Micrornas and Chromosomal Abnormalities in Cancer Cells

GA Calin and CM Croce

Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, Ohio State University, Columbus, OH, USA

Over the past five decades, a plethora of nonrandom viral (avian) oncogene homolog activation in Burkitt’s chromosomal abnormalities have been consistently lymphoma (BL): about 80% of cases harbor the t(8:14) reported in malignant cells facilitating the identification that juxtaposes c-Myc on chromosome 8 with IgH of cancer-associated protein coding oncogenes and tumor enhancer elements on chromosome 14 with consequent suppressors. The genetic dissection of hot spots for mRNA and protein overproduction (Dalla-Favera chromosomal abnormalities in the age of the sequenced et al., 1982). In the remaining 20% of cases, the same human genome resulted in the discovery that microRNA effects are achieved by t(2;8) and t(8;22), placing c-Myc (miRNA) genes, encoding for a class of small noncoding adjacent to either kappa or lambda light chain loci and RNAs, frequently resides in such genomic regions. The enhancer elements (Croce et al., 1983). As MYC protein combination of nonrandom chromosomal abnormalities is a helix-loop-helix leucine zipper transcription factor, and other types of genetic alterations or epigenetic events consequently to its overexpression, the levels of several contribute to downregulation or overexpression of miR- dozens of genes involved in the cell cycle regulation, NAs. The consequent abnormal expression of miRNAs apoptosis, and differentiation of germinal center B cells affect cell cycle, survival and differentiation programs and are altered (reviewed in Sanchez-Beato et al. (2003)). selective targeting of these noncoding genes could provide Furthermore, the genomes of cancer cells contain novel therapeutic options for killing the malignant cells. abnormalities undetectable by any type of cytogenetic Oncogene (2006) 25, 6202–6210. doi:10.1038/sj.onc.1209910 investigation such as deletions and amplifications, sometimes in the range of several tens of kilobases, Keywords: microRNAs;chromosomal aberrations; defined experimentally by minimal regions of loss of cancer heterozygosity (LOH), and minimal amplicons. In spite of careful genomic dissection, some chromosomal abnormalities had unknown targets and the mechanisms by which these changes affect certain specific disorders remained unraveled for long time. For example, over- For several chromosomal abnormalities no culprit protein expression of BCL2 protein was reported in many types coding gene(s) was identified of human cancers including leukemias, lymphomas and carcinomas (Sanchez-Beato et al., 2003). In follicular Over the past five decades, a plethora of nonrandom lymphomas and in a fraction of diffuse B-cell lympho- chromosomal abnormalities have been consistently mas, the mechanism of B-cell leukemia/lymphoma reported in malignant cells of hematopoietic and solid 2 gene (Bcl-2) activation was found to be the transloca- tumors (Nowell and Hungerford, 1960;Rowley, 1973; tion t(14;18) (q32;q21), which places Bcl-2 gene under Croce and Nowell, 1985) facilitating the identification of the control of immunoglobulin heavy chain enhancers, cancer-associated protein coding oncogenes (OGs) and resulting in deregulated expression of the gene tumor suppressor gene(s) (TSG(s)) (Russo et al., 1988; (Tsujimoto et al., 1984, 1985). In the most frequent Bishop, 1991;Rabbits, 2001). Numerical and structural adult leukemia in the Western world, the B-cell chronic chromosomal aberrations have been systematically lymphocytic leukemia (CLL) (Chiorazzi et al., 2005), the reported in the Mitelman Database of Chromosome Aberrations in Cancer (for more information, see http:// malignant, mostly nondividing, B cells of CLL over- cgap.nci.nih.gov/Chromosomes/Mittelman). The data- express BCL2 protein (Kitada et al., 1998). However, base passed the mark of 50 000 cases in the July update, with the exception of less than 5% of cases in which Bcl-2 and reports for new abnormalities are accumulating at is juxtaposed to immunoglobulin loci (Adachi et al., high rate with the advance of cytogenetics techniques 1990), no mechanism was discovered to explain BCL2 such as spectral karyotype analysis. One classical deregulation in CLL. paradigm is represented by the c-Myc myelocytomatosis The answer to this unsolved puzzle came from a decade long study of the most frequent deleted genomic region in CLL at chromosomal band 13q14.3, that is Correspondence: Professor CM Croce at Ohio State University, Comprehensive Cancer Center, Wiseman Hall Room 385K, 400 12th easily detected by cytogenetics and is associated with the avenue, Columbus Ohio, OH 43210, USA. longest treatment-free interval (Dohner et al., 2000). E-mail: [email protected]. Hemizygous and/or homozygous loss at 13q14.3 occur Chromosomal abnormalities and miRNAs GA Calin and CM Croce 6203 in more than half of cases and constitute the most have been described in CLL (Figure 1). Until year 2001, frequent chromosomal abnormality in CLL (Bullrich a total of eleven genes have been identified and screened et al., 2001). 