Mechanisms driving fasting-induced protection from genotoxic injury in the small intestine.

Genotoxic agents such as doxorubicin (DXR) can cause damage to the intestines that can be ameliorated by fasting. How fasting is protective and the optimal timing of fasting and refeeding remain unclear. Here, our analysis of fasting/refeeding-induced global intestinal transcriptional changes revealed metabolic shifts and implicated the cellular energetic hub mechanistic target of rapamycin complex 1 (mTORC1) in protecting from DXR-induced DNA damage. Our analysis of specific transcripts and proteins in intestinal tissue and tissue extracts showed that fasting followed by refeeding at the time of DXR administration reduced damage and caused a spike in mTORC1 activity. However, continued fasting after DXR prevented the mTORC1 spike and damage reduction. Surprisingly, the mTORC1 inhibitor, rapamycin, did not block fasting/refeeding-induced reduction in DNA damage, suggesting that increased mTORC1 is dispensable for protection against the initial DNA damage response. In Ddit4-/- mice [DDIT4 (DNA-damage-inducible transcript 4) functions to regulate mTORC1 activity], fasting reduced DNA damage and increased intestinal crypt viability vs. ad libitum-fed Ddit4-/- mice. Fasted/refed Ddit4-/- mice maintained body weight, with increased crypt proliferation by 5 days post-DXR, whereas ad libitum-fed Ddit4-/- mice continued to lose weight and displayed limited crypt proliferation. Genes encoding epithelial stem cell and DNA repair proteins were elevated in DXR-injured, fasted vs. ad libitum Ddit4-/- intestines. Thus, fasting strongly reduced intestinal damage when normal dynamic regulation of mTORC1 was lost. Overall, the results confirm that fasting protects the intestines against DXR and suggests that fasting works by pleiotropic - including both mTORC1-dependent and independent - mechanisms across the temporally dynamic injury response.NEW & NOTEWORTHY New findings are 1) DNA damage reduction following a 24-h fast depends on the timing of postfast refeeding in relation to chemotherapy initiation; 2) fasting/refeeding-induced upregulation of mTORC1 activity is not required for early (6 h) protection against DXR-induced DNA damage; and 3) fasting increases expression of intestinal stem cell and DNA damage repair genes, even when mTORC1 is dysregulated, highlighting fasting's crucial role in regulating mTORC1-dependent and independent mechanisms in the dynamic recovery process.

Loss of ZNRF3/RNF43 Unleashes EGFR in Cancer.

ZNRF3 and RNF43 are closely related transmembrane E3 ubiquitin ligases with significant roles in development and cancer. Conventionally, their biological functions have been associated with regulating WNT signaling receptor ubiquitination and degradation. However, our proteogenomic studies have revealed EGFR as the most negatively correlated protein with ZNRF3/RNF43 mRNA levels in multiple human cancers. Through biochemical investigations, we demonstrate that ZNRF3/RNF43 interact with EGFR via their extracellular domains, leading to EGFR ubiquitination and subsequent degradation facilitated by the E3 ligase RING domain. Overexpression of ZNRF3 reduces EGFR levels and suppresses cancer cell growth in vitro and in vivo, whereas knockout of ZNRF3/RNF43 stimulates cell growth and tumorigenesis through upregulated EGFR signaling. Together, these data highlight ZNRF3 and RNF43 as novel E3 ubiquitin ligases of EGFR and establish the inactivation of ZNRF3/RNF43 as a driver of increased EGFR signaling, ultimately promoting cancer progression. This discovery establishes a connection between two fundamental signaling pathways, EGFR and WNT, at the level of cytoplasmic membrane receptor, uncovering a novel mechanism underlying the frequent co-activation of EGFR and WNT signaling in development and cancer.