MYC through miR-17-92 Suppresses Specific Target Genes to Maintain Survival, Autonomous Proliferation, and a Neoplastic State

SummaryThe MYC oncogene regulates gene expression through multiple mechanisms, and its overexpression culminates in tumorigenesis. MYC inactivation reverses turmorigenesis through the loss of distinguishing features of cancer, including autonomous proliferation and survival. Here we report that MYC via miR-17-92 maintains a neoplastic state through the suppression of chromatin regulatory genes Sin3b, Hbp1, Suv420h1, and Btg1, as well as the apoptosis regulator Bim. The enforced expression of miR-17-92 prevents MYC suppression from inducing proliferative arrest, senescence, and apoptosis and abrogates sustained tumor regression. Knockdown of the five miR-17-92 target genes blocks senescence and apoptosis while it modestly delays proliferative arrest, thus partially recapitulating miR-17-92 function. We conclude that MYC, via miR-17-92, maintains a neoplastic state by suppressing specific target genes

A quantitative PCR method to detect blood microRNAs associated with tumorigenesis in transgenic mice

MicroRNA (miRNA) dysregulation frequently occurs in cancer. Analysis of whole blood miRNA in tumor models has not been widely reported, but could potentially lead to novel assays for early detection and monitoring of cancer. To determine whether miRNAs associated with malignancy could be detected in the peripheral blood, we used real-time reverse transcriptase-PCR to determine miRNA profiles in whole blood obtained from transgenic mice with c-MYC-induced lymphoma, hepatocellular carcinoma and osteosarcoma. The PCR-based assays used in our studies require only 10 nanograms of total RNA, allowing serial mini-profiles (20 – 30 miRNAs) to be carried out on individual animals over time. Blood miRNAs were measured from mice at different stages of MYC-induced lymphomagenesis and regression. Unsupervised hierarchical clustering of the data identified specific miRNA expression profiles that correlated with tumor type and stage. The miRNAs found to be altered in the blood of mice with tumors frequently reverted to normal levels upon tumor regression. Our results suggest that specific changes in blood miRNA can be detected during tumorigenesis and tumor regression

Impact of Hydrodynamic Injection and phiC31 Integrase on Tumor Latency in a Mouse Model of MYC-Induced Hepatocellular Carcinoma

Hydrodynamic injection is an effective method for DNA delivery in mouse liver and is being translated to larger animals for possible clinical use. Similarly, phiC31 integrase has proven effective in mediating long-term gene therapy in mice when delivered by hydrodynamic injection and is being considered for clinical gene therapy applications. However, chromosomal aberrations have been associated with phiC31 integrase expression in tissue culture, leading to questions about safety.To study whether hydrodynamic delivery alone, or in conjunction with delivery of phiC31 integrase for long-term transgene expression, could facilitate tumor formation, we used a transgenic mouse model in which sustained induction of the human C-MYC oncogene in the liver was followed by hydrodynamic injection. Without injection, mice had a median tumor latency of 154 days. With hydrodynamic injection of saline alone, the median tumor latency was significantly reduced, to 105 days. The median tumor latency was similar, 106 days, when a luciferase donor plasmid and backbone plasmid without integrase were administered. In contrast, when active or inactive phiC31 integrase and donor plasmid were supplied to the mouse liver, the median tumor latency was 153 days, similar to mice receiving no injection.Our data suggest that phiC31 integrase does not facilitate tumor formation in this C-MYC transgenic mouse model. However, in groups lacking phiC31 integrase, hydrodynamic injection appeared to contribute to C-MYC-induced hepatocellular carcinoma in adult mice. Although it remains to be seen to what extent these findings may be extrapolated to catheter-mediated hydrodynamic delivery in larger species, they suggest that caution should be used during translation of hydrodynamic injection to clinical applications

Developmental Context Determines Latency of MYC-Induced Tumorigenesis

One of the enigmas in tumor biology is that different types of cancers are prevalent in different age groups. One possible explanation is that the ability of a specific oncogene to cause tumorigenesis in a particular cell type depends on epigenetic parameters such as the developmental context. To address this hypothesis, we have used the tetracycline regulatory system to generate transgenic mice in which the expression of a c-MYC human transgene can be conditionally regulated in murine hepatocytes. MYC's ability to induce tumorigenesis was dependent upon developmental context. In embryonic and neonatal mice, MYC overexpression in the liver induced marked cell proliferation and immediate onset of neoplasia. In contrast, in adult mice MYC overexpression induced cell growth and DNA replication without mitotic cell division, and mice succumbed to neoplasia only after a prolonged latency. In adult hepatocytes, MYC activation failed to induce cell division, which was at least in part mediated through the activation of p53. Surprisingly, apoptosis is not a barrier to MYC inducing tumorigenesis. The ability of oncogenes to induce tumorigenesis may be generally restrained by developmentally specific mechanisms. Adult somatic cells have evolved mechanisms to prevent individual oncogenes from initiating cellular growth, DNA replication, and mitotic cellular division alone, thereby preventing any single genetic event from inducing tumorigenesis

