Association of a microRNA/TP53 feedback circuitry with pathogenesis and outcome of B-cell chronic lymphocytic leukemia.

CONTEXT: Chromosomal abnormalities (namely 13q, 17p, and 11q deletions) have prognostic implications and are recurrent in chronic lymphocytic leukemia (CLL), suggesting that they are involved in a common pathogenetic pathway; however, the molecular mechanism through which chromosomal abnormalities affect the pathogenesis and outcome of CLL is unknown. OBJECTIVE: To determine whether the microRNA miR-15a/miR-16-1 cluster (located at 13q), tumor protein p53 (TP53, located at 17p), and miR-34b/miR-34c cluster (located at 11q) are linked in a molecular pathway that explains the pathogenetic and prognostic implications (indolent vs aggressive form) of recurrent 13q, 17p, and 11q deletions in CLL. DESIGN, SETTING, AND PATIENTS: CLL Research Consortium institutions provided blood samples from untreated patients (n = 206) diagnosed with B-cell CLL between January 2000 and April 2008. All samples were evaluated for the occurrence of cytogenetic abnormalities as well as the expression levels of the miR-15a/miR-16-1 cluster, miR-34b/miR-34c cluster, TP53, and zeta-chain (TCR)-associated protein kinase 70 kDa (ZAP70), a surrogate prognostic marker of CLL. The functional relationship between these genes was studied using in vitro gain- and loss-of-function experiments in cell lines and primary samples and was validated in a separate cohort of primary CLL samples. MAIN OUTCOME MEASURES: Cytogenetic abnormalities; expression levels of the miR-15a/miR-16-1 cluster, miR-34 family, TP53 gene, downstream effectors cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) and B-cell CLL/lymphoma 2 binding component 3 (BBC3), and ZAP70 gene; genetic interactions detected by chromatin immunoprecipitation. RESULTS: In CLLs with 13q deletions the miR-15a/miR-16-1 cluster directly targeted TP53 (mean luciferase activity for miR-15a vs scrambled control, 0.68 relative light units (RLU) [95% confidence interval {CI}, 0.63-0.73]; P = .02; mean for miR-16 vs scrambled control, 0.62 RLU [95% CI, 0.59-0.65]; P = .02) and its downstream effectors. In leukemic cell lines and primary CLL cells, TP53 stimulated the transcription of miR-15/miR-16-1 as well as miR-34b/miR-34c clusters, and the miR-34b/miR-34c cluster directly targeted the ZAP70 kinase (mean luciferase activity for miR-34a vs scrambled control, 0.33 RLU [95% CI, 0.30-0.36]; P = .02; mean for miR-34b vs scrambled control, 0.31 RLU [95% CI, 0.30-0.32]; P = .01; and mean for miR-34c vs scrambled control, 0.35 RLU [95% CI, 0.33-0.37]; P = .02). CONCLUSIONS: A microRNA/TP53 feedback circuitry is associated with CLL pathogenesis and outcome. This mechanism provides a novel pathogenetic model for the association of 13q deletions with the indolent form of CLL that involves microRNAs, TP53, and ZAP70.

Resveratrol decreases the levels of miR-155 by upregulating miR-663, a microRNA targeting JunB and JunD.

An inflammatory component is present in the microenvironment of most neoplastic tissues, including those not causally related to an obvious inflammatory process. Several microRNAs, and especially miR-155, play an essential role in both the innate and adaptative immune response. Resveratrol (trans-3,4',5-trihydroxystilbene) is a natural antioxidant with anti-inflammatory properties that is currently at the stage of preclinical studies for human cancer prevention. Here, we establish that, in human THP-1 monocytic cells as well as in human blood monocytes, resveratrol upregulates miR-663, a microRNA potentially targeting multiple genes implicated in the immune response. In THP-1 cells, miR-663 decreases endogenous activator protein-1 (AP-1) activity and impairs its upregulation by lipopolysaccharides (LPS), at least in part by directly targeting JunB and JunD transcripts. We further establish that the downregulation of AP-1 activity by resveratrol is miR-663 dependent and that the effects of resveratrol on both AP-1 activity and JunB levels are dose dependent. Finally, we show that resveratrol impairs the upregulation of miR-155 by LPS in a miR-663-dependent manner. Given the role of miR-155 in the innate immune response and the fact that it is upregulated in many cancers, our results suggest that manipulating miR-663 levels may help to optimize the use of resveratrol as both an anti-inflammatory and anticancer agent against malignancies associated with high levels of miR-155.

