Constitutive C-myc Expression Research Articles

Circulating CD133+/–CD34– Have Increased c-MYC Expression in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are hematopoietic stem cell (HSC)-originated diseases with clonal myeloproliferation. The constitutive activation of the JAK/STAT pathway is frequently detected in patients with Philadelphia chromosome-negative (Ph–) MPNs with an acquired JAK2V617F mutation. The c-MYC proto-oncogene is associated with malignant growth and cellular transformation, and JAK2V617F was previously shown to induce constitutive expression of c-MYC. This study examines the expressional profile of c-MYC in Ph– MPNs with JAK2V617F and highlights its hierarchical level of activation in circulating hematopoietic stem/progenitor cell (HSPC) subgroups. Mononuclear cells (MNCs) of Ph– MPNs were fluorochrome-labeled in situ with wild-type (wt) JAK2 or JAK2V617F mRNA gold nanoparticle technology and sorted simultaneously. Isolated populations of JAK2wt or JAK2V617F were evaluated for their c-MYC expressions. The MNCs of 14 Ph– MPNs were further isolated for the study of HSPC subgroups regarding their CD34 and CD133 expressions, evaluated for the presence of JAK2V617F, and compared to cord blood (CB) counterparts for the expression of c-MYC. The mRNA-labeled gold nanoparticle-treated MNCs were determined to have the highest ratio of c-MYC relative fold-change expression in the biallelic JAK2V617F compartment compared to JAK2wt. The relative c-MYC expression in MNCs of MPNs was significantly increased compared to CB (p=0.01). The circulating HSPCs of CD133+/–CD34− MPNs had statistically significantly elevated c-MYC expression compared to CB. This is the first study of circulating CD133+/–CD34− cells in Ph– MPNs and it has revealed elevated c-MYC expression levels in HSCs/endothelial progenitor cells (HSCs/EPCs) and EPCs. Furthermore, the steady increase in the expression of c-MYC within MNCs carrying no mutations and monoallelic or biallelic JAK2V617F transcripts was notable. The presence of JAK2V617F with respect to c-MYC expression in the circulating HSCs/EPCs and EPCs of MPNs might provide some evidence for the initiation of JAK2V617F and propagation of disease. Further studies are needed to clarify the implications of increased c-MYC expression in such populations.

New Drug Repositioning Candidates for T-ALL Identified Via Human/Murine Gene Signature Comparison.

T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive subtype of leukemia for which important progress in treatment efficiency have been made in the past decades to reach a cure rate of 75%–80% nowadays. It is nevertheless mandatory to find new targets and active molecules for innovative therapeutic strategies as relapse is associated with a very dismal outcome. We designed an experimental workflow to highlight the conserved core pathways associated with leukemogenesis by confronting the gene expression profiles (GEPs) of human T-ALL cases to the GEP of a murine T-ALL representative model, generated by the conditional deletion of the PTEN tumor suppressor gene in T cell precursors (tPTEN-/-). We identified 844 differentially expressed genes, common GEPs (cGEP) that were conserved between human T-ALL and murine signatures, and also similarly differentially expressed, compared to normal T cells. Using bioinformatic tools we highlighted in cGEPan upregulation of E2F, MYC and mTORC1. Next, using Connectivity Map (CMAP) and CMAPViz a visualization procedure for CMAP data that we developed, we selected in silico three FDA-approved, bioactive molecule candidates: α-estradiol (α-E), nordihydroguaiaretic acid (NDGA) and prochlorperazine dimaleate (PCZ). At a biological level, we showed that the three drugs triggered an apoptotic cell death in a panel of T-ALL cell lines, activated a DNA damage response and interfered with constitutive mTORC1 activation and c-MYC expression. This analysis shows that the investigation of conserved leukemogenesis pathways could be a strategy to reveal new avenues for pharmacological intervention.

Protein arginine methyltransferase 5 represses tumor suppressor miRNAs that down-regulate CYCLIN D1 and c-MYC expression in aggressive B-cell lymphoma.

