High Levels Of c-Myc Research Articles

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Activating and sustaining c-Myc by depletion of miR-144/451 gene locus contributes to B-lymphomagenesis

Hyper activity of protooncogene c-Myc is one of the hallmarks of highly aggressive lymphomas. However, the mechanism of how c-Myc is subjected to activation and amplification is still not well defined. In this study, we use gene knockout strategy to show that targeted depletion of a well-conserved microRNA gene locus miR-144/451 initiates tumorigenesis including B-lymphoma development in aged mice. This is due, at least in part, to the direct activation of the c-Myc gene by loss of miR-144/451 expression in hematopoietic cells. Moreover, oncoprotein c-Myc inversely regulates miR-144/451 expression by directly binding to the miR-144/451 promoter region, forming a miRNA-Myc positive feedback loop to safeguard the high level of c-Myc in B-lymphocytes. We also demonstrate that this miRNA-Myc crosstalk is disrupted in human diffuse large B-cell lymphomas with aberrant c-Myc expression. Therefore, our findings provide strong evidence, for the first time, that deficiency of miR-144/451 expression may play a bona fide role in derepression of silenced c-Myc, which contributes to tumor development including B-lymphomagenesis.

Differential cellular responses by oncogenic levels of c-Myc expression in long-term confluent retinal pigment epithelial cells.

c-Myc is a highly pleiotropic transcription factor known to control cell cycle progression, apoptosis, and cellular transformation. Normally, ectopic expression of c-Myc is associated with promoting cell proliferation or triggering cell death via activating p53. However, it is not clear how the levels of c-Myc lead to different cellular responses. Here, we generated a series of stable RPE cell clones expressing c-Myc at different levels, and found that consistent low level of c-Myc induced cellular senescence by activating AP4 in post-confluent RPE cells, while the cells underwent cell death at high level of c-Myc. In addition, high level of c-Myc could override the effect of AP4 on cellular senescence. Further knockdown of AP4 abrogated senescence-like phenotype in cells expressing low level of c-Myc, and accelerated cell death in cells with medium level of c-Myc, indicating that AP4 was required for cellular senescence induced by low level of c-Myc.

Coordinated Activities of Multiple Myc-dependent and Myc-independent Biosynthetic Pathways in Hepatoblastoma

Hepatoblastoma (HB) is associated with aberrant activation of the β-catenin and Hippo/YAP signaling pathways. Overexpression of mutant β-catenin and YAP in mice induces HBs that express high levels of c-Myc (Myc). In light of recent observations that Myc is unnecessary for long-term hepatocyte proliferation, we have now examined its role in HB pathogenesis using the above model. Although Myc was found to be dispensable for in vivo HB initiation, it was necessary to sustain rapid tumor growth. Gene expression profiling identified key molecular differences between myc+/+ (WT) and myc-/- (KO) hepatocytes and HBs that explain these behaviors. In HBs, these included both Myc-dependent and Myc-independent increases in families of transcripts encoding ribosomal proteins, non-structural factors affecting ribosome assembly and function, and enzymes catalyzing glycolysis and lipid bio-synthesis. In contrast, transcripts encoding enzymes involved in fatty acid β-oxidation were mostly down-regulated. Myc-independent metabolic changes associated with HBs included dramatic reductions in mitochondrial mass and oxidative function, increases in ATP content and pyruvate dehydrogenase activity, and marked inhibition of fatty acid β-oxidation (FAO). Myc-dependent metabolic changes included higher levels of neutral lipid and acetyl-CoA in WT tumors. The latter correlated with higher histone H3 acetylation. Collectively, our results indicate that the role of Myc in HB pathogenesis is to impose mutually dependent changes in gene expression and metabolic reprogramming that are unattainable in non-transformed cells and that cooperate to maximize tumor growth.

C-Myc suppresses miR-451⊣YWTAZ/AKT axis via recruiting HDAC3 in acute myeloid leukemia.

