ABSTRACT
c-Myc is a proto-oncogene controlling expression of multiple genes involved in cell growth and differentiation. Although the functional role of c-Myc as a transcriptional regulator has been intensively studied, targeting this protein in cancer remains a challenge. Here, we report a trimodal regulation of c-Myc function by the Ras effector, Ras-association domain family member 7 (RASSF7), a nonenzymatic protein modulating protein-protein interactions to regulate cell proliferation. Using HEK293T and HeLa cell lines, we provide evidence that RASSF7 destabilizes the c-Myc protein by promoting Cullin4B-mediated polyubiquitination and degradation. Furthermore, RASSF7 competed with MYC-associated factor X (MAX) in the formation of a heterodimeric complex with c-Myc and attenuated its occupancy on target gene promoters to regulate transcription. Consequently, RASSF7 inhibited c-Myc-mediated oncogenic transformation, and an inverse correlation between the expression levels of the RASSF7 and c-Myc genes was evident in human cancers. Furthermore, we found that RASSF7 interacts with c-Myc via its RA and leucine zipper (LZ) domains and LZ domain peptide is sufficient to inhibit c-Myc function, suggesting that this peptide might be used to target oncogenic c-Myc. These results unveil that RASSF7 and c-Myc are functionally linked in the control of tumorigenesis and open up potential therapeutic avenues for targeting the "undruggable" c-Myc protein in a subset of human cancers.
Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Cell Transformation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic , Proto-Oncogene Proteins c-myc/genetics , Transcription Factors/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/chemistry , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Binding Sites , Binding, Competitive , Cell Line , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Cullin Proteins/genetics , Cullin Proteins/metabolism , HCT116 Cells , HEK293 Cells , Humans , Models, Molecular , Polyubiquitin/genetics , Polyubiquitin/metabolism , Promoter Regions, Genetic , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Proteolysis , Proto-Oncogene Mas , Proto-Oncogene Proteins c-myc/chemistry , Proto-Oncogene Proteins c-myc/metabolism , Signal Transduction , Transcription Factors/chemistry , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
RAS proteins are major human oncogenes, and most of the studies are focused on enzymatic RAS effectors. Recently, nonenzymatic RAS effectors (RASSF, RAS association domain family) have garnered special attention because of their tumor-suppressive properties in contrast to the oncogenic potential of the classical enzymatic RAS effectors. Whereas most members of RASSF family are deregulated by promoter hypermethylation, RASSF8 promoter remains unmethylated in many cancers but the mechanism(s) of its down-regulation remains unknown. Here, we unveil E4BP4 as a critical transcriptional modulator repressing RASSF8 expression through histone methyltransferases, G9a and SUV39H1. In line with these observations, we noticed a negative correlation of RASSF8 and E4BP4 expression in primary breast tumor samples. In addition, our data provide evidence that E4BP4 attenuates RASSF8-mediated anti-proliferation and apoptosis, shedding mechanistic insights into RASSF8 down-regulation in breast cancers. Collectively, our study provides a better understanding on the epigenetic regulation of RASSF8 function and implicates the development of better treatment strategies.
Subject(s)
Basic-Leucine Zipper Transcription Factors/metabolism , Breast Neoplasms/metabolism , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Histocompatibility Antigens/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Methyltransferases/metabolism , Neoplasm Proteins/metabolism , Repressor Proteins/metabolism , Tumor Suppressor Proteins/biosynthesis , Basic-Leucine Zipper Transcription Factors/genetics , Breast Neoplasms/genetics , Female , HEK293 Cells , Histocompatibility Antigens/genetics , Histone-Lysine N-Methyltransferase/genetics , Humans , MCF-7 Cells , Methyltransferases/genetics , Neoplasm Proteins/genetics , Repressor Proteins/genetics , Tumor Suppressor Proteins/geneticsABSTRACT
Nucleolar GTP-binding protein (NGP-1) is overexpressed in various cancers and proliferating cells, but the functional significance remains unknown. In this study, we show that NGP-1 promotes G1 to S phase transition of cells by enhancing CDK inhibitor p21(Cip-1/Waf1) expression through p53. In addition, our results suggest that activation of the cyclin D1-CDK4 complex by NGP-1 via maintaining the stoichiometry between cyclin D1-CDK4 complex and p21 resulted in hyperphosphorylation of retinoblastoma protein at serine 780 (p-RB(Ser-780)) followed by the up-regulation of E2F1 target genes required to promote G1 to S phase transition. Furthermore, our data suggest that ribosomal protein RPL23A interacts with NGP-1 and abolishes NGP-1-induced p53 activity by enhancing Mdm2-mediated p53 polyubiquitination. Finally, reduction of p-RB(Ser-780) levels and E2F1 target gene expression upon ectopic expression of RPL23a resulted in arrest at the G1 phase of the cell cycle. Collectively, this investigation provides evidence that NGP-1 promotes cell cycle progression through the activation of the p53/p21(Cip-1/Waf1) pathway.
