Masking epistasis between MYC and TGF-ß pathways in antiangiogenesis-mediated colon cancer suppression.

BACKGROUND: The c-Myc oncoprotein is activated in the majority of colorectal cancers (CRCs), whereas the TGF-ß pathway is frequently affected by loss-of-function mutations, for example in SMAD2/3/4 genes. The canonical model places Myc downstream of inhibitory TGF-ß signaling. However, we previously demonstrated that Myc also inhibits TGF-ß signaling through the miR-17~92 microRNA cluster, raising the question about functional relationships between these two pathways. METHODS: We engineered a series of genetically complex murine and human CRC cell lines in which Myc and TGF-ß activities could be manipulated simultaneously. This was achieved through retroviral expression of the Myc-estrogen receptor fusion protein and through Smad4 short hairpin RNA knockdown. Cell lines thus modified were injected subcutaneously in immunocompromised mice, and the resultant tumors (n = 5-10 per treatment group) were analyzed for overall growth and neovascularization. Additionally, the distribution of MYC and TGF-ß pathway mutations was analyzed in previously profiled human CRC samples. RESULTS: In kras-mutated/trp53-deleted murine colonocytes, either Myc activation or TGF-ß inactivation increased tumor sizes and microvascular densities approximately 1.5- to 2.5-fold, chiefly through downregulation of thrombospondin-1 and related type I repeat-containing proteins. Combining Myc activation with TGF-ß inactivation did not further accelerate tumorigenesis. This redundancy and the negative effect of TGF-ß signaling on angiogenesis were also demonstrated using xenografts of human CRC cell lines. Furthermore, the analysis of the Cancer Genome Atlas data revealed that in CRC without microsatellite instability, overexpression of Myc and inactivation of Smads (including acquired mutations in SMAD2) are mutually exclusive, with odds ratio less than 0.1. CONCLUSIONS: In human CRC, gain-of-function alterations in Myc and loss-of-function alterations in TGF-ß exhibit a masking epistatic interaction and are functionally redundant.

Targeting of TGFß signature and its essential component CTGF by miR-18 correlates with improved survival in glioblastoma.

The miR-17∼92 cluster is thought to be an oncogene, yet its expression is low in glioblastoma multiforme (GBM) cell lines. This could allow unfettered expression of miR-17∼92 target genes such as connective tissue growth factor (CTGF; or CCN2), which is known to contribute to GBM pathogenesis. Indeed, microRNA-18a (but not other miR-17∼92 members) has a functional site in the CTGF 3' UTR, and its forced reexpression sharply reduces CTGF protein and mRNA levels. Interestingly, it also reduces the levels of CTGF primary transcript. The unexpected effects of miR-18a on CTGF transcription are mediated in part by direct targeting of Smad3 and ensuing weakening of TGFß signaling. Having defined the TGFß signature in GBM cells, we demonstrate a significant anti-correlation between miR-18 and TGFß signaling in primary GBM samples from The Cancer Genome Atlas. Most importantly, high levels of miR-18 combined with low levels of the TGFß metagene correlate with prolonged patient survival. Thus, low expression of the miR-17∼92 cluster, and specifically miR-18a, could significantly contribute to GBM pathogenesis.

p53-responsive miR-194 inhibits thrombospondin-1 and promotes angiogenesis in colon cancers.

Thrombospondin-1 (TSP-1) is an endogenous inhibitor of angiogenesis encoded by the THBS1 gene, whose promoter is activated by p53. In advanced colorectal cancers (CRC), its expression is sustained or even slightly increased despite frequent loss of p53. Here, we determined that in HCT116 CRC cells, p53 activates the THBS1 primary transcript, but fails to boost THBS1 mRNA or protein levels, implying posttranscriptional regulation by microRNAs (miRNA). In a global miRNA gain-of-function screen done in the Dicer-deficient HCT116 variant, several miRNAs negatively regulated THBS1 mRNA and protein levels, one of them being miR-194. Notably, in agreement with published data, p53 upregulated miR-194 expression in THBS1 retrovirus-transduced HCT116 cells, leading to decreased TSP-1 levels. This negative effect was mediated by a single miR-194 complementary site in the THBS1 3'-untranslated region, and its elimination resulted in TSP-1 reactivation, impaired angiogenesis in Matrigel plugs, and reduced growth of HCT116 xenografts. Conversely, transient overexpression of miR-194 in HCT116/THBS1 cells boosted Matrigel angiogenesis, and its stable overexpression in Ras-induced murine colon carcinomas increased microvascular densities and vessel sizes. Although the overall contribution of miR-194 to neoplastic growth is context dependent, p53-induced activation of this GI tract-specific miRNA during ischemia could promote angiogenesis and facilitate tissue repair.

