Abstract: Over the last decade, a growing number of non-coding transcripts have been found to have roles in gene regulation and RNA processing. The most well known small non-coding RNAs (ncRNAs) are the microRNAs (miRNAs), but the network of long and short non-coding transcripts is complex and is likely to contain as yet unidentified classes of molecules that form transcriptional regulatory networks. miRNAs and some other ncRNAs have been found to be involved in human tumorigenesis, revealing a new layer in the molecular architecture of cancer. Gene expression studies have shown that hundreds of miRNAs are deregulated in cancer cells, and functional studies have clarified that miRNAs are involved in all the molecular and biologic processes that drive tumorigenesis. Here, we summarize the recent advances in understanding miRNAs' and other ncRNAs' involvement in cancer and illustrate how this knowledge may be useful in medical practice. New diagnostic classifiers based on miRNAs will soon be available for medical practitioners, and even more importantly, miRNAs may become novel anti-cancer therapies.
Non-coding RNAs (ncRNAs) are RNAs that do not encode proteins (Eddy, 2001; Mattick, 2009). Until a decade ago, ncRNAs went unnoticed in the genome sequencing field because the central dogma of genetics was that RNA is the messenger between genes and the final proteins that they encode. However, recent improvements in high-throughput technology for gene expression assays have led to the discovery that most human transcriptional units are ncRNAs (Bertone et al., 2004; Birney et al., 2007). Moreover, newly discovered ncRNAs seem to play important roles in several gene regulation mechanisms (Mercer et al., 2009; Ponting et al., 2009).
Long ncRNAs and Cancer
ncRNAs can be classified by their length. Long ncRNAs (lncRNAs) are typically >200 nucleotides long. Small ncRNAs are processed from longer precursors and include Piwi-interacting RNAs, small interfering RNAs (siRNAs), microRNAs (miRNAs), and some bacterial regulatory RNAs (Brosnan and Voinnet, 2009). Many ncRNA classes have been extensively studied, and their regulatory roles in cell biology are broadly recognized. miRNAs are well-known ncRNA molecules that regulate various gene expressions at the RNA level. Likewise, lncRNAs such as ultraconserved genes (UCGs) and HOX antisense intergenic RNA (HOTAIR) have been characterized.
The deregulation of several ncRNA types has been reported in various human diseases. The most robust data support a role for ncRNAs in human carcinogenesis, providing a rationale for targeting these molecules as anticancer agents. However, to date, no clear association with cancer has been found for several ncRNA subgroups: miRNAs, UCGs, and HOTAIR. Therefore, in the present review, we focus on recent progress in understanding the association between miRNAs, UCGs, and HOTAIR and cancer.
lncRNAs range in size from approximately 200 nucleotides to over 100 kilobases. Like messenger RNAs (mRNAs), they seem to mostly be transcribed by RNA polymerase II, but many lncRNAs do not undergo the subsequent standard mRNA processing steps (Motamedi et al., 2004). This feature is intimately linked to their regulatory functions, which often involve nuclear retention close to transcription sites (Seidl et al., 2006). Although the function and mechanism of lncRNAs are largely unknown, substantial evidence suggests that they mirror protein-coding genes (Taft et al., 2010). lncRNAs have been implicated as principal players in imprinting and X-inactivation.
Recent large-scale genome sequencing has demonstrated that although lncRNAs make up a large portion of the mammalian transcriptome, their function has remained elusive (Mercer et al., 2009). However, further detailed investigations have shown that lncRNA is involved in fundamental cellular processes such as RNA processing, gene regulation, chromatin modification, gene transcription (controlling the RNA polymerase II activity and taking part as cofactors in RNA-protein complexes), and post-transcriptional gene regulation (splicing, editing, transportation, translation, and degradation) on the basis of RNA sequence complementarity interactions (Mercer et al., 2009; Orom et al., 2010).
lncRNAs’ mechanism of action is generally unknown, but it may be similar to that of an lncRNA termed HOX antisense intergenic RNA (HOTAIR). HOTAIR regulates the chromatin methylation state of the HOXD locus in trans through polycomb repressive complex 2 (PRC2). HOTAIR associates with PRC2 and lysine-specific demethylase 1/RE1-silencing transcription factor co-repressor 1/RE1-silencing transcription factor protein complexes, coordinates the chromatin targeting of these proteins, and couples histone H3K27 methylation and H3K4 demethylation for epigenetic gene silencing (Rinn et al., 2007; Tsai et al., 2010). HOTAIR has recently been reported to have a role in cancer metastasis (Gupta et al., 2010).
Another category of ncRNAs is the recently identified ultraconserved genes (UCGs), which are transcribed from ultraconserved regions (UCRs) (Calin et al., 2007) and have a tissue-specific expression pattern (Allen et al., 2004; Calin et al., 2007). Researchers became aware of UCRs when the alignments of human, rat, and mouse genomes demonstrated that despite 300 million years of negative selection, some genomic regions remained highly conserved (100% identity) (Bejerano et al., 2004; Mercer et al., 2009). In fact, UCRs are even more conserved than coding genes and are now believed to have fundamental functions in vertebrate evolution, including that of mammals (Barbarotto et al., 2008; Bejerano et al., 2004; Elgar and Vavouri, 2008; Mattick, 2009). UCRs are frequently found in fragile genomic regions that are usually associated with cancer, and UCG expression is aberrant in several human carcinomas and leukemias compared with that in normal tissue counterparts (Calin et al., 2007). Deregulated UCG signatures are cancer specific and have prognostic implications. Similar to miRNAs, UCGs can act as oncogenes or tumor suppressor genes, and UCG expression is controlled by miRNAs (Calin et al., 2007).
MicroRNAs and Cancer
miRNAs constitute a large class of phylogenetically conserved single-stranded RNA molecules of 19 to 25 nucleotides that are involved in post-transcriptional gene silencing. They arise from intergenic or intragenic (both exonic and intronic) genomic regions that are transcribed as long primary transcripts. Primary transcripts undergo two processing steps that produce the short “mature” molecule. The mature miRNA binds to specific regions of target mRNA transcripts and destabilizes the target mRNA transcript, blocks its translation, or both (for detailed reviews, see Ghildiyal and Zamore, 2009 and Brodersen and Voinnet, 2009). miRNAs not only regulate various developmental and physiologic processes but also are involved in cancer development (Figure 1).
