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The transcription factor and oncoprotein MYC is a potent driver of many human cancers and can regulate numerous biological activities that contribute to tumorigenesis. How a single transcription factor can regulate such a diverse set of biological programmes is central to the understanding of MYC function in cancer. In this Perspective, we highlight how multiple proteins that interact with MYC enable MYC to regulate several central control points of gene transcription. These include promoter binding, epigenetic modifications, initiation, elongation and post-transcriptional processes. Evidence shows that a combination of multiple protein interactions enables MYC to function as a potent oncoprotein, working together in a 'coalition model', as presented here. Moreover, as MYC depends on its protein interactome for function, we discuss recent research that emphasizes an unprecedented opportunity to target protein interactors to directly impede MYC oncogenesis.

Androgen receptor (AR) in prostate cancer (PCa) can drive transcriptional repression of multiple genes including MYC, and supraphysiological androgen is effective in some patients. Here, we show that this repression is independent of AR chromatin binding and driven by coactivator redistribution, and through chromatin conformation capture methods show disruption of the interaction between the MYC super-enhancer within the PCAT1 gene and the MYC promoter. Conversely, androgen deprivation in vitro and in vivo increases MYC expression. In parallel, global AR activity is suppressed by MYC overexpression, consistent with coactivator redistribution. These suppressive effects of AR and MYC are mitigated at shared AR/MYC binding sites, which also have markedly higher levels of H3K27 acetylation, indicating enrichment for functional enhancers. These findings demonstrate an intricate balance between AR and MYC, and indicate that increased MYC in response to androgen deprivation contributes to castration-resistant PCa, while decreased MYC may contribute to responses to supraphysiological androgen therapy., Androgen receptor in prostate cancer (PCa) transcriptionally represses multiple genes including MYC. Here, the authors suggest that increased MYC in response to androgen deprivation contributes to castration-resistant PCa, while decreased MYC may contribute to responses to supraphysiological androgen therapy.

LRIG1 has been reported to be a tumor suppressor in gastrointestinal tract and epidermis. However, little is known about the expression, regulation and biological functions of LRIG1 in prostate cancer (PCa). We find that LRIG1 is overexpressed in PCa, but its expression correlates with better patient survival. Functional studies reveal strong tumor-suppressive functions of LRIG1 in both AR

This paper is in recognition of the 100th birthday of Dr. Herbert Tabor, a true pioneer in the polyamine field for over 70 years, who served as the editor-in-chief of the Journal of Biological Chemistry from 1971 to 2010. We review current knowledge of MYC proteins (c-MYC, MYCN, and MYCL) and focus on ornithine decarboxylase 1 (ODC1), an important bona fide gene target of MYC, which encodes the sentinel, rate-limiting enzyme in polyamine biosynthesis. Although notable advances have been made in designing inhibitors against the “undruggable” MYCs, their downstream targets and pathways are currently the main avenue for therapeutic anticancer interventions. To this end, the MYC–ODC axis presents an attractive target for managing cancers such as neuroblastoma, a pediatric malignancy in which MYCN gene amplification correlates with poor prognosis and high-risk disease. ODC and polyamine levels are often up-regulated and contribute to tumor hyperproliferation, especially of MYC-driven cancers. We therefore had proposed to repurpose α-difluoromethylornithine (DFMO), an FDA-approved, orally available ODC inhibitor, for management of neuroblastoma, and this intervention is now being pursued in several clinical trials. We discuss the regulation of ODC and polyamines, which besides their well-known interactions with DNA and tRNA/rRNA, are involved in regulating RNA transcription and translation, ribosome function, proteasomal degradation, the circadian clock, and immunity, events that are also controlled by MYC proteins.

Chronic activation of the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARA) promotes MYC-linked hepatocellular carcinoma in mice. Recent studies have shown that MYC can function as an amplifier of transcription where MYC does not act as an ‘on-off’ switch for gene expression, but rather accelerates transcription rates at active promoters by stimulating transcript elongation. Considering the possibility that MYC may amplify the expression of PPARA target genes to potentiate cell proliferation and liver cancer, gene expression was analyzed from livers of wild-type and hepatocyte-specific Myc knockout mice (Myc(ΔHep)) treated with the PPARA agonist Wy-14643. A subset of PPARA target genes was amplified in the presence of MYC, including keratin 23 (Krt23). The induction of Krt23 was significantly attenuated in Myc(ΔHep) mice and completely abolished in Ppara-null mice. Reporter gene assays and chromatin immunoprecipitation confirmed direct binding of both PPARA and MYC to sites within the Krt23 promoter. Forced expression of KRT23 in primary hepatocytes induced cell cycle-related genes. These data indicate that PPARA activation elevates MYC expression which in turn potentiates the expression of select PPARA target genes involved in cell proliferation. Finally, KRT23 protein are highly elevated in human hepatocellular carcinomas. These results revealed that MYC-mediated transcriptional potentiation of select PPARA target genes, such as Krt23, may remove rate-limiting constraints on hepatocyte growth and proliferation leading to liver cancer.

The MYC oncogene is commonly altered across human cancers. Distinct from the normal MYC proto-oncogene, which is under tight transcriptional, translational, and post-translational control, deregulated oncogenic MYC drives imbalanced, non-linear amplification of transcription that results in oncogenic 'stress.' The term 'stress' had been a euphemism for our lack of mechanistic understanding, but synthesis of many studies over the past decade provides a more coherent picture of oncogenic MYC driving metastable cellular states, particularly altered metabolism, that activate and depend on cellular stress response pathways to allow for continued growth and survival. Both deregulated metabolism and these stress response pathways represent vulnerabilities that can be exploited therapeutically.

The bromodomain and extra-terminal domain inhibitors (BETi) are promising epigenetic drugs for the treatment of various cancers through suppression of oncogenic transcription factors. However, only a subset of colorectal cancer (CRC) cells response to BETi. We investigate additional agents that could be combined with BETi to overcome this obstacle. JQ1-resistant CRC cells were used for screening of the effective combination therapies with JQ1. RNA-seq was performed to explore the mechanism of synergistic effect. The efficacy of combinational treatment was tested in the CRC cell line- and patient-derived xenograft (PDX) models. In BETi-sensitive CRC cells, JQ1 also impaired tumor angiogenesis through the c-myc/miR-17-92/CTGF+THBS1 axis. CTGF knockdown moderately counteracted anti-angiogenic effect of JQ1 and led to partially attenuated tumor regression. JQ1 decreased c-myc expression and NF-κB activity in BETi-sensitive CRC cells but not in resistant cells. Bortezomib synergistically sensitized BETi-resistant cells to the JQ1 treatment, and JQ1+Bortezomib induced G2/M arrest in CRC cells. Mechanistically, inhibition of NF-κB by Bortezomib or NF-κB inhibitor or IKK1/2 siRNA all rendered BETi-resistant cells more sensitive to BETi by synergistic repression of c-myc, which in turn induces GADD45s’ expression, and by synergistic repression of FOXM1 which in turn inhibit G2/M checkpoint genes’ expression. Activation of NF-κB by IκBα siRNA induced resistance to JQ1 in BETi-sensitive CRC cells. Last, JQ1+Bortezomib inhibited tumor growth and angiogenesis in CRC cell line xenograft model and four PDX models. Our results indicate that anti-angiogenic effect of JQ1 plays a vital role in therapeutic effect of JQ1 in CRC, and provide a rationale for combined inhibition of BET proteins and NF-κB as a potential therapy for CRC.