The miR-17-92 microRNA cluster regulates multiple components of the TGF-ß pathway in neuroblastoma.

The miR-17-92 microRNA cluster is often activated in cancer cells, but the identity of its targets remains elusive. Using SILAC and quantitative mass spectrometry, we examined the effects of activation of the miR-17-92 cluster on global protein expression in neuroblastoma (NB) cells. Our results reveal cooperation between individual miR-17-92 miRNAs and implicate miR-17-92 in multiple hallmarks of cancer, including proliferation and cell adhesion. Most importantly, we show that miR-17-92 is a potent inhibitor of TGF-ß signaling. By functioning both upstream and downstream of pSMAD2, miR-17-92 activation triggers downregulation of multiple key effectors along the TGF-ß signaling cascade as well as direct inhibition of TGF-ß-responsive genes.

The myc-miR-17~92 axis blunts TGF{beta} signaling and production of multiple TGF{beta}-dependent antiangiogenic factors.

c-Myc stimulates angiogenesis in tumors through mechanisms that remain incompletely understood. Recent work indicates that c-Myc upregulates the miR-17∼92 microRNA cluster and downregulates the angiogenesis inhibitor thrombospondin-1, along with other members of the thrombospondin type 1 repeat superfamily. Here, we show that downregulation of the thrombospondin type 1 repeat protein clusterin in cells overexpressing c-Myc and miR-17∼92 promotes angiogenesis and tumor growth. However, clusterin downregulation by miR-17∼92 is indirect. It occurs as a result of reduced transforming growth factor-ß (TGFß) signaling caused by targeting of several regulatory components in this signaling pathway. Specifically, miR-17-5p and miR-20 reduce the expression of the type II TGFß receptor and miR-18 limits the expression of Smad4. Supporting these results, in human cancer cell lines, levels of the miR-17∼92 primary transcript MIR17HG negatively correlate with those of many TGFß-induced genes that are not direct targets of miR-17∼92 (e.g., clusterin and angiopoietin-like 4). Furthermore, enforced expression of miR-17∼92 in MIR17HG(low) cell lines (e.g., glioblastoma) results in impaired gene activation by TGFß. Together, our results define a pathway in which c-Myc activation of miR-17∼92 attenuates the TGFß signaling pathway to shut down clusterin expression, thereby stimulating angiogenesis and tumor cell growth.

Regulation of CLU gene expression by oncogenes and epigenetic factors implications for tumorigenesis.

In no other field has the function of clusterin (CLU) been more controversial than in cancer genetics. After more than 20 years of research, there is still uncertainty with regard to the role of CLU in human cancers. Some investigators believe CLU to be an oncogene, others-an inhibitor of tumorigenesis. However, owing to the recent efforts of several laboratories, the role of CLU in important cellular processes like proliferation, apoptosis, differentiation, and transformation is beginning to emerge. The "enigmatic" CLU is becoming less so. In this chapter, we will review the work of research teams interested in understanding how CLU is regulated by oncogenic signaling. We will discuss how and under what circumstances oncogenes and epigenetic factors modify CLU expression, with important consequences for mammalian tumorigenesis.

Clusterin, a haploinsufficient tumor suppressor gene in neuroblastomas.

