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In line with their broad-based effects, microRNAs (miRNAs), small non-coding RNA molecules ~22 nucleotides long that silence target mRNAs, are thought to act as oncogenes or tumor suppressor genes based on their inhibition of tumor-suppressive and oncogenic mRNAs, respectively. We and others previously showed that global downregulation of miRNAs, a common feature of human tumors, is functionally relevant to oncogenesis as impairment of miRNA biogenesis enhanced transformation in both cancer cells and a K-Ras-driven model of lung cancer. The dysregulation of miRNA biosynthesis in cancer emerges as a cancer-specific mechanism that enhances its tumorigenic capacity. These observations are further supported by the fact that frameshift mutations of TARBP2 occur in sporadic and hereditary carcinomas with microsatellite instability and that DICER1 mutations are associated with familial pleuropulmonary blastoma. Accordingly, it was reported that reduced expression of miRNA-processing factors is associated with poor prognosis in lung cancer and ovarian cancer. Recently we have also demonstrated the presence of Exportin 5 (XPO5) inactivating mutations in tumors with microsatellite instability. This observed genetic defect is responsible for nuclear retention of pre-miRNAs, thereby reducing miRNA processing. The characterized mutant form of the XPO5 protein lacks a C-terminal region that contributes to the formation of the pre-miRNA/XPO5/Ran-GTP ternary complex and the protein itself, as well as pre-miRNAs accumulating in the nucleus of cancer cells. Most importantly, the restoration of XPO5 function reverses the impaired export of pre-miRNAs and has tumor suppressor features. Our data suggest a cancer-specific mechanism to guide the subcellular distribution of miRNA precursors and prevent them from being processed to the active mature miRNA. The control of the miRNA biosynthesis pathway is emerging as an important mechanism in defining the spatiotemporal pattern of miRNA expression in cancer cells.
MicroRNAs (miRNAs) are regulatory RNAs that silence mRNAs in a sequence-specific manner. Originally discovered in Caenorhabditis elegans, almost a thousand human miRNAs are now known to repress target mRNAs. Biogenesis of miRNAs in mammalian systems involves multiple steps, including transcription of primary miRNA (pri-miRNA), cleavage of pri-miRNA to precursor miRNA (pre-miRNA), nucleocytoplasmic export of pre-miRNA, cleavage of pre-miRNA to miRNA duplex and formation of functional RISC.1,2 Pri-miRNAs are transcribed by RNA polymerase II and cleaved by the Microprocessor Complex, containing at least Drosha (RNAase III endonuclease) and DGCR8 in humans (a double-stranded RNA-binding protein). This complex recognizes the double-stranded RNA structure of the pri-miRNA and specifically cleaves at the base of the stem loop, releasing a ~70-nucleotide precursor pre-miRNA. This pre-miRNA is then exported through the Exportin-5 (XPO5) pathway into the cytoplasm. XPO5 protein in a complex with Ran protein recognizes and binds the pre-miRNA molecule, exporting it to the cytoplasm along a GTP to GDP gradient. Once in the cytoplasm it is further processed into a mature miRNA duplex by DICER1, a second RNase III endonuclease, together with its catalytic partner TAR Binding Protein (TRBP). The miRNA duplex is then loaded into a multi-complex, the RNA-induced silencing complex (RISC), which is comprised of at least TRBP, DICER1 and one Argonaute (Ago2 in humans). Most animal miRNAs exhibit imperfect homology with their targets, which inhibits translation, thereby controlling some of the most important cellular processes such as proliferation, apoptosis and development, amongst others.
