Review

"The microRNA network and tumor metastasis".

H Zhang ¹, Y Li ¹ and M Lai ^{1, @}

¹ Department of Pathology, School of Medicine, Zhejiang University, Zhejiang, PR China

^@ Correspondence: Professor M Lai, Department of Pathology, School of Medicine, Zhejiang University, Zhejiang 310058, PR China. E-mail: lmp@zju.edu.cn

Received 27 July 2009; Revised 1 September 2009; Accepted 15 October 2009; Published online 23 November 2009.

Metastasis is the most significant process affecting the clinical management of cancer patients and occurs in multiple sequential steps. However, the molecular pathways underlying each step still remain obscure. Recent research has shown that there is a microRNA (miRNA) network that functions as a regulator of tumor metastasis. In this paper, we review the role of miRNAs in tumor metastasis, including control of epithelial–mesenchymal transition, regulation of metastasis-associated genes and epigenetic alterations. More information on miRNAs will promote a better understanding of the molecular mechanism of metastasis.

Keywords:  microRNA,   tumor,   metastasis

Tumor metastasis is a significant factor in the clinical management of cancer, as most cancer mortality is associated with disseminated disease rather than the primary tumor (Hunter et al., 2008). However, the management of cancer is still at a primitive level because metastasis is a complex, multi-step process: primary tumor cells invade adjacent tissue, enter the systemic circulation (intravasate), translocate through the vasculature, arrest in distant capillaries, extravasate into the surrounding tissues and finally proliferate from initial microscopic growths (micrometastases) into macroscopic secondary tumors (Fidler, 2003). The traditional view of metastasis includes the process of clonal selection in which rare variant clones within the primary tumor become capable of completing the complex multi-step metastatic process (Fidler, 2003; Talmadge, 2007). Recently, expression profiling analyses have revealed various tumor metastasis genes and metastasis suppressor genes, which not only regulate the metastatic process (Dong et al., 1995, 1996; Bandyopadhyay et al., 2004; Nuyten and van de Vijver, 2006; Rinker-Schaeffer et al., 2006; Tomida et al., 2007; Albini et al., 2008; Kim et al., 2009; Lukes et al., 2009) but also maintain the microenvironment of tumor cells, and initiate the process of epithelial–mesenchymal transition (EMT).

MicroRNAs (miRNAs) are small, highly conserved noncoding RNAs that control gene expression posttranscriptionally, either by the degradation of target mRNAs or by the inhibition of protein translation. Hundreds of miRNAs, many of them evolutionarily conserved, have been identified (http://microrna.sanger.ac.uk), and some of these molecules exhibit highly specific, regulated patterns of expression in various kinds of organs and organisms (Chen et al., 2004; Landgraf et al., 2007; Neilson et al., 2007; Merkerova et al., 2008). Moreover, these novel 19–22 nucleotide molecules may also be involved in a wide range of pivotal biological processes in both plants and animals, such as cell growth, development, proliferation, differentiation and death (Ambros, 2003; Carrington and Ambros, 2003; Miska, 2005). More than 50% of annotated human miRNA genes are located in fragile chromosomal regions that are susceptible to amplification, deletion or translocation in the process of tumor development (Calin et al., 2004). In addition, recent evidence indicates that some miRNAs can function either as oncogenes or as tumor suppressors, and expression profiling has revealed characteristic miRNA signatures in certain human cancers (Lu et al., 2005; Calin and Croce, 2006; Roldo et al., 2006). In effect, biochemical and molecular biological studies are providing fundamental insights into the molecular role of miRNA in tumor metastasis, and networks of miRNAs may regulate much of this whole process. The review presented here focuses on the relationship between miRNA and EMT, metastasis-associated genes and the epigenetic modification of tumor metastasis, and highlights the various pathways of miRNA involved in these processes.

miRNA and EMT:

Epithelial–mesenchymal transition describes the molecular reprogramming and phenotypic changes characterizing the conversion of polarized immotile epithelial cells to motile mesenchymal cells. This process allows the remodeling of tissues during embryonic development, and is implicated in the promotion of tumor invasion and metastasis (Thiery, 2002; Acloque et al., 2008). Recent years have witnessed a completely new evaluation of EMT, namely, one regulated by miRNAs.

miRNAs lead to tumor cell-intrinsic modifications that are involved in the EMT

Some research has focused on the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429), whose downregulation is believed to be the essential feature of EMT. Hurteau et al. (2007) and Christoffersen et al. (2007) have implicated ZEB1 and ZEB2 (zinc finger E-box-binding homeobox 1 and 2) as targets of miR-200c and miR-200b, respectively: ZEB1 and ZEB2 induce EMT by repressing E-cadherin transcription and promoting vimentin transcription. Other studies have provided more detailed information on downregulation of the miR-200 family and miR-205 as an important step in tumor progression and metastasis. Gregory et al. (2008) showed that the miR-200 family may be the downstream molecules of the transforming growth factor-b (TGF-b) pathway in EMT. Moreover, ectopic expression of the miR-200 family or miR-205 leads to downregulation of ZEB1 and ZEB2, upregulation of E-cadherin and mesenchymal–epithelial transition (MET) in cells that had previously undergone EMT (Gregory et al., 2008; Korpal et al., 2008; Park et al., 2008). Therefore, the miR-200 family can reduce EMT by repressing ZEB1 and ZEB2 and promoting E-cadherin, so as to prevent tumor metastasis.