Cancer prevention and therapy through the modulation of the tumor microenvironment

Cancer arises in the context of an in vivo tumor microenvironment. This microenvironment is both a cause and consequence of tumorigenesis. Tumor and host cells co-evolve dynamically through indirect and direct cellular interactions, eliciting multiscale effects on many biological programs, including cellular proliferation, growth, and metabolism, as well as angiogenesis and hypoxia and innate and adaptive immunity. Here we highlight specific biological processes that could be exploited as targets for the prevention and therapy of cancer. Specifically, we describe how inhibition of targets such as cholesterol synthesis and metabolites, reactive oxygen species and hypoxia, macrophage activation and conversion, indoleamine 2,3-dioxygenase regulation of dendritic cells, vascular endothelial growth factor regulation of angiogenesis, fibrosis inhibition, endoglin, and Janus kinase signaling emerge as examples of important potential nexuses in the regulation of tumorigenesis and the tumor microenvironment that can be targeted. We have also identified therapeutic agents as approaches, in particular natural products such as berberine, resveratrol, onionin A, epigallocatechin gallate, genistein, curcumin, naringenin, desoxyrhapontigenin, piperine, and zerumbone, that may warrant further investigation to target the tumor microenvironment for the treatment and/or prevention of cancer

Cell Cycle Re-Entry and Mitochondrial Defects in Myc-Mediated Hypertrophic Cardiomyopathy and Heart Failure

While considerable evidence supports the causal relationship between increases in c-Myc (Myc) and cardiomyopathy as a part of a “fetal re-expression” pattern, the functional role of Myc in mechanisms of cardiomyopathy remains unclear. To address this, we developed a bitransgenic mouse that inducibly expresses Myc under the control of the cardiomyocyte-specific MHC promoter. In adult mice the induction of Myc expression in cardiomyocytes in the heart led to the development of severe hypertrophic cardiomyopathy followed by ventricular dysfunction and ultimately death from congestive heart failure. Mechanistically, following Myc activation, cell cycle markers and other indices of DNA replication were significantly increased suggesting that cell cycle-related events might be a primary mechanism of cardiac dysfunction. Furthermore, pathological alterations at the cellular level included alterations in mitochondrial function with dysregulation of mitochondrial biogenesis and defects in electron transport chain complexes I and III. These data are consistent with the known role of Myc in several different pathways including cell cycle activation, mitochondrial proliferation, and apoptosis, and indicate that Myc activation in cardiomyocytes is an important regulator of downstream pathological sequelae. Moreover, our findings indicate that the induction of Myc in cardiomyocytes is sufficient to cause cardiomyopathy and heart failure, and that sustained induction of Myc, leading to cell cycle re-entry in adult cardiomyocytes, represents a maladaptive response for the mature heart

Loss of Dnmt3b function upregulates the tumor modifier Ment and accelerates mouse lymphomagenesis

DNA methyltransferase 3B (Dnmt3b) belongs to a family of enzymes responsible for methylation of cytosine residues in mammals. DNA methylation contributes to the epigenetic control of gene transcription and is deregulated in virtually all human tumors. To better understand the generation of cancer-specific methylation patterns, we genetically inactivated Dnmt3b in a mouse model of MYC-induced lymphomagenesis. Ablation of Dnmt3b function using a conditional knockout in T cells accelerated lymphomagenesis by increasing cellular proliferation, which suggests that Dnmt3b functions as a tumor suppressor. Global methylation profiling revealed numerous gene promoters as potential targets of Dnmt3b activity, the majority of which were demethylated in Dnmt3b–/– lymphomas, but not in Dnmt3b–/– pretumor thymocytes, implicating Dnmt3b in maintenance of cytosine methylation in cancer. Functional analysis identified the gene Gm128 (which we termed herein methylated in normal thymocytes [Ment]) as a target of Dnmt3b activity. We found that Ment was gradually demethylated and overexpressed during tumor progression in Dnmt3b–/– lymphomas. Similarly, MENT was overexpressed in 67% of human lymphomas, and its transcription inversely correlated with methylation and levels of DNMT3B. Importantly, knockdown of Ment inhibited growth of mouse and human cells, whereas overexpression of Ment provided Dnmt3b+/+ cells with a proliferative advantage. Our findings identify Ment as an enhancer of lymphomagenesis that contributes to the tumor suppressor function of Dnmt3b and suggest it could be a potential target for anticancer therapies