About 50% of annotated human miRNAs are mapped in fragile regions of chromosomes, which are areas of the genome that are associated with various human cancers (Calin et al., 2004). Genomic instability that disrupts miRNA-target gene regulation has been associated with an increasing number of cancer types (Selcuklu et al., 2009). Global miRNA processing inhibition can lead to increased tumorigenicity and transformation (Kumar et al., 2007). miRNAs have been shown to act as oncogenes (promoting malignant potential) and tumor suppressor genes (blocking malignant potential) and induce all of the six hallmarks of malignant cells (Hanahan and Weinberg, 2000; Negrini et al., 2009).
Self-sufficiency in growth signals
To become independent from external growth factor signals and evade tissue homeostasis, tumor cells must activate different pathways to sustain cell proliferation and survival. The mammalian RAS proteins H-RAS, K-RAS, and N-RAS modulate many major proliferation pathways in cells and their deregulation has crucial consequences in tumor development. In fact, RAS-activating mutations are commonly observed in tumor cells. RAS oncogenic signaling also occurs in response to decreased levels of let-7, a well-documented post-transcriptional RAS regulator (Johnson et al., 2005) that is inversely correlated with RAS expression in solid and hematologic malignancies. Low let-7 expression has been shown to indicate poor prognosis (short post-operative survival) in lung cancer patients (Esquela-Kerscher and Slack, 2006) and head and neck squamous cell carcinoma patients (Childs et al., 2009).
In general, let-7 acts as a tumor suppressor by targeting oncogenes such as RAS and HMGA2 and is downregulated in many solid organ cancers (Boyerinas et al., 2010). Let-7 down-modulation can also help tumor cells grow in an anchorage-independent manner and not undergo apoptosis after they lose contact with the basal membrane. Low let-7 expression levels in tumors likely exert this effect (Mayr et al., 2007; Schultz et al., 2008) by de-repressing the pleiothropic architectural transcription factor HMGA2, a major let-7 target that contributes to multiple differentiation programs (Lee and Dutta, 2007). The clinical relevance of let-7’s anti-proliferative effects was recently demonstrated by let-7-induced tumor regression in in vivo murine lung cancer models (Esquela-Kerscher et al., 2008; Kumar et al., 2008); this evidence is promising for the development of let-7-based anti-cancer therapies.
miR-21, one of the most frequently upregulated miRNAs in solid tumors, also participates in RAS oncogenic signaling. miR-21 is transcriptionally induced by AP1 downstream of RAS and exerts its oncogenic effect by keeping phosphatase and tensin homolog and PDCD4 in check (Talotta et al., 2009).
Because PDCD4 negatively controls AP1, AP1-induced miR-21 represents a positive feedback loop that sustains AP1 activity in response to RAS (Talotta et al., 2009). miR-21 overexpression enhances tumorigenesis, and genetic deletion partially protects against tumor formation in transgenic mice with loss-of-function and gain-of-function miR-21 alleles and non-small cell lung cancer. miR-21 drives tumorigenesis by inhibiting negative RAS/mitogen-activated protein kinase/extracellular signal-regulated kinase pathway regulators and inhibiting apoptosis (Hatley et al., 2010).
Insensitivity to anti-growth signals
The retinoblastoma (RB) pathway is one of the major cell cycle regulatory routes that is altered in almost all human cancer cells (Malumbres and Barbacid, 2001). RB is a transcriptional repressor that inhibits cell cycle transcription factors of the E2F family, resulting in proliferative arrest and inhibition of the expression of genes required for cell cycle progression. E2F transcription factors play a critical role in controlling cell cycle progression by regulating the timely expression of genes required for DNA synthesis at the G1/S phase boundary.
The p16INK4/RB/E2F pathway is defective in a wide range of human tumors, resulting in deregulated and hyperactive E2F in transformed cells and uncontrolled cell proliferation (Emmrich and Putzer, 2010). E2F transcription factor activities are also controlled at the post-transcriptional level by a series of miRNAs. miR-17-5p, miR-20a, miR-106b, and miR-92 were found to inhibit E2F1 (O’Donnell et al., 2005; Petrocca et al., 2008), whereas miR-20a was shown to inhibit E2F2 and E2F3 (Sylvestre et al., 2007). Furthermore, E2F-activating transcription factors have been shown to regulate the expression of clustered microRNAs (Petrocca et al., 2008; Sylvestre et al., 2007; Woods et al., 2007). Unbound E2Fs increase miR-17~92 and miR-106b~25 expression; in turn, these keep E2F levels in check and participate in the delicate equilibrium between cell proliferation and apoptosis, which is affected by alterations in E2F levels. In addition, two miRNAs of the miR-17~92 cluster, miR-17-5p and miR-20a, have been reported to negatively regulate E2F1 (O’Donnell et al., 2005). Upregulated miRNAs from these clusters target apoptotic and growth inhibitory proteins such as BIM and p21 (Petrocca et al., 2008).
The miR-17~92 cluster is also essential for integrating signals during the G1 phase of the cell cycle and determining whether a signal should be interpreted as proliferative or apoptotic (Coller et al., 2007). In physiologic conditions, the miR-17~92 cluster can limit MYC activation by dampening the E2F positive feedback loop. In tumors with MYC activation, the miR-17~92 cluster protects cells from MYC-induced apoptotic E2F responses, leading to uncontrolled cellular proliferation. miR-17-5p levels were found to be inversely correlated with E2F1 levels in colon cancer tumor samples, suggesting that these miRNAs promote malignancy in tissue by rendering cells insensitive to E2F1’s apoptotic function (Diaz et al., 2008).
