Flavia Pichiorri

miR-221&222 regulate TRAIL resistance and enhance tumorigenicity through PTEN and TIMP3 downregulation.

Lung and liver cancers are among the most deadly types of cancer. Despite improvements in treatment over the past few decades, patient survival remains poor, underlining the need for development of targeted therapies. MicroRNAs represent a class of small RNAs frequently deregulated in human malignancies. We now report that miR-221&222 are overexpressed in aggressive non-small cell lung cancer and hepatocarcinoma cells, as compared with less invasive and/or normal lung and liver cells. We show that miR-221&222, by targeting PTEN and TIMP3 tumor suppressors, induce TRAIL resistance and enhance cellular migration through the activation of the AKT pathway and metallopeptidases. Finally, we demonstrate that the MET oncogene is involved in miR-221&222 activation through the c-Jun transcription factor.

MicroRNA Signatures Associated with Cytogenetics and Prognosis in Acute Myeloid Leukemia

MicroRNAs (miRNAs) are small RNAs of 19 to 25 nucleotides that are negative regulators of gene expression. To determine whether miRNAs are associated with cytogenetic abnormalities and clinical features in acute myeloid leukemia (AML), we evaluated the miRNA expression of CD34(+) cells and 122 untreated adult AML cases using a microarray platform. After background subtraction and normalization using a set of housekeeping genes, data were analyzed using Significance Analysis of Microarrays. An independent set of 60 untreated AML patients was used to validate the outcome signatures using real-time polymerase chain reaction. We identified several miRNAs differentially expressed between CD34(+) normal cells and the AML samples. miRNA expression was also closely associated with selected cytogenetic and molecular abnormalities, such as t(11q23), isolated trisomy 8, and FLT3-ITD mutations. Furthermore, patients with high expression of miR-191 and miR-199a had significantly worse overall and event-free survival than AML patients with low expression (overall survival: miR-191, P = .03; and miR-199a, P = .001, Cox regression). In conclusion, miRNA expression in AML is closely associated with cytogenetics and FLT3-ITD mutations. A small subset of miRNAs is correlated with survival.

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.

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.

mRNA/microRNA gene expression profile in microsatellite unstable colorectal cancer

BackgroundColorectal cancer develops through two main genetic instability pathways characterized by distinct pathologic features and clinical outcome.ResultsWe investigated colon cancer samples (23 characterized by microsatellite stability, MSS, and 16 by high microsatellite instability, MSI-H) for genome-wide expression of microRNA (miRNA) and mRNA. Based on combined miRNA and mRNA gene expression, a molecular signature consisting of twenty seven differentially expressed genes, inclusive of 8 miRNAs, could correctly distinguish MSI-H versus MSS colon cancer samples. Among the differentially expressed miRNAs, various members of the oncogenic miR-17-92 family were significantly up-regulated in MSS cancers. The majority of protein coding genes were also up-regulated in MSS cancers. Their functional classification revealed that they were most frequently associated with cell cycle, DNA replication, recombination, repair, gastrointestinal disease and immune response.ConclusionThis is the first report that indicates the existence of differences in miRNA expression between MSS versus MSI-H colorectal cancers. In addition, the work suggests that the combination of mRNA/miRNA expression signatures may represent a general approach for improving bio-molecular classification of human cancer.

Modulation of different clusterin isoforms in human colon tumorigenesis

Clusterin is a ubiquitous secretory heterodimeric disulfide-linked glycoprotein, which is implicated in several physiological processes, including immune regulation, cell adhesion and morphological transformation, lipid transportation, tissue remodelling, membrane recycling and cell–cell interactions. A large number of studies have focused their interest on clusterin gene products as mediators of cell cycle progression and cell death induction, although data on the different isoforms and their role in the different cell processes are still obscure. Recently, an increased clusterin expression in breast cancer has been reported. In order to elucidate the role of clusterin in tumor progression and whether one of its isoforms is preferentially expressed in tumorigenesis, we examined its presence throughout the different steps of human colon carcinoma, one of the best-characterized models of human tumor progression. The immunohistochemical observation of 30 bioptic and surgical colon specimens demonstrated a cell compartment clusterin translocation from the nucleus to the cytoplasm directly related to tumor progression. In fact, a nuclear localization found in healthy colonic mucosa is consistent with the involvement of the proapoptotic nuclear form in the regulation of cell cycle progression and in cell death induction. The progression towards high-grade and metastatic carcinoma leads to cytoplasmic clusterin distribution. Protein extracts from freshly isolated cells of the same patients confirm in high-grade carcinomas with metastatic nodes the complete loss of the proapoptotic nuclear form and a cytoplasmic overexpression of the highly glycosylated form. Data obtained from in vitro experiments confirm that this form is released in the extracellular space and corresponded to the fully glycosylated one. These data suggest that the controversial data on clusterin function in tumors may be related to the pattern shift of its isoform production. As the secreted form of clusterin is correlated to cell matrix formation, cell membrane remodeling and cell–cell adhesion, the overexpression of this form in highly aggressive tumors and metastatic nodes could be a potential new prognostic and predictive marker for colon carcinoma aggressiveness.

