How do stem cells decode proto-oncogenic Ras signaling to eliminate hyper-proliferative cells in vivo?

About This Grant

PROJECT SUMMARY Most adult organs are composed of differentiated and stem cells and are maintained at a constant size through a process of dynamic turnover: organs lose differentiated cells as they age and eventually die, but are replen- ished by stem cell divisions. However, while stem cell divisions are crucial for normal homeostatic turnover, excessive divisions can result in hyperplasia and ultimately cancer. Therefore, a key problem faced by adult organs is how to promote enough stem cell divisions to replace aging cells without generating so many cells that the organ becomes hyperplastic. Studying how this balance is robustly maintained in healthy organs is crucial for understanding how it breaks down in the early stages of tumor development. Throughout metazoan tissues, stem cells are triggered to divide through activation the Ras-Raf-MEK-ERK signaling cascade. Unsur- prisingly, activating mutations in this pathway are found in ~30% of human tumors. In particular, over half of colorectal cancers are characterized by mutations in the RAS gene. However, expression of these mutants in the intestinal cells of mice and flies is insufficient to generate colorectal tumors. What explains this apparent contradiction? Evidence from multiple in vivo systems suggests that when Ras-Raf-MEK-ERK signaling ex- ceeds physiological levels, safeguard mechanisms are evoked to counteract nascent hyperplasia and preserve tissue homeostasis. Yet, how stem cells distinguish overactive signaling from physiological signaling and how tissues suppress hyperplasia remains poorly understood. Here, I will study these questions using the Drosoph- ila adult midgut, a stem cell-based organ that is functionally equivalent to the vertebrate intestine. While Ras signaling underlies homeostatic stem cell proliferation in this organ, my preliminary data shows that sustained expression of constitutively active Ras mutants in progenitor cells generates a biphasic response where stem cell proliferation on short time scales (12-24 hours) is paradoxically followed by a striking decrease in the num- ber of progenitors over longer timescales (24-72 hours). I hypothesize that this depletion of progenitors is trig- gered by a “persistence detector” mechanism that distinguishes between physiological and proto-oncogenic Ras signaling based on the duration of activity. Using a combination of advanced genetic tools, high-resolution confocal microscopy, single-cell RNA-sequencing, and pharmacological manipulation I aim to understand 1) How differential activation dynamics for different genes modules downstream of Ras generate a biphasic re- sponse to RasV12 expression, 2) What dynamic feature of RasV12 signaling distinguishes it from physiologi- cal Ras signaling, and 3) How interactions between different cell trigger the later phase decrease in progenitor cells in response to RasV12 expression. Overall, my findings will shed light on the fundamental mechanisms for decoding signaling pathway activity and may ultimately inform approaches for treating cancer.