Myc-dependent Cell Research Articles

377 - Myc-dependent cell cycle progression through the activation of CDK1 and phosphorylation of p27

377 - Myc-dependent cell cycle progression through the activation of CDK1 and phosphorylation of p27

Proteasomal Inhibition by Ixazomib Induces CHK1 and MYC-Dependent Cell Death in T-cell and Hodgkin Lymphoma.

Proteasome-regulated NF-κB has been shown to be important for cell survival in T-cell lymphoma and Hodgkin lymphoma models. Several new small-molecule proteasome inhibitors are under various stages of active preclinical and clinical development. We completed a comprehensive preclinical examination of the efficacy and associated biologic effects of a second-generation proteasome inhibitor, ixazomib, in T-cell lymphoma and Hodgkin lymphoma cells and in vivo SCID mouse models. We demonstrated that ixazomib induced potent cell death in all cell lines at clinically achievable concentrations. In addition, it significantly inhibited tumor growth and improved survival in T-cell lymphoma and Hodgkin lymphoma human lymphoma xenograft models. Through global transcriptome analyses, proteasomal inhibition showed conserved overlap in downregulation of cell cycle, chromatin modification, and DNA repair processes in ixazomib-sensitive lymphoma cells. The predicted activity for tumor suppressors and oncogenes, the impact on "hallmarks of cancer," and the analysis of key significant genes from global transcriptome analysis for ixazomib strongly favored tumor inhibition via downregulation of MYC and CHK1, its target genes. Furthermore, in ixazomib-treated lymphoma cells, we identified that CHK1 was involved in the regulation of MYC expression through chromatin modification involving histone H3 acetylation via chromatin immunoprecipitation. Finally, using pharmacologic and RNA silencing of CHK1 or the associated MYC-related mechanism, we demonstrated synergistic cell death in combination with antiproteasome therapy. Altogether, ixazomib significantly downregulates MYC and induces potent cell death in T-cell lymphoma and Hodgkin lymphoma, and we identified that combinatorial therapy with anti-CHK1 treatment represents a rational and novel therapeutic approach. Cancer Res; 76(11); 3319-31. ©2016 AACR.

PCYT1A in MYC-Driven Lymphoma: Role on Aberrant Choline Metabolism and Therapeutic Targeting By Berberine

Malignant lymphoma is a heterogenous group of diseases varied from pathological features, immunophenotypes and genetic characteristics. Although modern chemotherapy has remarkably improved the prognosis of the patients, targeted therapy on actionable tumor biomarkers, irrespective of pathological subtypes, remains great interests in lymphoma.MYC was initially uncovered as the target of the t(8;14)(q24;q32) chromosome translocation in Burkitt lymphoma and subsequently identified as a master regulator in lymphoma progression. Here in patients with diffuse large B-cell lymphoma (DLBCL, n=108) and peripheral T-cell lymphoma (PTCL, n=40), MYC gene expression was significantly higher than that of reactive hyperplasia, in consistent with increased MYC protein expression (>40% by immunohistochemistry, Figure 1A-B). Moreover, pharmacological silencing of MYC using specific MYC inhibitor 10058-F4 induced B- and T-lymphoma cell growth inhibition, G0/G1 cell cycle arrest, apoptosis and necroptosis (Figure 1C-D). Therefore, MYC was overexpressed and biologically functional in main lymphoma subtypes like DLBCL and PTCL.However, how to specifically target the unique dependencies of MYC-addicted malignant lymphocytes without influencing their normal counterparts warrants further investigation. Growing evidences showed that activation of oncogene reprograms tumor cell metabolism. Thus, serum metabolomic analysis was performed in 33 DLBCL patients, 24 PTCL patients and 50 healthy volunteers referred as the normal control. The results showed that dysregulation of choline metabolism occurred in both subtypes of lymphoma (Figure 2A-B). Importantly, using gene expression profile in frozen tissue samples of 9 DLBCL and 8 PTCL patients, distinct gene pattern of choline-related metabolic pathways was revealed according to MYC expression (Figure 2C). As mechanism of action, further luciferase and ChIP assay demonstrated that MYC modulated choline metabolism through transcriptionally activating choline-related enzyme phosphate cytidylyltransferase 1 choline alpha (PCYT1A) (Figure 2D-E).Increased expression of PCYT1A was observed in DLBCL and PTCL patients with higher tumor MYC expression, in accordingly with lower serum downstream choline metabolite phosphatidylcholine (Figure 3A-B) and International Prognostic Index indicating intermediate-high and high-risk. Meanwhile, molecular silencing of PCYT1A by siRNA inhibited lymphoma cell growth through inducing necroptotic cell death (Figure 3C). Lipid-lowering agent Berberine is an isoquinoline alkaloid isolated from several Chinese herbal medicines and exhibits anti-cancer effect in solid tumors. Interestingly, MYC-overexpressing lymphoma cells, resistant to chemotherapeutic agent doxorubicin, were sensitive to Berberine, which could downregulate PCYT1A expression, inhibit lymphoma cell growth and induce lymphoma cell necroptosis (Figure 3D-F).Collectively, our data proved a direct link between MYC oncoprotein with aberrant choline metabolism in DLBCL and PTCL, highlighting the potential clinical utility of targeting MYC-dependent tumor cell metabolism in lymphoma. As a lipid-modifying enzyme, PCYT1A was controlled by MYC and attributed to dysregulation of choline metabolism. Functioned as a sensitive biomarker of lymphoma progression, PCYT1A could be targeted by lipid-lowering agent Berberine, providing a novel therapeutic strategy in treating MYC-driven lymphoma. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract A23: Identification of KJ-Pyr-9 as a potent MYC inhibitor

