MicroRNA (miRNA)-encoding small non-coding RNA have been recognized as important regulators of a number of biological processes that inhibit the expression of hundreds of genes. Accumulating evidence also indicates the involvement of miRNA alterations in various types of human cancer, including lung cancer, which has long been the leading cause of cancer death in economically well-developed countries, including Japan. We previously found that downregulation of members of the tumor-suppressive let-7 miRNA family and overexpression of the oncogenic miR-17-92 miRNA cluster frequently occur in lung cancers, and molecular insight into how these miRNA alterations may contribute to tumor development has been rapidly accumulating. The present review summarizes recent advances in elucidation of the molecular functions of these miRNA in relation to their roles in the pathogenesis of lung cancer. Given the crucial roles of miRNA alterations, additional studies are expected to provide a better understanding of the underlying molecular mechanisms of disease development, as well as a foundation for novel strategies for cancer diagnosis and treatment of this devastating disease. (Cancer Sci 2011; 102: 9–17)
Lung cancer is the leading cause of cancer death in most economically developed countries, including Japan. Solid evidence indicates that the disease develops from accumulations of various genetic and epigenetic alterations(1,2) resulting in alterations of gene expression profiles, which are tightly associated with the clinicopathological features of lung cancer. MicroRNA (miRNA) in the human genome were only recently discovered(3) and accumulating evidence clearly indicates their roles in various crucial aspects of gene expression regulation. We initiated a search for miRNA that are dysregulated in lung cancer, which resulted in the discovery of major representative miRNA involved in lung cancer development with either tumor suppressive or oncogenic roles. These miRNA are members of the let-7 miRNA family and among the most representative type of tumor suppressor miRNA,(4) along with the miR-17-92 miRNA cluster, which is recognized as a typical oncogene-type miRNA.(5) There is a number of high-quality review articles dealing with the general roles of miRNA alterations in carcinogenesis,(6–9) thus in the present review we specifically focus on recent advances related to let-7 and miR-17-92, with special emphasis on their relationships to lung carcinogenesis.
Discovery of miRNA in lower eukaryotes and humans
miRNA are evolutionally conserved approximately 22 nucleotide-long short non-coding RNA molecules. As of March 2010, 721 hairpin miRNA precursors and 1007 mature miRNA in the human genome had been deposited into the primary database (miRBase: http://www.mirbase.org/index.shtml). The genes first recognized to encode miRNA were lin-4 and let-7, both of which were originally identified as heterochronic mutant genes and affect the progression of larval stages during the development of C. elegans.(10–12) As C. elegans develops, lin-4 is upregulated in the first larval (L1) stage and suppresses expression of lin-14, thus promoting progression from the L1 to L2 stage. Subsequent downregulation of a second lin-4 target, lin-28, is required for execution of the L3 larval stage. In contrast, let-7 is expressed later in development and required for execution of the larval to adult (L/A) switch, which occurs at the end of the L4 stage. Mutations of lin-4 and let-7 have effects on the differentiation of seam stem cells, resulting in reiteration of the larval stages.(10–13)
miRNA are generated from long precursor transcripts and have an imperfectly matched stem-loop structure. These primary transcripts (pri-miRNA) are first processed into hairpin RNA (pre-miRNA) by a nuclear ribonuclease, Drosha, then transported to the cytoplasm and processed by a second ribonuclease, Dicer. Subsequently, the single stranded miRNA (mature miRNA) are incorporated into a miRNA-induced silencing complex (miRISC) and interact with “seed” sequence-matched recognition sites at the 3′ UTR of target mRNA. These miRNA–mRNA interactions result in inhibition of expression of the target genes at the post-transcriptional level through translational inhibition and mRNA destabilization.(14,15) Each miRNA directly represses, albeit mildly in general, hundreds of target genes, most of which contain conserved seed sequence(s) at the 3′ UTR. Because a large number of miRNA is present in the human genome, more than 60% of human protein-coding genes are targeted by miRNA,(16) suggesting that miRNA abnormalities may cause a wide spectrum of alterations in gene expressions.