Loss of substantia nigra dopaminergic neurons results in Parkinson disease (PD). Degenerative PD usually presents in the seventh decade whereas genetic disorders, including mutations in PARK2, predispose to early onset PD. PARK2 encodes the parkin E3 ubiquitin ligase which confers pleotropic effects on mitochondrial and cellular fidelity and as a mediator of endoplasmic reticulum (ER) stress signaling. Although the majority of studies investigating ameliorative effects of parkin focus on dopaminergic neurons we found that astrocytes are enriched with parkin. Furthermore, astrocytes deficient in parkin display stress-induced elevation of nucleotide-oligomerization domain receptor 2 (NOD2), a cytosolic receptor integrating ER stress and inflammation. Given the neurotropic and immunomodulatory role of astrocytes we reasoned that parkin may regulate astrocyte ER stress and inflammation to control neuronal homeostasis. We show that, in response to ER stress, parkin knockdown astrocytes exhibit exaggerated ER stress, JNK activation and cytokine release, and reduced neurotropic factor expression. In coculture studied we demonstrate that dopaminergic SHSY5Y cells and primary neurons with the presence of parkin depleted astrocytes are more susceptible to ER stress and inflammation-induced apoptosis than wildtype astrocytes. Parkin interacted with, ubiquitylated and diminished NOD2 levels. Additionally, the genetic induction of parkin ameliorated inflammation in NOD2 expressing cells and knockdown of NOD2 in astrocytes suppressed inflammatory defects in parkin deficient astrocytes and concurrently blunted neuronal apoptosis. Collectively these data identify a role for parkin in modulating NOD2 as a regulatory node in astrocytic control of neuronal homeostasis.

Breast cancer stem cells (BCSCs) constitute a small fraction of the primary tumor that can self-renew and become a drug-resistant cell population, thus limiting the treatment effects of chemotherapeutic drugs. The present study evaluated the cytotoxic effects of five phytochemicals including 6-gingerol (6-G), 6-shogaol (6-S), 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-HF), nobiletin (NOL), and pterostilbene (PTE) on MCF-7 breast cancer cells and BCSCs. The results showed that 6-G, 6-S, and PTE selectively killed BCSCs and had high sensitivity for BCSCs isolated from MCF-7 cells that expressed the surface antigen CD44(+)/CD24(-). 6-S and PTE induced cell necrosis phenomena such as membrane injury and bleb formation in BCSCs and inhibited mammosphere formation. In addition, 6-S and PTE increased the sensitivity of isolated BCSCs to chemotherapeutic drugs and significantly increased the anticancer activity of paclitaxel. Analysis of the underlying mechanism showed that 6-S and PTE decreased the expression of the surface antigen CD44 on BCSCs and promoted β-catenin phosphorylation through the inhibition of hedgehog/Akt/GSK3β signaling, thus decreasing the protein expression of downstream c-Myc and cyclin D1 and reducing BCSC stemness.

Marek׳s disease virus (MDV) is a representative alpha herpes virus able to induce rapid-onset T-cell lymphoma in its natural host and regarded as an ideal model for the study of virus-induced tumorigenesis. Recent studies have shown that the mdv1-miR-M4-5p, a viral analog of cellular miR-155, is critical for MDV׳s oncogenicity. However, the precise mechanism whereby it was involved in MD lymphomagenesis remained unknown. We have presently identified the host mRNA targets of mdv1-miR-M4-5 and identified the latent TGF-β binding protein 1 (LTBP1) as a critical target for it. We found that during MDV infection, down-regulation of LTBP1 expression by mdv1-miR-M4-5p led to a significant decrease of the secretion and activation of TGF-β1, with suppression of TGF-β signaling and a significant activation of expression of c-Myc, a well-known oncogene which is critical for virus-induced tumorigenesis. Our findings reveal a novel and important mechanism of how mdv1-miR-M4-5p potentially contributes to MDV-induced tumorigenesis.

Progression of non-alcoholic fatty liver disease (NAFLD) in the context of metabolic syndrome (MetS) is only partially explored due to the lack of preclinical models. In order to study the alterations in hepatic metabolism that accompany this condition, we developed a model of MetS accompanied by the onset of steatohepatitis (NASH) by challenging golden hamsters with a high-fat diet low in vitamin E and selenium (HFD), since combined deficiency results in hepatic necroinflammation in rodents. Metabolomics and transcriptomics integrated analyses of livers revealed an unexpected accumulation of hepatic S-Adenosylmethionine (SAM) when compared with healthy livers likely due to diminished methylation reactions and repression of GNMT. SAM plays a key role in the maintenance of cellular homeostasis and cell cycle control. In agreement, analysis of over-represented transcription factors revealed a central role of c-myc and c-Jun pathways accompanied by negative correlations between SAM concentration, MYC expression and AMPK phosphorylation. These findings point to a drift of cell cycle control toward senescence in livers of HFD animals, which could explain the onset of NASH in this model. In contrast, hamsters with NAFLD induced by a conventional high-fat diet did not show SAM accumulation, suggesting a key role of selenium and vitamin E in SAM homeostasis. In conclusion, our results suggest that progression of NAFLD in the context of MetS can take place even in a situation of hepatic SAM excess and that selenium and vitamin E status might be considered in current therapies against NASH based on SAM supplementation.

Malignant neoplasms evolve in response to changes in oncogenic signalling(1). Cancer cell plasticity in response to evolutionary pressures is fundamental to tumour progression and the development of therapeutic resistance(2,3). Here we elucidate the molecular and cellular mechanisms of cancer cell plasticity in a conditional oncogenic Kras mouse model of pancreatic ductal adenocarcinoma (PDAC), a malignancy that displays considerable phenotypic diversity and morphological heterogeneity. In this model, stochastic extinction of oncogenic Kras signalling and emergence of Kras-independent escaper populations (cells that acquire oncogenic properties) are associated with de-differentiation and aggressive biological behaviour. Transcriptomic and functional analyses of Kras-independent escapers reveal the presence of Smarcb1–Myc-network-driven mesenchymal reprogramming and independence from MAPK signalling. A somatic mosaic model of PDAC, which allows time-restricted perturbation of cell fate, shows that depletion of Smarcb1 activates the Myc network, driving an anabolic switch that increases protein metabolism and adaptive activation of endoplasmic-reticulum-stress-induced survival pathways. Elevated protein turnover renders mesenchymal sub-populations highly susceptible to pharmacological and genetic perturbation of the cellular proteostatic machinery and the IRE1-α–MKK4 arm of the endoplasmic-reticulum-stress-response pathway. Specifically, combination regimens that impair the unfolded protein responses block the emergence of aggressive mesenchymal subpopulations in mouse and patient-derived PDAC models. These molecular and biological insights inform a potential therapeutic strategy for targeting aggressive mesenchymal features of PDAC.

