Micro (mi)RNAs are small, highly conserved noncoding RNAs that control gene expression post-transcriptionally either via the degradation of target mRNAs or the inhibition of protein translation. Each miRNA is believed to regulate the expression of multiple mRNA targets, and many miRNAs have been linked to the initiation and progression of human cancer. miRNAs control various activities of the immune system and different stages of hematopoietic development, and their misexpression is the cause of various blood malignancies. Certain miRNAs have oncogenic activities, whereas others have the potential to act as tumor suppressors. Because they control fundamental processes such as differentiation, cell growth and cell death, the study of the role of miRNAs in human neoplasms holds great promise for novel forms of therapy. Here, we summarize the role of miRNAs and their targets in contributing to human cancers and their function as regulators of apoptotic pathways and the immune system.
Micro RNAs (miRNAs) are small noncoding RNAs of about 19–22 nucleotides (nt) that regulate protein expression by posttranscriptional silencing. The relevance of this class of novel small RNA regulators has only become clear over the past few years. However, it was realized early on that miRNAs are fundamental regulators of cellular processes that have physiological significance, and hence deregulation of various miRNAs is emerging as an important contributor to many human diseases including cancer. It was long known in the Caenorhabditis elegans community that a decrease in the expression of the heterochronic gene lin-14 that regulates developmental timing was critical for early larval stage transition, and that the lin-4 gene was required to reduce lin-14 levels and allow the larval transition to occur (Chalfie et al., 1981). The mechanism of repression was not known until groundbreaking work by two groups showed that repression occurred through complementary binding of the small RNA lin-4 to the 3′-UTR (untranslated region) of the lin-14 mRNA (Lee et al., 1993; Wightman et al., 1993). Subsequent discovery of the small non-coding RNA let-7 in C. elegans as a critical regulator in the determination of developmental cellular fate (Reinhart et al., 2000), and concurrent discovery of conserved sequence and temporal expression patterns of let-7-related genes across multiple species (Pasquinelli et al., 2000), suggested that these miRNAs may act as fundamental developmental regulators. In addition, these findings opened the floodgates of information to the understanding that miRNAs act as key participants in cellular differentiation. Later work revealed or suggested the role of miRNAs in neuronal patterning (Johnston and Hobert, 2003), lineage commitment in hematopoiesis (Chen et al., 2004), tissue homeostasis (Cui et al., 2006; Plaisance et al., 2006; Rodriguez et al., 2007) and apoptosis (Baehrecke, 2003; Cimmino et al., 2005). Currently, there are 678 mature human miRNA sequences listed in the miRNA registry (http://www.microrna.sanger.ac.uk/sequences) with approximately 1000 predicted miRNAs, each potentially targeting approximately 200 genes (Lewis et al., 2003). Lewis et al. (2005) further refined the identification of miRNA target sites and found that 5300 of 17 850 genes in their data set contained conserved miRNA target sites. Therefore, the possibility exists that >30% of the human genome may be under the translational regulation of miRNAs.
The emerging role of miRNAs in such a diverse and fundamental set of cellular mechanisms clearly suggests that proper control of these regulatory elements is essential for the maintenance of a non-pathologic state. A basic understanding of cancer formation revolves around the oncogene/tumor suppressor model of carcinogenesis—with RAS as an example of a classic oncogene and p53 the most common example of a tumor suppressor that inhibits cellular proliferation and induces apoptosis. Despite the simplicity of this model, it provides a solid foundation for an understanding of how two main classes of proteins function in the development of cancer. We are now beginning to understand miRNAs as master regulators that, in some cases, can act as either an oncogene or a tumor suppressor, whereas in other cases, they can affect both phases of tumorigenesis. Loss of let-7, for example, contributes to multiple facets of tumor progression by wholesale alteration of differentiation status. As our understanding of miRNA biology continues to increase, we will likely need to comprehend miRNA function more on a systems level, where linear relationships between miRNAs and target genes are less important than changes in the overall gene expression pattern induced by altered miRNA levels.
miRNAs negatively regulate protein expression at the point of protein translation. In the following, we will cover the basic pathway of miRNA biogenesis and maturation as well as the mechanism of translational repression. These topics have been reviewed in detail elsewhere (Bartel, 2004; Kim, 2005; Engels and Hutvagner, 2006; Zeng, 2006). miRNA biogenesis requires several posttranscriptional processing steps to yield the functional mature miRNA. Primary miRNAs (pri-miRNAs) are generally >1 kb transcriptional products of RNA polymerase-II and contain a 5′ 7-methylguanosine cap and 3′ poly-A tail (Cullen, 2004). Key to miRNA production is a hairpin within an imperfectly base-paired double-stranded RNA. The hairpin of the pri-miRNA is recognized by the nuclear RNAse-III enzyme Drosha and its cofactor DGCR8. The Drosha/DGCR8 complex cleaves the pri-miRNA to an approximately 70 nt double-stranded RNA hairpin pre-miRNA with a 2 nt 3′ overhang. The 2 nt 3′ overhang is recognized by exportin-5 and the Ran-GTPase, mediating the pre-miRNA nuclear export (Zeng and Cullen, 2006).