BACKGROUND: Clusterin expression in various types of human cancers may be higher or lower than in normal tissue, and clusterin may promote or inhibit apoptosis, cell motility, and inflammation. We investigated the role of clusterin in tumor development in mouse models of neuroblastoma. METHODS: We assessed expression of microRNAs in the miR-17-92 cluster by real-time reverse transcription-polymerase chain reaction in MYCN-transfected SH-SY5Y and SH-EP cells and inhibited expression by transfection with microRNA antisense oligonucleotides. Tumor development was studied in mice (n = 66) that were heterozygous or homozygous for the MYCN transgene and/or for the clusterin gene; these mice were from a cross between MYCN-transgenic mice, which develop neuroblastoma, and clusterin-knockout mice. Tumor growth and metastasis were studied in immunodeficient mice that were injected with human neuroblastoma cells that had enhanced (by clusterin transfection, four mice per group) or reduced (by clusterin short hairpin RNA [shRNA] transfection, eight mice per group) clusterin expression. All statistical tests were two-sided. RESULTS: Clusterin expression increased when expression of MYCN-induced miR-17-92 microRNA cluster in SH-SY5Y neuroblastoma cells was inhibited by transfection with antisense oligonucleotides compared with scrambled oligonucleotides. Statistically significantly more neuroblastoma-bearing MYCN-transgenic mice were found in groups with zero or one clusterin allele than in those with two clusterin alleles (eg, 12 tumor-bearing mice in the zero-allele group vs three in the two-allele group, n = 22 mice per group; relative risk for neuroblastoma development = 4.85, 95% confidence interval [CI] = 1.69 to 14.00; P = .005). Five weeks after injection, fewer clusterin-overexpressing LA-N-5 human neuroblastoma cells than control cells were found in mouse liver or bone marrow, but statistically significantly more clusterin shRNA-transfected HTLA230 cells (3.27%, with decreased clusterin expression) than control-transfected cells (1.53%) were found in the bone marrow (difference = 1.74%, 95% CI = 0.24% to 3.24%, P = .026). CONCLUSIONS: We report, to our knowledge, the first genetic evidence that clusterin is a tumor and metastasis suppressor gene.

Lin-28B transactivation is necessary for Myc-mediated let-7 repression and proliferation.

Direct control of microRNA (miRNA) expression by oncogenic and tumor suppressor networks results in frequent dysregulation of miRNAs in cancer cells and contributes to tumorigenesis. We previously demonstrated that activation of the c-Myc oncogenic transcription factor (Myc) broadly influences miRNA expression and in particular leads to widespread miRNA down-regulation. miRNA transcripts repressed by Myc include several with potent tumor suppressor activity such as miR-15a/16-1, miR-34a, and let-7 family members. In this study, we have investigated mechanisms downstream of Myc that contribute to miRNA repression. Consistent with transcriptional down-regulation, Myc activity results in the decreased abundance of multiple miRNA primary transcripts. Surprisingly, however, primary transcripts encoding several let-7 miRNAs are not reduced in the high Myc state, suggesting a posttranscriptional mechanism of repression. The Lin-28 and Lin-28B RNA binding proteins were recently demonstrated to negatively regulate let-7 biogenesis. We now show that Myc induces Lin-28B expression in multiple human and mouse tumor models. Chromatin immunoprecipitation and reporter assays reveal direct association of Myc with the Lin-28B promoter resulting in transcriptional transactivation. Moreover, we document that activation of Lin-28B is necessary and sufficient for Myc-mediated let-7 repression. Accordingly, Lin-28B loss-of-function significantly impairs Myc-dependent cellular proliferation. These findings highlight an important role for Lin-28B in Myc-driven cellular phenotypes and uncover an orchestration of transcriptional and posttranscriptional mechanisms in Myc-mediated reprogramming of miRNA expression.

c-Myb oncoprotein is an essential target of the dleu2 tumor suppressor microRNA cluster.

The dleu2 tumor suppressor locus encodes two microRNAs, miR-15a and miR-16, which are thought to play an important role in B-cell neoplasms. However, relatively little is known about proteins that regulate or are regulated by this microRNA cluster. Here we demonstrate that the Pax5 oncoprotein downregulates the dleu2 gene and at the same time boosts expression of its own heterodimeric partner c-Myb. Interestingly, c-Myb upregulation occurs primarily at a post-transcriptional level, suggesting that it might be a target for microRNAs such as miR-15a/16. Indeed, miR-15a/16 have predicted binding sites in the c-Myb 3'-UTR and through them diminish protein output in luciferase sensor assays. Moreover, forced overexpression of miR-15a/16 reduces endogenous c-Myb levels and compromises Pax5 function. Conversely, restoration of c-Myb levels partly alleviates tumors suppressive effects of miR-15a/16, suggesting that c-Myb is a key downstream target of this microRNA cluster.