Aberrant activation of c-Myc plays an important oncogenic role via regulating a series of coding and non-coding genes in acute myeloid leukemia (AML). Histone deacetylases (HDACs) can remove acetyl group from histone and regulate gene expression via changing chromatin structure. Here, we found miR-451 is abnormally down-regulated in AML patient samples; c-Myc recruits HDAC3 to form a transcriptional suppressor complex, co-localizes on the miR-451 promoter, epigenetically inhibits its transcription and finally induces its downregulation in AML. Furthermore, our in vitro and in vivo results suggest that miR-451 functions as a tumor suppressor via promoting apoptosis and suppressing malignant cell proliferation. The mechanistic study demonstrated that miR-451 directly targets YWHAZ mRNA and suppresses YWHAZ/AKT signaling in AML. Knockdown of c-Myc results in restoration of miR-451 and inhibition of YWHAZ/AKT signaling. In AML patients, low level of miR-451 is negatively correlated with high levels of c-Myc and YWHAZ, while c-Myc level is positively related to YWHAZ expression. These results suggested that c-Myc⊣miR-451⊣YWHAZ/AKT cascade might play a crucial role during leukemogenesis, and reintroduction of miR-451 could be as a potential strategy for AML therapy.

A c-Myc/miR-17-5p feedback loop regulates metastasis and invasion of hepatocellular carcinoma.

The molecular mechanisms that control metastasis of hepatocellular cancer (HCC) are still poorly understood. It has been determined that microRNA (miRNA) expression has tissue and cell specific, and decreased expression of specific miRNA could induce tumor genesis or metastasis. In this study, we identified that miR-17-5p was expressed lower in high metastatic capability HCC cell lines HCCLM3 and MHCC97H than low metastatic HCC cell line HepG2 by real-time (RT)-PCR. Restoration of miR-17-5p could significantly repress the invasiveness and metastasis of MHCC97H cell line. Furthermore, we validated c-Myc as a downstream and functional target of miR-17-5p using luciferase reporter assay. Immunohistochemical assay revealed that the expression of c-Myc protein levels was significantly increased in cancerous tissues compared with para-tumor tissues. After clinical data analysis, we observed that the higher level of c-Myc was significantly associated with a reduced overall survival (p = 0.0209). Consistent with previous research, we also demonstrated that c-Myc could upregulate the expression of miR-17-5p. Taken together, our data indicated that there is a regulatory feedback loop between miR-17-5p and c-Myc, in which miR-17-5p could suppress some of the distinguishing features, invasion, and metastasis, of oncogenic c-Myc in HCC cells, and meanwhile, miR-17-5p is upregulated by c-Myc role as a transcription factor, although further studies are still needed.

Abstract B37: Targeting Myc-reprogrammed glutamine metabolism for treatment of neuroblastoma

Abstract Oncogenic Myc is known to alter cellular metabolism resulting in glutamine addiction, and the MYCN-amplified subclass of neuroblastoma is associated with extremely poor prognosis. Successful identification of therapeutic agents that are effective in neuroblastoma, particularly in tumors with MYCN amplification, could significantly increase patient survival. To explore the effect of inhibiting glutamine metabolism in neuroblastoma, we used the glutamine antagonist DON (6-diazo-5-oxo-norleucine). DON is an irreversible competitive inhibitor which alkylates enzymes that utilize glutamine. Although first studied in the 1950s as a possible cancer chemotherapeutic, DON was associated with severe nausea which limited its use in patients. DON remains a potent inhibitor, and a useful tool to study the effect of modulating glutamine metabolism in vivo. We tested the DON sensitivity of six human neuroblastoma cell lines (Kelly, IMR-32, SK-N-BE2, SK-N-AS, SK-N-FI, and SH-SY5Y) compared to immortalized BJ fibroblast control cells. DON effectively inhibited all neuroblastoma cell lines tested with minimal effect on BJ cells. Furthermore, MycN overexpression in SK-N-AS cells, a cell line which is not MYCN-amplified and expresses low levels of cMyc, was sufficient to make cells significantly more sensitive to DON. Next, we tested the effect of DON treatment on subcutaneous xenografts. For these experiments we used the cell lines SK-N-AS (not MYCN-amplified), SK-N-BE2 (MYCN-amplified with moderate MycN levels), and IMR-32 (MYCN-amplified with high MycN levels). Mice were treated twice per week by intraperitoneal injection with DON (50 or 100mg/kg) or water control. Treatment with 50mg/kg DON significantly inhibited tumor growth of all three cell lines, suggesting neuroblastoma tumors may be broadly sensitive to glutamine inhibitors. IMR-32 tumors were the most sensitive to DON treatment, leading to a 6-fold reduction in tumor size after 2.5 weeks of treatment. Tumor BrdU incorporation decreased following DON treatment, and IMR-32 tumors showed increased levels of cleaved caspase 3, a marker of apoptosis. At the higher 100mg/kg dose, DON treatment led to a more substantial decrease in BrdU incorporation, and at this dose BE2 tumors also showed significantly increased levels of cleaved caspase 3, which were not detected in AS tumors. These results suggest that MYCN-amplified tumors may be particularly sensitive to glutamine inhibition. To examine the mechanism of DON-induced cell death, we treated cells with the pan-caspase inhibitor QVD. QVD treatment for 48 or 72hr significantly rescued both MYCN-amplified cell lines (Kelly, IMR-32) and cells that express high levels of cMyc (SY5Y), while having minimal effect in the remaining neuroblastoma cell lines with low Myc levels. These results confirm that DON induces cell death partially by activating apoptosis in high Myc expressing cell lines. Next, we used shRNA to show that in Kelly cells DON-induced apoptosis occurs by signaling through Bax, but not Bak. Lastly, we tested DON in combination with pro-apoptotic compounds in vitro. We found that DON treatment combined with the Bcl-2 family inhibitor ABT-263 had synergistic effects on cell death in the MYCN-amplified cell lines Kelly and BE2. Our results suggest that combined targeting of glutamine metabolism while reducing the threshold for tumor cell apoptosis may be beneficial for neuroblastoma treatment. Citation Format: Rachelle R. Olsen, Michelle N. Mary-Sinclair, Zhirong Yin, Kevin W. Freeman. Targeting Myc-reprogrammed glutamine metabolism for treatment of neuroblastoma. [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 B37.

