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Akt is a serine threonine kinase with a major role in transducing survival signals and regulating proteins involved in apoptosis. To find new interactors of Akt involved in cell survival, we performed a two-hybrid screening in yeast using human full-length Akt c-DNA as bait and a murine c-DNA library as prey. Among the 80 clones obtained, two were identified as Bcl-w. Bcl-w is a member of the Bcl-2 family that is essential for the regulation of cellular survival, and that is up-regulated in different human tumors, such as gastric and colorectal carcinomas. Direct interaction of Bcl-w with Akt was confirmed by immunoprecipitation assays. Subsequently, we addressed the function of this interaction: by interfering with the activity or amount of Akt, we have demonstrated that Akt modulates the amount of Bcl-w protein. We have found that inhibition of Akt activity may promote apoptosis through the downregulation of Bcl-w protein and the consequential reduction in interaction of Bcl-w with proapoptotic members of the Bcl-2 family. Our data provide evidence that Bcl-w is a new member of the Akt pathway and that Akt may induce anti-apoptotic signals at least in part through the regulation of the amount and activity of Bcl-w.

MicroRNA (miRNA or miR) are an important class of regulators that participate in such biological functions as development, cell proliferation, differentiation, and apoptosis. The aim of this study was to elucidate the role of miRNA in cell proliferation using a unique cell system, namely thyroid cells that require thyrotropin for their growth. Here, we report the identification of a set of five specific miRNA (miR-1, miR-28-A, miR-290-5p, miR-296-3p, and miR-297a), whose down-regulation by thyrotropin is required for thyroid cell growth. In fact, overexpression of these miRNA negatively affects cell growth. We show that three of these miRNA target cAMP-responsive element binding protein (CREB)1, a thyrotropin-activated transcription factor, and that CREB1 binds the regulatory regions of the down-regulated miRNA. Hence, these data indicate that a synergistic loop involving thyrotropin, CREB1, and miRNA is required for thyroid cell proliferation.

The involvement of the MET oncogene in de novo and acquired resistance of non-small cell lung cancers (NSCLCs) to tyrosine kinase inhibitors (TKIs) has previously been reported, but the precise mechanism by which MET overexpression contributes to TKI-resistant NSCLC remains unclear. MicroRNAs (miRNAs) negatively regulate gene expression, and their dysregulation has been implicated in tumorigenesis. To understand their role in TKI-resistant NSCLCs, we examined changes in miRNA that are mediated by tyrosine kinase receptors. Here we report that miR-30b, miR-30c, miR-221 and miR-222 are modulated by both epidermal growth factor (EGF) and MET receptors, whereas miR-103 and miR-203 are controlled only by MET. We showed that these miRNAs have important roles in gefitinib-induced apoptosis and epithelial-mesenchymal transition of NSCLC cells in vitro and in vivo by inhibiting the expression of the genes encoding BCL2-like 11 (BIM), apoptotic peptidase activating factor 1 (APAF-1), protein kinase C ? (PKC-?) and sarcoma viral oncogene homolog (SRC). These findings suggest that modulation of specific miRNAs may provide a therapeutic approach for the treatment of NSCLCs.

Carcinoma of the thyroid gland is an uncommon cancer, but one of the most frequent malignancies of the endocrine system. Most thyroid cancers are derived from the follicular cells. Follicular carcinoma is considered more malignant than papillary thyroid carcinoma (PTC), and anaplastic thyroid cancer (ATC) is one of the most lethal human cancers. Even though several genetic lesions have been already described in human thyroid cancer, particularly in the papillary histotype, the mechanisms underlying the development of these neoplasias are still far from being completely elucidated. Some years ago, several studies were undertaken to analyze the expression of microRNAs (miRNAs or miRs) in thyroid carcinoma to evaluate a possible role of their deregulation in the process of carcinogenesis. These studies showed an aberrant microRNA expression profile that distinguishes unequivocally among PTC, ATC, and normal thyroid tissue. Here, other than summarizing the current findings on microRNA expression in human thyroid carcinomas, we discuss the mechanisms by which microRNA deregulation may play a role in thyroid carcinogenesis, and the possible use of microRNA knowledge in the diagnosis and therapy of thyroid neoplasms.

