MAD3 AND MAD4 - NOVEL MAX-INTERACTING TRANSCRIPTIONAL REPRESSORS THAT SUPPRESS C-MYC DEPENDENT TRANSFORMATION AND ARE EXPRESSED DURING NEURAL AND EPIDERMAL DIFFERENTIATION

The basic helix-loop-helix-leucine zipper (bHLHZip) protein Max associates with members of the Myc family, as well as with the related proteins Mad (Mad1) and Mxi1, Whereas both Myc:Max and Mad:Max heterodimers bind related E-box sequences, Myc:Max activates transcription and promotes proliferation while Mad:Max represses transcription and suppresses Myc dependent transformation, Here we report the identification and characterization of two novel Mad1-and Mxi1-related proteins, Mad3 and Mad4. Mad3 and Mad4 interact with both Max and mSin3 and repress transcription from a promoter containing CACGTG binding sites, Using a rat embryo fibroblast transformation assay, we show that both Mad3 and Mad4 inhibit c-Myc dependent cell transformation, An examination of the expression patterns of all mad genes during murine embryogenesis reveals that mad1, mad3 and mad4 are expressed primarily in growth-arrested differentiating cells. mxi1 is also expressed in differentiating cells, but is co-expressed with either c-myc, N-myc, or both in proliferating cells of the developing central nervous system and the epidermis, In the developing central nervous system and epidermis, downregulation of myc genes occurs concomitant with upregulation of mad family genes, These expression patterns, together with the demonstrated ability of Mad family proteins to interfere with the proliferation promoting activities of Myc, suggest that the regulated expression of Myc and Mad family proteins function in a concerted fashion to regulate cell growth in differentiating tissues

The MRN complex is transcriptionally regulated by MYCN during neural cell proliferation to control replication stress

The MRE11/RAD50/NBS1 (MRN) complex is a major sensor of DNA double strand breaks, whose role in controlling faithful DNA replication and preventing replication stress is also emerging. Inactivation of the MRN complex invariably leads to developmental and/or degenerative neuronal defects, the pathogenesis of which still remains poorly understood. In particular, NBS1 gene mutations are associated with microcephaly and strongly impaired cerebellar development, both in humans and in the mouse model. These phenotypes strikingly overlap those induced by inactivation of MYCN, an essential promoter of the expansion of neuronal stem and progenitor cells, suggesting that MYCN and the MRN complex might be connected on a unique pathway essential for the safe expansion of neuronal cells. Here, we show that MYCN transcriptionally controls the expression of each component of the MRN complex. By genetic and pharmacological inhibition of the MRN complex in a MYCN overexpression model and in the more physiological context of the Hedgehog-dependent expansion of primary cerebellar granule progenitor cells, we also show that the MRN complex is required for MYCN-dependent proliferation. Indeed, its inhibition resulted in DNA damage, activation of a DNA damage response, and cell death in a MYCN- and replication-dependent manner. Our data indicate the MRN complex is essential to restrain MYCN-induced replication stress during neural cell proliferation and support the hypothesis that replication-born DNA damage is responsible for the neuronal defects associated with MRN dysfunctions.Cell Death and Differentiation advance online publication, 12 June 2015; doi:10.1038/cdd.2015.81

Oesophageal adenocarcinoma is associated with a deregulation in the MYC/MAX/MAD network

Oesophageal adenocarcinoma, which arises from an acquired columnar lesion, Barrett's metaplasia, is rising in incidence more rapidly than any other cancer in the Western world. Elevated expression of c-MYC has been demonstrated in oesophageal adenocarcinoma; however, the expression of other members of the MYC/MAX/MAD network has not been addressed. The aims of this work were to characterise the expression of c-MYC, MAX and the MAD family in adenocarcinoma development and assess the effects of overexpression on cellular behaviour. mRNA expression in samples of Barrett's metaplasia and oesophageal adenocarcinoma were examined by qRT–PCR. Semi-quantitative immunohistochemistry and western blotting were used to examine cellular localisation and protein levels. Cellular proliferation and mRNA expression were determined in SEG1 cells overexpressing c-MYCER or MAD1 using a bromodeoxyuridine assay and qRT–PCR, respectively. Consistent with previous work expression of c-MYC was deregulated in oesophageal adenocarcinoma. Paradoxically, increased expression of putative c-MYC antagonists MAD1 and MXI1 was observed in tumour specimens. Overexpression of c-MYC and MAD proteins in SEG1 cells resulted in differential expression of MYC/MAX/MAD network members and reciprocal changes in proliferation. In conclusion, the expression patterns of c-MYC, MAX and the MAD family were shown to be deregulated in the oesophageal cancer model

The opposing transcriptional functions of Sin3a and c-Myc are required to maintain tissue homeostasis.

