Long Non-coding RNAs and Their Clinical Relevance in Cancer and Cancer Therapy

Long non-coding RNAs (lncRNAs) are clinically relevant in at least two major ways: as biomarkers for cancer or carcinogenesis and as actual targets for cancer therapy.

LncRNAs as biomarkers for cancer and carcinogenesis

LncRNAs can be biomarkers for cancer or carcinogenesis, and can yield insight into possible sensitivity or resistance to potential anti-cancer therapies. A well-known example includes the FDA-approved PROGENSA PCA3 assay testing in urinary samples used as part of the screening paradigm for prostate cancer. This is one demonstration of a testable form of lncRNA that exists with sufficient stability in a non-invasive biological sample, highlighting the key attributes of stability and ability to be assayed of a viable biomarker.

LncRNAs as targets for cancer therapy

A number of lncRNA genes have been found to be expressed at elevated levels which correlate with various cancers. To investigate a possible causal connection, there are several options to reduce or knockout expression in vivo, where the technology has already been proven to be effective in in vitro or cell culture settings. These approaches include (Gutschner et al.,2018):

  • siRNAs that are complementary to lncRNA which takes advantage of the RISC/argonaute system to degrade the target molecule
  • anti-sense oligos (ASOs), refined with chemical modifications to enhance activity or stability, which have been demonstrated to function by targeting lncRNAs to the endogenous RNase H systems for destruction
  • the use of targeting ribozymes, deoxyribozymes, or CRISPR/Cas9-derived technologies which have been proposed for silencing lncRNA.

The evolving field of RNA biology and lncRNA

Image credit: Parasramka et al. (2016)

Once relegated to the ‘junk’ DNA category, large segments of the genome and their associated transcripts have long been ignored, or thought of as unimportant. Only a few years ago it was demonstrated by Djebali et al. (2012) that up to 75% of the human genome is transcribed into processed RNA transcripts even though less than 2% of the genome encodes proteins. According to the latest GENCODE release, the human genome contains in the neighborhood of 20,000 protein-coding genes while the total number of annotated lncRNA genes is nearly 16,000 (Hu et al., 2018). However, this number is not a true final tally, and the real number may be considerably higher. In a 2017 paper, Hon et al. (2017), while attempting to create a highly accurate map of all lncRNAs 5’-ends, determined that there are likely over 25,000 lncRNA genes in humans and possibly many more depending upon the stringency of the algorithms used to determine them. Equally intriguing is the data that indicate lncRNA expression seems to be more cell-type and tissue specific than protein coding genes (Yan et al., 2015). LncRNA sequence conservation is also often, though not universally, lower between model organisms and humans. This may indicate that lncRNA is a later evolutionary arrival than protein-coding genes, and thus may be particularly relevant to the development of higher eukaryotes.

The fields of RNA and cancer biology have experienced numerous fascinating transformations in the last few decades. LncRNAs are at the center of one of the most intriguing stories in this regard, as they touch both domains. LncRNAs are operationally defined as transcripts with 200 nucleotides or more in length with either zero or very limited protein-coding potential. They have significant properties in common with mRNA, including being transcribed by RNA Polymerase II and being subject to RNA processing, such as capping, splicing, polyadenylation, and even RNA editing (Gutschner et al.,2018).