Abstract 486: BET protein inhibition by JQ1 blocks EWS-FLI1 activity in Ewing sarcoma

Abstract Ewing sarcoma is the second most common bone cancer in children, with an estimated 5-year survival rate of 20% for metastatic cases. These tumors harbor the characteristic chromosomal translocation t(11;22)(q24;q12), which leads to expression of the oncogenic chimeric transcription factor, EWS-FLI1. EWS-FLI1 drives Ewing sarcoma tumorigenesis, and is an attractive therapeutic target because it is expressed exclusively in tumor cells. However, directly targeting EWS-FLI1 has proved difficult. Thus, despite advances in our understanding of EWS-FLI1 function, treatment options remain limited, and to date, no targeted therapeutics have been developed. In this study, we investigated the effects of the epigenetic modifier drug, JQ1, on Ewing sarcoma pathogenesis. JQ1 is a small molecule inhibitor of the BET family of bromodomain proteins, which bind to acetylated histones and transcription factors to regulate gene expression. JQ1 selectively inhibits these acetylation-dependent BET protein interactions. Recent studies reveal that JQ1 has potent anti-neoplastic activity against multiple cancers, with c-MYC downregulation being a key mechanism by which it induces cell death in several tumor types. Because Ewing sarcoma cells express high levels of c-MYC, we predicted that they would be sensitive to the cytotoxic effects of JQ1. Our results demonstrate that JQ1 indeed inhibits growth of Ewing sarcoma cells, but unexpectedly, it does not function through inhibition of c-MYC. Using multiple patient-derived Ewing sarcoma cell lines, we find that JQ1 induces apoptosis as measured by PARP cleavage. Preliminary data further indicate that JQ1 suppresses growth of Ewing sarcoma xenografts in vivo. Mechanistic studies and microarray analysis reveal that JQ1 inhibits EWS-FLI1 activity, with multiple targets of the oncogene being rapidly downregulated upon JQ1 treatment. Studies are currently underway to identify the specific BET family members required for EWS-FLI1 function, and to determine the mechanism by which they regulate EWS-FLI1 activity. Given the emergence of numerous JQ1-derivative compounds targeting specific BET proteins, it is hopeful that this class of compounds may ultimately be used as targeted therapies for the treatment of Ewing sarcoma. Citation Format: Krista L. Bledsoe, Aaron Stonestrom, Stephan Kadauke, Laura Quick, Robert Young, Gerd A. Blobel, Margaret M. Chou. BET protein inhibition by JQ1 blocks EWS-FLI1 activity in Ewing sarcoma. [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 486. doi:10.1158/1538-7445.AM2015-486

Abstract PR02: Myc-Ras cooperation can overwhelm tumor suppressive mechanisms within lung adenocarcinomas