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.

Inactivation of Myc in murine two-hit B lymphomas causes dormancy with elevated levels of interleukin 10 receptor and CD20: implications for adjuvant therapies.

Overexpression of c-Myc and inactivation of p53 are hallmarks of human Burkitt's lymphomas. We had previously showed that transduction of murine p53-null bone marrow cells with a Myc-encoding retrovirus is sufficient for B lymphomagenesis. To address the role of Myc in tumor sustenance, we generated lymphomas induced by the Myc-estrogen receptor fusion protein (MycER). Engrafted hosts were continuously treated with the ER ligand 4-hydroxytamoxifen (4-OHT) to allow tumor formation. Subsequent inactivation of MycER via 4-OHT deprivation resulted in tumor stasis but only partial regression. At the cellular level, dormant neoplastic lymphocytes withdrew from mitosis and underwent further B-cell differentiation. Concomitantly, they up-regulated genes involved in lymphocyte proliferation and survival, most notably interleukin 10 receptor alpha (IL10Ralpha) and CD20, the target for antibody therapy with Rituxan. We found that overexpression of IL10Ralpha affords significant proliferative advantages and in 4-OHT-deprived animals correlates with eventual tumor relapse. Both dormant and relapsing tumors maintain IL10Ralpha expression suggesting that they might be sensitive to emerging drugs targeting the IL-10 pathway. Up-regulation of CD20 following Myc inactivation was also observed in immortalized human lymphocytes. Importantly, in this system, Myc(OFF)CD20(HIGH) cells were more prone to Rituxan-induced apoptosis than Myc(ON)CD20(MED). Thus, targeting Myc, while moderately effective on its own, shapes the phenotype of dormant neoplastic cells and sensitizes them to adjuvant molecular therapies.

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

Tumor necrosis factor alpha (TNF-alpha)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF-alpha 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.

Myc-transformed epithelial cells down-regulate clusterin, which inhibits their growth in vitro and carcinogenesis in vivo.

Effective treatment of malignant carcinomas requires identification of proteins regulating epithelial cell proliferation. To this end, we compared gene expression profiles in murine colonocytes and their c-Myc-transformed counterparts, which possess enhanced proliferative potential. A surprisingly short list of deregulated genes included the cDNA for clusterin, an extracellular glycoprotein without a firmly established function. We had previously demonstrated that in organs such as skin, clusterin expression is restricted to differentiating but not proliferating cell layers, suggesting a possible negative role in cell division. Indeed, its transient overexpression in Myc-transduced colonocytes decreased cell accumulation. Furthermore, clusterin was down-regulated in rapidly dividing human keratinocytes infected with a Myc-encoding adenovirus. Its knockdown via antisense RNA in neoplastic epidermoid cells enhanced proliferation. Finally, recombinant human clusterin suppressed, in a dose-dependent manner, DNA replication in keratinocytes and other cells of epithelial origin. Thus, clusterin appears to be an inhibitor of epithelial cell proliferation in vitro. To determine whether it also affects neoplastic growth in vivo, we compared wild-type and clusterin-null mice with respect to their sensitivity to 7, 12-dimethylbenz(a)anthracene /12-Otetradecanoylphorbol-13-acetate (DMBA/TPA)-induced skin carcinogenesis. We observed that the mean number of papillomas/mouse was higher in clusterin-null animals. Moreover, these papillomas did not regress as readily as in wild-type mice and persisted beyond week 35. The rate of progression toward squamous cell carcinoma was not altered, although those developing in clusterin-null mice were on average better differentiated. These data suggest that clusterin not only suppresses epithelial cell proliferation in vitro but also interferes with the promotion stage of skin carcinogenesis.