let-7 alterations in lung cancer
In 2004, we reported that expression levels of the let-7 family members are generally reduced in lung cancer when compared with those in normal lung tissues, indicating an association with poor prognosis in surgically treated patients who have tumors with low levels of let-7 expression.(4) That study was the first report of let-7 alterations in any type of cancer, as well as of the relationship of cancer patient prognosis with any type of miRNA alteration. Perhaps more importantly, our experimental finding that the introduction of let-7 into a lung cancer cell line with a low level of let-7 expression significantly inhibited the growth of lung cancer cells was the first direct indication that the miRNA expression level has an effect on the biological behavior of cancer cells. Subsequently, Slack’s group identified K-ras as a target of let-7 and showed that antisense-mediated inhibition of let-7 increased cancer cell division, whereas overexpression of let-7 induced cell-cycle arrest in cancer cell lines.(17) Together, these findings observed in human lung cancer cells appear to be consistent with the roles of let-7 in C. elegans, as seam cells in let-7 mutants fail to exit the cell cycle and reiterate the larval stage, showing dysregulation of the cell cycle and cell growth. The significance of reduced let-7 expression in lung carcinogenesis was further supported in studies of genetically engineered mice. Jacks’ group showed that let-7 suppressed tumor initiation in an autochthonous non-small cell lung cancer (NSCLC) model of K-RasG12D transgenic mice, which was effectively rescued by ectopic expression of K-RasG12D lacking the 3′ UTR.(18)let-7 also inhibited in vitro and in vivo growth of K-RasG12D-expressing murine lung cancer cells and human lung cancer xenografts.(19) Inhibitory effects of let-7 against human lung cancer development have also been supported by circumstantial evidence reported by Chin et al.,(20) who sequenced let-7 complementary sites (LCS) in the KRAS 3′ UTR from NSCLC cases and found that the single nucleotide polymorphism (SNP) at LCS6 was significantly associated with NSCLC patients who smoked <40 pack-years. Interestingly, they also showed that this SNP results in KRAS overexpression in vitro.
Each miRNA is thought to regulate tens or hundreds of protein coding genes, thus it is reasonable to speculate that let-7 downregulates other growth-promoting genes, such as oncogenes (Fig. 1). Indeed, HMGA2, which encodes a non-histone chromosomal high-mobility group protein with a putative oncogenic function, has been shown to be under the control of let-7.(21) In several types of malignancy, the HMGA2 gene locus is disrupted by chromosomal translocation and loses its 3′ UTR that harbors multiple let-7 recognition sites, while HMGA2 promotes anchorage-independent growth.(22) In mice, Hmga2 promotes self-renewal of fetal and young-adult neural stem cells, partly by decreasing p16Ink4a/p19Arf expression, while let-7 expression, which increases with age, negatively affects Hmga2 expression and self-renewal capacity.(23) Other targets of let-7 include various cell-cycle-related genes such as cyclin D2, CDK6 and CDC25A,(24) as well as various oncofetal genes, including insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1, also called IMP-1/CRD-BP) and IGF2BP2/IMP-2,(25) which are known to bind various mRNA and regulate their translation, leading to stabilization of crucial mRNA such as Myc.(25)
Shell et al.(26) also reported the importance of let-7 in cancer classification. Cancer cell lines can be divided into two groups, epithelial type (II) and mesenchymal type (I), suggesting a progression from type II to type I through epithelial–mesenchymal transition (EMT).(27) Shell et al.(26) found that cancer cell lines that exhibit epithelial-type characteristics express higher levels of let-7 than those with mesenchymal features, and suggested that loss of let-7 expression may be a marker for less differentiated and advanced cancer. Also, a joint study conducted by the laboratories of Croce and Harris reported associations of miRNA profiles with survival of patients with lung adenocarcinomas, and showed that high expression of miR-155 and low expression of let-7a-2 were strongly associated with poor survival.(28) An association of reduced let-7a level with unfavorable postoperative prognosis in patients with NSCLC was also reported by Yu et al. using quantitative RT-PCR-based analysis, in which a poorer prognosis was shown to be associated with reduced let-7 and miR-221 expression, as well as with increased levels of miR-137, miR-372 and miR-182*.(29) Interestingly, a search for miRNA differentially altered between lung cancer patients who never smoked and those who were smokers showed that downregulation of let-7c and miR-138 was preferentially present in the never-smoked patients.(30)