We showed that Emicro-MiR-155 transgenic mice develop acute lymphoblastic leukemia/high-grade lymphoma. Most of these leukemias start at approximately 9 months irrespective of the mouse strain. They are preceded by a polyclonal pre-B-cell proliferation, have variable clinical presentation, are transplantable, and develop oligo/monoclonal expansion. In this study, we show that in these transgenic mice the B-cell precursors have the highest MiR-155 transgene expression and are at the origin of the leukemias. We determine that Src homology 2 domain-containing inositol-5-phosphatase (SHIP) and CCAAT enhancer-binding protein beta (C/EBPbeta), 2 important regulators of the interleukin-6 signaling pathway, are direct targets of MiR-155 and become gradually more down-regulated in the leukemic than in the preleukemic mice. We hypothesize that miR-155, by down-modulating Ship and C/EBPbeta, initiates a chain of events that leads to the accumulation of large pre-B cells and acute lymphoblastic leukemia/high-grade lymphoma.

Altered expression of microRNAs (miRNAs) has been detected in cancer, suggesting that these small non-coding RNAs can act as oncogenes or tumor suppressor genes. In the present study, we investigated the expression of miRNA-17-5p, miRNA-18a, miRNA-20a, miRNA-92a, miRNA-146a, miRNA-146b and miRNA-155 by real-time quantitative RT-PCR in a panel of melanocyte cultures and melanoma cell lines and explored the possible role of miRNA-155 in melanoma cell proliferation and survival. The analyzed miRNAs were selected on the basis of previous studies strongly supporting their involvement in cancer development and/or progression. We found that miRNA-17-5p, miRNA-18a, miRNA-20a, and miRNA-92a were overexpressed, whereas miRNA-146a, miRNA-146b and miRNA-155 were down-regulated in the majority of melanoma cell lines with respect to melanocytes. Ectopic expression of miRNA-155 significantly inhibited proliferation in 12 of 13 melanoma cell lines with reduced levels of this miRNA and induced apoptosis in 4 out of 4 cell lines analyzed. In conclusion, our data further support the finding of altered miRNA expression in melanoma cells and establish for the first time that miRNA-155 is a negative regulator of melanoma cell proliferation and survival.

HMGA1 (high-mobility-group A1) proteins are architectural transcription factors that are found overexpressed in embryogenesis and malignant tumours. We have shown previously that they have a role in lymphopoiesis, since the loss of HMGA1 expression leads to an impairment of T-cell development and to an increase in B-cell population. Since RAGs (recombination activating genes) are key regulators of lymphoid differentiation, in the present study we investigate whether RAG2 expression is dependent on HMGA1 activity. We show that RAG2 gene expression is up-regulated in Hmga1-/- ES (embryonic stem) cells and EBs (embryoid bodies) as well as in yolk sacs and fibroblasts from Hmga1-/- mice, suggesting that HMGA1 proteins control RAG2 gene expression both in vitro and in vivo. We show that the effect of HMGA1 on RAG2 expression is direct, identify the responsible region in the RAG2 promoter and demonstrate binding to the promoter in vivo using chromatin immunoprecipitation. Since RAG2 is necessary for lymphoid cell development, our results suggest a novel mechanism by which HMGA1 might regulate lymphoid differentiation.

