Additional file 1: Figure S1. Cell apoptosis was analyzed in HN6 and HN13 transfected cells. (A) Cleaved Caspase-3 was utilized to detect the cell apoptosis in HN6 and HN13 cells. (B) Flow cytometry was used to determine the early and late stages apoptotic cells

Gang Li (34549)

Heming Wu (306767)

Laikui Liu (3477614)

Wei Zhang (405)

Wenyi Gu (38121)

Xiaomeng Song (414568)

Xu Ding (517035)

Yang Zheng (185762)

Yunong Wu (593737)

Zhao Wang (145195)

Cancer Cell International

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MOESM1 of A novel Notch1 missense mutation (C1133Y) in the Abruptex domain exhibits enhanced proliferation and invasion in oral squamous cell carcinoma

Abstract Background Notch1 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). Methods We 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. Results We 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. Conclusions Taken 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

Yang Zheng

Zhao Wang

Xu Ding

Wei Zhang

Gang Li

Laikui Liu

Heming Wu

Wenyi Gu

Yunong Wu

Xiaomeng Song

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

Notch1 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).We 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.We 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.Taken 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

Zheng, Yang

Wang, Zhao

Ding, Xu

Zhang, Wei

Li, Gang

Liu, Laikui

Wu, Heming

Gu, Wenyi

Wu, Yunong

Song, Xiaomeng

University of Queensland eSpace

https://figshare.com/articles/MOESM1_of_A_novel_Notch1_missense_mutation_C1133Y_in_the_Abruptex_domain_exhibits_enhanced_proliferation_and_invasion_in_oral_squamous_cell_carcinoma/5768730

MOESM1 of A novel Notch1 missense mutation (C1133Y) in the Abruptex domain exhibits enhanced proliferation and invasion in oral squamous cell carcinoma

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University of Queensland eSpace