The retinoblastoma (RB) signaling network is frequently altered in advanced bladder cancer (BLCA). We investigated the potential of CDK4/6 as a therapeutic target and determined biomarkers for patient stratification.

Genetic alterations were analyzed using public databases (TCGA, COSMIC and CCLE). Effects of CDK4/6-inhibitors, PD-0332991 or LY2835219, were examined using 10 BLCA cell lines by immunoblotting, cell viability, apoptosis and cell cycle progression. Efficacy of PD-0332991 and cisplatin combination was analyzed using the combination index (CI). Gene expression level was determined by quantitative polymerase chain reaction (qPCR). CMV promoter regulated recombinant RB was used for reconstitution. Three-dimensional xenografts were grown on the chicken chorioallantoic membrane (CAM) and analyzed by measuring tumor weight and immunohistochemical expression of total RB and Ki-67.

PD-0332991 treatment reduced proliferation of RB positive BLCA cell lines and was synergistic in combination with cisplatin. PD-0332991 or LY2835219 treatment reduced phosphorylation, total protein and transcript level of RB. 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 RB negative cells reconstituted with recombinant RB, PD-0332991 affected only phosphorylation and not total RB level. These cells remained resistant to treatment. In three-dimensional RB positive xenografts, treatment resulted in reduced tumor weight and decreased expression of total RB and Ki-67.

We provide preclinical evidence that CDK4/6 inhibition is a potential therapeutic strategy for RB positive BLCA that probably acts by negatively regulating RB transcription.