Fine tuning of let-7 expression level and cancer
In addition to the cancer-related genes described above, let-7 appears to have another interesting target (Fig. 1). We found that let-7 directly downregulates Dicer through binding sites at the 3′ UTR.(31) Dicer is an essential endonuclease required at the final processing step in miRNA biogenesis that includes let-7. Overexpression of let-7 reduces the expression of Dicer as well as that of a large number of other mature miRNA, whereas antisense-mediated inhibition of let-7 leads to upregulation of Dicer expression associated with increased expression levels of mature miRNA.(31) The existence of three conserved let-7 target sites within the open reading frame in Dicer was also reported,(32) although they appear to be less efficient than the 3′ UTR binding sites (Tokumaru S and Takahashi T, unpublished observation 2008). Therefore, the existence of let-7-mediated negative regulation of Dicer may provide a basis for the tightly regulated equilibration of expression levels of Dicer and let-7, as well as of other miRNA. Interestingly, let-7 appears to be a constituent of another regulatory loop within the miRNA processing steps (Figs 1,2). Lin28 was shown to be a direct target for let-7-mediated inhibition, while it in turn inhibits Drosha- and/or Dicer-mediated processing of let-7.(33,34) Both Lin28 and a homologue, Lin28B, are overexpressed in approximately 15% of primary human tumor samples in association with reduced expression of the entire let-7 family, as well as with a poor clinical prognosis.(35) Furthermore, negative regulation of the let-7 family by Lin28 and Lin28B involves induction of uridylation of the pre-let-7 3′-terminus.(36) In addition, Lin28 proteins may directly recruit the uridylating enzyme TUTase4 (TUT4),(37) also known as zinc finger, CCHC domain containing 11 (Zcchc11),(38) to pre-let-7. The terminal uridylation of pre-let-7 blocks Dicer processing and also promotes its decay, while a tetra-nucleotide sequence motif (GGAG) in the terminal loop is recognized by Lin28. Thus, other miRNA with the same loop sequence motif may also be regulated via the same mechanism. Indeed, Zcchc11 has been shown to uridylate miR-26a targeting IL-6 and stabilize IL-6 transcripts.(39)
It is notable that reduced Dicer expression appears to be involved in tumor development. We previously reported an association of reduced expression of Dicer with poor prognosis in lung cancer patients.(40) Consistent with that finding in human lung cancer, Jacks’ group reported that knockdown of Dicer1 accelerated the tumorigenicity and invasiveness of a mouse lung adenocarcinoma cell line, while conditional deletion of Dicer1 enhanced tumor development in a K-Ras-induced mouse model of lung cancer.(41)
Maintenance of stemness in relation to let-7 expression
Oct4, Sox2 and Nanog, core regulators of ES cell differentiation, co-occupy the promoters of differentiation-related transcriptional factors and also several miRNA, suggesting miRNA plays a role in regulation of differentiation.(42) A number of mature miRNA are not expressed in ES or P19 EC cells, whereas they are expressed at the late embryonic stage. Lin-28 binds conserved nucleotides in the loop region of let-7 precursors,(43) and effectively blocks their cleavage by the Drosha-DGCR8 microprocessor in the nucleus(33,43) and by Dicer in the cytoplasm(34) of embryonic stem cells (Fig. 1). In neuronal stem (NS) cells, which are more differentiated than ES cells, Lin-28 is downregulated by mir-125 (lin-4 homologue) and let-7, which allows pre-let-7 processing to proceed. Suppression of let-7 or mir-125 activity in NS cells leads to upregulation of Lin-28 and loss of pre-let-7 processing activity, suggesting that let-7, mir-125 and Lin-28 participate in an autoregulatory circuit that controls miRNA processing during NS cell commitment.(34) Thus, Lin28 functions as a negative regulator of miRNA biogenesis, and may play a central role in blocking miRNA-mediated differentiation in stem cells and certain cancers (Figs 1,2).