More intriguingly and by contrast, ZEB1 and ZEB2 can also directly suppress transcription of the miR-200 family by binding to E-box or Z-box in their putative common promoter (Bracken et al., 2008; Burk et al., 2008). Findings from miRNA expression microarrays and real-time PCR showed a significant increase in the miR-200 family after knockdown of ZEB1 in undifferentiated pancreatic, colorectal and breast cancer cell lines. These interesting findings render the miRNA regulation of tumor cell EMT to be a more complex process (Figure 1). The miR-200 family is clustered at two locations in the genome (miR-200b, miR-200a and miR-429 located in chromosome 1; miR-200c and miR-141 in chromosome 12), which provides a possible explanation for their posttranscriptional corepression of ZEB1 and ZEB2. However, the miR-200 family is in return negatively regulated by ZEB1 and ZEB2. Moreover, TGF-b, which induces ZEB1 and ZEB2 expression, is also inhibited by miR-141 at the translational level, and consequently, a reciprocal negative feedback loop between the miR-200 family and ZEB1/ZEB2 is generated to maintain the equilibrium between EMT and MET. If some signal, such as a mutation in the miRNA gene or their target gene, breaks down the balance, the loop might as well reinforce expression of the miRNAs or ZEB1/ZEB2, and thus reinduce an epithelial or mesenchymal phenotype. This might explain the considerable phenotypic heterogeneity often seen within individual tumors and metastases. Feedback loops feature in a number of genetic pathways involving miRNAs, in which they seem to enhance the functionality and robustness of gene networks. Otherwise, because ZEB1 is crucial for TGF-b-mediated EMT in various steps in organogenesis (Liu et al., 2008), the reciprocal negative feedback loop between ZEB1/ZEB2 and miR-200 family may also be important in specifying an epithelial or a mesenchymal state not only during embryonic development but also in tumorigenesis.

Figure 1: Pathways of reciprocal negative feedback mediated by the miR-200 family in regulating epithelial–mesenchymal transition (EMT).

Figure 1: Pathways of reciprocal negative feedback mediated by the miR-200 family in regulating epithelial–mesenchymal transition (EMT).

In the nucleus, the pre-miR-200 family (include pre-miR-200a, pre-miR-200b, pre-miR-200c, pre-miR-429 and pre-miR-141) are transcribed and processed from miRNA genes in chromatin 1 and 12. These pre-miRNAs are then exported to the cytoplasm in which they are further processed into mature microRNAs (miRNAs), which are incorporated into the multiple-protein nuclease complex, the RNA-induced silencing complex (RISC). These RISCs inhibit the expression of zinc finger E-box-binding homeobox 1 (ZEB1) and ZEB2 by directly binding 3'-untranslated regions (UTRs) of their mRNAs; moreover, miR-141 can also indirectly inhibit their expression by silencing transforming growth factor-b (TGF-b) posttranscriptionally. However, on one hand the ZEB1 and ZEB2 enter the nucleus to inhibit the pre-miR-200 family transcription and on the other hand, they can activate Vimentin and inactivate E-cadherin. Therefore, miR-200 family inhibits the ZEB1 and ZEB2 so as to prevent the EMT and metastasis by activating E-cadherin and inactivating Vimentin.

Apart from the miR-200 family, some other miRNAs have important regulatory roles in EMT. miR-155, encoding within a region known as Bic (B-cell integration cluster) and identified originally as a frequent integration site for avian leucosis virus (Tam et al., 1997; Lagos-Quintana et al., 2002), can not only regulate the generation of immunoglobulin class-switched plasma cells (Vigorito et al., 2007) but also augment the epithelial cell plasticity as part of EMT by targeting RhoA (Kong et al., 2008). In addition, Valastyan et al. (2009) depicted how miR-31, a pleiotropically acting miRNA, inhibited breast cancer metastasis, and a few pro-metastatic genes had been verified as its targets, such as Fzd3, ITGA5, M-RIP, Matrix metalloproteinases 16 (MMP16), RDX and RhoA. Among these targets, Fzd3, ITGA5, RDX or RhoA can reverse, at least partially, miR-31-dependent metastasis-relevant phenotypes; however, M-RIP and MMP16 not only failed to reverse the phenotypes but also their silence cannot reduce cell invasion and motility in vitro. Therefore, miR-31 impedes metastasis through posttranscriptionally silencing Fzd3, ITGA5, RDX and RhoA.

