retinoblastoma xenografts, treatment resulted in reduced tumor weight and decreased expression of total retinoblastoma and Ki-67.
We provide preclinical evidence that CDK4/6 inhibition is a potential therapeutic strategy for retinoblastoma positive bladder cancer that probably acts by negatively regulating retinoblastoma transcription.
视网膜母细胞瘤信号网络在晚期膀胱癌中常发生改变。我们研究了细胞周期蛋白依赖性激酶4/6（CDK4/6）作为治疗靶点的潜力，并确定了用于患者分层的生物标志物。
使用公共数据库分析基因改变，包括癌症基因组图谱（TCGA）、癌症体细胞突变目录（COSMIC）和癌细胞系百科全书（CCLE）。通过免疫印迹、细胞活力、凋亡和细胞周期进程检测CDK4/6抑制剂PD-0332991或LY2835219对10种膀胱癌细胞系的影响。使用联合指数分析PD-0332991与顺铂联合用药的疗效。通过定量聚合酶链反应测定基因表达水平。使用巨细胞病毒启动子调控的重组视网膜母细胞瘤进行重组。在鸡胚绒毛尿囊膜上培养三维异种移植瘤，并通过测量肿瘤重量以及总视网膜母细胞瘤和Ki-67的免疫组化表达进行分析。
PD-0332991治疗可降低视网膜母细胞瘤阳性膀胱癌细胞系的增殖，并与顺铂联合具有协同作用。PD-0332991或LY2835219治疗可降低视网膜母细胞瘤的磷酸化水平、总蛋白水平和转录水平。治疗导致E2F靶基因表达（CCNA2和CCNE2）降低，细胞周期从G0/G1期进展至S期，但不影响细胞凋亡。在用重组视网膜母细胞瘤重组的视网膜母细胞瘤阴性细胞中，PD-0332991仅影响磷酸化水平，而不影响总视网膜母细胞瘤水平。这些细胞对治疗仍具有抗性。在三维视网膜母细胞瘤异种移植瘤中，治疗导致肿瘤重量减轻，总视网膜母细胞瘤和Ki-67的表达降低。
我们提供了临床前证据，表明抑制CDK4/6是视网膜母细胞瘤阳性膀胱癌的一种潜在治疗策略，其可能通过负向调节视网膜母细胞瘤转录发挥作用。
