A novel Notch1 missense mutation (C1133Y) in the Abruptex domain exhibits enhanced proliferation and invasion in oral squamous cell carcinoma

Abstract

BackgroundNotch1 has been regarded as a fundamental regulator in tissue differentiation and stem cell properties. Recently, Notch1 mutations have been reported intensively both in solid tumors and in hematopoietic malignancies. However, little is known about the biological effect and the clinical implication of these reported mutations. Previously, we discovered several missense mutations in the Notch1 receptor in a Chinese population with oral squamous cell carcinoma (OSCC).MethodsWe selected a ‘hotspot’ mutation in the Abruptex domain (C1133Y). The expression of Notch1 was determined by western blot and real-time qPCR in OSCC cell lines transfected with pcDNA3.1-Notch1WT, pcDNA3.1-Notch1C1133Y, or pcDNA3.1 empty vector. CCK-8 assays were used to assess cell proliferation. Flow cytometry and western blot were used to confirm the alteration of cell cycle after transfection. Transwell assays and the detection of Epithelial-to-mesenchymal transition (EMT) markers were used to determine the invasive ability. The effects of Notch1 C1133Y mutation were analyzed by Immunofluorescence staining and the expression of EGFR-PI3K/AKT signaling.ResultsWe demonstrated that Notch1C1133Y mutation inactivated the canonical Notch1 signaling. We identified an oncogenic phenotype of this mutation by promoting cell proliferation, invasion and by inducing EMT in OSCC cell lines. We found that the Notch1C1133Y mutation exhibited a decreased S1-cleavage due to the impaired transport of Notch1 protein from the endoplasmic reticulum (ER) to the Golgi complex, which was consistent with the observation of the failure of the Notch1C1133Y mutated receptor to present at the cell surface. Importantly, the mutated Notch1 activated the EGFR-PI3K/AKT signaling pathway, which has been confirmed as an overwhelming modulator in OSCC.ConclusionsTaken together, our findings revealed for the first time a novel Notch1 mutation that enhances proliferation and invasion in OSCC cell lines. The Notch1 C1133Y mutation impairs the processing of notch1 protein and the critical links between the mutated Notch1 and the activated EGFR-PI3K/AKT signaling pathway.Electronic supplementary materialThe online version of this article (10.1186/s12935-017-0496-5) contains supplementary material, which is available to authorized users.

Multifactorial Contribution of Notch Signaling in Head and Neck Squamous Cell Carcinoma

“…The downstream target Hes1 might directly downregulate expression of PTEN, increasing PI3K activity [155] (Figure 4). In a screening for Notch1 inactivating mutation, the oncogenic phenotype was associated with activation of the EGF-PI3K/AKT pathway and resulted in increased cell proliferation, EMT, and invasion in OSCC cell lines [156].…”

Expression and oncogenic properties of membranous Notch1 in oral leukoplakia and oral squamous cell carcinoma

“…As Notch1 has been suggested as a tumor suppressor in several studies ( 23 , 45 , 46 ), it is possible that membranous Notch1 accumulation is simply an alternative mechanism to inactivate the gene function. However, a recent study revealed that the Notch signaling pathway was active in about one third of Caucasian patients with HNSCC ( 34 ), suggesting an oncogenic role of Notch signaling in a significant proportion of patients with HNSCC in both Chinese and Caucasian populations, although the mechanisms of tumorigenesis could be different ( 47 ). Further studies are warranted to determine how membranous Notch1 accumulation leads to oral tumorigenesis and OSCC progression.…”

Isoform specific FBXW7 mediates NOTCH1 Abruptex mutation C1133Y deregulation in oral squamous cell carcinoma

“…Human OSCC cell lines (HN4, HN6, HN13, and CAL27) were provided as previously described 17,30 . HOK cells were purchased from the American Type Culture Collection (ATCC).…”

“…Although FBXW7 has been described to be involved in NOTCH1 protein degradation, no literatures have reported the interplay between FBXW7β and mutant NOTCH1 in the Abruptex domain yet. We have previously found that the NOTCH1 C1133Y mutation led to the retention of NOTCH1 protein in the endoplasmic reticulum and reduced the transport of full-length NOTCH1 protein from endoplasmic reticulum to the Golgi apparatus 17 . Matsumoto et al 31,32 reported that FBXW7β includes a supposed transmembrane domain and suggested that it substantially penetrates the ER membrane.…”

“…Similarly, mutations in the extracellular domain (such as Abruptex domain) may result in attenuated NOTCH1 signaling activation. In order to gain further insight into the regulation of NOTCH1 mutation, we offered point mutation (C1133Y) in the previous study 17 . We found that this mutation prevented the canonical NOTCH1 signaling activation, producing a loss of NOTCH1 function.…”