Journal News

MCP: Deciphering
the RAF inhibitor paradox

Saddiq Zahari
June 01, 2017

The mitogen-activated protein kinase signaling pathway, called MAPK for short, is a crucial pathway for cellular growth and division and is activated aberrantly in roughly 30 percent of cancers, which is why scientists have sought for drugs that inhibit it. In a study published in the journal Molecular & Cellular Proteomics, researchers at Université de Montréal aimed to delineate the changes that underlie a paradoxical effect seen in cancers that have mutations in a central member of the MAPK pathway. The lead investigator, Pierre Thibault, says, “This research will bring us closer to a real understanding of the biological response of cells to environmental cues and to therapeutic agents.”

In cancer cells with RAS mutations, vemurafenib activates the MAPK pathway. The research team treated cells with vemurafenib and then subjected them to phosphoproteome analysis. They took snapshots every five minutes for an hour.Image courtesy of Pierre Thibault

One class of drugs, a group of inhibitors against the kinase B-RAF, has been effective in stemming the aberrant activation of the MAPK pathway. B-RAF frequently is mutated in cancers; in fact, the V600E mutation of B-RAF is highly prevalent in melanomas and thyroid cancers. Vemurafenib, a drug approved by the U.S. Food and Drug Administration, inhibits this mutant B-RAF. It is especially efficacious in treating cancers with the V600E mutation.

But there’s a paradox. In cancer cells with mutations in an upstream activator of B-RAF called RAS, vemurafenib and inhibitors like it provoke the opposite response — they activate the MAPK pathway instead. This causes drug resistance in tumors and limits the efficacy of the inhibitors. Thibault characterizes this paradoxical pathway activation as “a textbook example of unpredicted events caused by pharmacological agents.”

In collaboration with Marc Therrien at Université de Montréal, Thibault and his team used high-resolution mass spectrometry to profile the temporal changes in the phosphorylation levels of all proteins in the cell, the phosphoproteome, that occur when cancer cells are treated with vemurafenib. They coupled high-resolution mass spectrometry to a method called stable isotope labeling of amino acids in cell culture, or SILAC, which labels cells with heavy versions of amino acids to quantify accurately the changes in protein phosphorylation between the treated and control cells.

A phosphoproteome profile at a time point provides a snapshot of the signaling picture at that time point. However, this static picture limits the information obtained, which is particularly problematic when trying to understand a highly dynamic event, such as cellular signaling activation. So the investigators painstakingly took snapshots every five minutes up to one hour after inhibitor treatment. By doing so, the researchers managed to understand how phosphorylation of proteins changes over time.

The investigators identified 37,910 phosphorylation sites. By comparing the kinetic profiles of two cell lines that harbor either an RAS or a B-RAF mutation, the researchers uncovered which proteins were modulated in response to vemurafenib. They identified 660 phosphorylation sites that displayed temporal response in line with the paradoxical effects in the mutated RAS cancer cells.

From the 660 phosphorylation sites, they discovered a number of novel targets of ERK2, the downstream effector of B-RAF. One of these is TEAD3, a transcription factor involved in the Hippo pathway, another important pathway in cancer, suggesting a direct link between the MAPK pathway and the Hippo pathway. Another novel target found was MKL1, a cofactor involved in cancer metastasis. Through a series of biochemical experiments, the investigators discovered a novel mechanism of MKL1 phosphorylation by ERK2.

“This approach will greatly assist researchers who are developing better inhibitors of B-RAF to inhibit oncogenic signaling in colorectal cancer and other cancer types without adverse paradoxical activation,” says Thibault. He also believes that their approach will be of use to answer questions in basic biology, such as the effects of sustained versus transient ERK activation and how these effects influence the fate of the cell. Thibault says, “Our approach will allow us to tackle these fundamental questions in several cellular contexts.”

Saddiq Zahari

Saddiq Zahari is the editor for manuscript integrity at MCP.

Join the ASBMB Today mailing list

Sign up to get updates on articles, interviews and events.

Latest in Science

Science highlights or most popular articles

Rodents in space Keeping bone and muscle strong on the ISS
News

Rodents in space Keeping bone and muscle strong on the ISS

September 19, 2020

Researchers helped mice stay mighty with an experiment to counter the effects of microgravity. The gene treatment might also enhance muscle and bone health on Earth — and in humans.

Understanding the impact of Type 1 diabetes susceptibility genes
Research Spotlight

Understanding the impact of Type 1 diabetes susceptibility genes

September 17, 2020

Starting in eighth grade, a series of mentors who saw something special in Sharifa Love–Rutledge helped her stay on the path to being a researcher — and becoming a mentor to others.

Re-creating coagulation in a lab
Journal News

Re-creating coagulation in a lab

September 15, 2020

Threatened arthropods are in the crossfire of medical and conservation efforts, but new research could benefit horseshoe crabs and humans alike.

Decoy receptor neutralizes coronavirus in cell cultures
News

Decoy receptor neutralizes coronavirus in cell cultures

September 13, 2020

To keep COVID-19 from infecting tissues once they’re exposed, a new study led by Erik Procko suggests luring the virus with an engineered, free-floating receptor protein that binds the virus and blocks infection.

When plants and their microbes are not in sync, the results can be disastrous
News

When plants and their microbes are not in sync, the results can be disastrous

September 06, 2020

Sheng-Yang He’s lab has discovered that plants can develop dysbiosis, a condition similar to inflammatory bowel disease in humans, with severe consequences.

A zest for synthetic biology
Feature

A zest for synthetic biology

September 02, 2020

Metabolic engineers seek to overcome the challenges of mass-producing commodity chemicals, such as limonene, an oil from orange peels.