ABSTRACT
BACKGROUND: Cyclin-dependent kinases (CDKs) control cell cycle progression, RNA transcription and apoptosis, making them attractive targets for anticancer drug development. Unfortunately, CDK inhibitors developed to date have demonstrated variable efficacy. METHODS: We generated drug-resistant cells by continuous low-dose exposure to a model pyrazolo[1,5-a]pyrimidine CDK inhibitor and investigated potential structural alterations for optimal efficacy. RESULTS: We identified induction of the ATP-binding cassette (ABC) transporters, ABCB1 and ABCG2, in resistant cells. Assessment of features involved in the ABC transporter substrate specificity from a compound library revealed high polar surface area (>100 Å(2)) as a key determinant of transporter interaction. We developed ICEC-0782 that preferentially inhibited CDK2, CDK7 and CDK9 in the nanomolar range. The compound inhibited phosphorylation of CDK substrates and downregulated the short-lived proteins, Mcl-1 and cyclin D1. ICEC-0782 induced G2/M arrest and apoptosis. The permeability and cytotoxicity of ICEC-0782 were unaffected by ABC transporter expression. Following daily oral dosing, the compound inhibited growth of human colon HCT-116 and human breast MCF7 tumour xenografts in vivo by 84% and 94%, respectively. CONCLUSION: We identified a promising pyrazolo[1,5-a]pyrimidine compound devoid of ABC transporter interaction, highly suitable for further preclinical and clinical evaluation for the treatment of cancer.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP-Binding Cassette Transporters/metabolism , Cyclin-Dependent Kinases/antagonists & inhibitors , Neoplasm Proteins/metabolism , Protein Kinase Inhibitors/pharmacology , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/genetics , Cell Division/drug effects , Cell Division/genetics , Cell Line, Tumor , Cell Membrane Permeability/drug effects , Cell Membrane Permeability/genetics , Cyclin D1/genetics , Cyclin-Dependent Kinases/metabolism , Down-Regulation/drug effects , Drug Resistance, Neoplasm , Female , G2 Phase/drug effects , G2 Phase/genetics , HCT116 Cells , Humans , MCF-7 Cells , Mice , Mice, Inbred BALB C , Mice, Nude , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Neoplasm Proteins/genetics , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacokinetics , Pyrimidines/pharmacokinetics , Pyrimidines/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Estrogen receptor alpha (ERalpha) is a ligand-inducible transcription factor that acts to regulate gene expression by binding to palindromic DNA sequence, known as the estrogen response element, in promoters of estrogen-regulated genes. In breast cancer ERalpha plays a central role, where estrogen-regulated gene expression leads to tumor initiation, growth and survival. As an approach to silencing estrogen-regulated genes, we have studied the activities of a fusion protein between ERalpha and the promyelocytic leukemia zinc-finger (PLZF) protein, a transcriptional repressor that acts through chromatin remodeling. To do this, we have developed lines from the estrogen-responsive MCF-7 breast cancer cell line in which the expression of the fusion protein PLZF-ERalpha is conditionally regulated by tetracycline and shows that these feature long-term silencing of the expression of several well-characterized estrogen-regulated genes, namely pS2, cathepsin-D and the progesterone receptor. However, the estrogen-regulated growth of these cells is not inhibited unless PLZF-ERalpha expression is induced, an observation that we have confirmed both in vitro and in vivo. Taken together, these results show that PLZF-ERalpha is a potent repressor of estrogen-regulated gene expression and could be useful in distinguishing estrogen-regulated genes required for the growth of breast cancer cells.