13q14.3 deletions also occur in B50% of for alterations at the DNA and/or RNA level in mantle cell lymphoma, in up to 40% of multiple sporadic and familial cases of CLL. However, detailed myeloma, and in 60% of prostate cancers (Dong et al., genetic analysis, including extensive LOH, mutation and 2001), suggesting that one or more TSG(s) at 13q14.3 expression studies, failed to demonstrate the consistent may be involved in the pathogenesis of these human involvement of any of the genes located in the deleted tumors. CLL have relatively few consistent chromo- region (described in Calin et al. (2005c) and Migliazza somal abnormalities, suggesting that because of the et al. (2001)). specificity and frequency of observed deletions at In order to decipher the nature of elusive TSG(s) at 13q14.3 they are of pathologic significance. Several 13q14.3, in 2001 we generated somatic cell hybrids groups have used positional cloning to identify the gene between mouse LMTK-(thymidine kinase deficient) and or genes targeted by the deletions. A region of more human CLL cells carrying 13q14.3 translocations and/ than 1 Mb has been fully sequenced and charac- or deletions and, using these hybrids, we identified a terized in detail (Bullrich et al., 2001). One of the 30-kb region of deletion between exons 2 and 5 of LEU2 difficulties first encountered during analysis of this locus gene (Calin et al., 2002). The same region was involved was the lack of a clear definition of the minimal region in translocations observed in CLL patients (Calin et al., of loss. In fact, various and relatively large (between 2002). We continued to search for genes within the 130 and 550 kb) minimal regions of LOH at 13q14.3 region using publicly available sequence information and databases. This resulted in the discovery that a cluster of two noncoding microRNA (miRNA) genes, miR-15a and miR-16-1, is located precisely in the deleted region and is involved in the analysed translocation (Figure 1) (Calin et al., 2002). Further detailed investigation showed that both genes are deleted or downregulated in approximately 68% of CLL cases as compared with CD5 þ lymphocytes from healthy donors (Calin et al., 2002). Furthermore, a rare mutation identified in two CLL patients including one from a family with individuals having CLL and breast cancer, and possibly affecting the expression of these genes was found to be associated with the loss of the normal allele in the leukemic cells (Calin et al., 2005a). Fully explaining the tumor-suppressor function of miR-15 and miR-16, it was proved that the levels of Figure 1 The 13q14.3 deletions in CLL cells target the miR15a/ both genes inversely correlates with the BCL2 protein miR-16-1 cluster. The upper panel shows several minimal regions of deletion, the one identified by cell hybrids experiments and expression, and that BCL2 repression by miR-15a containing the cluster of miR-15a/miR-16-1 being right-dashed. and miR-16-1 induces apoptopsis in leukemia cells The genomic markers as well as the gene orientation are indicated. (Cimmino et al., 2005). The lower panel shows the genes located inside or very close to the deleted region (the gene symbols are as in NCBI at http:// www.ncbi.nlm.nih.gov/entrez). Of note, all the genes are located in the centromeric half of the analysed genomic region, while no gene miRNAs are located at cancer-associated genomic regions in the large genomic desert of the telomeric 750 kb was found. This region is rich in noncoding RNAs (ncRNAs) because four RNAs The genetic dissection of hot spots for chromosomal without correspondent protein have been found. DLEU1 and abnormalities in the age of the sequenced human DLEU2 are members of the large ncRNAs subfamily (with transcripts of about 0.6–6 and 14–18 kb, respectively), while genome showed that miRNAs, a small noncoding miR-15a/miR-16-1 are members of the small ncRNAs subfamily RNA class of genes, frequently resides in such genomic of miRNAs (with precursors of about 70 nc). The genes in blue are regions (Calin et al., 2004b). Ambros group (Lee et al., protein coding, while the red ones are noncoding, meaning 1993) first described miRNAs in 1993 in Caenorhabditis coding for RNAs without an open reading frame (ORF). For the identification of the minimal region of deletion, somatic cell elegans. At present days, over 3500 members of this new hybrids were generated following conventional methods and class of small noncoding RNAs (ncRNAs), have been selected in hypoxanthine–aminopterin–thymidine. We generated identified in the last four years in vertebrates, flies, 15 fusion clones isolated from fusion of a CLL case (CLL-B) worms and plants, and also in viruses (Ambros, 2003; carrying a t(2;13) (q32; q14) translocation and one clone was Bartel, 2004;Griffiths-Jones et al., 2006). The miRNA isolated from fusion of another CLL case (CLL-A) carrying a t(2;13) (q12; q13) translocation. Twelve CLL-B derived clones functions are ubiquitous, raging from the control of leaf carried a full complement of both chromosomes 13 and 2, whereas and flower development in plants to the modulation of three carried the derivative chromosome 13 and a full complement hematopoietic lineage differentiation in mammals (Chen of chromosome 2. The single clone from CLL-A carried a et al., 2004). Several groups have uncovered roles for chromosome 13 with a small deletion at 13q14.3 and no part of chromosome 2. (Modified from Calin and Croce, 2006a and miRNAs in the coordination of cell proliferation and Pekarsky et al., 2005, with kind permission of Springer Science and cell death during development, and in stress resistance Business Media). and fat metabolism (for reviews see Ambros (2003);