Once in the cytoplasm, the pre-miRNA is further digested by Dicer, the RNAse III enzyme of the RNA silencing pathway, yielding the ∼22 nt mature miRNA that enters the RNA-induced silencing complex (RISC) complex to mediate mRNA translational repression. Translational repression is a consequence of the miRNA/RISC complex binding to a target site in the 3′-UTR of the targeted mRNA that is complementary to the 5′-miRNA seed sequence, but usually not completely complementary along the entire miRNA/mRNA complex (Lai, 2002). Binding of the RISC/miRNA complex to the 3′-UTR of a target mRNA results in steric hindrance of the translational machinery and a consequent inability to translate the message. In the case of perfect complementarity between an miRNA and its target, the mRNA is degraded through the RNA silencing mechanism in a process distinct from miRNA-mediated translational repression (Engels and Hutvagner, 2006). The location, number and proximity of the miRNA binding sites along a target mRNA are all relevant in the translational repression, and this allows for prediction and ranking of putative miRNA targets based solely on sequence information (Grimson et al., 2007; Nielsen et al., 2007).
miRNAs in hematopoiesis and immune function
The study of miRNAs is now moving beyond descriptions of target analysis and toward a global understanding of how miRNAs impact entire developmental and differentiation processes. Several groups have targeted components within the miRNA-processing pathway and determined the effects of general loss of miRNA on development, differentiation, homeostasis and tumorigenesis. Particularly, it was found that Dicer-null mouse embryos fail to survive beyond embryonic day 7.5 and that Dicer is required for murine stem cell differentiation (Bernstein et al., 2003). Furthermore, knocking down Dicer implicated miRNAs in the maintenance of skeletal muscle development (O’Rourke et al., 2007), transformation and tumorigenesis (Kumar et al., 2007).
Hematopoiesis represents an elegant developmental model to observe normal changes in miRNA expression and to test the effects of miRNA levels on differentiation and tumorigenesis. There are a number of miRNAs that are prominently expressed in hematopoietic cells (for example, miR-142, miR-146, miR-150, miR-155, miR-181 and miR-223) (Chen et al., 2004; Ramkissoon et al., 2006). Differential expression of miRNAs during hematopoiesis is most likely involved in the regulation of hematopoietic differentiation. One such observation implicates miR-221 and miR-222 in maintaining hematopoietic stem cells by preventing the formation of early erythroblasts by directly targeting the receptor c-kit (Felli et al., 2005). Thus, downmodulation of these miRNAs is believed to allow the formation of early erythroblasts. Furthermore, the introduction of miR-221 or miR-222 also leads to an increase in cell death at a time point consistent with that of the terminal maturation of erythrocytes. This mechanism may be an evolutionarily conserved response to protect cells from temporal misregulation and de-differentiation during erythropoiesis. Contrary to increasing cell death, miR-221 and miR-222 were shown to directly target p27Kip1 in a prostate tumor cell line model, resulting in proliferation and increased oncogenesis (Galardi et al., 2007; Gillies and Lorimer, 2007; le Sage et al., 2007). Therefore, it appears that these miRNAs, which help maintain a normal stem cell state during erythropoiesis, act as oncogenes when misexpressed in a prostate tumor model, supporting the notion that miRNAs most likely have unique temporal regulatory roles during both differentiation and normal physiologic responses.
The descriptions of miR-181a and miR-150 clearly support the multifunctional physiologic roles of miRNAs. miR-181a directs hematopoietic stem cells toward differentiation into B cells (Chen et al., 2004). In addition, it was shown to modulate the intrinsic sensitivity of T cells to antigen by targeting a number of phosphatases that are known negative regulators of T-cell responses (Li et al., 2007). Furthermore, miR-150, found to exist at high levels in mature B and T cells but not their progenitors (Zhou et al., 2007), is required for mature B-cell development and the attenuation of the humoral immune response by translational repression of c-Myb (Xiao et al., 2007). The physiologic expression of mir-150 has a significant impact on limiting antibody production upon activation. Therefore, miR-181a and miR-150 have at least dual roles in normal physiologic processes.
Elevated c-Myc expression is common in many cancers (Grandori et al., 2000). A hallmark of Burkitt's lymphoma is a chromosomal translocation juxtaposing the c-myc and immunoglobulin genes and yielding high c-MYC expression (Janz, 2006). Furthermore, c-Myc is known to drive the expression of the pro-tumorigenic miR-17-92 cluster of miRNAs (O’Donnell et al., 2005; Dews et al., 2006), and a high level of c-Myc also leads to a decrease of multiple miRNAs known to have anti-tumorigenic properties (Chang et al., 2008). Chang et al. found that a majority of the c-Myc-repressed miRNAs provided a selective disadvantage when stably re-expressed in Myc3 cells, a B-cell lymphoma cell line, or in 38B9 cells, an Abl1-transformed pro-B cell line, and analysed 3 weeks after injection in an SCID mouse model.
