How chemotherapy drug delays enzymes that aid growth of cancer cells

NEW YORK: A recent Cornell University study sheds fresh light on how the chemotherapy medication etoposide slows and destroys critical enzymes that drive cancer cell development. The study, done in Michelle Wang's lab in the College of Arts and Sciences, will help researchers better comprehend a variety of cancer inhibitors. Michelle Wang is the James Gilbert White Distinguished Professor of Physical Sciences and a Howard Hughes Medical Institute Investigator. The approaches developed by the researchers will also enable the development of sensitive screening tools for discovering pharmacological pathways that can improve patient care.

The group's paper, 'Etoposide Promotes DNA Loop Trapping and Barrier Formation by Topoisomerase II', was published on January 30 in Nature Chemical Biology. The co-lead authors are research specialist Tung Le and postdoctoral researcher Meiling Wu. For 40 years, etoposide has been a trusted chemotherapeutic for treating a variety of cancers. Etoposide succeeds by targeting Type IIA eukaryotic topoisomerases, enzymes - also known as topo IIs -- that enable the replication of cancer cells.

At the centre of that replication process are the long, entangled, helical-coiled strands of DNA. In order for cancer to spread, these strands need to be untangled, rotated and copied by motor proteins. Topo IIs are well-suited for the job. They perform an elaborate kind of rope trick that relaxes the supercoiled DNA by cutting it, very quickly passing another DNA strand through its middle, and then reconnecting the cut DNA back together. All of that is done without damaging the DNA's delicate genetic structure -- an incredible, and incredibly fast, feat of biology that happens in the body roughly 300 billion times a day. Etoposide's great virtue is that it can stabilize a DNA double-stranded break before anything is reconnected, thereby preventing the cancer cell from replicating. However, the intricacies of how etoposide interacts with DNA's structure have remained murky.

"We normally ask: What is the best way to study molecular machinery that takes place on DNA?" Wang said, adding, "To understand how those enzymes work, we want to mimic what might be happening in the cell. Motor proteins pull on the DNA or apply a force on the DNA. So we said, OK, we can apply a force and see what happens." Wang's lab used three different single-molecule manipulation techniques to observe etoposide's effect on three topo IIs, which were provided by collaborators led by professor James Berger of Johns Hopkins University: yeast topoisomerase II, human topoisomerase II alpha and human topoisomerase II beta.

"DNA topology, conceptually and in terms of torsional mechanical properties, is really hard for people to grasp," Wang said, adding, "There were very few ways to study it. But we happen to have just the right tools. And the reason we have the right tools is that for the last 20 years, we've been working on developing them. These tools and this problem just happened to converge at the right time." First, the researchers used optical tweezers to stretch DNA into various configurations, demonstrating how etoposide compacts, releases and breaks it and creates DNA loops. This loop-trapping behaviour surprised everyone as it revealed a new impact of etoposide that was not previously known. It implies that etoposide could promote topo II to significantly alter DNA structure and topology in vivo.

Then the team used optical tweezers to unzip double-stranded DNA into two single strands for high-resolution mapping of protein interactions with the DNA, and so mimicked the motor removal of a bound protein. The findings suggest that etoposide could convert topo II into a strong roadblock of DNA-processing types of machinery. Their third technique is a version of magnetic tweezers in which they twisted DNA with a bound topo II and watched the topo II relax the DNA at a steady rate. When they added etoposide, they found the chemical staggered this pattern, introducing pauses that correlate with the trapping of supercoiled loops.

By capturing the different ways etoposide enhances these actions and interferes with topo II function, the researchers now have a quantitative system for characterizing how other topoisomerase drugs behave. "I think this gives us a set of tools that would allow us to study many different kinds of topoisomerases and other kinds of drugs in a very comprehensive way," Wang said, adding, "Everything we do mimics what happens in vivo. We just do it in a mechanically controlled fashion. This is why it's so powerful."