We developed stromal- and epithelial-specific cre-transgenic mice to directly visualize epithelial-mesenchymal transition (EMT) during cancer progression in vivo. Using three different oncogene-driven mouse mammary tumor models and cell-fate mapping strategies, we show in vivo evidence for the existence of EMT in breast cancer and show that myc can specifically elicit this process. Hierarchical cluster analysis of genome-wide loss of heterozygosity reveals that the incidence of EMT in invasive human breast carcinomas is rare, but when it occurs it is associated with the amplification of MYC. These data provide the first direct evidence for EMT in breast cancer and suggest that its development is favored by myc-initiated events.

Conversion of somatic cells to pluripotency by defined factors is a long and complex process that yields embryonic-stem-cell-like cells that vary in their developmental potential. To improve the quality of resulting induced pluripotent stem cells (iPSCs), which is important for potential therapeutic applications, and to address fundamental questions about control of cell identity, molecular mechanisms of the reprogramming process must be understood. Here we discuss recent discoveries regarding the role of reprogramming factors in remodelling the genome, including new insights into the function of MYC, and describe the different phases, markers and emerging models of reprogramming.

The Myc family proteins are thought to be involved in transcription because they have both a carboxy-terminal basic-helix-loop-helix-zipper (bHLH-Z) domain, common to a large class of transcription factors, and an amino-terminal fragment which, for c-Myc, has transactivating function when assayed in chimaeric constructs. In addition, c-, N- and L-Myc proteins heterodimerize, in vitro and in vivo, with the bHLH-Z protein Max. In vitro, Max homodimerizes but preferentially associates with Myc, which homodimerizes poorly. Furthermore Myc-Max heterodimers specifically bind the nucleotide sequence CACGTG with higher affinity than either homodimer alone. The identification of Max and the specific DNA-binding activities of Myc and Max provides an opportunity for directly testing the transcriptional activities of these proteins in mammalian cells. We report here that Myc overexpression activates, whereas Max overexpression represses, transcription of a reporter gene. Max-induced repression is relieved by overexpression of c-Myc. Repression requires the DNA-binding domain of Max, whereas relief of repression requires the dimerization and transcriptional activation activities of Myc. Both effects require Myc-Max-binding sites in the reporter gene.

Mouse erythroleukaemia cells (also called Friend cells) can be isolated from the spleen of certain strains of mice that have been infected with the Friend virus complex. The cells resemble proerythroblasts and, when exposed to dimethyl sulphoxide (DMSO) or a variety of other chemicals, can be induced to undergo a programme of differentiation which closely resembles the final stages of normal erythropoiesis. This includes the cessation of proliferation and large increases in the production of messenger RNA for both alpha- and beta-globin. In addition, DMSO induces a rapid (less than 2 h) decrease in c-myc mRNA levels. The c-myc oncogene is expressed in the majority of proliferating normal cells and altered expression of the gene has been implicated in the genesis of a wide variety of tumours. To study the influence of oncogene activation on differentiation, we have transfected viral-promoter-driven c-myc genes into mouse erythroleukaemia cells. Constitutive c-myc expression was found to block DMSO-induced differentiation.

Aberrant oncogene activation induces cellular senescence, an irreversible growth arrest that acts as a barrier against tumorigenesis. To identify microRNAs (miRNAs) involved in oncogene-induced senescence, we examined the expression of miRNAs in primary human TIG3 fibroblasts after constitutive activation of B-RAF. Among the regulated miRNAs, both miR-34a and miR-146a were strongly induced during senescence. Although members of the miR-34 family are known to be transcriptionally regulated by p53, we find that miR-34a is regulated independently of p53 during oncogene-induced senescence. Instead, upregulation of miR-34a is mediated by the ETS family transcription factor, ELK1. During senescence, miR-34a targets the important proto-oncogene MYC and our data suggest that miR-34a thereby coordinately controls a set of cell cycle regulators. Hence, in addition to its integration in the p53 pathway, we show that alternative cancer-related pathways regulate miR-34a, emphasising its significance as a tumour suppressor.