The miR-17–92 cluster is a group of miRNAs (consisting of miR-17-5p, miR-18, miR-19a, miR-20 and miR-92) located on chromosome 13q31 that is transcribed as a polycistronic unit. miR-17-92 members have been described to target p21, a critical regulator of G1–S phase checkpoint, as well as the pro-apoptotic factor BIM, allowing cells to bypass the DNA damage checkpoint and obtain a survival advantage. The 17–92 miRNA cluster also targets anti-angiogenic factors Tsp1 and connective tissue growth factor (CTGF), resulting in an increase in angiogenesis in solid tumors. Recently, it was shown that this cluster regulates monocyte differentiation by targeting the transcription factor AML1 (Fontana et al., 2007), which is required for the expression of the macrophage colony stimulating factor (M-CSF) receptor. Taken together, these studies suggest that elevated c-Myc levels can contribute to multiple levels of tumorigenesis by direct modulation of miR-17-92 levels.
miR-223 has been shown to be involved in the differentiation of granulocytes (Fazi et al., 2005), and the transcription of miR-223 is competitively regulated by nuclear factor I/A (NFIA) and CCAAT/enhancer binding protein α (CEBPA); NFIA transcriptionally represses the expression of miR-223, whereas CEBPA causes the upregulation of miR-223 in response to differentiation induced by retinoic acid. Interestingly, miR-223 specifically targets NFIA favoring its own expression upon induced differentiation. Furthermore, CEBPA binding to the miR-223 promoter is required for the retinoic acid response, suggesting that miR-223 has a significant role in induced differentiation and that NFIA levels are critical in blocking this process.
In most cases, the functions of miRNAs were identified by studying predicted targets, and only a few examples exist in which a functional role was established without knowing a putative target. An example of a miRNA that was studied both in terms of its regulation and its targets is miR-146a, which has an important function in the innate immune system. miR-146a was found to be upregulated in cells treated with lipopolysaccharide (LPS) through the activation of toll-like receptor (TLR) 4 and concurrent activation of the nuclear factor-κB pathway (Taganov et al., 2006). Interestingly, two of its main predicted targets, interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK1) and TRAF6, are both signaling components of the TLRs. Both were confirmed to be direct targets of miR-146a, placing this miRNA into a negative feedback loop that limits inflammatory signaling and could potentially cause chronic inflammation or become detrimental through the induction of septic shock if it became deregulated.
Another TLR-responsive gene, miR-155, was identified to respond to the stimulation of TLR3 on macrophages (O’Connell et al., 2007) and immunoglobulin heavy chain enhancer (Eμ-miR-155 transgenic mice were hypersensitive to LPS/D-galactosamine-induced septic shock; Tili et al., 2007). Similar to miR-146a, evidence suggests that the putative targets of miR-155 are components of the TLR signaling machinery. Because miR-155 is best known through its involvement in the development of B-cell malignancies (see below), this discovery adds to the connection between inflammation and cancer that has recently attracted significant attention (Karin et al., 2006). In addition to its function in monocytes, miR-155 was shown to have a physiological function in B and T cells (Thai et al., 2007). miR-155-deficient mice have a defect in the germinal center (GC) reaction and a concurrent weak T-cell-dependent antibody response. It was suggested that this might be due to an impaired production of tumor necrosis factor α (TNFα) by the B cells that is required for a proper GC response. In the T-cell compartment, these mice were skewed toward TH2 differentiation, as their T cells produced more IL-4 and less interferon-γ than T cells from control mice (Thai et al., 2007). These data illustrate that miRNAs play important roles in the development of various blood-derived cell types and often are part of complex feedback regulatory networks. Interestingly, in many reported cases, it is not the miRNA that drives differentiation, but rather miRNAs are expressed to prevent de-differentiation and/or maintain pools of cells at certain stages of differentiation. Hence, their downregulation seems to assist in driving cells down certain de-differentiation paths (Georgantas et al., 2007).
OncomiRs as tumor suppressors and oncogenes
We have just described the important role of various miRNAs in immune function and hematopoietic fate determination, and have suggested that the deregulation of this control system can lead to oncogenesis and other pathologies. Not surprisingly, multiple pieces of evidence point at miRNAs playing a profound role in cancer formation and progression (Table 1). Gene expression array analyses as well as real-time PCR analyses of cancers reveal cancer specific changes in the expression of miRNAs, when compared with noncancerous tissues (Liu et al., 2004; Lu et al., 2005; Volinia et al., 2006). In addition, an expression analysis of 217 miRNAs in various human cancers clearly described the developmental origin of tumors based on miRNA profile, and it also identified a global downregulation of miRNAs in tumors regardless of cell type (Lu et al., 2005). It has also been found that the induction of miRNAs coincides with normal cellular differentiation, supporting the hypothesis that miRNA loss leads to a de-differentiation process and less-differentiated tumors (Lu et al., 2005). Interestingly, Lu et al. showed that the expression levels of the 217 miRNAs allowed for a better stratification of cancer material, when compared with information obtained from a gene array analysis of about 16 000 mRNAs. The prognostic power of these miRNA profiles is quite striking and strongly argues for the ultimate importance of miRNAs in developing and maintaining cellular fates. It is then quite easy to envisage how gain or loss of a single miRNA or a set of miRNAs in either differentiated adult tissues or more differentiated tumors could both induce and further neoplastic progression.