The oncomiR-17~92 is upregulated in many lymphoproliferative disorders (Zanette et al., 2007) and solid cancers, not only through MYC activation but also through genomic amplification, BCR-ABL regulation (Venturini et al., 2007), or retroviral insertion (Cui et al., 2007). The inhibited proapoptotic phenotypes of the miR-106b~25 family were found to be induced in gastric cancer (Petrocca et al., 2008). Therefore, miR-106b~25, like miR-17~92, seems to prevent excessively high E2F1 expression levels that can cause tumor cell apoptosis. In contrast to miR-17~92, in which the growth-promoting effect is mediated by E2F1 inhibition, miR-330-3p acts as a tumor suppressor; it was found to negatively regulate E2F1 expression in prostate cancer cells (Lee et al., 2009).
Apoptosis is a physiologic self-destruction cellular mechanism that leads to the removal of unwanted cells. p53 is the most widely studied transcription factor; it controls the expression of several genes to promote tumor suppression. p53 responds to diverse cellular stress and regulates target genes involved in DNA integrity, cell cycle arrest at the G1/S checkpoint, and apoptosis. It is the most commonly mutated gene in human cancer. miRNAs have been found to function downstream of p53. miRNAs such as miR-34, miR-192/215, miR-107, and miR-145 are known transcriptional targets of p53 (Boominathan, 2010). miR-15a and miR-16-1 are clustered on human chromosome 13q14, which is frequently lost or downregulated in B-cell chronic leukemia and several solid tumor types (Calin et al., 2008). The cancer-associated genomic region 1p36, which is lost or rearranged in many tumor types, including those from neural, epithelial, and hematopoietic tissues, contains the candidate tumor suppressor miR-34a.
The miR-34 family is composed of three evolutionarily conserved members: miR-34a, miR-34b, and miR-34c. Global gene expression microarray analyses have revealed that the transcriptome induced by miR-34 overexpression is similar to that observed with p53 induction and that miR-34a functions as a potent suppressor of cell proliferation by modulating E2F signals (Bommer et al., 2007; Chang et al., 2007; He et al., 2007). Indeed, miR-34a was subsequently found to be directly induced by p53, contributing to p53-mediated cell death by promoting apoptosis, and is essential to the correct execution of p53-dependent cellular responses. However, miR-34a pro-apoptotic effects seem to be cell type dependent; in fact, miR-34a levels increased in a stress-induced renal carcinogenesis rat model, and its inhibition affected tumor cell proliferation (Dutta et al., 2007).
Other major pro-apoptotic miRNAs with reduced expression in tumors include the miR-15a/16-1 cluster, which represses anti-apoptotic BCL2 protein and activates the intrinsic apoptotic program APAF-1-caspase-9-poly (ADP-ribose) polymerase (Calin et al., 2008).
Limitless replicative potential
Cellular senescence is a physiologic withdrawal from the cell cycle in response to a multitude of different stress stimuli, including oncogene activation, and involves telomerase deregulation. Various stresses can induce senescence, but p53 and RB have been identified as critical pathways common to the initiation, execution, and maintenance of senescence-associated growth arrest. This irreversible growth arrest is triggered by critically short, unprotected telomeres that induce a DNA damage-like signal (Palm and de Lange, 2008) and is executed by two important cell cycle inhibitors, p21 and p16 (Ben-Porath and Weinberg, 2005). Because senescent cells cannot re-enter the cell cycle, cellular senescence may inhibit the carcinomatous transformation of mutated cells and thus to contribute to cancer prevention. However, some cell types persist in senescent tissues and are not eliminated by apoptosis or the immune system; their altered functional profiles may alter tissue microenvironments to promote cancer (Krtolica and Campisi, 2002; Rodier et al., 2007). miRNA’s relevance in oncogene-induced premature senescence has been addressed with a genetic miRNA-screening library: miR-373 and miR-372 have been identified as capable of allowing the transformation of primary cells that harbor oncogenic RAS and wild-type p53 by neutralizing p53-mediated CDK inhibition; this is accomplished by suppressing LATS2 (Voorhoeve et al., 2006).
p53-activated miRNAs are also important in senescence: the miR-34 family participates in the senescence program (Kumamoto et al., 2008) by modulating (at least in miR-34a’s case) the E2F signaling pathway (Tazawa et al., 2007). Furthermore, 15 miRNAs have been found to be downregulated in senescent cells and breast cancers harboring wild-type p53, which demonstrated that these miRNAs are repressed by p53 in an E2F1-mediated manner (Brosh et al., 2008).
Recently, telomerase was found to be a direct target of regulation by miRs such as miR-138, miR-143, and miR-146a. The discovery of these miRs will help us understand the connections between telomerase expression, senescence, and cellular aging processes (Bonifacio and Jarstfer, 2010).
Tumor cells turn on an “angiogenic switch” to produce large amounts of pro-angiogenic factors and promote neovascularization. Hypoxia adaptation is an essential cellular response that is controlled by vascular endothelial growth factor (VEGF), which is also regulated by the oxygen-sensitive master transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 expression is controlled by specific miRNAs; it, in turn, controls the expression of other miRNAs, which fine-tune adaptation to low oxygen tension.
In tumor progression, hypoxia has been found to contribute to the modulation of miRNA expression, partly by direct HIF-1 transcriptional activation of specific miRNAs (Kulshreshtha et al., 2008). These miRNAs have dual functions: they participate in the angiogenic process while helping the cell engage anti-apoptotic programs that sustain survival (e.g., miR-26, miR-107, and miR-210 inhibit caspase 3 activation).
The endothelial cell-restricted miRNA miR-126 is an essential regulator of developmental angiogenesis (Wang et al., 2008). miR-126 expression was found to be enriched in endothelial cells during angiogenesis and to repress negative regulators of the VEGF pathway (SPRED1 and PIK3R2) (Fish et al., 2008; Wang et al., 2008), thus playing opposite roles according to cell context. miRNAs that are downregulated in hypoxic conditions, such as miR-16, miR-15b, miR-20a, and miR-20b, can also directly modulate VEGF expression levels. This creates a positive feed-forward loop in which hypoxia-repressed miRNAs reinforce the expression levels of a potent pro-angiogenic hypoxia-induced growth factor, such as VEGF (Hua et al., 2006).
