c-Myc Function Research Articles

Abstract 4902: Targeting APC mutant colon cancer cells by chloroquine

Abstract The adenomatous polyposis coli (APC) tumor suppressor is mutated in approximately 80% of sporadic colorectal cancers (CRC) where it is recognized as the critical initiating event. Genetic studies revealed that c-Myc is the crucial mediator of the early stages of tumor development following APC loss. One of the key functions of c-Myc is to repress p21 and thus switches to p53-mediated apoptosis in cancer cells. We therefore hypothesize that p21 might be an attractive target for killing APC-deficient human colon cancer cells preferentially. Here we first utilized p21-deficient human colon cancer cell line (p21−/−HCT116) to elucidate the role of p21 in colon cancer cells for apoptosis. Chloroquine (CQ), a small molecule inhibitor of autophagy, was identified to preferentially initiate p53-PUMA-dependent apoptosis in p21−/−HCT116 cells, but not in isogenic p21+/+HCT116 cells. Although CQ inhibits the late stages of autophagy by targeting lysosmoal degradation, however, the molecular basis of CQ-mediated cancer cell death remains unclear. We found that blockade of the early stage of autophagy by individual knockdown of genes involved in autophagy (Atg5, Atg7, Beclin1 and ULK1) attenuated CQ-induced accumulation of autophagosomes and apoptosis in p21−/−HCT116 cells. In addition, we showed that reactive oxygen species (ROS)-initiated autophagy was essential for CQ-mediated cancer cell death. These results suggest that inhibition of autophagy at a late stage by CQ can be detrimental to cancer cell survival when autophagy is initiated at early stage. To determine the specificity of p21 in mediating CQ-induced cancer cell death, we demonstrated that knockdown of p21 in p21+/+HCT116 cells sensitized, while ectopic expression of p21 in p21−/−HCT116 cells inhibited, CQ-induced apoptosis. Furthermore, administration of CQ in vivo significantly suppressed tumor growth in xenograft mouse model bearing the p21−/−HCT116 cells, but not the p21+/+HCT116 cells. Our data strongly support that inactivation of p21-mediated anti-apoptotic event is required for CQ to kill cancer cells and inhibit tumor growth in vivo. To examine whether what was observed in p21−/−HCT116 cells was also true for killing of APC mutant colon cancer cells due to suppression of p21 through Myc activation upon APC loss, we took shRNA-mediated knockdown approaches to effectively deplete APC in p21+/+HCT116 cells. Treatment of cancer cells with CQ specifically caused apoptosis and blocked p21 induction in APC depleted cancer cells, which correlated with a selective inhibition of tumor growth in xenograft model. Given that CQ has been safely used in patients for many years as anti-malarial and anti-rheumatoid therapies, our findings provide compelling rationale to consider clinic translational trials in colon cancer patients carrying APC mutations using lysosomotropic agents such as CQ. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4902. doi:1538-7445.AM2012-4902

Phosphoinositide 3-kinase/AKT/mTORC1/2 signaling determines sensitivity of Burkitt's lymphoma cells to BH3 mimetics.

Burkitt's lymphoma (BL), driven by translocation and overexpression of the c-MYC gene, is an aggressive, highly proliferative lymphoma, and novel therapeutic strategies are required to overcome drug resistance following conventional treatments. The importance of the prosurvival BCL-2 family member BCL-X(L) in BL cell survival suggests that antagonistic BH3-mimetic compounds may have therapeutic potential. Here, we show that treatment of BL cell lines with ABT-737 induces caspase-3/7 activation and apoptosis with varying potency. Using selective inhibitors, we identify phosphoinositide 3-kinase (PI3K) as a proproliferative/survival pathway in BL cells and investigate the potential of combined pharmacologic inhibition of both the BCL-2 family and PI3K signaling pathway. PI3K/AKT inhibition and ABT-737 treatment induced synergistic caspase activation, augmented BL cell apoptosis, and rendered chemoresistant cells sensitive. Targeting mTORC1/2 with PP242 was also effective, either as a monotherapy or, more generally, in combination with ABT-737. The combined use of a dual specificity PI3K/mTOR inhibitor (PI 103) with ABT-737 proved highly efficacious. PI 103 treatment of BL cells was associated with an increase in BIM/MCL-1 expression ratios and loss of c-MYC expression. Furthermore, blocking c-MYC function using the inhibitor 10058-F4 also induced apoptosis synergistically with ABT-737, suggesting that maintenance of expression of BCL-2 family members and/or c-MYC by the PI3K/AKT/mTOR pathway could contribute to BL cell survival and resistance to ABT-737. The combined use of BH3 mimetics and selective mTORC1/2 inhibitors may therefore be a useful novel therapeutic approach for the treatment of B-cell malignancy, including chemoresistant lymphomas.