Abstract We have identified a novel small-molecule inhibitor of MYC, KJ-Pyr-9, from a Kröhnke pyridine library. KJ-Pyr-9 functions as an inhibitor of MYC in biochemical assays, in cells and in xenograft experiments. In vitro, KJ-Pyr-9 binds to MYC as well as MYC-MAX dimers and can disrupt binding of MYC-MAX dimers with E-box DNA. In cells, MYC-MAX dimerization can be disrupted in cells as demonstrated by protein fragment complementation assays. KJ-Pyr-9 disrupts the transcriptional targets of MYC. The P493-6 cell line has tetracycline regulatable MYC expression. For this cell line, the transcriptional targets of MYC can be readily observed upon the addition of 0.1 ug/mL doxycycline. Both doxycycline and KJ-Pyr-9 negatively regulate MYC transcriptional signatures as determined by GSEA. The growth of a variety of different MYC-dependent cell lines is inhibited, including MYC-translocated Burkitt's lymphoma cell lines expressing high levels of MYC as well as solid tumor cell lines expressing moderate amounts of MYC. In vivo, KJ-Pyr-9 effectively blocks the growth of a xenotransplant of MYC-amplified human cancer cells. Acknowledgements: This work was supported by the National Cancer Institute under award number R01 CA078230 (PKV), The Skaggs Institute for Chemical Biology (KDJ) and by the Austrian Science Fund (FWF) grant P23652 (KB). This is manuscript 25089 of The Scripps Research Institute. Citation Format: Jonathan R. Hart, Klaus Bister, Kim D. Janda, Peter K. Vogt. Identification of KJ-Pyr-9 as a potent MYC inhibitor. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A23.

Abstract 965: Identification of KJ-Pyr-9 as a potent MYC inhibitor

Abstract We have identified a novel small-molecule inhibitor of MYC, KJ-Pyr-9, from a Kröhnke pyridine library. KJ-Pyr-9 functions as an inhibitor of MYC in biochemical assays, in cells and in xenograft experiments. In vitro, KJ-Pyr-9 binds to MYC as well as MYC-MAX dimers and can disrupt binding of MYC-MAX dimers with E-box DNA. In cells, MYC-MAX dimerization can be disrupted in cells as demonstrated by protein fragment complementation assays. KJ-PYR-9 disrupts the transcriptional targets of MYC. The P493-6 cell line has tetracycline regulatable MYC expression. For this cell line, the transcriptional targets of MYC can be readily observed upon the addition of 0.1 ug/mL doxycycline. Both doxycycline and KJ-PYR-9 negatively regulate MYC transcriptional signatures as determined by GSEA. The growth of a variety of different MYC-dependent cell lines are inhibited, including MYC translocated Burkitt's lymphoma cell lines expressing high levels of MYC as well as solid tumor cell lines expressing moderate amounts of MYC. In vivo, KJ-Pyr-9 effectively blocks the growth of a xenotransplant of MYC-amplified human cancer cells. Citation Format: Jonathan R. Hart, Klaus Bister, Kim D. Janda, Peter K. Vogt. Identification of KJ-Pyr-9 as a potent MYC inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 965. doi:10.1158/1538-7445.AM2015-965

Reversible linkage of two distinct small molecule inhibitors of Myc generates a dimeric inhibitor with improved potency that is active in myc over-expressing cancer cell lines.

