CDK4/6 Inhibition Controls Proliferation of Bladder Cancer and Transcription of RB1.

Abstract

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.