The Max b-HLH-LZ Can Transduce into Cells and Inhibit c-Myc Transcriptional Activities

The inhibition of the functions of c-Myc (endogenous and oncogenic) was recently shown to provide a spectacular therapeutic index in cancer mouse models, with complete tumor regression and minimal side-effects in normal tissues. This was achieved by the systemic and conditional expression of omomyc, the cDNA of a designed mutant of the b-HLH-LZ of c-Myc named Omomyc. The overall mode of action of Omomyc consists in the sequestration of Max and the concomitant competition of the Omomyc/Max complex with the endogenous c-Myc/Max heterodimer. This leads to the inhibition of the transactivation of Myc target genes involved in proliferation and metabolism. While this body of work has provided extraordinary insights to guide the future development of new cancer therapies that target c-Myc, Omomyc itself is not a therapeutic agent. In this context, we sought to exploit the use of a b-HLH-LZ to inhibit c-Myc in a cancer cell line in a more direct fashion. We demonstrate that the b-HLH-LZ domain of Max (Max*) behaves as a bona fide protein transduction domain (PTD) that can efficiently transduce across cellular membrane via through endocytosis and translocate to the nucleus. In addition, we show that the treatment of HeLa cells with Max* leads to a reduction of metabolism and proliferation rate. Accordingly, we observe a decrease of the population of HeLa cells in S phase, an accumulation in G1/G0 and the induction of apoptosis. In agreement with these phenotypic changes, we show by q-RT-PCR that the treatment of HeLa cells with Max* leads to the activation of the transcription c-Myc repressed genes as well as the repression of the expression of c-Myc activated genes. In addition to the novel discovery that the Max b-HLH-LZ is a PTD, our findings open up new avenues and strategies for the direct inhibition of c-Myc with b-HLH-LZ analogs.

C-MYC and SIRT1 locked in a vicious cycle

The c-MYC proto-oncogene represents a nodal point of gene regulation, as it integrates numerous mitogenic incoming signals and converts them into enhanced synthesis of the c-MYC transcription factor, which orchestrates the expression of hundreds of genes [1]. Deregulation of c-MYC, as it is found in ~50% of all human cancers, not only induces cellular proliferation and growth but also leads to activation of the p53 tumor suppressor protein [2], which mediates apoptosis and/or senescence. This intrinsic fail-safe mechanism prevents expansion of cells harboring dangerous oncogenic mutations. The necessity to bypass this barrier is thought to explain why p53 is frequently mutated in human cancers of all types. However, ectopic expression of c-MYC may immortalize certain primary cells in the presence of wild-type p53, suggesting that c-MYC circumvents the action of p53 by alternative mechanisms. We hypothesized that c-MYC may activate a factor, which abrogates or down-regulates p53 function directly. An attractive candidate for this function was SIRT1, an NAD+-dependent deacetylase, which has been shown to inhibit the function of p53 [3]. Recently, four papers showed that c-MYC and SIRT1 regulate each other via feedback loops [4-7]. The three more recent publications suggest the existence of a positive feedback loop between c-MYC and SIRT1 [4-6], whereas a study published earlier entertains a negative feedback [7]. The positive feedback suggests an oncogenic role of SIRT1, whereas the negative feedback implies a tumor suppressive activity of SIRT1. In our hands activation of conditional c-MYC alleles resulted in a robust increase in SIRT1 protein levels and activity. This was mediated on one hand via the direct induction of the NAMPT gene, which encodes nicotinamide phosphoribosyltransferase, the rate limiting enzyme of the NAD+ salvage pathway. The resulting increased NAD+ levels are known to mediate the activation of SIRT1 [8]. A second mode of SIRT1 activation was through sequestration of the SIRT1 inhibitor DBC1 (deleted in breast cancer 1) by c-MYC. In c-MYC-immortalized cells the increased levels and enzymatic activity of SIRT1 were necessary to prevent senescence and c-MYC-induced apoptosis. Furthermore, c-MYC is bound and deacetylated by SIRT1 which resulted in reduced K48- and increased K63-ubiquitin linkage, and ultimately increased c-MYC stability and activity. In cancer cells deregulation of this reciprocal activation between c-MYC and SIRT1 presumably contributes to evasion of senescence and apoptosis [6]. Increased expression of SIRT1 mRNA in cells with elevated c-MYC or N-MYC levels suggested that c-MYC may transactivate the SIRT1 gene [5, 7]. Although we could not detect an increase in SIRT1 mRNA after c-MYC activation [6], a concomitant induction of SIRT1 mRNA and post-transcriptional activation of the SIRT1 enzyme by c-MYC, as described above, may act synergistically. In the three studies describing a positive feedback, this involves a direct physical interaction between c-MYC and SIRT1, leading to increased c-MYC function [4-6]. Only Yuan et al. observed that the interaction with SIRT1 reduced c-MYC stability and inhibited c-MYC functions, such as transformation and transactivation [7]. The region of MYC interacting with SIRT1 was reported to be either the C-terminus of c-MYC [4] or MYC-Box I within the N-terminus of N-MYC [5]. Although three studies document deacetylation of c-MYC as a result of the SIRT1/c-MYC interaction, there are considerable discrepancies with respect to the functional consequences of c-MYC deacetylation. In agreement with our own observations obtained for c-MYC, Marshall et al. report that the N-MYC protein is stabilized in the presence of SIRT1. However, this is mediated indirectly through a novel transcriptional repressor complex consisting of N-MYC and SIRT1, which down-regulates expression of mitogen-activated protein kinase phosphatase 3 (MKP3) and thereby increases ERK-mediated phosphorylation of N-MYC at serine 62. Mao et al. did not observe significant effects of SIRT1 on c-MYC protein levels. Furthermore, they found that SIRT1 binds to the C-terminus of c-MYC, which mediates heterodimerization with MAX. Interestingly, SIRT1 expression increased the c-MYC/MAX association and also enhanced c-MYC transcriptional activity. Taken together, the majority of the reported analyses (3 of 4) describe a positive feedback between c-MYC and SIRT1. Multiple mechanisms of c-MYC activation seem to be involved in this feedback. Indeed, the SIRT1-mediated increase in c-MYC expression and function by enhanced ubiquitin 63 linkage, serine 62 phosphorylation and c-MYC/MAX heterodimerization may occur in parallel and act synergistically. In conjunction with the inactivation of p53 and/or other tumor suppressive SIRT1 substrates, SIRT1 therefore seems to act pro-tumorigenic at least in the context of cells and tumors, which display alterations in c-MYC-activating pathways or c-MYC itself.