We have previously isolated the coding sequence for a novel substrate for tyrosine kinases, eps8, from NIH3T3 fibroblasts. Eps8 was phosphorylated in vivo by several receptor tyrosine kinases (RTKs) and, upon overexpression, was able to enhance EGFR-mediated mitogenic signaling in NIH3T3 cells. To gain understanding of eps8 function as well as its role in normal and neoplastic proliferation, we cloned the human eps8 coding sequence and studied expression of the human RNA and protein, evolutionary conservation, and chromosomal location. In addition to a previously identified SH3 domain, the predicted amino acid sequence of human eps8 revealed a non-random distribution of prolines, clustered in a way to suggest SH3-binding sites and a putative PH domain. Eps8 was expressed in all epithelial and fibroblastic lines examined and in some, but not all, hematopoietic cells. An essential function of eps8 in cell growth regulation was underscored by its conservation during evolution, where eps8-related sequences were detected as early as in Saccharomyces cerevisiae. Finally, the human EPS8 locus was mapped to chromosome 12q23-q24.

Employing an expression cloning approach for tyrosine kinase substrates, we have previously isolated the coding sequence for a novel putative EGFR substrate, eps15, from NIH3T3 fibroblasts. Eps15 displayed a receptor-specific pattern of tyrosine phosphorylation in vivo and was able to transform NIH3T3 cells upon overexpression. To gain understanding of eps15 function as well as its role in normal and neoplastic proliferation, we cloned the human eps15 coding sequence and studied expression of the human RNA and protein, evolutionary conservation, and chromosomal location. The close structural similarity of human eps15 with the murine homologue is indicated by 89% and 90% identity of nucleotide and predicted amino acid sequences, respectively. Using the human eps15 coding sequence as probe, we demonstrate that eps15 is member of a gene family that is highly conserved during evolution. An essential function of eps15 in cell growth regulation is underscored by our observation of ubiquitous expression at the transcript and the protein level in normal and malignant human cells. The human EPS15 locus maps to chromosome 1p31-p32, a region involved in deletion in neuroblastoma, translocations in acute lymphoblastic leukemia, and exhibiting a fragile site.

Loss of heterozygosity involving the short arm of chromosome 3 has been reported in gastric and other human tumors. We have cloned and mapped a candidate tumor suppressor gene, FHIT (fragile histidine triad), to this chromosomal region (3p14.2). To investigate the role of FHIT gene alterations in the development of gastric carcinoma, we examined 8 gastric carcinoma-derived cell lines and 32 primary adenocarcinoma samples by Southern blot analysis. We also analyzed the integrity of FHIT transcripts by reverse transcription-PCR. The occurrence of alterations in the FHIT gene and its transcript correlated with the absence of Fhit protein expression by immunoblot analysis in the cancer cell lines. Four of eight cell lines showed deletion or rearrangement within the FHIT gene, together with the absence of the wild-type transcript and the Fhit protein. Among the primary gastric carcinomas, rearrangement of the FHIT gene and/or aberrant reverse transcription-PCR products were detected in 17 of 32 (53%) tumors, and 20 of 30 (67%) samples exhibited an absence of Fhit protein expression. Gastric cancer is thought to develop from carcinogenic exposure, possibly explaining the high frequency of abnormalities in the FHIT gene, a fragile locus exhibiting susceptibility to carcinogen-induced alterations. The consequent absence or reduction of Fhit protein expression is consistent with the proposal that the FHIT gene is a preferential target of environmental carcinogens and that FHIT inactivation plays a role in the development of gastric cancer.

Chromosomal region 11q23 participates in a number of reciprocal translocations with specific regions of chromosomes 4, 9, 19, and others. These translocations are associated with acute lymphocytic leukemia and acute myelomonocytic, monocytic, and myelogenous leukemia. From a yeast artificial chromosome containing human DNA derived from 11q23 we cloned a DNA fragment which can be used as a probe to detect rearrangements in leukemic cells from the majority of patients with the t(4;11), t(9;11), and t(11;19) translocations. The breakpoints cluster in a small DNA region of less than 5.8 kilobases.

