Background:Oesophgeal adenocarcinoma (OAC) is the most frequent cause of cancer death. POLO-like kinase 1 (PLK1) is overexpressed in broad spectrum of tumors and has prognostic value in many cancers including esophageal cancer, suggesting its potential as a therapeutic target. p53, the guardian of genome is the most important tumor suppressors that represses the promoter of PLK1, whereas tumor cells with inactive p53 are arrested in mitosis due to DNA damage. PLK1 expression has been linked to the elevated p53 expression and has been shown to act as a biomarker that predicts poor prognosis in OAC.Objective:The aim of the present study was identification of PLK1 associated phosphorylation targets in p53 mutant and p53 normal cells to explore the downstream signaling evets.Methods:Here we develop a proof-of-concept phospho-proteomics approach to identify possible biomarkers that can be used to identify mutant p53 or wild-type p53 pathways. We treated PLK1 asynchronously followed by mass spectrometry data analysis. Protein networking and motif analysis tools were used to identify the significant clusters and potential biomarkers.Results:We investigated approximately 1300 potential PLK1-dependent phosphopeptides by LCMS/ MS. In total, 2216 and 1155 high confidence phosphosites were identified in CP-A (p53+) and OE33 (p53-) cell lines owing to PLK1 inhibition. Further clustering and motif assessment uncovered many significant biomarkers with known and novel link to PLK1.Conclusion:Taken together, our study suggests that PLK1 may serve as a potential therapeutic target in human OAC. The data highlight the efficacy and specificity of small molecule PLK1 kinase inhibitors to identify novel signaling pathways in vivo.