Further support for the relevance of miRNAs in cancer came from studies that demonstrated that half of the known miRNAs are located in regions of chromosomal instability associated with cancer (Calin et al., 2004), and that a high proportion of genomic loci containing miRNA genes shows DNA copy number alterations (Zhang et al., 2006). As we have described above, specific roles of miRNAs in certain cancers have been established, and many miRNA targets are emerging that allow one to explain or predict some of the activities of miRNAs in cancer. Oncogenic miRNAs include miR-155, miR-17-5p and miR-21, whereas miRNAs with tumor-suppressing activities include miR-15a, miR-16–1 and let-7 (Table 1). Both oncogenic-and tumor-suppressing miRNAs are collectively called oncomiRs because of their respective roles in cancer progression (Esquela-Kerscher and Slack, 2006). One key pathway in which various miRNAs play the role of both tumor suppressor and oncogene is that of apoptosis.
Apoptosis in immune system maintenance
Apoptosis is of key importance in maintaining tissue homeostasis, developmental tissue remodeling, the defense against microbial and viral infections and in preventing tumor formation. Apoptosis also plays essential roles in various processes in the immune system, such as lymphocyte development, homeostatic control of immune response and also in the elimination of virally infected or tumor cells. During lymphocyte development, autoreactive T and B lymphocytes are eliminated by apoptosis in the primary lymphoid organs, the thymus and bone marrow, respectively, in a process termed negative selection. This process is important for preventing autoimmunity.
Apoptosis induction is initiated intracellularly upon various cellular insults mediated through the mitochondria (the intrinsic pathway) or through transmembrane receptors (death receptors) that initiate apoptosis at the cell surface (the extrinsic pathway). In the intrinsic apoptosis pathway, sentinel proteins sense changes in the endoplasmic reticulum (ER stress), the nucleus (DNA damage), or directly at the mitochondria, which are the central command center of the apoptotic process. Key regulators of mitochondrial apoptotic activities are members of the BCL-2 protein family. They can be roughly divided into pro- and anti-apoptotic members and are characterized by the presence of BCL-2 homology (BH) domains. The multidomain-containing members that function directly on the outer mitochondrial membrane include pro-apoptotic BAX and BAK (containing BH1-BH3) and anti-apoptotic BCL-2 and BCL-XL (containing BH1–BH4). Upstream of these proteins are a number of very diverse pro-apoptotic sensor proteins containing only the BH3 domain (e.g., BIM, BID, BAD and NOXA). These proteins are activated by various pro-apoptotic stimuli and all act by either activating BAX/BAK or inhibiting BCL-2/BCL-XL (Coultas and Strasser, 2003).
Among the BH3-only proteins, BIM, in particular, has been well recognized to play a critical role in the apoptosis of autoreactive T cells during negative selection in the thymus. Bim knockout mice accumulate three- to five-fold excess numbers of lymphoid and myeloid cells, and activated T cells from these mice are abnormally resistant to cytokine withdrawal-induced apoptosis (Bouillet et al., 1999). BIM has also been demonstrated to be essential for the deletion of autoreactive B lymphocytes in both bone marrow and periphery (Enders et al., 2003).
Once activated, mitochondria release factors, including cytochrome c and SMAC/Diablo, that act by triggering the formation of the apoptosome (comprising cytochrome c, APAF-1, the initiator caspase 9 and dATP/ATP) and by inhibiting apoptosis inhibitors of the IAP family (Salvesen and Duckett, 2002), respectively. Both of these events contribute to the activation of the executioner caspases 3 and 7. In the extrinsic apoptosis pathway, surface receptors of the death receptor family that include TNFRI, CD95 (Fas/APO-1) and the TNF-related apoptosis inducing ligand (TRAIL) receptors DR4 and DR5 (Schulze-Osthoff et al., 1998), once triggered by their cognate ligands, recruit adaptor proteins (FADD and TRADD) to form a high-order receptor signaling structure that is collectively called the death-inducing signaling complex (DISC) (Peter and Krammer, 2003). Additional components of the DISC are the initiator caspases 8 and 10, and the caspase-8/10 regulator c-FLIP. Once activated, caspase-8 cleaves a number of substrates including the executioner caspase 3 and the BH3 protein BID (Barry et al., 2000; Waterhouse et al., 2005).
The involvement of death receptors in negative selection has not been consistent and is somewhat controversial. TRAIL-deficient mice displayed increased numbers of immature thymocytes and accelerated autoimmune disease (Lamhamedi-Cherradi et al., 2003), but no defects in lymphoid homeostasis or in T-cell function (Sedger et al., 2002; Cretney et al., 2003). Although negative selection is intact in CD95/CD95L (Singer and Abbas, 1994; Adachi et al., 1996), TNFR1 (Pfeffer et al., 1993), TNFR2 (Erickson et al., 1994) or TNFR1 and TNFR2 double-deficient mice (Page et al., 1998), there has been evidence supporting roles of TNF and CD95 in negative selection in some models (Castro et al., 1996; Kishimoto et al., 1998). Death receptor-mediated apoptosis does not appear to play a critical role in thymic central tolerance; however, genetic deletion of members of the BCL-2 family, especially BIM in mice, has shown BIM to be a critical mediator of apoptosis for negative selection in the thymus (Bouillet et al., 2002; Strasser et al., 2008). Recently, studies have shown that BIM functions together with the death receptor CD95 in the apoptotic process during T-cell clonal contraction or peripheral deletion during acute and chronic infection (Hughes et al., 2008; Hutcheson et al., 2008; Weant et al., 2008).