We describe the successful application of a novel approach for generating dimeric Myc inhibitors by modifying and reversibly linking two previously described small molecules. We synthesized two directed libraries of monomers, each comprised of a ligand, a connector, and a bioorthogonal linker element, to identify the optimal dimer configuration required to inhibit Myc. We identified combinations of monomers, termed self-assembling dimeric inhibitors, which displayed synergistic inhibition of Myc-dependent cell growth. We confirmed that these dimeric inhibitors directly bind to Myc blocking its interaction with Max and affect transcription of MYC dependent genes. Control combinations that are unable to form a dimer do not show any synergistic effects in these assays. Collectively, these data validate our new approach to generate more potent and selective inhibitors of Myc by self-assembly from smaller, lower affinity components. This approach provides an opportunity for developing novel therapeutics against Myc and other challenging protein:protein interaction (PPI) target classes.

Abstract 2957: Inhibition of glutaminase induces slows tumor growth cell autonomously and promotes survival in a MYC driven hepatocellular carcinoma mouse model

Abstract Glutamine catabolism has been shown to be critical in many cancer types, particularly in MYC dependent tumor types. Given that MYC dependent cell lines have been shown to be dependent on glutamine for growth and survival, we hypothesize that inhibiting glutamine metabolism could slow the growth of MYC driven tumors in vivo. Small molecule (BPTES) inhibition of kidney-type glutaminase (GLS) is being developed as an anti-cancer therapy, yet basic questions remained unanswered about glutamine inhibition. The BPTES insensitive liver-type glutaminase (GLS2) has been proposed to act as a tumor suppressor in liver cancer cell lines. We have found that hepatocellular carcinomas downregulate GLS2 and upregulate GLS compared to surrounding tissue in both humans and mice, leaving hepatocellular carcinomas potentially sensitive to GLS inhibition. BPTES slows the growth of hepatocellular carcinoma cell lines in vitro. Here were report the first study that demonstrates the ability of BPTES to slow tumor progression in genetic cancer models in immunocompetent mice, showing the BPTES prolongs survival of mice in a MYC driven hepatocellular carcinoma model. However, we could not determine from these data if the anti-cancer effects of GLS inhibition in vivo were systemic or cell autonomous, causing us to switch to a xenograft system. Using prostate and lymphoma cell lines overexpressing the BPTES resistant GLS K325A mutant, we show that BPTES functions through on-target inhibition of GLS in vitro. We then used a p493-6 B-cell lymphoma cell line to determine the cell autonomous effects of BPTES in vivo. P493-6 xenograft growth was slowed by BPTES treatment, while p493 xenografts overexpressing either wild type GLS or BPTES resistant GLS K325A were not slowed by BPTES treatment in vivo. We then set out to develop an alternative method to perform tumor specific knock down GLS in vivo to obtain genetic evidence of the role of GLS in tumor cells, developing a human specific vivo-morpholino that targets GLS for nonsense-mediated decay. Consistent with the effect of BPTES on tumor cells, the human GLS targeting vivo-morpholino slowed p493-6 xenograft growth in vivo. As the vivo-morpholino knockdown of GLS was xenograft specific, it demonstrates that GLS inhibition slows cancer growth through cancer cell autonomous methods and not though systemic changes that alter the tumor niche. In summary, we find that BPTES exhibits on target GLS inhibition in vivo to prolong survival in mouse hepatocellular carcinoma mouse models. Citation Format: Zachary Stine, Yan Xiang, Jinsong Xia, Ping Gao, Ramani Dinavahi, Chi V. Dang. Inhibition of glutaminase induces slows tumor growth cell autonomously and promotes survival in a MYC driven hepatocellular carcinoma mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2957. doi:10.1158/1538-7445.AM2014-2957

Abstract 1629: Identification and biological characterization of a novel class of small molecules to inhibit c-myc transcription

