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Showing papers by "Carlo M. Croce published in 2011"


Journal ArticleDOI
TL;DR: A role for miRNAs in lung tissues and plasma as molecular predictors of lung cancer development and aggressiveness is indicated and has theoretical and clinical implication for lung cancer management.
Abstract: The efficacy of computed tomography (CT) screening for early lung cancer detection in heavy smokers is currently being tested by a number of randomized trials. Critical issues remain the frequency of unnecessary treatments and impact on mortality, indicating the need for biomarkers of aggressive disease. We explored microRNA (miRNA) expression profiles of lung tumors, normal lung tissues and plasma samples from cases with variable prognosis identified in a completed spiral-CT screening trial with extensive follow-up. miRNA expression patterns significantly distinguished: (i) tumors from normal lung tissues, (ii) tumor histology and growth rate, (iii) clinical outcome, and (iv) year of lung cancer CT detection. Interestingly, miRNA profiles in normal lung tissues also displayed remarkable associations with clinical features, suggesting the influence of a permissive microenvironment for tumor development. miRNA expression analyses in plasma samples collected 1–2 y before the onset of disease, at the time of CT detection and in disease-free smokers enrolled in the screening trial, resulted in the generation of miRNA signatures with strong predictive, diagnostic, and prognostic potential (area under the ROC curve ≥ 0.85). These signatures were validated in an independent cohort from a second randomized spiral-CT trial. These results indicate a role for miRNAs in lung tissues and plasma as molecular predictors of lung cancer development and aggressiveness and have theoretical and clinical implication for lung cancer management.

655 citations


Journal ArticleDOI
24 Nov 2011-Oncogene
TL;DR: Data show that methylation-dependent tissue-specific regulation of the lncRNA MEG3 by miR-29a may contribute to HCC growth and highlight the inter-relationship between two classes of non-coding RNA, miRNAs and lncRNAs, and epigenetic regulation of gene expression.
Abstract: The human genome is replete with long non-coding RNAs (lncRNA), many of which are transcribed and likely to have a functional role. Microarray analysis of >23 000 lncRNAs revealed downregulation of 712 (∼3%) lncRNA in malignant hepatocytes, among which maternally expressed gene 3 (MEG3) was downregulated by 210-fold relative to expression in non-malignant hepatocytes. MEG3 expression was markedly reduced in four human hepatocellular cancer (HCC) cell lines compared with normal hepatocytes by real-time PCR. RNA in situ hybridization showed intense cytoplasmic expression of MEG3 in non-neoplastic liver with absent or very weak expression in HCC tissues. Enforced expression of MEG3 in HCC cells significantly decreased both anchorage-dependent and -independent cell growth, and induced apoptosis. MEG3 promoter hypermethylation was identified by methylation-specific PCR and MEG3 expression was increased with inhibition of methylation with either 5-Aza-2-Deoxycytidine, or siRNA to DNA Methyltransferase (DNMT) 1 and 3b in HCC cells. MiRNA-dependent regulation of MEG3 expression was studied by evaluating the involvement of miR-29, which can modulate DNMT 1 and 3. Overexpression of mir-29a increased expression of MEG3. GTL2, the murine homolog of MEG3, was reduced in liver tissues from hepatocyte-specific miR-29a/b1 knock-out mice compared with wild-type controls. These data show that methylation-dependent tissue-specific regulation of the lncRNA MEG3 by miR-29a may contribute to HCC growth and highlight the inter-relationship between two classes of non-coding RNA, miRNAs and lncRNAs, and epigenetic regulation of gene expression.

587 citations


Journal ArticleDOI
TL;DR: By transactivating expression of miRNAs that repress expression of the ZEB1 and ZEB2 transcription factors, p53 inhibits the epithelial–mesenchymal transition.
Abstract: p53 suppresses tumor progression and metastasis. Epithelial–mesenchymal transition (EMT) is a key process in tumor progression and metastasis. The transcription factors ZEB1 and ZEB2 promote EMT. Here, we show that p53 suppresses EMT by repressing expression of ZEB1 and ZEB2. By profiling 92 primary hepatocellular carcinomas (HCCs) and 9 HCC cell lines, we found that p53 up-regulates microRNAs (miRNAs), including miR-200 and miR-192 family members. The miR-200 family members transactivated by p53 then repress ZEB1/2 expression. p53-regulated miR-192 family members also repress ZEB2 expression. Inhibition or overexpression of the miRNAs affects p53-regulated EMT by altering ZEB1 and ZEB2 expression. Our findings indicate that p53 can regulate EMT, and that p53-regulated miRNAs are critical mediators of p53-regulated EMT.

