Rami I. Aqeilan

MiR-15a and miR-16-1 cluster functions in human leukemia

MicroRNAs (miRNAs) are short noncoding RNAs regulating gene expression that play roles in human diseases, including cancer. Each miRNA is predicted to regulate hundreds of transcripts, but only few have experimental validation. In chronic lymphocytic leukemia (CLL), the most common adult human leukemia, miR-15a and miR-16-1 are lost or down-regulated in the majority of cases. After our previous work indicating a tumor suppressor function of miR-15a/16-1 by targeting the BCL2 oncogene, here, we produced a high-throughput profiling of genes modulated by miR-15a/16-1 in a leukemic cell line model (MEG-01) and in primary CLL samples. By combining experimental and bioinformatics data, we identified a miR-15a/16-1-gene signature in leukemic cells. Among the components of the miR-15a/16-1 signature, we observed a statistically significant enrichment in AU-rich elements (AREs). By examining the Gene Ontology (GO) database, a significant enrichment in cancer genes (such as MCL1, BCL2, ETS1, or JUN) that directly or indirectly affect apoptosis and cell cycle was found.

MicroRNAs regulate critical genes associated with multiple myeloma pathogenesis

Progress in understanding the biology of multiple myeloma (MM), a plasma cell malignancy, has been slow. The discovery of microRNAs (miRNAs), a class of small noncoding RNAs targeting multiple mRNAs, has revealed a new level of gene expression regulation. To determine whether miRNAs play a role in the malignant transformation of plasma cells (PCs), we have used both miRNA microarrays and quantitative real time PCR to profile miRNA expression in MM-derived cell lines (n = 49) and CD138+ bone marrow PCs from subjects with MM (n = 16), monoclonal gammopathy of undetermined significance (MGUS) (n = 6), and normal donors (n = 6). We identified overexpression of miR-21, miR-106b∼25 cluster, miR-181a and b in MM and MGUS samples with respect to healthy PCs. Selective up-regulation of miR-32 and miR-17∼92 cluster was identified in MM subjects and cell lines but not in MGUS subjects or healthy PCs. Furthermore, two miRNAs, miR-19a and 19b, that are part of the miR-17∼92 cluster, were shown to down regulate expression of SOCS-1, a gene frequently silenced in MM that plays a critical role as inhibitor of IL-6 growth signaling. We also identified p300-CBP-associated factor, a gene involved in p53 regulation, as a bona fide target of the miR106b∼25 cluster, miR-181a and b, and miR-32. Xenograft studies using human MM cell lines treated with miR-19a and b, and miR-181a and b antagonists resulted in significant suppression of tumor growth in nude mice. In summary, we have described a MM miRNA signature, which includes miRNAs that modulate the expression of proteins critical to myeloma pathogenesis.

Biological Functions of miR-29b Contribute to Positive Regulation of Osteoblast Differentiation

Bone tissue arises from mesenchymal cells induced into the osteoblast lineage by essential transcription factors and signaling cascades. MicroRNAs regulate biological processes by binding to mRNA 3′-untranslated region (UTR) sequences to attenuate protein synthesis. Here we performed microRNA profiling and identified miRs that are up-regulated through stages of osteoblast differentiation. Among these are the miR-29, miR-let-7, and miR-26 families that target many collagens and extracellular matrix proteins. We find that miR-29b supports osteoblast differentiation through several mechanisms. miR-29b decreased and anti-miR-29b increased activity of COL1A1, COL5A3, and COL4A2 3′-UTR sequences in reporter assays, as well as endogenous gene expression. These results support a mechanism for regulating collagen protein accumulation during the mineralization stage when miR-29b reaches peak levels. We propose that this mechanism prevents fibrosis and facilitates mineral deposition. Our studies further demonstrate that miR-29b promotes osteogenesis by directly down-regulating known inhibitors of osteoblast differentiation, HDAC4, TGFβ3, ACVR2A, CTNNBIP1, and DUSP2 proteins through binding to target 3′-UTR sequences in their mRNAs. Thus, miR-29b is a key regulator of development of the osteoblast phenotype by targeting anti-osteogenic factors and modulating bone extracellular matrix proteins.