Oncogene Chromosomal abnormalities and miRNAs GA Calin and CM Croce 6204 Bartel (2004) and Harfe (2005)). Functionally, it was chromosome 17 and c-Myc, meaning that the regulatory shown that miRNAs reduce the levels of many of their elements of this miRNA are likely involved in the target transcripts as well as the amount of protein overexpression of MYC (Calin et al., 2004b). The encoded by these transcripts (Lim et al., 2005). clinical consequences were dramatic for the patient, In order to check if the genetic association between leading to an aggressive acute prolymphocytic leukemia miR-15a and miR-16-1 with a nonrandom cytogenetic that presumably derived from precursors of an indolent abnormality occurring in a region that frequently lymphoma carrying a rearranged Bcl-2 gene (Gauwerky contains a homozygous deletion (HD) is simply by et al., 1989). Apart from the involvement in the t(8;17) chance, we decided to investigate at a genome-wide level breakpoint in acute leukemia, miR-142-3p and miR-142- the association between various cytogenetic and mole- 5p are also within the 17q23 minimal amplicon described cular abnormalities and the location of miRNA genes. in breast cancer (Barlund et al., 2000) and near the As in 2003 few human miRNA genes were reported, FRA17B site, a target for HPV16 integration in cervical we initiated the bioinformatics study by cloning ‘in tumors (Calin et al., 2004b). silico’ all the human homologous miRNAs correspond- Other examples of miRNA genes located near break- ing to the genes discovered at that time in various point regions include miR-180 at 1 kb from the MN1 species (mainly mouse, Drosophila and Caenorhabditis) gene (meningioma (disrupted in balanced translocation) 1) and obtained a database of 186 human miRNAs. As the involved in a t(4;22) in meningioma that inactivates second step, we generated several databases containing MN1 gene and possibly the miRNA gene located in the the exact genomic positions of markers used for cloning same position. Also, in a patient with precursor B-cell cancer-associated genomic regions (CAGR), including acute lymphoblastic leukemia an insertion of miR-125b-1 157 minimal regions of loss-of-heterozygosity (LOH), into a rearranged immunoglobulin heavy chain locus suggestive of the presence of TSG(s), 37 minimal regions was described, possibly an early step in leukemogesis of amplification, suggestive of the presence of OG(s), 45 (Sonoki et al., 2005). Arguing for the importance of common breakpoint regions in or near possible OG(s) miRNA aberrations by chromosomal rearrangements, or TSG(s) and 29 fragile sites (FRA). A substantial come the identification of a similar mechanism in body of evidence supports the proposal that at least the development of a tumor of another species, the some common chromosomal FRA predispose to DNA woodchuck hepatocellular carcinoma. Sequence com- instability in cancer cells (Richards, 2001;Arlt et al., parisons showed the woodchuck homolog of human 2003). Indeed, FRA are preferential sites of sister miR-122a is in sense orientation with the ORF of hcr chromatid exchange, translocation, deletion, amplifica- gene from woodchuck, a noncoding gene involved in a tion or integration of plasmid DNA and tumor- rearrangement with c-Myc in a woodchuck hepato- associated viruses such as human papilloma virus cellular carcinoma (Etiemble et al., 1989). Further sup- (HPV). The results were surprising and suggested a porting the suggested role of miR-122a in cancer, we larger than thought involvement of miRNAs in cancer. found that the human miR-122a is located in the Overall, we found that more than half of miRNA genes minimal amplicon around MALT1 in aggressive mar- were located in CAGRs, including 65 miRNAs located ginal zone lymphoma (MZL) and about 160 kb from the exactly in minimal regions of LOH, and 15 miRNAs in breakpoint region of translocation t(11;18) in mucosa- minimal regions of amplification, described in a variety associated lymphoid tissue (MALT) lymphoma (Calin of tumors, including lung, breast, ovarian, colon, gastric et al., 2004b). and hepatocellular carcinoma as well as leukemias and Various regions frequently altered in cancer, but lymphomas. In addition, looking at 113 FRA scattered without clear culprit genes, were shown to contain in human karyotype, we found that 61 miRNAs are clusters of miRNAs. For example, in breast carcinomas located in the same cytogenetic positions with FRA two different regions of loss at 11q23, independent from (Figure 2) (Calin et al., 2004b). ataxia telangiectasia-mutated (ATM) locus have been The first report linking a chromosomal breakpoint studied extensively: the first spans about 2 Mb between with the genomic location of miRNAs was done by loci D11S1347–D11S927, the second is located between Croce group a couple of decades ago (Gauwerky et al., loci D11S1345–D11S1316 and is estimated at about 1989). By analyzing the oncogene rearrangements 1Mb (di Iasio et al., 1999). Candidate TSG(s) were not involving c-Myc in leukemia cells, a masked t(8;17) found in spite of extensive effort, except the PPP2R1B translocation that resulted in a high activation of the (protein phosphatase 2, regulatory subunit A, beta oncogene was identified. The sequence analysis revealed isoform) gene, involved in o10% of cases (Wang et al., that c-Myc from chromosome 8 was truncated at the 1998;Calin et al., 2002). Both minimal LOH regions end of first exon (which is noncoding) and the coding contain numerous miRNAs: the cluster miR-34b/ region joined the regulatory elements of a gene located miR-34c in the first and the cluster miR-125b-1/let-7a-2/ on chromosome 17, called BCL3 (B-cell leukemia/ miR-100 in the second. High frequency of LOH at lymphoma 3) and transcribed as a 1.7 kb message in a 17p13.3 and relatively low frequency of TP53 mutation variety of hematopoietic cell lines. In spite of extensive in cases of hepatocellular carcinomas (HCC), lung genomic search, BCL3 remained an elusive entity until cancers and astrocytomas indicate the presence of other the identification of the human miRNAs. After 15 years TSGs involved in the development of these tumors. from the initial discovery, it was found that miR-142 We have found that one minimal LOH region described gene is located 50 nt from the t(8;17) break involving in HCC and located telomeric to TP53 (tumor protein