Inactivation of APC is a strongly predisposing event in the development of colorectal cancer, prompting the search for vulnerabilities specific to cells that have lost APC function. Signalling through the mTOR pathway is known to be required for epithelial cell proliferation and tumour growth, and the current paradigm suggests that a critical function of mTOR activity is to upregulate translational initiation through phosphorylation of 4EBP1 (refs 6, 7). This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP1 (ref. 8), would be ineffective in limiting cancer progression in APC-deficient lesions. Here we show in mice that mTOR complex 1 (mTORC1) activity is absolutely required for the proliferation of Apc-deficient (but not wild-type) enterocytes, revealing an unexpected opportunity for therapeutic intervention. Although APC-deficient cells show the expected increases in protein synthesis, our study reveals that it is translation elongation, and not initiation, which is the rate-limiting component. Mechanistically, mTORC1-mediated inhibition of eEF2 kinase is required for the proliferation of APC-deficient cells. Importantly, treatment of established APC-deficient adenomas with rapamycin (which can target eEF2 through the mTORC1-S6K-eEF2K axis) causes tumour cells to undergo growth arrest and differentiation. Taken together, our data suggest that inhibition of translation elongation using existing, clinically approved drugs, such as the rapalogs, would provide clear therapeutic benefit for patients at high risk of developing colorectal cancer.

Deregulated expression of MYC is a driver of colorectal carcinogenesis, necessitating novel strategies to inhibit MYC function. The ubiquitin ligase HUWE1 (HECTH9, ARF-BP1, MULE) associates with both MYC and the MYC-associated protein MIZ1. We show here that HUWE1 is required for growth of colorectal cancer cells in culture and in orthotopic xenograft models. Using high-throughput screening, we identify small molecule inhibitors of HUWE1, which inhibit MYC-dependent transactivation in colorectal cancer cells, but not in stem and normal colon epithelial cells. Inhibition of HUWE1 stabilizes MIZ1. MIZ1 globally accumulates on MYC target genes and contributes to repression of MYC-activated target genes upon HUWE1 inhibition. Our data show that transcriptional activation by MYC in colon cancer cells requires the continuous degradation of MIZ1 and identify a novel principle that allows for inhibition of MYC function in tumor cells. See also: FX Schaub & JL Cleveland (December 2014)

The role of MYC proteins in somatic stem and progenitor cells during development is poorly understood. We have taken advantage of a chick in vivo model to examine their role in progenitor cells of the developing neural tube. Our results show that depletion of endogenous MYC in radial glial precursors (RGPs) is incompatible with differentiation and conversely, that overexpression of MYC induces neurogenesis independently of premature or upregulated expression of proneural gene programs. Unexpectedly, the neurogenic function of MYC depends on the integrity of the polarized neural tissue, in contrast to the situation in dissociated RGPs where MYC is mitogenic. Within the polarized RGPs of the neural tube, MYC drives differentiation by inhibiting Notch signaling and by increasing neurogenic cell division, eventually resulting in a depletion of progenitor cells. These results reveal an unexpected role of MYC in the control of stemness versus differentiation of neural stem cells in vivo.

Interactions between thymic epithelial cells (TEC) and developing thymocytes are essential for T cell development, but molecular insights on TEC and thymus homeostasis are still lacking. Here we identify distinct transcriptional programs of TEC that account for their age-specific properties, including proliferation rates, engraftability and function. Further analyses identify Myc as a regulator of fetal thymus development to support the rapid increase of thymus size during fetal life. Enforced Myc expression in TEC induces the prolonged maintenance of a fetal-specific transcriptional program, which in turn extends the growth phase of the thymus and enhances thymic output; meanwhile, inducible expression of Myc in adult TEC similarly promotes thymic growth. Mechanistically, this Myc function is associated with enhanced ribosomal biogenesis in TEC. Our study thus identifies age-specific transcriptional programs in TEC, and establishes that Myc controls thymus size., Thymic epithelial cells (TEC) are essential for the maturation of functional T cells, while thymus size is proportional to the overall output efficiency. Here the authors show, using transcriptome analyses, that mouse fetal TEC maintain a Myc-dependent genetic program to ensure a rapid increase in thymus size, and thereby expedited T cell generation.

// Justin M. Roberts 1 , Rebeca San Martin 1 , D. Badrajee Piyarathna 1, 2 , James G. MacKrell 3 , Guilherme V. Rocha 3 , Jeffery A. Dodge 3 , Cristian Coarfa 1, 2 , Venkatesh Krishnan 3 , David R. Rowley 1 and Nancy L. Weigel 1, 4 1 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA 2 Alkek Center for Molecular Discovery, Baylor College of Medicine, Houston, TX, USA 3 Lilly Research Labs, Eli Lilly and Company, Indianapolis, IN, USA 4 Scott Department of Urology, Baylor College of Medicine, Houston, TX, USA Correspondence to: Nancy L. Weigel, email: nweigel@bcm.edu Keywords: vitamin D, prostate cancer, CYP24A1, TMPRSS2:ERG, VCaP Received: November 30, 2016 Accepted: May 04, 2017 Published: May 18, 2017 ABSTRACT Whether vitamin D is chemopreventive and/or has potential therapeutically in prostate cancer is unresolved. One confounding factor is that many prostate cancers express a TMPRSS2:ERG fusion gene whose expression is increased both by androgens and by vitamin D receptor (VDR) activation. Two challenges that limit VDR agonist use clinically are hypercalcemia and the cooperation of VDR with ERG to hyper-induce the 1α,25-dihydroxyvitamin D 3 metabolizing enzyme, CYP24A1, thus reducing VDR activity. Using the VCaP TMPRSS2:ERG positive cell line as a model, we found that a nonsecosteroidal CYP24A1 resistant VDR agonist, VDRM2, substantially reduces growth of xenograft tumors without inducing hypercalcemia. Utilizing next generation RNA sequencing, we found a very high overlap of 1,25D(OH) 2 D 3 and VDRM2 regulated genes and by drawing upon previously published datasets to create an ERG signature, we found activation of VDR does not induce ERG activity above the already high basal levels present in VCaP cells. Moreover, we found VDR activation opposes 8 of the 10 most significant ERG regulated Hallmark gene set collection pathways from Gene Set Enrichment Analysis (GSEA). Thus, a CYP24A1 resistant VDR agonist may be beneficial for treatment of TMPRSS2:ERG positive prostate cancer; one negative consequence of TMPRSS2:ERG expression is inactivation of VDR signaling.