It is clear that miRNA processing is regulated in a complex manner, and we are only beginning to understand its true nature. In principle, miRNA abundance could be controlled at the transcription of the pri-miRNA, during any of the biogenesis steps, or with the turnover of the mature miRNA. Early studies by several laboratories found that mature miRNA expression does not always correlate with expression of the pri-miRNA, especially in the context of oncogenesis.3–8 However, PCR and northern blot have been used to demonstrate that the expression of mature miRNAs is generally correlated with pri-miRNA expression in normal tissues.9–12 On the other hand, several studies have shown that miRNAs are present at the level of the precursor but are not processed to the mature miRNA in cancer cells and primary tumors. For example northern blot analysis demonstrated that the precursors of miR-143 and miR-145 were expressed in colorectal tissues and tumors; however, the mature miRNA was detectable only in normal colorectal tissue.13 Pre-miR-138-2 was shown by northern blot and in situ hybridization to be expressed in HeLa cells, but the mature miR-138 was undetectable.4 Also, it has been reported that a wide discrepancy exists between the levels of precursor and mature miRNA, suggesting that unknown mechanisms that control processing play a critical role in regulating the expression of the active, mature miRNA.7 It was also demonstrated that mutations14 or SNPs15 within the miRNA precursors can interfere with miRNA processing. Although we believe this to be an unlikely explanation of the lack of miRNA processing, the widespread nature of the phenomenon makes it important to demonstrate that cancer cells avoid being controlled by miRNAs that impair their biosynthesis.
Therefore, impairment of these biosynthesis checkpoint control mechanisms of mature miRNAs in cancer cells could lead to an abnormal expression profile of these small non-coding RNAs, thus enhancing the tumorigenic process.
The causes of the aberrant miRNA expression pattern in cancer may be due to DNA copy number amplification or deletion,16 inappropriate transactivation,17 genetic mutation18 or epigenetic mechanisms.19,20 Indeed, some miRNA loci often show genomic instability in cancer, and it was also reported that c-Myc broadly repressed the transcription of miRNA genes.21,22
The widespread downregulation of miRNAs is often observed in human malignancies, including breast, prostate and ovarian cancers.23–26 In addition, global repression of miRNA biogenesis by suppression of the key components of miRNA processing machinery, such as Drosha, DGCR8, DICER1, TRBP and XPO5, promotes cellular transformation and tumorigenesis.27–31 While the mechanism(s) remain to be fully elucidated, it suggests that miRNAs might have an intrinsic function in tumor suppression and its downregulation eventually accelerates oncogenesis.
Moreover, it has become evident that some characteristic aspects of cancer-related biological processes, including drug resistance, tumor angiogenesis and metastasis, are associated with miRNA function.32,33 Therefore, understanding the mechanisms behind miRNAs dysregulation in human cancer and their functional consequences might provide new insights for improving the classification, prognosis prediction and treatment of cancer.
A distinctive stage in evolution was the formation of the cell nucleus, which stores the genetic information encoded by DNA. This compartmentalization resulted in the segregation of key steps in the synthetic pathways from DNA to protein. A similarity exists with the biogenesis of miRNA molecules. The appropriate subcellular localization of the pre-miRNA is essential to their complete processing, their function and regulation. Compartmentalization can control access to binding partners, concentrate cofactors or temporarily segregate components of the pathway away from the rest of the cellular environment. The exquisite spatiotemporal control of miRNA abundance is made possible, in part, by regulation of the miRNA biogenesis pathway. While pri-miRNA processing is a nuclear event, pre-miRNA processing occurs in the cytoplasm. Thus, pre-miRNAs need to transit from the nucleus into the cytoplasm, a process that requires the nuclear export receptor XPO5.34,35