miRNAs lead to modifications of the extracellular matrix that are involved in the EMT

The urokinase-type plasminogen activator (uPA) encodes a serine protease involved in the degradation of the extracellular matrix (ECM) and possibly tumor cell migration and proliferation. So far, two miRNAs have been identified to posttranscriptionally silence uPA. One is miR-193b, which suppresses the invasive and migratory capacity of breast cancer cell by inhibiting the expression of uPA (Li et al., 2009b), and the other is miR-23b, it can also reduce migratory ability in hepatocellular carcinoma by targeting uPA and c-MET (Salvi et al., 2009). Thus, miR-193b and miR-23b may mediate uPA to degrade the ECM and to regulate tumor metastasis.

Matrix metalloproteinases are a family of more than 28 enzymes that can degrade the ECM and change cell–cell and cell–ECM interactions to induce EMT. Recently, some miRNAs were found to regulate metastasis by targeting MMPs. Liu et al. (2009b) used microarrays to identify a panel of differentially expressed miRNAs that contributed to the metastasis of oral tongue squamous cell carcinoma. Among them, miR-222 was considered as an anti-metastatic miRNA, and at same time, MMP1 and SOD2 (superoxide dismutase 2) were identified as its targets using luciferase reporter gene assays. More interestingly, SOD2 knockdown by small interfering RNA also led to the downregulation of MMP1 expression. Consequently, miR-222 regulates tumor metastasis not only by the direct targeting of MMP1 but also by the indirect controlling of MMP1 gene expression by targeting SOD2. Another representative example is that miR-146b inhibits glioma and breast caner metastasis by silencing MMP16 (Hurst et al., 2009; Xia et al., 2009). However, miR-146a might not simply be involved in the innate immune response and modulation (Taganov et al., 2006; Dai et al., 2008), it seems to arrest EMT and inhibit invasion and migration (Hurst et al., 2009; Liu et al., 2009a). Taken together, these current studies provide a new challenging insight into the possibility that miRNAs may be the regulating bridge between tumor metastasis and the immune state. The understanding of this linkage offers potential opportunities for tumor treatment because the immune state of tumor patients is associated with prognosis.

miRNA and metastasis-associated genes

Microarray studies have identified sets of genes whose expression in primary tumors correlates with metastasis and poor prognosis. These metastasis-associated genes have important roles in tumor invasion and metastasis, and miRNA networks act as upstream regulators of these genes in tumorigenesis and metastasis. An increasing number of pro-metastatic miRNAs (Table 1) and anti-metastatic miRNAs (Table 2) from miRNA profiling of tumor versus non-tumor tissue have been identified as regulatory inhibitors of metastasis genes or metastasis suppressor genes.

Table 1 - Pro-metastatic miRNAs regulating tumor metastasis.

Table 1 - Pro-metastatic miRNAs regulating tumor metastasis.

Table 2 - Anti-metastatic miRNAs regulating tumor metastasis.
 

MicrioRNA-10b downregulation in breast cancer was first reported by Iorio et al. (2005), but Ma et al. (2007) showed a new molecular pathway, in which miR-10b promoted breast tumor metastasis. The ectopic expression of miR-10b in non-invasive breast cancer cells conferred invasive and metastatic properties in vitro and in vivo without affecting the viability or proliferation of these cells. To elucidate the mechanism, they validated miR-10b as an essential element in Twist-induced metastasis, and Twist was shown by ChIP (chromatin immunoprecipitation) assay to promote miR-10b directly. But the breast cancer metastasis suppressor 1 (BRMS1) was validated to suppress pro-metastatic miRNAs (miR-10b, miR-520c and miR-373), concurrently, it increased expression of anti-metastasis miRNA (miR-146a, miR-146b and miR-335) (Edmonds et al., 2009). Furthermore, the HoxD10 (homeobox transcription factor) that promotes or maintains a differentiated phenotype in epithelial cells was identified as a target of miR-10b. In turn, the downregulation of HoxD10 induced the expression of RhoC, a well-characterized G-protein pro-metastatic gene product (Clark et al., 2000; Myers et al., 2002; Carrio et al., 2005). Although miR-10b overexpression does not correlate with distant metastases or poor prognosis in breast cancer clinically (Gee et al., 2008), the BRMS1/Twist/miR-10b/HOXD10/RHOC inter-relation is certainly a first example of metastasis-related interplay between miRNAs and cancer metastasis-associated genes.