Abstract The ability of Myc and Ras to cooperate during tumorigenesis was first observed over thirty years ago. Numerous mechanisms have been implicated in their collaboration. For instance, Myc can act to circumvent Ras-induced growth arrest. Alternatively, Ras has been shown to prevent Myc-induced apoptosis by activating the anti-apoptotic kinase Akt. Early experiments in primary rodent cells demonstrated that neither Ras nor Myc alone was sufficient for transformation, whereas when expressed together, transformation occurred. However, these transformation events were rare, suggesting additional co-operating mutations were also required. These and other investigations ultimately led to the concept of ‘intrinsic tumor suppression’, whereby the effects of activation of an individual oncogene is limited by their dual potential to invoke cell cycle arrest or apoptosis, which limit their other oncogenic functions. The dogma suggests that cancers only arise in oncogene expressing cells when anti-apoptotic/cell arrest mutations are acquired and the disruption of these pathways leads to net proliferation. There is increasing evidence that the activation of intrinsic tumor suppressive mechanisms depend on increased oncogenic flux resulting from changes in the level of oncogene expression. For example, high expression of oncogenic Ras results in premature growth arrest in MEFs, whereas an endogenous level does not. To address this phenomenon with regards to Myc, we previously generated a novel knock-in mouse (R26c-MycER) in which the c-MycERTAM protein was inserted into the relatively ubiquitous, albeit low expressing Rosa26 locus. In contrast to high levels of Myc expressed in classical transgenic models, low-level c-Myc expression did not trigger intrinsic tumor suppression mechanisms in most tissues, suggesting that low levels of oncogenic Myc may be a more efficient initiator of oncogenesis than over expressed Myc, as it might not require cooperating loss of tumor suppressive pathways. We have now created a R26CAG-c-MycER allele that expresses a 5-10 fold higher constitutive expression of MycERTAM than the R26c-MycER allele. Together with R26c-MycER mice we have been able to generate an allelic series of mice that allow the control, in a spatial and temporal fashion, the activity of de-regulated c-Myc at low, medium and high levels. When we sporadically activated c-MycERTAM within the lung epithelial cells, only very few high-level expressing c-MycERTAM and none of the low-level mice developed tumors within 18 weeks suggesting Myc alone is insufficient for tumorigenesis irrespective of oncogenic level. However, in the presence of an endogenous level of oncogenic K-RasG12D, lung adenomas are evident in mice expressing both low and high levels of c-Myc. Mice expressing the highest levels of c-Myc developed adenocarcinomas with extremely short latency and have a greatly reduced survival when compared to mice with lower levels of c-Myc. As expected, low level c-Myc did not activate intrinsic tumor suppression via the Arf/p53 pathways within K-Ras driven lesions and these tumors grew faster than lesions with oncogenic K-Ras alone. However, tumors expressing high levels of c-Myc did engage a number of key tumor suppressors and apoptosis increased as a function of c-Myc level, despite the continued growth and extremely short latency of the adenomas/adenocarcinomas in these mice. Thus, when combined with oncogenic K-Ras, high c-Myc cooperation results in rapid tumorigenesis whereby tumors are still able to grow prolifically despite the apparent activation of the surveillance mechanisms. Further analysis is currently underway to determine the specific survival advantage oncogenic K-Ras offers within this system. This abstract is also presented as Poster B20. Citation Format: Catherine H. Wilson, Deborah L. Burkhart, Jinyang Li, Trevor D. Littlewood, Gerard I. Evan. Myc-Ras cooperation can overwhelm tumor suppressive mechanisms within lung adenocarcinomas. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr PR02. doi: 10.1158/1557-3125.RASONC14-PR02

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

MiR‐494 acts as an anti‐oncogene in gastric carcinoma by targeting c‐myc

We recently showed that miR-494 was downregulated in gastric carcinoma (GC). The objectives of this study were to determine the role of miR-494 in GC malignancy and to identify its target genes. Real-time polymerase chain reaction was employed to quantify the expression level of miR-494 and c-myc in gastric cancer tissues. Bioinformatics was used to predict the downstream target genes of miR-494, which were confirmed by luciferase and RNA immunoprecipitation assays. Cell functional analyses and a xenograft mouse model were used to evaluate the role of miR-494 in malignancy. miR-494 was downregulated in human GC tissues and in GC cells and was negatively correlated with c-myc expression. High level of c-myc or low level of miR-494 correlated with poor prognosis. The miR-494-binding site in the c-myc 3' untranslated region was predicted using TargetScan and was confirmed by the luciferase assay. Additionally, c-myc and miR-494 were enriched in coimmunoprecipitates with tagged Argonaute2 proteins in cells overexpressing miR-494. Furthermore, a miR-494 mimic significantly downregulated endogenous c-myc expression, which may contribute to the delayed G1/S transition, decreased synthesis phase bromodeoxyuridine incorporation, and impaired cell growth and colony formation; on the other hand, treatment with a miR-494 inhibitor displayed the opposite effects. Reduced tumor burden and decreased cell proliferation were observed following the delivery of miR-494 into xenograft mice. miR-494 is downregulated in human GC and acts as an anti-oncogene by targeting c-myc. miR-494 plays a role in the pathogenesis of gastric cancer in a recessive fashion.