485 citations


Journal ArticleDOI
TL;DR: It is demonstrated that osteoblastogenesis is limited by an elaborate network of functionally tested miRNAs that directly target the osteogenic master regulator Runx2, and their effects can be reversed by the corresponding anti-miRNAs.
Abstract: Lineage progression in osteoblasts and chondrocytes is stringently controlled by the cell-fate–determining transcription factor Runx2. In this study, we directly addressed whether microRNAs (miRNAs) can control the osteogenic activity of Runx2 and affect osteoblast maturation. A panel of 11 Runx2-targeting miRNAs (miR-23a, miR-30c, miR-34c, miR-133a, miR-135a, miR-137, miR-204, miR-205, miR-217, miR-218, and miR-338) is expressed in a lineage-related pattern in mesenchymal cell types. During both osteogenic and chondrogenic differentiation, these miRNAs, in general, are inversely expressed relative to Runx2. Based on 3′UTR luciferase reporter, immunoblot, and mRNA stability assays, each miRNA directly attenuates Runx2 protein accumulation. Runx2-targeting miRNAs differentially inhibit Runx2 protein expression in osteoblasts and chondrocytes and display different efficacies. Thus, cellular context contributes to miRNA-mediated regulation of Runx2. All Runx2-targeting miRNAs (except miR-218) significantly impede osteoblast differentiation, and their effects can be reversed by the corresponding anti-miRNAs. These findings demonstrate that osteoblastogenesis is limited by an elaborate network of functionally tested miRNAs that directly target the osteogenic master regulator Runx2.

378 citations


Journal ArticleDOI
03 Mar 2011-Oncogene
TL;DR: This first in-depth investigation into the role of miR-221/222 in acquired fulvestrant resistance demonstrates that these two ‘oncomirs’ may represent promising therapeutic targets for treating hormone-independent, SERD-resistant breast cancer.
Abstract: Fulvestrant is a selective estrogen receptor downregulator (SERD) and highly effective antagonist to hormone-sensitive breast cancers following failure of previous tamoxifen or aromatase inhibitor therapies. However, after prolonged fulvestrant therapy, acquired resistance eventually occurs in the majority of breast cancer patients, due to poorly understood mechanisms. To examine a possible role(s) of aberrantly expressed microRNAs (miRNAs) in acquired fulvestrant resistance, we compared antiestrogen-resistant and -sensitive breast cancer cells, revealing the overexpression of miR-221/222 in the SERD-resistant cell lines. Fulvestrant treatment of estradiol (E2)- and fulvestrant-sensitive MCF7 cells resulted in increased expression of endogenous miR-221/222. Ectopic upregulation of miR-221/222 in estrogen receptor-α (ERα)-positive cell lines counteracted the effects of E2 depletion or fulvestrant-induced cell death, thus also conferring hormone-independent growth and fulvestrant resistance. In cells with acquired resistance to fulvestrant, miR-221/222 expression was essential for cell growth and cell cycle progression. To identify possible miR-221/222 targets, miR-221- or miR-222- induced alterations in global gene expression profiles and target gene expression at distinct time points were determined, revealing that miR-221/222 overexpression resulted in deregulation of multiple oncogenic signaling pathways previously associated with drug resistance. Activation of β-catenin by miR-221/222 contributed to estrogen-independent growth and fulvestrant resistance, whereas TGF-β-mediated growth inhibition was repressed by the two miRNAs. This first in-depth investigation into the role of miR-221/222 in acquired fulvestrant resistance, a clinically important problem, demonstrates that these two 'oncomirs' may represent promising therapeutic targets for treating hormone-independent, SERD-resistant breast cancer.

320 citations


Journal ArticleDOI
05 Jan 2011-JAMA
TL;DR: 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 and its downstream effectors.
Abstract: 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.

280 citations


Journal ArticleDOI
TL;DR: In insights about how micro- RNAs act as oncogenes and tumor suppressor genes are provided and how these findings can be applied to optimize recent miRNA-based technologies and make them suitable for clinical applications are provided.
Abstract: It has been demonstrated that all the known processes involved in cancer, including apoptosis, proliferation, survival, and metastasis, are regulated by small regulatory noncoding RNAs consisting of approximately 19-25 nucleotides; these are named microRNAs (miRNAs). Both loss and gain of miRNA function contribute to cancer development through the upregulation and silencing, respectively, of different target genes. Experimental evidence indicates that the use of miRNA mimics or anti-microRNAs may represent a powerful therapeutic strategy to interfere with key molecular pathways involved in cancer. This review provides insights about how micro- RNAs act as oncogenes and tumor suppressor genes and how these findings, along with our increasing understanding of miRNA regulation, can be applied to optimize recent miRNA-based technologies and make them suitable for clinical applications.

