Research published online today in Nature Cell Biology explains how a non-coding RNA encourages cancer growth and metastasis. Specifically, scientists from the Medical University of South Carolina (MUSC) found that PNUTS lncRNA soaks up a microRNA that prevents epithelial-to-mesenchymal transition, which is a key feature of tumor growth and metastasis.
The contribution of lncRNAs to tumor progression and the regulatory mechanisms driving their expression are areas of keen interest to Philip H. Howe, Ph.D., chair of the department of biochemistry & molecular biology, and the Hans and Helen Koebig Endowed Chair in Oncology at the MUSC Hollings Cancer Center. Howe and his team found that a pre-RNA for a protein called PNUTS can be alternatively spliced to form a lncRNA that contributes to cancer progression. The PNUTS lncRNA does not encode a protein, but rather soaks up a specific microRNA that is usually tasked with preventing epithelial-to-mesenchymal transition.
Determining how this complex process unfolds required examination of a ribonucleoprotein called hnRNP E1, which binds to pre-RNA and suppresses alternative splicing. Howe’s team knew that TGF-beta, which is released in large amounts by tumor cells, could prevent its binding, potentially allowing alternate forms to be made.
Computer models predicted that this ribonucleoprotein could bind to PNUTS pre-RNA on its alternative splicing site. In lung and breast cancer cell lines, specially designed RNA probes confirmed that this exact splicing site was more exposed when the ribonucleoprotein was knocked down and that those cells had more PNUTS lncRNA. When cells were exposed to TGF-beta over time, PNUTS lncRNA was made in increasing amounts. The team found that the ribonucleoprotein was bound more tightly with the alternative splice site. In normal conditions, this allowed PNUTS protein to be made, but in tumors, the alternative splice site became exposed and more lncRNA was made instead.
In order to confirm exactly how PNUTS lncRNA could encourage tumor formation, additional computer simulations were conducted and they predicted that, based on its sequence, there were seven potential locations on the PNUTS lncRNA for microRNA-205 to bind. This microRNA binds and destroys a transcriptional regulator called ZEB1 that encourages cells to unstick from one another and spread, a major step that allows epithelial-to-mesenchymal transition to occur. As predicted, without those potential binding locations, the lncRNA and the microRNA were unable to bind together. This helped cells stick together and spread less, even with TGF-beta added to push them to spread.
When it appeared that PNUTS lncRNA was soaking up microRNA-205, which freed up ZEB1 to encourage cells to act more like tumors, the group stuck fluorescent molecules to ZEB1 to track it and found that more of it was present when there were more PNUTS lncRNA. In addition, preclinical models revealed that breast and lung tumors grew faster and larger when their cells contained more PNUTS lncRNA.
Now Howe and his team are conducting additional experiments to find other such long non-coding RNAs that follow this same mechanism in cancer, with the goal of developing therapies to target them. "My prediction is that this mechanism didn't evolve to make just one long non-coding RNA," he says. "There are probably others that are generated in this same fashion."