Tumor stem cells, postulated to be the source cells for malignancies, have been identified in several cancers using cell-surface expression of markers including CD133, a pentaspan membrane protein. CD133+ve cells form neurospheres, exhibit self-renewal and differentiation, and are tumorigenic. However, despite its association with stem cells, a causal relationship of CD133 to tumorigenesis remains to be defined. Hypothesizing that specific epigenetic and transcription factors implicated in driving the stem cell state may concurrently regulate CD133 expression in stem cells, we analyzed the structure and regulation of CD133 promoter in glioma stem cells and glioma cell lines. Initially, a minimal promoter region was identified by analyzing the activity of CD133 promoter-driven luciferase-expressing 5'-and 3'-deletion-constructs upstream of the transcription start site. This region contained a CpG island that was hypermethylated in CD133-ve glioma stem cells (GSC) and glioma cells but unmethylated in CD133+ve ones. Of several predicted TF-binding sites in this region, the role of tandem Sp1 (-242 and -221) and two Myc (-541 and -25)-binding sites were examined. Overexpression of Sp1 or Myc increased CD133 minimal promoter-driven luciferase activity and CD133 levels in GSC and in glioma cell line. Mithramycin, a Sp1 inhibitor, decreased minimal promoter activity and downregulated CD133 levels in GSC. Gel-shift assays demonstrated direct binding of Sp1 to their predicted sites that was competitively inhibited by oligonucleotide-binding-site sequences and supershifted by anti-Sp1 confirming the interaction. Sp1 and Myc-antibody chromatin immunoprecipitation (ChIP) analysis in GSC showed enrichment of regions with Sp1 and Myc-binding sites. In CD133-ve cells, ChIP analysis showed binding of the methyl-DNA-binding proteins, MBD1, MBD2 and MeCP2 to the methylated CpG island and repression of transcription. These results demonstrate that Sp1 and Myc regulate CD133 transcription in GSC and that promoter methylation and methyl-DNA-binding proteins cause repression of CD133 by excluding transcription-factor binding.

Eukaryotes control nearly every cellular process in part by modulating the transcription of genes encoded by their nuclear genome. However, these cells are faced with the added complexity of possessing a second genome, within the mitochondria, which encodes critical components of several essential processes, including energy metabolism and macromolecule biosynthesis. As these cellular processes require gene products encoded by both genomes, cells have adopted strategies for linking mitochondrial gene expression to nuclear gene expression and other dynamic cellular events. Here we discuss examples of several mechanisms that have been identified, by which eukaryotic cells link extramitochondrial signals to dynamic alterations in mitochondrial transcription. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

Deregulated expression of the MYC oncoprotein contributes to the genesis of many human tumours, yet strategies to exploit this for a rational tumour therapy are scarce. MYC promotes cell growth and proliferation, and alters cellular metabolism to enhance the provision of precursors for phospholipids and cellular macromolecules. Here we show in human and murine cell lines that oncogenic levels of MYC establish a dependence on AMPK-related kinase 5 (ARK5; also known as NUAK1) for maintaining metabolic homeostasis and for cell survival. ARK5 is an upstream regulator of AMPK and limits protein synthesis via inhibition of the mammalian target of rapamycin 1 (mTORC1) signalling pathway. ARK5 also maintains expression of mitochondrial respiratory chain complexes and respiratory capacity, which is required for efficient glutamine metabolism. Inhibition of ARK5 leads to a collapse of cellular ATP levels in cells expressing deregulated MYC, inducing multiple pro-apoptotic responses as a secondary consequence. Depletion of ARK5 prolongs survival in MYC-driven mouse models of hepatocellular carcinoma, demonstrating that targeting cellular energy homeostasis is a valid therapeutic strategy to eliminate tumour cells that express deregulated MYC.

In the field of induced potency and fate reprogramming, it remains unclear what the best starting cell might be and to what extent a cell need be transported back to a more primitive state for translational purposes. Reprogramming a committed cell back to pluripotence to then instruct it toward a particular specialized cell type is demanding and may increase risks of neoplasia and undesired cell types. Precursor/progenitor cells from the organ of therapeutic concern typically lack only one critical attribute—the capacity for sustained self-renewal. We speculated that this could be induced in a regulatable manner such that cells proliferate only in vitro and differentiate in vivo without the need for promoting pluripotence or specifying lineage identity. As proof-of-concept, we generated and tested the efficiency, safety, engraftability, and therapeutic utility of “induced conditional self-renewing progenitor (ICSP) cells” derived from the human central nervous system (CNS); we conditionally induced self-renewal efficiently within neural progenitors solely by introducing v- myc tightly regulated by a tetracycline (Tet)- on gene expression system. Tet in the culture medium activated myc transcription and translation, allowing efficient expansion of homogeneous, clonal, karyotypically normal human CNS precursors ex vivo; in vivo, where Tet was absent, myc was not expressed, and self-renewal was entirely inactivated (as was tumorigenic potential). Cell proliferation ceased, and differentiation into electrophysiologically active neurons and other CNS cell types in vivo ensued upon transplantation into rats, both during development and after adult injury—with functional improvement and without neoplasia, overgrowth, deformation, emergence of non-neural cell types, phenotypic or genomic instability, or need for immunosuppression. This strategy of inducing self-renewal might be applied to progenitors from other organs and may prove to be a safe, effective, efficient, and practical method for optimizing insights gained from the ability to reprogram cells.

MYC family oncoproteins (MYC, N‐MYC and L‐MYC) function as basic helix‐loop‐helix‐leucine zipper (bHLH‐Zip) transcription factors that are activated (i.e., overexpressed) in well over half of all human malignancies (Boxer & Dang, 2001; Beroukhim et al, 2010). In this issue of EMBO Molecular Medicine, Eilers and colleagues (Peter et al, 2014) describe a novel approach to disable MYC, whereby inhibition of the ubiquitin ligase HUWE1 stabilizes MIZ1 and leads to the selective repression of MYC‐activated target genes.

Gene expression profiles of CD34+ cells were compared between 51 MDS patients and 7 controls. The most up-regulated genes in patients included HBG2, HBG1, CYBRD1, HSPA1B, ANGPT, and MYC, while 13 genes related to B-lymphopoiesis showed down-regulation. We observed in advanced MDS patients decreased expression of genes involved in cell cycle control, DNA repair and increased expression of proto-oncogenes, angiogenic and anti-apoptic genes. The results suggest that increased cell proliferation and resistance to apoptosis together with a loss of cell cycle control, damaged DNA repair and altered immune response may play an important role in malignant clone expansion in MDS.

