Shifting liver cancer cells away from migratory state could reduce their drug resistance

Whether a liver cancer cell is primed to grow or move affects its ability to resist cancer drugs, according to new work from scientists at Fred Hutchinson Cancer Research Center and the University of Washington. The team identified key molecules that orchestrate these different cell states in hepatocellular carcinoma, or HCC. In lab dishes, experimental compounds that target these molecules can shift drug-resistant HCC cells toward drug sensitivity.

“We found new [molecular] players that are important in liver cancer and mediate resistance to therapy,” said Hutch systems biologist Dr. Taran Gujral, who co-led the project with Dr. Shao-En Ong at UW. “We found we can restore their sensitivity [to therapy] with drugs.”

In the study, published August 4 in the journal Cell Systems, the team also identified molecular signatures associated with sensitivity to specific drugs. The researchers hope their findings are a step toward bringing personalized oncology to patients with HCC.

A deadly liver cancer with few drug targets

Liver cancer is the fourth-most deadly cancer worldwide. Hepatocellular carcinoma, or HCC, makes up about 90% of liver cancer cases, and the diagnosis rate for it in the U.S. has tripled since the 1980s.

Advanced HCC is difficult to treat and so far it’s eluded the reach of personalized medicine, said Gujral. These tumors have few mutations that can be targeted with current drugs and there’s no way to match a patient with the best drug for his or her tumor. Oncologists prescribing these meds are taking a shot in the dark.

But most of the current drugs for HCC have something in common: They target the activity of specific enzymes, which are proteins that catalyze chemical reactions. This suggested to Gujral and Ong that enzymes might hold the key to unlocking the problem of drug resistance in HCC. With postdoctoral fellow Dr. Martin Golkowski, who led the work in Ong’s lab, and senior scientist Dr. Marina Chan, who contributed experiments from Gujral’s lab, they chose to study a class of enzymes called kinases. These enzymes often play central roles in turning on the molecular programs that make cellular process like growth, movement and division possible. Kinases themselves often turn “on” and “off,” switching between active and inactive states.

Gujral is an expert in the use of kinase inhibitors, molecules that block kinase activity, to untangle kinases’ roles in cellular processes. Ong had developed a method to measure protein abundance and activation state. With Golkowski and Chan, they combined their expertise to examine the role of kinases in drug resistance in HCC.

Cancer cells hijack normal developmental processes for their own ends

One of the main goals of Gujral’s research program is to untangle the complex problem of cancer-cell drug resistance. As part of this, he works to understand a phenomenon known as the epithelial-to-mesenchymal transition, or EMT, when specialized stationary cells acquire new characteristics that allow them to move through the body and make them harder to kill.

Epithelial cells are cells with specialized functions. The epithelial cells in the liver are hepatocytes, which detoxify the blood. HCC originates from hepatocytes. When the liver is injured, such as by viral infection, hepatocytes start to lose their specialized abilities and their tight connections to each other. They change shape and take on the ability to migrate, becoming more mesenchymal.

“I like to think of it as a ‘go or grow,’” Gujral said. “If the cell is dividing, it's not going to move at the same time.”

The epithelial-to-mesenchymal transition is an important part of normal embryonic development, as cells migrate to the correct areas of the body to begin growing into specialized tissues. It can also occur during wound healing, as cells move to fill in the gap.

“But cancer cells also use some of the components of the EMT for a completely different purpose — for acquiring the ability to invade surrounding tissue and metastasize [spread through the body],” Gujral said. “Most importantly, [the EMT] has been associated with drug resistance.”

Reversing the EMT reverses drug resistance

To examine the role of kinases in drug resistance, the team screened 17 HCC cell lines (cancer cells that can grow indefinitely in lab dishes) for differences in the amount and activity of all the kinases in each.

They found that HCC cells with mesenchymal characteristics were more likely to be drug resistant than those with epithelial characteristics. So they mapped the kinases that are turned on in mesenchymal HCC cells and turned off in epithelial HCC cells, and vice versa.

The new approach confirmed the importance in the EMT of a kinase Gujral had previously identified, Frizzled2, and identified several new kinases, including AXL and NUAK1 and 2, that also help coordinate the mesenchymal state.

The team was able to sensitize drug-resistant HCC cell lines by genetically removing or pharmacologically blocking AXL and other kinases associated with the mesenchymal state.

Identifying signatures of drug resistance and sensitivity

The investigators also tested each cell line’s vulnerability to kinase inhibitor drugs currently used in HCC treatment. They found that kinase activity better predicted drug sensitivity than the mere presence of the kinase. HCC cells with an active kinase were much more likely to be sensitive to drugs targeting that kinase than cells with an inactive version of the kinase.

“It's better to get closer to the source of the drug target if you want to predict the response to the drug,” Gujral said.

The team also collaborated with Dr. Ray Yeung, a liver cancer surgeon at UW Medicine, to examine kinase activity in tumor biopsy samples and identify molecular signatures that associate with sensitivity to specific drugs.

Continuing to seek drug targets that reduce drug resistance

So why don’t doctors use kinase activity assays to match their patients to the drugs most likely to help them? Well, they’re tricky to do, Gujral said. Unlike DNA — which, for the purposes of clinical testing, is nearly unchanging — or protein amounts, which take hours or days to change, the proteins can turn off or on in milliseconds, Gujral said. It's tough to create a clinical test when faced with a need for delicate, lightning-fast handling of tissue samples.

But there’s hope — work like Gujral’s could help identify key “controller” kinases like AXL or Frizzled that orchestrate the large cellular programs that make cells drug-resistant. Because such kinases are maintaining the program, their activation state is likely to be more stable, he said.

Gujral is working with researchers at the National Cancer Institute to seek out more drugs that are active against mesenchymal-promoting kinases like Frizzled and AXL.

The findings suggest that drugs which reverse the EMT, combined with drugs that kill epithelial-like tumor cells, could be someday be effective against HCC.

And by mapping the kinases in mesenchymal and epithelial cells, Gujral and his collaborators learned about the parallel molecular roads HCC cells used to arrive at the same mesenchymal state. This knowledge could help scientists figure out how to preempt cancer’s ability to find new routes to drug resistance when a drug blocks one path, he said.

This work was funded by the National Institutes of Health, the Sidney Kimmel Foundation, the German Research Foundation and a Research Scholar Grant from the American Cancer Society.