Dll4/Notch1 signalling pathway is required in collective invasion of salivary adenoid cystic carcinoma

Abstract

High expression of δ‑like ligand 4 (Dll4) is reportedly related to the invasion, metastasis, and clinical prognosis of various malignant tumours. Our previous study revealed that collective cell invasion was a common pattern in salivary adenoid cystic carcinoma (SACC). However, the roles of the Dll4/Notch1 signalling pathway in the collective invasion of SACC remain unclear. The present study revealed that Dll4 expression was higher at the invasive front of SACC, and that this upregulation was associated with solid tumour type, high TNM grade, and high rates of metastasis and recurrence. Furthermore, the expression levels of Notch1 and Dll4 were positively correlated at the invasive front, and a three‑dimensional (3D) culture model revealed that leader cells showed high expression of Dll4, while follower cells showed high expression of Notch1. Moreover, silencing of Dll4 expression using small interfering RNA reduced the migration, invasion, and collective invasion of SACC cells, and these abilities were rescued by Notch1 overexpression. Finally, SACC collective invasion was increased via the Dll4/Notch1 signalling pathway in experiments that involved a stiff 3D gel, hypoxia and co‑culture with human endothelial cells. These findings indicated that the Dll4/Notch1 signalling pathway may be involved in the collective invasion of SACC, which may help to provide possible targets for the treatment of SACC.

Keywords: salivary adenoid cystic carcinoma; δ-like ligand 4; Notch1; collective invasion; migration and invasion.

Figure 1.

Immunohistochemical expression of Dll4 and Notch1 in the central and invasive areas of SACC tissues. (A) Representative images of Dll4 and Notch1 expression at the invasive front and interior of SACC tissues, with a normal gland used as a negative control. The red dotted line indicates the boundary between the tumour invasive front and the peri-carcinomatous tissue. (B) The percentage of tumour cells with Dll4 expression and the percentage of tumour cells with Notch1 expression at the invasive front and interior of SACC tissues. (C) Spearman's correlation coefficient was performed to analyse the correlation between Dll4 and Notch1 expression at the collective invasive front of SACC (r=0.704, P<0.001). ***P<0.001. SACC, salivary adenoid cystic carcinoma; Dll4, δ-like ligand 4; H&E, haematoxylin and eosin.

Figure 2.

Dll4 and Notch1 expression can be used as markers of leader and follower SACC cells as determined using a 3D culture. (A) SACC cells were subjected to three methods of 3D culture: (Aa) Soft agar suspension in 96-well plates (magnification, ×200), (Ab) the hanging drop technique (magnification, ×40) and (Ac) using 96-well ultra-low attachment plates (magnification, ×40). (B) Immunofluorescence results are shown for Dll4 expression (red) and Notch1 expression (green) in the 3D cultures (magnification, ×200). SACC, salivary adenoid cystic carcinoma; Dll4, δ-like ligand 4; 3D, three-dimensional.

Figure 3.

Effects of gel stiffness on SACC collective invasion and Dll4/Notch1 expression. The SACC-83 sphere in three-dimensional culture was examined at various gel stiffness levels, and the invasion area is indicated by the red line. IFC results are shown for Dll4 and Notch1 expression according to the different gel stiffness levels (magnification, ×40). SACC, salivary adenoid cystic carcinoma; Dll4, δ-like ligand 4; IFC, immunofluorescence.

Figure 4.

Dll4 knockdown inhibits the migration and invasion of SACC cells. (A) The relative Dll4 mRNA expression in transfected cells was evaluated using RT-qPCR. (B) The migration of SACC cells transfected with Dll4 siRNA-3 or control siRNA was determined using a wound healing assay (magnification, ×100). (C) The invasion of SACC cells transfected with Dll4 siRNA-3 or control siRNA was measured using a Transwell assay (magnification, ×100). (D) The tube formation ability of HUVECs treated with CM of SACC cells was quantified by measuring the number of nodes and the number of meshes (magnification, ×100). (E) In SACC cells, RT-qPCR revealed that Dll4 knockdown was associated with the downregulation of Dll4, Notch1, Hey1, Hes1, Twist1, MMP9 and CXCR4 mRNA expression. The data are presented as the mean ± standard deviation (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. control siRNA or as indicated. SACC, salivary adenoid cystic carcinoma; Dll4, δ-like ligand 4; CM, conditioned medium; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; siRNA, small interfering RNA; HUVECs, human umbilical vein endothelial cells; Hey1, hairy/enhancer-of-split related with YRPW motif protein 1; Hes1, transcription factor HES-1; Twist1, Twist-related protein 1; CXCR4, C-X-C chemokine receptor type 4.

Figure 5.

Hypoxia regulates SACC collective invasion via the Dll4/Notch1 signalling pathway. (A) Immunofluorescence results for Dll4 and Notch1 expression in SACC cells with or without Dll4 knockdown under hypoxia (magnification, ×40). (B) The migration of SACC cells transfected with Dll4 siRNA-3 or control siRNA under hypoxia (magnification, ×100). (C) The invasion of SACC cells transfected with Dll4 siRNA-3 or control siRNA under hypoxia (magnification, ×100). (D) The relative mRNA expression levels of Dll4, Notch1, Hey1, Hes1, Twist1, MMP9 and HIF1-α in SACC cells transfected with Dll4 siRNA-3 or control siRNA under hypoxia. The data are presented as the mean ± standard deviation (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. control siRNA + Hypoxia. SACC, salivary adenoid cystic carcinoma; Dll4, δ-like ligand 4; siRNA, small interfering RNA; Hey1, hairy/enhancer-of-split related with YRPW motif protein 1; Hes1, transcription factor HES-1; Twist1, Twist-related protein 1; HIF1-α, hypoxia-inducible factor 1-α.

Figure 6.

HUVECs regulates SACC collective invasion via the Dll4/Notch1 signalling pathway. (A) Immunofluorescence results for Dll4 expression (red) and Notch1 expression (green) in SACC cells transfected with Dll4 siRNA-3 or control siRNA during co-culture with HUVECs (magnification, ×40). (B) The migration of SACC cells transfected with Dll4 siRNA-3 or control siRNA during co-culture with HUVECs (magnification, ×100). (C) The invasion of SACC cells transfected with Dll4 siRNA-3 or control siRNA during co-culture with HUVECs (magnification, ×100). (D) The relative mRNA expression levels of Dll4, Notch1, Hey1, Hes1, Twist1, MMP9 and VEGFA in SACC cells transfected with Dll4 siRNA-3 or control siRNA during co-culture with HUVECs. The data are presented as the mean ± standard deviation (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. control siRNA + HUVECs. SACC, salivary adenoid cystic carcinoma; Dll4, δ-like ligand 4; siRNA, small interfering RNA; HUVECs, human umbilical vein endothelial cells; Hey1, hairy/enhancer-of-split related with YRPW motif protein 1; Hes1, transcription factor HES-1; Twist1, Twist-related protein 1.