Protein arginine methyltransferase-5 (PRMT5) is overexpressed in aggressive B-cell non-Hodgkin's lymphomas, including mantle cell lymphoma and diffuse large B-cell lymphoma, and supports constitutive expression of CYCLIN D1 and c-MYC. Here, we combined ChIP analysis with next-generation sequencing to identify microRNA (miRNA) genes that are targeted by PRMT5 in aggressive lymphoma cell lines. We identified enrichment of histone 3 dimethylation at Arg-8 (H3(Me2)R8) in the promoter regions of miR33b, miR96, and miR503. PRMT5 knockdown de-repressed transcription of all three miRNAs, accompanied by loss of recruitment of epigenetic repressor complexes containing PRMT5 and either histone deacetylase 2 (HDAC2) or HDAC3, enhanced binding of co-activator complexes containing p300 or CREB-binding protein (CBP), and increased acetylation of specific histones, including H2BK12, H3K9, H3K14, and H4K8 at the miRNA promoters. Re-expression of individual miRNAs in B-cell lymphoma cells down-regulated expression of PRMT5, CYCLIN D1, and c-MYC, which are all predicted targets of these miRNAs, and reduced lymphoma cell survival. Luciferase reporter assays with WT and mutant 3'UTRs of CYCLIN D1 and c-MYC mRNAs revealed that binding sites for miR33b, miR96, and miR503 are critical for translational regulation of the transcripts of these two genes. Our findings link altered PRMT5 expression to transcriptional silencing of tumor-suppressing miRNAs in lymphoma cells and reinforce PRMT5's relevance for promoting lymphoma cell growth and survival.

Expression of p27 and c-Myc by immunohistochemistry in breast ductal cancers in African American women.

Proteins p27 and c-Myc are both key players in the cell cycle. While p27, a tumor suppressor, inhibits progression from G1 to S phase, c-Myc, a proto-oncogene, plays a key role in cell cycle regulation and apoptosis. The objective of our study was to determine the association between expression of c-Myc and the loss of p27 by immunohistochemistry (IHC) in the four major subtypes of breast cancer (BC) (Luminal A, Luminal B, HER2, and Triple Negative) and with other clinicopathological factors in a population of 202 African-American (AA) women. Tissue microarrays (TMAs) were constructed from FFPE tumor blocks from primary ductal breast carcinomas in 202 AA women. Five micrometer sections were stained with a mouse monoclonal antibody against p27 and a rabbit monoclonal antibody against c-Myc. The sections were evaluated for intensity of nuclear reactivity (1-3) and percentage of reactive cells; an H-score was derived from the product of these measurements. Loss of p27 expression and c-Myc overexpression showed statistical significance with ER negative (p < 0.0001), PR negative (p < 0.0001), triple negative (TN) (p < 0.0001), grade 3 (p = 0.038), and overall survival (p = 0.047). There was no statistical significant association between c-Myc expression/p27 loss and luminal A/B and Her2 overexpressing subtypes. In our study, a statistically significant association between c-Myc expression and p27 loss and the triple negative breast cancers (TNBC) was found in AA women. A recent study found that constitutive c-Myc expression is associated with inactivation of the axin 1 tumor suppressor gene. p27 inhibits cyclin dependent kinase2/cyclin A/E complex formation. Axin 1 and CDK inhibitors may represent possible therapeutic targets for TNBC.

C-Myc co-ordinates mRNA cap methylation and ribosomal RNA production.

The mRNA cap is a structure added to RNA pol II transcripts in eukaryotes, which recruits factors involved in RNA processing, nuclear export and translation initiation. RNA guanine-7 methyltransferase (RNMT)–RNA-activating miniprotein (RAM), the mRNA cap methyltransferase complex, completes the basic functional mRNA cap structure, cap 0, by methylating the cap guanosine. Here, we report that RNMT–RAM co-ordinates mRNA processing with ribosome production. Suppression of RNMT–RAM reduces synthesis of the 45S ribosomal RNA (rRNA) precursor. RNMT–RAM is required for c-Myc expression, a major regulator of RNA pol I, which synthesises 45S rRNA. Constitutive expression of c-Myc restores rRNA synthesis when RNMT–RAM is suppressed, indicating that RNMT–RAM controls rRNA production predominantly by controlling c-Myc expression. We report that RNMT–RAM is recruited to the ribosomal DNA locus, which may contribute to rRNA synthesis in certain contexts.