How the proto-oncogene c-Myc balances the processes of stem-cell self-renewal, proliferation and differentiation in adult tissues is largely unknown. We explored c-Myc's transcriptional roles at the epidermal differentiation complex, a locus essential for skin maturation. Binding of c-Myc can simultaneously recruit (Klf4, Ovol-1) and displace (Cebpa, Mxi1 and Sin3a) specific sets of differentiation-specific transcriptional regulators to epidermal differentiation complex genes. We found that Sin3a causes deacetylation of c-Myc protein to directly repress c-Myc activity. In the absence of Sin3a, genomic recruitment of c-Myc to the epidermal differentiation complex is enhanced, and re-activation of c-Myc-target genes drives aberrant epidermal proliferation and differentiation. Simultaneous deletion of c-Myc and Sin3a reverts the skin phenotype to normal. Our results identify how the balance of two transcriptional key regulators can maintain tissue homeostasis through a negative feedback loop

Upstream ORF affects MYCN translation depending on exon 1b alternative splicing

<p>Abstract</p> <p>Background</p> <p>The <it>MYCN </it>gene is transcribed into two major mRNAs: one full-length (<it>MYCN) </it>and one exon 1b-spliced (<it>MYCN</it>^{Δ1<it>b</it>}) mRNA. But nothing is known about their respective ability to translate the MYCN protein.</p> <p>Methods</p> <p>Plasmids were prepared to enable translation from the upstream (uORF) and major ORF of the two <it>MYCN </it>transcripts. Translation was studied after transfection in neuroblastoma SH-EP cell line. Impact of the upstream AUG on translation was evaluated after directed mutagenesis. Functional study with the two <it>MYCN </it>mRNAs was conducted by a cell viability assay. Existence of a new protein encoded by the <it>MYCN</it>^{Δ1<it>b </it>}uORF was explored by designing a rabbit polyclonal antibody against a specific epitope of this protein.</p> <p>Results</p> <p>Both are translated, but higher levels of protein were seen with <it>MYCN</it>^{Δ1<it>b </it>}mRNA. An upstream ORF was shown to have positive cis-regulatory activity on translation from <it>MYCN </it>but not from <it>MYCN</it>^{Δ1<it>b </it>}mRNA. In transfected SH-EP neuroblastoma cells, high MYCN dosage obtained with <it>MYCN</it>^{Δ1<it>b </it>}mRNA translation induces an antiapoptotic effect after serum deprivation that was not observed with low MYCN expression obtained with <it>MYCN </it>mRNA. Here, we showed that MYCNOT: <it>MYCN </it>Overlap Transcript, a new protein of unknown function is translated from the upstream AUG of <it>MYCN</it>^{Δ1<it>b </it>}mRNA.</p> <p>Conclusions</p> <p>Existence of upstream ORF in <it>MYCN </it>transcripts leads to a new level of MYCN regulation. The resulting MYCN dosage has a weak but significant anti-apoptotic activity after intrinsic apoptosis induction.</p

