ABSTRACT
Developing therapeutics to effectively inhibit the MYC oncoprotein would mark a key advance towards cancer patient care as MYC is deregulated in over 50% of human cancers. MYC deregulation is correlated with aggressive disease and poor patient outcome. Despite strong evidence in mouse models that inhibiting MYC would significantly impact tumour cell growth and patient survival, traditional approaches have not yet yielded the urgently needed therapeutic agents that directly target MYC. MYC functions through its interaction with MAX to regulate gene transcription by binding to E-box DNA response elements of MYC target genes. Here we used a structure-based strategy to design ME47, a small minimalist hybrid protein (MHP) able to disrupt the MAX:E-box interaction/binding and block transcriptional MYC activity. We show that inducing ME47 expression in established tumour xenografts inhibits tumour growth and decreases cellular proliferation. Mechanistically, we show by chromatin immunoprecipitation that ME47 binds to E-box binding sites of MYC target genes. Moreover, ME47 occupancy decreases MYC:DNA interaction at its cognate E-box binding sites. Taken together, ME47 is a prototypic MHP inhibitor that antagonizes tumour cell growth in vitro and in vivo and inhibits the interaction of MYC with DNA E-box elements. These results support ME47's role as a MYC inhibitor and suggest that MHPs provide an alternative therapeutic targeting system that can be used to target transcription factors important in human diseases, including cancer.
Subject(s)
E-Box Elements/genetics , Nucleotide Motifs/genetics , Proto-Oncogene Proteins c-myc/metabolism , Recombinant Fusion Proteins/metabolism , Triple Negative Breast Neoplasms/metabolism , Xenograft Model Antitumor Assays/methods , Animals , Binding, Competitive , Cell Line, Tumor , Chromatin Immunoprecipitation , HEK293 Cells , Humans , Mice, Inbred NOD , Mice, SCID , Peptide Fragments/genetics , Peptide Fragments/metabolism , Promoter Regions, Genetic/genetics , Protein Binding , Proto-Oncogene Proteins c-myc/genetics , Recombinant Fusion Proteins/genetics , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathology , Tumor Burden/geneticsABSTRACT
OBJECTIVE: To analyze the function and mechanism of miR-214 in regulating breast cancer cell proliferation. MATERIALS AND METHODS: MiR-214 expression level was measured by quantitative reverse transcription Polymerase Chain Reaction (qRT-PCR). The protein level of ß-catenin, PCNA and WNT targets (cyclin D1 and c-Myc) was evaluated by Western blot analysis. The effects of miR-214 on cell proliferation and cisplatin sensitivity were assessed by Cell Counting Kit-8 (CCK-8) assay and/or 5-bromo-2'-deoxyuridine (BrdU) assay. The effect of miR-214 on ß-catenin and WNT signaling activity was tested by luciferase reporter assay. RESULTS: MiR-214 was significantly downregulated in breast cancer tissues and was inversely correlated with ß-catenin expression. Forced expression of miR-214 in breast cancer cell line MCF-7 led to a decrease in cell proliferation and an increase in cisplatin sensitivity. Moreover, forced expression of miR-214 decreased the activity of WNT luciferase reporter and the luciferase reporter containing the 3'-untranslated region (3'UTR) of ß-catenin, whereas antisense inhibitor of miR-214 showed an opposite effect. Finally, miR-214 decreased the expression of ß-catenin and multiple WNT target genes. CONCLUSIONS: MiR-214 is downregulated and serves as a novel tumor suppressor in breast cancer. Forced expression of miR-214 in breast cancer cells diminished cancer cell survival possibly through inhibiting WNT signaling by direct repression of ß-catenin.