NOTCH inhibition promotes bronchial stem cell renewal and epithelial barrier integrity after irradiation

Abstract

Hyperactivity of the NOTCH pathway is associated with tumor growth and radiotherapy resistance in lung cancer, and NOTCH/γ-secretase inhibitors (GSIs) are a potential therapeutic target. The therapeutic outcome, however, is often restricted by the dose-limiting toxicity of combined treatments on the surrounding healthy tissue. The NOTCH signaling pathway is also crucial for homeostasis and repair of the normal airway epithelium. The effects of NOTCH/γ-secretase inhibition on the irradiation of normal lung epithelium are unknown and may counteract antitumor activity.Here we, therefore, investigated whether normal tissue toxicity to radiation is altered upon NOTCH pathway inhibition. We established air-liquid interface pseudostratified and polarized cultures from primary human bronchial epithelial cells and blocked NOTCH signaling alone or after irradiation with small-molecule NOTCH inhibitor/ GSI. We found that the reduction in proliferation and viability of bronchial stem cells (TP63+) in response to irradiation is rescued with concomitant NOTCH inhibition. This correlated with reduced activation of the DNA damage response and accelerated repair by 24 hours and 3 days postirradiation. The increase in basal cell proliferation and viability in GSI-treated and irradiated cultures resulted in an improved epithelial barrier function. Comparable results were obtained after in vivo irradiation, where the combination of NOTCH inhibition and irradiation increased the percentage of stem cells and ciliated cells ex vivo. These encourage further use of normal patient tissue for toxicity screening of combination treatments and disclose novel interactions between NOTCH inhibition and radiotherapy and opportunities for tissue repair after radiotherapy.

Targeting Notch to Maximize Chemotherapeutic Benefits: Rationale, Advanced Strategies, and Future Perspectives

“…It is worth mentioning that combining Notch inhibitors with radiotherapy had quite contrasting consequences for airway epithelium regeneration in experimental works. Radiation damages the DNA of progenitor cells, and Notch signaling inactivation increases the phosphorylation of ATM and CHK2 and alters DNA damage response, but it could result both in decreased and improved renewal capacity of basal stem cells in different conditions [608,609].…”

The Radiation-Induced Regenerative Response of Adult Tissue-Specific Stem Cells: Models and Signaling Pathways

“…The air–liquid interface system (ALI system) is an example of how better models allow more in-depth studies [ 8 ]. ALI is a transwell system that allows differentiation of the upper airway basal lung SCs into a polarized mucociliary pseudostratified epithelium in vitro and enables the study of the basal and luminal cell response to radiation [ 9 ].…”

“…Indeed, this system better resembles the in vivo structure of the lung and allows the coculture with different cell types such as tumor cells, fibroblasts, or immune cells. For instance, lung SC response to radiation was studied by Giuranno et al using this model [ 8 , 10 ]. Inhibition of the Notch SC pathway was shown to increase lung SC self-renewal upon IR, contributing to a more intact epithelium as assessed in a completely differentiated setting [ 8 ].…”

“…For instance, lung SC response to radiation was studied by Giuranno et al using this model [ 8 , 10 ]. Inhibition of the Notch SC pathway was shown to increase lung SC self-renewal upon IR, contributing to a more intact epithelium as assessed in a completely differentiated setting [ 8 ]. This effect was not observed in 2D models, which do not permit the culture of the differentiated cells, suggesting the contribution of the luminal cells to SC response to RT [ 10 ].…”

“…The role of the Notch pathway in the lung SC in response to RT has been investigated in only a few papers [ 8 , 10 ]. Giuranno et al [ 8 ] showed that Notch inhibition increased the proliferation of the irradiated primary lung SCs, reduced DNA damage, and contributed to a more intact epithelium ( Figure 2 b).…”