The MYB related loci, AMYB and BMYB, were localized to specific human chromosome regions by Southern blot analysis of their segregation patterns in a panel of rodent-human hybrid DNAs using radiolabeled AMYB and BMYB probes. The AMYB locus was present in hybrids retaining the chromosome region 8cen----8q22 and was absent in hybrids which had lost this chromosome region. The presence of the BMYB locus in rodent-human hybrids correlated with, and only with, chromosome region Xq13. Chromosomal in situ hybridization refined the localization of AMYB to region 8q22-23 and confirmed the localization of BMYB to region Xq13. Chromosome region 8q22 is involved in recurrent translocations in malignant lymphoma and in acute myeloid leukemia (AML-M2); therefore AMYB is a candidate for involvement in such translocations. A region on Xq13 is also involved in chromosomal abnormalities in acute myeloid leukemia and myelodysplasias.

We have analyzed cells of the B lineage for expression of the Tp44 antigen, a 44,000 homodimer detected by monoclonal antibody 9.3 on approximately 80% of mature human T lymphocytes. Previous evidence has suggested that Tp44 may function as a receptor for accessory signals in T cell activation. High level Tp44 expression was observed on plasmacytomas grown in cell culture and on plasma cells from bone marrow biopsies of multiple myeloma patients. This antigen is not present on resting B cells from either peripheral blood or lymphoid organs, or on any other B cell tumor. The growth kinetics and Ig production in plasmacytomas are not affected by the binding of antibody 9.3. Moreover, the Tp44 molecule is co-expressed with PCA-1, an antigen characteristic of plasma cells, on peripheral blood B cells stimulated in vitro to differentiate toward plasma cells. Tp44 may represent a later stage of B cell differentiation than PCA-1 because unlike the PCA-1 antigen, this molecule could not be detected on any EBV-transformed cell line or Burkitt's lymphoma lines. The m.w. of the Tp44 molecule expressed on plasma cells and on T cells is identical, as determined by immunoprecipitation of radioiodinated cell surface proteins with monoclonal antibody 9.3. This antigen might be useful in studying the mechanism of growth and differentiation of human B cells, the heterogeneity within plasma cell populations, and B cell interactions with other components of the immune system.

We have analyzed the organization and expression of the immunoglobulin heavy and light chain gene in the human myeloblastic leukemic sublines, ML1, ML2, and ML3, and in the human myeloid leukemic cell lines, HL-60, U937, THP1, and K562. ML1, ML2, and ML3 cells, despite a predominant granulocytic phenotype, express a rearrangement of the immunoglobulin heavy chain gene that typically occurs during the early stages of the B cell differentiation pathway. No rearrangement was found in any of the other cell lines tested. These findings strongly support the notion that, at least in some cases, acute myeloid leukemia (AML) cells represent highly atypical cells with profoundly altered gene expression, rather than cells arrested at a well-defined stage of the myeloid lineage.

Rearrangements of the HMGI-C gene have frequently been detected in human benign tumors of mesenchymal origin, including lipomas. The HMGI-C protein has three AT-hook domains and an acidic COOH-terminal tail. The HMGI-C modifications consist in the loss of the C-tail and the fusion with ectopic sequences. Recent results show that the loss of the COOH-terminal region, rather than the acquisition of new sequences, is sufficient to confer to HMGI-C the ability to transform NIH3T3 cells. Therefore, transgenic mice carrying a HMGI-C construct (HMGI-C/T), containing only the three AT-hook domains, were generated. The HMGI-C/T mice showed a giant phenotype, together with a predominantly abdominal/pelvic lipomatosis, suggesting a pivotal role of the HMGI-C truncation in the generation of human lipomas.

