Notch1 O-GlcNAcylation drives tumor stemness and mechanoadaptation to a stiff microenvironment and promotes chordoma recurrence
Chengjie Lian, Weiyan Peng, Peiqiang Su, Yan Ye, Jialing Liu, Dongsheng Huang, Xuejuan Sun, Yi Pu, Zhiheng Liao, Xudong Wang, Zhu Qiu, Shanshan Wu, Lei Liu
Chengjie Lian, Weiyan Peng, Peiqiang Su, Yan Ye, Jialing Liu, Dongsheng Huang, Xuejuan Sun, Yi Pu, Zhiheng Liao, Xudong Wang, Zhu Qiu, Shanshan Wu, Lei Liu
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Research Article Cell biology Oncology

Notch1 O-GlcNAcylation drives tumor stemness and mechanoadaptation to a stiff microenvironment and promotes chordoma recurrence

Abstract

Chordomas are rare malignant osseous neoplasms with a striking rate of recurrence. Primary chordomas typically originate from embryonic notochord remnants, whereas recurrent chordomas usually stem from tumor cells infiltrating bone or cartilage after surgery. Clinically, the recurrent chordomas exhibit a stiffer extracellular microenvironment (ECM) than primary tumors. Intriguingly, this study identified cytoskeleton rearrangement, stress fiber reorganization, enhanced stemness, and Notch signaling activation in recurrent chordoma tissues or cell lines surviving stiff substrates, indicating the critical roles of mechanical remodeling and tumor stemness in stiffness resistance. We propose a potentially novel recurrence model where tumor cells experience mechanoadaptive organization, which enables them to resist stiff microenvironment-induced cell death. O-GlcNAcylation of Notch1 intracellular domain (NICD1) is central to this process. Mechanistically, the stiff ECM-driven ligand-independent phosphorylation of EPHA2 sequentially activated LYN kinase and subsequently triggered O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) activity by phosphorylating Y989 and Y418, critical residues for OGT glycosyltransferase activity; this induced NICD1 O-GlcNAcylation at T2063, T2090, and S2162, specifically promoting transcription of mechanical and stemness-related genes. MIR31 deletion upregulated LYN, enhancing stiffness perception and promoting O-GlcNAc addition to NICD1, finally resulting in mechanoadaptation- and tumor stemness–driven recurrence. Consequently, MIR31 deletion is a potential biomarker for recurrence and patient stratification in Notch- or OGT-targeted therapies.

Authors

Chengjie Lian, Weiyan Peng, Peiqiang Su, Yan Ye, Jialing Liu, Dongsheng Huang, Xuejuan Sun, Yi Pu, Zhiheng Liao, Xudong Wang, Zhu Qiu, Shanshan Wu, Lei Liu

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Figure 6

EPHA2 perceives ECM stiffness to induce LYN phosphorylation.

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EPHA2 perceives ECM stiffness to induce LYN phosphorylation. (A and B) I...
(A and B) IP, Duolink PLA, and IF to identify OGT tyrosine phosphorylation, global tyrosine phosphorylation, and LYN phosphorylation in MUG-Chor1 cells cultured on substrates with different stiffnesses. Col is for type I collagen. Scale bar: 50 μm. (C) IP was used to identify the interaction between LYN and EPHA2 in MUG-Chor1 cells cultured on substrates with different stiffnesses. (D) The effects of EHPA2 inhibitor ALW II-41-27 (10 nM) on phosphorylation of LYN and EPHA2 in MUG-Chor1 cells cultured on substrates with different stiffnesses were assessed. (E) Phosphorylation of EPHA2 S897 along with Notch signaling activity in response to different stiffnesses were determined by IF. Scale bar: 50 μm. (F and G) IP was used to determine effects of high stiffness with EHPA2 inhibitor on the interaction between OGT and LYN and OGT tyrosine phosphorylation. Type I collagen (4 mg/mL). (H) IP was used to assess the effect of LYN kinase suppression on ALW II-41-27–mediated OGT tyrosine phosphorylation inhibition. (I) Glycosyltransferase activity of WT or mutated OGT was determined using the UDP-Glo Glycosyltransferase Assay. (J) The effects of silencing OGT or treatment of DON on NICD1 O-GlcNAcylation in MUG-Chor1 cells cultured on substrates with different stiffnesses were assessed. (K) The effects of MLR-1023 and ALW II-41-27 on NICD1 ubiquitination in cells cultured on substrates with different stiffnesses were determined by IP. (L) The effects of sustained EPHA2 S897 phosphorylation on F-actin, VCL, and focal adhesions were determined in the presence or absence of Notch1 silencing. Scale bar: 10 μm. (M) IHC was used to determine expression of COL1A1, EPHA2 p-S897, and LYN p-Y507 in primary and recurrent clinical chordoma tissues (n = 20 pairs). Scale bar: 100 μm. Data in B and I are presented as mean ± SD. Data in M are presented as mean (minimum to maximum). Statistical analysis was performed using paired Student’s t test (M) and 2-way ANOVA (B and I). *P < 0.05.