The c-MYC oncoprotein, the NAMPT enzyme, the SIRT1-inhibitor DBC1, and the SIRT1 deacetylase form a positive feedback loop

Silent information regulator 1 (SIRT1) represents an NAD(+)-dependent deacetylase that inhibits proapoptotic factors including p53. Here we determined whether SIRT1 is downstream of the prototypic c-MYC oncogene, which is activated in the majority of tumors. Elevated expression of c-MYC in human colorectal cancer correlated with increased SIRT1 protein levels. Activation of a conditional c-MYC allele induced increased levels of SIRT1 protein, NAD(+), and nicotinamide-phosphoribosyltransferase (NAMPT) mRNA in several cell types. This increase in SIRT1 required the induction of the NAMPT gene by c-MYC. NAMPT is the rate-limiting enzyme of the NAD(+) salvage pathway and enhances SIRT1 activity by increasing the amount of NAD(+). c-MYC also contributed to SIRT1 activation by sequestering the SIRT1 inhibitor deleted in breast cancer 1 (DBC1) from the SIRT1 protein. In primary human fibroblasts previously immortalized by introduction of c-MYC, down-regulation of SIRT1 induced senescence and apoptosis. In various cell lines inactivation of SIRT1 by RNA interference, chemical inhibitors, or ectopic DBC1 enhanced c-MYC-induced apoptosis. Furthermore, SIRT1 directly bound to and deacetylated c-MYC. Enforced SIRT1 expression increased and depletion/inhibition of SIRT1 reduced c-MYC stability. Depletion/inhibition of SIRT1 correlated with reduced lysine 63-linked polyubiquitination of c-Myc, which presumably destabilizes c-MYC by supporting degradative lysine 48-linked polyubiquitination. Moreover, SIRT1 enhanced the transcriptional activity of c-MYC. Taken together, these results show that c-MYC activates SIRT1, which in turn promotes c-MYC function. Furthermore, SIRT1 suppressed cellular senescence in cells with deregulated c-MYC expression and also inhibited c-MYC-induced apoptosis. Constitutive activation of this positive feedback loop may contribute to the development and maintenance of tumors in the context of deregulated c-MYC.

