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Wnt signaling modulates the response to DNA damage in the Drosophila wing imaginal disc by regulating the EGFR pathway [1]

['Ben Ewen-Campen', 'Department Of Genetics', 'Blavatnik Institute', 'Harvard Medical School', 'Boston', 'Massachusetts', 'United States Of America', 'Norbert Perrimon', 'Howard Hughes Medical Institute']

Date: 2024-07

Despite the deep conservation of the DNA damage response (DDR) pathway, cells in different contexts vary widely in their susceptibility to DNA damage and their propensity to undergo apoptosis as a result of genomic lesions. One of the cell signaling pathways implicated in modulating the DDR is the highly conserved Wnt pathway, which is known to promote resistance to DNA damage caused by ionizing radiation in a variety of human cancers. However, the mechanisms linking Wnt signal transduction to the DDR remain unclear. Here, we use a genetically encoded system in Drosophila to reliably induce consistent levels of DNA damage in vivo, and demonstrate that canonical Wnt signaling in the wing imaginal disc buffers cells against apoptosis in the face of DNA double-strand breaks. We show that Wg, the primary Wnt ligand in Drosophila, activates epidermal growth factor receptor (EGFR) signaling via the ligand-processing protease Rhomboid, which, in turn, modulates the DDR in a Chk2-, p53-, and E2F1-dependent manner. These studies provide mechanistic insight into the modulation of the DDR by the Wnt and EGFR pathways in vivo in a highly proliferative tissue. Furthermore, they reveal how the growth and patterning functions of Wnt signaling are coupled with prosurvival, antiapoptotic activities, thereby facilitating developmental robustness in the face of genomic damage.

Funding: This work was supported by the National Institutes of Health (5R24OD026435 to NP) and the Charles King Postdoctoral Fellowship (to BEC), and NP is an HHMI Investigator. No funder played any role in study design, data collection or analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Ewen-Campen, Perrimon. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, using a CRISPR/Cas9-based approach to genetically inflict consistent levels of DNA damage in vivo, we demonstrate that loss-of-function of canonical Wnt signaling in the larval wing disc sensitizes these cells to DNA damage and biases them towards apoptosis. In contrast, Wg overexpression biases them away from apoptosis. We show that this function is mediated via expression of rhomboid (rho), which encodes a protease required for processing and secretion of ligands of the epidermal growth factor receptor (EGFR) pathway, and that the effects of Wg loss-of-function can be rescued by activation of the EGFR pathway. This Wnt-mediated effect on the DDR requires the highly conserved components of the DDR Chk2, p53, and E2F1, and the proapoptotic factor hid. Altogether, we demonstrate that in the Wg signaling promotes cell survival in the face of DNA damage during development of the Drosophila wing.

In the fruit fly Drosophila, signaling via the major Wnt ligand Wingless (encoded by the wg gene, the fly ortholog of WNT1) plays critical roles in growth and patterning, including in the larval precursor of the adult wing, the wing imaginal disc [ 23 ]. In addition, wg has been implicated in radioresistance in one particular context in this tissue, a region of the disc termed the “frown,” which displays remarkable resistance to DNA damage [ 8 , 9 ]. The “frown” refers to a band of cells fated to become the hinge between the adult wing and notum, which can withstand high levels of IR without undergoing apoptosis, in a process that requires Wg signaling and JAK/STAT signaling, and which is mediated by regulation of the proapoptotic gene reaper [ 8 , 9 ]. These damage-resistant cells then contribute to the regeneration of the wing pouch following damage-induced apoptosis. In addition, a recent study using a specialized Gal4 system driven by the effector caspase Drice has shown that modulating Wnt signaling in the wing disc can affect both the apoptotic response to high levels of radiation and the ability for cells to survive low levels of caspase activation [ 24 ]. However, there remain open questions regarding the mechanisms connecting Wnt to the DDR and regarding whether Wnt signaling promotes resistance to DNA damage in other cellular contexts.

A variety of mechanisms linking Wnt signaling to the DDR have been proposed in mammalian systems. One study in cultured CRC cells demonstrated the direct transcriptional activation of the critical DNA repair component Lig4 by the transcription factor TCF downstream of Wnt signaling, in a process independent of p53 status [ 15 ]). Other studies have identified a converse phenomenon: down-regulation of Wnt signaling via p53 or E2F activity in response to DNA damage [ 17 – 22 ]. Altogether, it remains unclear whether these observed interactions between Wnt signaling and the DDR are generalizable or conserved in different contexts.

Among the numerous cell signaling pathways implicated in modulating the DDR, the Wnt signaling pathway has been shown to interact with the DDR in a variety of contexts. The Wnt pathway is a highly conserved cell signaling pathway with critical functions in development, in adult stem cell populations, and in tissue homeostasis, and its dysregulation is implicated in a variety of diseases [ 12 – 14 ]. In humans, excess Wnt signaling correlates with radioresistance in a variety of tissue contexts and cancers (reviewed [ 15 ]). For example, in human colorectal cancer (CRC), activated Wnt signaling is considered the key driver of cancer progression, and functional studies have also demonstrated that Wnt signaling promotes radioresistance in CRC [ 15 , 16 ]. When human CRC cells are sorted based on levels of a Wnt reporter into Wnt high and Wnt low populations, Wnt high cells display significant resistance to radiation, and treatment with an inhibitor of the β-catenin/TCF interaction increases radiosensitivity [ 15 ]. In addition, treating nontumorigenic epithelial cell lines with Wnt pathway agonists leads to resistance to irradiation (IR) and to chemoradiotherapy [ 16 ].

In response to DNA damage, eukaryotic cells activate a highly conserved intracellular signaling pathway known as the DNA damage response (DDR) [ 1 , 2 ]. This complex pathway allows cells to detect genomic damage and to mount an appropriate cellular response, from pausing the cell cycle and repairing DNA damage, to entering senescence, to undergoing apoptosis [ 1 , 2 ]. However, while many of the molecular components of the DDR are highly conserved across eukaryotic evolution, there is profound variation in how different cells respond to DNA damage based on such factors as signaling pathway status, tissue context, cell cycling status, development stage, and more [ 3 – 10 ]. Facing the same type and amount of DNA damage, cells in different contexts can vary widely in their propensity to undergo apoptosis in the face of DNA damage. For example, aberrant signaling pathway activity in many tumor types leads to a phenomenon known as radioresistance, in which tumor cells survive levels of DNA damage caused by radiation therapy that would induce apoptosis in similar nontumorous cells [ 11 ]. In contrast, some cell types are exquisitely sensitive to DNA damage. Human pluripotent stem cells, for example, have a remarkably low tolerance for DNA damage and undergo apoptosis in response to double-strand breaks (DSBs) caused by CRISPR/Cas9, which are routinely withstood by many other cell types [ 6 ]. In comparison with our understanding of the DDR pathway itself, much less is known about what drives differential sensitivity to genome damage in vivo.

Results

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002547

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