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alterations, such as SNPs, in cis-regulatory ele-ments can alter target gene regulation by either creating or destroying miRNA target sites9. Indeed, SNPs occurring at cis-regulatory ele-ments have been shown to either enhance or inhibit the recognition of a miRNA, causing aberrant gene expression and disease pheno-types10,11. 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 compo-nent to Crohn’s disease, as an individual’s risk is directly associated with the amount of first- and second-degree relatives with the disease. Genetic mapping in people with this disorder has uncovered numerous mutations—in par-ticular, a silent mutation in IRGM (encoding immunity-related GTPase family, M), a gene required for autophagy of intracellular bac-teria that is highly prevalent in individuals of European descent12. Brest et al.2 found that the protective IRGM variant, but not the risk-associated 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 vari-ant 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 accumu-lation of intracellular bacteria and disease pro-gression.

The observation from the study of Brest et al.2 provides direct evidence that a silent muta-tion can, in fact, have a function in disease, and it can do so by creating an aberrant cis-regu-latory 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 specifi-cally expressed in the inflamed mucosa to tar-get 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 impor-tance in human disease.

COMPETING FINANCIAL INTERESTSThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturemedicine/.

1. Easton, D.F. et al. Am. J. Hum. Genet. 81, 873–883 (2007).

2. Brest, P. et al. Nat. Genet. 43, 242–245 (2011).3. Pastinen, T. & Hudson, T.J. Science 306, 647–650

(2004).4. Pastinen, T. et al. Hum. Mol. Genet. 15 Spec No 1,

R9–R16 (2006).5. Bartel, D.P. Cell 116, 281–297 (2004).6. Esquela-Kerscher, A. & Slack, F.J. Nat. Rev. Cancer 6,

259–269 (2006).7. van Rooij, E. et al. Science 316, 575–579 (2007).8. Bartel, D.P. & Chen, C.Z. Nat. Rev. Genet. 5, 396–400

(2004).9. Saunders, M.A. et al. Proc. Natl. Acad. Sci. USA 104,

3300–3305 (2007).10. Abelson, J.F. et al. Science 310, 317–320 (2005).11. Chin, L.J. et al. Cancer Res. 68, 8535–8540

(2008).12. McCarroll, S.A. et al. Nat. Genet. 40, 1107–1112

(2008).13. Chen, C. et al. J. Cell. Mol. Med. 15, 14–23 (2011).

■ BeNCh To BeDSIDe

Understanding cancer gene dependencyCarlo M Croce

Carlo M. Croce is in the Department of Molecular

Virology, Immunology and Medical Genetics,

The Ohio State University School of Medicine,

Columbus, Ohio, USA.

e-mail: carlo.croce@osumc.edu

Most human cancers are driven by somatic genetic alterations, which often involve onco-genes and tumor suppressor genes1. For exam-ple, chromosome translocations involving the MYC oncogene on chromosome 8 and one of three immunoglobulin loci dysregulate the MYC oncogene, leading to the development of Burkitt’s lymphoma1. The BCR-ABL fusion

gene resulting from the Philadelphia chromo-some causes elevation of the activity of the Abl tyrosine kinase and is responsible for chronic myelogenous leukemia (CML)1. Because such driver gene alterations are essential for tumor cell growth, survival or both, their reversion can lead to selective death of cancer cells1.

Specific drugs, such as imatinib in the case of CML, can target the consequences of such genetic alterations2. Treatment with imatinib leads to complete remission in more than 95% of patients with CML in the chronic phase with few side effects2, indicating that targeting the activated Abl tyrosine kinase can selectively

result in the death of cancer cells with consid-erable benefit for the patients. Given that the malignancy is caused by the expression of the BCR-ABL fusion gene, the results suggest that reverting cancer ‘drivers’ can lead to tumor regression and possibly cure.

The expression of additional cancer genes can be altered during tumor progression and can contribute to tumor growth and spreading; therefore, targeting these additional alterations may have some effect. But such alterations may not be present in every cell within a tumor. Another possibility is that cooperation of can-cer genes may be required for or may speed up

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CML with imatinib, and those obtained in the context of a malignancy driven (initiated) by miRNA dysregulation, such as the case of miR-21, clearly indicate that targeting the initiators of malignant transformation should be a priority (Fig. 1). Targeting other alterations occurring during the progression of malignancy might not be as useful.

Unfortunately, for most of the pharmaceuti-cal industry a cancer target is a cancer target, independently of its role in tumor develop-ment. It is doubtful that targeting players that are involved in one of the multiple steps of tumor progression will have the same efficacy as targeting early events, possibly the initiation events, leading to cancer.

Although for several malignancies, such as CML, follicular lymphoma and CLL, the initial drivers have been identified, for many of the most common solid tumors, this may not be the case. Thus, a lot of work should be done to try to identify the earliest driver of such malignancies, providing better targets for the development of effective targeted therapies.COMPETING FINANCIAL INTERESTSThe author declares no competing financial interests.

1. Croce, C.M. N. Engl. J. Med. 358, 502–511 (2008).2. Gambacorti-Passerini, C. et al. J. Natl. Cancer Inst.

103, 553–561 (2011).3. Calin, G.A. et al. Proc. Natl. Acad. Sci. USA 99,

15524–15529 (2002).4. Calin, G.A. et al. Proc. Natl. Acad. Sci. USA 101,

2999–3004 (2004).5. Johnson, S.M. et al. Cell 120, 635–647 (2005).6. Cimmino, A. et al. Proc. Natl. Acad. Sci. USA 102,

13944–13949 (2005).7. Calin, G.A. & Croce, C.M. Nat. Rev. Cancer 6, 857–866

(2006).8. Costinean, S. et al. Proc. Natl. Acad. Sci. USA 103,

7024–7029 (2006).9. Cialfrè, S.A. et al. Biochem. Biophys. Res. Commun.