// Farzaneh Moshiri 1, 2 , Alessandro Salvi 3 , Laura Gramantieri 4 , Angelo Sangiovanni 5 , Paola Guerriero 1 , Giuseppina De Petro 3 , Cristian Bassi 1 , Laura Lupini 1 , Arash Sattari 2 , Douglas Cheung 2 , Dario Veneziano 2 , Giovanni Nigita 2 , Ram C. Shankaraiah 1 , Nazario Portolani 6 , Paolo Carcoforo 1 , Francesca Fornari 4, 7 , Luigi Bolondi 4, 7 , Antonio Frassoldati 8 , Silvia Sabbioni 9 , Massimo Colombo 5 , Carlo M. Croce 2 and Massimo Negrini 1 1 Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy 2 Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA 3 Department of Molecular and Translational Medicine, Division of Biology and Genetics, University of Brescia, Brescia, Italy 4 Center for Applied Biomedical Research, St. Orsola-Malpighi University Hospital, Bologna, Italy 5 Gastroenterology and Hepatology Division, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, University of Milano, Milan, Italy 6 Department of Medical and Surgical Sciences, Surgical Clinic, University of Brescia, Brescia, Italy 7 Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy 8 Oncology Division, University Hospital of Ferrara, Cona (FE), Italy 9 Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy Correspondence to: Massimo Negrini, email: ngm@unife.it Carlo M. Croce, email: Carlo.Croce@osumc.edu Keywords: hepatocellular carcinoma; cirrhosis; circulating microRNA; diagnostic biomarkers Received: March 09, 2017 Accepted: November 05, 2017 Epub: February 27, 2018 Published: March 16, 2018 ABSTRACT Hepatocellular carcinoma (HCC) is the most common liver cancer and second leading cause of cancer related death worldwide. Most HCCs occur in a damaged cirrhotic background and it may be difficult to discriminate between regenerative nodules and early HCCs. No dependable molecular biomarker exists for the early detection of HCC. MicroRNAs (miRNAs) have attracted attention as potential blood-based biomarkers. To identify circulating miRNAs with diagnostic potential in HCC, we performed preliminary RNAseq studies on plasma samples from a small set of HCC patients, cirrhotic patients and healthy controls. Then, out of the identified miRNAs, we investigated miR-101-3p, miR-106b-3p, miR-1246 and miR-411-5p in plasma of independent HCC patients’ cohorts. The use of droplet digital PCR (ddPCR) confirmed the aberrant levels of these miRNAs. The diagnostic performances of each miRNA and their combinations were measured using Receiver Operating Characteristic (ROC) curve analyses: a classifier consisting of miR-101-3p, miR-1246 and miR-106b-3p produced the best diagnostic precision in plasma of HCC vs. cirrhotic patients (AUC = 0.99). A similar performance was found when the levels of miRNAs of HCC patients were compared to healthy controls (AUC = 1.00). We extended the analyses of the same miRNAs to serum samples. In serum of HCC vs. cirrhotic patients, the combination of miR-101-3p and miR-106b-3p exhibited the best diagnostic accuracy with an AUC = 0.96. Thus, circulating miR-101-3p, miR-106b-3p and miR-1246, either individually or in combination, exhibit a considerable potential value as diagnostic biomarkers of HCC.

Akt is a serine-threonine kinase that has an important role in transducing survival signals. Akt also regulates a number of proteins involved in the apoptotic process. To find new Akt interactors, we performed a two-hybrid screening in yeast using full-length Akt cDNA as bait and a human cDNA heart library as prey. Among 200 clones obtained, two of them were identified as coding for the c-FLIP(L) protein. c-FLIP(L) is an endogenous inhibitor of death receptor-induced apoptosis through the caspase-8 pathway. Using co-immunoprecipitation experiments of either transfected or endogenous proteins, we confirmed the interaction between Akt and c-FLIP(L). Furthermore, we observed that c-FLIP(L) overexpression interferes with Gsk3-β phosphorylation levels. Moreover, through its effects on Gsk3β, c-FLIP(L) overexpression in cancer cells induced resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This effect was mediated by the regulation of p27(Kip1) and caspase-3 expression. These results indicate the existence of a new mechanism of resistance to TRAIL in cancer cells, and unexpected functions of c-FLIP(L).

