Duonan Yu

Augmentation of tumor angiogenesis by a Myc-activated microRNA cluster

Human adenocarcinomas commonly harbor mutations in the KRAS and MYC proto-oncogenes and the TP53 tumor suppressor gene. All three genetic lesions are potentially pro-angiogenic, as they sustain production of vascular endothelial growth factor (VEGF). Yet Kras-transformed mouse colonocytes lacking p53 formed indolent, poorly vascularized tumors, whereas additional transduction with a Myc-encoding retrovirus promoted vigorous vascularization and growth. In addition, VEGF levels were unaffected by Myc, but enhanced neovascularization correlated with downregulation of anti-angiogenic thrombospondin-1 (Tsp1) and related proteins, such as connective tissue growth factor (CTGF). Both Tsp1 and CTGF are predicted targets for repression by the miR-17-92 microRNA cluster, which was upregulated in colonocytes coexpressing K-Ras and c-Myc. Indeed, miR-17-92 knockdown with antisense 2′-O-methyl oligoribonucleotides partly restored Tsp1 and CTGF expression; in addition, transduction of Ras-only cells with a miR-17-92–encoding retrovirus reduced Tsp1 and CTGF levels. Notably, miR-17-92–transduced cells formed larger, better-perfused tumors. These findings establish a role for microRNAs in non–cell-autonomous Myc-induced tumor phenotypes.

Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma

Autophagy is a lysosome-dependent degradative pathway frequently activated in tumor cells treated with chemotherapy or radiation. Whether autophagy observed in treated cancer cells represents a mechanism that allows tumor cells to survive therapy or a mechanism for initiating a nonapoptotic form of programmed cell death remains controversial. To address this issue, the role of autophagy in a Myc-induced model of lymphoma generated from cells derived from p53ER(TAM)/p53ER(TAM) mice (with ER denoting estrogen receptor) was examined. Such tumors are resistant to apoptosis due to a lack of nuclear p53. Systemic administration of tamoxifen led to p53 activation and tumor regression followed by tumor recurrence. Activation of p53 was associated with the rapid appearance of apoptotic cells and the induction of autophagy in surviving cells. Inhibition of autophagy with either chloroquine or ATG5 short hairpin RNA (shRNA) enhanced the ability of either p53 activation or alkylating drug therapy to induce tumor cell death. These studies provide evidence that autophagy serves as a survival pathway in tumor cells treated with apoptosis activators and a rationale for the use of autophagy inhibitors such as chloroquine in combination with therapies designed to induce apoptosis in human cancers.

A GATA-1-regulated microRNA locus essential for erythropoiesis

MicroRNAs (miRNAs) control tissue development, but their mechanism of regulation is not well understood. We used a gene complementation strategy combined with microarray screening to identify miRNAs involved in the formation of erythroid (red blood) cells. Two conserved miRNAs, miR 144 and miR 451, emerged as direct targets of the critical hematopoietic transcription factor GATA-1. In vivo, GATA-1 binds a distal upstream regulatory element to activate RNA polymerase II-mediated transcription of a single common precursor RNA (pri-miRNA) encoding both mature miRNAs. Zebrafish embryos depleted of miR 451 by using antisense morpholinos form erythroid precursors, but their development into mature circulating red blood cells is strongly and specifically impaired. These results reveal a miRNA locus that is required for erythropoiesis and uncover a new regulatory axis through which GATA-1 controls this process.

Insights into GATA-1-Mediated Gene Activation versus Repression via Genome-wide Chromatin Occupancy Analysis

The transcription factor GATA-1 is required for terminal erythroid maturation and functions as an activator or repressor depending on gene context. Yet its in vivo site selectivity and ability to distinguish between activated versus repressed genes remain incompletely understood. In this study, we performed GATA-1 ChIP-seq in erythroid cells and compared it to GATA-1-induced gene expression changes. Bound and differentially expressed genes contain a greater number of GATA-binding motifs, a higher frequency of palindromic GATA sites, and closer occupancy to the transcriptional start site versus nondifferentially expressed genes. Moreover, we show that the transcription factor Zbtb7a occupies GATA-1-bound regions of some direct GATA-1 target genes, that the presence of SCL/TAL1 helps distinguish transcriptional activation versus repression, and that polycomb repressive complex 2 (PRC2) is involved in epigenetic silencing of a subset of GATA-1-repressed genes. These data provide insights into GATA-1-mediated gene regulation in vivo.