Abstract The transcription factor c-myc, deregulated in ∼70% of all cancer cases, facilitates tumor initiation and promotion most frequently in colon, breast, lung, ovarian, and lymphoid malignancies, and acts as a universal amplifier of aberrant growth-related cell signaling. Due to its helix-loop helix topology, short protein half-life, and rapid replenishment within the cancer cell, c-myc was long considered to be ‘undruggable.’ Recently, much attention has focused on inhibiting c-myc either through indirect pathway modulation, or on the transcriptional level. 85-90% of c-myc gene expression is controlled by a G-quadruplex (G4) DNA structure found in the nuclease hypersensitive element III(1) region (NHEIII1) of its promoter, which, when stabilized with a chemical agent, would theoretically inhibit myc transcription by preventing normal polymerase function. In this study, we report the identification and biological characterization of a novel chemical class to directly bind the c-myc G4 DNA. The chemical motif discussed here was identified from a highly selective (hit rate of 0.16%, with few false positives) small molecule microarray screen of 20,000 compounds. A panel of the 12 most promising hits was chosen for biological and biochemical secondary validation in a variety of in vitro and cell based assays to confirm mechanism of action and functional effects in biological systems. Of note, numerous compounds from the initial panel of hits exhibited effects with a range of potency in comparison with untreated and non quadruplex forming nucleic acid controls, thereby validating the original screening method. For prioritization, hits were further evaluated for their capacity to preferentially induce myc-dependent cell death in a tetracycline controlled c-myc stable cell line. A top lead was identified, and structure-activity relationship was preliminarily evaluated with a small panel of analogues, leading to a final compound with three times greater potency. The chosen compound exhibited strong dose-dependent cell killing activity, in human multiple myeloma with high levels of c-myc, with an IC50 in the single digit micromolar range. Additionally, the compound reduced c-myc gene expression in a series of B cell neoplasms to near the limit of detection, both transcriptionally and translationally. The mechanism of action was confirmed in a Burkitt's Lymphoma model through a quadruplex promoter-specific comparative analysis of myc transcript copy numbers in the presence or absence of treatment. Continuing studies will focus on further structural optimization of the ligand, a thorough examination of its genome-wide effects, and tolerability in animal models. Citation Format: Kenneth M. Felsenstein, John K. Simmons, Peter Gareiss, Beverly A. Mock, John ‘Jay’ S. Schneekloth. Identification and biological characterization of a novel class of small molecules to inhibit c-myc transcription. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1629. doi:10.1158/1538-7445.AM2014-1629

Antagonistic effects of MYC and hypoxia in channeling glucose and glutamine into de novo nucleotide biosynthesis

Background Cell proliferation requires up regulation of nucleotide biosynthesis for making new DNA and RNA to support protein bio-synthesis. MYC is a major transcription factor that regulates metabolic processes essential for cell division, and is overexpressed in many cancers. The nutrient sources and integration of the metabolic pathways for nucleotide biosynthesis that enable MYC-dependent cell division are poorly defined. Using Stable Isotope Resolved Metabolomics (SIRM) we have determined the fate of atoms from C6-glucose, C5, N2-glutamine, or H-glycine into nucleotides under varied conditions of MYC expression in the MYC-inducible P493-6 B-lymphocyte [2] and several lung cell lines.

Targeting cyclin-dependent kinase 1 (CDK1) but not CDK4/6 or CDK2 is selectively lethal to MYC-dependent human breast cancer cells

BackgroundAlthough MYC is an attractive therapeutic target for breast cancer treatment, it has proven challenging to inhibit MYC directly, and clinically effective pharmaceutical agents targeting MYC are not yet available. An alternative approach is to identify genes that are synthetically lethal in MYC-dependent cancer. Recent studies have identified several cell cycle kinases as MYC synthetic-lethal genes. We therefore investigated the therapeutic potential of specific cyclin-dependent kinase (CDK) inhibition in MYC-driven breast cancer.MethodsUsing small interfering RNA (siRNA), MYC expression was depleted in 26 human breast cancer cell lines and cell proliferation evaluated by BrdU incorporation. MYC-dependent and MYC-independent cell lines were classified based on their sensitivity to siRNA-mediated MYC knockdown. We then inhibited CDKs including CDK4/6, CDK2 and CDK1 individually using either RNAi or small molecule inhibitors, and compared sensitivity to CDK inhibition with MYC dependence in breast cancer cells.ResultsBreast cancer cells displayed a wide range of sensitivity to siRNA-mediated MYC knockdown. The sensitivity was correlated with MYC protein expression and MYC phosphorylation level. Sensitivity to siRNA-mediated MYC knockdown did not parallel sensitivity to the CDK4/6 inhibitor PD0332991; instead MYC-independent cell lines were generally sensitive to PD0332991. Cell cycle arrest induced by MYC knockdown was accompanied by a decrease in CDK2 activity, but inactivation of CDK2 did not selectively affect the viability of MYC-dependent breast cancer cells. In contrast, CDK1 inactivation significantly induced apoptosis and reduced viability of MYC-dependent cells but not MYC- independent cells. This selective induction of apoptosis by CDK1 inhibitors was associated with up-regulation of the pro-apoptotic molecule BIM and was p53-independent.ConclusionsOverall, these results suggest that further investigation of CDK1 inhibition as a potential therapy for MYC-dependent breast cancer is warranted.

