Causes and consequences of microRNA dysregulation in cancer Abstract: Over the past years it has become clear that alterations in the expression of microRNA genes contribute to the pathogenesis of human malignancies. {These can be caused by various mechanisms: deletions, amplifications or mutations in the miRNA loci, epigenetic silencing, or dysregulation of transcription factors targeting miRNA. Malignant cells show dependence on this dysregulation of miRNA genes, which control or are controlled by the dysregulation of several protein-coding oncogenes and tumor supressor genes. These facts provide important opportunities for other types of treatments, especially miRNA-based therapies.} Hulusi Onur Kuzucu - 2007103937

What are miRNA? 1993, Victor Ambros Small non-protein-coding RNA Undergo several modifications Incorporated into RISC Negative regulators; inhibit translation, promote degredation So what are miRNA? In 1993, Victor Ambros and his colleagues discovered a gene called lin-4 that affected development in C. Elegans. The product of this gene was small non-protein-coding RNA, later to be called as micro RNA. miRNA are produced by a mechanism that involves several processing steps done in the nucleus and the cytosol. The miRNA genes have their own promoter and regulatory units and usually lie in anti-sense orientation to the genes. The mature miRNA is an active part of RISC, RNA-induced silencing complex. Upon binding to a complementary site on an mRNA – usually in the 3’ UTR region – it inhibits the translation of the mRNA and promotes degredation. miRNA have been found to regulate over 1/3 of mRNAs and have important roles in many fundamental processes.

miRNA as Cancer genes Chronic Lymphocytic Leukaemia (CLL) Alterations in 13q14, non-protein-coding region miR-15a & miR-16-1 General alterations of miRNA genes Dysregulation of miRNA expression When looked at the tumor suppressor gene at 13q14, a chromosomal region frequently lost in chronic lymphocytic leukaemia (CLL) (B-CLL is a disease in which antibody-production-defected B-cells multiply in bone marrow and blood, where they crowd out the healthy cells), it is seen that protein-coding genes were not specifically altered. Instead, a small deletion in the non-protein-coding region of 13q14 was found. This deletion caused loss of miR-15a and miR-16-1. This loss was observed in most indolent CLLs. This fact lead researchers to map the chromosomal location of all known miRNA genes, and suprisingly, they were all located in chromosomal regions that were frequently subject to alterations in many types of human cancers. These changes were also observed in tumors without cytogenetic abnormalities, suggesting that the pathways dysregulated in cancer may affect and dysregulate miRNA expression, as miRNAs could be downstream targets and effectors of many regulatory pathway. {Members of let-7 family of microRNAs were lost in multiple different malignancies. The miR-17-92 cluster was found to be overexpressed in many different tumors. }

This figure shows the locations of miRNA-gene-containing regions in the human chromosomes that are involved in cancer. Many of these regions undergo rearrangements and deletions. {miR-15-a & miR-16-1 --- 13q14 miR-29-a & miR-29-b --- 7q32 Let-7 --- 3p2, 9q22, 11q23-q24, 21q21 All these regions are involved in deletions in a range of solid malignancies.}

miRNA as Tumor suppressors Heterogeneity Loss of miR-15a & miR-16-1 in indolent CLLs Loss of miRNA at 11q23 and TP53, leading to aggressive cells No rigid sequence of genetic events Mutations affect miRNA function Every malignancy is heterogeneous, which creates a challenge to understand the events that initiate cancer development. It is important to target the earliest genetic alterations, as they will be present in all of the cancer cells. The loss of miR-15a and miR-16-1 in indolent CLLs and aggressive CLLs originated from the indolent forms tells us that such genetic alterations must occur very early during the development of cancer, however, it is seen that the sequence of genetic events leading to cancer development is not rigid. Most indolent CLLs develop a 13q14 deletion, and over time, the miRNA genes at 11q23 or the tumor protein 53 gene at 17p13 are lost, causing them to be aggressive. However, some aggressive CLLs may start with a 11q23 deletion and then acquire a 13q14 deletion. These facts suggest that both alterations are necessary for the development of aggressive CLL. Not only deletions but mutations may also affect miRNA function. Some of these mutations that occurred in germ line confirmed that the loss of miR-15a and miR-16-1 contributes to the development of CLL, and their loss leads to a familial form of CLL. {This was also seen in TCL1 (T-cell lymphoma) transgenic mouse.} {Discoveries by Slack et al., of let-7 family negatively regulating the expression of RAS, and Cimmino et al., of miR-15a & miR-16-1 targeting BCL2 which inhibits apoptosis, showed that the loss of miRNAs could contribute to the malignant transformation through the dysfunction of cellular oncogenes. Later, let-7 family was found to target another gene called HMGA2 that is dysregulated in various tumors. Although miRNAs have many different targets, only a small number of them have a crucial role in cancer pathogenesis. Various alterations in the expression of specific protein targets lead to the same phenotype.}

