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Cancer Res. Author manuscript; available in PMC 2011 Jul 1.
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PMCID: PMC3057914

Long intergenic non-coding RNAs – New links in cancer progression


The process of cancer metastasis consists of a series of sequential and complex steps. Here we give a perspective on recent results of non-coding transcription in cancer progression, focusing on the emerging role of long intergenic non-coding RNAs (lincRNAs). LincRNAs target chromatin modification complexes or RNA binding proteins to alter gene expressing programs. Similar to miRNAs, lincRNAs exhibit distinct gene expression patterns in primary tumors and metastases. We discuss how lincRNAs can be utilized for cancer diagnosis, prognosis, and serve as potential therapeutic targets.


Most deaths from cancer are due to metastasis (1) and the main barrier to the treatment of metastases is the biological heterogeneity of cancer cells in the primary neoplasm and in metastases. Full understanding of gene regulation network during this transition is essential but still far beyond completion. Intensive investigations over the last few decades focused on the protein-coding genes in pathogenesis of cancer. The human genome contains only ~20,000 protein-coding genes, representing <2% of the total genome while a substantial fraction of the human genome can be transcribed, yielding many short or long non-coding RNAs (ncRNAs) with limited protein-coding capacity (2). Is ncRNA the missing piece of cancer jigsaw puzzle? One of the emerging themes of non-coding transcripts is microRNAs (miRNAs), a class of small regulatory RNAs, mediate post-transcriptional silencing of specific target mRNAs (3). The identification of miRNA, miR-34, as the direct target of the tumor suppressor gene p53 (4), revealed for the first time that ncRNAs function in this crucial tumor-suppressor pathway. Numerous miRNA expression profiling and functional studies also associate miRNAs with cancer progression, diagnosis, prognosis and treatments (5, 6). Therefore, the interplay between proteins and ncRNAs is anticipated in cancer biology, and ncRNAs may be the missing links of well known oncogenic and tumor suppressor networks. The other class of newly discovered ncRNAs is long intergenic non-coding RNAs (lincRNAs, ranging from several hundred to tens of thousands of bases). Here we provide an update and perspective on recent advances in lincRNA mechanisms in cancer progression.

LincRNA expression and cancer

There are over 3,000 human lincRNAs, but less than 1% has been characterized (79). Recent studies show that lincRNAs are exquisitely regulated during development and in response to diverse signaling cues (8), and can be misexpressed in solid tumors and leukemias (10). Numerous HOX lincRNAs were found differently expressed between primary breast carcinomas and distant metastases (11), and many p53-dependent lincRNAs are also identified in respond to DNA damage (12). It has been found that several lincRNAs can control transcriptional alteration, implying that the difference of lincRNA profiling between normal and cancer cells is not merely the secondary effect of cancer transformation and lincRNAs are strongly associated with cancer progression (13). Thus, differential expression of lincRNAs may be profiled to aid cancer diagnosis, prognosis and select potential therapeutics.

Although lincRNAs may have impacts on human diseases (10, 13, 14), the basis of their molecular mechanisms is still largely unknown. Several lincRNAs can control gene expression by direct recruitment of histone modifying enzymes to chromatin (9). Chromatin modification and DNA methylation are key epigenetic events which are fundamentally disturbed during the development of cancer. Epigenetic alterations are potentially stable and heritable, which can occur at a much greater rate than DNA mutations in somatic cells (15). Dysregulated lincRNAs may affect epigenetic information and produce a cellular growth advantage; its selection may result in the progressive and uncontrolled growth of a tumor. One such lincRNA, HOTAIR is highly expressed in breast cancer metastases and in primary tumors predisposed to future metastases. Importantly, enforced expression of HOTAIR can target Polycomb Repressive Complex 2 (PRC2, comprised of histone H3 lysine 27 methylase EZH2, SUZ12, and EED) genome-wide to alter H3K27 methylation and gene expression patterns, and increased cancer invasiveness and metastasis in vivo. Loss of HOTAIR or PRC2 components inhibits cancer invasiveness, indicating potentially direct roles for lincRNAs in modulating cancer progression (11). HOTAIR is the first lincRNA identified functioning in trans (16), but other antisense lincRNAs can silence genes in cis. For example, ANRIL is antisense transcribed from tumor suppressor genes CDKN2B and CDKN2A, where ANRIL interact with CBX7 (subunit of PRC1) resulting heterochromatin formation and gene silencing (1719).