Dual Inhibition of Histone Deacetylases and Phosphoinositide 3-Kinase Enhances Therapeutic Activity Against B Cell Lymphoma

Oncogenic co-operation between c-Myc and activated phosphoinositide 3-kinase (PI3K) signaling pathways is crucial in lymphomagenesis, providing an opportunity for developing mechanism-based therapy to disrupt this co-operative survival mechanism. Combining constitutive c-Myc expression with constitutive PI3K activity in mouse germinal center B (GCB) cells resulted in Burkitt lymphoma-like tumors. Furthermore, analysis of primary human Burkitt lymphoma (BL) tissue sections revealed that two-thirds of the cases expressed high levels of phosphorylated AKT and S6 proteins, indicative of PI3K and mTORC1 activation. Prior attempts to develop small molecule inhibitors that specifically and directly target c-Myc protein have been unsuccessful. However, c-Myc cellular protein abundance can be decreased by using epigenetic modifying drugs (HDAC inhibitors or bromodomain/BET inhibitors) that are known to inhibit c-Myc transcription. Several investigators attempted disrupting c-Myc and PI3K cooperation by combining HDAC inhibitors and PI3K pathway inhibitors and synergic activity was demonstrated in DLBCL irrespective of subtype. In this study, we assessed the efficacy of CUDC-907, a new rationally designed dual inhibitor of PI3K and HDACs, in a panel of lymphoma cell lines. CUDC-907 treatment resulted in a dose- and time-dependent growth inhibition and cell death of DLBCL cell lines, irrespective of the cell of origin. CUDC-907 treatment down-regulated the phosphorylation of PI3K downstream targets, including AKT, PRAS40, S6, and 4EBP1, increased histone 3 acetylation, and decreased Myc protein levels. SILAC-based quantitative mass spectrometry demonstrated that CUDC-907 treatment decreased the protein levels of several components of the B cell receptor (BCR) and Toll like receptor (TLR) pathways, including BTK, SYK, and MyD88 proteins. These cellular changes were associated with an inhibition of NF-kB activation. CUDC-907 demonstrated in vivo efficacy with no significant toxicity in a human DLBCL xenograft mouse model. Collectively, these data provide a mechanistic rationale for evaluating CUDC-907 for the treatment of patients with c-Myc and PI3K-dependent lymphomas. DisclosuresNo relevant conflicts of interest to declare.

Developments in Burkitt&amp;#39;s lymphoma: novel cooperations in oncogenic MYC signaling

Burkitt’s lymphoma (BL) is an aggressive disorder associated with extremely high rates of cell proliferation tempered by high levels of apoptosis. Despite the high levels of cell death, the net effect is one of rapid tumor growth. The tumor arises within the germinal centers of secondary lymphoid tissues and is identifiable by translocation of the c-MYC gene into the immunoglobulin gene loci, resulting in deregulation of the proto-oncogene. Many of the major players involved in determining the development of BL have been characterized in human BL cell lines or in mouse models of MYC-driven lymphomagenesis. Both systems have been useful so far in characterizing the role of tumor suppressor genes (for example, p53), prosurvival signaling pathways, and members of the B-cell lymphoma-2 family of apoptosis regulators in determining the fate of c-MYC overexpressing B-cells, and ultimately in regulating lymphoma development. Signaling through phosphoinositide (PI)3-kinase stands out as being critical for BL cell survival. Recurrent mutations in ID3 or TCF3 (E2A) that promote signaling through PI3-kinase have recently been identified in human BL samples, and new therapeutic strategies based on coordinately targeting both the prosurvival factor, B-cell lymphoma-XL, and the PI3-kinase/AKT/mammalian target of rapamycin (mTOR) signaling pathway to synergistically induced BL apoptosis have been proposed. Now, engineering both constitutive c-MYC expression and PI3-kinase activity, specifically in murine B-cells undergoing the germinal center reaction, has revealed that there is synergistic cooperation between c-MYC and PI3-kinase during BL development. The resulting tumors phenocopy the human malignancy, and acquire tertiary mutations also present in human tumors. This model may, therefore, prove useful in further studies to identify functionally relevant mutational events necessary for BL pathogenesis. This review discusses these cooperating interactions, the possible influence of BL tumor-associated viruses, and highlights potential new opportunities for therapeutic intervention.