Since these findings, other miRNAs that functions as tumor suppressors have been discovered. Several of the miRNA described as TS are found to be deleted or mutated in malignancies. In some cases, they are silenced by methylation.

miRNA as Oncogenes Overexpression of miRNA miR-155 – 21q23 Amplification in various BCL miR-17-92 cluster – C13orf25 Promote proliferation Transactivation by MYC Affects E2F transcription factors Downregulates PTEN, p21, BIM Not all miRNA are downregulated. Overexpression of several miRNAs were observed in various human tumors. miR-155 and the miR-17-92 cluster are among the first miRNAs that were found to be overexpressed; they are amplified at the DNA level in various BCLs. miR-155 is located in a host non-protein-coding genomic region located on 21q23. The mechanism of dysregulation of miR-155 was not known at time of this review. The miR-17-92 cluster, which contains six miRNA genes (17, 18a, 19a, 20a, 19b-1, 92a-1) is located in the non-protein-coding gene C13orf25 at 13q31.3. Overexpression of this cluster was observed in a wide range of tumors. These miRNAs promote proliferation, inhibit apoptosis, induce tumor angiogenesis and cooperate with MYC (myelocytomatosis viral oncogene), the gene which transactivates this cluster. This cluster affects cell cycle through its regulation of E2F transcription factors. The E2F gene family encode proteins that activate multiple genes involved in cell cycle progression from G1 to S phase. This cluster also downregulates important targets such as the tumor suppressors PTEN, p21 and BIM (Bcl2-interacting mediator). {Although the function of the cluster is known, the roles of each individual miRNAs are yet to be discovered.}

Several other miRNAs have been found to be overexpressed Several other miRNAs have been found to be overexpressed. These miRNAs target important tumor suppressors. miRNAs shouldn’t be labeled as oncogenes or tumor suppressor genes unless the tissue is specified. For example, miR-221 and miR-222 act both as an oncogene and as a tumor suppressor gene. {miR-21 was found to be overexpressed in most solid tumors and in many cancers. Inhibition of miR-21 by antisense oligonucleotides caused apoptosis in liver and breast cancer cells. miR-221 and miR-222 were also found to be in many solid malignancies.}

The miR-17-92 cluster is frequently amplified in lymphoma The miR-17-92 cluster is frequently amplified in lymphoma. Recent studies have shown that this cluster is essential for B cell proliferation. This cluster promotes cell cycle progression and proliferation, while repressing BIM, PTEN and p21. This cluster has homologues in two other clusters, MCM7 (minichromosome maintenance complex component 7) on chromosome 7, and X chromosome. MYC induces the expression of MCM7 and C13orf25, in addition to E2F1. E2F1 regulates these two clusters. MYC also promotes BIM. Inactivation of TGF (transforming growth factor) -ß tumor suppressor pathway is an important step in the development of tumors. Clusters miR-106b-25 and miR-17-92 function as key modulators of TGF-ß signaling, interfering with cell cycle arrest and apoptosis. Normally, these two clusters manage the pace of TGF-ß response by repressing BIM and p21 expression in proliferating cells and allowing expression as cells differentiate and undergo cell cycle arrest and apoptosis. Dysregulation in this regulatory mechanism would provide an escape from TGF-ß-induced arrest and apoptosis. {Ventura et al. shows that double knockout of these miRNA genes have much more severe phenotype compared to single knockout mice.}

miRNA dysregulation by transcription factors miR-34 family and p53 TS – positive ERα tumors & miR-221, miR-222 – negative Dysregulated miRNA genes, targets and mechanism of action Recent reports indicate that the miR-34 family is induced by the p53 tumor suppressor, and suggest that some p53 effects could be regulated by these miRNA. Authors compared the expression levels of p53 and miR-34 family members and observed correlation. Experiments showed that p53 binds to the miR-34 promoters. In breast cancer, it was seen that ERα- (oestrogen receptor-α-negative) tumors arise from ERα+ precursors. High levels of miR-221 and miR-222 were found in ERα- cells. Overexpression of these miRNA suppressed ERα protein in ERα+ cells. ERα was also found to negatively regulate expression of miR-221 and miR-222 by promoter binding. Therefore, miRNA can be dysregulated by transcription factors, and genetic or epigenetic alterations that result in the dysregulation of transcription factors can cause miRNA dysregulation, leading to malignant transformation. Volinia et al. have observed that some miRNA genes are dysregulated in not only one tumor type but in many, suggesting that these miRNAs may be downstream targets of pathways that are dysregulated in cancer. This also means that the mechanism of action of an oncogene is partly miRNA dysregulation. {If we look at the MYC transcription factor, which upregulates miR-17-92 cluster and downregulates miR-15a and miR-16-1, we can see that overexpression of mir-17-92 causes downregulation of tumor suppressors and underexpression of miR-15a and miR-16-1 causes upregulation of oncogenes. Genetic or epigenetic changes in protein-coding cancer genes or in miRNA genes may have similar consequences.}