LncRNAs play interesting roles in gene expression and cancer

LncRNAs not only appear to be ubiquitous in eukaryotic biology but have the capacity to play many interesting roles in gene expression. As Hu and team demonstrated (2018), lncRNA can use a host of both cis- and trans-regulation mechanisms to modulate gene expression: LncRNAs can regulate gene expression by acting as assembly points for transcription factors. Another example is their ability to  regulate the activity of various chromatin modification complexes by acting as a scaffold at target sites in the genome. In several cases, including the MALAT1 gene, lncRNAs appear to play roles in subnuclear architecture to either activate or repress target genes by association with particular domains within the nucleus (Koopp and Mendeell, 2018). In other instances, lncRNAs can act as molecular decoys or guides for proteins that bind to DNA or other RNAs. At the post-transcriptional level, lncRNAs can act as mRNA regulators to change either translation or mRNA stability, and they have been reported to modulate  alternative splicing of mRNA. In addition, lncRNAs can act as microRNA sponges. It should be noted that in some cases of cis-regulation of target genes, the lncRNA product itself may not be crucial. Instead, it is the actual process of transcription of the lncRNA gene that represents the critical step in affecting nearby gene expression (Koopp and Mendell, 2018).

With so many mechanisms to influence gene expression, it is not surprising that lncRNA molecules can play roles in various types of cancer. In a recent review, Gutschner and team (2018) described the impact of lncRNAs and their mis-regulation and therefore contribution to the development of various cancers. Examples include:

  • the relatively well characterized example PCA3 can bind to an mRNA target which is normally a tumor suppressor(Salameh et al., 2015)
  • the lncRNA, HOTAIR, which interacts with a histone modification complex to epigenetically silence a collection of tumor suppressor genes (Kim et al., 2013), or
  • the lncRNA MALAT1 which is actually highly conserved amongst mammals and regulates both metastasis-associated and cell-motility related genes (Gutschner et al.,2018).

LncRNA goes commercial – companies that are betting on lncRNA

The commercial sector of lncRNA is relatively new as reflected by the fact that there are not yet many companies working in this sector. Some of the emerging interesting actors or products in this area include:

Translate Bio (formerly RaNA Therapeutics) focuses on the discovery and development of therapeutics that target or work via RNA. In 2016, the company issued a patent that is based on blocking interactions of lncRNA with chromatin modification complexes. Translate Bio focuses on a variety of diseases including Cystic Fibrosis, OTC deficiency and rare liver, lung or CNS diseases.

 

RXI Pharmaceuticals, in collaboration with Biogazelle, has designed a ‘self-delivering’ RNAi (sd-rxRNA) system to specifically silence multiple lncRNAs.

 

 

 

Ocean Ridge Biosciences, an RNA sequencing and microarray and genomic analysis company, offers lncRNA adapted RNA-seq and analysis services.

 

 

Bio-Rad Laboratories recently announced a lncRNA quantification workflow – an RT-qPCR workflow optimized for lncRNA discovery and validation – without the need for RNA-seq.

 

Arraystar offers an entire series of microarray products designed to evaluate lncRNA expression levels in human, mouse, or rat.

 

 

 

References

Djebali et al. Landscape of transcription in human cells. (2012) Nature, 6;489(7414):101-8.

Gutschner et al. From biomarkers to therapeutic targets-the promises and perils of long non-coding RNAs in cancer. (2018) Cancer Metastasis Rev., 37(1):83-105.

Hu et al. The role of long noncoding RNAs in cancer: the dark matter matters. (2018) Curr Opin Genet Dev., 48:8-15.

Hon et al. An atlas of human long non-coding RNAs with accurate 5′ ends. (2017) Nature, 9;543(7644):199-204.

Kim et al. HOTAIR is a negative prognostic factor and exhibits pro-oncogenic activity in pancreatic cancer. (2013) Oncogene, 32(13):1616-25.

Koopp and Mendell, Functional Classification and Experimental Dissection of Long Noncoding RNAs. (2018) Cell, 25;172(3):393-407.

Parasramka et al. Long non-coding RNAs as novel targets for therapy in hepatocellular carcinoma (2016) Pharmacol Ther., 161:67-78.

Salameh et al. PRUNE2 is a human prostate cancer suppressor regulated by the intronic long noncoding RNA PCA3 (2015) Proc Natl Acad Sci USA,112(27):8403-8.

Yan et al. Comprehensive Genomic Characterization of Long Non-coding RNAs across Human Cancers. (2015) Cancer Cell, 12;28(4):529-540.