In addition to Lin-28, the zinc finger protein Lin41 is also a target of let-7 and involved in the regulatory network that controls pluripotency.(44) Lin41 interacts with Dicer and the Ago family at P-body, and acts as an E3 ubiquitin ligase, mediating the ubiquitylation of Ago2.(44) Therefore, Lin41 negatively regulates let-7 activity and co-operates with Lin28 in stem cells (Figs 1,2). These findings indicate the importance of Lin-28 to maintain pluripotency and are consistent with the finding that Lin-28 is included in a cocktail of reprogramming factors (Oct3/4, Sox-2, Nanog, Lin28) to create induced pluripotent stem (iPS) cells from adult human fibroblasts.(45) In addition, Myc directly associates with the Lin28B promoter to induce Lin-28B expression, resulting in let-7 repression. Accordingly, Lin-28B loss-of-function significantly impairs Myc-dependent cellular proliferation.(46) The self-renewing progenitor population in mouse mammary epithelial cells shows a unique miRNA signature of high expression levels of miR-205 and miR-22, and depletion of let-7 and miR-93, while enforced let-7 expression was shown to induce loss of the self-renewing population, suggesting negative regulation of tissue progenitor maintenance by let-7.(47)
Other lines of evidence suggest the involvement of let-7 in carcinogenesis in relation to its function to regulate differentiation. For example, let-7 expression is markedly reduced in mammospheres/tumor-initiating cells of breast cancer and increased along with cell differentiation.(48) Conversely, forced expression of let-7 has been shown to reduce cellular proliferation and mammosphere formation, as well as in vivo tumor formation and metastasis. Interestingly, silencing of H-RAS reduced the self-renewal of mammospheres but had no effect on differentiation, while that of HMGA2 enhanced differentiation but did not affect self-renewal, suggesting that both H-RAS and HMGA2 are major target genes of let-7 and de-repression of both is involved separately in tumorigenesis.(48) In addition, those findings indicate an important role for let-7 and its regulation in the regulation of pluripotency. In contrast to let-7, several miRNA such as the members of the miR-290 family are expressed specifically in ES cells(49) and positively regulate ES cell self-renewal.(50,51) Dgcr8-deficient ES cells are unable to suppress self-renewal, because of defective biogenesis of miRNA. However, introduction of let-7 can suppress self-renewal and induce differentiation, whereas miR-294, an ES cell-specific miRNA, blocks the suppression of self-renewal by let-7, suggesting that let-7 and ES cell-specific miRNA alternatively regulate ES cell fate, that is, self-renewal versus differentiation.(51) Our recent miRNA microarray analysis findings showed that lung adenocarcinomas are grouped into four major clusters with distinct miRNA expression profiles. Along the same line, it is interesting that one of the clusters with characteristically low let-7 and high miR-17-92 expression levels was related to a significantly worse prognosis, and those patients exhibited significantly higher dysregulation of ES cell-related gene sets (Arima C and Takahashi T, manuscript in preparation).