Oncogene Chromosomal abnormalities and miRNAs GA Calin and CM Croce 6205 miRs located at Fragile sites A miRs located in other genomic regions B C C L D A D A D B E E A A A B E C J B F F B K B D D G G G F K H E F I D C C C I J G E

B B C A C C D B I D A C J G E F F C H E D F A D A G B F H E A B D C EB G A I E D F EF

A A A A B E B C C B A D D BC B EC D

B A B B A

Figure 2 Correlation between FRA and miRNAs. A karyotype showing the position of 113 FRA and 186 miRNAs is presented. The 61 miRNA genes located in the same chromosomal band as the FRA are marked with red. It is very probable that this is an underestimation, because the mapping of the unstable FRA regions is not complete and the identification of miRNAs is still an open field. Red arrow shows frequently observed FRA (reproduced from Calin et al, 2004b). p53) gene between markers D17S1866 and D17S1574 (Zabarovsky et al., 2002). All these data taken together (Zhao et al., 2003) harbor three miRNAs: miR-22, strongly support the fact that the association between miR-132 and miR-212, while between ENO3 and TP53 miRNAs and chromosomal abnormalities in cancer cells (Tsuchiya et al., 2000) we found miR-195 (Calin et al., is not casual, leading to the possibility that more 2004b). examples will be identified in the near future. Homozygous deletions in cancer suggest the presence of TSGs (Huebner et al., 1998) and several miRNA genes are located in homozygously deleted regions Chromosomal abnormalities as a cause of disturbed without known culprit. In addition to miR-15a and miRNAs expression miR-16a located at 13q14.3 HD region in B-CLL, the cluster miR-99a/let-7c/miR-125b-2 mapped in a 21p11.1 Several arguments suggest that the highly significant region of HD in lung cancers and miR-32 at 9q31.2 in a association between the location of miRNAs and region of HD in various types of cancer. Among the chromosomal or molecular genomic aberrations is not seven regions of LOH and HD on the short arm of without consequences. First, this association is specific chromosome 3 three of them harbor miRNAs: miR-26a for the miRNA class of genes, as is not true for several in region AP20, miR-138-1 in region 5 at 3p21.3 and the other classes of genes (e.g., the RNA splicing family of cluster let-7g/miR-135-1 in region 3 at 3p21.1–p21.2 genes) (Table 1). Second, considering the additional

Oncogene Chromosomal abnormalities and miRNAs GA Calin and CM Croce 6206 Table 1 miRNAs are located at cancer-associated genomic regionsa Region of interest microRNAs (186 genes) RNA splicing genes (80 genes)

IRR 95% CI IRR P IRR 95% CI IRR P

Fragile sites vs nonfragile sites 9.12 6.22, 13.38 o0.001 1.49 0.60, 3.77 0.39 Ampliﬁed region vs nonampliﬁed 3.97 2.31, 6.83 o0.001 —b —b —b LOH vs non-LOH regions 4.08 2.99, 5.56 o0.001 0.63 0.02, 21.25 0.80

Abbreviations: FRA, fragile sites;miRNAs, microRNAs. aTo test hypotheses related to the incidence of miRNAs and RNA splicing genes and association with FRA, ampliﬁed regions in cancer, and deleted regions in cancer, we utilized random effect Poisson regression models as described in Calin et al. (2004b). bToo few events, likelihood-based models are numerically unstable. Bold represents statistically signiﬁcant P-values.