miR-15a and miR-16-1 in cancer: discovery, function and future perspectives

MicroRNAs (miRNAs) encoded by the miR-15/16 cluster are known to act as tumor suppressors. Expression of these miRNAs inhibits cell proliferation, promotes apoptosis of cancer cells, and suppresses tumorigenicity both in vitro and in vivo. miR-15a and miR-16-1 function by targeting multiple oncogenes, including BCL2, MCL1, CCND1, and WNT3A. Down-regulation of these miRNAs has been reported in chronic lymphocytic lymphoma (CLL), pituitary adenomas, and prostate carcinoma. This review summarizes the discovery, functions, and clinical relevance of these miRNAs in cancer, particularly CLL.

MicroRNA gene expression during retinoic acid-induced differentiation of human acute promyelocytic leukemia

MicroRNAs (miRNAs) are small non-coding RNAs of 19–25 nucleotides that are involved in the regulation of critical cell processes such as apoptosis, cell proliferation and differentiation. However, little is known about the role of miRNAs in granulopoiesis. Here, we report the expression of miRNAs in acute promyelocytic leukemia patients and cell lines during all-trans-retinoic acid (ATRA) treatment by using a miRNA microarrays platform and quantitative real time–polymerase chain reaction (qRT–PCR). We found upregulation of miR-15a, miR-15b, miR-16-1, let-7a-3, let-7c, let-7d, miR-223, miR-342 and miR-107, whereas miR-181b was downregulated. Among the upregulated miRNAs, miR-107 is predicted to target NFI-A, a gene that has been involved in a regulatory loop involving miR-223 and C/EBPa during granulocytic differentiation. Indeed, we have confirmed that miR-107 targets NF1-A. To get insights about ATRA regulation of miRNAs, we searched for ATRA-modulated transcription factors binding sites in the upstream genomic region of the let-7a-3/let-7b cluster and identified several putative nuclear factor-kappa B (NF-κB) consensus elements. The use of reporter gene assays, chromatin immunoprecipitation and site-directed mutagenesis revealed that one proximal NF-κB binding site is essential for the transactivation of the let-7a-3/let-7b cluster. Finally, we show that ATRA downregulation of RAS and Bcl2 correlate with the activation of known miRNA regulators of those proteins, let-7a and miR-15a/miR-16-1, respectively.

Downregulation of p53-inducible microRNAs 192, 194, and 215 impairs the p53/MDM2 autoregulatory loop in multiple myeloma development.

In multiple myeloma (MM), an incurable B cell neoplasm, mutation or deletion of p53 is rarely detected at diagnosis. Using small-molecule inhibitors of MDM2, we provide evidence that miR-192, 194, and 215, which are downregulated in a subset of newly diagnosed MMs, can be transcriptionally activated by p53 and then modulate MDM2 expression. Furthermore, ectopic re-expression of these miRNAs in MM cells increases the therapeutic action of MDM2 inhibitors in vitro and in vivo by enhancing their p53-activating effects. In addition, miR-192 and 215 target the IGF pathway, preventing enhanced migration of plasma cells into bone marrow. The results suggest that these miRNAs are positive regulators of p53 and that their downregulation plays a key role in MM development.

miRNA Signatures Associate with Pathogenesis and Progression of Osteosarcoma

Osteosarcoma remains a leading cause of cancer death in adolescents. Treatment paradigms and survival rates have not improved in two decades. Driving the lack of therapeutic inroads, the molecular etiology of osteosarcoma remains elusive. MicroRNAs (miRNAs) have demonstrated far-reaching effects on the cellular biology of development and cancer. Their role in osteosarcomagenesis remains largely unexplored. Here we identify for the first time an miRNA signature reflecting the pathogenesis of osteosarcoma from surgically procured samples from human patients. The signature includes high expression of miR-181a,miR-181b, and miR-181c as well as reduced expression of miR-16, miR-29b, and miR-142-5p. We also demonstrate that miR-181b and miR-29b exhibit restricted expression to distinct cell populations in the tumor tissue. Further, higher expression of miR-27a and miR-181c* in pre-treatment biopsy samples characterized patients who developed clinical metastatic disease. In addition, higher expression of miR-451 and miR-15b in pre-treatment samples correlated with subsequent positive response to chemotherapy. In vitro and in vivo functional validation in osteosarcoma cell lines confirmed the tumor suppressive role of miR-16 and the pro-metastatic role of miR-27a. Furthermore, predicted target genes for miR-16 and miR-27a were confirmed as down-regulated by real-time PCR. Affymetrix array profiling of cDNAs from the osteosarcoma specimens and controls were interrogated according to predicted targets of miR-16, miR142-5p, miR-29b, miR-181a/b, and miR-27a. This analysis revealed positive and negative correlations highlighting pathways of known importance to osteosarcoma, as well as novel genes. Thus, our findings establish a miRNA signature associated with pathogenesis of osteosarcoma as well as critical pre-treatment biomarkers of metastasis and responsiveness to therapy.