Pluripotent cells, such as embryonic stem cells, are invaluable tools for research and can potentially serve as a source of cell- and tissue-replacement therapy. Rejection after transplantation of cells and tissue derived from embryonic stem cells is a significant obstacle to their clinical use. Recently, human somatic cells have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Human iPS cells are a potential source of patient-specific pluripotent stem cells that would bypass immune rejection. iPS cells can also be used to study diseases for which there are no adequate human in vitro or animal models. In this protocol, we describe how to establish primary human fibroblasts lines and how to derive iPS cells by retroviral transduction of reprogramming factors. Overall, it takes 2 months to complete reprogramming human primary fibroblasts starting from biopsy.

In mammalian cells, Id proteins coordinate proliferation and differentiation. Id2 is a dominant-negative antagonist of basic helix-loop-helix transcription factors and proteins of the retinoblastoma (Rb) family. Here we show that Id2-Rb double knockout embryos survive to term with minimal or no defects in neurogenesis and haematopoiesis, but they die at birth from severe reduction of muscle tissue. In neuroblastoma, an embryonal tumour derived from the neural crest, Id2 is overexpressed in cells carrying extra copies of the N-myc gene. In these cells, Id2 is in molar excess of the active form of Rb. The overexpression of Id2 results from transcriptional activation by oncoproteins of the Myc family. Cell-cycle progression induced by Myc oncoproteins requires inactivation of Rb by Id2. Thus, a dual connection links Id2 and Rb: during normal cell-cycle, Rb prohibits the action of Id2 on its natural targets, but oncogenic activation of the Myc-Id2 transcriptional pathway overrides the tumour-suppressor function of Rb.

The c-myc proto-oncogene product, Myc, is a transcription factor that binds thousands of genomic loci. Recent work suggested that rather than up- and downregulating selected groups of genes, Myc targets all active promoters and enhancers in the genome (a phenomenon termed 'invasion') and acts as a general amplifier of transcription. However, the available data did not readily discriminate between direct and indirect effects of Myc on RNA biogenesis. We addressed this issue with genome-wide chromatin immunoprecipitation and RNA expression profiles during B-cell lymphomagenesis in mice, in cultured B cells and fibroblasts. Consistent with long-standing observations, we detected general increases in total RNA or messenger RNA copies per cell (hereby termed 'amplification') when comparing actively proliferating cells with control quiescent cells: this was true whether cells were stimulated by mitogens (requiring endogenous Myc for a proliferative response) or by deregulated, oncogenic Myc activity. RNA amplification and promoter/enhancer invasion by Myc were separable phenomena that could occur without one another. Moreover, whether or not associated with RNA amplification, Myc drove the differential expression of distinct subsets of target genes. Hence, although having the potential to interact with all active or poised regulatory elements in the genome, Myc does not directly act as a global transcriptional amplifier. Instead, our results indicate that Myc activates and represses transcription of discrete gene sets, leading to changes in cellular state that can in turn feed back on global RNA production and turnover.

Oncogene-induced replicative stress activates an Atr- and Chk1-dependent response, which has been proposed to be widespread in tumors. We explored whether the presence of replicative stress could be exploited for the selective elimination of cancer cells. To this end, we evaluated the impact of targeting the replicative stress-response on cancer development. In mice (Mus musculus), the reduced levels of Atr found on a mouse model of the Atr-Seckel syndrome completely prevented the development of Myc-induced lymphomas or pancreatic tumors, both of which showed abundant levels of replicative stress. Moreover, Chk1 inhibitors were highly effective in killing Myc-driven lymphomas. By contrast, pancreatic adenocarcinomas initiated by K-Ras(G12V) showed no detectable evidence of replicative stress and were nonresponsive to this therapy. Besides its impact on cancer, Myc overexpression aggravated the phenotypes of Atr-Seckel mice, revealing that oncogenes can modulate the severity of replicative stress-associated diseases.