Crystal Structure of the Minimalist Max-E47 Protein Chimera

Max-E47 is a protein chimera generated from the fusion of the DNA-binding basic region of Max and the dimerization region of E47, both members of the basic region/helix-loop-helix (bHLH) superfamily of transcription factors. Like native Max, Max-E47 binds with high affinity and specificity to the E-box site, 5′-CACGTG, both in vivo and in vitro. We have determined the crystal structure of Max-E47 at 1.7 Å resolution, and found that it associates to form a well-structured dimer even in the absence of its cognate DNA. Analytical ultracentrifugation confirms that Max-E47 is dimeric even at low micromolar concentrations, indicating that the Max-E47 dimer is stable in the absence of DNA. Circular dichroism analysis demonstrates that both non-specific DNA and the E-box site induce similar levels of helical secondary structure in Max-E47. These results suggest that Max-E47 may bind to the E-box following the two-step mechanism proposed for other bHLH proteins. In this mechanism, a rapid step where protein binds to DNA without sequence specificity is followed by a slow step where specific protein:DNA interactions are fine-tuned, leading to sequence-specific recognition. Collectively, these results show that the designed Max-E47 protein chimera behaves both structurally and functionally like its native counterparts

Hypoxia induces differential translation of enolase/MBP-1

<p>Abstract</p> <p>Background</p> <p>Hypoxic microenvironments in tumors contribute to transformation, which may alter metabolism, growth, and therapeutic responsiveness. The α-enolase gene encodes both a glycolytic enzyme (α-enolase) and a DNA-binding tumor suppressor protein, c-myc binding protein (MBP-1). These divergent α-enolase gene products play central roles in glucose metabolism and growth regulation and their differential regulation may be critical for tumor adaptation to hypoxia. We have previously shown that MBP-1 and its binding to the c-myc P₂promoter regulates the metabolic and cellular growth changes that occur in response to altered exogenous glucose concentrations.</p> <p>Results</p> <p>To examine the regulation of α-enolase and MBP-1 by a hypoxic microenvironment in breast cancer, MCF-7 cells were grown in low, physiologic, or high glucose under 1% oxygen. Our results demonstrate that adaptation to hypoxia involves attenuation of MBP-1 translation and loss of MBP-1-mediated regulation of c-myc transcription, evidenced by decreased MBP-1 binding to the c-myc P₂promoter. This allows for a robust increase in c-myc expression, "early c-myc response", which stimulates aerobic glycolysis resulting in tumor acclimation to oxidative stress. Increased α-enolase mRNA and preferential translation/post-translational modification may also allow for acclimatization to low oxygen, particularly under low glucose concentrations.</p> <p>Conclusions</p> <p>These results demonstrate that malignant cells adapt to hypoxia by modulating α-enolase/MBP-1 levels and suggest a mechanism for tumor cell induction of the hyperglycolytic state. This important "feedback" mechanism may help transformed cells to escape the apoptotic cascade, allowing for survival during limited glucose and oxygen availability.</p

Identification of Novel High-Frequency DNA Methylation Changes in Breast Cancer

Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (∼200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis

Mammary Involution and Breast Cancer Risk: Transgenic Models and Clinical Studies

Postlactational involution is the process following weaning during which the mammary gland undergoes massive cell death and tissue remodeling as it returns to the pre-pregnant state. Lobular involution is the process by which the breast epithelial tissue is gradually lost with aging of the mammary gland. While postlactational involution and lobular involution are distinct processes, recent studies have indicated that both are related to breast cancer development. Experiments using a variety of rodent models, as well as observations in human populations, suggest that deregulation of postlactational involution may act to facilitate tumor formation. By contrast, new human studies show that completion of lobular involution protects against subsequent breast cancer incidence

miR-210: fine-tuning the hypoxic response

Hypoxia is a central component of the tumor microenvironment and represents a major source of therapeutic failure in cancer therapy. Recent work has provided a wealth of evidence that noncoding RNAs and, in particular, microRNAs, are significant members of the adaptive response to low oxygen in tumors. All published studies agree that miR-210 specifically is a robust target of hypoxia-inducible factors, and the induction of miR-210 is a consistent characteristic of the hypoxic response in normal and transformed cells. Overexpression of miR-210 is detected in most solid tumors and has been linked to adverse prognosis in patients with soft-tissue sarcoma, breast, head and neck, and pancreatic cancer. A wide variety of miR-210 targets have been identified, pointing to roles in the cell cycle, mitochondrial oxidative metabolism, angiogenesis, DNA damage response, and cell survival. Additional microRNAs seem to be modulated by low oxygen in a more tissue-specific fashion, adding another layer of complexity to the vast array of protein-coding genes regulated by hypoxia