Treasures within the genetic ‘junks’: cancer therapeutics of lncRNA

Illustrated by Jennifer Broza

As the curtain fell on the Human Genome project in 2003, all the spotlight focused on those tiny double helixes and abundant information hidden within. While it thrilled the scientists that they finally had the blueprint of life all laid out on the table, further sequencing endeavors revealed a perplexing fact: less than 2 percent of the genome codes for proteins, which are the essential building blocks and functional units in our bodies. The other 98 percent of DNA is transcribed but serves no apparent or immediate functions. They were thus dismissed as “transcription noises” and referred to as “junk DNA.” 

However, as scientists investigated our genome further, these “junks” gradually earned their place in the arena. Although they do not code for proteins, they code for non-coding RNAs (ncRNAs), which plays an essential role in various cellular processes, including transcriptional regulation, post-transcriptional modifications, cell cycle arrest and chromatin regulation. Long non-coding RNAs (lncRNAs), defined as ncRNAs that are longer than 200 nucleotides, constitute a major class of ncRNA. Discovery of their regulatory pathways has increased attention to its implication in cancers, in which these crucial steps go awry. Studies regarding lncRNA and cancer spiked in the last decade, generating extensive evidence proving its role as an accomplice in cancer progression (2). Lnc2Cancer database, for example, documented associations between 1,614 human lncRNAs and 165 human cancer subtypes in more than 6500 papers.

Interestingly, lncRNAs modulate cancer from various aspects. By tuning the tumor suppressors, lncRNAs can either drive or halt the development of tumors. One of the most investigated tumor suppressors is p53, which works as a transcription factor and activates the expression of multiple genes related to cell cycle arrest and apoptosis. Studies have identified various lncRNAs that interact with p53 and contribute to tumorigenesis. For example, maternally expressed gene 3 (MEG3) is an imprinted maternal gene, and its RNA interacts with the p53 DNA binding domain, increasing p53 stability and transcriptional activity. In cervical tumors, increased expression of MEG3 reduces the tumor size, indicating its strong tumor-suppressing activity (3).  

Another point of lncRNA modulation happens in tumor metastasis, which is a crucial step in cancer progression and drastically impacts the prognosis of the patients. The benign disguise of the tumor is torn down as tumor cells begin to disseminate across the body. On the cellular level, one key step of metastasis is the epithelial-to-mesenchymal transition (EMT). Epithelial cells are specialized cells located on body surfaces (like our skin) and inner lining of organs, connecting closely to each other via junctions; in contrast, mesenchymal cells are unspecialized mobile cells unbonded with one another (4). During EMT, these cells reprogram their gene expression, break away from other cells and rearrange their cytoskeleton to increase their mobility (5). Within a decade of research on lncRNAs, we already discovered a significant amount of them modulating metastatic gene expression at all levels. For example, high expression of HOX transcript antisense intergenic RNA (HOTAIR) is a biomarker for metastatic progression and poor survival in breast, colorectal and prostate cancers (2; 3). HOTAIR does not affect the genetic expression of any genes near-by, but recruits polycomb repressive complex 2 (PRC2, a histone methylase) to modify (methylate) the chromatin at a specific locus. This repressive mark is associated with differentiation in embryonic cells and can help maintain a mesenchymal cellular phenotype (2,6). In other words, overexpression of HOTAIR helps cancer cells migrate and settle down in new colonies, facilita facilitating cancer metastasis. Another prominent example is Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). It plays an active role in metastasis as it taps into diverse regulatory processes from epigenetic marking to splicing control. As one mechanism, its interaction with serine/arginine-rich (SR) pre-mRNA splicing factors 1 (SRSF1) may lead to accumulation of its splicing targets, indirectly contributing to cancer cell survival and metastasis (6).  

Reaching here, you may be thinking: “Good to know, but how are we going to use the information? How to target RNAs?” Well, since we are aiming at these unconventional molecules, we also need novel forms of drugs, such as nucleic acid therapeutics. The intervention of lncRNAs’ formation can happen in multiple stages: 1) inhibition of lncRNAs transcription by modifying the DNA or blocking the promoter region. This stage can be achieved using the CRISPR technique; 2) initiating RNA degradation pathways; 3) blocking the interaction of lncRNA with protein complexes to render it unfunctional. Nucleic acid therapeutics can invoke RNA interference mechanisms and thus mainly tap into stage 2). Specifically, siRNA is a type of double-stranded RNA. It can recruit Dicer (an RNase-III enzyme) to cleave it into short single-stranded RNA, which combines with other proteins to form RISC (RNA induced silencing complex). In RISC, the single-stranded RNA serves as the “sight” of this weapon, binding specifically to specific RNA sequences, and RISC is responsible for the “attack” — cleaving the targeted RNA. Harnessing this pathway, the scientists artificially synthesized siRNA, chemically modified it to reduce unwanted nuclease cleavage, and introduced them into cancer cells (7). Although this technique has been primarily used to develop knockout or knockdown models, it has already shown clinical potential. For instance, siRNA treatments against the MALAT1 and HOTAIR lncRNA transcripts that we mentioned above inhibited cancer invasion and reduced viability in different cancer models (7, 8). 

Antisense oligonucleotides (ASOs) are another type of nucleic acid therapeutics option. ASOs in their simplest form are short single-stranded DNAs, but they are modified and combined with RNA to elevate their functionality and stability. They bind to RNAs, either hindering their expression physically or triggering the RNase H cleavage pathway. Arun, Diermeier, & Spector (2018) showed an almost 80% relative reduction in metastasis in certain breast cancer mouse model via the delivery of MALAT1 of ASOs. Despite these exciting experimental results, drug delivery presents a daunting hurdle that scientists need to surmount. These nucleic acids may have trouble finding their way through the cell membrane or being trapped in other “organs” of the cell without reaching the target RNA. Fortunately, several methods have been developed to facilitate delivery, such as lipid-based nanoparticle delivery, conjugate-based delivery and polymer-based delivery (8). Another critical issue is to ensure that these alien molecules do not accidentally invoke immune responses. Further research has been delving into these issues and conceiving chemical modifications and molecular structures that better the experimental outcome (7).  

As an accomplice of tumor genesis, progression and resistance, lncRNA rises as a promising therapeutic target. The combination of therapeutics targeting lncRNA and conventional drugs may prolong the lives of the patients, even potentially curing their malignancies. Admittedly, many mechanisms of lncRNAs are still elusive, and the behaviors of some have discovered to be contradictory in different cancers (3). Nevertheless, these gold within the ‘junks’ in our genome gives us new insights into the complex mechanism of cancer, signaling the opening of a new chapter of cancer therapeutics.

Edited by: Frank Lin
Illustrated by: Jennifer Broza

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