HIV-Associated Burkitt Lymphoma: Case Report and Literature Review

IntroductionHIV associated Burkitt lymphoma (BL) is a highly aggressive lymphoma. Compared to other AIDS related non-Hodgkin lymphoma's its clinical presentation is less predictable and characterized by rapid clinical changes. The paradigm shift to treat HIV associated disease with highly effective intensive chemotherapy regimens while managing immunosuppression has made it possible to achieve remission and long-term survival. Southern Louisiana has one the of the highest incidence rates of HIV and cancers in the US. The authors present a challenging case of a 33-year old HIV+ male from southern Louisiana with advanced stage BL. The patient presented with advanced disease and a poor performance status. His initial providers recommended palliation only and significant education was needed to allow aggressive treatment. The patient tolerated induction chemotherapy and remains in remission. This is a case based review of local and nationwide epidemiology and the trial data supporting different treatment options for HIV associated Burkitt lymphoma. CaseOur patient is a 33 year-old male who presented with severe cachexia, poor performance status and clinical symptomatic cord compression. He was diagnosed with advanced stage Burkitt lymphoma including bone marrow involvement. His CD4 count was 277 with an HIV viral load of 571,000 copies/mL. Although he had a poor performance status and his primary team favored palliation only he underwent induction chemotherapy with 4 cycles of infusional dose adjusted R-EPOCH with IT CNS chemoprophylaxis. HAART therapy with efavirenz/emtricitabine/tenofovir was started during his initial admission. He tolerated this regimen well, clinically dramatically improved being independent in his ADL/IADLs, and remains in remission. DiscussionThe pathogenesis of HIV-associated Burkitt lymphoma is linked to oncogenic virus co-infection (40% EBV association) and chronic HIV antigen stimulation that provoke expansion of polyclonal B-cells. This leads to oncogenic chromosomal translocations - most notably t(8;14) causing constitutive expression of oncogene c-Myc. HIV patients have a 60 to 200-fold increased incidence of NHL. Relative risk for BL in HIV patients is 261-fold higher compared to the general population. Unlike the nationwide reported 19 per 100,000 person-years, data from a large HIV clinic in southern Louisiana continues to show an incidence rate of 23 per 100,000 person-years. Louisiana continues to have comparatively high incidence rates in HIV (4th in US) and cancer (11th in US) with the highest rates in southern Louisiana parishes. It is important for community physicians, especially in these areas, to feel more confident when treating these HIV associated malignancies.Historically highly immunosuppressive chemotherapy has been avoided in HIV patients with lymphoma as studies in the 1990's showed increased toxicity. The addition of the monoclonal C20 antibody rituximabin the treatment of AIDS associated NHLs was also controversial, as trial results showed increase infectious death rates. However, these clinical trials confirmed similar death rates from infections in individuals with a CD4 count >50. In addition concomitant use of HAART with chemotherapy has improved overall survival. Although the more dose intense Burkitt regimens have not been studied extensively in HIV-associated BL, CODOX-M/IVAC and DA-EPOCH +/- Rituximab (depending on CD4 count) have shown good efficacy and tolerance and are treatment regimens of choice this disease. A recent trial with 14 patients with HIV associated BL receiving RD-CODOXM/IVAC had 86% progression free survival rate at 1 year; patients receiving concomitant HAART had 100% PFS rate at 1 year. In another trial, 10 HIV associated BL patients receiving DA-EPOCH-R showed a 100% overall survival rate at 57 months. ConclusionHIV associated Burkitt lymphoma should be treated with HAART and high dose chemotherapy. Long-term remission has been shown with regimens including CODOX-M/IVAC and DA-EPOCH +/-Rituximab (R use discouraged in low CD4 counts). This case based report outlines the relatively high prevalence of disease in Southern Louisiana and emphasizes that remission is possible in patients with poor performance status and advanced disease. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

A c-Myc and surface CD19 signaling amplification loop promotes B cell lymphoma development and progression in mice.