Both intrinsic and extrinsic apoptotic pathways converge on the level of caspase-3 activation, which then goes on to cleave various intracellular substrates that cause the typical changes observed in apoptotic cells. The ability to evade the grips of the apoptotic pathway has been described as a hallmark of cancer, and all neoplasms contain disruptions of the apoptotic machinery. As >30% of the human genome is now believed to be under the control of miRNAs, and considering the fundamental role of miRNAs in important differentiation and homeostatic processes, it became an intriguing possibility that a relationship existed between miRNAs and the apoptotic machinery.
Apoptosis-regulating miRNAs (‘apoptomiRs’)
The first miRNA described as an apoptotic regulator was the Drosophila gene bantam (Brennecke et al., 2003). Bantam was found to directly regulate the pro-apoptotic factor hid, suppressing the apoptotic activity and allowing proliferation to occur. Furthermore, miR-14 was found to suppress pro-apoptotic activity of reaper during the eye formation (Xu et al., 2003). Although spatially and temporally regulated as observed in other cell-fate-determining miRNAs, both bantam and miR-14 are described to function as anti-apoptotic miRNAs. In the following, we will give examples of miRNAs that have been shown to regulate cell death in mammalian cells.
Chromosome 13q14 is a region deleted in more than half of B-cell chronic lymphocytic leukemia (CLL). The identification of miR-15 and miR-16 as being the tumor suppressors at this chromosomal location represented the first link between miRNAs and cancer (Calin et al., 2002). Groundbreaking studies by Cimmino et al. (2005) looking into tumorigenic effects of the miR-15a/miR-16-1 deletion demonstrated that the expression of miR-15a and miR-16-1 inversely correlated with BCL-2 expression in CLL cells, providing the first description of direct miRNA mediation of apoptotic activity in human tissue. The 3′-UTR of BCL2 contains a target site for each of these miRNAs, and miR-15a/16-1 controls the expression of BCL-2. Post-translational regulation of BCL-2 by miR-15a and miR-16-1 opens the door to potential therapeutic development of miRNAs in BCL-2 overexpressing tumors. Another link between miRNAs and CLL is the finding that the TCL1 oncogene, which is often found in aggressive CLL, is regulated by miR-29 and miR-181b (Pekarsky et al., 2006).
In contrast to miR-15/miR-16, whose expression is often reduced in cancers, the miR-17-92 cluster is overexpressed in many cancers including B-cell lymphoma (Ota et al., 2004; Hayashita et al., 2005; He et al., 2005b). Recently, it was demonstrated that the inhibition of two miRNAs in this cluster, miR-17-5p and miR-20a, sensitized lung cancer cells to apoptotic insults (Matsubara et al., 2007), which suggests that these miRNAs may be useful as a potential therapeutic modality. miR-20a negatively regulates E2F1 (O’Donnell et al., 2005), E2F2 and E2F3 (Sylvestre et al., 2007), and all three E2Fs regulate the expression of miR-17-92 through binding to its promoter, suggesting an autoregulatory feedback loop. The E2F transcriptional networks are believed to link cell cycle progression to apoptosis, and perturbation of their various levels can induce either proliferation or apoptosis depending on the cellular context. miR-20a overexpression protects cancer cell lines from apoptosis, suggesting that overexpression of the miR-17-92 cluster promotes oncogenesis, in part, by perturbing the E2F network in a way that induces proliferation and inhibits apoptosis (Sylvestre et al., 2007).
Recently, the identification of MCL-1 regulation by miR-29a,b and c expanded what is known about miRNA regulation within the BCL-2 family (Mott et al., 2007). MCL-1, a member of the BCL-2 family of anti-apoptotic proteins, is upregulated in malignant cells, whereas miR-29b is downregulated in cancer. The mechanism of sensitization of cancer cells to TRAIL-induced apoptosis differs among cell types and is regulated by multiple downstream factors. Enforced expression of miR-29b renders tumor cells more sensitive to the apoptosis-inducing activity of TRAIL, suggesting that the miR-29b/MCL-1 connection is functionally important and could be exploited for cancer therapy. Recently, it was demonstrated that acquired resistance to TRAIL therapy involves c-Myc-dependent upregulation of both MCL-1 and cIAP2 (Ricci et al., 2007), suggesting that targeting MCL-1 using miR-29 could therefore be a viable treatment option in this situation.
It seems clear that tight regulation of anti-apoptotic function through miRNAs is critical in development and other cellular processes. Interestingly, a database search using TargetScan (http://www.targetscan.org) for potential miRNA regulators of BCL-2 family members found a notable lack of conserved miRNAs that could regulate the pro-apoptotic Bcl-2 members (with the exception of BIM), but revealed that a majority of the anti-apoptotic BCL-2 members maintain conserved potential miRNA-binding sites (Table 2). Pro-survival members have on average six miRNA target sites in their 3′-UTRs as compared with 2.2 sites found in pro-apoptosis members. BCL-2, BCL-W and MCL-1 have multiple conserved miRNA target sites in their 3′-UTR. BCL-XL only has three such sites, and this could be due to the fact that the alternative splice form BCL-XS, which shares the same 3′-UTR with BCL-XL, has pro-apoptotic activities. This may indicate that evolutionary pressure favored the fine-tuning of the anti-apoptotic process over the pro-apoptotic process as part of the regulation of development and tissue homeostasis. Consistent with this hypothesis is an analysis of the 3′-UTRs of all caspases that revealed that most caspases are not predicted miRNA targets with the exception of caspase-3 (Table 2). It is interesting to note that caspase-3 has been shown to have nonapoptotic functions as well (Algeciras-Schimnich et al., 2002), which could be regulated by miRNAs.