As described previously, HOXD10 may be a key in the cancer metastatic miRNA pathway. In addition, it still positively regulates miR-7 (Reddy et al., 2008), which was found to control epidermal growth factor receptor signaling and promote photoreceptor differentiation, inducing cell cycle arrest and cell death in some cancer cell lines (Kefas et al., 2008; Webster et al., 2009). miRNA expression profiling revealed that miR-7 was involved in tumor aggressiveness, invasion and metastasis in urothelial carcinomas and breast cancer (Foekens et al., 2008; Veerla et al., 2009). Reddy et al. (2008) found that miR-7 inhibited p21-activated kinase 1 (Pak1) that has the potential to promote tumor invasion and metastasis. Accordingly, the HoxD10/miR-7/Pak1 and BRMS1/Twist/miR-10b/HOXD10/RHOC pathways form a cascade effect for tumor metastasis (Figure 2).

Figure 2. Regulation of tumor metastasis by miR-10b and miR-7. BRMS1/Twist/miR-10b/HOXD10/RHOC and HoxD10/miR-7/Pak1 pathways form a cascade effect for tumor metastasis.

Figure 2. Regulation of tumor metastasis by miR-10b and miR-7. BRMS1/Twist/miR-10b/HOXD10/RHOC and HoxD10/miR-7/Pak1 pathways form a cascade effect for tumor metastasis.

BRMS1, breast cancer metastasis suppressor 1; Pak1, p21-activated kinase 1.

Previous studies have shown that miR-21 expression is increased in many solid tumors, such as glioblastoma (Ciafre et al., 2005), breast (Iorio et al., 2005), lung, prostate, colon and stomach cancer (Volinia et al., 2006). But miR-21 seems to produce a more marked effect on tumor metastasis. The level of miR-21 expression correlates significantly with advanced clinical stage, metastasis and poor prognosis in these tumors (Krichevsky and Gabriely, 2009). Moreover, miR-21 was found to stimulate cell invasion and metastasis in different tumor models (breast cancer, colon cancer and gliomas, for example) both in vitro and in vivo (Asangani et al., 2008; Gabriely et al., 2008; Zhu et al., 2008). Many targets of miR-21 have been identified by bioinformatic prediction and molecular biological assay, which include some metastasis-associated genes directly regulating tumor metastasis. Tropomyosin 1, identified as a target gene of miR-21 by proteomics (Zhu et al., 2007), was associated with actin in all cell types studied. It serves as an actin-binding protein and stabilizes microfilaments, and the downregulation of tropomyosin 1 promotes cell transformation and tumor metastasis (Boyd et al., 1995). Programmed cell death 4 (PDCD4) is a potentially important target of miR-21, not only because it has been experimentally validated in colon cancer (Asangani et al., 2008), breast cancer (Frankel et al., 2008; Zhu et al., 2008; Wickramasinghe et al., 2009) and cholangiocarcinoma (Selaru et al., 2009) but also because PDCD4 can conversely inhibit miR-21 by controlling AP-1 (JunB, c-Jun and Fra-1) activity in RAS transformation (Fujita et al., 2008; Talotta et al., 2009). Similar to PDCD4, NFIB, as the target of miR-21, also represses transcription of pri-miR-21 (Fujita et al., 2008). Besides the autoregulatory loop, the miR-21 was still regulated by several tumor metastasis-associated genes. TGF-b, EGF, BMP2 and BMP4 induce the expression of miR-21 in a posttranscriptional step (Davis et al., 2008; Gabriely et al., 2008; Papagiannakopoulos et al., 2008); nevertheless, BMP6 inhibits miRNA-21 expression in breast cancer by repressing ZEB1 and AP-1 (Du et al., 2009). With the reciprocal negative feedback loop between ZEB1/ZEB2 and miR-200, these upstream regulators, auto-regulators and their targets, such as PTEN (Meng et al., 2007; Asangani et al., 2008), maspin (Zhu et al., 2008), MARCKS (Li et al., 2009a), RECK (Gabriely et al., 2008), TiMP3 (Selaru et al., 2009) and SPRY2 (Sayed et al., 2008) create a delicate miR-21 feedback network to regulate tumor metastasis, as shown in Figure 3.

Figure 3. miR-21 network and feedback regulation in tumor metastasis.

Figure 3. miR-21 network and feedback regulation in tumor metastasis.