277 citations


Journal ArticleDOI
TL;DR: The findings offer a preclinical proof of efficacy for chol-anti-miR-221 in a valid orthotopic mouse model of HCC, suggesting that this targeted agent could benefit treatment for patients with advanced HCC.
Abstract: Patients with advanced hepatocellular carcinoma (HCC) face a dismal prognosis because of a lack of any effective therapies. To address this situation, we conducted a preclinical investigation of the therapeutic efficacy of oligonucleotides directed against the oncogenic microRNA miR-221, which has been implicated in HCC. Of 9 chemistries evaluated, we determined that a 2'-O-methyl phosphorothioate-modified anti-miR-221 oligonucleotide was most effective at reducing proliferation in vitro. A cholesterol-modified isoform of anti-miR-221 (chol-anti-miR-221) exhibited improved pharmacokinetics and liver tissue distribution compared with unmodified oligonucleotide. Chol-anti-miR-221 significantly reduced miR-221 levels in liver within a week of intravenous administration and in situ hybridization studies confirmed accumulation of the oligonucleotide in tumor cells in vivo. Within the same period, chol-anti-miR-221 reduced tumor cell proliferation and increased markers of apoptosis and cell-cycle arrest, elevating the tumor doubling time and increasing mouse survival. Taken together, our findings offer a preclinical proof of efficacy for chol-anti-miR-221 in a valid orthotopic mouse model of HCC, suggesting that this targeted agent could benefit treatment for patients with advanced HCC.

225 citations


Journal ArticleDOI
TL;DR: The small molecule enoxacin, a fluoroquinolone used as an antibacterial compound, enhances the production of miRNAs with tumor suppressor functions by binding to the miRNA biosynthesis protein TAR RNA-binding protein 2 (TRBP).
Abstract: MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression at the posttranscriptional level and are critical for many cellular pathways. The disruption of miRNAs and their processing machineries also contributes to the development of human tumors. A common scenario for miRNA expression in carcinogenesis is emerging that shows that impaired miRNA production and/or down-regulation of these transcripts occurs in many neoplasms. Several of these lost miRNAs have tumor-suppressor features, so strategies to restore their expression globally in malignancies would be a welcome addition to the current therapeutic arsenal against cancer. Herein, we show that the small molecule enoxacin, a fluoroquinolone used as an antibacterial compound, enhances the production of miRNAs with tumor suppressor functions by binding to the miRNA biosynthesis protein TAR RNA-binding protein 2 (TRBP). The use of enoxacin in human cell cultures and xenografted, orthotopic, and metastatic mouse models reveals a TRBP-dependent and cancer-specific growth-inhibitory effect of the drug. These results highlight the key role of disrupted miRNA expression patterns in tumorigenesis, and suggest a unique strategy for restoring the distorted microRNAome of cancer cells to a more physiological setting.

221 citations


Journal ArticleDOI
TL;DR: A critical role is identified for TUC338 in regulation of transformed cell growth and of transcribed ultraconserved ncRNA as a unique class of genes involved in the pathobiology of HCC.
Abstract: Although expression of non–protein-coding RNA (ncRNA) can be altered in human cancers, their functional relevance is unknown. Ultraconserved regions are noncoding genomic segments that are 100% conserved across humans, mice, and rats. Conservation of gene sequences across species may indicate an essential functional role, and therefore we evaluated the expression of ultraconserved RNAs (ucRNA) in hepatocellular cancer (HCC). The global expression of ucRNAs was analyzed with a custom microarray. Expression was verified in cell lines by real-time PCR or in tissues by in situ hybridization using tissue microarrays. Cellular ucRNA expression was modulated with siRNAs, and the effects on global gene expression and growth of human and murine HCC cells were evaluated. Fifty-six ucRNAs were aberrantly expressed in HepG2 cells compared with nonmalignant hepatocytes. Among these ucRNAs, the greatest change was noted for ultraconserved element 338 (uc.338), which was dramatically increased in human HCC compared with noncancerous adjacent tissues. Although uc.338 is partially located within the poly(rC) binding protein 2 (PCBP2) gene, the transcribed ncRNA encoding uc.338 is expressed independently of PCBP2 and was cloned as a 590-bp RNA gene, termed TUC338. Functional gene annotation analysis indicated predominant effects on genes involved in cell growth. These effects were experimentally demonstrated in both human and murine cells. siRNA to TUC338 decreased both anchorage-dependent and anchorage-independent growth of HCC cells. These studies identify a critical role for TUC338 in regulation of transformed cell growth and of transcribed ultraconserved ncRNA as a unique class of genes involved in the pathobiology of HCC.