FOXO3a regulates reactive oxygen metabolism by inhibiting mitochondrial gene expression

Forkhead transcription factors of the O class (FOXOs) are important targets of the phosphatidylinositol 3-kinase/Akt pathway, and are key regulators of the cell cycle, apoptosis and response to oxidative stress. FOXOs have been shown to have tumour suppressor function and are important for stem cell maintenance. We have performed a detailed analysis of the transcriptional programme induced in response to Forkhead-box protein O3a (FOXO3a) activation. We observed that FOXO3a activation results in the repression of a large number of nuclear-encoded genes with mitochondrial function. Repression of these genes was mediated by FOXO3a-dependent inhibition of c-Myc. FOXO3a activation also caused a reduction in mitochondrial DNA copy number, expression of mitochondrial proteins, respiratory complexes and mitochondrial respiratory activity. FOXO3a has been previously implicated in the detoxification of reactive oxygen species (ROS) through induction of manganese-containing superoxide dismutase (SOD2). We observed that reduction in ROS levels following FOXO3a activation was independent of SOD2, but required c-Myc inhibition. Hypoxia increases ROS production from the mitochondria, which is required for stabilisation of the hypoxia-inducible factor-1α (HIF-1α). FOXO3a activation blocked the hypoxia-dependent increase in ROS and prevented HIF-1α stabilisation. Our data suggest that FOXO factors regulate mitochondrial activity through inhibition of c-Myc function and alter the hypoxia response.

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

How the proto-oncogene c-Myc balances the processes of stem cell self-renewal, proliferation and differentiation in adult tissues is largely unknown. We explored c-Myc’s transcriptional roles at the Epidermal Differentiation Complex (EDC) a locus essential for skin maturation. Binding of c-Myc can simultaneously recruit (Klf4, Ovo1-1) and displace (C/EBPα, Mxi1 and Sin3A) specific sets of differentiation-specific transcriptional regulators to EDC genes. We found that Sin3A causes de-acetylation of c-Myc protein to directly repress c-Myc activity. In the absence of Sin3A, genomic recruitment of c-Myc to the EDC is enhanced, and re-activation of c-Myc-target genes drives aberrant epidermal proliferation and differentiation. Simultaneous deletion of c-Myc and Sin3A reverts the skin phenotype to normal. Our results identify how the balance of two transcriptional key regulators can maintain tissue homeostasis via a negative feedback loop.

Inhibition of c-Myc Expression and Function in Hematologic Malignancies

Abstract 1409MYC is among the most prolific oncogenes in cancer, yet pharmacologic strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have undertaken to target c-Myc transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative co-activator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain-containing proteins as transcriptional regulatory factors for c-Myc. BET inhibition with JQ1 rapidly downregulates c-Myc transcription, followed by depletion of chromatin-bound c-Myc and genome-wide downregulation of Myc-dependent target genes. In translational model systems of multiple myeloma and Burkitt's lymphoma, both canonical MYC -dependent hematologic malignancies, JQ1 treatment produces a potent antiproliferative effect associated with cell cycle arrest. In multiple myeloma, adhesion to bone marrow stroma is associated with upregulation of BRD4, a BET bromomdomain coactivator protein. Inhibition of Myc function with JQ1 leads to impaired adhesion to stroma and cellular senescence, a classical Myc-specific phenotype. Mechanistically, JQ1 treatment depletes BRD4 from IgH enhancers, leading to prompt and robust downregulation of MYC transcription. In vivo efficacy of JQ1 in two disseminated models of multiple myeloma and in a Burkitt's lymphoma human xenograft establishes the therapeutic rationale for BET bromodomain inhibition in these diseases. Together, these studies identify a mechanistic rationale for targeting c-Myc in human cancer, and potentially other undruggable oncogenes driven by immunoglobulin rearrangement.Note: G.C.I. and J.E.D. have made equal contributions to this research; C.S.M. and J.E.B. are jointly senior authors Disclosures:Richardson:Millennium: Advisory Board; Celgene: Advisory Board; Johnson & Johnson: Advisory Board; Novartis: Advisory Board; Bristol Myers Squibb: Advisory Board. Ghobrial:Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Noxxon:; Millennium:; Celgene:; Novartis:. Anderson:Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Mitsiades:Millennium Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Kosan: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centocor: Consultancy, Honoraria; Amnis Therapeutics: Consultancy, Honoraria; PharmaMar:; OSI Pharmaceuticals: Research Funding; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding; Genzyme: Research Funding; Johnson & Johnson: Research Funding. Bradner:Acetylon Pharmaceuticals: Scientific Founder; SHAPE Pharmaceuticals: Scientific Founder; Tensha Therapeutics: Scientific Founder.