Quantitative determination of c-myc facilitates the assessment of prognosis of OSCC patients

Myc genes are a family of proto-oncogenes whose proteins are implicated in the regulation of cell proliferation, differentiation and apoptosis, and in regulating the activity of genes involved in cell division. The aim of the present study was to establish a quantitative description of the expression of c-myc and evaluate its relationship with other clinical and prognostic factors, as well as to establish a multivariate survival prediction model. This is a retrospective study of 68 patients diagnosed with oral squamous cell carcinoma (OSCC). We constructed a tissue microarray for investigating the expression of c-myc by immunohistochemistry. Statistical analyses were carried out, and a multivariate model that predicts survival was established. The average expression of c-myc was 50.32 (SD, 26.05) with a range from 6.60 to 99.48; similar for initial and advanced tumor stages. Non-smoking patients had higher levels of c-myc, showing statistically significant differences (Kruskal-Wallis χ2=5.975; p=0.05). We found no statistically significant relationship between the quantitative expression of c-myc and any other clinical or pathological parameters. For each unit of increase of c-myc, the risk increased by 1.15 (p<0.001; HR, 1.150; 95% CI, 1062-1245). Further study of this protein, which may have a significant diagnostic, prognostic and therapeutic value is warranted. Its determination can be valuable when used together with other markers to assess the prognosis of OSCC patients.

Toll-like receptor agonists induce apoptosis in mouse B-cell lymphoma cells by altering NF-κB activation

Toll-like receptor 9 (TLR9) recognizes microbial DNA containing unmethylated cytosyl guanosyl (CpG) sequences, induces innate immune responses, and facilitates antigen-specific adaptive immunity. Recent studies report that in addition to stimulating innate immunity, TLR9 ligands induce apoptosis of TLR9 expressing cancer cells. To understand the mechanism of TLR9-induced apoptosis, we compared the effects of CpG containing oligodeoxynucleotides (CpG ODN) on a mouse B-cell lymphoma line, CH27, with those on mouse splenic B cells. CpG ODN inhibited constitutive proliferation and induced apoptosis in the CH27 B-cell lymphoma line. In contrast, CpG ODN-treated primary B cells were stimulated to proliferate and were rescued from spontaneous apoptosis. The induction of apoptosis required the ODNs to contain the CpG motif and the expression of TLR9 in lymphoma B cells. A decrease in Bcl-xl expression and an increase in Fas and Fas ligand expression accompanied lymphoma B-cell apoptosis. Treatment with the Fas ligand-neutralizing antibody inhibited CpG ODN-induced apoptosis. CpG ODN triggered a transient NF-κB activation in the B-cell lymphoma cell line, which constitutively expresses a high level of c-Myc, while CpG ODN induced sustained increases in NF-κB activation and c-Myc expression in primary B cells. Furthermore, an NF-κB inhibitor inhibited the proliferation of the CH27 B-cell lymphoma line. Our data suggest that the differential responses of lymphoma and primary B cells to CpG ODN are the result of differences in NF-κB activation. The impaired NF-κB activation in the CpG ODN-treated B-cell lymphoma cell line alters the balance between NF-κB and c-Myc, which induces Fas/Fas ligand-dependent apoptosis.