205 citations


Journal ArticleDOI
TL;DR: The findings reveal a potential and appealing role for miRs as cancer therapeutic targets and aims at providing the molecular bases for the understanding of the potential role of miRs in the translational and clinical setting.

PatentDOI
TL;DR: A mouse model and uses there of for detecting, treating, characterizing, and diagnosing various diseases are described in this paper, where a mouse model is used to detect, treat, characterise, and diagnose various diseases.
Abstract: A mouse model and uses there of for detecting, treating, characterizing, and diagnosing various diseases are described.

Journal ArticleDOI
TL;DR: A review of the current knowledge of miRNAs in both solid and hematological malignancies with a focus on their potential application as therapeutic targets in cancer.

Journal ArticleDOI
TL;DR: Functional and molecular studies uncovered mechanisms that linked deregulated miRNAs to cancer-associated pathways, thereby placing their deregulation in a more rational framework and improving knowledge concerning the molecular basis of HCC.
Abstract: The role of microRNAs (miRNAs) in human tumorigenesis has been demonstrated by gene profiling and functional studies. In hepatocellular carcinoma (HCC), consistently deregulated miRNAs were identified. Their aberrant expression revealed relations shared with other types of cancer and others unique to HCC, namely the down-regulation of miR-122. Most importantly, functional and molecular studies uncovered mechanisms that linked deregulated miRNAs to cancer-associated pathways, thereby placing their deregulation in a more rational framework. These results improved our knowledge concerning the molecular basis of HCC and helped to increase our understanding about the great clinical potential behind these small molecules. In fact, a number of studies proved that miRNAs may have clinical relevance as bio-pathologic markers for HCC classification, prognostic stratification, early diagnosis or follow-up of patients. Additionally, the demonstration that miRNAs themselves or anti-miRNA oligonucleotides could be successfully used for in vivo modulation of miRNA actions has shown significant potentials in molecularly targeted therapy. In this context, the liver represents an organ of election to test therapeutic possibilities associated with miRNAs.

Journal ArticleDOI
15 Sep 2011-Blood
TL;DR: In this paper, the expression values of miR-181b, the most dysregulated microRNA, decreased in patients with a progressive (P <.001, training and validation sets) but not in samples of patients having a stable disease (P =.3, training set; P =.2, validation set) over time.

Journal ArticleDOI
TL;DR: It is established that FFPE samples can be used to reveal the tissue of origin of metastatic cancers by using microRNA expression profiling and suggested that the approach, if applied, could provide strong indications for CUPs, whose correct diagnosis is presently undefined.
Abstract: Cancer of unknown primary (CUP) represents a common and important clinical problem. There is evidence that most CUPs are metastases of carcinomas whose primary site cannot be recognized. Driven by the hypothesis that the knowledge of primary cancer could improve patient’s prognosis, we investigated microRNA expression profiling as a tool for identifying the tissue of origin of metastases. We assessed microRNA expression from 101 formalin-fixed, paraffin-embedded (FFPE) samples from primary cancers and metastasis samples by using a microarray platform. Forty samples representing ten different cancer types were used for defining a cancer-type-specific microRNA signature, which was used for predicting primary sites of metastatic cancers. A 47-miRNA signature was identified and used to estimate tissue-of-origin probabilities for each sample. Overall, accuracy reached 100% for primary cancers and 78% for metastases in our cohort of samples. When the signature was applied to an independent published dataset of 170 samples, accuracy remained high: correct prediction was found within the first two options in 86% of the metastasis cases (first prediction was correct in 68% of cases). This signature was also applied to predict 16 CUPs. In this group, first predictions exhibited probabilities higher than 90% in most of the cases. These results establish that FFPE samples can be used to reveal the tissue of origin of metastatic cancers by using microRNA expression profiling and suggest that the approach, if applied, could provide strong indications for CUPs, whose correct diagnosis is presently undefined.