Frameshift mutations occur in protein-coding sequences, which may render affected proteins nonfunctional and thus drive carcinogenesis through the inactivation of tumor suppressor genes. We have reported frameshift mutations in the XPO5 gene in two MSI+ cell lines and primary tumors.31 The mutations found in exon 32 alter and truncate the protein sequence and prevent XPO5 from associating with its pre-miRNA cargo and exiting the nucleus (Fig. 1). In XPO5 heterozygous mutant cells, less pre-miRNA was hence accessible to processing by the cytoplasmic machinery, resulting in decreased mature miRNA levels and enhanced tumorigenicity. Restoration of XPO5 wild-type protein levels in the defective cells rescued pre-miRNA export and processing defects and had tumor suppressor features. These cancer cells exhibited impaired miRNA processing but failed to lose the wild-type XPO5 allele. Recent work has suggested that other components of the miRNA biogenesis pathway, DICER1 and TARBP2, are haploinsufficient tumor suppressors.28–30 Moreover, biallelic deletion was found to impair cell viability, hence preventing the phenomenon of loss-of-heterozygosity (LOH).28–30 This is also the case for XPO5, where mimicking LOH by RNA interference against the XPO5 wild-type transcript rendered cells unviable.31 In addition, the miRISC components AGO2, TNRC6A and TNRC6C can also be mutated in MSI+ cancers,36 although the functional consequences remain to be evaluated. The presence of mutations in the miRNA pathway genes, including TARBP2 and XPO5 genes, in MSI+ cancer samples has also been reported in this separate study of Korean patients.36
The mutated status of this central component turned out to be crucial in malignant transformation since it clogged pre-miRNA flow between the nucleus and cytoplasm. The disruption of this process resulted in a dramatic deregulation of cellular functions that triggered tumorigenesis. Additionally, we also characterized a minimal region in XPO5 required for pre-miRNA binding and/or recognition and therefore for nuclear envelope transversion. Others have reported that XPO5 is expressed at low levels in many tumor types.37 Therefore, the XPO5 heterozygous mutational event found in human cancer clogs the miRNA-nuclear export complex in the nucleus and prevents the export of pre-miRNAs to the cytoplasm and further processing. Nonetheless, analysis of miRNA array data shows that there are pre-miRNAs that the processing does not seem to alter by this impairment of the export machinery. Recent work in C. elegans has suggested additional nuclear export pathways for pre-miRNAs that could explain why some miRNA levels remained unaffected by this mutation.38 Many pre-miRNAs are also targeted by ADARs at various stages of their processing, and the modification can also prevent export of pre-miRNAs.
Similar to what we demonstrated, the expression of over 200 precursor and mature miRNAs demonstrated a wide discrepancy between in a high percentage of human primary tumors.10 Our results are also in line with the finding that, after profiling 225 precursor and mature miRNAs in 22 human primary tumors and 16 pancreatic and liver tissues/tumors, many of the miRNAs analyzed are processed to the precursor but these precursors are retained in the nucleus.7 However, this study did not provide any possible explanation that could shed light on the mechanism underlying this pre-miRNA processing blockage and nuclear retention. In light of our results we can now speculate about possible impairment at the level of the nucleocytoplasmic pre-miRNA export machinery in some of the analyzed samples. Two other groups have also reported the accumulation of let7 miRNA precursors at various stages during fruit fly and sea urchin development.39,40 Although this could represent a defect in RNA processing, it is also possible that the nuclear export of the let7 precursor, and hence access to cytoplasmic DICER1 and TRBP, are developmentally regulated.
Recent reports show that states of increased proliferation or cellular transformation are associated with widespread occurrence of the production of mRNAs with shortened 3′UTR and fewer miRNA target sites, indicating that a global switch of the use of miRNA-mediated gene regulation could occur as part of a general program for cellular proliferation and transformation.41,42 These findings are consistent with the enhancement of miRNA maturation under DNA damage and the previous findings on widespread decrease of miRNAs in cancer. Avoidance of regulation of gene expression by miRNAs might be a general feature of cancer cells, and a regulatory layer by miRNAs may have an essential function in tumor suppression. Accordingly, it was reported that reduced expression of miRNA processing factors is associated with poor prognosis in lung and ovarian cancer.42,43
Overall, there is increasing evidence from the genetic28,30,31 and functional27,29 standpoints to suggest a role for miRNA processing machinery genes, such as DICER1, TRBP, DROSHA and XPO5, as tumor suppressor genes in human cancer.