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The MYC oncogene is not only deregulated in cancer through abnormally high levels of expression, but also through oncogenic lesions in upstream signalling cascades. Modelling MYC deregulation using signalling mutants is a productive research strategy. For example, the MYC threonine-58 to alanine substitution mutant (T58A) within MYC-homology box 1 is more transforming than wild-type (WT) MYC, because of decreased apoptosis and increased protein stability. Understanding the regulatory mechanisms controlling T58 phosphorylation has led to new approaches for the development of MYC inhibitors. In this manuscript, we have extensively characterized a MYC signalling mutant in which six lysine residues near the highly conserved MYC homology box IV and basic region have been substituted to arginines (6KR). Previous literature suggests these lysines can undergo both ubiquitylation and acetylation. We show MYC 6KR is able to fully rescue the slow growth phenotype of HO15.19 MYC-null fibroblasts, and promote cell cycle entry of serum-starved MCF10A cells. Remarkably, 6KR increased anchorage-independent colony growth compared with WT MYC in both SH-EP and MCF10A cells. Moreover, it was also more potent in promoting xenograft tumour growth of Rat1A and SH-EP cells. Combined, our data identify this region and these six lysines as important residues for the negative regulation of MYC-induced transformation. Mechanistically, we demonstrate that, unlike T58A, the increased transformation is not a result of increased protein stability or a reduced capacity for 6KR to induce apoptosis. Through expression analysis and luciferase reporter assays, we show that 6KR has increased transcriptional activity compared with WT MYC. Combined, through a comprehensive evaluation across multiple cell types, we identify an important regulatory region within MYC. A better understanding of the full scope of signalling through these residues will provide further insights into the mechanisms contributing to MYC-induced tumorigenesis and may unveil novel therapeutic strategies to target Myc in cancer. [ABSTRACT FROM AUTHOR]

The ability of Myc to promote cellular transformation is well established; however, a better understanding of the mechanisms through which Myc mediates tumorigenesis is essential for the development of therapeutic approaches to target this potent oncoprotein. Structure-function studies in rodent fibroblast cells have provided the basis for much of our current understanding of these mechanisms. To build on these approaches, we have characterized three novel human cell line models of Myc-dependent transformation: MCF10A, SH-EP Tet21/N-Myc, and LF1/TERT/LT/ST cells. We have also evaluated Myc family proteins (c-Myc and L-Myc), a naturally occurring isoform of Myc (MycS), and a set of N-terminal domain mutants (ΔMBII, W135E, T58A) for their ability to promote anchorage-independent growth in these models. Taken together, these results provide the field with three new human cell-based models to study Myc activity, highlight the importance of cellular context, and challenge the paradigm that the ability of Myc to promote tumorigenesis is exclusively MBII-dependent. [ABSTRACT FROM AUTHOR]

c-myc nullizygous fibroblasts (KO cells) were used to compare the abilities of c-myc, N-myc and L-myc oncoproteins to accelerate growth, promote apoptosis, revert morphology, and regulate the expression of previously described c-myc target genes. All three myc oncoproteins were expressed following retroviral transduction of KO cells. The proteins all enhanced the growth rate of KO cells and significantly shortened the cell cycle transition time. They also accelerated apoptosis following serum deprivation, reverted the abnormal KO cell morphology, and modulated the expression of previously described c-myc target genes. In most cases, Lmyc was equivalent to c-myc and N-myc in restoring all of the c-myc-dependent activities. These findings contrast with the previously reported weak transforming and transactivating properties of L-myc. Myc oncoproteins may thus impart both highly similar as well as dissimilar signals to the cells in which they are expressed. [ABSTRACT FROM AUTHOR]

The growth arrest and DNA damage inducible (gadd) genes are induced by various genotoxic and non-genotoxic stresses such as serum starvation, ultraviolet irradiation and treatment with alkylating agents. Their coordinate induction is a growth arrest signal which may play an important role in the response of cells to DNA damage. Conversely, c-myc is a strong proliferative signal, and overexpression of Myc is frequently observed in cancer cells. We have found that ectopic expression of v-myc in RAT-1 cells results in an attenuated induction of the three major gadd transcripts by methyl methanesulfonate (MMS), and almost completely blocks the response to ultraviolet (UV) radiation. Myc acts in part by reducing the stress-responsiveness of the gadd45 promoter, as a c-myc expression vector strongly suppressed activation of gadd45-reporter constructs. This activity of Myc localizes to a recently described GC-rich binding site within the gadd45 promoter. These results indicate that a coordinate down-regulation of the gadd gene response is one mechanism by which Myc can circumvent growth arrest and contribute to the neoplastic phenotype. [ABSTRACT FROM AUTHOR]