Malignant B cells responding to external stimuli are likely to gain a growth advantage in vivo. These cells may therefore maintain surface CD19 expression to amplify transmembrane signals and promote their expansion and survival. To determine whether CD19 expression influences this process, Eμ-Myc transgenic (c-Myc(Tg)) mice that develop aggressive and lethal B cell lymphomas were made CD19 deficient (c-Myc(Tg)CD19⁻/⁻). Compared with c-Myc(Tg) and c-Myc(Tg)CD19⁺/⁻ littermates, the median life span of c-Myc(Tg)CD19⁻/⁻ mice was prolonged by 81-83% (p < 0.0001). c-Myc(Tg)CD19⁻/⁻ mice also lived 42% longer than c-Myc(Tg) littermates following lymphoma detection (p < 0.01). Tumor cells in c-Myc(Tg) and c-Myc(Tg)CD19⁻/⁻ mice were B lineage derived, had a similar phenotype with a large blastlike appearance, invaded multiple lymphoid tissues, and were lethal when adoptively transferred into normal recipient mice. Importantly, reduced lymphomagenesis in c-Myc(Tg)CD19⁻/⁻ mice was not due to reductions in early B cell numbers prior to disease onset. In mechanistic studies, constitutive c-Myc expression enhanced CD19 expression and phosphorylation on active sites. Reciprocally, CD19 expression in c-Myc(Tg) B cells enhanced c-Myc phosphorylation at regulatory sites, sustained higher c-Myc protein levels, and maintained a balance of cyclin D2 expression over that of cyclin D3. These findings define a new and novel c-Myc:CD19 regulatory loop that positively influences B cell transformation and lymphoma progression.

Synergy between PI3K Signaling and MYC in Burkitt Lymphomagenesis

In Burkitt lymphoma (BL), a germinal center B-cell-derived tumor, the pro-apoptotic properties of c-MYC must be counterbalanced. Predicting that survival signals would be delivered by phosphoinositide-3-kinase (PI3K), a major survival determinant in mature B cells, we indeed found that combining constitutive c-MYC expression and PI3K activity in germinal center B cells of the mouse led to BL-like tumors, which fully phenocopy human BL with regard to histology, surface and other markers, and gene expression profile. The tumors also accumulate tertiary mutational events, some of which are recurrent in the human disease. These results and our finding of recurrent PI3K pathway activation in human BL indicate that deregulated c-MYC and PI3K activity cooperate in BL pathogenesis.

Burkitt Lymphoma: Much More than MYC

Chromosomal translocations causing deregulated c-MYC expression are detectable in most Burkitt lymphoma cases. However, little is known about the additional lesions necessary for lymphomagenesis. Now, two independent studies, one of which was performed by Sander etal. in this issue of Cancer Cell, identify constitutive PI3K signaling and CyclinD3 mutations as cooperating lesions in both mice and humans. The results have directly actionable therapeutic implications.

Abstract 275: Burkitt lymphoma cells express GLI1 but are not responsive to Hedgehog signaling

Squamous cell carcinoma (SCC) is an epithelial malignancy involving many anatomical sites and is the most common cancer capable of metastatic spread. Development of early diagnosis methods and novel therapeutics are important for prevention and mortality reduction. In this effort, numerous molecular alterations have been described in SCCs. SCCs share many phenotypic and molecular characteristics, but they have not been extensively compared. This article reviews SCC as a disease, including: epidemiology, pathology, risk factors, molecular characteristics, prognostic markers, targeted therapy, and a new approach to studying SCCs. Through this comparison, several themes are apparent. For example, HPV infection is a common risk factor among the four major SCCs (NMSC, HNSC, ESCC, and NSCLC) and molecular abnormalities in cell-cycle regulation and signal transduction predominate. These data reveal that the molecular insights, new markers, and drug targets discovered in individual SCCs may shed light on this type of cancer as a whole.

Metastable Pluripotent States in NOD-Mouse-Derived ESCs

Embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of blastocysts, whereas epiblast stem cells (EpiSCs) are derived from the postimplantation epiblast and display a restricted developmental potential. Here we characterize pluripotent states in the nonobese diabetic (NOD) mouse strain, which prior to this study was considered "nonpermissive" for ESC derivation. We find that NOD stem cells can be stabilized by providing constitutive expression of Klf4 or c-Myc or small molecules that can replace these factors during in vitro reprogramming. The NOD ESCs and iPSCs appear to be "metastable," as they acquire an alternative EpiSC-like identity after removal of the exogenous factors, while their reintroduction converts the cells back to ICM-like pluripotency. Our findings suggest that stem cells from different genetic backgrounds can assume distinct states of pluripotency in vitro, the stability of which is regulated by endogenous genetic determinants and can be modified by exogenous factors.

