How a non-coding RNA encourages cancer growth and metastasis

A pro-tumor environment in the cell can encourage a gene to produce an alternative form of RNA that enables cancer to spread, report researchers at the Medical University of South Carolina in the journal Nature Cell Biology. 

MUSC researchers determined for the first time how an alternative form of RNA that does not encode a protein is formed and encourages tumors to spread. The finding, published August 21, may explain why certain breast and lung cancers grow and spread.

Two different kinds of RNA interact with each other to promote the growth of cancer. Neither RNA provides the code to make a protein, as many RNAs do. Instead, they push epithelial cells in the lung and breast to form tumors. Understanding how non-coding RNAs such as these work is a key challenge facing biologists around the world, because they could become an important source of new biomarkers and drug targets.

In cells, genes are expressed when DNA is copied to RNA to provide the code for a protein. Right after DNA is copied, the RNA must be cut and assembled into its final form. Sometimes it is cut and assembled in several different ways, in a process known as alternative splicing. These alternative forms of RNA can provide the code for slightly different proteins, but scientists are discovering that many types of RNA do not, instead performing different functions. Cells use different types of RNA to control how much of a given protein is made, which then helps control how cells behave. MicroRNAs, for example, do not provide the code for proteins, but instead home in on those that do and help degrade them.  

It is another class, called long non-coding RNAs (lncRNA), that are of particular interest to Philip H. Howe, Ph.D., chairman of the Department of Biochemistry and Molecular Biology at MUSC and the Hans and Helen Koebig Endowed Chair in Oncology at the MUSC Hollings Cancer Center.

Less is known about this type of RNA than the many others. Howe and his research team found that the RNA that provides the code for a protein called PNUTS can be spliced into its alternative form, an lncRNA that contributes to cancer progression. The PNUTS lncRNA does not encode a protein, but rather soaks up like a sponge a certain microRNA that prevents cells in the lung and breast from becoming more like tumor cells. 

Howe’s group connected a number of dots to explain how this happens. First, they found that breast cancer cells had more PNUTS lncRNA content than normal breast cells, which was a good initial sign that the lncRNA could be involved with cancer development. Those cells were also more likely to form tumors that spread. 

They next examined a protein that suppresses alternative splicing and that they thought could prevent this lncRNA from forming. Importantly, they knew that TGF-beta, a molecule that is released in large amounts by tumor cells to help them grow and spread, could prevent this suppressive protein from doing its job. In other words, TGF-beta could potentially allow alternate forms of RNA such as lncRNA to be made. Computer models predicted that this would happen, but the researchers ran experiments to confirm their suspicions. In lung and breast cancer cells, specially-designed RNA tracking probes confirmed that when the suppressive protein was removed from those cells, they had more PNUTSlncRNA. When those cells were exposed to TGF-beta over time, the suppressive protein was also blocked, and PNUTS lncRNA was made in increasing amounts.  

Yet the group wanted to confirm exactly how PNUTS lncRNA could encourage tumors to form. Additional computer simulations predicted that, based on its genetic sequence, there were seven potential locations on the PNUTS lncRNA where a microRNA called microRNA-205 could bind. This microRNA helps reduce the levels of a protein called ZEB1 that encourages cells to unstick from one another and spread like tumors. 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 when TGF-beta was added to push them to spread. 

To be sure that this was true, the group stuck fluorescent molecules to ZEB1 to track it and found that more of it was present when there was more PNUTS lncRNA. It appeared that PNUTS lncRNA was soaking up microRNA-205 like a sponge, which freed up ZEB1 to encourage cells to act more like tumors.

Finally, preclinical experiments revealed that breast and lung tumors grew faster and larger when their cells contained more PNUTS lncRNA. By connecting all of the dots, Howe’s group showed that one gene can make either a protein-coding RNA or a long non-coding RNA. With TGF-beta, the lncRNA soaked up microRNA-205, freeing up ZEB to drive cells to unstick from one another and spread, a critical event in the development of cancer and metastasis.

Howe suspects that the suppressive protein might serve the same function as many cancer drugs that stop tumors from making RNA. But they showed that the protein that suppressed RNA was itself blocked by TGF-beta, allowing alternative RNA to be made and cancer to spread. This may be one theory to explain why those cancer drugs may work for a short time before a cancer returns more aggressively and is resistant to therapy. 

This is the first study to show exactly how TGF-beta drives cancer through formation of a long non-coding RNA. Howe and his team are conducting 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,” said Howe. “There are probably others that are generated in this same fashion.”