Journal ArticleDOI
TL;DR: In this article, a miRNA microarray analysis comparing native esophageal mucosa with all the phenotypic lesions seen in the Barrett's carcinogenic process was performed to test the hypothesis that a specific miRNA expression signature characterizes BM development and progression.
Abstract: Barrett's esophagus (BE) is characterized by the native stratified squamous epithelium (N) lining the esophagus being replaced by a columnar epithelium with intestinal differentiation (Barrett's mucosa; BM). BM is considered as the main risk factor for esophageal adenocarcinoma (Barrett's adenocarcinoma; BAc). MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting messenger RNAs and they are reportedly dysregulated in BM. To test the hypothesis that a specific miRNA expression signature characterizes BM development and progression, we performed miRNA microarray analysis comparing native esophageal mucosa with all the phenotypic lesions seen in the Barrett's carcinogenic process. Specimens were collected from 14 BE patients who had undergone esophagectomy, including: 14 with N, 14 with BM, 7 with low-grade intraepithelial neoplasia, 5 with high-grade intra-epithelial neoplasia and 11 with BAc. Microarray findings were further validated by quantitive real-time polymerase chain reaction and in situ hybridization analyses using a different series of consecutive cases (162 biopsy samples and 5 esophagectomies) of histologically proven, long-segment BE. We identified a miRNA signature of Barrett's carcinogenesis consisting of an increased expression of 6 miRNAs and a reduced expression of 7 miRNAs. To further support these results, we investigated target gene expression using the Oncomine database and/or immunohistochemical analysis. We found that target gene expression correlated significantly with miRNA dysregulation. Specific miRNAs are directly involved in BE progression to cancer. miRNA profiling significantly expands current knowledge on the molecular history of Barrett's carcinogenesis, also identifying molecular markers of cancer progression.

Journal ArticleDOI
TL;DR: A perspective on emerging concepts in the clinical application of miRNA and other non‐coding RNAs as biomarkers in cancer with an eye on the eventual integration of both miRNAs and otherNon-coding RNA biology into the understanding of cancer pathogenesis and treatment is provided.

Journal ArticleDOI
TL;DR: This pilot study suggested that analysis of microRNA expression profile offers diagnostic utility, and provide pathogenetic and prognostic discrimination in MDS.
Abstract: The myelodysplastic syndromes (MDS) display both haematological and biological heterogeneity with variable leukaemia potential. MicroRNAs play an important role in tumour suppression and the regulation of self-renewal and differentiation of haematopoietic progenitors. Using a microarray platform, we evaluated microRNA expression from 44 patients with MDS and 17 normal controls. We identified a thirteen microRNA signature with statistically significant differential expression between normal and MDS specimens (P < 0·01), including down-regulation of members of the leukaemia-associated MIRLET7 family. A unique signature consisting of 10 microRNAs was closely associated with International Prognostic Scoring System (IPSS) risk category permitting discrimination between lower (Low/Intermediate-1) and higher risk (Intermediate-2/High) disease (P < 0·01). Selective overexpression of MIR181 family members was detected in higher risk MDS, indicating pathogenetic overlap with acute myeloid leukaemia. Survival analysis of an independent cohort of 22 IPSS lower risk MDS patients revealed a median survival of 3·5 years in patients with high expression of MIR181 family compared to 9·3 years in patients with low MIR181 expression (P = 0·002). Our pilot study suggested that analysis of microRNA expression profile offers diagnostic utility, and provide pathogenetic and prognostic discrimination in MDS.

Journal ArticleDOI
15 Oct 2011-Cancer
TL;DR: In this article, the authors integrate the transcriptome and microRNome in AML cells, and identify previously unidentified functional miRNA-mRNA interactions in the context of AML.
Abstract: Our results indicate that by integrating the transcriptome and microRNome in AML cells, it is possible to identify previously unidentified putative functional miRNA-mRNA interactions in AML.

Journal ArticleDOI
TL;DR: The very similar phenotype shared by all mutant lines implies that all four genes are involved and required for expansion of MLL-AF4 associated leukemic cells in mice, and down-regulation of any of them is not compensated by the others.
Abstract: Rearrangements of the MLL (ALL1) gene are very common in acute infant and therapy-associated leukemias. The rearrangements underlie the generation of MLL fusion proteins acting as potent oncogenes. Several most consistently up-regulated targets of MLL fusions, MEIS1, HOXA7, HOXA9, and HOXA10 are functionally related and have been implicated in other types of leukemias. Each of the four genes was knocked down separately in the human precursor B-cell leukemic line RS4;11 expressing MLL-AF4. The mutant and control cells were compared for engraftment in NOD/SCID mice. Engraftment of all mutants into the bone marrow (BM) was impaired. Although homing was similar, colonization by the knockdown cells was slowed. Initially, both types of cells were confined to the trabecular area; this was followed by a rapid spread of the WT cells to the compact bone area, contrasted with a significantly slower process for the mutants. In vitro and in vivo BrdU incorporation experiments indicated reduced proliferation of the mutant cells. In addition, the CXCR4/SDF-1 axis was hampered, as evidenced by reduced migration toward an SDF-1 gradient and loss of SDF-1–augmented proliferation in culture. The very similar phenotype shared by all mutant lines implies that all four genes are involved and required for expansion of MLL-AF4 associated leukemic cells in mice, and down-regulation of any of them is not compensated by the others.