Role of Pirh2 in Mediating the Regulation of p53 and c-Myc

Ubiquitylation is fundamental for the regulation of the stability and function of p53 and c-Myc. The E3 ligase Pirh2 has been reported to polyubiquitylate p53 and to mediate its proteasomal degradation. Here, using Pirh2 deficient mice, we report that Pirh2 is important for the in vivo regulation of p53 stability in response to DNA damage. We also demonstrate that c-Myc is a novel interacting protein for Pirh2 and that Pirh2 mediates its polyubiquitylation and proteolysis. Pirh2 mutant mice display elevated levels of c-Myc and are predisposed for plasma cell hyperplasia and tumorigenesis. Consistent with the role p53 plays in suppressing c-Myc-induced oncogenesis, its deficiency exacerbates tumorigenesis of Pirh2−/− mice. We also report that low expression of human PIRH2 in lung, ovarian, and breast cancers correlates with decreased patients' survival. Collectively, our data reveal the in vivo roles of Pirh2 in the regulation of p53 and c-Myc stability and support its role as a tumor suppressor.

C-MYC Functions as a Molecular Switch to Alter the Response of Human Mammary Epithelial Cells to Oncostatin M

Cytokines play an important role in creating an inflammatory microenvironment, which is now considered a hallmark of cancer. Although tumor cells can exploit cytokine signaling to promote growth, invasion, and metastasis, the response of normal and premalignant epithelial cells to cytokines present in a developing tumor microenvironment remains unclear. Oncostatin M (OSM), an IL-6 family cytokine responsible for STAT3 activation, has been implicated in cancer development, progression, invasion, and metastasis. Paradoxically, OSM can also suppress the growth of normal cells and certain tumor-derived cell lines. Using isogenic human mammary epithelial cells (HMEC) at different stages of neoplastic transformation, we found that OSM signaling suppressed c-MYC expression and engaged a p16- and p53-independent growth arrest that required STAT3 activity. Inhibition of STAT3 activation by expressing a dominant-negative STAT3 protein or a STAT3-shRNA prevented the OSM-mediated arrest. In addition, expression of c-MYC from a constitutive promoter also abrogated the STAT3-mediated arrest, and strikingly, cooperated with OSM to promote anchorage-independent growth (AIG), a property associated with malignant transformation. Cooperative transformation by c-MYC and OSM required PI3K and AKT signaling, showing the importance of multiple signaling pathways downstream of the OSM receptor in defining the cellular response to cytokines. These findings identify c-MYC as an important molecular switch that alters the cellular response to OSM-mediated signaling from tumor suppressive to tumor promoting.

Abstract B63: Synergistic combination between resveratrol and tanshinone I for effective prevention of pancreatic cancer

Abstract Pancreatic cancer is the fourth most common cause of cancer-related mortality in the United States. Pancreatic cancer is often dubbed the silent killer because it seldom causes symptoms until advanced. As a result, few pancreatic cancers are operable at diagnosis. Furthermore, pancreatic cancer is often resistant to conventional radiotherapy or chemotherapy, and the morbidity rate almost equals the mortality rate. Therefore, search for effective regimens with moderate side effects for pancreatic cancer therapy and prevention is an urgent necessity. Chinese herbal medicines have been used for centuries with demonstrated safety profiles, thus their bioactive ingredients may be effective candidates for the prevention and/or treatment of pancreatic cancer. Danshen (Salvia miltiorrhiza) has been widely adopted in the traditional Chinese medicinal preparations for treatment of cardiovascular disease with minimal side effects. In the present study, we evaluated the effect of several major Danshen components, tanshinone IIA (T2A), tanshinone I (T1) and cryptotanshinone (CT) on pancreatic cancer cells. We found that CT, T2A and T1 all showed inhibitory effects on the growth of human pancreatic cancer cell lines MIA PaCa-2 and ASPC-1 with IC50 around 30μM, 3–8μM and 3–5μM, respectively. The drug-resistant human pancreatic cancer cell line PANC-1 showed less sensitivity to CT and T2A treatment but was very sensitive to T1 treatment with IC50 around 3.5μM. The potent T1 activity against pancreatic cancer cells was associated with downregulation of several protein markers related to cell cycle (cyclin D and cyclin B) and anti-apoptosis (Bcl2 and survivin), c-myc and Aurora A and B kinases in both MIA PaCa-2 and PANC-1 cells. To further identify more potent synergistic combination effect of natural bioactive compounds against pancreatic cancer, we determined the anti-growth activity of the combination between T1 and resveratrol, a natural compound found in the skin of red grape and other fruits. T1 and resveratrol combinations consistently showed synergistic effect on inhibiting the growth of MIA PaCa-2, ASPC-1 and PANC-1 cells. Protein assays showed that the synergistic combination effect of T1 and resveratrol was associated with combined downregulation of c-myc and Aurora A protein levels, suggesting that T1 and resveratrol may inhibit the growth of pancreatic cancer cells in part via combined inhibition on the functions of c-myc and Aurora A. Aurora A is the kinase that plays a critical role in controlling mitosis and cell growth. We further confirmed if Aurora A was a functional target for the synergistic combination effect of T1 and resveratrol. Knockdown of Aurora A dramatically inhibited PANC-1 cell growth and decreased the T1 and resveratrol combination activity, suggesting that Aurora A could be one of the targets by the T1 and resveratrol combination effect. Aurora A gene promoter contains the c-myc binding site, suggesting that Aurora A may be regulated by c-myc and the inhibition of c-myc expression may be one of the causes of reduced Aurora A expression following T1 treatment. To confirm this, we treated PANC-1 cells with a c-myc inhibitor and found decreased Aurora A gene expression in a time-dependent manner, indicating the regulation of c-myc on Aurora A gene expression. Our results suggest that the T1 and resveratrol combination may serve as a novel chemopreventive regimen for effective prevention of pancreatic cancer. Supported in part by DOD (PC073988) and NCI/NIH (R21CA127794) Citation Information: Cancer Prev Res 2011;4(10 Suppl):B63.

