The unexpected complexities of TERT, a key cancer driver

Telomerase reverse transcriptase (TERT), an enzyme associated with nearly all malignant human cancers, is even more diverse and unconventional than previously realized, new CU Boulder research finds. 

Telomeres, the protective ends of chromosomes, help to maintain genomic stability. In most normal adult human cells, the telomeres eventually shorten beyond a critical length, bringing a cell’s life to its natural end. In almost all human cancers, however, telomerase reactivates, leading to cell proliferation and tumor formation. This is a key part of what makes cancer cells “immortal.”

As TERT is the component of telomerase that is required for cancer development, it has become an attractive target for cancer therapeutics in recent decades. 

“TERT has great importance in cancer progression and hence there is a great interest in studying its expression regulation,” said Gabrijela Dumbovic, a post-doctoral researcher in the Department of Biochemistry and co-lead author of the CU Boulder study.

The study, published recently in the journal Proceedings of the National Academy of Sciences, used powerful, high-magnification imaging techniques to reveal differences in how TERT is produced on the single-cell level, in individual cancer cells. Previously, studies relied on general averages of TERT production within whole tissue samples or large cell populations, without investigating potential cell-to-cell variation.

About a year ago, researchers at the BioFrontiers Institute began discussing ways to apply ribonucleic acid (RNA) localization imaging protocols to better classify how TERT RNA was being made within individual cancer cells. (RNA typically serves as a messenger for turning DNA genetic information into proteins, though it is also important for coding and regulating gene expression and catalyzing certain biochemical reactions.) What began as a casual conversation grew into an interdisciplinary effort, combining experience in RNA imaging from the laboratory of Professor John Rinn of CU Boulder’s Department of Biochemistry and the BioFrontiers Institute with innovative telomerase research led by Distinguished Professor Thomas Cech, a Howard Hughes Medical Institute (HHMI) Investigator and Nobel laureate.

“We have a great community of scholars in the Caruthers Biotechnology Building,” Cech explained. “Our students and postdoctoral fellows are encouraged to talk freely about their work, so collaborations are forged naturally and frequently.”

The researchers used a microscopy technique known as single-molecule RNA fluorescent in situ hybridization (“smFISH,” for short) to visualize individual RNA molecules that coded for TERT in separate cancer cells. Looking at the microscope images, they counted the number of TERT RNA molecules – which appeared as tiny fluorescent spots – in different locations within each cell. 

“Previous studies were mostly focused on studying TERT expression in a population of cells. We took a different approach and actually could visualize TERT RNA levels, and RNA distribution on a single-cell level,” said Dumbovic.

The authors also found that while most cells in our bodies have two copies of a given gene (one from our mother and one from our father), the cancer cells frequently had more than two copies of the TERT gene. Such gene amplification is common in cancer cells, which have relatively unstable genomes. 

“The TERT gene has made all these extra copies of itself,” Rinn said. “Selfishly, it wants to replicate itself, and cancer wants to hijack that mechanism to keep the its cells alive indefinitely. That’s something we can only see with this kind of imaging.”

Intriguingly, the study also found that when looking at where TERT messenger RNA resides within a given cell, a high amount (over 80 percent in some instances) stays quarantined in the nucleus, rather than the expected cytoplasm, raising yet another mystery for future study. Typically, messenger RNA, which is made in the cell’s nucleus, is exported from the nucleus to be turned into protein in the cell’s cytoplasm. 

“RNA imaging has continually shed new insights into biology by providing an important layer of information of where a gene is in the cell. When we took the ‘molecular picture’ of the TERT gene, we’re struck by the unexpected and sometimes substantial amount of TERT RNA in the nucleus where it can’t function to make protein,” explained Rinn. “This opens up a new layer of regulation where the molecules of TERT RNA are when considering its abundance in cancer and other disease states.”

This surprising pattern of nuclear localization was also observed in healthy cells that produce TERT, specifically human induced pluripotent stem cells (iPSCs). “This suggests that the nuclear localization is not a behavior specific to the diseased cancer cells,” said Teisha Rowland, co-lead author of the study and former post-doctoral research in the lab of Thomas Cech. Rowland is now Director of the new Stem Cell Research and Technology Resource Center in the Department of Molecular, Cellular, and Developmental Biology.

“It was generally assumed that TERT mRNA localizes to the cytoplasm, which is needed for it to make a protein product, but now we have a different perspective. Now, we want to understand why TERT RNA is retained in the nucleus,” Dumbovic said. “Is there a stimulus that causes it to move, or to stay?”