The miR-17-92 cluster represents a prototypical polycistronic miRNA gene that encodes 6 mature miRNAs: miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a, all of which are processed from a single primary transcript (Bonauer et al., 2009). The miR-17-92 cluster targets HIF-1α (Taguchi et al., 2008). The miR-17-92 cluster itself also has oncogenic activity, affecting cell cycle progression, apoptosis, and tumor angiogenesis. Individual miRs in the cluster have different effects on angiogenesis. Pro-angiogenesis activity is mainly attributable to miR-17-5p, miR-18a, and miR-19a (Suarez et al., 2008).
Invasion and metastasis
The metastatic process involves multiple steps and genes that allow cells to detach from the primary tumor, enter the blood or lymphatic vasculature, and spread to distant organs. Epithelial-to-mesenchymal transition describes the biologic process that facilitates the conversion of epithelial cells to cells with a more mesenchymal phenotype; such cells have more migratory and invasive properties. The dysregulation of epithelial-to-mesenchymal transition can give cells migratory and invasive properties that promote the dissemination of tumor cells and metastasis.
Recent studies have shown that members of the miR-200 family (miR-141, miR-429, miR-200a, miR-200b, and miR-200c) are essential regulators of differentiation and epithelial cell character in a wide array of cell types and tissues (Dykxhoorn et al., 2009; Hurteau et al., 2009). In addition, miR-200 regulates epithelial cell character through the targeted silencing of the zinc finger E box-binding homeobox (Zeb) family of transcriptional factors Zeb1 and Zeb2. Zeb1 and Zeb2 are master regulators of the mesenchymal phenotype and repress the transcription of genes containing E-box elements in their promoters, including E-cadherin. miR-205 is also significantly down-modulated during epithelial-to-mesenchymal transition.
miR-10b upregulation promotes invasion and metastasis. Twist, a metastasis-promoting transcription factor, was found to induce miR-10b expression, whereas HOXD10, a homeobox transcription factor that promotes or maintains a differentiated phenotype in epithelial cells, was found to be an miR-10b target that is expressed at low levels in metastatic tumors. Consequently, RhoC, a G-protein involved in metastasis that is repressed by HOXD10, was found to be strongly expressed in response to miR-10b expression (Ma et al., 2007). HOTAIR overexpression in breast cancer cell lines results in increased cell invasion in vitro and metastasis in vivo. This increased metastatic potential in HOTAIR overexpression is accompanied by an altered chromatin state; PRC2 is recruited to 854 genes that normally do not bind to PRC2 in epithelial cells, resulting in the downregulation of multiple metastasis suppressor genes, including HOXD10 (Gupta et al., 2010).
miR-21 does not only control cell survival and proliferation; it is also a master regulator of the metastatic process by directly modeling the cell cytoskeleton via TPM1 suppression (Zhu et al., 2007; Zhu et al., 2008) and by indirectly regulating the expression of the pro-metastatic UPAR (via maspin and PDCD4 direct suppression) (Zhu et al., 2008), matrix metalloproteinases (via RECK and TIMP3) (Gabriely et al., 2008), and phosphatase and tensin homolog direct suppression (Meng et al., 2007) (Figure 1).
Circulating miRNAs as Novel Biomarkers
miRNAs are considered powerful markers for early detection, prognosis, response, and recurrence surveillance because they are widely involved in oncogenesis. miRNA-21 was the first miRNA reported to be highly abundant in the sera of diffuse large B-cell lymphoma patients; serum miR-21 levels were associated with relapse-free survival and may be a diagnostic biomarker for diffuse large B cell lymphoma (Lawrie et al., 2008). miR-21 expression was evaluated in solid tumor and serum. Overexpressed miR-21 was associated with poor survival and poor therapeutic outcome in colon adenocarcinoma (Schetter et al., 2008) and poor prognosis and liver metastasis in pancreatic endocrine and acinar tumors (Roldo et al., 2006). miR-21 inhibition increased sensitivity to gemcitabine-based treatment, whereas miR-21 expression increased in response to 5-fluorouracil (Rossi et al., 2007). Among lncRNAs, HOTAIR overexpression was significantly correlated with metastasis and poor prognosis in primary breast cancer patients than already known clinicopathologic risk factors (Gupta et al., 2010).
miRNAs’ utility as diagnostic markers was further revealed by studies in human plasma and serum because serum can be obtained in a less invasive manner than can tissue. In colorectal cancer, miR-17-3p and miR-92 were significantly upregulated in plasma samples from colorectal cancer patients and significantly reduced after surgery (Ng et al., 2009). miR-92 distinguished among colorectal cancer, gastric cancer, inflammatory bowel disease, and healthy colons (Ng et al., 2009). Recently, plasma miR-29a and miR-92a were found to have significant diagnostic value in advanced colorectal neoplasia (Huang et al., 2010). In breast cancer, circulating miR-195 and let-7a levels after curative resection were similar to those of controls (Heneghan et al., 2010). In renal cell carcinoma patients, circulating mRNA levels were predictive of malignancy and survival (Feng et al., 2008). Because most current approaches to cancer screening are invasive and unable to detect early-stage disease, it is important to determine when tumor-related circulating miRNAs and lncRNAs can be detected in the bloodstream during disease development.
RNA Inhibition by miRNAs
RNA-based gene therapy can be used to treat cancer in two ways: a) by using RNA or DNA molecules as therapy against the mRNA of genes involved in cancer pathogenesis and b) by directly targeting the ncRNAs that participate in pathogenesis. The use of miRNAs is still being evaluated preclinically; no clinical or toxicologic studies have been published. It was recently reported that therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model: hepatocellular carcinoma cells exhibit reduced expression of miR-26a and systemic administration of miR-26a in a mouse model of hepatocellular carcinoma using adeno-associated virus results in inhibition of cancer cell proliferation, induction of tumor-specific apoptosis, and dramatic protection from disease progression without toxicity (Kota et al., 2009).