several hundreds of cloned miRNAs carefully recorded Brenton (2005);Chen (2005);Croce and Calin (2005); at miRBase (http://microrna.sanger.ac.uk/cgi-bin/ Gregory and Shiekhattar (2005);Calin et al. (2005b); sequences/) (Griffiths-Jones et al., 2006) or predicted Esquela-Kerscher and Slack (2006);Hammond (2006); miRNAs as extensively published in the last years Hwang and Mendell (2006) and Calin and Croce (Berezikov et al., 2005;Xie et al., 2005), the association (2006b)). The combination of nonrandom chromosomal remain highly significant. The meaning of this finding is abnormalities and other types of genetic or epigenetic that this is not a peculiarity of the first set of cloned events could contribute to downregulation or over- miRNAs, that are also the most extensively and highly expression of miRNAs (Figure 3). The dimensions of expressed in human cells (GAC, JP Hagan and CMC, miRNAs are quite small in respect with the cDNAs of unpublished data). Third, it was shown by Northern protein coding genes, and therefore the mutations in blottings, that the genomic location influence the miRNA transcripts should be rare events. A combina- miRNA expression: miR-16a expression (located at tion of LOH þ mutation was reported as inactivating 13q14.3) was low or absent in the majority of B-CLL ‘two-hit’ events in two cases of CLL (Calin et al., 2005a), cases while the expression of miR-26a (at 3p21) and while a naturally occurring sequence polymorphism in miR-99a (at 21q11.2), both regions not involved in an miRNA precursor that results in reduced processing B-CLL, was at relatively equal levels in all cases. By and lower levels of mature miRNA expression and contrast, both miR-26a and miR-99a are not expressed function has been reported (Gottwein et al., 2006). An or are expressed at low levels in lung cancer cell lines, independent study on human cancer cell lines showed and this correlate with their location in regions of LOH/ that heterozygous genetic variants in miRNA precursors HD in lung tumors/cell lines. On the other hand, the are common, but are unlikely to have physiologic expression of miR-16a was the same in the majority of significance (Diederichs and Haber, 2006). Further cell lines as in normal lung (Calin et al., 2004b). Fourth, expanding the ways in which miRNA expression could an extensive study of high-resolution array-based be altered, it was proved that DNA demethylation and comparative genomic hybridization on 227 human histone deacetylase inhibition can activate expression ovarian cancer, breast cancer and melanoma specimens, of miR-127 that may act as a tumor suppressor by clearly proved that regions hosting miRNAs exhibit targeting the BCL6 (B-cell leukemia/lymphoma 6) high-frequency genomic alterations in human cancer. proto-oncogene (Saito et al., 2006). Strengthening the importance of these findings is the Up to date, two large profiling studies using tumors of fact that miRNA copy changes correlate with miRNA various histotypes were published (Lu et al., 2005; expression (Zhang et al., 2006). The analyses of the same Volinia et al., 2006). Lu et al. (2005) using a bead-based histotype, breast ductal carcinomas, performed by two flow-cytometric profiling technology performed on a set independent groups using distinct techniques, revealed of 334 including multiple human cancers mainly overlapping sets of miRNAs that are differentially leukemias, found that miRNA expression profiles expressed (Iorio et al., 2005) and miRNAs that are classify human cancers reflecting the developmental located in genomic regions with DNA copy number lineage and differentiation state of the tumors. Of gains or losses (Zhang et al., 2006) compared to normal important diagnostic consequences, the miRNA-based breast tissues. As shown in Table 2, the majority of classifier is far better in establishing