Mutations, drugs drive cancer by blurring growth signals

Healthy cells rely on the central Ras/Erk growth signaling pathway (also known as the Ras/MAPK pathway) to interpret external cues about how and when to grow, divide, and migrate, but defects in how these messages are communicated can cause cells to grow out of control and aggressively invade other parts of the body. Such mutations are found in the majority of human cancers, making treatments for Ras/Erk defects a "holy grail" of cancer research.

Decades of study have led scientists to believe that Ras/Erk-driven cancers occur when mutations cause one or more components of the pathway to get stuck in a pro-growth state. Researchers have labored to develop targeted treatments that flip these broken switches back off, but so far most have failed to make it through clinical trials. Now, using a high-throughput technique developed at UCSF that allows scientists to take control of Ras/Erk signaling using pulses of light, and then quickly read out resulting genomic activity, researchers have made a surprising discovery about this extensively studied pathway.

Optogenetics -- in which light-sensitive proteins are genetically engineered into cells in order to make them respond to pulses of light -- has been a transformative laboratory technique in neuroscience, allowing researchers to control and study electrical activity patterns within networks of neurons with exquisite precision. By using the same approach to explore patterns of chemical communication within individual cells, the new research has revealed that some Ras/Erk mutations may trigger cancer by altering the timing, rather than the intensity, of cellular growth signals. The new study also shows that this blurring of signal timing may explain why some targeted drugs designed to shut off defective Ras/Erk signaling can paradoxically activate the pathway instead potentially raising the risk of new tumor formation.

"This new technique is like a diagnostic instrument that we hook up to a diseased cell, which lets us stimulate and interrogate the cell with many light-based stimuli to see how it responds," said UCSF synthetic biologist Wendell Lim, PhD, one of the study's senior authors. "Using this approach, we were able to identify cancer cells that have certain defects in how they process signals, behaviors that lead to cell proliferation in response to signals that normally are filtered by the cell circuits."

UCSF medical oncologist and cancer biologist Trever Bivona, MD, PhD, and Princeton molecular biologist Jared Toettcher, PhD, formerly a postdoctoral researcher in Lim's lab, were co-senior authors of the new study. The study's lead author was Lukasz Bugaj, PhD, of the University of Pennsylvania, also formerly a postdoctoral researcher in Lim's lab.

Optogenetics Reveals Corruption of Cellular Growth Signaling by Mutations, Drugs

The Ras/Erk pathway is complex, but at its core is a cascade of four proteins -- Ras, Raf, Mek, and Erk -- that activate one another like a chain of falling dominoes in response to growth signals from outside the cell. Ras sits at the cell membrane and receives incoming signals, then passes them along to Raf and Mek, which process and amplify them, until finally Erk (also called MAP Kinase or MAPK) transports the signal into the cell nucleus, where it can activate the appropriate genetic programs.

Previously, researchers had little understanding of how the timing of growth signals impacted cells' behavior. To address this question, the new research made use of a novel optogenetic tool which was developed by Toettcher as a post-doc in the Lim lab. This tool, OptoSOS, can be engineered into cells to trigger Ras activity in response to precisely timed pulses of light.

To track cells' responses to different patterns of Ras activation, the researchers engineered the OptoSOS system into multiple lines of healthy and cancerous cells, and placed different groups of these cells into an array of small wells in a laboratory dish. By illuminating this dish with a specially designed device -- dubbed the optoPlate -- the team was able to rapidly stimulate hundreds of different experimental groups of cells with a variety of test patterns, and simultaneously read out their responses under a microscope.

These techniques revealed that healthy cells respond selectively to long-lasting growth signals, while ignoring signals that flicker on and off -- presumably considering them to be irrelevant "noise." In contrast, the researchers found that certain non-small cell lung cancer (NSCLC) cell lines appear to misinterpret these intermittent noisy signals as stronger, sustained signals, triggering excessive growth and tumor formation.

"Cancer biologists expect oncogenic mutations to turn a pathway on to a constant, high level," Toettcher said."Our work shows that there is a second option, where mutant cells can still sense external inputs but alter the dynamics of their response."

This misreading of signals appears to occur because of a specific type of mutation in the protein B-Raf corrupts the timing of incoming growth signals, the researchers found, causing short pulses of Ras activation to reverberate for longer within an affected cell -- similar to how the "sustain" pedal on a piano causes individual notes to be drawn out and blur together.

When the researchers activated Ras in healthy cells with a brief pulse of OptoSOS stimulation, Erk would turn on and off again with only about a two-minute lag. In contrast, in B-Raf mutant cells, it took Erk activity 20 minutes to dissipate following OptoSOS stimulation, and further experiments showed that this resulted in activation of downstream genetic programs associated with cell growth and proliferation.

The researchers also showed that some targeted cancer drugs that are intended to shut down overactive components of the Ras/Erk signaling pathway may blur the fidelity of signaling much as B-Raf mutations do. Specifically, they found that vemurafenib and SB590885 -- part of a class of drugs called paradox activating B-Raf inhibitors -- significantly slowed how long it took Ras/Erk activity to shut off following OptoSOS stimulation, which could help researchers understand these drugs' known risk of triggering new tumor formation in patients.

"This research teaches us about a previously underappreciated dimension to oncogenic signaling and suggests that the timing of growth signaling could play an important role in a wider variety of human cancers," Bivona said. "There may be future diagnostic and therapeutic opportunities that leverage the ability to detect aspects of signal corruption on a functional level that are not apparent by merely sequencing the cancer genome with the descriptive approaches that are currently standard in the field."

Lim added, "We can now use interrogative tools like optogenetics to achieve a much more quantitative and systematic understanding of how cellular circuits work and how they break. This approach may be able to help us uncover what goes wrong in many diseases involving malfunctioning decision-making circuits in cells, ranging from cancer to autoimmunity."