LincRNAs may carry out many of their functions by acting as modular scaffolds fro protein-chromatin interaction (20, 21). Tsai et al. recently discovered that HOTAIR bridges together PRC2 complex with the LSD1 H3K4 demethylase complex, and recruits both complexes to target genes to coordinately alter several histone modifications and enforce gene silencing (21). As another example, TLS protein can be allosteric modified by interacting with ncRNACCND1, transcribing from 5’ regulatory regions of CCND1 in response to DNA damage signals. The conformation change of TLS induces gene-specific TLS–CBP/p300 interactions which in turn inhibit CCND1 transcription (22). Another lincRNA MALAT-1, highly expressed in many different tumors, can interact with SR splicing factors and modulates their distribution to nuclear speckles. MALAT1 then regulates alternative splicing by controlling the functional levels of SR splicing factors (23). LincRNAs can also affect global gene changes in response to signals to classic transcription factors. For example, p53 induces lincRNA-p21, which in turn represses numerous genes globally by recruiting the repressor protein hnRNP-K (12).

Dozens of lincRNAs are differentially expressed in cancers —sometimes by hundreds to thousands of folds—among primary tumors with different clinical courses, and indeed in multivariate analysis with clinical and pathologic risk factors, HOTAIR RNA can serve as a significant and independent predictor of eventual metastasis and death (11). Risk stratification by mRNA or miRNA profiling typically requires dozens if not hundreds of RNA species, and in this regard, the ability of a single species of lincRNA, HOTAIR, to achieve these results is promising. These examples suggest that HOTAIR and other lincRNAs may be used as a novel prognostic tool in cancer management.

Mutations and alternative RNA structural variation

Misexpression of lincRNAs in cancer naturally raises the question of structural variations in lincRNA genes, either germline or somatic, that may contribute to cancer predisposition. To date, analysis of DNA copy number variations and rearrangements have focused on protein coding genes, although many non-coding sequences are included in recurrent structural aberrations of cancer genomes. Similarly, genome-wide association studies (GWAS) of cancer susceptibility have mainly identified single nucleotide polymorphisms in noncoding portions of the genome, some of which may be transcribed (24). For instance, a region upstream of the 9p21 locus--encoding cyclin-dependent kinase inhibitors CDKN2B, CDKN2A and p53 activator ARF-- is associated with cancer as well as cardiovascular disease and diabetes, and this region is transcribed to produce ANRIL, a long noncoding transcript that is implicated in controlling chromatin modification of the locus (1719). Unlike mutations in protein coding genes where certain single nucleotide mutations (such as a premature stop codon or frame shift) can completely abrogate protein function, structural variations in lincRNAs may have more subtle effects, thus more difficult to verify in experimentally (25). Better annotation of lincRNA expression patterns and primary structures should improve the detection and interpretation of genome aberrations that affect lincRNAs. Moreover, because known oncogenes and tumor suppressor genes are often dysregulated by multiple mechanisms, lincRNAs that are misexpressed in cancers should be the starting point for possible structural alterations in lincRNA genes.

Targeting Small Non-coding RNAs associated with Cancer

A major goal in developing cancer therapeutics is the identification of molecular targets that are specific for cancer cells (26). The differential expression and biological importance of specific ncRNAs in cancer suggest that ncRNAs may be useful targets (27). Here we briefly discuss efforts to target miRNAs, and then speculate on the potential of targeting long non-coding RNAs for cancer treatment (Figure 1).

Figure 1
Targeting cancer specific ncRNAs

MicroRNAs have been effectively targeted in preclinical models by small molecules and exogenously introduced complimentary RNA sequences (28). High-throughput screens, both in vitro and in vivo, have identified small molecule that affect several steps in miRNA biogenesis, including pre-miRNA processing or interacting directly with the miRNA, thus blocking miRNA hybridization with its endogenous 3’-UTR sequences (28). For example, a small molecule inhibitor of miR-122 can selectively induce apoptosis in liver cancer cells (29). Screens involving small molecules often are initiated with little prior chemical knowledge of a successful binder. Because every RNA sequence is different, generalizing the chemical functionality of anti-miRNA molecules is difficult. Further, although the studies such as those mentioned above are leading to promising compounds, their modest binding affinity, often in the micromolar range (30), suggests the need for improvement. A more targeted approach, by exploiting the miRNA sequence directly, may be more advantageous.

Antagomirs are synthetic RNA molecules that are designed to directly hybridize with miRNAs, thus potentially limiting the availability of the miRNA for Argonaut loading and 3’-UTR hybridization. Antagomirs are chemically modified to prevent premature degradation by RNases, thereby increasing their half-life in vivo (31). Krützfeldt et al. first demonstrated that antagomirs can inhibit specific miRNA function in living mice (32). A recent study by the Weinberg lab demonstrated the utility of antagiomirs in preventing the onset of metastasis. Systematic treatment of tumor-bearing mice with antagomirs targeting miR-10b, increased the expression of its target, HOXD10, a tumor suppressor gene that is down-regulated in metastasis (33). Intravenous administration of the antagomir did not reduce primary mammary tumor growth, however, strikingly suppressed the formation of lung metastases, in a sequence specific manner (34). These studies preliminarily suggest that antagomirs may be powerful molecules to disrupt the miRNA pathway and may be potential chemotherapeutics. While our understanding of the potential of lincRNAs as therapeutic targets is just beginning, it is conceivable that the avenues that have been explored for other non-coding RNAs could be utilized to target LincRNA-protein interactions. In this next section, we will highlight some of the potential avenues that can be tailored to LincRNAs and their protein partners.