With the goal of identifying miRNAs that are relevant for proliferation or cell death, Cheng et al. (2005) individually knocked down 90 human miRNAs in HeLa and A549 cells. MiR-21 was identified among miRNAs that were identified as regulators of apoptosis. Subsequently, miR-21 was shown to be highly overexpressed in breast tumors compared with normal breast tissue, and the inhibition of miR-21 was shown to inhibit tumor growth (Si et al., 2007). Further work identified PTEN and tropomyosin 1 as direct targets of miR-21 in cancer cell lines (Zhu et al., 2007; Meng et al., 2007a). In glioblastoma, miR-21 was described as an anti-apoptotic factor (Chan et al., 2005), and a combination treatment of anti-miR-21 and TRAIL was able to eradicate apoptosis-resistant glioma cells in vitro and in vivo (Corsten et al., 2007). A recent study linked miR-21 to the pro-apoptotic tumor suppressor p53 and identified programmed cell death 4 (PDCD4) as a direct target (Frankel et al., 2008). The fundamental significance of miR-21 is most likely best documented by the fact that it is the only miRNA that was consistently upregulated in six out of six solid human cancers (breast, colon, lung, pancreas, prostate and stomach), when compared with matching noncancerous tissues (Volinia et al., 2006). Hence, this single miRNA provides a significant survival advantage to cancer cells upon its upregulation, and this may be a fundamental mechanism utilized by cancers to elude apoptotic cell death.
Other apoptosis regulators targeted by miRNAs include HSP60 and HSP70 (targeted by miR-1) and caspase-9 (targeted by miR-133), and both of these miRNAs are predominantly expressed in cardiac and skeletal muscles (Xu et al., 2007). miR-1 and miR-133 have opposing affects on apoptosis, with miR-1 being pro-apoptotic and miR-133 being anti-apoptotic. Interestingly, miR-1 and miR-133 are transcribed together from the same chromosomal locus, suggesting that their relative levels might be regulated at a post-transcriptional level, and that it is the post-transcriptional regulation of these miRNAs that determines the eventual physiological outcome within cardiac and skeletal muscle tissues.
A key regulator in the activation of the intrinsic apoptosis pathway in response to DNA damage, cellular stress and/or improper mitotic stimulation is p53. As a transcriptional regulator, p53 both activates and represses gene expression, and is the most frequently dysregulated protein in cancer with a majority of cancers harboring functional loss of p53. Five groups independently identified miR-34 as a target for p53 (Bommer et al., 2007; Chang et al., 2007; He et al., 2007; Raver-Shapira et al., 2007; Tarasov et al., 2007). He et al. (2007) described that miR-34 expression correlates with p53 expression. Using a p53-inducible system, they reported that the miR-34 family of miRNAs is directly regulated by p53, and that miR-34 mediates growth arrest in multiple cell lines via direct 3′-UTR regulation of cell cycle regulatory factors, such as cyclin-E2 (CCNE2), cyclin-dependent kinase 4 (CDK4) and the hepatocyte growth factor receptor (c-Met). Ectopic expression of miR-34 also resulted in a decrease of phospho-Rb, supporting the hypothesis that miR-34 regulates CDK4 and CCNE2. Furthermore, miR-34a was shown to directly target E2F3. In addition, miR-34a resulted in an increase in caspase-dependent death when introduced into two cell lines (Welch et al., 2007) and contributed to an increase in p53-mediated apoptosis (Chang et al., 2007; Raver-Shapira et al., 2007). miR-34 was therefore described as a general sensitizer to apoptosis mainly through its link to p53, although its targets are still speculative. Interestingly, the other main activity of p53 to induce growth arrest, and cellular senescence also seems to involve miR-34a, as it was recently demonstrated that HCT116 colon cancer cells that were suppressed in their growth upon the introduction of miR-34a acquired a senescence-like phenotype (Tazawa et al., 2007).
It remains to be determined how many of the activities of p53 depend on regulation by miR-34a. For instance, tumor protein p53-induced nuclear protein (TP53INP1) is a stress-induced p53 target gene that induces caspase-3-dependent apoptosis. TP53INP1 was shown to be dramatically reduced in its expression in pancreatic ductal adenocarcinoma (Gironella et al., 2007). TP53INP1 was demonstrated to be a direct target of miR-155, and consistently miR-155 is often overexpressed in pancreatic cancer (Lee et al., 2007; Szafranska et al., 2007). Other miRNAs functionally connected to p53 are miR-372 and miR-373, which were identified as oncogenes in testicular germ cell tumors, and target the tumor-suppressor LATS2 and neutralize p53-mediated CDK inhibition (Voorhoeve et al., 2006). Taken together, this body of evidence demonstrates that multiple miRNA families target p53, which is universally known as one of the most important tumor suppressors with apoptosis-inducing activities. As with the processes of differentiation and cell fate determination, it has become quite clear that miRNAs play a fundamental role in regulating key cogs of the apoptotic machinery.