Multi-upstream regulators and multi-targets of miR-21 constitute a delicate network to regulate tumor metastasis with miR-200 reciprocal negative feedback loop. TGF-b, transforming growth factor-b; ZEB, zinc finger E-box-binding homeobox.

miR-373 and miR-520c, which belong to the same miRNA family, have been identified as metastasis-promoting miRNAs through a genetic screening of a non-metastatic human breast tumor cell line (Huang et al., 2008). miR-373 was previously identified as a potential oncogene in testicular germ-cell tumors: it suppressed the oncogene-induced p53 pathway and cooperated with oncogenic RAS to promote cellular transformation, in part through direct inhibition of the tumor suppressor, LATS2 (Voorhoeve et al., 2006). miR-520c was discovered by sequencing the genes of miRNAs in cells (Bentwich et al., 2005), but has not yet been functionally characterized. Huang et al. (2008) showed that miR-373 and miR-520c promoted a migratory and invasive phenotype in vitro and in vivo by overexpressing themselves in MCF cells transplanted into nude mice. CD44, encoding a cell surface receptor for hyaluronan, is lost in breast cancer with high metastatic potential and acts as a metastatic suppressor in prostate and colon cancer. Recently, CD44 was identified as the common target of miR-373 and miR-520c (Huang et al., 2008; Yang et al., 2009). Moreover, miR-373 inversely correlated with CD44 expression, and was overexpressed in breast carcinomas, especially in those with lymph node metastasis (Huang et al., 2008). These clinical data further supported miR-373 as a metastasis-associated miRNA. Another tumor metastasis-promoting miRNA, miR-182, was recently identified in melanoma. MiR-182 is a member of a miRNA cluster in a chromosomal locus (7q31-34) with a much-amplified gene copy number in melanoma. Its ectopic expression stimulates migration of melanoma cells in vitro and in vivo by directly repressing microphthalmia-associated transcription factor and FOXO3 (Segura et al., 2009). Recently, a report (Zhang et al., 2009) showed a novel pathway mediated by NF-kB (nuclear factor-kB) that promoted the metastasis of hepatitis B virus-related hepatocellular cancer. Besides its transcriptional regulation of invasion-related factors, such as MMP9 and vascular endothelial growth factor, miR-143 was directly regulated by NF-kB to repress fibronectin type III domain containing 3B(FNDC3B) gene expression. Furthermore, the downregulation of FNDC3B enhanced the invasion and migration capability of hepatocarcinoma cell. Therefore, NF-kB/miR-143/FNDC3B pathway is an important complement to the NF-kB-mediated metastasis network.

Some miRNAs have been identified to be involved in angiogenesis, but few miRNAs were confirmed to regulate metastatic tumor angiogenesis. At present, although no data suggests that miR-378 can regulate tumor metastasis, it can promote tumorigenesis and angiogenesis by targeting the tumor suppressors, SuFu and Fus-1 (Lee et al., 2007). Chen and Gorski (2008) showed that miR-130a regulated the angiogenic phenotype of vascular endothelial cells through its ability to inhibit the expression of the homeodomain gene, GAX, and the antiangiogenic homeobox gene, HOXA5. The miR-17-92 cluster, including miR-17, miR-18a, miR-19a, miR-20a, miR-19b and miR-92a, showed potent tumor angiogenesis-promoting activity (Venturini et al., 2007). Myc can activate miR-17-92 cluster to enhance tumor vessel growth in a non-cell-autonomous manner in RAS-expressing cells (Dews et al., 2006). The connective tissue growth factor and the potent angiogenesis-inhibitor, thrombospondin-1, were identified as their targets (Dews et al., 2006). But the role of these miRNAs in tumor metastasis remains to be determined. So we speculate that they may facilitate tumor dissemination and metastasis by regulating vascular function.

Anti-metastatic miRNAs regulate tumor metastasis

Wang et al. (2008) identified miR-183 as a negative regulator of lung cancer metastasis by screening with a miRNA array. The target gene, Ezrin, which was confirmed by luciferase reporter gene assay, has a role in controlling the actin cytoskeleton, cell adhesion and motility. Another metastasis suppressor, miR-335, was found to upregulate in neonatal mouse and fetal human lung as a maternally imprinted miRNA cluster located on human chromosome 14q32.31 (Williams et al., 2007). However, Tavazoie et al. (2008) found that miR-335, miR-126 and miR-206 were potential metastasis suppressor miRNAs in human breast cancer by performing array-based miRNA profiling. The miRNAs were consistently downregulated in metastatic foci, and restoring their expression significantly decreased the number of metastatic foci. The low miR-335 or miR-126 expression in human primary tumors was significantly associated with poor metastasis-free survival, which provided clinical relevance to their findings. To identify putative metastasis genes, the authors analysed the transcriptional profile between lung metastasis cells and cells with restored miR-335 expression. SOX4, TNC, PTPRN2, MERTK were further verified as miR-335 target genes, and the knockdown of these genes can diminish in vitro invasive ability and in vivo metastatic potential. The downregulation of miR-126 was also found in lung cancer (Yanaihara et al., 2006). miR-126 can decrease adhesion, migratory and invasive capacity of lung caner cell lines through posttranscriptional silencing Crk (Crawford et al., 2008). At the time of submission of this paper, Song et al. (2009) found that miR-206 can activate apoptosis, and inhibit cancer cell migration and foci formation by targeting NOTCH3. Moreover, the discovery that miR-206 expression was downregulated in estrogen receptor-+-positive human breast cancer (Kondo et al., 2008) supports miR-206 as a anti-metastatic miRNA.