The Myc oncogene regulates the expression of several components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, RNA polymerase III and ribosomal DNA. Whether and how increasing the cellular protein synthesis capacity affects the multistep process leading to cancer remains to be addressed. Here we use ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in Emu-Myc/+ transgenic mice to normal levels, and show that the oncogenic potential of Myc in this context is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc-overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a new mechanism that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation used to regulate the expression of selective messenger RNAs. We show that an aberrant increase in cap-dependent translation downstream of Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site (IRES)-dependent translation that is required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic-specific expression of the endogenous IRES-dependent form of Cdk11 (also known as Cdc2l and PITSLRE), which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in Emu-Myc/+ mice. When accurate translational control is re-established in Emu-Myc/+ mice, genome instability is suppressed. Our findings demonstrate how perturbations in translational control provide a highly specific outcome for gene expression, genome stability and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post-genomic level.

Activation of eukaryotic gene transcription involves the recruitment by DNA-binding activators of multiprotein histone acetyltransferase (HAT) and Mediator complexes. How these coactivator complexes functionally cooperate and the roles of the different subunits/modules remain unclear. Here we report physical interactions between the human HAT complex STAGA (SPT3-TAF9-GCN5-acetylase) and a "core" form of the Mediator complex during transcription activation by the MYC oncoprotein. Knockdown of the STAF65gamma component of STAGA in human cells prevents the stable association of TRRAP and GCN5 with the SPT3 and TAF9 subunits; impairs transcription of MYC-dependent genes, including MYC transactivation of the telomerase reverse transcriptase (TERT) promoter; and inhibits proliferation of MYC-dependent cells. STAF65gamma is required for SPT3/STAGA interaction with core Mediator and for MYC recruitment of SPT3, TAF9, and core Mediator components to the TERT promoter but is dispensable for MYC recruitment of TRRAP, GCN5, and p300 and for acetylation of nucleosomes and loading of TFIID and RNA polymerase II on the promoter. These results suggest a novel STAF65gamma-dependent function of STAGA-type complexes in cell proliferation and transcription activation by MYC postloading of TFIID and RNA polymerase II that involves direct recruitment of core Mediator.

The acetyl-transferase Tip60 might influence tumorigenesis in multiple ways. First, Tip60 is a co-regulator of transcription factors that either promote or suppress tumorigenesis, such as Myc and p53. Second, Tip60 modulates DNA-damage response (DDR) signalling, and a DDR triggered by oncogenes can counteract tumour progression. Using E(mu)-myc transgenic mice that are heterozygous for a Tip60 gene (Htatip) knockout allele (hereafter denoted as Tip60+/- mice), we show that Tip60 counteracts Myc-induced lymphomagenesis in a haplo-insufficient manner and in a time window that is restricted to a pre- or early-tumoral stage. Tip60 heterozygosity severely impaired the Myc-induced DDR but caused no general DDR defect in B cells. Myc- and p53-dependent transcription were not affected, and neither were Myc-induced proliferation, activation of the ARF-p53 tumour suppressor pathway or the resulting apoptotic response. We found that the human TIP60 gene (HTATIP) is a frequent target for mono-allelic loss in human lymphomas and head-and-neck and mammary carcinomas, with concomitant reduction in mRNA levels. Immunohistochemical analysis also demonstrated loss of nuclear TIP60 staining in mammary carcinomas. These events correlated with disease grade and frequently concurred with mutation of p53. Thus, in both mouse and human, Tip60 has a haplo-insufficient tumour suppressor activity that is independent from-but not contradictory with-its role within the ARF-p53 pathway. We suggest that this is because critical levels of Tip60 are required for mounting an oncogene-induced DDR in incipient tumour cells, the failure of which might synergize with p53 mutation towards tumour progression.

Interaction with Max via the helix-loop-helix/leucine zipper (HLH-LZ) domain is essential for Myc to function as a transcription factor. Myc is commonly upregulated in tumours, however, its activity can also be potentiated by virally derived mutations. vMyc, derived from the virus, MC29 gag-Myc, differs from its cellular counterpart by five amino acids. The N-terminal mutation stabilizes the protein, however, the significance of the other mutations is not known. We now show that vMyc can sustain longer deletions in the LZ domain than cMyc before complete loss in transforming activity, implicating the viral mutations in contributing to Myc:Max complex formation. We confirmed this both in vitro and in vivo, with loss of Max binding correlating with a loss in the biological activity of Myc. A specific viral mutation, isoleucine383>leucine (I383>L) in helix 2 of the HLH domain, extends the LZ domain from four to five heptad repeats. Significantly, introduction of I383>L into a Myc mutant that is defective for Max binding substantially restored its ability to complex with Max in vitro and in vivo. We therefore propose that this virally derived mutation is functional by significantly contributing to establishing a more hydrophobic interface between the LZs of Myc and Max.

TGF-beta is a multifunctional cytokine known to exert its biological effects through a variety of signaling pathways of which Smad signaling is considered to be the main mediator. At present, the Smad-independent pathways, their interactions with each other, and their roles in TGF-beta-mediated growth inhibitory effects are not well understood. To address these questions, we have utilized a human breast cancer cell line MCF10CA1h and demonstrate that p38 MAP kinase and Rho/ROCK pathways together with Smad2 and Smad3 are necessary for TGF-beta-mediated growth inhibition of this cell line. We show that Smad2/3 are indispensable for TGF-beta-mediated growth inhibition, and that both p38 and Rho/ROCK pathways affect the linker region phosphorylation of Smad2/3. Further, by using Smad3 mutated at the putative phosphorylation sites in the linker region, we demonstrate that phosphorylation at Ser203 and Ser207 residues is required for the full transactivation potential of Smad3, and that these residues are targets of the p38 and Rho/ROCK pathways. We demonstrate that activation of the p38 MAP kinase pathway is necessary for the full transcriptional activation potential of Smad2/Smad3 by TGF-beta, whereas activity of Rho/ROCK is necessary for both down-regulation of c-Myc protein and up-regulation of p21waf1 protein, directly interfering with p21waf1 transcription. Our results not only implicate Rho/ROCK and p38 MAPK pathways as necessary for TGF-beta-mediated growth inhibition, but also demonstrate their individual contributions and the basis for their cooperation with each other.

