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, 24 (3), 619-633

The NOTCH4- HEY1 Pathway Induces Epithelial-Mesenchymal Transition in Head and Neck Squamous Cell Carcinoma

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The NOTCH4- HEY1 Pathway Induces Epithelial-Mesenchymal Transition in Head and Neck Squamous Cell Carcinoma

Takahito Fukusumi et al. Clin Cancer Res.

Abstract

Purpose: Recently, several comprehensive genomic analyses demonstrated NOTCH1 and NOTCH3 mutations in head and neck squamous cell carcinoma (HNSCC) in approximately 20% of cases. Similar to other types of cancers, these studies also indicate that the NOTCH pathway is closely related to HNSCC progression. However, the role of NOTCH4 in HNSCC is less well understood.Experimental Design: We analyzed NOTCH4 pathway and downstream gene expression in the TCGA data set. To explore the functional role of NOTCH4, we performed in vitro proliferation, cisplatin viability, apoptosis, and cell-cycle assays. We also compared the relationships among NOTCH4, HEY1, and epithelial-mesenchymal transition (EMT)-related genes using the TCGA data set and in vitro assays.Results:HEY1 is specifically upregulated in HNSCC compared with normal tissues in the TCGA data set. NOTCH4 is more significantly related to HEY1 activation in HNSCC in comparison with other NOTCH receptors. NOTCH4 promotes cell proliferation, cisplatin resistance, inhibition of apoptosis, and cell-cycle dysregulation. Furthermore, NOTCH4 and HEY1 upregulation resulted in decreased E-cadherin expression and increased Vimentin, Fibronectin, TWIST1, and SOX2 expression. NOTCH4 and HEY1 expression was associated with an EMT phenotype as well as increased invasion and cell migration.Conclusions: In HNSCC, the NOTCH4-HEY1 pathway is specifically upregulated, induces proliferation and cisplatin resistance, and promotes EMT. Clin Cancer Res; 24(3); 619-33. ©2017 AACR.

Conflict of interest statement

Disclosure of Potential Conflict of Interest

The authors declare no potential conflicts of interest.