Journal ArticleDOI
TL;DR: Evidence is provided that the anticancer agent benzyl isothiocyanate (BITC) has the ability to modulate the level of miRNAs such as miR-221 andMiR-375, known to be abnormally expressed in pancreatic cancer patients, to switch hyperproliferative pancreas with preneoplastic lesions to a hypoproliferative state.
Abstract: The deregulated presence or absence of microRNAs (miRNAs) might play an important role in molecular pathways leading to neoplastic transformation. At present, it is also thought that the approaches to interfere miRNA functions should be helpful for developing novel therapeutic opportunities for human cancer. In this study, we provide evidence that the anticancer agent benzyl isothiocyanate (BITC) has the ability to modulate the level of miRNAs such as miR-221 and miR-375, known to be abnormally expressed in pancreatic cancer patients. Interestingly, ectopic expression of miR-375 or the enforced silencing of miR-221 in cultured pancreatic cancer cells attenuates cell viability and sensitizes antiproliferative action of BITC. We also show that the expression of putative tumor suppressor miR-375 is more abundant in nonpathological mice pancreata than those with Kras(G12D)-driven pancreatic intraepithelial neoplasia (PanIN). To the contrary, the expression of oncogenic miR-221 is significantly elevated in the mouse pancreas with PanIN lesions. Although miR-375 has been shown to be aberrantly expressed in pancreatic cancer patients, there has not been a comprehensive study to investigate the molecular pathways targeted by this miRNA in pancreatic cancer cells. Further analysis by gene expression microarray revealed that IGFBP5 and CAV-1, potential biomarkers of pancreatic cancer, were significantly downregulated in cells transfected with miR-375. Correlatively, elevated expression of IGFBP5 and CAV-1 was evident in the mouse pancreas with preneoplastic lesions in which the expression of miR-375 wanes. Taken together, our findings suggest that anticancer agent BITC might target the expression of miR-221 and miR-375 to switch hyperproliferative pancreatic cancer cells to a hypoproliferative state.

Journal ArticleDOI
08 Sep 2011-Blood
TL;DR: A recurrent heterozygous somatic mutation in the DNA-binding domain of IRF4 detected in 7 of 457 chronic lymphocytic leukemia patients (1.5%) is reported, and it is found thatIRF4 mRNA expression is higher in the patients with the mutation.

Journal ArticleDOI
TL;DR: This review provides a state-of-the-art knowledge on the implications of microRNA (miRNA) dysregulation in lymphoid malignancies and demonstrates that miRNAs can effectively be used as tumoral biomarkers with implications for diagnosis, prognosis, and prediction of response to therapy.
Abstract: MicroRNAs (miRNAs) are non-coding RNAs (ncRNAs) that regulate gene expression and affect several biological processes, spanning from development, differentiation, cell cycle regulation to senescence and metabolism (1-5). The biogenesis of miRNAs has been described in several excellent reviews (6, 7). The full spectrum of miRNAs expressed in a specific cell type (the miRNome) varies between normal and pathologic tissues (8), and specific signatures of deregulated miRNAs harbor diagnostic and prognostic implications (9, 10). The first link between miRNAs and cancer came from the discovery of loss of miR-15a and miR-16-1 in chronic lymphocytic leukemia (11) and from the discovery that these ncRNAs are frequently located in cancer associated genomic regions (CAGRs), which include minimal regions of amplification, loss of heterozygosity, common breakpoint regions in or near oncogenes (OGs) and tumor suppressor genes (TSGs), and fragile sites (preferential sites of chromatid exchange, deletion, translocation, amplification or integration of plasmid DNA and tumor-associated viruses) (12). Since then, a plethora of studies have investigated the aberrancies of the miRNome in almost all types of human cancer, both solid tumors and hematological malignancies (for reviews see (10, 13-18)). In particular, while some miRNAs act mainly as TSGs, others exert a predominantly oncogenic function, although labeling a miRNA as exclusively a TSG or an OG is inappropriate, given that the overall function of any given miRNA seems to depend on its cellular context. The present review will focus on the most recent discoveries in the field of miRNAs and lymphoid malignancies, which led to the identification of new pathogenetic molecular networks with prognostic significance and potential therapeutic implications.

Journal ArticleDOI
TL;DR: It is demonstrated that expression of miR-483 can be induced independently of IGF2 by the oncoprotein β-catenin through an interaction with the basic helix–loop–helix protein upstream stimulatory transcription factor 1.
Abstract: hsa-mir-483 is located within intron 2 of the IGF2 gene. We have previously shown oncogenic features of miR-483-3p through cooperation with IGF2 or by independently targeting the proapoptotic gene BBC3/PUMA. Here we demonstrate that expression of miR-483 can be induced independently of IGF2 by the oncoprotein β-catenin through an interaction with the basic helix–loop–helix protein upstream stimulatory transcription factor 1. We also show that β-catenin itself is a target of miR-483-3p, triggering a negative regulatory loop that becomes ineffective in cells harboring an activating mutation of β-catenin. These results provide insights into the complex regulation of the IGF2/miR-483 locus, revealing players in the β-catenin pathway.