// Giuseppina Roscigno 1, 2 , Cristina Quintavalle 1, 2 , Elvira Donnarumma 3 , Ilaria Puoti 1 , Angel Diaz-Lagares 4 , Margherita Iaboni 1 , Danilo Fiore 1 , Valentina Russo 1 , Matilde Todaro 5 , Giulia Romano 6 , Renato Thomas 7 , Giuseppina Cortino 7 , Miriam Gaggianesi 5 , Manel Esteller 4 , Carlo M. Croce 6 , Gerolama Condorelli 1, 2 1 Department of Molecular Medicine and Medical Biotechnology, “Federico II” University of Naples, Naples, Italy 2 IEOS-CNR, Naples, Italy 3 IRCCS-SDN, Naples, Italy 4 Epigenetic and Cancer Biology Program (PEBC) IDIBELL, Hospital Duran I Reynals, Barcelona, Spain 5 Department of Surgical and Oncological Sciences, Cellular and Molecular Pathophysiology Laboratory, University of Palermo, Palermo, Italy 6 Department of Molecular Virology, Immunology and Medical Genetics, Human Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA 7 Department of Surgical and Oncology, Clinica Mediterranea, Naples, Italy Correspondence to: Gerolama Condorelli, e-mail: gecondor@unina.it Keywords: microRNAs, breast cancer, cancer stem cells, DNMT Received: June 15, 2015 Accepted: October 09, 2015 Published: October 19, 2015 ABSTRACT Cancer stem cells (CSCs) are a small part of the heterogeneous tumor cell population possessing self-renewal and multilineage differentiation potential as well as a great ability to sustain tumorigenesis. The molecular pathways underlying CSC phenotype are not yet well characterized. MicroRNAs (miRs) are small noncoding RNAs that play a powerful role in biological processes. Early studies have linked miRs to the control of self-renewal and differentiation in normal and cancer stem cells. We aimed to study the functional role of miRs in human breast cancer stem cells (BCSCs), also named mammospheres. We found that miR-221 was upregulated in BCSCs compared to their differentiated counterpart. Similarly, mammospheres from T47D cells had an increased level of miR-221 compared to differentiated cells. Transfection of miR-221 in T47D cells increased the number of mammospheres and the expression of stem cell markers. Among miR-221’s targets, we identified DNMT3b. Furthermore, in BCSCs we found that DNMT3b repressed the expression of various stemness genes, such as Nanog and Oct 3/4 , acting on the methylation of their promoters, partially reverting the effect of miR-221 on stemness. We hypothesize that miR-221 contributes to breast cancer tumorigenicity by regulating stemness, at least in part through the control of DNMT3b expression.

Glioblastoma is the most frequent brain tumor in adults and is the most lethal form of human cancer. Despite the improvements in treatments, survival of patients remains poor. To define novel pathways that regulate susceptibility to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in glioma, we have performed genome-wide expression profiling of microRNAs (miRs). We show that in TRAIL-resistant glioma cells, levels of different miRs are increased, and in particular, miR-30b/c and -21. We demonstrate that these miRs impair TRAIL-dependent apoptosis by inhibiting the expression of key functional proteins. T98G-sensitive cells treated with miR-21 or -30b/c become resistant to TRAIL. Furthermore, we demonstrate that miR-30b/c and miR-21 target respectively the 3' untranslated region of caspase-3 and TAp63 mRNAs, and that those proteins mediate some of the effects of miR-30 and -21 on TRAIL resistance, even in human glioblastoma primary cells and in lung cancer cells. In conclusion, we show that high expression levels of miR-21 and -30b/c are needed to maintain the TRAIL-resistant phenotype, thus making these miRs as promising therapeutic targets for TRAIL resistance in glioma.Oncogene advance online publication, 10 September 2012; doi:10.1038/onc.2012.410.