Direct repression of FLIP expression by c-myc is a major determinant of TRAIL sensitivity

ABSTRACT Tumor necrosis factor alpha (TNF-α)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF-α family of death receptor ligands and holds great therapeutic potential as a tumor cell-specific cytotoxic agent. Using a panel of established tumor cell lines and normal cells, we found a significant difference between the number of TRAIL-sensitive cells expressing high levels of c-myc and TRAIL-resistant cells expressing low levels of c-myc (P < 0.05, n = 19). We also found a direct linear correlation between c-myc levels and TRAIL sensitivity in TRAIL-sensitive cell lines (r = 0.94, n = 6). Overexpression of c-myc or activation of a myc-estrogen receptor (ER) fusion sensitized TRAIL-resistant cells to TRAIL. Conversely, small interfering RNA (siRNA)-mediated knockdown of c-myc significantly reduced both c-myc expression and TRAIL-induced apoptosis. The gene encoding the inhibitor of caspase activation, FLICE inhibitory protein (FLIP), appears to be a direct target of c-myc-mediated transcriptional repression. Overexpression of c-myc or activation of myc-estrogen receptor (ER) decreased FLIP levels both in cell culture and in mouse models of c-myc-induced tumorigenesis, while knocking down c-myc using siRNA increased FLIP expression. Chromatin immunoprecipitation and luciferase reporter analyses showed that c-myc binds and represses the human FLIP promoter. c-myc expression enhanced TRAIL-induced caspase 8 cleavage and FLIP cleavage at the death-inducing signaling complex. Combined siRNA-mediated knockdown of FLIP and c-myc resensitized cells to TRAIL. Therefore, c-myc down-regulation of FLIP expression provides a universal mechanism to explain the ability of c-myc to sensitize cells to death receptor stimuli. In addition, identification of c-myc as a major determinant of TRAIL sensitivity provides a potentially important screening tool for identification of TRAIL-sensitive tumors.

Activation of Transferrin Receptor 1 by c-Myc Enhances Cellular Proliferation and Tumorigenesis

ABSTRACT Overexpression of transferrin receptor 1 (TFRC1), a major mediator of iron uptake in mammalian cells, is a common feature of human malignancies. Therapeutic strategies designed to interfere with tumor iron metabolism have targeted TFRC1. The c-Myc oncogenic transcription factor stimulates proliferation and growth by activating thousands of target genes. Here we demonstrate that TFRC1 is a critical downstream target of c-Myc. Using in vitro and in vivo models of B-cell lymphoma, we show that TFRC1 expression is activated by c-Myc. Chromatin immunoprecipitation experiments reveal that c-Myc directly binds a conserved region of TFRC1. In light of these findings, we sought to determine whether TFRC1 is required for c-Myc-mediated cellular proliferation and cell size control. TFRC1 inhibition decreases cellular proliferation and results in G1 arrest without affecting cell size. Consistent with these findings, expression profiling reveals that TFRC1 depletion alters expression of genes that regulate the cell cycle. Furthermore, enforced TFRC1 expression confers a growth advantage to cells and significantly enhances the rate of c-Myc-mediated tumor formation in vivo. These findings provide a molecular basis for increased TFRC1 expression in human tumors, illuminate the role of TFRC1 in the c-Myc target gene network, and support strategies that target TFRC1 for cancer therapy.

miR-451 protects against erythroid oxidant stress by repressing 14-3-3ζ

The bicistronic microRNA (miRNA) locus miR-144/451 is highly expressed during erythrocyte development, although its physiological roles are poorly understood. We show that miR-144/451 ablation in mice causes mild erythrocyte instability and increased susceptibility to damage after exposure to oxidant drugs. This phenotype is deeply conserved, as miR-451 depletion synergizes with oxidant stress to cause profound anemia in zebrafish embryos. At least some protective activities of miR-451 stem from its ability to directly suppress production of 14-3-3zeta, a phospho-serine/threonine-binding protein that inhibits nuclear accumulation of transcription factor FoxO3, a positive regulator of erythroid anti-oxidant genes. Thus, in miR-144/451(-/-) erythroblasts, 14-3-3zeta accumulates, causing partial relocalization of FoxO3 from nucleus to cytoplasm with dampening of its transcriptional program, including anti-oxidant-encoding genes Cat and Gpx1. Supporting this mechanism, overexpression of 14-3-3zeta in erythroid cells and fibroblasts inhibits nuclear localization and activity of FoxO3. Moreover, shRNA suppression of 14-3-3zeta protects miR-144/451(-/-) erythrocytes against peroxide-induced destruction, and restores catalase activity. Our findings define a novel miRNA-regulated pathway that protects erythrocytes against oxidant stress, and, more generally, illustrate how a miRNA can influence gene expression by altering the activity of a key transcription factor.