AID and RAG1 do not contribute to lymphomagenesis in Eμ c-myc transgenic mice

DNA breaks caused by recombination-activating gene 1 (RAG1) and activation-induced cytidine deaminase (AID) induce c-myc/immunoglobulin (Ig) heavy chain chromosomal translocations and thereby stimulate lymphomagenesis. However, constitutive expression of c-myc alone is not sufficient to induce lymphomas. Because RAG1 and AID activity occurs outside of Ig genes, we assessed whether these enzymes provide the secondary genetic lesions in Emu c-myc transgenic mice to promote lymphoma development. We found that the tumor incidence and tumor phenotype in Emu c-myc transgenic mice is similar in AID+/+, AID+/- and AID-/- backgrounds in both specific pathogen-free and conventional animal facilities, indicating that AID does not contribute to lymphoma development in Emu c-myc transgenic mice. To examine the role of RAG proteins in Emu c-myc mice, we examined Emu c-myc transgenic mice that harbor the Ig-HEL heavy- and light-chain transgenes, and thus have reduced RAG expression in B cells. We found that tumor incidence was not affected by these Ig transgenes. However, we found that RAG1-/- Emu c-myc mice exhibited accelerated tumor development compared to controls. This data combined with our finding that Emu c-myc mice lived longer in the conventional facility than in the specific pathogen-free facility suggest an immune-mediated activity that suppresses lymphoma development.

Induction of Constitutive High-Level Expression of c-Myc in 32D Cells by Mycoplasmas is Associated with Their Ability to Prevent Apoptosis and Induce Malignant Transformation

Our previous studies showed that infections by mycoplasmas could prevent apoptosis and induce the malignant transformation of mammalian cells. Other studies indicate that c-Myc plays an important role in promoting apoptosis and malignant transformation of cells. To understand the role of c-Myc in the mycoplasma-induced apoptosis prevention and malignant cell transformation, 32D cells, an IL-3 dependent cell line, were infected and transformed by different species of mycoplasmas. The expression of Myc and ras gene families, apoptosis and the cell cycle during the infection and transformation were examined. Results showed that c-Myc expression was significantly increased in mycoplasma-transformed 32D cells. Withdrawal of IL-3 substantially decreased c-Myc expression in non-infected 32D cells and led to cell cycle arrest at the G1 phase followed by rapid apoptosis. Infection by M. fermentans or M. penetrans not only alleviated the sharp decrease of c-Myc expression, rescued 32D cells from cell-cycle arrest and prevented apoptosis in IL-3-free culture, but also induced autonomous growth of 32D cells. Although M. hominis and M. salivarium had the ability neither to prevent apoptosis nor to induce malignant transformation, they were still able to rescue the cells from cell cycle arrest at the G1 phase. The expression of ras family did not change significantly during the infection and transformation. These results suggest that constitutive expression of c-Myc appears to be associated with the continuous growth and malignant transformation of 32D cells induced by M. fermentans and M. penetrans, but not with rescuing the cell cycle arrest by the mycoplasmas.

The Synergistic Action of the microRNA-17 Cluster with c-MYC in Aggressive Cancer Development.

Malignant lymphoma frequently shows genomic amplification of 13q31-32, which harbor c13orf25, a host of mature microRNA-17, 18, 19a, 19b, 20a, and 92–1 (Ota et al., Cancer Res 2004). It has been reported that Eμ-myc mice enforced by over-expression of the miR-17-19b, could develop tumor significantly earlier than those without the expression (He et al., Nature 2005), and that overexpression of a proto-oncogene, c-MYC can up-regulate miR-17 cluster via binding directory upstream of miR-17–92 locus (O' Donnell et al., Nature 2005). These findings suggest that the miR-17 cluster and c-MYC synergistically contribute to cancer development. However, it is still unclear how c-myc acts synergistically in tumorigenesis with the miR-17 cluster, and which genes are essentially regulated by the miR-17 cluster. In this study, we inserted 674 or 755 bp fragments spanning a given miRNA-genomic region in a modified PMXs vector such that they are placed under the control of a LTR promoter, and thus established Rat-1 and NIH3T3 cells which stably expressed miR-17–18-19a–20a-19b-92 or miR-17–18-19a-20-19b (miR-17–92/miR-17-19b). We demonstrated that the miR-17 cluster is highly upregulated under the constitutive expression of c-myc, and that a rat fibroblast (Rat-1) stably expressing both the miR-17 cluster and c-myc (miRs+myc) shows a full-blown neoplastic transformation phenotype. Because the transformation of Rat-1 cells could be due to the suppression of tumor suppressor gene(s), we next conducted screening for predicted tumor related gene(s) by a Western blot analysis with lysate from Rat-1 transfectants. Our examined predicted tumor suppressor genes of miR-17 cluster were p130 (RBR2), PTEN, SOSC-3, Smad2, MeCp2, and TGF-β type II receptor (TβRII). Among these, we could detect significant reduced expression of TβRII protein in the miRs+myc transfectant, although no other predicted targets showed such a significant difference. The expression levels of TβRII gene in Rat-1 and NIH3T3 transfected with miRs+myc were also repressed in comparison to those of the other transfectants. These results demonstrate that a combined effect of RNA destruction and translation inhibition is used by the miR-17 cluster to silence TβRII. To further substantiate TβRII as a direct target of miR-17 cluster, we cloned its 3′UTR sequence downstream of the firefly luciferase gene (pGL3-TβRII). As a control, a construct with the mutated target sequence was also made. Transfection experiments showed a significant repression of the luciferase activity in the wild type construct in comparison to mutated TβRII, thus suggesting that TβRII is direct downstream target of the miR-17 cluster. Up-regulation of miR-17 cluster and c-myc might contribute to tumorigenesis via the down regulation of both the TβRII-Smads and the c-myc-Miz-1-p15INK4b pathways, respectively. Furthermore, we demonstrated that aggressive B-cell lymphoma frequently represents both c-MYC rearrangement and the upregulation of the miR-17 cluster via 13q31 amplification. These results suggest that the deregulation of the miR-17 cluster and c-MYC synergistically contribute to the aggressive clinicopathological features of cancers.