FoxO3A promotes metabolic adaptation to hypoxia by antagonizing Myc function

Exposure of metazoan organisms to hypoxia engages a metabolic switch orchestrated by the hypoxia-inducible factor 1 (HIF-1). HIF-1 mediates induction of glycolysis and active repression of mitochondrial respiration that reduces oxygen consumption and inhibits the production of potentially harmful reactive oxygen species (ROS). Here, we show that FoxO3A is activated in hypoxia downstream of HIF-1 and mediates the hypoxic repression of a set of nuclear-encoded mitochondrial genes. FoxO3A is required for hypoxic suppression of mitochondrial mass, oxygen consumption, and ROS production and promotes cell survival in hypoxia. FoxO3A is recruited to the promoters of nuclear-encoded mitochondrial genes where it directly antagonizes c-Myc function via a mechanism that does not require binding to the consensus FoxO recognition element. Furthermore, we show that FoxO3A is activated in human hypoxic tumour tissue in vivo and that FoxO3A short-hairpin RNA (shRNA)-expressing xenograft tumours are decreased in size and metabolically changed. Our findings define a novel mechanism by which FoxO3A promotes metabolic adaptation and stress resistance in hypoxia.

Kinetic profiling of the c-Myc transcriptome and bioinformatic analysis of repressed gene promoters

Mammalian c-Myc is a member of a small family of three related proto-oncogenic transcription factors. c-Myc has an unusually broad array of regulatory functions, which include roles in cell cycle and apoptosis, a variety of metabolic functions, cell differentiation, senescence, and stem cell maintenance. c-Myc modulates the expression of a very large number of genes, but the magnitude of the majority of the regulatory effects is only 2-fold or less. c-Myc can both activate and repress the promoters of its target genes. Identification of genes directly regulated by c-Myc has been an enduring question in the field. We report here microarray expression profiling of a high resolution time course of c-Myc induction, using fibroblast cells in which c-Myc activity can be modulated from null to physiological. The c-Myc transcriptome dataset presented is the largest reported to date with 4,186 differentially regulated genes (1,826 upregulated, 2,360 downregulated, 1% FDR). The gene expression patterns fit well with the known biological functions of c-Myc. We describe several novel findings and present tools for further data mining. Although the mechanisms of transcriptional activation by c-Myc are well understood, how c-Myc represses an even greater number of genes remains incompletely described. One mechanism involves the binding of c-Myc to other, positively acting transcription factors, and interfering with their activities. We identified rapid-response genes likely to be direct c-Myc targets, and analyzed the promoters of the repressed genes to identify transcription factors that could be targets of c-Myc repression.

The expression and role of c-Myc in mouse hair follicle morphogenesis and cycling

Although the function of c-Myc has been clarified in many tissues, until now its expression and role in hair follicle morphogenesis and the hair cycle remains unknown. In this study we detected c-Myc expression pattern in the process of mouse hair follicle development and normal cycle. We found that during hair follicle morphogenesis, the stage-specific expression of c-Myc was detected in mouse skin and was predominantly localized to the hair follicle epithelium. c-Myc expression was also consistently found in mouse skin throughout the hair follicle cycle. Through the in vivo injection of c-Myc inhibitory peptide and c-Myc expression plasmid, we also investigated the direct effects of c-Myc on the hair follicle structures during the hair follicle cycle. Our results showed that c-Myc inhibitory peptide significantly restrained the development of anagen hair follicles, while the injection of plasmid DNA encoding c-Myc in vivo clearly promoted anagen development. Our data indicate that c-Myc may play an important role in the proliferation and differentiation of the hair follicle keratinocytes during hair follicle development. c-Myc also was shown to participate in the regulation of the mouse hair growth cycle and could promote the proliferation of the hair matrix keratinocytes as well as the differentiation of the inner root sheath.