the correct diagnosis miRNAs causally linked to human tumorigenesis are of poorly differentiated samples than the protein cod- located in genomic regions altered in cancer and the ing genes messenger RNA classifier (Po5 Â 10–11 vs genomic abnormality is concordant with the expression P ¼ 0.47). Volinia et al. (2006) described a detailed deregulation (genomic deletion for downregulation and analysis by microarray in 540 samples from six solid amplification for upregulation, respectively). types of the most frequent human cancers and found a If the location of miRNAs is relevant to tumorigene- common signature composed by 61 miRNAs, mainly sis, then structural or functional alterations of miRNAs overexpressed in respect with correspondent normal should be identified in various types of cancers. A tissues. Supporting the roles of these genes in tumo- growing number of reports are providing such evidence rigenesis, it was found that the predicted targets and suggest that abnormal expression of miRNAs is for the differentially expressed miRNAs are signifi- central to cancer pathogeny (reviewed in McManus cantly enriched for protein-coding TSG(s) and OG(s) (2003);Berezikov and Plasterk (2005);Caldas and (Po0.0001).

Oncogene Table 2 Oncogenic and suppressor miRNAs are targeted by chromosomal rearrangements Putative function MiRNA Location Chromosomal Expression in cancer Molecular consequencesa Suggestive References rearrangements

Suppressors miR-16-1/15a cluster 13q14.3, intron 4 of Deletion of 13q14.3 band or Downregulated in the Exogenous restoration in leu- Calin et al. (2002); DLEU2 LOH in hematopoietic and majority of B-CLLs and in kemia cells induces apoptosis Eis et al. (2005); solid cancers the majority of DLBCLs by directly reducing levels of Cimmino et al. (2005) antiapoptotic BCL2 miR-127 14q32.31 LOH in solid cancers Reduced expression in Translationally downregulation Saito et al. (2006) prostate and bladder cancers of the transcriptional repressor BCL6 miR-145/143 cluster 5q32, intergenic Deletion of 5q32 band Reduced expression in colon Unknown Michael et al. (2003); or LOH in MDS adenomas and carcinomas Iorio et al. (2005) (5q-syndrome) and in breast cancers let-7 family Various LOH in lung cancers Reduced expression in lung let-7 regulates RAS oncogene Takamizawa et al. (2004); expression in lung tumors Johnson et al. (2005); Yanaihara et al. (2006) miR-17/92 cluster 13q31.3, intron 3 Negative regulatory feed-back O’Donnell et al. (2005) C13orf25 loop c-Myc – miR-17-5p-miR- 20a – E2F1 AClnadC Croce CM and Calin GA miRNAs and abnormalities Chromosomal Oncogenic miR-17/92 cluster 13q31.3, intron 3 AMPLIF in follicular Overexpressed in malignant The miRNAs cluster, but not Ota et al. (2004);Hayashita C13orf25 lymphomas lymphomas and lung cancers the host C13orf25 gene, en- et al. (2005);Tagawa and hances cell proliferation Seto (2005) miR-21 7q23.2, 3’UTR AMPLIF in neuroblastoma Elevated levels in Increased apoptotic cell death Chan et al. (2005);Iorio VMP1 and breast, colon, lung glioblastomas, breast, colon, after knockdown in et al. (2005);Meng et al. cancers lung, pancreas, prostate, glioblastoma cells;miR-21 (2006);Volinia et al. (2006) stomach cancers and modulated gemcitabine induced cholangiocarcinomas apoptosis by PTEN miR-155 21q21.3, exon 3 of Not reported High expression of precursor Unknown Metzler et al. (2004);Eis ncRNA BIC or active form in pediatric et al. (2005);Iorio et al. BL, in Hodgkin, primary (2005);Kluiver et al. (2005); mediastinal and DLBCL Tam and Dahlberg (2006); lymphomas;overexpressed in Volinia et al. (2006); breast, colon and lung cancers Yanaihara et al. (2006) miR-372, miR-373 19q13.42, intergenic Not reported High expression in testicular Proliferation of p53 wild-type Voorhoeve et al. (2006) germ cell tumors cancer cells sensible to DNA-damaging agents