The let-7 miRNA family is one of the tumor suppressing miRNAs that can inhibit both tumorigenesis and metastasis. The first direct piece of evidence for a role of let-7 in cancer was the observation that let-7 was reduced in a significant number of lung cancer cell lines and primary human lung cancer tissues (Takamizawa et al., 2004). Several important oncogenes have been identified as the targets of let-7, including RAS, MYC and HMGA2 (Johnson et al., 2005; Mayr et al., 2007; Chang et al., 2008). These target genes, making use of the RAS–MEK pathway contribute to EMT, though let-7 miRNA-mediated HMGA2 downregulation had no effect on the prevention of the transformed phenotype in pancreatic cancer cells (Watanabe et al., 2009). Another let-7 miRNA-mediated tumor metastasis-regulating pathway was addressed recently: Raf kinase inhibitory protein, which inhibits mitogen-activated protein kinase signaling cascades, can decrease transcription of LIN28 by Myc. Suppression of LIN28 enabled it to enhance let-7 processing in breast cancer cells, allowing the elevated let-7 expression to inhibit HMGA2 that activated pro-invasive and pro-metastatic genes. As let-7 targets RAS, the upstream activator of Raf-1, a positive feedback loop emerged to control tumor invasion and metastasis (Dangi-Garimella et al., 2009). Strikingly, Yu et al. (2007) have now provided an evidence for upregulation of let-7 in breast cancer stem cells. As targets of let-7, H-RAS and HMGA2 were downregulated when let-7 was overexpressed. However, there was a significant difference in the biological function between H-RAS and HMGA2: silencing H-RAS in a cancer stem cell-enriched breast cancer cell line reduced self-renewal, but had no effect on differentiation. By contrast, silencing HMGA2 enhanced differentiation, but did not affect self-renewal. Cancer stem cell has the capacity of unlimited self-renewal and can differentiate into multiple cell types, allowing them to repopulate tumors after therapy and seed metastasis to distant sites. More intriguingly, the MYC was posttranscriptionally regulated by let-7, but it can also control the expression of let-7 (or other miRNAs) at the level of transcription through repressing RNA polymerase-II to prevent pri-let-7 RNA transcription (Sampson et al., 2007; Chang et al., 2008). Similar to MYC, LIN28, as a target of let-7, can also prevent either pri-let-7 and/or pre-let-7 from processing mature let-7 in murine embryonic stem cells and undifferentiated cancer stem cells (Rybak et al., 2008). Consequently, let-7, MYC and LIN28 interact to make an autoregulatory feedback loop (Figure 4).

Figure 4. The autoregulatory feedback loop of let-7, MYC and LIN28.

Figure 4. The autoregulatory feedback loop of let-7, MYC and LIN28.

The let-7 micro RNA (miRNA) inhibits tumor metastasis through silencing MYC, RAS and HMGA2; however, MYC can also inhibit let-7 in transcriptional and posttranscriptional level by regulating RNA polymerase II (RNA pol II) and Lin28. CSC, cancer stem cell.

Recently, several other miRNAs were proposed to suppress tumor metastasis. Tsuchiya et al. (2009) showed that miR-338-3p and miR-451 contributed to the formation of epithelial basolateral polarity by facilitating translocalization of 1-integrin to the basolateral membrane in an epithelial differentiation model. Moreover, miR-451 can also regulate cancer proliferation and metastasis through targeting macrophage migration inhibitory factor (Bandres et al., 2009). Another tumor suppressor miRNA, miR-122, negatively impacts hepatocellular carcinoma intrahepatic metastasis by suppressing angiogenesis, and exerts its action through regulation of the disintegrin and metalloprotease 17 (ADAM17) (Tsai et al., 2009). The gene expression data (Coulouarn et al., 2009) showed that the hepatocyte nuclear factors (HNF1A, HNF3A and HNF3B) may transcriptionally activate miR-122 by interacting with chromatin. These pro-metastatic and anti-metastatic miRNAs comprise a complex network controlling tumor metastasis.