Antagomirs are therapeutic RNA molecules that were originally designed to inhibit miRNAs (Krutzfeldt et al., 2007). The proof-of-principle for antagomirs’ anti-cancer activity was demonstrated in a neuroblastoma model (Fontana et al., 2008) in which miR-17-92 cluster overexpression augmented the in vivo tumorigenesis of MYCN-unamplified neuroblastoma cells. Tumors that had been subcutaneously induced in nude mice were treated with antagomir-17-5p for two weeks; this resulted in tumor growth inhibition and, in 30% of cases, complete regression. The control antagomir had no effect on tumor development.
The safety and efficacy of locked nucleic acid-mediated microRNA silencing has been assessed in mouse and African green monkey models (Elmen et al., 2008). Efficient miR-122 silencing was achieved with three doses of 10 mg/kg locked nucleic acid-anti-miR, leading to a long-lasting and reversible decrease in total plasma cholesterol and no evidence of associated toxicities or histopathologic changes in the liver. Thus, by determining the feasibility, safety, and efficacy of anti-miRNA oligonucleotides in a preclinical setting, this study established the basis for their use in clinical trials.
miRNAs are undoubtedly important in cancer. The main advances in this competitive field will be in at least three areas: 1) identifying functional feed-back and feed-forward loops involving microRNAs gene targets, which will provide a wider picture of how cancer cells malignantly transform, multiply, and invade; 2) identifying the clinical conditions under which miRNAs and lncRNAs will be useful markers for early diagnosis, improved prognosis, and treatment; and 3) defining the best conditions for the use of miRNAs and their inhibitors as anti-cancer agents. One challenge will be identifying which patients will benefit from such advanced therapies.
G.A.C. is supported as a Fellow at The University of Texas MD Anderson Research Trust, as a University of Texas System Regents Research Scholar, and by the CLL Global Research Foundation. Work in Dr. Calin’s laboratory is supported in part by the National Institutes of Health, a Department of Defense Breast Cancer Idea Award, Developmental Research Awards in Breast Cancer, MD Anderson’s Ovarian Cancer, Brain Cancer, and Leukemia SPOREs, a CTT/3I-TD grant, a 2009 Seena Magowitz-Pancreatic Cancer Action Network AACR Pilot Grant, and the Arnold Foundation. We would like to thank Ann Sutton (Department of Scientific Publications, MD Anderson Cancer Center) for her help with the editing of this manuscript.
The authors report no conflicts of interest.
George A. Calin, M.D., Ph.D., Department of Experimental Therapeutics and Department of Cancer Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.
Allen TA, Von Kaenel S, Goodrich JA, Kugel JF. The SINE-encoded mouse B2 RNA represses mRNA transcription in response to heat shock. Nat Struct Mol Biol 11(9):816-821, 2004.
Barbarotto E, Schmittgen TD, Calin GA. MicroRNAs and cancer: profile, profile, profile. Int J Cancer 122(5):969-977, 2008.
Bejerano G, Pheasant M, Makunin I, Stephen S, Kent WJ, Mattick JS, Haussler D. Ultraconserved elements in the human genome. Science 304(5675):1321-1325, 2004.
Ben-Porath I, Weinberg RA. The signals and pathways activating cellular senescence. Int J Biochem Cell Biol 37(5):961-976, 2005.
Bertone P, Stolc V, Royce TE, Rozowsky JS, Urban AE, Zhu X, Rinn JL, Tongprasit W, Samanta M, Weissman S, Gerstein M, Snyder M. Global identification of human transcribed sequences with genome tiling arrays. Science 306(5705):2242-2246, 2004.
Birney E, Stamatoyannopoulos JA, Dutta A, Guigo R, Gingeras TR, Margulies EH, Weng Z, Snyder M, Dermitzakis ET, Thurman RE, Kuehn MS, Taylor CM, Neph S, Koch CM, Asthana S, Malhotra A, Adzhubei I, Greenbaum JA, Andrews RM, Flicek P, et al. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447(7146):799-816, 2007.
Bommer GT, Gerin I, Feng Y, Kaczorowski AJ, Kuick R, Love RE, Zhai Y, Giordano TJ, Qin ZS, Moore BB, Macdougald OA, Cho KR, Fearon ER. p53-mediated activation of miRNA34 candidate tumor-suppressor genes. Curr Biol 17(15):1298-1307, 2007.
Bonauer A, Carmona G, Iwasaki M, Mione M, Koyanagi M, Fischer A, Burchfield J, Fox H, Doebele C, Ohtani K, Chavakis E, Potente M, Tjwa M, Urbich C, Zeiher AM, Dimmeler S. MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice. Science 324(5935):1710-1713, 2009.
Bonifacio LN, Jarstfer MB. MiRNA profile associated with replicative senescence, extended cell culture, and ectopic telomerase expression in human foreskin fibroblasts. PLoS One 5(9):pii:e12519, 2010.
Boominathan L. The guardians of the genome (p53, TA-p73, and TA-p63) are regulators of tumor suppressor miRNAs network. Cancer Metastasis Rev 29(4):613-639, 2010.
Boyerinas B, Park SM, Hau A, Murmann AE, Peter Me. The role of let-7 in cell differentiation and cancer. Endocr Relat Cancer 17(1):F19-F36, 2010.
Brodersen P, Voinnet O. Revisiting the principles of microRNA target recognition and mode of action. Nat Rev Mol Cell Biol 10(2):141-148, 2009.
Brosh R, Shalgi R, Liran A, Landan G, Korotayev K, Nguyen GH, Enerly E, Johnsen H, Buganim Y, Solomon H, Goldstein I, Madar S, Goldfinger N, Borresen-Dale AL, Ginsberg D, Harris CC, Pilpel Y, Oren M, Rotter V. p53-repressed miRNAs are involved with E2F in a feed-forward loop promoting proliferation. Mol Syst Biol 4:229, 2008.
Brosnan CA, Voinnet O. The long and the short of noncoding RNAs. Curr Opin Cell Biol 21(3):416-425, 2009.
