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

Defects in cellular signaling pathways, like those in some cancer cells, are often thought to result in increased or decreased steady-state signals that promote or inhibit cell proliferation. But Bugaj et al. show that dynamic changes in the duration or frequency of a signal can also alter cellular responses (see the Perspective by Kolch and Kiel). They took precise control of signaling in cultured human or mouse cells with a light-controlled mechanism for activating and inactivating the guanosine triphosphate Ras. Known cancer mutations in components of the Ras-activated signaling pathway or inhibitors of particular pathway components altered signal timing and readouts. The modified dynamics changed transcriptional outcomes and could inappropriately support cell proliferation. The ability to probe responses of signaling networks in this way may enhance understanding of biological regulation and reveal new therapeutic targets.Science, this issue p. eaao3048; see also p. 844 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.Optogenetic profiling of cancer cells reveals perturbed signal transmission dynamics that can drive improper proliferation.Optogenetic stimulation of Ras allows precise profiling of the fidelity of Ras-Erk pathway signaling in normal and cancer cells. We found that cancer cells with certain BRAF mutations have dramatically altered signal transmission dynamics compared with normal cells. These altered dynamics lead to a loss of temporal input resolution, so that the cancer cell may now misinterpret nonproliferative pulsatile input patterns as a trigger to proliferate.The Ras-Erk (extracellular signal-regulated kinase) pathway encodes information in its dynamics; the duration and frequency of Erk activity can specify distinct cell fates. To enable dynamic encoding, temporal information must be accurately transmitted from the plasma membrane to the nucleus. We used optogenetic profiling to show that both oncogenic B-Raf mutations and B-Raf inhibitors can cause corruption of this transmission, so that short pulses of input Ras activity are distorted into abnormally long Erk outputs. These changes can reshape downstream transcription and cell fates, resulting in improper decisions to proliferate. These findings illustrate how altered dynamic signal transmission properties, and not just constitutively increased signaling, can contribute to cell proliferation and perhaps cancer, and how optogenetic profiling can dissect mechanisms of signaling dysfunction in disease.

Science 2018

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