Abbreviations: B-CLL, B-cell chronic lymphocytic leukemia;BIC, noncoding RNA gene;BL, Burkitt lymphoma;DLBCL, diffuse large B-cell lymphoma;D LEU2, noncoding RNA gene;MDS, myelodysplastic syndrome;miRNAs, microRNAs;VMP1, vacuole membrane protein 1. aMolecular consequences with proven cancer relevance, as reported by the references in the right column, were presented. Oncogene 6207 Chromosomal abnormalities and miRNAs GA Calin and CM Croce 6208

Figure 3 Consequences of the cooperation between chromosomal and molecular alterations affecting miRNA loci in human cancers. Some of the well-characterized molecular models for miRNA involvement in cancers are presented. The effects of deletions (del), mutations (mut), promoter hypermethylation (hypermethyl), amplification (amplif) or translocations (t) are presented. The diagrams are simplified and did not account for all the molecular complexity, as combinatorial effects of multiple miRNAs acting on the same target or multiple targets influenced by the same miRNA were proposed. Furthermore, only the main functional consequences were presented. The promoter regions are presented as orange triangles and the structural miRNA genes as blue circles. C-Myc gene is presented as a blue rectangle.

After the identification of specific miRNA signatures programs, such as cell cycle and survival or differentia- in CLL (Calin et al., 2004a, 2005a), miRNAs differen- tion programs. A growing list of examples of cause– tially expressed between tumors and normal counter- effect relations explaining the intimate involvement of parts were identified for several tumor types like miRNA in cancer is now available (Table 2). Therefore, lymphoma (He et al., 2005b), breast cancer (Iorio miRNAs might be potential targets for therapy, as et al., 2005), lung cancer (Yanaihara et al., 2006), knocking down overexpressed miRNAs or expressing thyroid papillary carcinoma (He et al., 2005a), glioblas- silenced miRNAs in cancer cells might contribute to toma (Ciafre et al., 2005), hepatocellular carcinoma tumor killing. While the pathogenetic mechanisms and (Murakami et al., 2006), colorectal carcinoma the therapeutic consequences of deregulation of various (Cummins et al., 2006) and pancreatic tumors (Roldo miRNAs (such as let-7 family, the cluster miR-17-5p/ et al., 2006). miR-92 or the cluster miR-15a/miR-16-1) were subject to It is logical to question why this association between extensive reviews (see Morris and McManus (2005); CAGR and miRNAs is of significant pathogenetic Czech (2006);Slack and Weidhans (2006) and Calin and importance for tumorigenesis. The answer could be that Croce (2006a, b), we will briefly focus only on two very genomic aberrations in primary tumors preferentially recent findings. involve the genomic loci where miRNA resides (Calin The list of oncogenic miRNAs includes miR-21, the et al., 2004b;Zhang et al., 2006). The consequence is cluster miR-17-5p-miR-92, miR-155 and the cluster miR- that the clones having genomic aberrations involving 372/miR-373 (Table 2). MiR-21 is located in 30UTR of miRNA loci have biological advantages consequent to VMP1 (vacuole membrane protein 1) gene at chromo- disturbed miRNAs expression. some 17q23.2, a region amplified in neuroblastomas and in breast, colon and lung cancers. Consequently, miR-21 was found as the only miRNA overexpressed in all six Abnormal miRNAs expression in cancer can be types of solid cancers (breast, colon, lung, prostate and therapeutically exploited stomach carcinomas and pancreas exocrine tumors) analysed in the study by Volinia et al. (2006), as well as If the abnormalities of genomic loci containing miRNAs in glioblastomas (Chan et al., 2005;Ciafre et al., 2005). are causal for human cancer, then the abnormal Knockdown of miR-21 in cultured glioblastoma cells expression of these genes should affect fundamental cell triggers apoptosis by a caspase-dependent mechanism