miR-17-92 overexpression in lung cancer
Our initial discovery of frequent downregulation of let-7 and its biological and clinicopathological involvement in lung cancer prompted us to search for miRNA conversely overexpressed in lung cancers.(4) Consequently, we found frequent and marked overexpression, with occasional gene amplification, of clustered miRNA (miR-17-92) within intron 3 of the C13orf25 gene at 13q31.3 in lung cancer samples, especially those with a small cell lung cancer (SCLC) histology.(5) Stimulatory activity by this miRNA cluster toward lung cancer cell growth was observed, while antisense-mediated inhibition of miR-17-5p and miR-20a, constituents of miR-17-92, induced apoptosis in miR-17-92-overexpressing lung cancer cell lines, suggesting an addiction to continued overexpression of miR-17-92 for cancer development. In contrast to our approach, Hammond et al initiated a study based on evidence suggesting the involvement of the C13orf25 genomic region in B-cell lymphomas, as previously reported by Ota et al.(52) in the results of detailed array CGH analysis. Consequently, they identified overexpression of miR-17-92 in occasional association with gene amplification in B-cell lymphomas(53) and showed that introduction of miR-17-92 into hematopoietic stem cells in Eμ-myc transgenic mice significantly accelerated formation of lymphoid malignancies. MYC transactivates expression of the miR-17-92 miRNA cluster,(54) while members of the myc gene family have been shown to be frequently amplified and/or overexpressed in SCLC.(55) Interestingly, our previous studies of miR-17-92 and the myc gene family in lung cancers suggested the existence of two potential mechanisms that lead to overexpression of the miR-17-92 cluster, that is, gene amplification of the miRNA cluster itself and increased expression of the myc gene family, with or without gene amplification. It is also important to note that a significant role of the miR-17-92 cluster in tumorigenesis is also supported by frequent retrovirus integration-mediated activations of mouse miR-17-92(56) and paralogous miR-160a-363(57) in mouse tumors.
Overexpression of E2F1 induces inappropriate entry into the S-phase, resulting in apoptosis induction.(58) MYC and E2F1 positively regulate each other, while MYC-induced miR-17-92 negatively regulates E2F1,(54) suggesting possible fine tuning of the E2F1 expression level for correct regulation of S-phase entry. In addition to Myc, the E2F family also transactivates miR-17-92, which exerts a negative feedback loop, resulting in suppression of E2F family expression.(59,60) Therefore, the expression levels of MYC, the E2F family and miR-17-92 are finely regulated by each other, suggesting their crucial roles in cell-cycle regulation (Fig. 3). miR-17-92 is preferentially overexpressed in lung cancers with neuroendocrine features, especially in SCLC, which is known to exhibit overexpression of members of the MYC gene family with frequent gene amplification. We reported that survival of lung cancer cell lines with miR-17-92 overexpression relies on the continued expression of miR-17-92.(61) Interestingly, we also found frequent accumulation of constitutively phosphorylated H2AX (γ-H2AX), which reflects persistent DNA damage, preferentially in SCLC. Small cell lung cancers almost invariably carry inactivated retinoblastoma (RB) and p53, which conceivably contributes to elicit dysregulated cell-cycle progress, leading to replication-dependent DNA double-strand breaks. In fact, in NSCLC cells with wild-type RB, knockdown of RB induced γ-H2AX foci formation and growth inhibition in NSCLC cells with wild-type RB, which was canceled by overexpression of miR-20a. In addition, suppression of miR-20a with antisense-oligonucleotides further induced γ-H2AX foci formation in a miR-17-92-overexpressing SCLC cell line.(62) RB disruption also induces ROS, which are negatively regulated by miR-20a. Therefore, miR-17-92 overexpression may serve as a fine-tuning influence to counterbalance the generation of DNA damage in RB-inactivated SCLC cells, thus reducing excessive DNA damage to a tolerable level and consequently leading to genetic instability (Fig. 4).(62) These findings are consistent with the report by Pickering et al.,(63) who showed the role of miR-17/miR-20a in the cell-cycle regulation of fibroblasts. Inhibition of miR-17/miR-20a leads to G1 checkpoint activation due to an accumulation of DNA double-strand breaks, resulting from premature temporal accumulation of the E2F1 transcription factor. Thus, Myc-regulated miR-17/miR-20a appears to play a role in controlling the precise timing of E2F1expression and circumventing the G1 checkpoint caused by E2F1 accumulation, which is perturbed in cancer overexpressing miR-17-92. It is also important to note that the consequences of coupling between the E2F/Myc positive feedback and E2F/Myc/miR-17-92 negative feedback loops have been analyzed using a mathematical model, which predicted that miR-17-92 plays a critical role in regulating the position of the on–off switch related to E2F/Myc protein levels.(64) Cyclin D1 may also be involved in this miR-17/miR-20a negative feedback loop in breast cancer.(65)
