Cancer’s Lifelines

The long and short of telomeres
Summer 2017

In this image, prepared by Pitt’s Roderick O’Sullivan, you can see actual, individual chromosomes; it looks like a petri dish of blue bowties. On the ends of each chromosome, notice the tiny, glowing dots. Each dot represents one telomere. We can even tell the lagging strand (green) apart from the leading strand (red).



Like us, our cells are mortal. Each time they divide, the tiny, protective caps at the ends of our chromosomes—our telomeres—get shorter and shorter, sort of like a fuse. When enough of the fuses burn out, the body senses that the cell has run its course and moves into self-destruct protocol. This is the cellular circle of life, a perfectly natural event that happens innumerable times within your body each day. It’s probably happening right now.

For cancer cells, however, mortality is often sidestepped; they are masters at gaming the body’s built-in self-destruct apparatus. One way that cancer cells (about 85 percent of them) do this is by producing telomerase, an enzyme used in our own stem cells to rebuild telomeres. “Once the cell is able to maintain its chromosomes on its own, then it becomes quite dangerous,” says Patricia Opresko, a molecular biologist and associate professor of environmental and occupational health in the Graduate School of Public Health and in the University of Pittsburgh Cancer Institute (UPCI). This is because the cancer cell can now live through things normal cells can’t. And as it does so, it gets stronger. 

“The problem with biological systems is they all look the same from a distance. Everything’s basically just bags of water, protein, and nucleic acids, and there’s not a lot of specific contrast you can get out of that unless you add something to it,” says Marcel Bruchez. To overcome this, Bruchez has rigged up 14 pairings of proteins and fluorescent dyes (engineered bits of human antibodies) that allow us to label bewilderingly small targets. What’s more, when these fluorogen-activating proteins, or FAPs, are exposed to even short bursts of light, they generate higher energy oxygen molecules (singlet oxygen), which cause oxidative damage. Here we can see cancer cells, and only cancer cells, labeled in red. These same cells exposed to light and destroyed with precision (yellow), while neighboring cells (blue on the left) are left completely unharmed.






“Cancer cells accumulate all these chromosomal changes and rearrangements that they can potentially take advantage of to evade the things we throw at them, like chemotherapeutics and ionizing radiation,” says Opresko. 

What’s interesting is that cancer cells don’t typically use telomerase to create long, youthful telomere fuses. Instead, they create short, stubby telomeres just long enough to keep the Bureau of Programmed Cell Death off their backs. And it’s this weakness scientists hope to exploit. Remember, shorter telomeres are just a breath away from self-destruction. So if researchers could figure out a way to inhibit telomerase production in cancer cells, it shouldn’t take long for those fuses to burn out on their own.

Unfortunately, telomerase is only one pathway to tumor immortality.

In just the past two decades, scientists have discovered that some cancers can perform a sort of chromosomal alley-oop, where a short telomere can actually fix itself by copying and pasting what it needs from a long telomere found elsewhere in the cell. This is what’s known as homologous recombination. Homologous recombination is crucial for conjuring up new sperm and egg cells, by the way. It’s also employed by the 15 percent of cancers that use what’s known as the alternative lengthening of telomeres, or ALT.

“A lot of these cancers are the really, really bad ones,” says Roderick O’Sullivan, a Pitt assistant professor of pharmacology and chemical biology, who specializes in ALT. “They tend to be resistant to chemotherapy. And usually, by the time you identify them, [it’s] too late.”

What’s particularly frustrating is that while some cancers are typically telomerase-based and others are ALT-based, the two can also work in tandem. A patient suffering from pancreatic cancer can have some cells wielding telomerase and some using ALT. Even in a single tumor, both pathways can exist. And what’s worse, O’Sullivan says, is that if you inhibit telomerase production in some cancer cells, you can actually activate ALT.  

But even immortal cells have weaknesses. And thanks to Opresko, O’Sullivan, and other Pitt people, we’re learning more about these vulnerabilities every day.

In fact, one way to hamstring these little nasties may be to hit them with a weapon as ancient as life itself—oxidation.

You see, life has been battling with oxidative stress and its constant, caustic bombardments to cellular machinery for perhaps 4 billion years. In fact, the advent of oxygen in the atomosphere is considered one of Earth’s first major polluting events.

“It’s no coincidence that life didn’t crawl out of the muck until it had evolved strong pathways for limiting oxidative damage,” says Ben Van Houten, Richard M. Cyert Professor of Molecular Oncology in the Department of Pharmacology and Chemical Biology.

