Cisplatin-based chemotherapy has been deployed as the first-line therapy for advanced bladder cancer for over 30 years. However, even after the chemotherapy, the disease specific survival of patients with regional lymph node invasion or distant metastasis is still quite poor. Target therapy, which is designed to selectively inhibit the cellular molecular pathways necessary for cancer viability and progression, has been deployed as a novel therapeutic strategy in a wide range of cancer since 1990s. Unfortunately, though targeting the immune checkpoint could be a promising therapy in a small ratio of advanced BCs, so far no other target therapy has been approved by FDA or evaluated in phase III clinical trial for BC therapy.
The CDK4/6-RB1-E2F pathway, which regulates the transition of cell cycle from G1 phase to S phase, is frequently disrupted in 93% of advanced bladder cancer. Our previous work has characterized the CDK4/6-RB1 pathway as a potential therapy target in BC testing the effects of CDK4/6 inhibitors in vitro and in vivo. In this project, we further studied molecular mechanisms induced by CDK4/6 inhibitor PD-0332991 and analyzed the roles of these molecules in therapy response.
A prerequisite for response to CDK4/6 inhibitors was the expression of RB1, since RB1-negative cells, which were either RB1 mutant or established via RB1 knockdown, were resistant to CDK4/6 inhibition. We also observed that the therapy response of RB1-positive cell lines, T24 and RT112, to PD-0332991 correlated with a synchronous robust reduction in protein level of both total and phosphorylated RB1. The mechanism underlying the down-regulation on RB1 upon CDK4/6 inhibition was characterized next. First, we revealed a transcriptional repression on RB1 upon the treatment by RT-qPCR. Second, we observed that proteasome inhibition partially rescue the down-regulation of RB1, indicating a proteasomal degradation of RB1 upon the treatment. MDM2 was identified as a mediator that promoted the proteasomal degradation of RB1 upon CDK4/6 inhibition. Silencing of MDM2 by siRNA interfered with the degradation on RB1 and the therapy response in a time-dependent manner. Furthermore, recombinant HA-tagged wild type RB1 (RB1-WT), mutant RB1 lacking the C-terminal 42 amino acids (RB1-C42) and mutant RB1 with all CDKs phosphorylation sites mutated (RB1-CDK) were introduced into BC cells to compare their stability in response to PD-0332991. We observed that the de-phosphorylation of RB1 is not a prerequisite for its further degradation. But the MDM2-binding to RB1 is required for its degradation upon CDK4/6 inhibition. Besides, analysis on the functional and biochemical effects of prolonged CDK4/6 inhibition revealed a partial recovery of cell cycle progression and re-phosphorylation of RB1.The effects were also accompanied by a precise differential regulation on protein levels of E2F family.
Overall, this work on molecular mechanisms induced by CDK4/6 inhibition raised a contradiction between two opposite observations. Therapy response correlated with a decrease of RB1, while the expression of RB1 is required for therapy response, indicating that the role of RB1 in CDK4/6 inhibition is complicated and still not thoroughly interpreted. This complexity is also consistent with the fact that RB1 is not a reliable biomarker to predict therapy response in clinical trials. Both transcriptional repression and MDM2-dependent proteasomal degradation are involved in this down-regulation of RB1 upon CDK4/6 inhibition. In addition, a partial adaptive resistance to prolonged treatment with mono-CDK4/6 inhibition is another challenge for therapy.
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Cisplatin-based chemotherapy has been deployed as the first-line therapy for advanced bladder cancer for over 30 years. However, even after the chemotherapy, the disease specific survival of patients with regional lymph node invasion or distant metastasis is still quite poor. Target therapy, which is designed to selectively inhibit the cellular molecular pathways necessary for cancer viability and progression, has been deployed as a novel therapeutic strategy in a wide range of cancer since 1990...
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