Cancer mutations and targeted drugs can disrupt dynamic signal encoding by the Ras-Erk pathway

Authors: L. J. Bugaj, A. J. Sabnis, A. Mitchell, J. E. Garbarino, J. E. Toettcher, T. G. Bivona, W. A. Lim


Introduction: Signaling pathways, such as the Ras-Erk (extracellular signal-regulated kinase) pathway, encode information through both their amplitude and dynamics. Differences in signal duration and frequency can lead to distinct cellular output decisions. Thus, temporal signals must be faithfully transmitted from the plasma membrane (Ras) to the nucleus (Erk) to properly control the cell’s response. Because the Ras-Erk pathway regulates important cell decisions such as proliferation, changes to dynamic signal transduction properties could result in improper cell decisions and dysfunction. However, it has been difficult to examine whether corruption of signal transmission dynamics is associated with diseases such as cancer.

Rationale: We used optogenetic stimulation of the Ras-Erk pathway to quantitatively screen whether cancer mutations and drug treatments alter the fidelity of dynamic signal transmission. Most cancer-associated mutations in the Ras-Erk pathway are thought to drive cancer by inducing constitutive pathway activation—a high basal amplitude of activity. We explored whether cancer cells might also have altered dynamic properties that could contribute to disease. We used live-cell microscopy and new high-throughput optogenetic devices to systematically measure cell responses to a broad range of dynamic input stimulus patterns. We could detect subtle but important perturbations in pathway signal transmission properties by monitoring how these upstream stimulus patterns (generated by use of Ras-activating optoSOS) altered pathway output at the downstream levels of signaling, gene expression, and cell proliferation.

Results: We found that cells that harbor particular B-Raf mutations (in the kinase P-loop) exhibit substantially corrupted dynamic signal transmission properties. In particular, the kinetics of Ras-Erk pathway inactivation are substantially slowed (half-time for signal decay is 10-fold longer). In these cancer cells, the active Erk output signal remains abnormally high for ~20 min after Ras input activity (optoSOS) is withdrawn (compared with 1 to 2 min for normal cells). Mutants or drugs that enhance B-Raf dimerization led to similar slow pathway deactivation. We could pinpoint B-Raf as the node responsible for altered transmission by using a combination of small molecular inhibitors and optogenetic stimulation at alternative input points.

Elongated pathway decay kinetics resulted in physiologically important cellular misinterpretation of dynamic inputs. In response to pulsatile inputs with intermediate frequencies, the perturbed cells responded with transcriptional profiles typically observed with sustained inputs. This signal misinterpretation propagated to proliferative decisions, resulting in aberrant cell-cycle entry in response to otherwise nonproliferative pulsatile inputs. These changes in pathway transmission shift the threshold of temporal input patterns that can drive cell proliferation, so that a space of inert input patterns that are normally filtered by the pathway can now drive proliferation.

Conclusion: Cancer mutations and targeted drugs can corrupt dynamic transmission properties in signaling pathways, shifting cellular response thresholds and changing cell decisions in a potentially pathological manner. Optogenetic approaches, especially in a high-throughput format, can be a powerful tool with which to systematically profile how a cell transmits and interprets information. We anticipate that further understanding the landscape of such functional alterations may help us mechanistically understand, stratify, and treat diseases that involve corrupted cellular decision-making.

Source: Science, 2018; 361 (6405): eaao3048