Other targets of miR-17-92 related to cancer
Each miRNA may potentially influence more than 100 target mRNA. Accordingly, a search for targets of miR-17-92, which are actually affected in immortalized lung epithelial cells by the components of this miRNA cluster, was conducted through global expression profiling using differential tagging with iTRAQ™ reagent, followed by multidimensional liquid chromatography and tandem mass spectrometry analysis, which resulted in identification of HIF-1α as a target for miR-17-92 (Fig. 4).(66) Interestingly, an intricate and finely tuned circuit involving c-myc, HIF-1α and miR-17-92 exists and plays a role in cancer cell proliferation under normoxia in a cellular context-dependent manner without interfering with the robust induction of HIF-1α for cellular adaptation to hypoxia. Yan et al.(67) recently reported that hypoxia reduced miR-17-92 expression in colon cancer cells through p53-mediated repression by its direct binding to the promoter of miR-17-92 and consequential competition with the TATA-binding protein (TBP). They also showed that forced expression of miR-17-92 markedly inhibited hypoxia-induced apoptosis, whereas antisense-mediated inhibition of miR-17-5p and miR-20a sensitized the cells to hypoxia-induced apoptosis, indicating that p53-mediated repression of miR-17-92 expression is likely to have an important function in hypoxia-induced apoptosis. In contrast, we did not detect a readily noticeable change in miR-17-92 expression under hypoxia in an immortalized normal bronchial epithelial cell line,(66) suggesting that there might be different effects depending on the cellular contexts.
Additional targets for miR-17-92 have been reported in studies that used various systems (Fig. 3). Transgenic mice carrying the miR-17-92 transgene conditionally active in lymphocytes showed increased proliferation and reduced activation-induced cell death of lymphocytes, resulting in lethal lymphoproliferative and autoimmune diseases.(68) That study also found that miR-17-92 miRNA suppressed the expression of Pten and Bim, both of which contribute to the phenotype. BIM, a proapoptotic BCL2 family member, functionally inhibits anti-apoptotic BCL2 family members through physical interaction and plays an essential role in apoptosis induction during lymphocyte differentiation. PTEN encoding phosphatidylinositol-3,4,5-trisphosphate (PIP3) 3-phosphatase inhibits activation of the PDK1–AKT signaling pathway through inhibition of PIP3 generation and is frequently inactivated by mutations in several cancer types. Disruption of both genes induces lymphoproliferative and autoimmune diseases, suggesting that the lethal phenotype is attributable mainly to repression of PTEN and Bim by miR-17-92.(68) Meanwhile, disruption of miR-17-92 leads to upregulation of Bim and inhibits B cell development during the transition from pro-B to pre-B.(69) Another tumor suppressor gene, CDKN1A (p21Waf1/Cip1), is also repressed by miR-17, miR-20a and miR-106b.(62,70,71)
It has also been shown that miR-17-92 is involved in regulation of angiogenesis. Although K-Ras-transformed colonocytes were shown to form indolent and poorly vascularized tumors, transduction of the Myc gene caused upregulation of miR-17-92 in K-Ras-colonocytes and neovascularization in related tumors, in association with downregulation of anti-angiogenic thrombospondin-1 (Tsp1) and a related protein, connective tissue growth factor (CTGF).(72) In addition, antisense-mediated suppression of miR-17-92 expression partly restored Tsp1 and CTGF expressions, while transduction of miR-17-92 reduced Tsp1 and CTGF levels, resulting in larger, better-perfused tumors.(72) Similarly, vascular endothelial growth factor (VEGF) induced expression of miR-17-92 in endothelial cells, which was shown to be via miR-18a-mediated inhibition of Tsp1expression.(73) These results suggest a possible role of miR-17-92 overexpression in tumor angiogenesis in lung cancer. In contrast to these reports on the proangiogenic effects of miR-17-92, forced overexpression of miR-92a in endothelial cells was shown to block angiogenesis both in vitro and in vivo through repression of several proangiogenic proteins (integrin α-subunits, etc.).(74) In contrast, a different network was observed in chronic lymphocytic leukemia (CLL), as upregulation of miR-92 was found to contribute to repression of von Hippel-Lindau tumor suppressor gene (VHL) expression under a normoxic condition in CLL cells, which led to reduced ubiquitination and degradation of HIF-1α, and consequential autocrine stimulation of VEGF secretion.(75) Such overexpression of miR-17-92 observed in lung cancer may contribute to angiogenesis. Therefore, miR-17-92 may regulate the angiogenic network positively or negatively in a cellular context-dependent manner.