B cell–specific loss of histone 3 lysine 9 methylation in the V H locus depends on Pax5

Immunoglobulin heavy chain rearrangement (VH-to-DJH) occurs only in B cells, suggesting it is inhibited in other lineages. Here we found that in the mouse VH locus, methylation of lysine 9 on histone H3 (H3-K9), a mark of inactive chromatin, was present in non–B lineage cells but was absent in B cells. As others have shown that H3-K9 methylation can inhibit V(D)J recombination on engineered substrates, our data support the idea that H3-K9 methylation inhibits endogenous VH-to-DJH recombination. We also show that Pax5, a transcription factor required for B cell commitment, is necessary and sufficient for the removal of H3-K9 methylation in the VH locus and provide evidence that one function of Pax5 is to remove this inhibitory modification by a mechanism of histone exchange, thus allowing B cell–specific VH-to-DJH recombination.

Oncogenic BRAF regulates β-Trcp expression and NF-κB activity in human melanoma cells

Mutational activation of BRAF is a frequent event in human malignant melanomas suggesting that BRAF-dependent signaling is conducive to melanoma cell growth and survival. Previously published work reported that melanoma cells exhibit constitutive anti-apoptotic nuclear factor κB (NF-κB) transcription factor activation triggered by proteolysis of its inhibitor IκB. IκB degradation is dependent upon its phosphorylation by the IκB kinase (IKK) complex and subsequent ubiquitination facilitated by β-Trcp E3 ubiquitin ligase. Here, we report that melanocytes expressing a conditionally oncogenic form of BRAFV600E exhibit enhanced β-Trcp expression, increased IKK activity and a concomitant increase in the rate of IκBα degradation. Conversely, inhibition of BRAF signaling using either a broad-spectrum Raf inhibitor (BAY 43-9006) or by selective knock-down of BRAFV600E expression by RNA interference in human melanoma cells leads to decreased IKK activity and β-Trcp expression, stabilization of IκB, inhibition of NF-κB transcriptional activity and sensitization of these cells to apoptosis. Taken together, these data support a model in which mutational activation of BRAF in human melanomas contributes to constitutive induction of NF-κB activity and to increased survival of melanoma cells.

An Essential Role of Th1 Responses and Interferon-gamma in Infection-Mediated Suppression of Neoplastic Growth

We had previously demonstrated that in mice acute toxoplasmosis leads to systemic inhibition of angiogenesis and, consequently, strong suppression of neoplastic growth. Here we investigated the role of Th1 cytokines, in particular interferon gamma (IFNg), in this phenomenon. Besides toxoplasma, neoplastic growth was readily blocked during acute infection with other Th1 response-inducing pathogens such as Listeria monocytogenes and lymphocytic choriomeningitis virus (LCMV). In contrast, chronic infection with LCMV (when Th1 responses were strongly suppressed) and acute infection with Schistosoma mansoni (when Th2 responses predominated) afforded no anti-tumor protection. To corroborate the involvement of Th1 cytokines in infection-mediated suppression of neoplastic growth, we utilized mice deficient in interleukin-10 (IL10), a suppressor of Th1 responses. When challenged with B16 cells concomitantly with toxoplasma infection, both IL10-null and wild type mice exhibited resistance to neoplastic growth. However, tumors borne by IL10-null animals were even smaller than those borne by their wild type counterparts. This enhanced resistance correlated with dramatically elevated levels of circulating IFNg, a principal Th1 cytokine. Furthermore, while interleukin-12 and tumor necrosis factor g were dispensable for tumor suppression, in animals deficient in IFNg production or signaling, tumor growth and neovascularization were markedly enhanced. Interestingly, the enhancement was also apparent in uninfected animals suggesting that IFNg and its anti-angiogenic effects underlie both infection-dependent and -independent tumor surveillance.