'Gain' of supernumerary copies of the 8q24.21 chromosomal region has been shown to be common in many human cancers and is associated with poor prognosis. The well-characterized myelocytomatosis (MYC) oncogene resides in the 8q24.21 region and is consistently co-gained with an adjacent 'gene desert' of approximately 2 megabases that contains the long non-coding RNA gene PVT1, the CCDC26 gene candidate and the GSDMC gene. Whether low copy-number gain of one or more of these genes drives neoplasia is not known. Here we use chromosome engineering in mice to show that a single extra copy of either the Myc gene or the region encompassing Pvt1, Ccdc26 and Gsdmc fails to advance cancer measurably, whereas a single supernumerary segment encompassing all four genes successfully promotes cancer. Gain of PVT1 long non-coding RNA expression was required for high MYC protein levels in 8q24-amplified human cancer cells. PVT1 RNA and MYC protein expression correlated in primary human tumours, and copy number of PVT1 was co-increased in more than 98% of MYC-copy-increase cancers. Ablation of PVT1 from MYC-driven colon cancer line HCT116 diminished its tumorigenic potency. As MYC protein has been refractory to small-molecule inhibition, the dependence of high MYC protein levels on PVT1 long non-coding RNA provides a much needed therapeutic target.

The correct balance between self‐renewal and expansion of stem cells versus differentiation is essential to maintain tissue homeostasis and prevent neoplastic transformation. The MYC proto‐oncogene family (comprising MYC , MYCN and MYCL ) is a family of proteins that plays a crucial role in the decision of stem and progenitor cells to expand or undergo differentiation. MYC proteins are transcription factors that act through activation and repression of a wide variety of target genes and, when deregulated, promote oncogenic transformation of most cell types [1]. There is overwhelming support to the notion that MYC is essential to preserve the stem cell state and inhibit differentiation. This is also true in the developing nervous system where MYC ablation leads to premature neuronal differentiation, whereas MYC accumulation locks cells in an undifferentiated and abnormally proliferative state [2], [3]. In this issue of EMBO reports , Zinin et al [4] present, however, an unexpected pro‐neurogenic function of MYC in neural progenitors that diverges from the established mitogenic and anti‐differentiation functions of this family of proteins. Through loss‐ and gain‐of‐function experiments in the developing chick neural tube, the authors identify a novel function of MYC in neuronal differentiation of radial glial precursors (RGPs), the multipotent progenitors in the neurogenic areas of the brain. Concurrent silencing of the two MYC family members that are expressed in the developing neural tube (MYC and MYCN) led to inhibition of neuronal differentiation. Rather than what might have been surmised from the established …

Human central nervous system (CNS) cell lines would substantially facilitate drug discovery and basic research by providing a readily renewable source of human neurons. We isolated clonal human CNS cell lines that had been immortalized with a tetracycline (Tc)-responsive v-myc oncogene; addition of Tc to the growth medium suppressed the oncoprotein rapidly and virtually completely, allowing differentiation to proceed. Two classes of bipotent precursor cells were immortalized: the first class had a default differentiation pathway of neurons only, and the second class had a default differentiation pathway of neurons and astrocytes. We found that after exposure to different external signals in vitro, the environment is capable of redirecting the fate of a particular cell, even in the case of the bipotent precursor cell whose default differentiation pathway was neurons only. These data suggest that extrinsic cues can prevail over intrinsic determinants in directing cell fate in the human CNS.

Tissue mechanics regulate development and homeostasis and are consistently modified in tumor progression. Nevertheless, the fundamental molecular mechanisms through which altered mechanics regulate tissue behavior and the clinical relevance of these changes remain unclear. We demonstrate that increased matrix stiffness modulates microRNA expression to drive tumor progression through integrin activation of β-catenin and MYC. Specifically, in human and mouse tissue, increased matrix stiffness induced miR-18a to reduce levels of the tumor suppressor phosphatase and tensin homolog (PTEN), both directly and indirectly by decreasing levels of homeobox A9 (HOXA9). Clinically, extracellular matrix stiffness correlated directly and significantly with miR-18a expression in human breast tumor biopsies. miR-18a expression was highest in basal-like breast cancers in which PTEN and HOXA9 levels were lowest, and high miR-18a expression predicted poor prognosis in patients with luminal breast cancers. Our findings identify a mechanically regulated microRNA circuit that can promote malignancy and suggest potential prognostic roles for HOXA9 and miR-18a levels in stratifying patients with luminal breast cancers. © 2014 Nature America, Inc. All rights reserved.

Cell cycle progression is controlled by sequential activation of cyclin-dependent kinases (CDKs), which are often deregulated in cancer. Consequently numerous pharmacological inhibitors of CDKs have been developed with the aim of treating cancers. The article briefly reviews CDK inhibitors and their use to treat cancers, with specific focus on the use of biomarkers and drugs combination to improve their therapeutic efficacy.

We are interested in identifying the transcriptional targets of the Myc oncoproteins. To this end, we have fused Myc of the MC29 retrovirus with the rat glucocorticoid receptor. This chimeric protein requires dexamethasone to undergo nuclear translocation and achieve an active conformation. We employed a differential hybridization approach to identify mRNAs that are induced or repressed in infected avian fibroblasts in response to dexamethasone. This screen yielded one mRNA underrepresented in the dexamethasone-treated cells. In Myc-transformed cell clones, its level decreases 6-fold as early as 4 h and more than 30-fold after 32 h of exposure to the hormone. This mRNA was also down-regulated by recombinant Myc retroviruses in rodent fibroblasts, including those refractory to transformation. Sequence analysis revealed that it is homologous to the 3′ untranslated regions of the mammalian thrombospondin-1 genes. Using an anti-thrombospondin antibody, we confirmed that rodent cells overexpressing Myc produce very small amounts of this protein. Also, they do not support efficient expression of a reporter gene driven by the thrombospondin-1 promoter. Thus, thrombospondin-1 is a bona fide target of Myc. Moreover, its silencing might pertain to the transforming activity of Myc, since in several systems thrombospondin-1 exhibits tumor suppressor properties, presumably due to its negative effect on neovascularization.

A regulatable retroviral vector in which the v-myc oncogene is driven by a tetracycline-controlled transactivator and a human cytomegalovirus minimal promoter fused to a tet operator sequence was used for conditional immortalization of adult rat neuronal progenitor cells. A single clone, HC2S2, was isolated and characterized. Two days after the addition of tetracycline, the HC2S2 cells stopped proliferating, began to extend neurites, and expressed the neuronal markers tau, NeuN, neurofilament 200 kDa, and glutamic acid decarboxylase in accordance with the reduced production of the v-myc oncoprotein. Differentiated HC2S2 cells expressed large sodium and calcium currents and could fire regenerative action potentials. These results suggest that the suppression of the v-myc oncogene may be sufficient to make proliferating cells exit from cell cycles and induce terminal differentiation. The HC2S2 cells will be valuable for studying the differentiation process of neurons.

