Notch signaling determines cell fates in mouse intestine. Notch receptors contain multiple epidermal growth factor-like (EGF) repeats modified by O-glycans that regulate Notch signaling. Conditional deletion of protein O-fucosyltransferase 1 ( Pofut1 ) substantially reduces Notch signaling and markedly perturbs lineage development in mouse intestine. However, mice with inactivated Pofut1 are viable, whereas complete elimination of Notch signaling in intestine is lethal. Here we investigate whether residual Notch signaling enabled by EOGT permits mice lacking Pofut1 in intestine to survive. Mice globally lacking Eogt alone were grossly unaffected in intestinal development. In contrast, mice lacking both Eogt and Pofut1 died at ∼28 days after birth with greater loss of body weight, a greater increase in the numbers of goblet and Paneth cells, and greater downregulation of Notch target genes, compared to Pofut1 deletion alone. These data establish that both O-fucose and O-GlcNAc glycans are fundamental to Notch signaling in the intestine and provide new insights into roles for O-glycans in regulating Notch ligand binding. Finally, EOGT and O-GlcNAc glycans provide residual Notch signaling and support viability in mice lacking Pofut1 in the intestine.

Identifying biological roles for glycosyltransferases is a continuing challenge and important for defining morbidities associated with congenital disorders of glycosylation. Here we investigate the consequences to intestinal development of conditionally deleting Lfng alone or Lfng, Mfng and Rfng together in a mixed or Eogt-null genetic background. Each Fringe transfers N-acetylglucosamine (GlcNAc) to fucose (Fuc) attached to Ser or Thr by POFUT1 in a consensus sequence found in certain epithelial growth factor-like (EGF) repeats. EOGT transfers GlcNAc directly to Ser/Thr in a separate consensus sequence of an EGF repeat. Notch receptors and Notch ligands contain the largest number of EGF repeats with consensus sites for these O-glycans. Conditional deletion of Pofut1 in mouse intestine causes similar developmental defects to deletion of Notch1 and Notch2 or Dll1 and Dll4. LFNG also contributes to optimal Notch signaling in mouse intestine. In this work, we generated Lfng[F/F]:Villin-Cre and Lfng[F/F]Mfng[-/-]Rfng[-/-]:Villin-Cre mice in which extension of O-Fuc on EGF repeats was respectively inhibited or prevented in intestinal epithelium. Conditional deletion of either Lfng alone or all three Fringe activities together led to defective intestinal development with a marked increase in goblet and Paneth cells, increased crypt width and reduced villus length. Unexpectedly, in mice globally lacking EOGT, conditional inactivation of the three Fringe genes did not lead to defective intestinal development. Thus, the absence of EOGT prevented disruption of development in Fringe-null intestine, identifying a novel role for EOGT in regulating intestinal development.

Identifying biological roles for glycosyltransferases is a continuing challenge and important for defining morbidities associated with congenital disorders of glycosylation. Here we investigate the consequences to intestinal development of conditionally deleting Lfng alone or Lfng, Mfng and Rfng together in a mixed or Eogt -null genetic background. Each Fringe transfers N-acetylglucosamine (GlcNAc) to fucose (Fuc) attached to Ser or Thr by POFUT1 in a consensus sequence found in certain epithelial growth factor-like (EGF) repeats. EOGT transfers GlcNAc directly to Ser/Thr in a separate consensus sequence of the EGF repeat. Notch receptors and Notch ligands contain the largest number of EGF repeats with consensus sites for these O-glycans. Conditional deletion of Pofut1 in mouse intestine causes similar developmental defects to deletion of Notch1 and Notch2 or Dll1 and Dll4 . LFNG also contributes to optimal Notch signaling in mouse intestine. In this work, we generated Lfng [F/F]: Villin -Cre and Lfng [F/F] Mfng [-/-] Rfng [-/-]: Villin -Cre mice in which extension of O-Fuc on EGF repeats was inhibited or prevented in intestinal epithelium. Conditional deletion of either Lfng alone or all three Fringe activities together led to defective intestinal development with a marked increase in goblet and Paneth cells, increased crypt width and reduced villus length. Unexpectedly, in mice globally lacking EOGT, conditional inactivation of the three Fringe genes did not lead to defective intestinal development. Thus, the absence of EOGT prevented disruption of development in Fringe-null intestine, identifying a novel role for EOGT in regulating intestinal development.

Cancer stemness is a pivotal driver of tumor initiation, treatment resistance, and tumor cell survival. Cancer stem cells (CSCs), though constituting only a small fraction of primary tumor cells, are progressively enriched during metastatic progression: from circulating tumor cells traveling in the bloodstream, to disseminated tumor cells lodged in the lung vasculature, to extravasated tumor cells that have entered tissue parenchyma. However, whether CSCs have an intrinsic advantage for extravasation over cancer non-stem cells (CnSCs), or simply their increased representation in circulation renders them more likely to extravasate, remains unresolved. MenaINV, an invasive isoform of the actin regulatory protein Mena, promotes tumor cell transendothelial migration in primary and secondary sites, yet the direct mechanistic link between stemness and MenaINV in lung metastasis remains unresolved. Here, using a validated fluorescent stemness reporter (SORE6) to identify CSCs, we found that CSCs display elevated MenaINV expression relative to CnSCs. High-resolution intravital imaging showed that CSCs extravasate efficiently into lung parenchyma and survive at higher levels, robustly forming metastatic lesions, while CnSCs show limited extravasation, low survival, and poor colonization. Mechanistically, MenaINV disruption in CSCs specifically impaired extravasation without affecting survival, demonstrating that MenaINV is the key extravasation effector downstream of stemness, whereas stemness-associated factors independently confer survival advantages. Moreover, reintroduction of MenaINV in CnSCs restores their extravasation ability upon which extravasated CnSCs reactivate stem program and form metastases. Overall, we discovered a hierarchical framework where stemness regulates both survival and extravasation capacity, with MenaINV as the key CSC extravasation effector. Significance: This study reveals how breast cancer stem cells achieve metastatic dominance through separable pathways: MenaINV-dependent extravasation and MenaINV-independent survival, providing rationale for targeting stem program to improve patient outcome.