A Common Met Polymorphism Harnesses Her2 Signaling to Drive Aggressive Squamous Cell Carcinoma

Abstract

as well as from patients who have undergone surgical resection at NUH. Germline DNA was obtained retrospectively from Pharmacogenetics DNA Bank and prospectively from patients with metastatic LUSC, HNSCC and NPC. All samples were processed with approval from National Healthcare Group's Domain Specific Review Board (DSRB) Committee, and under the guideline of Institutional Review Board (IRB) at National University of Singapore (NUS, Singapore). Written informed consent was obtained in all cases from patients at time of enrolment. Mass spectrometry analysis.Samples were treated by in-gel digestion prior to MS analysis. In brief, samples were reduced in 10 mM DTT for 1h at 56°C followed by alkylation with 55mM iodoacetamide (Sigma) for 45 min in the dark. Tryptic digest was performed in 50 mM ammonium bicarbonate buffer with 2 μg trypsin at 37°C overnight. Peptides were desalted on StageTips and analysed by nanoflow liquid chromatography on an EASY-nLC 1200 system coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). Peptides were separated on a C18-reversed phase column (25 cm long, 75 μm inner diameter) packed in-house with ReproSil-Pur C18-QAQ 1.9 μm resin (Dr Maisch). The column was mounted on an Easy Flex Nano Source and temperature controlled by a column oven (Sonation) at 40°C. A 105-min gradient from 2 to 40% acetonitrile in 0.5% formic acid at a flow of 225 nl/min was used. Spray voltage was set to 2.4 kV. The Q Exactive HF was operated with a TOP20 MS/MS spectra acquisition method per MS full scan. MS scans were conducted with 60,000 and MS/MS scans with 15,000 resolution. The raw files were processed with MaxQuant version 1.5.2.8 1 with preset standard settings for SILAC labelled samples and the re-quantify option was activated.Carbamidomethylation was set as fixed modification while methionine oxidation and protein N-acetylation were considered as variable modifications. Search results were filtered with a false discovery rate of 0.01. Cell viability assay.Approximately 3,000 cells/well in complete medium were seeded into clear-bottom black 96well plates 24 hours prior to drug treatment. Indicated compounds or rSema were added in serial dilution for 72 hours. The viability of cells were assayed using CellTiter-Glo luminescent reagent (Promega, #G7572). The luminescence signals were detected using TECAN Infinite M1000 pro multi-mode plate reader using an integration time of 1000 ms.The relative luminescence units from treated wells were normalized against the readings from DMSO control and expressed as percentage cell viability and presented as mean ± SD relative to DMSO-treated controls. IC50 values were calculated by curve-fitting using GraphPad Prism software, and expressed as mean value ± SD. RNAseq, EMT scoring, hierarchical clustering and pathway analysis.RNAseq and gene expression analyses were performed by Novogene. In brief, isogenic H2170 cell lines of wild type (WT) and MET N375S genotypes were subjected to Illumina HiSeqTM2500 for RNA-seq. CASAVA v1.8 was used to convert the...

Molecular mechanism(s) of regulation(s) of c-MET/HGF signaling in head and neck cancer

“…EGFR is the commonly expressed receptor in among 80-90% of HNSCC tumors and has also been associated with poor overall and progression free survival. Over expression of other tyrosine kinase receptors such as HER-2, c-MET has also been widely reported in the case of HNSCC enabling resistance towards receptor targeting monoclonal antibody therapies against HNSCC [60]. Studies reported that MET gene (N375S) polymorphism has the binding affinity for HER-2 and enables the interaction with HER-2 in a ligand independent fashion.…”

“…Studies reported that MET gene (N375S) polymorphism has the binding affinity for HER-2 and enables the interaction with HER-2 in a ligand independent fashion. This interaction transduces potent proliferative and pro-metastatic characteristics in HNSCC through HER-2 signaling and is associated with poor prognosis [60]. PI3K-AKT-mTOR pathway has been the common drive for HNSCC development as it is the most altered oncogenic pathway in HNSCC [61].…”