Targeted deletion of Wwox reveals a tumor suppressor function.

The WW domain-containing oxidoreductase (WWOX) spans the second most common fragile site of the human genome, FRA16D, located at 16q23, and its expression is altered in several types of human cancer. We have previously shown that restoration of WWOX expression in cancer cells suppresses tumorigenicity. To investigate WWOX tumor suppressor function in vivo, we generated mice carrying a targeted deletion of the Wwox gene and monitored incidence of tumor formation. Osteosarcomas in juvenile Wwox−/− and lung papillary carcinoma in adult Wwox+/− mice occurred spontaneously. In addition, Wwox+/− mice develop significantly more ethyl nitrosourea-induced lung tumors and lymphomas in comparison to wild-type littermate mice. Intriguingly, these tumors still express Wwox protein, suggesting haploinsuffiency of WWOX itself is cancer predisposing. These results indicate that WWOX is a bona fide tumor suppressor.

The E3 ubiquitin ligase Itch controls the protein stability of p63

p63, a member of the p53 family of transcription factors, plays an important role in epithelial development, regulating both cell cycle and apoptosis. Even though p63 activity is regulated mainly at the posttranslational level, the control of p63 protein stability is far from being fully understood. Here, we show that the Hect (homologous to the E6-associated protein C terminus)-containing Nedd4-like ubiquitin protein ligase Itch binds, ubiquitylates, and promotes the degradation of p63. The physical interaction occurs at the border between the PY and the SAM (sterile α motif) domains; a single Y504F mutation significantly affects p63 degradation. Itch and p63 are coexpressed in the epidermis and in primary keratinocytes where Itch controls the p63 protein steady-state level. Accordingly, p63 protein levels are significantly increased in Itch knockout keratinocytes. These data suggest that Itch has a fundamental role in the mechanism that controls endogenous p63 protein levels and therefore contributes to regulation of p63 in physiological conditions.

WW Domain–Containing Proteins, WWOX and YAP, Compete for Interaction with ErbB-4 and Modulate Its Transcriptional Function

The WW domain-containing oxidoreductase, WWOX, is a tumor suppressor that is deleted or altered in several cancer types. We recently showed that WWOX interacts with p73 and AP-2gamma and suppresses their transcriptional activity. Yes-associated protein (YAP), also containing WW domains, was shown to associate with p73 and enhance its transcriptional activity. In addition, YAP interacts with ErbB-4 receptor tyrosine kinase and acts as transcriptional coactivator of the COOH-terminal fragment (CTF) of ErbB-4. Stimulation of ErbB-4-expressing cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) results in the proteolytic cleavage of its cytoplasmic domain and translocation of this domain to the nucleus. Here we report that WWOX physically associates with the full-length ErbB-4 via its first WW domain. Coexpression of WWOX and ErbB-4 in HeLa cells followed by treatment with TPA results in the retention of ErbB-4 in the cytoplasm. Moreover, in MCF-7 breast carcinoma cells, expressing high levels of endogenous WWOX, endogenous ErbB-4 is also retained in the cytoplasm. In addition, our results show that interaction of WWOX and ErbB-4 suppresses transcriptional coactivation of CTF by YAP in a dose-dependent manner. A mutant form of WWOX lacking interaction with ErbB-4 has no effect on this coactivation of ErbB-4. Furthermore, WWOX is able to inhibit coactivation of p73 by YAP. In summary, our data indicate that WWOX antagonizes the function of YAP by competing for interaction with ErbB-4 and other targets and thus affect its transcriptional activity.