The Arabidopsis thaliana CBF cold response pathway plays a central role in cold acclimation. It is characterized by rapid cold induction of genes encoding the CBF1-3 transcription factors, followed by expression of the CBF gene regulon, which imparts freezing tolerance. Our goal was to further the understanding of the cis-acting elements and trans-acting factors involved in expression of CBF2. We identified seven conserved DNA motifs (CM), CM1 to 7, that are present in the promoters of CBF2 and another rapidly cold-induced gene encoding a transcription factor, ZAT12. The results presented indicate that in the CBF2 promoter, CM4 and CM6 have negative regulatory activity and that CM2 has both negative and positive activity. A Myc binding site in the CBF2 promoter was also found to have positive regulatory effects. Moreover, our results indicate that members of the calmodulin binding transcription activator (CAMTA) family of transcription factors bind to the CM2 motif, that CAMTA3 is a positive regulator of CBF2 expression, and that double camta1 camta3 mutant plants are impaired in freezing tolerance. These results establish a role for CAMTA proteins in cold acclimation and provide a possible point of integrating low-temperature calcium and calmodulin signaling with cold-regulated gene expression.

Embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of blastocysts, whereas epiblast stem cells (EpiSCs) are derived from the postimplantation epiblast and display a restricted developmental potential. Here we characterize pluripotent states in the nonobese diabetic (NOD) mouse strain, which prior to this study was considered "nonpermissive" for ESC derivation. We find that NOD stem cells can be stabilized by providing constitutive expression of Klf4 or c-Myc or small molecules that can replace these factors during in vitro reprogramming. The NOD ESCs and iPSCs appear to be "metastable," as they acquire an alternative EpiSC-like identity after removal of the exogenous factors, while their reintroduction converts the cells back to ICM-like pluripotency. Our findings suggest that stem cells from different genetic backgrounds can assume distinct states of pluripotency in vitro, the stability of which is regulated by endogenous genetic determinants and can be modified by exogenous factors.

Apoptosis inducing factor (AIF) is a novel apoptotic effector protein that induces chromatin condensation and large-scale ( approximately 50 kbp) DNA fragmentation when added to purified nuclei in vitro. Confocal and electron microscopy reveal that, in normal cells, AIF is strictly confined to mitochondria and thus colocalizes with heat shock protein 60 (hsp60). On induction of apoptosis by staurosporin, c-Myc, etoposide, or ceramide, AIF (but not hsp60) translocates to the nucleus. This suggests that only the outer mitochondrial membrane (which retains AIF in the intermembrane space) but not the inner membrane (which retains hsp60 in the matrix) becomes protein permeable. The mitochondrio-nuclear redistribution of AIF is prevented by a Bcl-2 protein specifically targeted to mitochondrial membranes. The pan-caspase inhibitor Z-VAD. fmk does not prevent the staurosporin-induced translocation of AIF, although it does inhibit oligonucleosomal DNA fragmentation and arrests chromatin condensation at an early stage. ATP depletion is sufficient to cause AIF translocation to the nucleus, and this phenomenon is accelerated by the apoptosis inducer staurosporin. However, in conditions in which both glycolytic and respiratory ATP generation is inhibited, cells fail to manifest any sign of chromatin condensation and advanced DNA fragmentation, thus manifesting a 'necrotic' phenotype. Both in the presence of Z-VAD. fmk and in conditions of ATP depletion, AIF translocation correlates with the appearance of large-scale DNA fragmentation. Altogether, these data are compatible with the hypothesis that AIF is a caspase-independent mitochondrial death effector responsible for partial chromatinolysis.

Approximately 5% to 10% of diffuse large B-cell lymphomas (DLBCLs) harbor an MYC oncogene rearrangement (MYC+). The prognostic significance of MYC+ DLBCL was determined in an unselected population of patients with newly diagnosed DLBCL treated with rituximab in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy (R-CHOP). Using a Vysis break-apart fluorescence in situ hybridization probe, 12 of 135 (8.8%) cases of MYC+ DLBCL were identified that had no defining high-risk features. MYC+ DLBCL was associated with an inferior 5-year progression-free survival (66% vs 31%, P = .006) and overall survival (72% vs 33%, P = .016). Multivariate analysis confirmed the prognostic importance of MYC for both progression-free survival (hazard ratio = 3.28; 95% confidence interval, 1.49-7.21, P = .003) and overall survival (hazard ratio = 2.98; 95% confidence interval, 1.28-6.95, P = .011). Cases of MYC+ DLBCL also had a higher risk of central nervous system relapse (P = .023), independent of other risk factors. The diagnosis of MYC+ DLBCL is likely underappreciated; and given the lack of defining risk factors, fluorescence in situ hybridization for MYC rearrangements should be performed in all patients with DLBCL. In the R-CHOP treatment era, MYC+ DLBCLs have an inferior prognosis. Treatment regimens similar to those used in Burkitt lymphoma may be more appropriate in this patient population and need to be prospectively tested.