Modulation of mismatch repair and genomic stability by miR-155.

Inactivation of mismatch repair (MMR) is the cause of the common cancer predisposition disorder Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer (HNPCC), as well as 10-40% of sporadic colorectal, endometrial, ovarian, gastric, and urothelial cancers. Elevated mutation rates (mutator phenotype), including simple repeat instability [microsatellite instability (MSI)] are a signature of MMR defects. MicroRNAs (miRs) have been implicated in the control of critical cellular pathways involved in development and cancer. Here we show that overexpression of miR-155 significantly down-regulates the core MMR proteins, hMSH2, hMSH6, and hMLH1, inducing a mutator phenotype and MSI. An inverse correlation between the expression of miR-155 and the expression of MLH1 or MSH2 proteins was found in human colorectal cancer. Finally, a number of MSI tumors with unknown cause of MMR inactivation displayed miR-155 overexpression. These data provide support for miR-155 modulation of MMR as a mechanism of cancer pathogenesis.

An ets2-driven transcriptional program in tumor-associated macrophages promotes tumor metastasis.

Tumor-associated macrophages (TAM) are implicated in breast cancer metastasis, but relatively little is known about the underlying genes and pathways that are involved. The transcription factor Ets2 is a direct target of signaling pathways involved in regulating macrophage functions during inflammation. We conditionally deleted Ets in TAMs to determine its function at this level on mouse mammary tumor growth and metastasis. Ets2 deletion in TAMs decreased the frequency and size of lung metastases in three different mouse models of breast cancer metastasis. Expression profiling and chromatin immunoprecipitation assays in isolated TAMs established that Ets2 repressed a gene program that included several well-characterized inhibitors of angiogenesis. Consistent with these results, Ets2 ablation in TAMs led to decreased angiogenesis and decreased growth of tumors. An Ets2-TAM expression signature consisting of 133 genes was identified within human breast cancer expression data which could retrospectively predict overall survival of patients with breast cancer in two independent data sets. In summary, we identified Ets2 as a central driver of a transcriptional program in TAMs that acts to promote lung metastasis of breast tumors.

Epigenetics, miRNAs, and human cancer: a new chapter in human gene regulation.

Cancer is a genetic and epigenetic disease. MicroRNAs (miRNAs), a class of small noncoding RNAs, have been shown to be deregulated in many diseases including cancer. An intertwined connection between epigenetics and miRNAs has been supported by the recent identification of a specific subgroup of miRNAs called "epi-miRNAs" that can directly and indirectly modulate the activity of the epigenetic machinery. The complexity of this connection is enhanced by the epigenetic regulation of miRNA expression that generates a fine regulatory feedback loop. This review focuses on how epigenetics affects the miRNome and how the recently identified epi-miRNAs regulate the epigenome in human cancers, ultimately contributing to human carcinogenesis.

Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-alpha stimulation and their possible roles in regulating the response to endotoxin shock.

We report here that miR-155 and miR-125b play a role in innate immune response. LPS stimulation of mouse Raw 264.7 macrophages resulted in the up-regulation of miR-155 and down-regulation of miR-125b levels. The same changes also occurred when C57BL/6 mice were i.p. injected with LPS. Furthermore, the levels of miR-155 and miR-125b in Raw 264.7 cells displayed oscillatory changes in response to TNF-alpha. These changes were impaired by pretreating the cells with the proteasome inhibitor MG-132, suggesting that these two microRNAs (miRNAs) may be at least transiently under the direct control of NF-kappaB transcriptional activity. We show that miR-155 most probably directly targets transcript coding for several proteins involved in LPS signaling such as the Fas-associated death domain protein (FADD), IkappaB kinase epsilon (IKKepsilon), and the receptor (TNFR superfamily)-interacting serine-threonine kinase 1 (Ripk1) while enhancing TNF-alpha translation. In contrast, miR-125b targets the 3'-untranslated region of TNF-alpha transcripts; therefore, its down-regulation in response to LPS may be required for proper TNF-alpha production. Finally, Emu-miR-155 transgenic mice produced higher levels of TNF-alpha when exposed to LPS and were hypersensitive to LPS/d-galactosamine-induced septic shock. Altogether, our data suggest that the LPS/TNF-alpha-dependent regulation of miR-155 and miR-125b may be implicated in the response to endotoxin shock, thus offering new targets for drug design.