Inhibiton of mTOR by RAD001 Restores Normal B Cell Differentiation and Prevents B Cell Lymphomas in Eμ-MYC Mice.

The c-Myc proto-oncogene encodes a bHLH-LZ transcription factor that regulates proliferation, differentiation and apoptosis. Deregulated expression of c-MYC is a frequent finding in a wide variety of human cancers, including B cell lymphoma. One emerging function of c-MYC is the regulation of ribosome biogenesis, protein synthesis and metabolism i.e. cell growth. mTOR, a key downstream signal transduction molecule in the PI3K/AKT growth regulatory pathway, is amenable to pharmacological inhibition by rapamycin analogues such as RAD001. We hypothesized that control of cell growth by c-MYC is important for its ability to regulate differentiation and act as an oncogene and that RAD001, by limiting cell growth, would attenuate the transforming properties of c-MYC. In Eμ-myc mice the c-myc transgene is under the control of the immunoglobulin heavy chain enhancer (Eμ). Constitutive expression of c-MYC results in a polyclonal expansion of B cell precursors followed by lymphoma development. In the current study ‘pre-lymphomatous' Eμ-myc mice were randomized to receive RAD001 5mg/kg (n=20) or placebo (n=18) 6 days per week from 4 weeks of age. Peripheral blood B cells were analyzed by surface marker expression after 2, 4 and 8 weeks of therapy. Mice were monitored weekly for the development of lymphadenopathy. 2 weeks of treatment with RAD001 significantly reduced the numbers of B cells in the blood of Eμ-myc mice compared to placebo (1.37±0.13 ×103/μL in the RAD001 arm versus 3.41±0.64 ×103/μL in the placebo arm, p<0.05). In particular, there was preferential suppression of less mature circulating B cell precursors over mature B cells by RAD001 resulting in B cell developmental subset profiles more closely approaching those of wild-type mice (Figure 1). Treatment with RAD001 was associated with improved lymphoma-free survival; 13/14 lymphoma-free mice (92.9%) versus 6/11 (54.6%) in the placebo group in an interim analysis of mice that had received at least 60 days of therapy. These results indicate that RAD001 can firstly oppose the expansion of B cell precursors and secondly reduce the incidence of malignant transformation induced by deregulated expression of c-MYC in B lymphocytes. These findings have implications for the application of mTOR inhibitors in the treatment or prevention of malignancies associated with MYC. [Display omitted]

Review of: c-Myc suppresses p21WAF1/CIP1expression during oestrogen signalling and antioestrogen resistance in human breast cancer cells