Myc-Driven Overgrowth Requires Unfolded Protein Response-Mediated Induction of Autophagy and Antioxidant Responses in Drosophila melanogaster

Autophagy, a lysosomal self-degradation and recycling pathway, plays dual roles in tumorigenesis. Autophagy deficiency predisposes to cancer, at least in part, through accumulation of the selective autophagy cargo p62, leading to activation of antioxidant responses and tumor formation. While cell growth and autophagy are inversely regulated in most cells, elevated levels of autophagy are observed in many established tumors, presumably mediating survival of cancer cells. Still, the relationship of autophagy and oncogenic signaling is poorly characterized. Here we show that the evolutionarily conserved transcription factor Myc (dm), a proto-oncogene involved in cell growth and proliferation, is also a physiological regulator of autophagy in Drosophila melanogaster. Loss of Myc activity in null mutants or in somatic clones of cells inhibits autophagy. Forced expression of Myc results in cell-autonomous increases in cell growth, autophagy induction, and p62 (Ref2P)-mediated activation of Nrf2 (cnc), a transcription factor promoting antioxidant responses. Mechanistically, Myc overexpression increases unfolded protein response (UPR), which leads to PERK-dependent autophagy induction and may be responsible for p62 accumulation. Genetic or pharmacological inhibition of UPR, autophagy or p62/Nrf2 signaling prevents Myc-induced overgrowth, while these pathways are dispensable for proper growth of control cells. In addition, we show that the autophagy and antioxidant pathways are required in parallel for excess cell growth driven by Myc. Deregulated expression of Myc drives tumor progression in most human cancers, and UPR and autophagy have been implicated in the survival of Myc-dependent cancer cells. Our data obtained in a complete animal show that UPR, autophagy and p62/Nrf2 signaling are required for Myc-dependent cell growth. These novel results give additional support for finding future approaches to specifically inhibit the growth of cancer cells addicted to oncogenic Myc.

EGFR Mutation-Induced Alternative Splicing of Max Contributes to Growth of Glycolytic Tumors in Brain Cancer

Alternative splicing contributes to diverse aspects of cancer pathogenesis including altered cellular metabolism, but the specificity of the process or its consequences are not well understood. We characterized genome-wide alternative splicing induced by the activating EGFRvIII mutation in glioblastoma (GBM). EGFRvIII upregulates the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 splicing factor, promoting glycolytic gene expression and conferring significantly shorter survival in patients. HnRNPA1 promotes splicing of a transcript encoding the Myc-interacting partner Max, generating Delta Max, an enhancer of Myc-dependent transformation. Delta Max, but not full-length Max, rescues Myc-dependent glycolytic gene expression upon induced EGFRvIII loss, and correlates with hnRNPA1 expression and downstream Myc-dependent gene transcription in patients. Finally, Delta Max is shown to promote glioma cell proliferation in vitro and augment EGFRvIII expressing GBM growth in vivo. These results demonstrate an important role for alternative splicing in GBM and identify Delta Max as a mediator of Myc-dependent tumor cell metabolism.