Abstract 3444: Enforced expression of MycN and C-Myc induces different medulloblastoma subtypes

Abstract Medulloblastoma (MB), a tumor of the cerebellum, is the most common malignant brain tumor of childhood. MBs are classified histologically and molecularly into four distinct major subgroups, two of which correspond to the deregulation of major developmental pathways, Sonic Hedgehog (SHH) and WNT. Among the two other subgroups, one correlates with high levels of C-MYC expression. Using orthotopic transplantation in the cerebellum or cortex of naïve recipient mice, we showed that enforced expression of MycN in cerebellar granule neuron progenitors (CGNPs) isolated from the cerebellum of postnatal (P) day 6-7 Cdkn2c-/-, Trp53-/-, Atoh1-GFP mice induced MBs with a SHH “signature”. Interestingly, overexpression of C-Myc in the same CGNPs induced MBs with Large Cell Anaplastic (LCA) features and mRNA and microRNA profiles distinct from the SHH subgroup. In vitro analysis revealed that tumor cells were resistant to Shh antagonists, such as cyclopamine and BMP4. Ongoing experiments are focusing on understanding the differences between MycN and C-Myc function in CGNPs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3444. doi:10.1158/1538-7445.AM2011-3444

Sequential and Coordinated Actions of c-Myc and N-Myc Control Appendicular Skeletal Development

BackgroundDuring limb development, chondrocytes and osteoblasts emerge from condensations of limb bud mesenchyme. These cells then proliferate and differentiate in separate but adjacent compartments and function cooperatively to promote bone growth through the process of endochondral ossification. While many aspects of limb skeletal formation are understood, little is known about the mechanisms that link the development of undifferentiated limb bud mesenchyme with formation of the precartilaginous condensation and subsequent proliferative expansion of chondrocyte and osteoblast lineages. The aim of this study was to gain insight into these processes by examining the roles of c-Myc and N-Myc in morphogenesis of the limb skeleton.Methodology/Principal FindingsTo investigate c-Myc function in skeletal development, we characterized mice in which floxed c-Myc alleles were deleted in undifferentiated limb bud mesenchyme with Prx1-Cre, in chondro-osteoprogenitors with Sox9-Cre and in osteoblasts with Osx1-Cre. We show that c-Myc promotes the proliferative expansion of both chondrocytes and osteoblasts and as a consequence controls the process of endochondral growth and ossification and determines bone size. The control of proliferation by c-Myc was related to its effects on global gene transcription, as phosphorylation of the C-Terminal Domain (pCTD) of RNA Polymerase II, a marker of general transcription initiation, was tightly coupled to cell proliferation of growth plate chondrocytes where c-Myc is expressed and severely downregulated in the absence of c-Myc. Finally, we show that combined deletion of N-Myc and c-Myc in early limb bud mesenchyme gives rise to a severely hypoplastic limb skeleton that exhibits features characteristic of individual c-Myc and N-Myc mutants.Conclusions/SignificanceOur results show that N-Myc and c-Myc act sequentially during limb development to coordinate the expansion of key progenitor populations responsible for forming the limb skeleton.

C-MYC expression sensitizes medulloblastoma cells to radio- and chemotherapy and has no impact on response in medulloblastoma patients

BackgroundTo study whether and how c-MYC expression determines response to radio- and chemotherapy in childhood medulloblastoma (MB).MethodsWe used DAOY and UW228 human MB cells engineered to stably express different levels of c-MYC, and tested whether c-MYC expression has an effect on radio- and chemosensitivity using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay, clonogenic survival, apoptosis assays, cell cycle analysis, and western blot assessment. In an effort to validate our results, we analyzed c-MYC mRNA expression in formalin-fixed paraffin-embedded tumor samples from well-documented patients with postoperative residual tumor and compared c-MYC mRNA expression with response to radio- and chemotherapy as examined by neuroradiological imaging.ResultsIn DAOY - and to a lesser extent in UW228 - cells expressing high levels of c-MYC, the cytotoxicity of cisplatin, and etoposide was significantly higher when compared with DAOY/UW228 cells expressing low levels of c-MYC. Irradiation- and chemotherapy-induced apoptotic cell death was enhanced in DAOY cells expressing high levels of c-MYC. The response of 62 of 66 residual tumors was evaluable and response to postoperative radio- (14 responders (CR, PR) vs. 5 non-responders (SD, PD)) or chemotherapy (23 CR/PR vs. 20 SD/PD) was assessed. c-MYC mRNA expression was similar in primary MB samples of responders and non-responders (Mann-Whitney U test, p = 0.50, ratio 0.49, 95% CI 0.008-30.0 and p = 0.67, ratio 1.8, 95% CI 0.14-23.5, respectively).Conclusionsc-MYC sensitizes MB cells to some anti-cancer treatments in vitro. As we failed to show evidence for such an effect on postoperative residual tumors when analyzed by imaging, additional investigations in xenografts and larger MB cohorts may help to define the exact function of c-MYC in modulating response to treatment.