Paralogous clusters of miR-17-92
In the mammalian genome, there are three paralogous miRNA clusters; miR-17-92, miR-106a-363 and miR-106b-25, among which the miR-17-92 and miR-106b-25 clusters have similar expression patterns in adult mice, while the expression level of the miR-106a-363 cluster is generally undetectably low.(69)miR-106b-25 is localized in an intron of the MCM7 gene, which is involved in licensing of DNA replication, and is transcriptionally regulated by E2F1 and MYC, similar to miR-17-92. miR-106b-25 was reported to be overexpressed in gastric cancer,(76) while it has also been shown that overexpression of miR-106b-25 modulates transforming growth factor (TGF)-β-induced cell-cycle arrest and apoptosis through inhibition of CDKN1A (p21Waf1/Cip1) and BIM, respectively.(76) However, a large body of evidence points to crucial involvement of miR-17-92 in tumor development among these three paralogous miRNA clusters. Along this line, it is interesting that only the miR-17-92 cluster contains miR-18 and miR-19, which are absent in other miRNA clusters, while miR-19 was suggested to be crucially involved as a key oncogenic miRNA in mice models of lymphoma development through inhibition of PTEN expression and consequent activation of AKT-mTOR and apoptosis repression.(77,78)
miR-17-92 in lung development
There are several lines of evidence that support the notion that miRNA are crucially involved in lung development.(69,79–81) Dicer deficiency induces branching arrests without epithelial growth arrest, resulting in a few large epithelial pouches. Therefore, miRNA processed by Dicer appear to play important roles in regulating lung epithelial morphogenesis.(79) miRNA expression profiling analysis has also shown that miR-17-92 clusters are abundantly expressed at the early stages of lung development, while the expression level declines as development proceeds. In contrast, the let-7 miRNA family has an inverse expression pattern and becomes predominant at the late stage.(80,82) Since the expression pattern suggests a physiological role of miR-17-92 in the early development of the lung, SPC-miR-17-92 transgenic mice were produced, which demonstrated expansion of the distal epithelial progenitors and increases in neuroendocrine cell clusters, indicating that miR-17-92 promotes a high level of proliferation and an undifferentiated phenotype of normal lung epithelial progenitors. Meanwhile, disruption of miR-17-92 clusters was shown to cause lethal abnormalities, including lung hypoplasia, ventricular septal defects and inhibition of B cell development.(69) In contrast, ablation of either miR-106b-25 or miR-106a-363 had no obvious phenotypic consequences. Interestingly, combined disruption of both miR-106b-25 and miR-17-92 resulted in a more severely lethal phenotype,(69) suggesting an additive effect of miR-106b-25. Crucial roles of miR-17, miR-20a and miR-106b, all of which are highly expressed at the pseudo-glandular stage of embryonic lung development, were also reported by Carraro et al.(81) In that study, expression of these miR-17 family members was suppressed in explants of isolated lung epithelium, and experimental results showed that these miRNA modulate FGF10-induced budding morphology by specifically targeting the signal transducer and activator of transcription 3 (STAT3), as well as mitogen-activated protein kinase 14 (MAPK14), which are FGF10-FGFR2β downstream signal mediators.(81) These results indicate a tight relationship between oncogenic properties and physiological functions of miR-17-92 in the lung.