Cohesin is best known as a multi-subunit protein complex that holds together replicated sister chromatids from S phase until G2. Cohesin also has an important role in the regulation of gene expression. We previously demonstrated that the cohesin complex positively regulates expression of the oncogene MYC. Cell proliferation driven by MYC contributes to many cancers, including breast cancer. The MYC oncogene is estrogen-responsive and a transcriptional target of estrogen receptor alpha (ERα). Estrogen-induced cohesin binding sites coincide with ERα binding at the MYC locus, raising the possibility that cohesin and ERα combine actions to regulate MYC transcription. The objective of this study was to investigate a putative role for cohesin in estrogen induction of MYC expression. We found that siRNA-targeted depletion of a cohesin subunit, RAD21, decreased MYC expression in ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231) breast cancer cell lines. In addition, RAD21 depletion blocked estradiol-mediated activation of MYC in ER-positive cell lines, and decreased ERα binding to estrogen response elements (EREs) upstream of MYC, without affecting total ERα levels. Treatment of MCF7 cells with estradiol caused enrichment of RAD21 binding at upstream enhancers and at the P2 promoter of MYC. Enriched binding at all sites, except the P2 promoter, was dependent on ERα. Since RAD21 depletion did not affect transcription driven by an exogenous reporter construct containing a naked ERE, chromatin-based mechanisms are likely to be involved in cohesin-dependent MYC transcription. This study demonstrates that ERα activation of MYC can be modulated by cohesin. Together, these results demonstrate a novel role for cohesin in estrogen-mediated regulation of MYC and the first evidence that cohesin plays a role in ERα binding.

Glioblastoma multiform (GBM) are devastating brain tumors containing a fraction of multipotent stem-like cells which are highly tumorigenic. These cells are resistant to treatments and are likely to be responsible for tumor recurrence. One approach to eliminate GBM stem-like cells would be to force their terminal differentiation. During development, neurons formation is controlled by neurogenic transcription factors such as Ngn1/2 and NeuroD1. We found that in comparison with oligodendrogenic genes, the expression of these neurogenic genes is low or absent in GBM tumors and derived cultures. We thus explored the effect of overexpressing these neurogenic genes in three CD133(+) Sox2(+) GBM stem-like cell cultures and the U87 glioma line. Introduction of Ngn2 in CD133(+) cultures induced massive cell death, proliferation arrest and a drastic reduction of neurosphere formation. Similar effects were observed with NeuroD1. Importantly, Ngn2 effects were accompanied by the downregulation of Olig2, Myc, Shh and upregulation of Dcx and NeuroD1 expression. The few surviving cells adopted a typical neuronal morphology and some of them generated action potentials. These cells appeared to be produced at the expense of GFAP(+) cells which were radically reduced after differentiation with Ngn2. In vivo, Ngn2-expressing cells were unable to form orthotopic tumors. In the U87 glioma line, Ngn2 could not induce neuronal differentiation although proliferation in vitro and tumoral growth in vivo were strongly reduced. By inducing cell death, cell cycle arrest or differentiation, this work supports further exploration of neurogenic proteins to oppose GBM stem-like and non-stem-like cell growth.

Epstein-Barr virus (EBV) infection and latency has been associated with malignant diseases including nasopharyngeal carcinoma, Hodgkin lymphoma, Burkitt lymphoma, and immune deficiency associated lymphoproliferative diseases. EBV-encoded latent membrane protein 2A (LMP2A) recruits Lyn and Syk kinases via its SH2-domain binding motifs, and modifies their signaling pathways. LMP2A transgenic mice develop hyperproliferative bone marrow B cells and immature peripheral B cells through modulation of Lyn kinase signaling. LMP2A/λ-MYC double transgenic mice develop splenomegaly and cervical lymphomas starting at 8 weeks of age. We reasoned that targeting Lyn in LMP2A-expressing B cells with dasatinib would provide a therapeutic option for EBV-associated malignancies. Here, we show that dasatinib inhibits B cell colony formation by LMP2A transgenic bone marrow cells, and reverses splenomegaly and tumor growth in both a pre-tumor and a syngeneic tumor transfer model of EBV-associated Burkitt lymphoma. Our data support the idea that dasatinib may prove to be an effective therapeutic molecule for the treatment of EBV-associated malignancies.

MicroRNAs are regulators of gene expression. We used miRNA-seq by the Illumina platform to quantify and compare the temporal miRNA expression profiles in the brain of a short-lived (GRZ) and a longer-lived strain (MZM) of the annual fish Nothobranchius furzeri. We used fuzzy-c-means clustering to group miRNAs with similar profiles. In MZM, we found tumor suppressors with known negative interactions with MYC and/or positive interactions with TP53 among up-regulated miRNAs (e.g. miR-23a, miR-26a/b, miR-29a/b and miR-101a) in aged animals. Conversely, we found oncogenes which are MYC targets among down-regulated miRNAs (miR-7a, members of miR cluster 17∼92). These latter were previously shown to be regulated in human replicative aging. In addition, three regulated miRNAs (miR-181c, miR-29a and miR-338) are known to be age-regulated and to globally contribute to regulation of their targets in the human brain. Therefore, there appears to be a degree of evolutionarily conservation in age-dependent miRNA expression between humans and N. furzeri. GRZ showed specific regulation of some miRNAs, notably a marked up-regulation of miR-124, a miRNA important for neuronal differentiation. The two strains differ in their miRNA expression profiles already at sexual maturity. Short lifespan in GRZ could therefore be--at least partially--due to dysregulated miRNA expression.

A major challenge for further development of drug screening procedures, cell replacement therapies and developmental studies is the identification of expandable human stem cells able to generate the cell types needed. We have previously reported the generation of an immortalized polyclonal neural stem cell (NSC) line derived from the human fetal ventral mesencephalon (hVM1). This line has been biochemically, genetically, immunocytochemically and electrophysiologically characterized to document its usefulness as a model system for the generation of A9 dopaminergic neurons (DAn). Long-term in vivo transplantation studies in parkinsonian rats showed that the grafts do not mature evenly. We reasoned that diverse clones in the hVM1 line might have different abilities to differentiate. In the present study, we have analyzed 9 hVM1 clones selected on the basis of their TH generation potential and, based on the number of v-myc copies, v-myc down-regulation after in vitro differentiation, in vivo cell cycle exit, TH+ neuron generation and expression of a neuronal mature marker (hNSE), we selected two clones for further in vivo PD cell replacement studies. The conclusion is that homogeneity and clonality of characterized NSCs allow transplantation of cells with controlled properties, which should help in the design of long-term in vivo experiments. © 2012 Ramos-Moreno et al., Spanish Ministry of Economy and Competitiveness; Comunidad Autónoma Madrid; Instituto Salud Carlos III; European Union (Excell, NMP4-SL-2008 214706); Foundation Ramon Areces

c-MYC was found to repress expression of the c-myc promoter in a transient assay system. Deletion constructs showed the cis-acting element(s) were common to both the P1 and P2 promoters. Further analysis involving replacement of the P2 TATA box and initiator elements with the SV40 late promoter initiator localized the cis-acting elements to the core promoter.