Phosphorylation Regulates c-Myc's Oncogenic Activity in the Mammary Gland

Expression of the c-Myc oncoprotein is affected by conserved threonine 58 (T58) and serine 62 (S62) phosphorylation sites that help to regulate c-Myc protein stability, and altered ratios of T58 and S62 phosphorylation have been observed in human cancer. Here, we report the development of 3 unique c-myc knock-in mice that conditionally express either c-Myc(WT) or the c-Myc(T58A) or c-Myc(S62A) phosphorylation mutant from the constitutively active ROSA26 locus in response to Cre recombinase to study the role of these phosphorylation sites in vivo. Using a mammary-specific Cre model, we found that expression of c-Myc(WT) resulted in increased mammary gland density, but normal morphology and no tumors at the level expressed from the ROSA promoter. In contrast, c-Myc(T58A) expression yielded enhanced mammary gland density, hyperplastic foci, cellular dysplasia, and mammary carcinoma, associated with increased genomic instability and suppressed apoptosis relative to c-Myc(WT). Alternatively, c-Myc(S62A) expression reduced mammary gland density relative to control glands, and this was associated with increased genomic instability and normal apoptotic function. Our results indicate that specific activities of c-Myc are differentially affected by T58 and S62 phosphorylation. This model provides a robust platform to interrogate the role that these phosphorylation sites play in c-Myc function during development and tumorigenesis.

Viral-Mediated Noisy Gene Expression Reveals Biphasic E2f1 Response to MYC

Gene expression mediated by viral vectors is subject to cell-to-cell variability, which limits the accuracy of gene delivery. When coupled with single-cell measurements, however, such variability provides an efficient means to quantify signaling dynamics in mammalian cells. Here, we illustrate the utility ofthis approach by mapping the E2f1 response to MYC, serum stimulation, or both. Our results revealed an underappreciated mode of gene regulation: E2f1 expression first increased, then decreased as MYC input increased. This biphasic pattern was also reflected in other nodes of the network, including the miR-17-92 microRNA cluster and p19Arf. A mathematical model of the network successfully predicted modulation of the biphasic E2F response byserum and a CDK inhibitor. In addition to demonstrating how noise can be exploited to probe signaling dynamics, our results reveal how coordination of the MYC/RB/E2F pathway enables dynamic discrimination of aberrant and normal levels of growth stimulation.

Pancreatic Ductal Adenocarcinoma and Transcription Factors: Role of c-Myc

Deregulated expression/activation of transcription factors is a key event in the establishment and progression of human cancer. Furthermore, most oncogenic signaling pathways converge on sets of transcription factors that ultimately control gene expression patterns resulting in cancer development, progression, and metastasis. Ductal pancreatic adenocarcinoma (PDA) is the main type of pancreatic cancer and the fourth leading cause of cancer mortality in the Western world. The early stage of the disease is characterized by pancreatic intraepithelial neoplasia lesions bearing mutations in the K-RAS proto-oncogene, which progress to malignant PDA by accumulating additional mutations in the tumor suppressor gene CDKN2A (p16) and in SMAD4 and TP53 transcription factors. The involvement of other signaling pathways in PDA development and progression is an active area of research which may help to clarify the critical steps of this devastating disease. In this regard, several in vitro and in vivo data have demonstrated the contribution of the transcription factor c-Myc to pancreatic carcinogenesis although the molecular mechanisms are poorly understood. c-Myc is a proto-oncogene which has a pivotal function in growth control, differentiation and apoptosis and is known to act as a downstream transcriptional effector of many signaling pathways involved in these processes. It is regulated at multiple levels and its abnormal expression contributes to the genesis of many human tumors. This review focuses on the role of c-Myc in pancreatic embryonic development and homeostasis as well as its involvement on pancreatic tumorigenesis. Evidences showing that c-Myc function is highly dose and cell context dependent, together with its recently demonstrated ability to reprogram somatic cells towards a pluripotent stem cell-like state, indicate that the role of c-Myc in pancreas pathophysiology might have been previously underscored.