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Cancer becomes metastatic when it escapes a tumor in one part of the body, then travels through the lymph system or bloodstream to invade another part, which is how tumors that form in the breast can spread to the brain.
Adam Nguyen, a graduate student at Fred Hutch Cancer Center, has won a F31 training award from the National Institutes of Health to study a complex molecule called Tie2 that aids and abets cancer’s escape from blood vessels when it malfunctions.
Nguyen will use leading-edge electron microscopy to visualize the complete structure of Tie2 with all the tiny pockets and grooves where a drug could potentially nestle and make a difference.
As an undergraduate student, he was fascinated by the intricate life of a cell that could be seen with a microscope, but he’s more excited by the opportunity to see life’s structures at the near atomic scale.
“I couldn’t imagine seeing anything smaller than a cell. That was really a big deal to me,” Nguyen said. “Now I can visualize the proteins and individual molecules that make life possible.”
The Ruth L. Kirschstein Predoctoral Individual National Research Service Award, about $144,000 over three years, includes tuition assistance and a stipend that will help him complete his PhD at the University of Washington.
Structural biologist Melody Campbell, PhD, in the Basic Sciences Division will guide the project. UW biochemistry professor Hannele Ruohola-Baker, PhD, an expert in the cellular biology of Tie2, is also a co-sponsor.
Cancer’s escape hatch
The body’s cardiovascular system constantly remodels itself, constructing new blood vessels from existing vasculature in a process called angiogenesis.
Tie2 is a protein mostly active in endothelial cells, which line the inner surface of blood vessels and regulate how tightly these cells are packed together. This creates a barrier that separates circulating blood and surrounding tissue.
Because it’s essential for life, angiogenesis is highly regulated. But genetic mutations, inflammation, and cancer can result in leaky blood vessels.
“It allows cancerous cells to get out of those vessels to metastasize,” Campbell said.
The role of Tie2 in metastasis makes it a tempting target for a drug, but the protein’s complete structure isn’t well defined.
“There aren’t a lot of therapies that target this specific protein,” Nguyen said.
Tie2 proteins are long and skinny and stick part way into the cell, penetrating the cell’s membrane like a cocktail toothpick spearing a ham sandwich.
Part of Tie2 sticking out of the cell binds to complex molecules called angiopoietins, which initiate growth signals through the toothpick into the cell, triggering more signaling within the cell.
The Tie2 proteins are super tiny, and researchers want to look at them in isolation when they’re not spearing a cell, but that creates a problem.
The segment of the toothpick sticking through the bread — the cell’s membrane — interacts with fat instead of water like the rest of the protein. If that segment is exposed to water, it will become a gooey mess.
That makes it difficult to stabilize the whole protein outside of the cell, so previously researchers have had to chop Tie2 into segments and then guess from the structure of those segments how they fit back together.
But those assumptions make it difficult to find an effective drug.
“If we have a drug that binds to a single fragment, how does this event impact another segment that isn’t attached? Because of this, we are assuming what could be happening,” Nguyen said.
No more guessing
Nguyen and Campbell plan to use a technique that protects the membrane-piercing segment with a tiny donut of fat called a nanodisc, which mimics the cell’s membrane and makes it possible to visualize the entire protein in one piece.
Nguyen will prepare purified proteins girded with nanodiscs in a super thin layer of water that is frozen so fast that it resembles glass because crystals don’t have time to form.
He’ll use cryo-EM, which shoots an electron beam through the frozen samples to a detector that creates two-dimensional images of the proteins captured from many different angles. A series of computer programs averages and sharpens all those snapshots to generate a high-resolution, three-dimensional image of the protein.
Campbell, who is the scientific director of Fred Hutch’s Electron Microscopy Core, said it will require a lot of troubleshooting at every stage from tricking cells into overproducing enough of the protein to purifying the samples to fitting them with nanodiscs for cryo-EM.
Nguyen will have to figure it out as he goes along, but the payoff will be significant if it leads to a better understanding of how Tie2 receives and sends growth signals across the cell’s membrane — essential information for finding a drug that can close cancer’s escape hatch.
Instead of imaging the protein segment by segment and inferring how the segments fit together, he’ll capture the whole Tie2 protein, including the angiopoietins it binds to outside the cell.
“There will be no more guessing,” Nguyen said. “We will actually be able to visualize the full protein in its native-like context without any assumptions about how things are ordinated with each other.”