Calin GA, Cimmino A, Fabbri M, Ferracin M, Wojcik SE, Shimizu M, Taccioli C, Zanesi N, Garzon R, Aqeilan RI, Alder H, Volinia S, Rassenti L, Liu X, Liu CG, Kipps TJ, Negrini M, Croce CM. miR-15a and miR-16-1 cluster functions in human leukemia. Proc Natl Acad Sci U S A 105(13):5166-5171, 2008.
Calin GA, Liu CG, Ferracin M, Hyslop T, Spizzo R, Sevignani C, Fabbri M, Cimmino A, Lee EJ, Wojcik SE, Shimizu M, Tili E, Rossi S, Taccioli C, Pichiorri F, Liu X, Zupo S, Her lea V, Gramantieri L, Lanza G, et al. Ultraconserved regions encoding ncRNAs are altered in human leukemias and carcinomas. Cancer Cell 12(3):215-229, 2007.
Calin GA, Sevignani C, Dumitru CD, Hyslop T, Noch E, Yendamuri S, Shimizu M, Rattan S, Bullrich F, Negrini M, Croce CM. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci U S A 101(9):2999-3004, 2004.
Chang TC, Wentzel EA, Kent OA, Ramachandran K, Mullendore M, Lee KH, Feldmann G, Yamakuchi M, Ferlito M, Lowenstein CH, Arking DE, Beer MA, Maitra A, Mendell JT. Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. Mol Cell 26(5):745-752, 2007.
Childs G, Fazzari M, Kung G, Kawachi N, Brandwein-Gensler M, McLemore M, Chen Q, Burk RD, Smith RV, Prystowsky MB, Belbin TJ, Schlecht NF. Low-level expression of microRNAs let-7d and miR-205 are prognostic markers of head and neck squamous cell carcinoma. Am J Pathol 174(3):736-745 2009.
Coller HA, Forman JJ, Legesse-Miller A. “Myc’ed messages”: myc induces transcription of E2F1 while inhibiting its translation via a microRNA polycistron. PLoS Genet 3(8):e146, 2007.
Cui JW, Li YJ, Sarkar A, Brown J, Tan YH, Premyslova M, Michaud C, Iscove N, Wang GJ, Ben-David Y. Retroviral insertional activation of the FLI-3 locus in erythroleukemias encoding a cluster of microRNAs that convert EPO-induced differentiation to proliferation. Blood 110(7):2631-2640, 2007.
Diaz R, Silva J, Garcia JM, Lorenzo Y, Garcia V, Pena C, Rodriguez R, Munoz C, Garcia F, Bonilla F, Dominguez G. Deregulated expression of miR-106a predicts survival in human colon cancer patients. Genes Chromosomes Cancer 47(9):794-802, 2008.
Dutta KK, Zhong Y, Liu YT, Yamada T, Akatsuka S, Hu Q, Yoshihara M, Ohara H, Takehashi M, Shinohara T, Masutani H, Onuki J, Toyokuni S. Association of microRNA-34a overexpression with proliferation is cell type-dependent. Cancer Sci 98(12):1845-1852, 2007.
Dykxhoorn DM, Wu Y, Xie H, Yu F, Lal A, Petrocca F, Martinvalet D, Song E, Lim B, Lieberman J. miR-200 enhances mouse breast cancer cell colonization to form distant metastases. PLoS One 4(9):e7181, 2009.
Eddy SR. Non-coding RNA genes and the modern RNA world. Nat Rev Genet 2(12):919-929, 2001.
Elgar G, Vavouri T. Tuning in to the signals: noncoding sequence conservation in vertebrate genomes. Trends Genet 24(7):344-352, 2008.
Elmen J, Lindow M, Schutz S, Lawrence M, Petri A, Obad S, Lindholm M, Hedtjarn M, Hansen HF, Berger U, Gullans S, Kearney P, Sarnow P, Straarup EM, Kauppinen S. LNA-mediated microRNA silencing in non-human primates. Nature 452(7189):896-899, 2008.
Emmrich S, Putzer BM. Checks and balances: E2F-microRNA crosstalk in cancer control. Cell Cycle, epub ahead of print, Jul. 14, 2010.
Esquela-Kerscher A, Slack FJ. Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer 6(4):259-269, 2006.
Esquela-Kerscher A, Trang P, Wiggins JF, Patrawala L, Cheng A, Ford L, Weidhaas JB, Brown D, Bader AG, Slack FJ. The let-7 microRNA reduces tumor growth in mouse models of lung cancer. Cell Cycle 7(6):759-764, 2008.
Feng G, Li G, Gentil-Perret A, Tostain J, Genin C. Elevated serum-circulating RNA in patients with conventional renal cell cancer. Anticancer Res 28(1A):321-326, 2008.
Fish JE, Santoro MM, Morton SU, Yu S, Yeh RF, Wythe JD, Ivey KN, Bruneau BG, Stainier DY, Srivastava D. miR-126 regulates angiogenic signaling and vascular integrity. Dev Cell 15(2):272-284, 2008.
Fontana L, Fiori ME, Albini S, Cifaldi L, Giovinazzi S, Forloni M, Boldrini R, Donfrancesco A, Federici V, Giacomini P, Peschle C, Fruci D. Antagomir-17-5p abolishes the growth of therapy-resistant neuroblastoma through p21 and BIM. PLoS One 3(5):e2236, 2008.
Gabriely G, Wurdinger T, Kesari S, Esau CC, Burchard J, Linsley PS, Krichevsky AM. MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators. Mol Cell Biol 28(17):5369-5380, 2008.
Ghildiyal M, Zamore PD. Small silencing RNAs: an expanding universe. Nat Rev Genet 10(2):94-108, 2009.
Gupta RA, Shah N, Wang KC, Kim J, Horlings HM, Wong DJ, Tsai MC, Hung T, Argani P, Rinn JL Wang Y, Brzoska P, Kong B, Li R, West RB, Van De Vijver MJ, Sukumar S, Chang HY. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 464(7291):1071-1076, 2010.
Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 100(1):57-70, 2000.