MicroRNAs/TP53 feedback circuitry in glioblastoma multiforme

MicroRNAs (miRNAs) are increasingly implicated in regulating cancer initiation and progression. In this study, two miRNAs, miR-25 and -32, are identified as p53-repressed miRNAs by p53-dependent negative regulation of their transcriptional regulators, E2F1 and MYC. However, miR-25 and -32 result in p53 accumulation by directly targeting Mdm2 and TSC1, which are negative regulators of p53 and the mTOR (mammalian target of rapamycin) pathway, respectively, leading to inhibition of cellular proliferation through cell cycle arrest. Thus, there is a recurrent autoregulatory circuit involving expression of p53, E2F1, and MYC to regulate the expression of miR-25 and -32, which are miRNAs that, in turn, control p53 accumulation. Significantly, overexpression of transfected miR-25 and -32 in glioblastoma multiforme cells inhibited growth of the glioblastoma multiforme cells in mouse brain in vivo. The results define miR-25 and -32 as positive regulators of p53, underscoring their role in tumorigenesis in glioblastoma.

Fhit Interaction with Ferredoxin Reductase Triggers Generation of Reactive Oxygen Species and Apoptosis of Cancer Cells

Fhit protein is lost in most cancers, its restoration suppresses tumorigenicity, and virus-mediated FHIT gene therapy induces apoptosis and suppresses tumors in preclinical models. We have used protein cross-linking and proteomics methods to characterize a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes Hsp60 and Hsp10 that mediate Fhit stability and may affect import into mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin. Viral-mediated Fhit restoration increases production of intracellular reactive oxygen species, followed by increased apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape apoptosis, carrying serious oxidative DNA damage that may contribute to an increased mutation rate. Characterization of Fhit interacting proteins has identified direct effectors of the Fhit-mediated apoptotic pathway that is lost in most cancers through loss of Fhit.

Inactivation of the Wwox Gene Accelerates Forestomach Tumor Progression In vivo

The WWOX gene encodes a tumor suppressor spanning the second most common human fragile site, FRA16D. Targeted deletion of the Wwox gene in mice led to an increased incidence of spontaneous and ethyl nitrosourea-induced tumors. In humans, loss of heterozygosity and reduced or loss of WWOX expression has been reported in esophageal squamous cell cancers (SCC). In the present study, we examined whether inactivation of the Wwox gene might lead to enhanced esophageal/forestomach tumorigenesis induced by N-nitrosomethylbenzylamine. Wwox+/- and Wwox+/+ mice were treated with six intragastric doses of N-nitrosomethylbenzylamine and observed for 15 subsequent weeks. Ninety-six percent (25 of 26) of Wwox+/- mice versus 29% (10 of 34) of Wwox+/+ mice developed forestomach tumors (P = 1.3 x 10(-7)). The number of tumors per forestomach was significantly greater in Wwox+/- than in Wwox+/+ mice (3.2 +/- 0.34 versus 0.47 +/- 0.17; P < 0.0001). In addition, 27% of Wwox+/- mice had invasive SCC in the forestomach, as compared with 0% of wild-type controls (P = 0.002). Intriguingly, forestomachs from Wwox+/- mice displayed moderately strong Wwox protein staining in the near-normal epithelium, but weak and diffuse staining in SCC in the same tissue section, a result suggesting that Wwox was haploinsufficient for the initiation of tumor development. Our findings provide the first in vivo evidence of the tumor suppressor function of WWOX in forestomach/esophageal carcinogenesis and suggest that inactivation of one allele of WWOX accelerates the predisposition of normal cells to malignant transformation.