Transcriptome Sequencing of Tumor Subpopulations Reveals a Spectrum of Therapeutic Options for Squamous Cell Lung Cancer

BackgroundThe only therapeutic options that exist for squamous cell lung carcinoma (SCC) are standard radiation and cytotoxic chemotherapy. Cancer stem cells (CSCs) are hypothesized to account for therapeutic resistance, suggesting that CSCs must be specifically targeted. Here, we analyze the transcriptome of CSC and non-CSC subpopulations by RNA-seq to identify new potential therapeutic strategies for SCC.MethodsWe sorted a SCC into CD133− and CD133+ subpopulations and then examined both by copy number analysis (CNA) and whole genome and transcriptome sequencing. We analyzed The Cancer Genome Atlas (TCGA) transcriptome data of 221 SCCs to determine the generality of our observations.ResultsBoth subpopulations highly expressed numerous mRNA isoforms whose protein products are active drug targets for other cancers; 31 (25%) correspond to 18 genes under active investigation as mAb targets and an additional 4 (3%) are of therapeutic interest. Moreover, we found evidence that both subpopulations were proliferatively driven by very high levels of c-Myc and the TRAIL long isoform (TRAILL) and that normal apoptotic responses to high expression of these genes was prevented through high levels of Mcl-1L and Bcl-xL and c-FlipL—isoforms for which drugs are now in clinical development. SCC RNA-seq data (n = 221) from TCGA supported our findings. Our analysis is inconsistent with the CSC concept that most cells in a cancer have lost their proliferative potential. Furthermore, our study suggests how to target both the CSC and non-CSC subpopulations with one treatment strategy.ConclusionsOur study is relevant to SCC in particular for it presents numerous potential options to standard therapy that target the entire tumor. In so doing, it demonstrates how transcriptome sequencing provides insights into the molecular underpinnings of cancer propagating cells that, importantly, can be leveraged to identify new potential therapeutic options for cancers beyond what is possible with DNA sequencing.

MiR-221 and MiR-222 Patterns Characterize Burkitt Lymphoma in Human and Mouse Model

AbstractAbstract 1304Recently, a class of noncoding RNAs called microRNA (miRNAs) has emerged as critical gene regulators in cell growth, differentiation, disease and development. MiRNAs are 18–24 nucleotide long noncoding RNAs, which regulate gene expression by pairing with 3′ untranslated region (UTR) of target mRNA and inhibiting protein translation and/or inducing mRNA degradation. Deregulated miRNA expression is reported in various human diseases including lymphomas, suggesting an important role in their pathogenesis.According to WHO classification, Burkitt lymphoma (BL) is a rare, highly aggressive NHL composed of monomorphic medium-sized B cells with multiple nucleoli and numerous mitotic figures and is more common in children than in adults. The molecular feature of BL is the translocation that places MYC under the control of immunoglobulin gene regulatory elements. High levels of c-MYC have been clearly shown to have a tumour-promoting effect. However, there is recent evidence that infrequent cases may lack an identifiable MYC translocation, the explanation for which is still uncertain, though suggesting the existence of pathogenetic mechanisms alternative to genetic alterations.Over the past years miRNA signatures have been described to characterize and classify different types of BL or to investigate the expression of miRNAs possibly regulated by c-Myc in BL cases positive or negative for Myc translocation. However, it remained unclear the functional role of differentially expressed miRNAs and no further studies have been conducted. We performed miRNA expression profile to gain further insights into the molecular pathology of BL. We conducted array analysis on a set of 5 sporadic BL patients, 3 endemic BL patients, 9 reactive tissues and 11 cases of mononucleosis. Our profile is the first one that shows the different expression between BL cases and normal B cells whereas recent miRNA profiles have been conducted in BL compared to other B-NHL (B-CLL, MCL & FL). A common trend of miRNAs altered expression was also observed by NanoString analysis in 10 BL cell lines compared to 5 normal CD-19+ B cells. Among several miRNAs previously described be deregulated in BL we identified a severe down-regulation of miR-221, miR-222 in all classes of comparisons we analyzed. The down-regulation of miR-221 and miR-222 associated to BL has been also confirmed by q-RT-PCR method in a different cohort of BL patients (20) compared to the healthy controls (6). We found that interesting considering the up-regulation of miR-221 and miR-222 previously confirmed in a lot of solid tumors by multiple studies. We are investigating a different role of the cluster miR-222 and miR-221 in lymphomas that have a different process in carcinogenesis than solid tumors.In vivo models to study the lymphomagenesis of BL have been created but until now no one studied the importance of the miRNAs in vivo. We analyzed the expression of miR-221 and miR-222 in a Myc transgenic mouse model. The transgene construct consists of the Myc oncogene (c-myc) in association with the Emu immunoglobulin heavy chain enhancer and Myc promoter. Expression of the mouse Myc transgene is restricted to the B cell lineage. Previously it has been shown an increase of pre-B cells in the bone marrow throughout life of hemizygotes and a transient increase in large pre-B cells in the blood at 3–4 weeks of age; moreover spontaneous pre-B and B cell lymphomas reach an incidence of 50% at 15–20 weeks in hemizygous progeny of a wildtype female mated with a hemizygous male. We observed the development of Burkitt lymphoma within 10 weeks of birth in 14 out of 25 Eu-Myc transgenic mice and a premature death in 5 out or 25 transgenic mice within 6–8 weeks of birth without showing any enlarged lymph nodes. Transgenic mice with masses showed the same phenotype characterized by enlarged spleen (3 fold), lymphosarcomas associated with BL and enlarged lymph nodes around the neck area. B-cells have been negatively selected from enlarged lymph nodes and enlarged spleen. A qRT-PCR has been conducted to evaluate the miR-221 and miR-222 expression. The miRNA levels showed a down-regulation in B cells collected from the masses when compared to normal B cells derived from the spleen of WT mice.In conclusion, our study reveals new insights into the functional significance in loss of miR-221 and miR-222 expression in BL pathogenesis. Disclosures:No relevant conflicts of interest to declare.