Figures

Figure 1
Figure 1. TCGA data set analysis of HES/HEY and NOTCH4 relation
(A) NOTCH4 expression is compared between the HES (HES1+HES5) and HEY1 high/low group using the TCGA data set. Ratio is calculated by dividing the mRNA expression of the HES or HEY1 high group by that of the low group. (B) HES1, HES5 and HEY1 expression are compared between HNSCC and normal tissue using the TCGA data set. Ratio is calculated by dividing the mRNA expression of the tumor samples by that of the normal samples. Whiskers indicate the minimum and maximum values. P value is calculated by using Student’s t-test.
Figure 2
Figure 2. NOTCH activity, proliferation, cisplatin viability, apoptosis assay and cell cycle analysis of si-NOTCH4 cells
(A) pGreenFire1-Notch plasmid vector was transfected to SKN3 cell. Scale bar indicates 100 μm. (B) NOTCH activity assay of si-control and si-NOTCH4 cells. GFP positive cells have high NOTCH activity. (C) Proliferation assays. si-NOTCH4 cells are compared cell growths to si-control cells on day 3. (D) IC50 of cisplatin in parental, si-control and si-NOTCH4 cells. The IC50 differences between si-control and si-NOTCH4 cells are compared. (E) Apoptosis assays. Total apoptotic fraction is defined as the sum of early and late apoptosis cells. (F) Cell cycle analysis. Each cell cycle phase is compared between si-control and si-NOTCH4 cells. P value is calculated by using Student’s t-test. *: P < 0.05, **: P < 0.01.
Figure 2
Figure 2. NOTCH activity, proliferation, cisplatin viability, apoptosis assay and cell cycle analysis of si-NOTCH4 cells
(A) pGreenFire1-Notch plasmid vector was transfected to SKN3 cell. Scale bar indicates 100 μm. (B) NOTCH activity assay of si-control and si-NOTCH4 cells. GFP positive cells have high NOTCH activity. (C) Proliferation assays. si-NOTCH4 cells are compared cell growths to si-control cells on day 3. (D) IC50 of cisplatin in parental, si-control and si-NOTCH4 cells. The IC50 differences between si-control and si-NOTCH4 cells are compared. (E) Apoptosis assays. Total apoptotic fraction is defined as the sum of early and late apoptosis cells. (F) Cell cycle analysis. Each cell cycle phase is compared between si-control and si-NOTCH4 cells. P value is calculated by using Student’s t-test. *: P < 0.05, **: P < 0.01.
Figure 3
Figure 3. Comparison of EMT-related genes between the NOTCH4 high and low groups using the TCGA data set. Comparison of NOTCH4 expression between sphere and parental cells
(A) The NOTCH4 high group is defined as tumors with expression 1 standard deviation greater than the mean of normal tissue for NOTCH4. The other tumors are defined as the low group. The boxes represent the interquartile range (25th-75th), and horizontal lines inside the boxes indicate the median. Whiskers indicate the minimum and maximum values. Ratio is calculated by dividing the mRNA expression of the high group by the expression of the low group. (B) NOTCH4 expression is compared between parental and sphere cells using qRT-PCR. P value is calculated by using Student’s t-test. **: P < 0.01.
Figure 4
Figure 4. The expression of EMT-related genes in si-NOTCH4 cells
(A) EMT-related gene (E-cadherin, Vimentin, Fibronectin, TWIST1, and SOX2) expression in parental, si-control and si-NOTCH4 cells is measured by qRT-PCR. The expression differences between si-control and si-NOTCH4 cells are compared. P value is calculated by using Student’s t-test. *: P < 0.05, **: P < 0.01, N.S.: not significant. (B) Western blots of EMT-related genes in si-control and si-NOTCH4 of SKN3 and Cal27 cells. GAPDH antibody is used as a control.
Figure 4
Figure 4. The expression of EMT-related genes in si-NOTCH4 cells
(A) EMT-related gene (E-cadherin, Vimentin, Fibronectin, TWIST1, and SOX2) expression in parental, si-control and si-NOTCH4 cells is measured by qRT-PCR. The expression differences between si-control and si-NOTCH4 cells are compared. P value is calculated by using Student’s t-test. *: P < 0.05, **: P < 0.01, N.S.: not significant. (B) Western blots of EMT-related genes in si-control and si-NOTCH4 of SKN3 and Cal27 cells. GAPDH antibody is used as a control.
Figure 5
Figure 5. HES1/HEY1 expression in si-NOTCH4 cells and NOTCH4 expression in si-HEY1 cells
HES1 (A) and HEY1 (B) expression in parental, si-control and si-NOTCH4 cells. mRNA expression is measured by qRT-PCR. The expression differences between si-control and si-NOTCH4 cells are compared. (C) Western blots of HES1 and HEY1 in si-control and si-NOTCH4 cells. (D) NOTCH4 expression of parental, si-control and si-HEY1 cells. mRNA expression is measured by qRT-PCR. The expression differences between si-control and si-HEY1 cells are compared. (E) Western blots of NOTCH4 in si-control and si-HEY1 cells. GAPDH antibody is used as a control. P value is calculated by using Student’s t-test. *: P < 0.05, **: P < 0.01, N.S.: not significant.
Figure 5
Figure 5. HES1/HEY1 expression in si-NOTCH4 cells and NOTCH4 expression in si-HEY1 cells
HES1 (A) and HEY1 (B) expression in parental, si-control and si-NOTCH4 cells. mRNA expression is measured by qRT-PCR. The expression differences between si-control and si-NOTCH4 cells are compared. (C) Western blots of HES1 and HEY1 in si-control and si-NOTCH4 cells. (D) NOTCH4 expression of parental, si-control and si-HEY1 cells. mRNA expression is measured by qRT-PCR. The expression differences between si-control and si-HEY1 cells are compared. (E) Western blots of NOTCH4 in si-control and si-HEY1 cells. GAPDH antibody is used as a control. P value is calculated by using Student’s t-test. *: P < 0.05, **: P < 0.01, N.S.: not significant.
Figure 6
Figure 6. Comparison of HEY1 and EMT-related genes using TCGA data set. Comparison of HEY1 expression between sphere and parental cells
(A) Comparison of EMT-related genes between HEY1 high and low groups using the TCGA data set. The boxes represent the interquartile range (25th-75th), and horizontal lines inside the boxes indicate median. Whiskers indicate the minimum and maximum values. Ratio is calculated by dividing the mRNA expression of the high group by the expression of the low group. (B) HEY1 expression is compared between parental and sphere cells using qRT-PCR. P value is calculated by using Student’s t-test. **: P < 0.01.
Figure 7
Figure 7. HEY1 relates to EMT gene expression and EMT functions
(A) The comparisons of EMT-related gene (E-cadherin, Vimentin, Fibronectin, TWIST1 and SOX2) expressions among parental, si-control and si-HEY1 cells. The expression differences between si-control and si-HEY1 cells are compared. (B) Western blots of EMT-related genes in si-control and si-HEY1 cells. (C) Representative images of migration and invasion assays. The cell line is SCC090. (D, E) Migration and invasion assays in parental, si-control, si-NOTCH4 and si-HEY1 cells. The migration and invasion indexes were calculated by deviding the number of parental cells thorough the chamber. The differences between si-control and si-NOTCH4/si-HEY1 cells are compared. P value is calculated by using Student’s t-test. *: P < 0.05, **: P < 0.01, N.S.: not significant.
Figure 7
Figure 7. HEY1 relates to EMT gene expression and EMT functions
(A) The comparisons of EMT-related gene (E-cadherin, Vimentin, Fibronectin, TWIST1 and SOX2) expressions among parental, si-control and si-HEY1 cells. The expression differences between si-control and si-HEY1 cells are compared. (B) Western blots of EMT-related genes in si-control and si-HEY1 cells. (C) Representative images of migration and invasion assays. The cell line is SCC090. (D, E) Migration and invasion assays in parental, si-control, si-NOTCH4 and si-HEY1 cells. The migration and invasion indexes were calculated by deviding the number of parental cells thorough the chamber. The differences between si-control and si-NOTCH4/si-HEY1 cells are compared. P value is calculated by using Student’s t-test. *: P < 0.05, **: P < 0.01, N.S.: not significant.

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