miRNA in Cancer epigenetics Methylation of CpG islands, miR-127 miR-29 & DNMTs In cancer, the most studied epigenetic changes are the methylation of cytosines in the dinucleotide CpG in DNA. These methylations of tumor suppressor genes result in their silencing and contributes to malignant transformation. Saito et al. reported that miR-127 is silenced by promoter methylation in bladder tumors. miRNAs can also regulate methylation enzymes. miR-29 family target DNMT3A and DNMT3B (DNA methyltransferase), causing demethylation of the CpG islands in the promoter regions of tumor suppressor genes. {miR-127 targets the BLC6 oncogene, therefore its silencing may lead to BCL6’s overexpression.} {Introduction of miR-29 into AML (acute myeloid leukemia) cells resulted in the loss of expression of MCL1 and reactivation of p16. In AMLs, miR-29 cluster encoded at 7q32 is downregulated, with deletions of 7q.}

Conclusion miRNA therapy Different methods to introduce miRNAs Use of miR-15a, miR-16-1 and miR-29 on cancer cells, reversal of changes Focusing on miRNA dysregulation Identifying miRNAs and their targets Shifting from conventional chemotherapy to targeted therapies These observations open up a new range of therapies for cancer. The fact that several miRNA genes are dysregulated in multiple types of cancer suggests that crucial pathways may have miRNA genes as downstream targets. Therefore, miRNAs and anti-miRNAs may be considered as drugs that induce apoptosis and cell cycle arrest in cancer cells causing miRNA dysregulation. The miRNAs or anti-miRNAs may be introduced into tumors in different ways. They can be introduced into vectors, such as adenoviruses, to carry the miRNA to the tissue of interest. Some miRNA can be modified to increase their half-life in vivo, without the requirement of a vector. Some tissues (kidney, liver, spleen, bone marrow) readily take up miRNA. In other cases, it may be necessary to use vectors or other delivery systems such as liposomes. One challenge is to identify all targets of miRNAs involved in cancer and establish their contribution to malignant transformation. Another is to identify all miRNAs that are dysregulated in human cancers. Instead of focusing on specific alteratons in genes – which may be difficult to treat – we could focus on their downstream miRNA targets. If these miRNA targets are crucial in the proliferation and survival of cancer cells, then we can expect that use of miRNAs or anti-miRNAs will result in tumor suppression. These studies may provide additional information concerning the involvement of miRNA in cancer, and help shifting from conventional chemotherapy to targeted therapies in curing cancer. {After the discovery of miR-15a and miR-16-1 deletions in CLL, many laboratories have shown miRNA dysregulation in all tumors studied. This dysregulation can be caused by multiple mechanisms, and affect the regulation of many targets of miRNA. } {Experiments show that miR-15a and miR-16-1 can be used to suppress the growth of leukaemic cells that lack these miRNA. Members of miR-29 can also be used to suppress growth of human AML cells. Therapeutic experiments on mice suggest that these treatments have modest side effects, if any.}

Thank you References: Carlo M. Croce Causes and consequences of microRNA dysregulation in cancer Nature Reviews Genetics: October 2009 Vol 10 No 10 p704 | doi:10.1038/nrg2634 Lee, R. C., Feinbaum, R. L. & Ambros, V. The C. elegans heterochronic gene lin-4 encodes small RNAs with anti- sense complementarity to lin-14. Cell 75, 843–854 (1993). O’Donnell, K. A. et al. c-Myc-regulated microRNAs modulate E2F1 expression. Nature 435, 839–843 (2005). Raver-Shapira, N. et al. Transcriptional activation of miR-34a contributes to p53-mediated apoptosis. Mol. Cell 26, 731–743 (2007). Volinia, S. et al. MicroRNA expression signature of human solid tumors defines cancer gene targets. Proc. Natl Acad. Sci. USA 103, 2257–2261 (2006). Garzon, R. et al. MicroRNA signatures associated with cytogenetics and prognosis in acute myeloid leukemia. Blood 111, 3183–3189 (2008). Saito, Y. et al. Specific activation of microRNA-127 with downregulation of the proto- oncogene BCL6 by chromatin-modifying drugs in human cancer cells. Cancer Cell 9, 435–443 (2006).