Hatley ME, Patrick DM, Garcia MR, Richardson JA, Bassel-Duby R, Van Rooij E, Olson EN. Modulation of K-Ras-dependent lung tumorigenesis by MicroRNA-21. Cancer Cell 18(3):282-293, 2010.
He L, He X, Lim LP, De Stanchina E, Xuan Z, Liang Y, Xue W, Zender L, Magnus J, Ridzon D, Jackson AL, Linsley PS, Chen C, Lowe SW, Cleary MA, Hannon GJ. A microRNA component of the p53 tumour suppressor network. Nature 447(7148):1130-1134, 2007.
Heneghan HM, Miller N, Lowery AJ, Sweeney KJ, Newell J, Kerin MJ. Circulating microRNAs as novel minimally invasive biomarkers for breast cancer. Ann Surg 251(3):499-505, 2010.
Hua Z, Lv Q, Ye W, Wong CK, Cai G, Gu D, Ji Y, Zhao C, Wang J, Yang BB, Zhang Y. MiRNA-directed regulation of VEGF and other angiogenic factors under hypoxia. PLoS One 1:e116, 2006.
Huang Z, Huang D, Ni S, Peng Z, Sheng W, Du X. Plasma microRNAs are promising novel biomarkers for early detection of colorectal cancer. Int J Cancer 127(1):118-126, 2010.
Hurteau GJ, Carlson JA, Roos E, Brock GJ. Stable expression of miR-200c alone is sufficient to regulate TCF8 (ZEB1) and restore E-cadherin expression. Cell Cycle 8(13):2064-2069, 2009.
Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, Labourier E, Reinert KL, Brown D, Slack FJ. RAS is regulated by the let-7 microRNA family. Cell 120(5):635-647, 2005.
Kota J, Chivukula RR, O’Donnell KA, Wentzel EA, Montgomery CL, Hwang HW, Chang TC, Vivekanandan P, Torbenson M, Clark KR, Mendell JR, Mendell JT. Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model. Cell 137(6):1005-1017, 2009.
Krtolica A, Campisi J. Cancer and aging: a model for the cancer promoting effects of the aging stroma. Int J Biochem Cell Biol 34(11):1401-1414, 2002.
Krutzfeldt J, Kuwajima S, Braich R, Rajeev Kg, Pena J, Tuschl T, Manoharan M, Stoffel M. Specificity, duplex degradation and subcellular localization of antagomirs. Nucleic Acids Res 35(9):2885-2892, 2007.
Kulshreshtha R, Davuluri RV, Calin GA, Ivan M. A microRNA component of the hypoxic response. Cell Death Differ 15(4):667-671, 2008.
Kumamoto K, Spillare EA, Fujita K, Horikawa I, Yamashita T, Appella E, Nagashima M, Takenoshita S, Yokota J, Harris CC. Nutlin-3a activates p53 to both down-regulate inhibitor of growth 2 and up-regulate mir-34a, mir-34b, and mir-34c expression, and induce senescence. Cancer Res 68(9):3193-3203, 2008.
Kumar MS, Erkeland SJ, Pester RE, Chen CY, Ebert MS, Sharp PA, Jacks T. Suppression of non-small cell lung tumor development by the let-7 microRNA family. Proc Natl Acad Sci U S A 105(10):3903-3908, 2008.
Kumar MS, Lu J, Mercer KL, Golub TR, Jacks T. Impaired microRNA processing enhances cellular transformation and tumorigenesis. Nat Genet 39(5):673-677, 2007.
Lawrie CH, Gal S, Dunlop HM, Pushkaran B, Liggins AP, Pulford K, Banham AH, Pezzella F, Boultwood J, Wainscoat JS, Hatton CS, Harris AL. Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma. Br J Haematol 141(5):672-675, 2008.
Lee KH, Chen YL, Yeh SD, Hsiao M, Lin JT, Goan YG, Lu PJ. MicroRNA-330 acts as tumor suppressor and induces apoptosis of prostate cancer cells through E2F1-mediated suppression of Akt phosphorylation. Oncogene 28(38):3360-3370, 2009.
Lee YS, Dutta A. The tumor suppressor microRNA let-7 represses the HMGA2 oncogene. Genes Dev 21(9):1025-1030, 2007.
Ma L, Teruya-Feldstein J, Weinberg RA. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature 449(7163):682-688, 2007.
Malumbres M, Barbacid M. To cycle or not to cycle: a critical decision in cancer. Nat Rev Cancer 1(3):222-231, 2001.
Mattick JS. The genetic signatures of noncoding RNAs. PLoS Genet 5(4):e1000459, 2009.
Mayr C, Hemann MT, Bartel DP. Disrupting the pairing between let-7 and Hmga2 enhances oncogenic transformation. Science 315(5818):1576-1579, 2007.
Meng F, Henson R, Wehbe-Janek H, Ghoshal K, Jacob ST, Patel T. MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology 133(2):647-658, 2007.
Mercer TR, Dinger ME, Mattick JS. Long non-coding RNAs: insights into functions. Nat Rev Genet 10(3):155-159, 2009.
Motamedi MR, Verdel A, Colmenares SU, Gerber SA, Gygi SP, Moazed D. Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs. Cell 119(6):789-802, 2004.
Negrini M, Nicoloso MS, Calin GA. MicroRNAs and cancer — new paradigms in molecular oncology. Curr Opin Cell Biol 21(3):470-479, 2009.
Ng EK, Chong WW, Jin H, Lam EK, Shin VY, Yu J, Poon TC, Ng SS, Sung JJ. Differential expression of microRNAs in plasma of colorectal cancer patients: A potential marker for colorectal cancer screening. Gut 58(10):1375-1381, 2009.
O’Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT. c-Myc-regulated microRNAs modulate E2F1 expression. Nature 435(7043):839-843, 2005.
Orom UA, Derrien T, Beringer M, Gumireddy K, Gardini A, Bussotti G, Lai F, Zytnicki M, Notredame C, Huang Q, Guigo R, Shiekhattar R. Long noncoding RNAs with enhancer-like function in human cells. Cell 143(1):46-58, 2010.