The cellular proto-oncogene c-myc can acquire transforming potential by a number of different means, including retroviral transduction. The transduced allele generally contains point mutations relative to c-myc and is overexpressed in infected cells, usually as a v-Gag-Myc fusion protein. Upon synthesis, v-Gag-Myc enters the nucleus, forms complexes with its heterodimeric partner Max, and in this complex binds to DNA in a sequence-specific manner. To delineate the role for each of these events in fibroblast transformation, we introduced several mutations into the myc gene of the avian retrovirus MC29. We observed that Gag-Myc with a mutated nuclear localization signal is confined predominantly in the cytoplasm and only about 5% of the protein could be detected in the nucleus (less than the amount of endogenous c-Myc). Consequently, only a small fraction of Max is associated with Myc. However, cells infected with this mutant exhibit a completely transformed phenotype in vitro, suggesting that production of enough v-Gag-Myc to tie up all cellular Max is not needed for transformation. While the nuclear localization signal is dispensable for transformation, minimal changes in the v-Gag-Myc DNA-binding domain completely abolish its transforming potential, consistent with a role of Myc as a transcriptional regulator. One of its potential targets might be the endogenous c-myc, which is repressed in wild-type MC29-infected cells. Our experiments with MC29 mutants demonstrate that c-myc down-regulation depends on the integrity of the v-Myc DNA-binding domain and occurs at the RNA level. Hence, it is conceivable that v-Gag-Myc, either directly or circuitously, regulates c-myc transcription.

The RhoA GTPase is crucial in numerous biological functions and is linked to cancer metastasis. However, the understanding of the molecular mechanism responsible for RhoA transcription is still very limited. Here we show that RhoA transcription is orchestrated by the Myc-Skp2-Miz1-p300 transcriptional complex. Skp2 cooperates with Myc to induce RhoA transcription by recruiting Miz1 and p300 to the RhoA promoter independently of Skp1-Cullin-F-box protein containing complex (SCF)-Skp2 E3 ligase activity. Deficiency of this complex results in impairment in RhoA expression, cell migration, invasion, and breast cancer metastasis, recapitulating the phenotypes observed in RhoA knockdown, and RhoA restoration rescues the defect in cell invasion. Overexpression of the Myc-Skp2-Miz1 complex is found in metastatic human cancers and is correlated with RhoA expression. Our study provides insight into how oncogenic Skp2 and Myc coordinate to induce RhoA transcription and establishes a novel SCF-Skp2 E3-ligase-independent function for oncogenic Skp2 in transcription and cancer metastasis.

Avian myelocytomatosis virus MC29 induces a wide variety of neoplastic diseases in infected birds and transforms cells of the macrophage lineage as well as fibroblasts and epithelial cells. A biological and biochemical analysis, carried out on a series of in-frame insertion and deletion mutations within the gag-myc gene of MC29, revealed several mutations within the 5' portion of the v-myc gene that encode proteins either completely defective for transformation or compromised in their ability to transform chicken embryo fibroblasts but not macrophages. Mutations within the 3' end of the v-myc gene which disrupt sequences encoding the basic/helix-loop-helix region were defective for transformation of both fibroblasts and macrophages. Eight variants were cloned into the replication-competent avian expression vector RCAS. Analysis of cells infected with transformation-defective, replication-competent viruses confirmed the expression of functionally defective Myc proteins. Further, expression of the transformation defective variant dl91-137 in chicken fibroblasts inhibited subsequent transformation by wild-type MC29. The results reported herein support the hypothesis that Myc proteins function as regulators of transcription in a variety of cell types and clearly point out the necessity of putative regulatory domains within the amino-terminal half of the Myc protein.

The expression of ras and myc oncogene proteins was examined in formalin-fixed paraffin-embedded sections of male Sprague-Dawley rat liver at various times after liver regeneration was induced by either a necrogenic dose of CCl 4 or a 2/3 partial hepatectomy. Antibody specific for these proteins and immuno-histochemical (IHC) procedures were used to detect oncogene expression in the liver. Increased expression of both oncogene proteins was detected at times comparable to those reported by others using either Northern or Western blot methods. However, the IHC procedure provided additional information since oncogene expression was detected only in hepatocytes, mainly occupying liver lobule zones known to contain proliferating hepatocytes during regeneration. Also, ras and myc stain intensity of individual hepatocytes did not increase during regeneration. Rather, a greater number of hepatocytes were positive at several time points in the regeneration process. This suggested that the increased ras and myc expression detected in Northern and Western blot methods probably represents a greater number of hepatocytes expressing the oncogene proteins rather than increased expression by individual hepatocytes. Overall, this study provides additional information on the cells involved in the expression of the ras and myc oncogenes during regeneration and further substantiates their role in a normal physiological process.

Myc, a key regulator of cellular proliferation, differentiation and apoptosis, exerts its biological functions by activating or suppressing the transcription of specific sets of target genes. C/EBP transcription factors play important roles during differentiation of various cell types and have been identified as critical targets for v-Myc- and c-Myc-dependent suppression of myeloid and fat cell differentiation. Here, we have addressed the mechanism by which v-Myc suppresses the activity of C/EBPbeta. We show that v-Myc is recruited to the aminoterminal domain of C/EBPbeta and interferes with the cooperation of C/EBPbeta and the co-activator p300 by preventing C/EBPbeta-induced phosphorylation of p300. We have identified the protein kinase responsible for C/EBPbeta-induced phosphorylation of p300 as homeo-domain interacting protein kinase 2 (HIPK2) and show that v-Myc displaces the kinase from the C/EBPbeta-p300 complex. Overall, our findings that the modulation of the C/EBPbeta-induced phosphorylation of p300 as a new mechanism of transcriptional suppression by v-Myc.

Co-amplification at chromosomes 8p11-8p12 and 11q12-11q14 occurs often in breast tumors, suggesting possible cooperation between genes in these regions in oncogenesis. We used high-resolution array comparative genomic hybridization (array CGH) to map the minimal amplified regions. The 8p and 11q amplicons are complex and consist of at least four amplicon cores at each site. Candidate oncogenes mapping to these regions were identified by combining copy number and RNA and protein expression analyses. These studies also suggested that CCND1 at 11q13 induced expression of ZNF703 mapping at 8p12, which was subsequently shown to be mediated by the Rb/E2F pathway. Nine candidate oncogenes from 8p12 and four from 11q13 were further evaluated for oncogenic function. None of the genes individually promoted colony formation in soft agar or collaborated with each other functionally. On the other hand, FGFR1 and DDHD2 at 8p12 cooperated functionally with MYC, whereas CCND1 and ZNF703 cooperated with a dominant negative form of TP53. These observations highlight the complexity and functional consequences of the genomic rearrangements that occur in these breast cancer amplicons, including transcriptional cross-talk between genes in the 8p and 11q amplicons, as well as their cooperation with major pathways of tumorigenesis.