OX40 engagement stabilizes Mxd4 and Mnt protein levels in antigen‐stimulated T cells leading to an increase in cell survival

OX40 engagement on activated T cells leads to increased proliferation, expansion and survival of Ag-specific T cells. Direct ex vivo examination of Ag-stimulated murine T cells show that the Myc antagonists, Mxd4 and Mnt, are transiently upregulated and translocated to the nucleus following OX40 engagement and may be involved in suppressing cell death. Both Mxd4 and Mnt are upregulated following OX40 stimulation through increased protein stability and we identify a critical phosphorylation site in Mxd4 that controls Mxd4 stability. The upregulation of Mxd4 and Mnt contributes to OX40-mediated T-cell survival because siRNA knockdown of Mxd4 and Mnt led to increased cell death. We hypothesize the upregulation of c-Myc following OX40 engagement drives T-cell proliferation and that upregulation of Mxd4 and Mnt suppresses Myc-dependent cell death. Thus, Mxd4 and Mnt upregulation following OX40 engagement most likely increases T-cell survival.

Stabilizers of the Max Homodimer Identified in Virtual Ligand Screening Inhibit Myc Function

Many human cancers show constitutive or amplified expression of the transcriptional regulator and oncoprotein Myc, making Myc a potential target for therapeutic intervention. Here we report the down-regulation of Myc activity by reducing the availability of Max, the essential dimerization partner of Myc. Max is expressed constitutively and can form unstable homodimers. We have isolated stabilizers of the Max homodimer by applying virtual ligand screening (VLS) to identify specific binding pockets for small molecule interactors. Candidate compounds found by VLS were screened by fluorescence resonance energy transfer, and from these screens emerged a potent, specific stabilizer of the Max homodimer. In vitro binding assays demonstrated that the stabilizer enhances the formation of the Max-Max homodimer and interferes with the heterodimerization of Myc and Max in a dose-dependent manner. Furthermore, this compound interferes with Myc-induced oncogenic transformation, Myc-dependent cell growth, and Myc-mediated transcriptional activation. The Max-Max stabilizer can be considered a lead compound for the development of inhibitors of the Myc network.

Hypertrophic growth in cardiac myocytes is mediated by Myc through a Cyclin D2-dependent pathway

c-Myc (Myc) is highly expressed in developing embryos where it regulates body size by controlling proliferation but not cell size. However, Myc is also induced in many postmitotic tissues, including adult myocardium, in response to stress where the predominant form of growth is an increase in cell size (hypertrophy) and not number. The function of Myc induction in this setting is unproven. Therefore, to explore Myc's role in hypertrophic growth, we created mice where Myc can be inducibly inactivated, specifically in adult myocardium. Myc-deficient hearts demonstrated attenuated stress-induced hypertrophic growth, secondary to a reduction in cell growth of individual myocytes. To explore the dependence of Myc-induced cell growth on CycD2, we created bigenic mice where Myc can be selectively activated in CycD2-null adult myocardium. Myc-dependent hypertrophic growth and cell cycle reentry is blocked in CycD2-deficient hearts. However, in contrast to Myc-induced DNA synthesis, hypertrophic growth is independent of CycD2-induced Cdk2 activity. These data suggest that Myc is required for a normal hypertrophic response and that its growth-promoting effects are also mediated through a CycD2-dependent pathway.

A Novel Myc-target Gene, mimitin, That Is Involved in Cell Proliferation of Esophageal Squamous Cell Carcinoma

Myc is a ubiquitous mediator of cell proliferation that transactivates the expression of various genes through E-box sites. Here we report a novel gene, mimitin (Myc-induced mitochondrial protein), that encodes a mitochondrial protein with a molecular mass of 20 kDa. We demonstrated that the transcription of mimitin is directly stimulated by c-Myc. To investigate the role of Mimitin, its expression was suppressed by the RNA interference (RNAi) technique. Whereas specific inhibition of mimitin expression did not affect cell proliferation in human cervical carcinoma, colon adenocarcinoma, and hepatocarcinoma cell lines, it did suppress cell proliferation in human glioblastoma, esophageal squamous cell carcinoma (ESCC), and embryonic lung fibroblastic cells, with the greatest suppression efficiency in ESCC cells. To investigate whether mimitin is related to tumorigenesis in ESCC in vivo, the expression of Mimitin protein in ESCC tissues was studied. Mimitin was highly expressed in 80% (28 of 35) of ESCC tumors, suggesting that high expression of Mimitin is a characteristic feature of ESCC. The expression level of Mimitin was found to be correlated with that of c-Myc and cell proliferation, but not with the histopathological grade, stage of cancer, or age of patients. Taken together, these results suggest that the novel gene mimitin is a direct transcriptional target of c-Myc, and is involved in Myc-dependent cell proliferation at least in ESCC cells.