Oncogene Chromosomal abnormalities and miRNAs GA Calin and CM Croce 6209 (Chan et al., 2005). Studies in cholangiocarcinoma antiapoptotic factors such as BCL6 and consequent cells proved that one direct target of miR-21 is the apoptosis. TSG gene PTEN (phosphatase and tensin homolog (mutated in multiple advanced cancers 1)), that encodes a phosphatase that normally inhibits the survival/ Concluding remarks growth promoting activity of phosphoinositole 3-kinase (PI-3 kinase) signaling (Cully et al., 2006). It was proved Loss or amplification of miRNA genes by gross that miR-21 modulates gemcitabine-induced apoptosis cytogenetic abnormalities or minute molecular aberra- by PTEN-dependent activation of PI 3-kinase and tions has been observed in a variety of human activation of AKT/mTOR signaling (Meng et al., malignancies and more than half of the miRNoma was 2006). Together these two findings suggest that miR-21 proved to be located in such regions. Several genomic is an antiapoptotic and prosurvival factor and therefore, regions in which the hunting for culprits failed, have the therapeutically potential is obvious: inhibition of been shown to harbor clusters of miRNA genes. The this miRNA should result in cell death. As both consequence of such aberrations is altered expression of cholangiocarcinomas and glioblastomas are highly these small noncoding regulatory genes that influence aggressive tumors with a very bad prognosis, identifica- the levels of various protein coding genes that are key tion of potential new therapeutic agents is of high players in the survival, proliferation and differentiation priority. programs of both hematopoietic and solid tissue cells. The list of tumor-suppressors miRNAs includes the Thus, miRNAs might be potential targets for therapy, cluster miR-15a/miR-16-1, the cluster miR-145/miR- as knocking down overexpressed miRNAs or expressing 143, let-7 family and miR-127. MiR-127 is located on silenced miRNAs in cancer cells might contribute to chromosome 14q32.31, in a region involved in several tumor killing. types of translocations identified in hematological cancers and deleted by LOH in solid cancers. Conse- quently, its expression is reduced or absent in various Abbreviations types of cancers (Saito et al., 2006). While the treatment of cancer cells with the DNA demethylating Bcl-2, B-cell leukemia/lymphoma 2 gene;CAGR, cancer- agent 5-aza-20-deoxycytidine (5-Aza-CdR) has a small associated genomic regions;CLL, B-cell chronic lymphocitic influence on miRNA expression, the combined treat- leukemia;c-Myc, v-myc myelocytomatosis viral oncogene ment 5-Aza-CdR and the histone deacetylase (HDAC) homolog (avian) gene;HD, homozygous deletion;FRA, inhibitor 4-phenylbutyric acid (PBA) has significant fragile sites;LOH, loss of heterozygosity;miRNAs,micro- effects. The most differentially expressed miRNA after RNAs;ncRNAs, noncoding RNAs;OG(s), oncogene(s); the combined 5-Aza-CdR þ PBA administration in TSG(s), tumor-suppressor gene(s). human bladder cancer cells was miR-127. Further investigations showed that miR-127 can downregulate Acknowledgements the oncogene BCL6 by translational repression (Saito et al., 2006). BCL6 is a zinc-finger transcription We thank Terry Hyslop (Thomas Jefferson University, Philadelphia) for the statistical analyses presented here. repressor that modulates DNA damage-induced apop- Dr Croce is supported by Program Project Grants from the tosis in germinal center B cells (Phan and Dalla- National Cancer Institute and Dr Calin by a Kimmel Favera, 2004). Therefore, induction of miR-127 by Foundation Scholar award and by the CLL Global Research 5-Aza-CdR and PBA treatment in cancer cells might Foundation. We apologize to many colleagues whose work have an anticancer effect by downregulation of was not cited due to space limitations.

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