Transcriptional Amplification in Tumor Cells with Elevated c-Myc

Elevated expression of the c-Myc transcription factor occurs frequently in human cancers and is associated with tumor aggression and poor clinical outcome. The effect of high levels of c-Myc on global gene regulation is poorly understood but is widely thought to involve newly activated or repressed "Myc target genes." We report here that in tumor cells expressing high levels of c-Myc the transcription factor accumulates in the promoter regions of active genes and causes transcriptional amplification, producing increased levels of transcripts within the cell's gene expression program. Thus, rather than binding and regulating a new set of genes, c-Myc amplifies the output of the existing gene expression program. These results provide an explanation for the diverse effects of oncogenic c-Myc on gene expression in different tumor cells and suggest that transcriptional amplification reduces rate-limiting constraints for tumor cell growth and proliferation.

Role of activation‐induced cytidine deaminase in the progression of follicular lymphoma

Activation-induced cytidine deaminase (AID/AICDA) is required for somatic hypermutation and class-switch recombination of the immunoglobulin gene, and for c-myc translocation of germinal center-derived B-cell lymphoma. In the present study, we attempted to clarify the significance of AID associated with c-myc in the progression of follicular lymphoma (FL) using RT-PCR and quantitative real-time PCR. Tissues from the patients with grade 3 FL expressed relatively higher levels of c-myc and AID. The samples taken from a patient with FL who died within 2 years after the start of treatment showed either no or low expression of AID, despite expressing high levels of c-myc. In order to examine the role of AID expression in rapidly progressive FL, the full-length AID transcript was transfected into AID-negative cell lines established from different patients with rapidly progressive FL. This led to the establishment of AID-expressing transfectants with a low proliferation rate and a significantly increased incidence of G(0)/G(1) arrest compared with controls. Our results indicate that AID may act as a negative regulator of cell survival in FL when sufficient c-myc is expressed. Switch-off or low expression of AID after c-myc amplification may correlate with the clinical outcomes of FL.

The Role of Activation-Induced Cytidine Deaminase (AID/AICDA) in the Progression of Follicular Lymphoma

Abstract 5234Follicular lymphoma (FL), a entity with a cell origin in germinal center B lymphocytes, may develop into a progressive or transformed disease in some patients. This transformation is usually associated with acceleration of the clinical course. Transformed FLs generally retain the t(14;18) translocation, and it is believed that other genetic abnormalities are necessary in order for this transformation to occur. Secondary genetic events have been associated with this transformation such as c-myc amplification or mutation. Activation induced cytidine deaminase (AID/AICDA) is required for somatic hyper-mutation and class switch recombination of immunoglobulin gene, and also deeply involved in c-myc translocation of GCB-cell lymphoma. However, the role of AID in transformed FL has not been established. Here we tried to identify the significance of AID associated with c-myc in the progression of FL using mRNAs from tissue biopsy samples from patients with FL. The FLs we examined were divided into three groups: patients with grade 1–2 FL (n=15), patients with grade 3 FL who survived more than two years (n=14), and patients with rapidly progressive FL (RPFL) who died within two years after the start of treatment (n=6). AID and c-myc expression among those patients with FL were examined with RT-PCR and quantitative real-time PCR. Samples from the patients with grade 3 FL expressed relatively higher levels of the c-myc transcript than samples from the patients with grade 1–2 FL (p=.02) respectively. Additionally, nonsignificantly high levels of AID expression were observed in tissues from the patients with grade 3 FL. Interestingly, the samples taken from a patient with FL who died within two years after the start of treatment, showed either no or low expression of AID, despite expressing high levels of c-myc. These results suggested that overexpression of c-myc might be correlated with the biological transformation and clinical progression of FL; and also that AID might play a partial role in disease progression, and low expression of AID may be associated with the clinical features and outcome of patients with RPFL. In order to examine the role of AID expression in RPFL, the full-length AID gene was transfected into AID-negative and c-myc-positive cell lines established from patients with RPFL. AID-positive transfectants exhibited damping of cell proliferation proportional to the genetic level of AID expression, relative to the parental cells and the vector-only transfectants used as controls. Cell cycle analysis showed that the AID-positive transfectants had an increased cell population in G0/G1 phase and a reduced population in S phase, suggesting that G0/G1 arrest was responsible for the reduction in cell proliferation relative to the controls. These in vivo and in vitro findings suggest that AID may act as a negative regulator of cell survival in FL when sufficient c-myc is expressed. Switch-off or low expression of AID after c-myc amplification may be a potentially useful marker for prognostication of FL. Disclosures:No relevant conflicts of interest to declare.