Loss of the Max-interacting protein Mnt in mice results in decreased viability, defective embryonic growth and craniofacial defects: relevance to Miller-Dieker syndrome.

The Mnt gene encodes a Mad-family bHLH transcription factor located on human 17p13.3. Mnt is one of 20 genes deleted in a heterozygous fashion in Miller-Dieker syndrome (MDS), a contiguous gene syndrome that consists of severe neuronal migration defects and craniofacial dysmorphic features. Mnt can inhibit Myc-dependent cell transformation and is hypothesized to counterbalance the effects of c-Myc on growth and proliferation in vivo by competing with Myc for binding to Max and by repressing target genes activated by Myc : Max heterodimers. Unlike the related Mad family members, Mnt is expressed ubiquitously and Mnt/Max heterodimers are found in proliferating cells that contain Myc/Max heterodimers, suggesting a unique role for Mnt during proliferation. To examine the role of Mnt in vivo, we produced mice with null (Mnt(KO)) and loxP-flanked conditional knock-out (Mnt(CKO)) alleles of Mnt. Virtually all Mnt(KO/KO) mutants in a mixed (129S6 x NIH Black Swiss) or inbred (129S6) genetic background died perinatally. Mnt-deficient embryos exhibited small size throughout development and showed reduced levels of c-Myc and N-Myc. In addition, 37% of the mixed background mutants displayed cleft palate as well as retardation of skull development, a phenotype not observed in the inbred mutants. These results demonstrate an important role for Mnt in embryonic development and survival, and suggest that Mnt may play a role in the craniofacial defects displayed by MDS patients.

Id2 is dispensable for Myc-induced epidermal neoplasia.

We have previously described a transgenic mouse model of epidermal neoplasia wherein expression of a switchable form of c-Myc, MycER(TAM), is targeted to the postmitotic suprabasal keratinocytes of murine epidermis via the involucrin promoter. Sustained activation of c-MycER(TAM) results in a progressive neoplastic phenotype characterized by aberrant ectopic proliferation and delayed differentiation of suprabasal keratinocytes, culminating in papillomatosis. Transcription of the Id2 gene is regulated by Myc family proteins. Moreover, Id2 is implicated as a pivotal determinant of cell fate in multiple lineages and has a demonstrated role in mediating Myc-dependent cell proliferation in vitro through its interaction with retinoblastoma protein. Using Id2 nullizygous mice, we assessed in vivo the requirement for Id2 in mediating Myc-induced papilloma formation in skin. We show that absence of Id2 has no discernible impact on any measurable attribute of Myc function or on the timing or extent of eventual tumor formation. Thus, our data argue against any essential role for Id2 in mediating Myc action in vivo.

HMad4, c-Myc endogenous inhibitor, induces a replicative senescence-like state when overexpressed in human fibroblasts.

Mad family proteins have an antagonistic action on Myc-dependent cell proliferation and transformation. We isolated a human cDNA clone, human Mad4 (hMad4), encoding a polypeptide of 209 amino acid residues, exhibiting 90% identity with mouse Mad4. Northern blot analysis shows that hMad4 probe hybridizes to a 3.8 kb message; its expression is highest in quiescent human WI38 fibroblasts. Among tissues, hMad4 mRNA is most abundant in brain, lung, and muscle. Consistent with other members of the Mad family, hMad4 can repress the transactivation activity of Myc/Max heterodimers on an E-box chloramphenicol acteyl transferase (CAT) reporter plasmid; inhibition of both proliferation and clonogenic formation of hMad4-infected cells correlates with the in vitro reporter repression. Moreover, infection of young human fibroblasts induces a replicative senescence-like state. This phenotype was accompanied by s-beta-galactosidase and PAI-1 expression. These results suggest that hMad4 might be an important regulator of replicative senescence in human cells.

Complementation of Myc-dependent cell proliferation by cDNA expression library screening.

The targeted knockout of the c-myc gene from rat fibroblasts leads to a stable defect in cell proliferation. We used complex cDNA libraries expressed from retroviral vectors and an efficient sorting procedure to rapidly select for cDNAs that can restore the growth rate of c-myc deficient cells. All of the biologically active cDNAs contained either c-myc or N-myc, suggesting that no other cellular genes can effectively bypass the requirement for c-myc in fibroblast proliferation. This approach provides a powerful screening method for cell cycle changes in genetically defined systems.