Mechanisms of dysregulation of let-7 and miR-17-92 in cancer
Elucidation of the molecular mechanisms of miRNA dysregulation is of immense interest and should help to better explain the global picture of the molecular pathogenesis of cancer, which would eventually lead to development of therapeutic strategies targeting miRNA abnormalities. Transcriptional repression, epigenetic silencing and genetic alteration may play roles in the reduced expression of let-7, as has been shown following downregulation of protein-coding genes. Among the 11 let-7 family members, six are localized within cancer-associated genomic regions or in fragile sites,(83) while there are also lung cancer cell lines that harbor homozygous deletions of the let-7c cluster at 21q11.2- q21.1(84). Epigenetic silencing has been specifically reported in let-7a-3(85,86), although cancer-related epigenetic silencing has not been reported in other let-7 family members. Furthermore, expression of the let-7 family was reported to be under the influence of direct repression by c-MYC.(87)
Aberrations in miRNA processing also appear to be involved. let-7 biogenesis is controlled by multiple layers of regulation, including negative regulation by LIN28, as discussed above. Along this line, c-MYC overexpression indirectly suppresses the expression level of mature let-7 through induction of LIN28,(46) which inhibits the processing of let-7 precursors. LIN28/LIN28B have also been shown to be induced by overexpression of c-MYC(46,88) as well as NF-κB activation,(89) both of which are known to be frequent in lung cancers.
Overexpression of miR-17-92 appears to be caused by transcriptional activation and/or genetic amplification. The miR-17-92 cluster is transactivated by c-MYC,(54) E2F1/E2F3(59,60) and STAT3,(90) each of which are frequently activated in cancer. In addition, a paralogous cluster, miR-106b-25, is transcriptionally upregulated together with a host gene, MCM7, by E2F1.(76) Inactivation of p53, which is frequently present in various types of cancer including lung cancer, may also be involved, since transcription of the miR-17-92 cluster has been shown to be repressed by this tumor suppressor.(67) Furthermore, we previously reported occasional association of the gene amplification of miR-17-92 with its overexpression in lung cancers,(5) while our preliminary analysis of a CGH dataset at Sanger Institute (http://www.sanger.ac.uk) showed an association of focal amplification/gain of the miR-17-92 locus with SCLC histology and large cell carcinomas (data not shown), confirming our previous report.(5) Also, Re et al.(91) performed a genome-wide survey and reported the possible existence of feed-forward regulatory circuits involving microRNA and transcription factors, including those of let-7 and miR-17-92. Given that computing power continues to increase, future detailed investigations of genome-wide mRNA–miRNA networks using high-powered computing methods will be of particular interest and should provide in-depth insight into the molecular mechanisms of dysregulation of miRNA in cancer.
Findings thus far reported clearly point to crucial roles for let-7 and miR-17-92 in the pathogenesis and progression of lung cancer, as they appear to affect the machinery of two key cellular functions, stemness maintenance and cell-cycle regulation. Several relevant targets for let-7 and miR-17-92 have been identified, and suggested to play roles in cancer development. However, we are far from gaining a complete picture of the dysregulation involved in the complex regulatory networks related to these miRNA. In addition, the world of non-coding RNA is rapidly expanding. Recent reports have demonstrated a miRNA-like function of snoRNA(92) and a novel RNA decoy function of miRNA.(93) Each miRNA is thought to regulate hundreds of target mRNA, which in turn regulate multiple genes, including protein-coding genes and miRNA, while tens of thousands of non-coding RNA other than miRNA are known to be transcribed from the human genome. Thus, it would be reasonable to predict the future necessity of a radically different approach to elucidate the resultant unbelievably complex regulatory networks present in cells in both normal and cancerous conditions. Along this line, a cancer systems biology approach with the aid of ever evolving computing power may help to show how these indispensably informative pieces of an as yet unresolved puzzle fit into a comprehensive understanding of lung cancer biology. Therapeutic application of such acquired knowledge of miRNA alterations in cancer remains a daunting challenge, although additional information should ultimately lead us to the answers we seek.
We thank all members of our laboratories at Nagoya University and Aichi Cancer Center for their invaluable contributions, including helpful discussions and critical comments. In addition, we apologize for the incompleteness of the referencing due to space constraints. Studies performed in our laboratories were supported in part by grants from the Ministry of Education, Science, Sports and Culture, Japan, and from the Ministry of Health, Labour, and Welfare, Japan, as well as from the Princess Takamatsu Cancer Research Fund and Uehara Memorial Foundation.