Androgen suppresses proliferation of castration‐resistant LNCaP 104‐R2 prostate cancer cells through androgen receptor, Skp2, and c‐Myc

Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, this therapy is associated with several undesired side-effects, including increased risk of cardiovascular diseases. To study if termination of long-term androgen ablation and restoration of testosterone levels could suppress the growth of relapsed hormone-refractory prostate tumors, we implanted testosterone pellets in castrated nude mice carrying androgen receptor (AR)-positive LNCaP 104-R2 cells, which relapsed from androgen-dependent LNCaP 104-S cells after long-term androgen deprivation. 104-R2 tumor xenografts regressed after testosterone pellets were implanted. Of 33 tumors, 24 adapted to elevation of testosterone level and relapsed as androgen-insensitive tumors. Relapsed tumors (R2Ad) expressed less AR and prostate-specific antigen. We then studied the molecular mechanism underlying the androgenic regulation of prostate cancer cell proliferation. Androgen suppresses proliferation of 104-R2 by inducing G(1) cell cycle arrest through reduction of S-phase kinase-associated protein 2 (Skp2) and c-Myc, and induction of p27(Kip1). 104-R2 cells adapted to androgen treatment and the adapted cells, R2Ad, were androgen-insensitive cells with a slower growth rate and low protein level of AR, high levels of c-Myc and Skp2, and low levels of p27(Kip1). Nuclear AR and prostate-specific antigen expression is present in 104-R2 cells but not R2Ad cells when androgen is absent. Overexpression of AR in R2Ad cells regenerated an androgen-repressed phenotype; knockdown of AR in 104-R2 cells generated an androgen-insensitive phenotype. Overexpression of Skp2 and c-Myc in 104-R2 cells blocked the growth inhibition caused by androgens. We concluded that androgens cause growth inhibition in LNCaP 104-R2 prostate cancer cells through AR, Skp2, and c-Myc.

PP2A-B56α controls oncogene-induced senescence in normal and tumor human melanocytic cells

Oncoprotein C-MYC is overexpressed in human metastatic melanomas and melanoma-derived cells where it is required for suppression of oncogene-induced senescence (OIS). The genetic events that maintain high levels of C-MYC in melanoma cells and their role in OIS are unknown. Here, we report that C-MYC in cells from several randomly chosen melanoma lines was up-regulated at the protein level, and largely due to the increased protein stability. Of all known regulators of C-MYC stability, levels of B56α subunit of the PP2A tumor suppressor complex were substantially suppressed in all human melanoma cells compared to normal melanocytes. Accordingly, immuno-histochemical analysis revealed that the lowest and the highest amounts of PP2A-B56α were predominantly detected in metastatic melanoma tissues and in primary melanomas from patients with good clinical outcome, respectively. Importantly, PP2A-B56α overexpression suppressed C-MYC in melanoma cells and induced OIS, whereas depletion of PP2A-B56α in normal human melanocytes up-regulated C-MYC protein levels and suppressed BRAFV600E- and, less efficiently, NRASQ61R-induced senescence. Our data reveal a mechanism of C-MYC overexpression in melanoma cells and identify a functional role for PP2A-B56α in OIS of melanocytic cells.