Abstract

Radiation therapy for abdominal tumors is challenging because the small intestine is exquisitely radiosensitive. Unfortunately, there are no FDA-approved therapies to prevent or mitigate GI radiotoxicity. The EGLN protein family are oxygen sensors that regulate cell survival and metabolism through the degradation of hypoxia-inducible factors (HIFs). Our group has previously shown that stabilization of HIF2 through genetic deletion or pharmacologic inhibition of the EGLNs mitigates and protects against GI radiotoxicity in mice by improving intestinal crypt stem cell survival. Here we aimed to elucidate the molecular mechanisms by which HIF2 confers GI radioprotection. We developed duodenal organoids from mice, transiently overexpressed non-degradable HIF2, and performed bulk RNA sequencing. Interestingly, HIF2 upregulated known radiation modulators and genes involved in GI homeostasis, including Wnt5a. Non-canonical Wnt5a signaling has been shown by other groups to improve intestinal crypt regeneration in response to injury. Here we show that HIF2 drives Wnt5a expression in multiple duodenal organoid models. Luciferase reporter assays performed in human cells showed that HIF2 directly activates the WNT5A promoter via a hypoxia response element. We then evaluated crypt regeneration using spheroid formation assays. Duodenal organoids that were pre-treated with recombinant Wnt5a had a higher cryptogenic capacity after irradiation, compared to vehicle-treated organoids. Conversely, we found that Wnt5a knockout decreased the cryptogenic potential of intestinal stem cells following irradiation. Treatment with recombinant Wnt5a prior to irradiation rescued the cryptogenic capacity of Wnt5a knockout organoids, indicating that Wnt5a is necessary and sufficient for duodenal radioprotection. Taken together, our results suggest that HIF2 radioprotects the GI tract by inducing Wnt5a expression.

Highlights

  • Radiation is one of the four pillars of cancer care, with approximately half of cancer patients receiving radiotherapy at some point of their treatment regimen [1]

  • We transiently overexpressed a non-degradable HIF1 or HIF2 allele [25], which contain mutations in the two key prolyl residues that are hydroxylated by the EGLN proteins, rendering them stable even under normoxic conditions, using adenoviral particles (Figure 1A)

  • Our results confirmed that Stat6, Aqp8, and Nos2 were upregulated and that Wnt4 and Egr1 were downregulated in HIF2-overexpressing duodenal organoids compared to wild-type duodenal organoids, indicating that the RNA sequencing dataset is reliable (Figure 2C)

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Summary

Introduction

Radiation is one of the four pillars of cancer care, with approximately half of cancer patients receiving radiotherapy at some point of their treatment regimen [1]. Recent clinical trials have shown that dose-escalated radiotherapy using highly precise 3D conformal radiation techniques, such as stereotactic body radiotherapy and intensity-modulated radiotherapy, can improve outcomes [7,8,9], but these techniques still cannot fully avoid the small intestines [10]. These sophisticated techniques are not widely accessible, as they require specialized expertise that is limited to some academic centers. An alternative to reduce GI radiotoxicity in cancer patients is to use a radioprotector to prevent radiation-induced damage and/or to improve GI repair following radiotherapy [9]

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