The reciprocal regulation between miRNA and epigenetic modification in tumor metastasis

Epigenetic modifications include DNA methylation and covalent modification of histones. These alterations are reversible, but very stable, and have a significant impact on the regulation of gene expression, the contribution of which to cancer goes beyond the early stages of tumor transformation to affect metastasis. However, miRNAs have an important role in epigenetic modification, and are also regulated by epigenetic mechanisms in tumor metastasis.

Lujambio et al. (2008) used a pharmacological and genomic approach to induce a loss of DNA methylation associated with release of miRNA gene silencing in cancer cells, and discovered five hypermethylated miRNAs, such as miR-148a, the three members of the miR-9 family and the miR-34b/c cluster, which exhibited cancer-specific methylation. Among these methylated miRNAs, restoration of the expression of miR-148a and the miR-34b/c cluster affected invasive capacity both in vitro and in vivo. The miR-34b/c cluster inhibits cell motility, tumor growth and metastasis formation through downregulation of their oncogenic targets, such as MYC, E2F3 and cyclin-dependent kinase 6. More strikingly, reports from several laboratories (Bommer et al., 2007; Chang et al., 2007; Corney et al., 2007; He et al., 2007) have, almost simultaneously, come to a similar conclusion: those members of the miR-34 family are directly regulated by p53, which induces apoptosis, cell cycle arrest and senescence. These data reinforce the awareness that miR-34 genes are central mediators of p53 function. Therefore, miRNAs may be the key players in tumor development by being located centrally within the p53 tumor suppressor network, and there is a balanced state between epigenetic modification and p53 regulating the expression of miR-34.

The epigenetic inactivation of miR-148 has been found in several cancer cells, for example, in melanoma and breast cancer (Lehmann et al., 2008; Lujambio et al., 2008). DNA methylation-associated silencing of miR-148 contributes to the development of cancer metastasis by upregulating its target gene, TGIF2. Interestingly, the overexpression of miR-148 led to the reduction in both mRNA and protein levels of DNA methyltransferase 3B (DNMT3B), and Duursma et al. (2008) confirmed that certain splice variants of human DNMT3B mRNA were the direct targets of miR-148. miR-148 repressed expression of DNMT3B through binding with high homology to the region of the coding sequence of the DNMT3B transcript, but the mechanism remained unclear. In contrast to miR-148a, miR-101 modulates cancer epigenetics by repressing the polycomb group protein EZH2, which contributes to the epigenetic silencing of target genes and regulates the survival and metastasis of cancer cells (Varambally et al., 2008; Friedman et al., 2009). Not only does miR-101 suppress the expression and function of EZH2 in cancer cell lines but also there is an inverse correlation between miR-101 expression and EZH2 expression during cancer progression in human prostate tumors (Varambally et al., 2008). Varambally et al. (2008) found that loss of one or the other of the two genomic loci of miR-101 occurred more frequently in metastatic tumor cells. Therefore, the genomic loss leads to overexpression of EZH2 and concomitant dysregulation of epigenetic pathways, resulting in cancer progression and metastasis.

In addition, the miR-29 family, including 29a, 29b and 29c, may be one of the crucial modulators of epigenetic alteration in tumor metastasis. Fabbri et al. (2007) revealed an inverse correlation between miR-29s and DNMT3A and DNMT3B in lung cancer tissues, and identified DNMT3A and DNMT3B as their direct targets. The enforced expression of miR-29s in lung cancer cell lines recovered normal patterns of DNA methylation following hypermethylation, and induced reexpression of the methylation-silenced tumor suppressor genes such as FHIT and WWOX. Otherwise, miR-29a promotes disruption of epithelial polarity and metastasis by suppressing tristetraprolin in vitro and in vivo (Gebeshuber et al., 2009). However, miRNA 29c is downregulated so as to upregulate mRNAs encoding ECM proteins that are involved in metastasis in nasopharyngeal carcinomas (Sengupta et al., 2008). More intriguingly, miR-29s also enter the p53 network: they activate p53 by targeting p85a and CDC42 (Park et al., 2009). Accordingly, the reciprocal repression between miRNAs and abnormal methylation (see Figure 5) may contribute to tumor invasion and metastasis.

Figure 5. Interaction between microRNAs (miRNAs) and abnormal methylation to control metastasis.

Figure 5. Interaction between microRNAs (miRNAs) and abnormal methylation to control metastasis.

The reciprocal regulation between miRNAs and abnormal methylation through DNA methyltransferases (DNMTs), EZH2 and P53 pathway may contribute to tumor invasion and metastasis.

Increasing evidence is now available to suggest that miRNAs, which function as either oncogenes or tumor suppressor genes, have a significant role in tumor development and prognosis. A modest change of one miRNA will provoke a chain reaction and feedback pathways involving multiple miRNAs and affecting multiple target genes of the same or different pathways. Accordingly, the deregulation of one single miRNA is sufficient to trigger global alterations of genetic programs implicated in cell proliferation, differentiation, survival or invasiveness.

