A mutation in NOTCH1 ligand binding region detected in patients with oral squamous cell carcinoma reduces NOTCH1 oncogenic effect

Uchibori,Masahiro; Aoyama,Ken-Ichi; Ota,Yoshihide; Kajiwara,Kagemasa; Tanaka,Masafumi; Kimura,Minoru

doi:10.3892/or.2017.5870

A mutation in NOTCH1 ligand binding region detected in patients with oral squamous cell carcinoma reduces NOTCH1 oncogenic effect

Authors:
- Masahiro Uchibori
- Ken-Ichi Aoyama
- Yoshihide Ota
- Kagemasa Kajiwara
- Masafumi Tanaka
- Minoru Kimura
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Affiliations: Department of Oral and Maxillofacial Surgery, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan
Published online on: August 3, 2017 https://doi.org/10.3892/or.2017.5870
Pages: 2237-2242

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Abstract

NOTCH1 is known as an oncogenic or tumor suppressive gene in solid cancer. NOTCH1 mutations in oral squamous cell carcinoma (OSCC) frequently occur near the ligand-binding region. These mutations change the domain structure of this protein and affect the ligand binding activity. When NOTCH1 is activated by ligand binding, NOTCH1 intracellular domain (NICD) is cleaved from the cell membrane. This study investigated the functional change induced by a NOTCH1 mutation detected in OSCC clinical samples using stable transformant analysis. HEK293 cell lines expressing NOTCH1 wild-type (WT cells) or p.A465T NOTCH1 (A465T cells) were established. NOTCH1 expression was analyzed by flow cytometry, western blotting, and immunofluorescence using an anti-human NOTCH1 antibody. mRNA expression levels in WT and A465T cells were determined by quantitative real-time PCR (qPCR). Cell proliferation was analyzed by using cell growth assays and a xenograft tumor assay. Flow cytometry indicated that NOTCH1 expression on the cell membrane was lower in A465T cells than that in WT cells. NOTCH1 and NICD were both detected by western blot in WT and A465T cells. The immunofluorescence signal for NICD was detected in the nucleus of WT cells, while it was localized mainly in the cytoplasm of A465T cells. HES1 and HEY1 mRNA expression levels were lower in A465T than in WT cells. The cell growth of WT cells was significantly higher than that of HEK293 cells (3-fold, P<0.01), while that of A465T cells was significantly lower than that of HEK293 cells (37%, P<0.01). In a xenograft model, the tumor cell implantation rate of WT cells was 80%, while that of A465T cells was 0%. This study indicates that NOTCH1 acts as an oncogene and that the NOTCH1 mutation (p.A465T) in the ligand-binding region causes the loss of tumorigenicity by downregulating the NOTCH1 pathway.

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