Citation of original article:S. Mukherjee, S. E. Conrad.Journal of Biological Chemistry2005;280: 17616–17625.Abstract of the original article:Oestrogen rapidly induces expression of the proto-oncogene c-Myc. c-Myc is required for oestrogen-stimulated proliferation of breast cancer cells, and deregulated c-Myc expression has been implicated in antioestrogen resistance. In this report, we investigate the mechanism(s) by which c-Myc mediates oestrogen-stimulated proliferation and contributes to cell cycle progression in the presence of antioestrogen. The MCF-7 cell line is a model of oestrogen-dependent, antioestrogen-sensitive human breast cancer. Using stable MCF-7 derivatives with inducible c-Myc expression, we demonstrated that in antioestrogen-treated cells, the elevated mRNA and protein levels of p21WAF1/CIP1, a cell cycle inhibitor, decreased upon either c-Myc induction or oestrogen treatment. Expression of p21 blocked c-Myc-mediated cell cycle progression in the presence of antioestrogen, suggesting that the decrease in p21WAF1/CIP1is necessary for this process. Using RNA interference to suppress c-Myc expression, we further established that c-Myc is required for oestrogen-mediated decreases in p21WAF1/CIP1. Finally, we observed that neither c-Myc nor p21WAF1/CIP1is regulated by oestrogen or antioestrogen in an antioestrogen-resistant MCF-7 derivative. The p21 levels in the antioestrogen-resistant cells increased when c-Myc expression was suppressed, suggesting that loss of p21 regulation was a consequence of constitutive c-Myc expression. Together, these studies implicate p21WAF1/CIP1as an important target of c-Myc in breast cancer cells and provide a link between oestrogen, c-Myc, and the cell cycle machinery. They further suggest that aberrant c-Myc expression, which is frequently observed in human breast cancers, can contribute to antioestrogen resistance by altering p21WAF1/CIP1regulation.

Transformation of Human and Murine Fibroblasts without Viral Oncoproteins

Murine embryo fibroblasts are readily transformed by the introduction of specific combinations of oncogenes; however, the expression of those same oncogenes in human cells fails to convert such cells to tumorigenicity. Using normal human and murine embryonic fibroblasts, we show that the transformation of human cells requires several additional alterations beyond those required to transform comparable murine cells. The introduction of the c-Myc and H-RAS oncogenes in the setting of loss of p53 function efficiently transforms murine embryo fibroblasts but fails to transform human cells constitutively expressing hTERT, the catalytic subunit of telomerase. In contrast, transformation of multiple strains of human fibroblasts requires the constitutive expression of c-Myc, H-RAS, and hTERT, together with loss of function of the p53, RB, and PTEN tumor suppressor genes. These manipulations permit the development of transformed human fibroblasts with genetic alterations similar to those found associated with human cancers and define specific differences in the susceptibility of human and murine fibroblasts to experimental transformation.

C-Myc Suppresses p21WAF1/CIP1 Expression during Estrogen Signaling and Antiestrogen Resistance in Human Breast Cancer Cells

Estrogen rapidly induces expression of the proto-oncogene c-myc. c-Myc is required for estrogen-stimulated proliferation of breast cancer cells, and deregulated c-Myc expression has been implicated in antiestrogen resistance. In this report, we investigate the mechanism(s) by which c-Myc mediates estrogen-stimulated proliferation and contributes to cell cycle progression in the presence of antiestrogen. The MCF-7 cell line is a model of estrogen-dependent, antiestrogen-sensitive human breast cancer. Using stable MCF-7 derivatives with inducible c-Myc expression, we demonstrated that in antiestrogen-treated cells, the elevated mRNA and protein levels of p21(WAF1/CIP1), a cell cycle inhibitor, decreased upon either c-Myc induction or estrogen treatment. Expression of p21 blocked c-Myc-mediated cell cycle progression in the presence of antiestrogen, suggesting that the decrease in p21 is necessary for this process. Using RNA interference to suppress c-Myc expression, we further established that c-Myc is required for estrogen-mediated decreases in p21(WAF1/CIP1). Finally, we observed that neither c-Myc nor p21(WAF1/CIP1) is regulated by estrogen or antiestrogen in an antiestrogen-resistant MCF-7 derivative. The p21 levels in the antiestrogen-resistant cells increased when c-Myc expression was suppressed, suggesting that loss of p21 regulation was a consequence of constitutive c-Myc expression. Together, these studies implicate p21(WAF1/CIP1) as an important target of c-Myc in breast cancer cells and provide a link between estrogen, c-Myc, and the cell cycle machinery. They further suggest that aberrant c-Myc expression, which is frequently observed in human breast cancers, can contribute to antiestrogen resistance by altering p21(WAF1/CIP1) regulation.