MicroRNAs (miRNA) have rapidly emerged as modulators of gene expression in cancer in which they may have great diagnostic and therapeutic import. MicroRNA-199a-3p (miR-199a-3p) is downregulated in several human malignancies including hepatocellular carcinoma (HCC). Here, we show that miR-199a-3p targets mammalian target of rapamycin (mTOR) and c-Met in HCC cells. Restoring attenuated levels of miR-199a-3p in HCC cells led to G1-phase cell cycle arrest, reduced invasive capability, enhanced susceptibility to hypoxia, and increased sensitivity to doxorubicin-induced apoptosis. These in vitro findings were confirmed by an analysis of human HCC tissues, which revealed an inverse correlation linking miR-199a-3p and mTOR as well as a shorter time to recurrence after HCC resection in patients with lower miR-199a-3p expression. These results suggest that tactics to regulate mTOR and c-Met by elevating levels of miR-199a-3p may have therapeutic benefits in highly lethal cancers such as HCC. Cancer Res; 70(12); 5184–93. ©2010 AACR.

Deregulated cell proliferation and apoptosis play a major role in hepatocellular carcinoma (HCC). MicroRNAs participate in the modulation of key molecules linked to hepatocarcinogenesis. Purpose: This study aims to investigate the role of miR-221 in the modulation of Bmf, a proapoptotic BH3-only protein, and to characterize miR-221 contribution to hepatocarcinogenesis through modulation of apoptosis. Experimental Design: Transfection of miR-221 and anti-miR-221 in HCC-derived cell lines and luciferase reporter assay were used to assess Bmf as a target of miR-221. Modulation of miR-221 and Bmf expression contributed to characterize their role in anoikis. Primary HCC tissues were analyzed to assess the clinical relevance of in vitro findings. Results: Enforced miR-221 expression caused Bmf down-regulation, whereas anti-miR-221 induced its up-regulation. A luciferase reporter assay confirmed Bmf as a target of miR-221. Following matrix detachment, miR-221 silencing led to increased apoptotic cell death. The analysis of HCC tissues revealed an inverse correlation between miR-221 and Bmf expression and a direct correlation between Bmf and activated caspase-3, as a marker of apoptosis. High miR-221 levels were associated with tumor multifocality and reduced time to recurrence after surgery. Conclusions: Our results indicate that miR-221, by targeting Bmf, inhibits apoptosis. Moreover, in HCC, miR-221 overexpression is associated with a more aggressive phenotype. These findings, together with the previously reported modulation of CDKN1B/p27 and CDKN1C/p57, show that miR-221 simultaneously affects multiple pro-oncogenic pathways and suggest miR-221 as a potential target for nonconventional treatment against HCC. (Clin Cancer Res 2009;15(16):5073–81)

The identification of target mRNAs is a key step for assessing the role of aberrantly expressed microRNAs in human cancer. MiR-221 is upregulated in human hepatocellular carcinoma (HCC) as well as in other malignancies. One proven target of miR-221 is CDKN1B/p27, whose downregulation affects HCC prognosis. Here, we proved that the cyclin-dependent kinase inhibitor (CDKI) CDKN1C/p57 is also a direct target of miR-221. Indeed, downregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to miR-221 transfection into HCC-derived cells and a significant upregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to antimiR-221 transfection. A direct interaction of miR-221 with a target site on the 3' UTR of CDKN1C/p57 mRNA was also demonstrated. By controlling these two CDKIs, upregulation of miR-221 can promote growth of HCC cells by increasing the number of cells in S-phase. To assess the relevance of these studies in primary tumors, matched HCC and cirrhosis samples were assayed for miR-221, for CDKN1B/p27 and CDKN1C/p57 expression. MiR-221 was upregulated in 71% of HCCs, whereas CDKN1B/p27 and CDKN1C/p57 proteins were downregulated in 77% of cases. A significant inverse correlation between miR-221 and both CDKN1B/p27 and CDKN1C/p57 was found in HCCs. In conclusion, we suggest that miR-221 has an oncogenic function in hepatocarcinogenesis by targeting CDKN1B/p27 and CDKN1C/p57, hence promoting proliferation by controlling cell-cycle inhibitors. These findings establish a basis toward the development of therapeutic strategies aimed at blocking miR-221 in HCC.