Potential new cancer therapy-- lincRNAs

While targeting cancer-specific miRNAs has proven to be successful, designing molecules to inhibit long non-coding RNAs presents different opportunities and challenges. LincRNAs can be depleted by siRNAs, providing a straight-forward approach to inhibit the function of lincRNAs implicated in cancer. Gupta et al. showed that depletion of HOTAIR inhibited the matrix invasiveness of breast cancer cell line (11). In practice, we and others have found that one needs to screen more siRNAs to have successful knockdown of lincRNAs compared to mRNAs, possibly due to extensive secondary structures in lincRNAs (21).

Structural insights into riboswitches, the ribosome, RNase P, and catalytic RNAs are beginning to be exploited for the design of specific small molecule inhibitors targeting RNA and ribonucleoprotein (RNP) complexes (3537). For example, atomic structure of the bacterial ribosome, the most complex RNP structurally characterized to date, has been utilized to identify functional sites within rRNA for therapeutic targeting by antibiotics (38). These results underscore the importance of structural studies in the aid of therapeutic design against complex RNAs. Detailed structural studies are still forthcoming for the recently discovered lincRNAs associate with cancer.

LincRNAs like HOTAIR serve as a structural scaffold for protein complexes, and possess complex RNA structural motifs (21). The chromatin complexes that interact with lincRNAs are ubiquitous; therefore, targeting the proteins themselves may lead to deleterious phenotypes. However, targeting the RNA-protein interface, focusing on the cancer-specific transcript such as HOTAIR, may provide an inroad to cancer- specific therapeutics. Targeting transcripts that are the size of lincRNAs may seem like a daunting task, nevertheless, there is precedence for fragmenting large RNP complexes into more manageable sizes. This strategy has been applied to the design of ligands that bind expanded rCUG and rCAG repeat RNAs expressed in myotonic dystrophy type 1 (DM1) that interact with Muscleblind-like 1 (MBNL1) protein. In vitro identified ligands also inhibit the formation of RNA-protein complexes in mouse myoblasts (39). These results, and others, provide strong evidence that examining defined, functionally relevant RNA fragments in vitro can lead to the discovery of small molecule binders; the information gained can be used in the cellular context to disrupt RNA-protein interactions.

While similar investigations and principles that are used to target miRNAs with small molecules may be applied to lincRNAs, the strategy with antagomirs will not be the same. Because of the structural content of lincRNAs is expected to be vast, designing antagomirs based on primary sequence may yield unsuccessful results. However, utilizing a more unbiased approach may yield oligonucleotide sequences that bind to lincRNAs. Methods such as SELEX (systematic evolution of ligands by exponential enrichment) provide an unbiased means to identify high affinity binders of a selected macromolecule (40). Just recently, SELEX was utilized to identify RNA sequences that bind to pri-miRNAs (41). Unlike their mature products, pri-miRNAs are larger and contain substantial predicted secondary structure. Therefore, a similar approach may yield RNA sequences that can bind to lincRNAs at key lincRNA-protein interfaces, thereby disrupting the interaction. We suggest the term “antagolincs” for this yet to be established class of molecules. Once an RNA sequence is identified through SELEX, the same RNA sequence can be transformed into the more chemically stable construct similar to an antagomir. The administration of an antagolinc against HOTAIR would then lead to competitive inhibition of a chromatin remodeling complex, such as PRC2, by binding to the lincRNA. This RNA-RNA interaction may then normalize the chromatin state to inhibit cancer cell growth and metastasis.

Concluding Remarks

Just as dysregulation of miRNAs is now recognized as a universal feature of many types of cancer, many lincRNAs that affect cancer initiation, progression, and treatment are surely waiting to be discovered. Cancer-associated lincRNAs may provide new approaches to the diagnosis and treatment of cancer. Systematic identification of lincRNA expression patterns and characterization of lincRNAs and their associated proteins should pave the way for designer therapeutics that may target lincRNAs for cancer and other disease states.


Supported by grants from National Institutes of Health (R01-CA118750 to H.Y.C., T32-AR007422 to R.C.S.), California Institute for Regenerative Medicine (RN1-00529-1), American Cancer Society (RSG 07-084-01-MGO) and Susan G. Komen Foundation (to M.-C.T.). H.Y.C. is an Early Career Scientist of the Howard Hughes Medical Institute.


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