Inappropriate transcriptional repression involving histone deacetylases (HDACs) is a prominent cause for the development of leukemia. We now identify faulty expression of a specific mediator of transcriptional repression in a solid tumor. Loss of the adenomatosis polyposis coli (APC) tumor suppressor induces HDAC2 expression depending on the Wnt pathway and c-Myc. Increased HDAC2 expression is found in the majority of human colon cancer explants, as well as in intestinal mucosa and polyps of APC-deficient mice. HDAC2 is required for, and sufficient on its own to prevent, apoptosis of colonic cancer cells. Interference with HDAC2 by valproic acid largely diminishes adenoma formation in APC(min) mice. These findings point toward HDAC2 as a particularly relevant potential target in cancer therapy.

The Ogt gene encodes a glycosyltransferase that links N-acetylglucosamine to serine and threonine residues (O-GlcNAc) on nuclear and cytosolic proteins. Efforts to study a mammalian model of Ogt deficiency have been hindered by the requirement for this X-linked gene in embryonic stem cell viability, necessitating the use of conditional mutagenesis in vivo. We have extended these observations by segregating Ogt mutation to distinct somatic cell types, including neurons, thymocytes, and fibroblasts, the latter by an approach developed for inducible Ogt mutagenesis. We show that Ogt mutation results in the loss of O-GlcNAc and causes T-cell apoptosis, neuronal tau hyperphosphorylation, and fibroblast growth arrest with altered expression of c-Fos, c-Jun, c-Myc, Sp1, and p27. We further segregated the mutant Ogt allele to parental gametes by oocyte- and spermatid-specific Cre-loxP mutagenesis. By this we established an in vivo genetic approach that supports the ontogeny of female heterozygotes bearing mutant X-linked genes required during embryogenesis. Successful production and characterization of such female heterozygotes further indicates that mammalian cells commonly require a functional Ogt allele. We find that O-GlcNAc modulates protein phosphorylation and expression among essential and conserved cell signaling pathways.

A human gene encoding the c-myc purine-binding transcription factor PuF was identified by screening of a cervical carcinoma cell complementary DNA library with a DNA fragment containing PuF binding sites. The 17-kilodalton bacterially produced PuF was shown to have biological activity and properties similar to that of human PuF. DNA sequence analysis of recombinant PuF revealed perfect identity with the human nm23-H2 nucleoside diphosphate kinase gene, a potential negative regulator of cancer metastasis. These results provide a link between nm23 and the c-myc oncogene and suggest that the nm23 protein can function in vitro in the transcriptional regulation of c-myc expression.

The MYC onco-protein is a transcription factor that regulates cell proliferation, metabolism, protein synthesis, mitochondrial function and stem cell renewal. A region on chromosome 8q24 encompassing the MYC locus is amplified in prostate cancer, but this occurs mostly in advanced disease suggesting that MYC alterations occur late in prostate cancer. In contrast, MYC mRNA is elevated in most prostate cancers, even those of relatively low stage and grade (eg Gleason score 6) suggesting that MYC plays a role in initiation. However, since MYC protein levels are tightly regulated, elevated MYC mRNA does not necessarily imply elevated MYC protein. Thus, it is critical to determine whether MYC protein is elevated in human prostate cancer, and if so, at what stage of the disease this elevation occurs. Prior studies of MYC protein localization have been hampered by lack of suitable antibodies and controls. We utilized a new anti-MYC antibody coupled with genetically defined control experiments to localize MYC protein within human tissue microarrays consisting of normal, atrophy, PIN, primary adenocarcinoma, and metastatic adenocarcinoma. Nuclear overexpression of MYC protein occurred frequently in luminal cells of PIN, as well as in most primary carcinomas and metastatic disease. MYC protein did not correlate with gain of 8q24, suggesting alternative mechanisms for MYC overexpression. These results provide evidence that upregulation of nuclear MYC protein expression is a highly prevalent and early change in prostate cancer and suggest that increased nuclear MYC may be a critical oncogenic event driving human prostate cancer initiation and progression.