Nevertheless, the role of miRNAs in mediating tumor metastasis is only now starting to be addressed, and this may raise the curtain on the elucidation of the mechanism of tumor metastasis. So far, some miRNAs have been confirmed as regulating the invasion–metastasis cascade. However, along with the identification of more miRNA signatures of tumor metastasis, they will weave a more complex and interrelated tumor metastasis network by interacting with protein-coding genes. The network will become more complex and comprehensive, but the essence of tumor metastasis should be revealed more thoroughly.

Conflict of interest:

The authors declare no conflict of interest.

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In their comprehensive new Review, Drs. H Zhang , Y Li  and M Lai have discussed new functions for microRNAs beyond the usual effects on messenger RNAs. It is now apparent that microRNAs also participate in mutual positive and negative reciprocal reactions with gene transcription at a wide variety of genes and gene clusters, at various times, and for various durations. These widening actions correspond to a similar widening activity of multiple genes on the same or separate chromosomes, within clusters of active genes undergoing transcription. These have been shown to involve as many as 3 chromosomes on one DNA locus, for either short or longer intervals, and suggest that these Kissing gene sites are being activated or programmed by their visitors (Schoenfelder, S, et al, "Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells", Nature Genetics, January 1, 2010; 42(1): 53-61.)
We may be confronting a more complex cluster of changing gene sites, that are responding to other more distant genes within the same nucleus. The context of gene activity is thus broadend even within normal adult cells. And in many cancer cells, embryonic-exclusive genes are also expressed within adult cells.

1. Schoenfelder S, Sexton T, Chakalova L, Cope NF, Horton A, Andrews S, Kurukuti S, Mitchell JA, Umlauf D, Dimitrova DS, Eskiw CH, Luo Y, Wei C-L, Ruan Y, Bieker JJ, and Fraser P,
"Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells".

2. Junier I, Martin O, and  Képès F, (2010).
"Spatial and Topological Organization of DNA Chains Induced by Gene Co-localization".

3. Lanctôt C, Cheutin T, Cremer M, Cavalli G, Cremer T (2007). Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions. Nat. Rev. Genet. 8, 104-115.

4. Nicodemi M, Prisco A (2009)  Biophys J., 96: 2168.
"Thermodynamic Pathways to Genome Spatial Organization in the Cell Nucleus".

5. Frenster JH, and Hovsepian JA,  (2006)
"DNase-I Ultrastructural Probe Sites and Kissing Chromosomes".

6. Frenster JH, and Hovsepian JA, (2008 )
"Models of successive levels of resolution during individual gene transcription".

1. Each cell retains all of its embryonic genes for a lifetime.

2. Controls for embryonic genes are often absent in adults.

3. Uncontrolled embryonic genes can replicate wildly.

4.  Replicating genes participate in  intra-cellular competition.

5.  The basis for gene competition is selective transcription.

6.  MicroRNAs can reprogram embryomic transcription.

7.  Gene reprogramming can produce normal phenotypes.

8.  Normal phenotypes can by-pass chromosomal lesions.

9.  MicroRNA therapy may need to be permanent.

10. Transplantation of microRNAs could be preferred.

http://www.embryomas.net/

1. Pathways within cell genomes involve a flow of information.

2. Information can flow by direct contact or by third parties.

3. Direct contact within whole genomes is difficult to regulate.

4. DNA-DNA direct contects are influenced by agents.

5. Nuclear agents include hydrophilic ionic and hydrophobic conforming ligands.

6. Third parties within genomes involve RNAs and proteins.

7.  RNAs and proteins are easy to regulate or reverse.

8.  Information can be shared, lost, or transformed.

9. System information can be hidden during system isolation.

10.  Local information can be permanently lost during system entropy.

http://www.embryomas.net/

Links to Current Research in Euchromatin:
Links to Euchromatin Activator RNA Reviews:
Links to Euchromatin Activator RNA Research:
Links to Ultrastructural Probes of DNase I-Sensitive Sites:
Links to RNA as a Therapeutic Agent:
Links to Hodgkin Lymphoma Immuno-Pathology:
Links to Activated T-Lymphocyte Immunotherapy:
Links to Medical Systems Biology:
Links to Selective Gene Transcription:
Links to RNA-Induced Epigenetics:
Links to RNA-Induced Embryogenesis:
Links to RNA and Biological Causality:
Links to Reprogramming and Neoplasia:

A Brief History of Activator RNA:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA".
(PowerPoint Presentation).

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For Further Information and Feedback:

Jeannette A. Hovsepian, M.D.
E-mail: frensasc@ix.netcom.com
Phone:  +1 650 367 6483