What’s more, organisms developed ways to take the lemons of oxidative damage known as free radicals and turn them into lemonade. “Our bodies are bathed in DNA damage,” says Van Houten. “And actually, a little bit of damage is good for you.”

Despite their reputation for contributing to disease and aging, free radicals help us digest our food, kill microbes, and power our muscles and brains. Opresko has even found that, under certain conditions, free radicals can help lengthen telomeres.

But too many free radicals scratch away at our cells’ internal workings like trillions of tiny, raspy dogs at the back door.

It appears as though cancer cells have even more free radicals bouncing around inside them than regular cells. Cancer cells are really good at processing free radicals before they can cause too much oxidative damage. But if we could inhibit the cancer cell’s ability to do so, oxidative damage would theoretically build up to such levels that the cell could not survive.

The Opresko lab has adapted Marcel Bruchez’s fluorogen-activating protein technology (see caption above) to constantly bombard the telomeres of cancer cells with targeted oxidative damage to see how they hold up. Preliminary results: not well. “If you continually hammer away at those telomeres, they start to become really unhappy,” she says.

Another researcher, Pitt’s Li Lan, MD/PhD assistant professor of microbiology and molecular genetics, has been using a fluorescent protein from jellyfish called KillerRed to inflict oxidative damage at the sections of DNA that produce genes. Similarly, Van Houten wants to understand how oxidative damage mangles mitochondrial DNA and what role that might play in bringing down the beast that is cancer.

These scientists are in search of cancer’s Achilles’ heel (or heels). And though Van Houten stresses that what they are doing is basic science and not developing any immediate new treatments, he can’t help but be optimistic about the armory of techniques he and his colleagues have assembled to probe an adversary known for its ability to duck what medicine throws at it. “This work could change cancer therapy as we know it,” Van Houten says.

In this first row of images, Pitt’s Li Lan shows how the KillerRed dye (stained green) can be attached to sites of individual telomeres (marked with red dots). Blue is a nucleus. By breaking these components down in highly specific ways, Lan says, we can better understand how cancer cells build themselves back up. One way cancer cells repair damage is with an enzyme, a kinase called Nek7. In the second row of images, you can see the tiny dots of Nek7 glowing green, then the telomere-targeted damage caused by KillerRed (in red), and finally an overlay showing that Nek7 comes in to stabilize the telomere once it’s been damaged. No one knew this interaction existed until Lan brought it to light recently.






Patricia Opresko aimed to find out how a cancer line with traditionally short telomeres performed in comparison to a cancer line with uncharacteristically long telomeres when each was treated with inhibitors that prevented the cells from cleaning out telomere building blocks that had been damaged by free radicals. The cells with the short telomeres (shown left) appeared to suffer a great deal more than their long-telomered cousins (right). “Every red spot is marking some sort of damage,” says Opresko. Studies like this tell us that cancer cells with short telomeres are extremely vulnerable to manipulation of their antioxidant mechanism.





Pitt’s Edward Burton, a neurologist, has enabled scientists to witness previously unknown aspects of cellular machinations of neurodegeneration as they happen in living systems. He's interested in oxidative damage, which can cripple brain function. Now Burton is helping UPCI colleagues port Bruchez’s ingenious FAP tags into zebra fish—which have telomeres pretty close in size to those in humans. (Mice have really, really long telomeres, says Opresko.) Although the data for this project are very preliminary, Burton offered this image of a live, larval zebra fish that’s had its retina, brain, and peripheral nerves labeled with a green-fluorescent protein expressed with an approach that’s similar to how his team is labeling telomeres with FAP.








​Images: Roderick O’Sullivan Lab; Marcel Bruchez Lab; Li Lan Lab; Elise Fouquerel, Opresko Lab; Edward A. Burton.

Opresko Lab images reprinted with permission from Macmillan Publishers Ltd: Nature Structural and Molecular Biology, Vol 23 Issue 12, Elise Fouquerel et al. “Oxidative guanine base damage regulates human telomerase activity,” pp. 1092-1100. © 2017.

Burton image reprinted from Neuroscience Letters, Vol 449 Issue 3, Qing Bai et al, “Expression of a 12-kb promoter element derived from the zebrafish enolase-2 gene in the zebrafish visual system,” pp. 252–257, © 2009, with permission from Elsevier.