Abstract
Inactivating mutations in the EGF-like ligand binding domain of NOTCH1 are a prominent feature of the mutational landscape of oral squamous cell carcinoma (OSCC). In this study, we investigated NOTCH1 mutations in keratinocyte lines derived from OSCC biopsies that had been subjected to whole exome sequencing. One line, SJG6, was found to have truncating mutations in both NOTCH1 alleles, resulting in loss of NOTCH1 expression. Overexpression of the NOTCH1 intracellular domain (NICD) in SJG6 cells promoted cell adhesion and differentiation, while suppressing proliferation, migration and clonal growth, consistent with the previously reported tumour suppressive function of NOTCH1 in OSCC. Comparative gene expression profiling identified SERPINE1 as being downregulated on NICD overexpression and predicted an interaction between SERPINE1 and genes involved in cell proliferation and migration. Mechanistically, overexpression of NICD resulted in upregulation of ETV7/TEL2, which negatively regulates SERPINE1 expression. Knockdown of SERPINE1 phenocopied the effects of NICD overexpression in culture. Consistent with previous studies and our in vitro findings, there were inverse correlations between ETV7 and SERPINE1 expression and survival in OSCC primary tumours. Our results suggest that the tumour suppressive role of NOTCH1 in OSCC is mediated, at least in part, by inhibition of SERPINE1 via ETV7.
Highlights
Notch1 is a heterodimeric and multifunctional transmembrane receptor that regulates key cellular processes, including cell fate determination, maintenance of stem cells, cell survival, proliferation and apoptosis [1, 2]
Based on whole exome analysis of 15 oral squamous cell carcinoma (OSCC) and the cell lines derived from them (Supplementary Table 1), we identified a hierarchy of nonsynonymous tumour specific mutations that was representative of mutations found in larger OSCC cohorts [13]
The expression of all 4 NOTCH receptors in the three lines that harbour NOTCH1 mutations was compared with normal oral mucosal keratinocytes (OK) and two OSCC lines that lack NOTCH1 mutations (Supplementary Figure 1A)
Summary
Notch is a heterodimeric and multifunctional transmembrane receptor that regulates key cellular processes, including cell fate determination, maintenance of stem cells, cell survival, proliferation and apoptosis [1, 2]. Notch has an extracellular domain (NECD), transmembrane domain and intracellular domain (NICD) [3, 4]. Ligand mediated activation of Notch induces proteolytic cleavage and release of NICD, which translocates to the nucleus and activates transcription of downstream target genes [5]. NOTCH1 is frequently altered in cancer, resulting in aberrant activation or loss of signaling in a tissue and context dependent manner [6]. A comprehensive analysis of multiple head and neck squamous cell carcinoma (HNSCC) datasets demonstrated that 10–15% of HNSCC harbour inactivating NOTCH1 mutations [7,8,9]. The majority of NOTCH1 mutations occur in the EGF-like ligand binding domain of the NECD, and prevent ligand binding and downstream signaling [3]
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