In mammalian cells, the MYC oncoprotein binds to thousands of promoters. During mitogenic stimulation of primary lymphocytes, MYC promotes an increase in the expression of virtually all genes. In contrast, MYC-driven tumour cells differ from normal cells in the expression of specific sets of up- and downregulated genes that have considerable prognostic value. To understand this discrepancy, we studied the consequences of inducible expression and depletion of MYC in human cells and murine tumour models. Changes in MYC levels activate and repress specific sets of direct target genes that are characteristic of MYC-transformed tumour cells. Three factors account for this specificity. First, the magnitude of response parallels the change in occupancy by MYC at each promoter. Functionally distinct classes of target genes differ in the E-box sequence bound by MYC, suggesting that different cellular responses to physiological and oncogenic MYC levels are controlled by promoter affinity. Second, MYC both positively and negatively affects transcription initiation independent of its effect on transcriptional elongation. Third, complex formation with MIZ1 (also known as ZBTB17) mediates repression of multiple target genes by MYC and the ratio of MYC and MIZ1 bound to each promoter correlates with the direction of response.

Eukaryotic chromosomes are packaged in nuclei by many orders of folding. Little is known about how higher-order chromatin packaging might affect gene expression. SATB1 is a cell-type specific nuclear protein that recruits chromatin-remodeling factors and regulates numerous genes during thymocyte differentiation. Here we show that in thymocyte nuclei, SATB1 has a cage-like 'network' distribution circumscribing heterochromatin and selectively tethers specialized DNA sequences onto its network. This was shown by fluorescence in situ hybridization on wild-type and Satb1-null thymocytes using in vivo SATB1-bound sequences as probes. Many gene loci, including that of Myc and a brain-specific gene, are anchored by the SATB1 network at specific genomic sites, and this phenomenon is precisely correlated with proper regulation of distant genes. Histone-modification analyses across a gene-enriched genomic region of 70 kb showed that acetylation of histone H3 at Lys9 and Lys14 peaks at the SATB1-binding site and extends over a region of roughly 10 kb covering genes regulated by SATB1. By contrast, in Satb1-null thymocytes, this site is marked by methylation at H3 Lys9. We propose SATB1 as a new type of gene regulator with a novel nuclear architecture, providing sites for tissue-specific organization of DNA sequences and regulating region-specific histone modification.

It is widely thought that c-onc genes (or proto-oncogenes)--the cellular progenitors of retroviral transforming genes--are involved in cellular differentiation and/or proliferation. Such ideas originate primarily from the ability of v-onc genes and 'activated' c-onc genes to induce uncontrolled cellular proliferation, and their capacity to arrest or interfere with differentiation processes in some systems. Haematopoietic cell populations provide additional support for these ideas as c-myb RNA is present in cell lines corresponding to immature, but not mature, cell types, and elevated levels have been found in tissues that are active in haematopoiesis. We have now examined the effects of induced differentiation on c-onc gene expression in a murine myeloid leukaemia cell line, WEHI-3B ('D+' subline). Our results show that the expression of c-myb and c-myc, at the level of transcription, decreases only at late stages in the monocytic differentiation of WEHI-3B cells, while expression of c-fos increases markedly. We suggest that c-myb and c-myc do not themselves control myeloid differentiation, but that they function in the maintenance of the proliferative state of myeloid cells. The induction of c-fos may reflect its role in some macrophage-specific functions.