OncomiRs not (yet) directly connected to apoptosis
Transcripts of miR-155 and its host gene BIC accumulate in B-cell malignancies. Elevated miR-155 levels are found in diffuse large B-cell lymphoma (Eis et al., 2005), Hodgkin's lymphoma (Kluiver et al., 2005) and certain Burkitt's lymphomas (Kluiver et al., 2006), supporting its role in B-cell development and lymphomagenesis. Consistently, transgenic mice expressing mmu-miR-155 driven by the pre-B-cell-specific immunoglobulin transcriptional enhancer element (Eμ-mmu-miR-155) exhibit preleukemic pre-B-cell proliferation that result in B-cell malignancy (Costinean et al., 2006).
miRNAs have also been directly linked to metastasis formation. miR-10b was one of a few miRNAs whose expression was found to be elevated in metastatic breast cancer (Ma et al., 2007). Exogenous miR-10b increased both in vitro and in vivo invasion of established breast cancer cells combined with increased proliferation, whereas inhibition of miR-10b led to a 10-fold reduction in invasion. The bHLH transcription factor TWIST, a known inducer of epithelial-to-mesenchymal transition (EMT) and driving force of breast metastases (Yang et al., 2004), induces expression of miR-10b, and this induction indirectly upregulates levels of the prometastatic gene RHOC because miR-10b directly targets HOXD10 (Ma et al., 2007). These studies are important because they were the first to establish a connection between a particular miRNA and metastasis formation of a human cancer. Additional miRNAs such as miR-335 and miR-126 have since been identified as key metastasis suppressors in vivo (Tavazoie et al., 2008). Recently, the miR-200 family of miRNAs (which includes miR-200a, b, c, miR-141 and miR-429) was identified as both a marker for epithelial cells and a powerful master regulator of EMT. Its activity was found to be mainly mediated through targeting two E-box-binding E-cadherin repressors, ZEB1 and ZEB2 (Gregory et al., 2008; Park et al., 2008). Interestingly, ZEB1 has been shown to regulate T-cell differentiation, to repress IL-2 production and to regulate the expression of CD4 (Yasui et al., 1998; Brabletz et al., 1999; Postigo and Dean, 1999). It will be interesting to determine whether miR-200 also regulates T-cell development or activation.
The case of let-7
Although much of the research on the role of miRNAs in apoptosis regulation focuses on direct relationships between a miRNA and its putative apoptosis-regulating target and because miRNAs can regulate global differentiation pathways, it is likely that activities exerted by miRNAs on apoptosis pathways may often be indirect. Cells with gains or losses of specific miRNAs may be altered in a way that changes entire genetic programs and results in new phenotypes that have different apoptotic properties. In the following, we will review such a differentiation process that is regulated by miRNAs and affects apoptosis sensitivity of cells.
The receptor-mediated activation of apoptosis is well defined in the TNF receptor superfamily. CD95 receptor is the best-characterized member of the death receptors in this superfamily, and it induces apoptosis by two distinct mechanisms in Type I or Type II cells, the latter discriminated by the requirement of mitochondrial involvement. Interestingly, T-cell activation was shown to represent another case of a conversion from Type II to Type I (Scaffidi et al., 1999; Schmitz et al., 2003). Upon binding of its ligand (CD95L), CD95 forms the DISC. The characteristics of the Type I pathway are receptor internalization followed by the formation of large amount of DISC on endosomes and the activation of caspase-8 that directly cleaves the executioner caspase 3 (Scaffidi et al., 1998; Barnhart et al., 2003; Lee et al., 2006b). In contrast, the mitochondrial-dependent Type II pathway does not involve internalization of the receptor to the same extent, and only a small amount of active caspase-8 is generated, which is sufficient to cleave Bid causing it to translocate to the mitochondria where it induces the release of mitochondrial factors that amplify the apoptotic signal (Li et al., 1998; Luo et al., 1998). Another difference between Type I and Type II cells is their response to stimulation with CD95L and to Taxanes. Type II cells were found to be much more sensitive to apoptosis induction by soluble CD95L and growth inhibition by Taxanes than Type I cells (Algeciras-Schimnich et al., 2003). The difference in CD95 apoptosis sensitivity was used to classify the 60 cell lines of the drug-screening panel at the NCI (NCI60). Of 58 cell lines tested, 22 cell lines were found to be sensitive to CD95-mediated apoptosis—11 of them were classified as Type I and 11 as Type II (Algeciras-Schimnich et al., 2003). In a previous study, the NCI60 cells had been subjected to a gene chip analysis (Ross et al., 2000), revealing that the cell lines genetically fell into two major superclusters (SCs). SC1 cells were found to express genes that expressed a less differentiated and stromal phenotype, when compared with SC2 cells that expressed a gene signature consistent with a more differentiated phenotype. We recently identified the let-7 family as being preferentially expressed in Type II cells. Because let-7 is a marker for more differentiated tissues, this suggests that let-7 levels could directly affect the differentiation state of cancer cells and hence their sensitivity to death ligands and Taxanes.
