Jagged1 intracellular domain/SMAD3 complex transcriptionally regulates TWIST1 to drive glioma invasion

Abstract

Jagged1 (JAG1) is a Notch ligand that correlates with tumor progression. Not limited to its function as a ligand, JAG1 can be cleaved, and its intracellular domain translocates to the nucleus, where it functions as a transcriptional cofactor. Previously, we showed that JAG1 intracellular domain (JICD1) forms a protein complex with DDX17/SMAD3/TGIF2. However, the molecular mechanisms underlying JICD1-mediated tumor aggressiveness remains unclear. Here, we demonstrate that JICD1 enhances the invasive phenotypes of glioblastoma cells by transcriptionally activating epithelial-to-mesenchymal transition (EMT)-related genes, especially TWIST1. The inhibition of TWIST1 reduced JICD1-driven tumor aggressiveness. Although SMAD3 is an important component of transforming growth factor (TGF)-β signaling, the JICD1/SMAD3 transcriptional complex was shown to govern brain tumor invasion independent of TGF-β signaling. Moreover, JICD1-TWIST1-MMP2 and MMP9 axes were significantly correlated with clinical outcome of glioblastoma patients. Collectively, we identified the JICD1/SMAD3-TWIST1 axis as a novel inducer of invasive phenotypes in cancer cells.

Fig. 1. JICD1 derived from JAG1 induces tumor invasiveness.

A Immunofluorescence showing endogenous JAG1 expression in mouse brain sections after intracranial injections of U87MG and LN229. Scale bar = 100 µm. B Bar graph showing the percentage of JAG1 positive cells in eight different margin regions in mouse brain sections after intracranial injections of U87MG and LN229. ***p < 0.001. C Bar graph showing the percentage of nuclear JAG1 in eight different margin regions in mouse brain sections after intracranial injections of U87MG and LN229. The data are presented as mean ± S.E.M. ***p < 0.001. D Immunofluorescence showing HA-JICD1 expression of migrated cells in A172 and U87MG control and HA-JICD1-overexpressing cells using 3D cell culture chips. A bar graph representing the average migration distance (black, left axis) and number of migrated cells per four grooves (blue, right axis) in A172 and U87MG control and HA-JICD1-overexpressing cells. Scale bar = 200 µm. ***p < 0.001. E Immunofluorescence showing transplanted U87MG control and HA-JICD1-overexpressing cells in tumor margin regions. White arrow indicates single cell migration and yellow arrow shows collective migration. Scale bar = 250 µm.

Fig. 2. JICD1 transcriptional complex promotes gene expression related to EMT.

A mRNA expression of EMT-TFs and markers in A172 and U87MG control and HA-JICD1-overexpressing cells. B Protein expression of canonical EMT-TFs and markers in A172 and U87MG control and HA-JICD1-overexpressing cells. C Schematic diagram that represents JICD1 transcriptional complex with DDX17, SMAD3, and TGIF2. D, E The association between ectopic HA-JICD1 and endogenous DDX17 and SMAD3 in A172 (D) and U87MG (E) control and HA-JICD1 overexpressing cells.

Fig. 3. EMT induced by JICD1 is independent of TGF-β signaling.

A Bar graph representing the average migration distance (black, left axis) and number of migrated cells per four grooves (blue, right axis) in A172 and U87MG control and HA-JICD1-overexpressing cells with SB431542 treatment (A172: 10 μM, U87MG: 20 μM). ***p < 0.001. B mRNA expression of EMT-related genes in A172 and U87MG control and HA-JICD1-overexpressing cells with the SB431542 treatment. C Protein expression of canonical EMT-TFs and markers in A172 and U87MG control and HA-JICD1-overexpressing cells with SB431542 treatment. D Association between HA-DDX17 and FLAG-SMAD3 in HEK293T cells with SB431542 treatment. E Association between HA-DDX17 and FLAG-SMAD3 mutants in HEK293T cells.

Fig. 4. TWIST1, transcriptionally activated by JICD1 transcriptional complex, induces GBM invasion.

A A schematic diagram of a SMAD3 binding element (TWGTCTGV) in the TWIST1 promoter. B, C ChIP-PCR analysis of (B) HA-JICD1 and (C) SMAD3 engagement with the SMAD3 binding motif at the TWIST1 promoter in A172 and U87MG control and HA-JICD1-overexpressing cells. *p < 0.05. D mRNA expression of EMT-related genes in A172 and U87MG control and HA-JICD1-overexpressing cells with TWIST1 knockdown. E Bar graph representing the average migration distance (black, left axis) and number of migrated cells per four grooves (blue, right axis) in A172 and U87MG control and HA-JICD1-overexpressing cells with TWIST1 knockdown. *p < 0.05, ***p < 0.001.

Fig. 5. JICD1 promotes tumor invasion and aggressiveness via TWIST1 in vivo.

A Representative images of hematoxylin-eosin staining of mouse brain sections 59 days after intracranial injections of U87MG control and HA-JICD1-overexpressing cells with TWIST1 knockdown. Scale bar = 2500 µm. B Survival graph of tumor-bearing mice after intracranial injections of U87MG control and HA-JICD1-overexpressing cells with TWIST1 knockdown. Statistical significance was tested by log-rank test. *p < 0.05, **p < 0.01, ***p < 0.001. C Images showing the invasive margin regions in mouse brain sections after intracranial injections of U87MG control and HA-JICD1-overexpressing cells with TWIST1 knockdown. White arrow indicates single cell migration and yellow arrow shows collective migration. Scale bar = 100 µm.

Fig. 6. JICD1-TWIST1-MMP2 and MMP9 axes correlate with cancer aggressiveness in IDH wild-type GBM patients.

A Correlation of JAG1 with TWIST1 in patients with IDH wild-type GBM using the TCGA GBMLGG database. B Survival rate of patients with IDH wild-type GBM according to the expressions of JAG1 and TWIST1. The patients were divided into two groups: JAG1-high and TWIST1-high vs JAG1-low and TWIST1-low based on mRNA expression (mean ± SEM). P-values were calculated by a log-rank (Mantel-Cox) test. *p < 0.05. C, D Correlation of JAG1 with JICD1/SMAD3-TWIST1 downstream signaling molecules (MMP2 and MMP9) in patients with IDH wild-type GBM using the TCGA GBMLGG database. E, F Survival rate of patients with IDH wild-type GBM according to the expressions of JAG1, TWIST1, MMP2, and MMP9. The patients were divided into two groups: JAG1-high, TWIST1-high, and MMP2-high vs JAG1-low, TWIST1-low, and MMP2-low (E) and JAG1-high, TWIST1-high, and MMP9-high vs JAG1-low, TWIST1-low, MMP9-low (F) based on mRNA expression (mean ± SEM). P-values were calculated by a log-rank (Mantel-Cox) test. *p < 0.05, ***p < 0.001.

Fig. 7. JICD1/SMAD3-TWIST1 axis induces tumor invasion.

A graphic summary describing JICD1-induced cell migration and invasion. JICD1 is a transcription cofactor with inhibitory SMAD3. This complex promotes TWIST1 expression, which is crucial for canonical TGF-β signaling-independent cell migration and invasion.