Palm W, De Lange T. How shelterin protects mammalian telomeres. Annu Rev Genet 42:301-334, 2008.
Petrocca F, Visone R, Onelli MR, Shah MH, Nicoloso MS, De Martino I, Iliopoulos D, Pilozzi E, Liu CG, Negrini M, Cavazzini L, Volinia S, Alder H, Ruco LP, Baldassarre G, Croce CM, Vecchione A. E2F1-regulated microRNAs impair TGFbeta-dependent cell-cycle arrest and apoptosis in gastric cancer. Cancer Cell 13(3):272-286, 2008.
Ponting CP, Oliver PL, Reik W. Evolution and functions of long noncoding RNAs. Cell 136(4):629-641, 2009.
Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA, Goodnough LH, Helms JA, Farnham PJ, Segal E, Chang HY. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129(7):1311-1323, 2007.
Rodier F, Campisi J, Bhaumik D. Two faces of p53: aging and tumor suppression. Nucleic Acids Res 35(22):7475-7484, 2007.
Roldo C, Missiaglia E, Hagan JP, Falconi M, Capelli P, Bersani S, Calin GA, Volinia S, Liu CG, Scarpa A, Croce CM. MicroRNA expression abnormalities in pancreatic endocrine and acinar tumors are associated with distinctive pathologic features and clinical behavior. J Clin Oncol 24(29):4677-4684, 2006.
Rossi L, Bonmassar E, Faraoni I. Modification of miR gene expression pattern in human colon cancer cells following exposure to 5-fluorouracil in vitro. Pharmacol Res 56(3):248-253, 2007.
Schetter AJ, Leung SY, Sohn JJ, Zanetti KA, Bowman ED, Yanaihara N, Yuen ST, Chan TL, Kwong DL, Au GK Liu CG, Calin GA, Croce CM, Harris CC. MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma. JAMA 299(4):425-436, 2008.
Schultz J, Lorenz P, Gross G, Ibrahim S, Kunz M. MicroRNA let-7b targets important cell cycle molecules in malignant melanoma cells and interferes with anchorage-independent growth. Cell Res 18(5):549-557, 2008.
Seidl CI, Stricker SH, Barlow DP. The imprinted Air ncRNA is an atypical RNAPII transcript that evades splicing and escapes nuclear export. EMBO J 25(15):3565-3575, 2006.
Selcuklu SD, Yakicier MC, Erson AE. An investigation of microRNAs mapping to breast cancer related genomic gain and loss regions. Cancer Genet Cytogenet 189(1):15-23, 2009.
Suarez Y, Fernandez-Hernando C, Yu J, Gerber SA, Harrison KD, Pober JS, Iruela-Arispe ML, Merkenschlager M, Sessa WC. Dicer-dependent endothelial microRNAs are necessary for postnatal angiogenesis. Proc Natl Acad Sci U S A 105(37):14082-14087, 2008.
Sylvestre Y, De Guire V, Querido E, Mukhopadhyay UK, Bourdeau V, Major F, Ferbeyre G, Chartrand P. An E2F/miR-20a autoregulatory feedback loop. J Biol Chem 282(4):2135-2143, 2007.
Taft RJ, Pang KC, Mercer TR, Dinger M, Mattick JS. Non-coding RNAs: regulators of disease. J Pathol 220(2):126-139, 2010.
Taguchi A, Yanagisawa K, Tanaka M, Cao K, Matsuyama Y, Goto H, Takahashi T. Identification of hypoxia-inducible factor-1 alpha as a novel target for miR-17-92 microRNA cluster. Cancer Res 68(14):5540-5545, 2008.
Talotta F, Cimmino A, Matarazzo MR, Casalino L, De Vita G, D’Esposito M, Di Lauro R, Verde P. An autoregulatory loop mediated by miR-21 and PDCD4 controls the AP-1 activity in RAS transformation. Oncogene 28(1):73-84, 2009.
Tazawa H, Tsuchiya N, Izumiya M, Nakagama H. Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells. Proc Natl Acad Sci U S A 104(39):15472-15477, 2007.
Tsai MC, Manor O, Wan Y, Mosammaparast N, Wang JK, Lan F, Shi Y, Segal E, Chang HY. Long noncoding RNA as modular scaffold of histone modification complexes. Science 329(5992):689-693, 2010.
Venturini L, Battmer K, Castoldi M, Schultheis B, Hochhaus A, Muckenthaler MU, Ganser A, Eder M, Scherr M. Expression of the miR-17-92 polycistron in chronic myeloid leukemia (CML) CD34+ cells. Blood 109(10):4399-4405, 2007.
Voorhoeve PM, Le Sage C, Schrier M, Gillis AJ, Stoop H, Nagel R, Liu YP, Van Duijse J, Drost J, Griekspoor A, Zlotorynski E, Yabuta N, De Vita G, Nojima H, Looijenga LH, Agami R. A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell 124(6):1169-1181, 2006.
Wang S, Aurora AB, Johnson BA, Qi X, McAnally J, Hill JA, Richardson JA, Bassel-Duby R, Olson EN. The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis. Dev Cell 15(2):261-271, 2008.
Woods K, Thomson JM, Hammond SM. Direct regulation of an oncogenic micro-RNA cluster by E2F transcription factors. J Biol Chem 282(4):2130-2134, 2007.
Zanette DL, Rivadavia F, Molfetta GA, Barbuzano FG, Proto-Siqueira R, Falcao RP, Zago MA, Silva-Jr WA. miRNA expression profiles in chronic lymphocytic and acute lymphocytic leukemia. Braz J Med Biol Res 40(11):1435-1440, 2007.Zhu S, Si ML, Wu H, Mo YY. MicroRNA-21 targets the tumor suppressor gene tropomyosin 1 (TPM1). J Biol Chem 282(19):14328-14336, 2007.
Zhu S, Wu H, Wu F, Nie D, Sheng S, Mo YY. MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res 18(3):350-359, 2008.
[Discovery Medicine; ISSN: 1539-6509; Discov Med 11(58):245-254, March 2011.]