Induced pluripotent stem (iPS) cells have been derived from fibroblast, stomach, and liver cultures at extremely low frequencies by ectopic expression of the transcription factors Oct4, Sox2, c-myc, and Klf4, a process coined direct or in vitro reprogramming [1-8]. iPS cells are molecularly and functionally highly similar to embryonic stem cells (ESCs), including their ability to contribute to all tissues as well as the germline in mice. The heterogeneity of the starting cell populations and the low efficiency of reprogramming suggested that a rare cell type, such as an adult stem cell, might be the cell of origin for iPS cells and that differentiated cells are refractory to reprogramming. Here, we used inducible lentiviruses [9] to express Oct4, Sox2, c-myc, and Klf4 in pancreatic beta cells to assess whether a defined terminally differentiated cell type remains amenable to reprogramming. Genetically marked beta cells gave rise to iPS cells that expressed pluripotency markers, formed teratomas, and contributed to cell types of all germ layers in chimeric animals. Our results provide genetic proof that terminally differentiated cells can be reprogrammed into pluripotent cells, suggesting that in vitro reprogramming is not restricted to certain cell types or differentiation stages.

c-Myc is helix-loop-helix-leucine zipper (HLH-ZIP) oncoprotein that is frequently deregulated in human cancers. In order to bind DNA, regulate target gene expression, and function in a biological context, c-Myc must dimerize with another HLH-ZIP protein, Max. A large number of c-Myc target genes have been identified, and many of the encoded proteins are transforming. Such functional redundancy, however, complicates therapeutic strategies aimed at inhibiting any single target gene product. Given this consideration, we have instead attempted to identify ways by which c-Myc itself could be effectively disabled. We have used a yeast two-hybrid approach to identify low-molecular-weight compounds that inhibit c-Myc-Max association. All of the compounds prevented transactivation by c-Myc-Max heterodimers, inhibited cell cycle progression, and prevented the in vitro growth of fibroblasts in a c-Myc-dependent manner. Several of the compounds also inhibited tumor growth in vivo. These results show that the yeast two-hybrid screen is useful for identifying compounds that can be exploited in mammalian cells. More specifically, they provide a means by which structural analogs, based upon these first-generation Myc-Max inhibitors, can be developed to enhance antitumor efficacy.

The product of the c-myc proto-oncogene is a nuclear phosphoprotein whose normal cellular function has not yet been defined. c-Myc has a number of biochemical properties, however, that suggest that it may function as a potential regulator of gene transcription. Specifically, it is a nuclear DNA-binding protein with a short half-life, a high proline content, segments that are rich in glutamine and acidic residues, and a carboxyl-terminal oligomerization domain containing the leucine zipper and helix-loop-helix motifs that serve as oligomerization domains in known regulators of transcription, such as C/EBP, Jun, Fos, GCN4, MyoD, E12, and E47. In an effort to establish that c-Myc might regulate transcription in vivo, we sought to determine whether regions of the c-Myc protein could activate transcription in an in vitro system. We report here that fusion proteins in which segments of human c-Myc are linked to the DNA-binding domain of the yeast transcriptional activator GAL4 can activate transcription from a reporter gene linked to GAL4-binding sites. Three independent activation regions are located between amino acids 1 and 143, a region that has been shown to be required for neoplastic transformation of primary rat embryo cells in cooperation with a mutated ras gene. These results demonstrate that domains of the c-Myc protein can function to regulate transcription in a model system and suggest that alterations of Myc transcriptional regulatory function may lead to neoplastic transformation.

Germline determinants of gene expression in tumors are infrequently studied due to the complexity of transcript regulation caused by somatically acquired alterations. We performed expression quantitative trait locus (eQTL)-based analyses using the multi-level information provided in The Cancer Genome Atlas (TCGA). Of the factors we measured, cis-acting eQTLs accounted for 1.2% of the total variation of tumor gene expression, while somatic copy-number alteration and CpG methylation accounted for 7.3% and 3.3%, respectively. eQTL analyses of 15 previously reported breast cancer risk loci resulted in the discovery of three variants that are significantly associated with transcript levels (false discovery rate [FDR] < 0.1). Our trans-based analysis identified an additional three risk loci to act through ESR1, MYC, and KLF4. These findings provide a more comprehensive picture of gene expression determinants in breast cancer as well as insights into the underlying biology of breast cancer risk loci.