Journal ArticleDOI
16 Jun 2011-Oncogene
TL;DR: Onconase likely exerts its antitumor effect through specific microRNAs (miRNAs) through downregulation of nuclear factor kappa B by specific microRNA mimics or inhibitors and that interference of this pathway could have implications for MPM resistance to chemotherapy.
Abstract: Treatment of malignant pleural mesothelioma (MPM) with Ranpirnase (Onconase) results in disruption of protein translation and cell apoptosis We hypothesize that Onconase exerts an effect via downregulation of nuclear factor kappa B (NFKβ) by specific microRNAs (miRNAs) and that interference of this pathway could have implications for MPM resistance to chemotherapy Three immortalized MPM cell lines (H2959, H2373 and H2591) were exposed to Onconase at 0–20 μg/ml Cell counts were measured at 48 and 72 h Gene expression in miRNA-enriched RNA was validated by reverse transcription–PCR (RT–PCR) The functional implications of miRNA expression were evaluated by transfecting miRNA mimics or inhibitors into MPM cell lines, and performing Matrigel invasion, cell proliferation, soft agar colony formation and scratch closure assays Effects on NFKβ expression and downstream targets including ABC transporters, BCL-xl and IAP were assessed by RT–PCR and western blotting Treatment with 20 μg/ml of Onconase significantly decreased cell count and invasion Hsa-miR-17* was significantly upregulated and hsa-miR-30c was significantly downregulated by Onconase treatment in all cell lines Forced expression of hsa-miR-17* mimic and hsa-miR-30c inhibitor each significantly decreased functional activity of Onconase in all assays NFKB1 (p50) expression and downstream targets were also decreased with Onconase treatment, as well as with forced expression of miRNA mimic and inhibitors Onconase treatment caused a significant decrease in cell proliferation, invasion and in expression of certain miRNAs Recapitulation of the resultant miRNA expression pattern with hsa-miR-17* mimic and hsa-miR-30c inhibitor resulted in downregulation of NFKB1 and reduced malignant behavior in functional assays Thus, Onconase likely exerts its antitumor effect through these miRNAs

Journal ArticleDOI
TL;DR: The expression of miRNAs and their function in lymphomas is described and potential miRNA-based therapies in the diagnosis and treatment of lymphomas are discussed.
Abstract: The recent discovery of microRNAs (miRNAs) has introduced a new layer of complexity to the process of gene regulation. MiRNAs are essential for cellular function, and their dysregulation often results in disease. Study of miRNA expression and function in animal models and human lymphomas has improved our knowledge of the pathogenesis of this heterogeneous disease. In this paper, we attempt to describe the expression of miRNAs and their function in lymphomas and discuss potential miRNA-based therapies in the diagnosis and treatment of lymphomas.

Journal ArticleDOI
TL;DR: This research presents a novel probabilistic approach to estimating the response of the immune system to laser-spot assisted, 3D image analysis of central nervous system injury and shows clear patterns in response to treatment-related injuries.
Abstract: Comment on: Boeri M, et al. Proc Natl Acad Sci USA 2011; 108:3713-8.

Journal ArticleDOI
TL;DR: ZD activates alternative inflammation‐associated cancer pathways that fuel tumor progression and bypass the antitumor effect of Cox‐2 ablation, which has important clinical implications, as combination cancer therapy that includes Zn may improve efficacy.
Abstract: Zinc (Zn)-deficiency (ZD) is implicated in the pathogenesis of human oral-esophageal cancers. Previously, we showed that in ZD mice genetic deletion of cyclooxygenase-2 (Cox-2) enhances N-nitrosomethylbenzylamine-induced forestomach carcinogenesis. By contrast, Cox-2 deletion offers protection in Zn-sufficient (ZS) mice. We hypothesize that ZD activates pathways insensitive to COX-2 inhibition, thereby promoting carcinogenesis. This hypothesis is tested in a Cox-2−/− mouse tongue cancer model that mimics pharmacologic blockade of COX-2 by firstly examining transcriptome profiles of forestomach mucosa from Cox-2−/− and wild-type mice on a ZD vs. ZS diet, and secondly investigating the roles of identified markers in mouse forestomach/tongue preneoplasia and carcinomas. In Cox-2−/− mice exposed to the tongue carcinogen 4-nitroquinoline 1-oxide, dietary ZD elicited tongue/esophagus/forestomach carcinomas that were prevented by ZS. The precancerous ZD:Cox-2−/−vs. ZS:Cox-2−/− forestomach had an inflammatory signature with upregulation of the proinflammation genes S100a8 and S100a9. Bioinformatics analysis revealed overrepresentation of inflammation processes comprising S100a8/a9 and an nuclear factor (NF)-κB network with connectivity to S100A8. Immunohistochemistry revealed co-overexpression of S100A8, its heterodimeric partner S100A9, the receptor for advanced glycation end-products (RAGE), NF-κB p65, and cyclin D1, in ZD:Cox-2−/− forestomach/tongue preneoplasia and carcinomas, evidence for the activation of a RAGE-S100A8/A9 inflammatory pathway. Accumulation of p53 in these carcinomas indicated activation of additional inflammatory pathways. Zn-replenishment in ZD:Cox-2−/−mice reversed the inflammation and inhibited carcinogenesis. Thus, ZD activates alternative inflammation-associated cancer pathways that fuel tumor progression and bypass the antitumor effect of Cox-2 ablation. These findings have important clinical implications, as combination cancer therapy that includes Zn may improve efficacy.