Let-7 is downregulated in a number of human cancers such as lung, colon, or ovarian cancer, and it serves as a prognostic marker for disease outcome (Takamizawa et al., 2004; Johnson et al., 2005; Akao et al., 2006; Yanaihara et al., 2006; Shell et al., 2007). It was also suggested to be a tumor suppressor for lung cancer through targeting RAS (Johnson et al., 2005). More recently, high-mobility group A2 (HMGA2) was identified as a major target for let-7 (Hebert et al., 2007; Lee and Dutta, 2007; Mayr et al., 2007; Shell et al., 2007), and indeed HMGA2, which is not expressed in most adult tissues, is upregulated in various cancers, such as neuroblastoma (Giannini et al., 2000), pancreatic cancer (Abe et al., 2003), thyroid neoplasms (Chiappetta et al., 1995), squamous carcinoma (Miyazawa et al., 2004) and lung cancer (Sarhadi et al., 2006). Interestingly, HMGA2 is an early embryonic gene. Consistently, let-7 is one of the main miRNAs that is upregulated late during embryonic development (Lagos-Quintana et al., 2001; Abbott et al., 2005; Schulman et al., 2005), and this is conserved from C. elegans to humans (Pasquinelli et al., 2000). The physiological targets for let-7 are therefore most likely embryonic genes. We have recently demonstrated that let-7 is selectively expressed by Type II human cancer cells that are at an early stage of de-differentiation (Shell et al., 2007), and we proposed that let-7 controls a number of oncofetal genes to prevent the de-differentiation process of reverse embryogenesis that might give tumor cells properties of embryonic cells (Park et al., 2007). Recently, a set of 12 let-7-regulated oncofetal genes (LOGs) was identified using a genome-wide bioinformatics approach (Boyerinas et al., 2008). Overexpression of let-7 inhibits cancer growth (Takamizawa et al., 2004; Akao et al., 2006; Johnson et al., 2007) and its inhibition promotes growth (Kumar et al., 2007). Recently, it was shown that let-7 effectively suppresses cancer development in a mouse model of spontaneous lung cancer (Esquela-Kerscher et al., 2008; Kumar et al., 2008), giving hope that it could be useful for cancer therapy.
Another target for cancer therapy are the recently discovered regulators of let-7 processing LIN28 and LIN28B (which happens to be LOG3) (Newman et al., 2008; Viswanathan et al., 2008). The stem cell factor LIN28 (Yu et al., 2007) has been shown to selectively block the processing of most let-7 family members at the Drosha level and it is likely to be upregulated in many different cancers. So far, this has been shown for hepatocellular carcinoma in which LIN28B was found to be upregulated in more advanced cancer (Guo et al., 2006).
Although let-7 is downregulated in many human tumors, it cannot be viewed as a classical tumor suppressor gene. First, it consists of 12 individual genes transcribed from 8 chromosomal loci (Park et al., 2007), and although all 12 miRNAs are predicted to have a similar set of targets, it is not clear whether all 12 miRNAs have the same function. Secondly, miRNAs exert their function through targeting other genes, and it is therefore conceivable that, in certain tissues under certain conditions, let-7 could have a set of targets that have tumor-suppressing functions, whereas in other contexts it may not. In fact, let-7a-3 was described to be epigenetically silenced in a human lung cancer cell line and was suggested to have oncogenic function (Brueckner et al., 2007). In another study, it was shown that the pro-inflammatory cytokine IL-6 can contribute to tumor growth and drug resistance through the upregulation of let-7a (Meng et al., 2007b). Other examples of miRNAs with no clearly defined activity with respect to tumorigenesis are miR-21 and miR-24, which, depending on the cell type, can either promote or inhibit cell growth (Cheng et al., 2005). As such, a miRNA can only be considered a tumor suppressor in particular tissues where its expression inhibits the expression of genes considered to have oncogenic properties. It is therefore most appropriate to refer to miRNAs that are involved in cancer development in more general terms as oncomiRs.
It has become quite clear that microRNAs regulate a diverse set of cellular processes in both the immune system and solid tissues. From proper immune function, to hematopoietic lineage commitment, to embryonic development, to apoptosis and cell cycle regulation, many miRNAs act as master regulators that enforce particular patterns of gene expression by both directly and indirectly modulating the expression of target genes. The field of miRNA studies is no longer in its infancy, yet we are many years from developing a comprehensive understanding of the subject. In many cases, there is a direct relationship between a particular miRNA and expression of a target gene—miR15a and BCL2. However, what has become clear is the importance of context when attempting to determine the functional outcome of gain or loss of any particular miRNA. The relationship between a miRNA and a particular target may be more important in some situations than others—mir-21, for instance, can play the part of both tumor suppressor and oncogene depending on cellular context. As the current evidence suggests that many miRNAs play an essential role in differentiation and cell fate determination, we will most likely find that many miRNAs function similar to let-7, which seems to control a program of differentiation, and whose loss contributes to carcinogenesis by modulating both apoptotic and cell cycle pathways.
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Schickel, R., Boyerinas, B., Park, SM. et al. MicroRNAs: key players in the immune system, differentiation, tumorigenesis and cell death. Oncogene 27, 5959–5974 (2008). https://doi.org/10.1038/onc.2008.274
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