334, 1351–1358 (2005).10. Volinia, S. et al. Proc. Natl. Acad. Sci. USA 103, 2257–

2261 (2006).11. Medina, P.P., Nolde, M. & Slack, F.J. Nature 467,

86–90 (2010).

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tumorigenicity in some tumors. In this case, targeting several of them may be beneficial. Yet, as most tumors initiate from one cell (monoclo-nal)1, it is quite likely that the initial alteration, which is present in 100% of the cells of a given tumor, is the crucial target. During tumor pro-gression, however, numerous genes can become mutated, making the tumor less dependent on the initial genetic change. For example, CML can progress to an acute leukemia that is more resistant to imatinib treatment2.

As recently as the start of this decade, all cancer-associated genes, oncogenes and tumor suppressors were thought to be protein-coding genes. Yet, in 2002, a study showed that altera-tions in miRNA genes, which regulate gene expression by targeting mRNA and blocking its translation, causing its degradation or both, could also lead to malignancy3. In fact, the loss of miR-15a and miR-16-1 was found as the most common alteration in human chronic lympho-cytic leukemia (CLL)3.

Further studies indicated that many miRNA genes map precisely to regions of the human genome that are consistently altered, for example, by deletion or gene amplification, in a large vari-ety of human cancers, including lung cancer and lymphoma4. For instance, the members of the let-7 family of miRNAs mapping to chromosome regions involved in deletions in multiple human cancers have been shown to target the oncogene RAS (ref. 5). In addition, miR-15a and miR-16-1, which are lost in CLL, target and downregulate the expression of the BCL2 oncogene, which is overexpressed in CLL6. This evidence indicates that loss of these miRNA results in the consti-tutive overexpression of the targeted oncogenes and contributes to malignant transformation.

Another miRNA, miR-155, was found to be overexpressed and often amplified in the acti-vated B cell (ABC) form of human diffuse large B cell lymphoma7. Transgenic mice in which the overexpression of miR-155 was specifically targeted to B cells developed an acute lympho-blastic leukemia or high-grade lymphoma, sug-gesting that dysregulation of a single miRNA gene can cause cancer in mice and humans8. Given that the development of the malignancy took a few months, it seems possible that addi-tional genetic or epigenetic changes may be necessary for full malignant transformation.

In 2004, profiling of expression of miRNAs in human glioblastoma multiforme indicated that another miRNA, miR-21, is highly overexpressed in this tumor type9. Further investigations showed that miR-21 is the most commonly dys-regulated miRNA in a variety of human tumors, including leukemias and solid cancers, suggest-ing that this miRNA could function as an onco-gene in many types of human malignancies10.

In a recent study, Medina et al.11 presented

in vivo evidence of tumor addiction—depen-dency—to miR-21 in a mouse model of lym-phoma. The authors overexpressed miR-21 in a Cre- and doxycycline-dependent manner using the Rosa26 locus (miR-21LSL-Tetoff). miR-21 was overexpressed in tissues where nestin is expressed, including the hematopoietic compo-nent, and its expression could be silenced upon doxycycline treatment. Interestingly, these mice in the absence of doxycycline developed clinical signs of aggressive lymphomas, including spe-nomegaly and severe lymphadenopathy, and disruption of the normal tissue architecture of the spleen and thymus by invasive malignant B cells11. Tumors from miR-21LSL-Tetoff mice were transplantable into immunodeficient nonobese diabetic, severe combined immunodeficient mice, confirming their fully malignant nature11. These findings indicated that, in the context of B cells, upregulation of miR-21 leads to an aggressive malignancy and that such dysregu-lation is the initial and causal event in cancer development.

The tumors in miR-21LSL-Tetoff mice not exposed to doxycycline were found to be mono-clonal or oligoclonal, which suggests that addi-tional epigenetic and genetic changes might be required for malignancy1. But the miR-21LSL-Tetoff mice also showed that tumors continued to be dependent on the overexpression of miR-21, the driver. In fact, administering DOX to these mice bearing advanced lymphomas caused silencing of miR-21 overexpression, and, consequently, rapid regression of the B cell tumors.

Increasing evidence is showing that dysregu-lation of a single miRNA, such as miR-21, can cause malignant tumors. Notably, those tumors remained dependent on the overexpression of miR-21—the driver—and when the initiator or driver was shut off, the tumors regressed11. The striking results in the treatment of patients with

miRNAs

Protein-coding genes(oncogenes, tumor suppressors)

Human validation in different cancers

Loss of miRNAexpression or overexpressioncan be targeted by miRNAsor antimiRNAs, respectively

miRNAas biomarkers

Alteration of miRNAsin human cancers

Validation of miRNAs as cancer drivers or initiators; miR-21or miR-155 overexpression;miR-15a and miR-16-1 loss

OncogenicmiRNAs

(miR-155, miR21)

AntimiRNAs

Targeting drugs(i.e., imatinib)

Suppressor miRNAs(miR-15a, miR-16-1)

Figure 1 miRNAs targeting oncogenes and tumor suppressors as cancer drivers. In human malignancies, mRNAs can be overexpressed (miR-155 and miR-21) or lost (for example, miR-15a and miR-16-1). Their role in human cancer as cancer drivers can be validated in mouse models. New cancer therapeutics may include miRNAs and antimiRNAs to target early cancer events.

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