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 1

Mechanical properties of remodeling and increased tumor stemness were identified during chordoma recurrence.

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Mechanical properties of remodeling and increased tumor stemness were id...
(A–C) We performed mRNA microarray analysis on 6 matched primary-recurrent tumor pairs. Genes that were differentially expressed in at least 5 of these pairs were subsequently analyzed by GO enrichment and KEGG analysis. (D) IHC was performed to determine VCL and COL1A1 expression levels in chordoma tissues. Alcian blue/periodic acid–Schiff’s (AB/PAS) staining was utilized to detect proteoglycan levels. Green arrows show proteoglycan. Scale bar: 500 μm. (E) Young’s modulus of primary and recurrence chordoma tissues, as detected by using atomic force microscopy (AFM). (F) qRT-PCR was used to assess the expression of CSC-related genes in 9 paired primary and recurrent chordoma tissues. (G) Chordoma cells were cultured on PA gel substrates with different stiffnesses for 48 hours. The TUNEL assay was used to detect the effect of increased stiffness on cell death. (H and I) In tumor cells surviving high stiffness, phalloidin staining and IF were used to determine F-actin and VCL expression levels, respectively. (H) The colocalization areas indicate focal adhesions. Scale bar: 10 μm. (I) Expression of Notch signaling–, mechanical properties–, and tumor stemness–related genes were analyzed using qRT-PCR. (J) qRT-PCR was used to assess the expression of Notch signaling downstream genes in 10 paired primary and recurrent chordoma tissues. (K) Kaplan-Meier analysis of the recurrence-free survival of a cohort of 87 patients with chordoma using the log-rank test. NICD1 expression was determined using IHC, and the median value was used as the cut-off value. (L) Phalloidin staining and IF were used to assess the effects of the NICD1 ectopic expression on cytoskeleton. Red arrows show focal adhesions. Scale bar: 10 μm. (M) AFM determines Young’s modulus of cells ectopically expressing NICD1. (N) The effects of NICD1 overexpression on stiff substrate-mediated apoptosis were analyzed. (O) TUNEL assay results of cells cultured on type I collagen with periodically stress stimulation. The pressure parameter is set to 10% compression rate (achieve compression rate in 1 second, hold for 1 second, and then return to the initial state in 1 second) for a cumulative duration of 3 hours. TUNEL staining was performed at 48 hours. Data in J are presented as mean (minimum to maximum). Data in E, G, I, and M–O are presented as mean ± SD. Statistical analysis was performed using unpaired Student’s t test (E, M, and O) and 2-way ANOVA (G, I, and N). *P < 0.05.