Journal ArticleDOI
TL;DR: The observation from the study of Brest et al.2 provides direct evidence that a silent mutation can, in fact, have a function in disease, and it can do so by creating an aberrant cis-regulatory element for a miRNA.
Abstract: alterations, such as SNPs, in cis-regulatory elements can alter target gene regulation by either creating or destroying miRNA target sites9. Indeed, SNPs occurring at cis-regulatory elements have been shown to either enhance or inhibit the recognition of a miRNA, causing aberrant gene expression and disease phenotypes10,11. However, the interaction of specific miRNAs with cis-acting sequence variants and the environmental cues required for their expression are just coming to light. Crohn’s disease is an inflammatory bowel disease caused by the persistence of bacteria in the epithelial lining of the gastrointestinal tract. There seems to be an inherent genetic component to Crohn’s disease, as an individual’s risk is directly associated with the amount of firstand second-degree relatives with the disease. Genetic mapping in people with this disorder has uncovered numerous mutations—in particular, a silent mutation in IRGM (encoding immunity-related GTPase family, M), a gene required for autophagy of intracellular bacteria that is highly prevalent in individuals of European descent12. Brest et al.2 found that the protective IRGM variant, but not the riskassociated allele, is a target site for the miR-196 family, which normally results in a decrease in cellular IRGM protein amounts in inflamed epithelial cells. miR-196 was also overexpressed in inflamed epithelial mucosa of the gut in people with Crohn’s disease, but this did not result in decreased expression of cellular IRGM in the presence of the risk allele. miR-196 was first found to be crucial in development, and subsequently in cancer progression, through regulation of HOX genes13. The pathophysiological relevance of miR-196– mediated suppression of IRGM protective variant is that decreased amounts of IRGM were necessary to clear Crohn’s disease–associated adherent invasive E. coli from the gut through autophagy (Fig. 1). Thus, individuals with Crohn’s disease with the risk allele were unable to clear these bacteria, resulting in an accumulation of intracellular bacteria and disease progression. The observation from the study of Brest et al.2 provides direct evidence that a silent mutation can, in fact, have a function in disease, and it can do so by creating an aberrant cis-regulatory element for a miRNA. In the future, the risk of a particular silent mutation should be assessed by its ability to functionally regulate gene expression under different environmental conditions. The finding that miR-196 is specifically expressed in the inflamed mucosa to target IRGM and that, in this scenario, the silent mutation in IRGM becomes crucial in disease causation2 is the perfect proof of this concept. The identification of such functional variants may therefore not be possible by comparing their altered frequency in control and disease populations without understanding concurrent physiology of the patients. Although this work substantially advances our understanding of Crohn’s disease, it has much broader implications for the clinical assessment of silent mutations and other VUS in disease. For example, there are many silent mutations in BRCA1 and BRCA2 classified as ‘uninformative’ or ‘not clinically significant’ by the US National Institutes of Health Breast Cancer Information Core database and Myriad Genetics, as they are not predicted to alter BRCA protein sequence or function1. Yet many of the individuals with these VUS still develop breast and/or ovarian cancer. In light of the observations by Brest et al.2, clinicians and genetic counselors may need to reconsider their assessment of BRCA VUS, as well as SNPs disrupting miRNA binding sites outside of protein coding sequences. As it is possible that many of these silent mutations or noncoding region SNPs alter miRNA target sites and that their function will depend on miRNA expression alterations in response to physiologic conditions—similar to that of the IRGM risk-associated allele—it will be crucial to bring the study of such variants back to the bench to understand their importance in human disease.