Transcriptional Regulation And Function Analysis Of PLK1 In Esophageal Squamous Cell Carcinoma

Serine/threonine kinase Polo-like kinase 1 (PLKl) is pivotal for mammalian cell mitosis regulation and is found frequently overexpressed in various kinds of human tumors. We have previously shown that overexpression of PLK1 is an independent prognostic factor and that depletion of PLK1 activated the intrinsic apoptotic pathway in esophageal squamous cell carcinoma (ESCC).In the present study, we performed a proteomic analysis to elucidate the substrates and interacting proteins of PLK1. Based on the findings that phospho-dependent ligand recognition by the PBD is necessary for the targeting of PLK1 to specific substrates, GST-PBD was used as a bait to pull-down all the proteins associated with PBD in esophageal cancer cells. After analyzed by LC-MS/MS, a total of 213 proteins were identified potentially interacted with PBD. Co-immunoprecipitation (Co-IP) confirmed that IQGAP1, M2-PK and (3-catenin were all able to form complex with PLKl.Although PLKl has been recently reported to phosphorylateβ-catenin, the biological consequences of their interaction remain to be elucidated. Here we demonstrated an important role of PLKl in regulating degradation ofβ-catenin protein. GST Pull-Down and Co-IP confirmed that PLKl interacted with (3-catenin in esophageal squamous cell carcinoma (ESCC) cells. SiRNA-mediated knockdown of PLK1 and forced expression of its kinase dead mutant caused a reduction inβ-catenin protein level and its transcription activity. Ubiquitinated form of (3-catenin was increased and interaction between (3-catenin and GSK-3(3/(3-Trcp was enhanced upon PLK1 depletion, suggesting that the destruction process of (3-catenin was accelerated. Overexpression of (3-catenin in PLKl down-regulated cells restored their ability to grow in soft agar. In primary esophageal tumors, (3-catenin accumulation in cytoplasm was correlated with high level of PLK1 expression. Combined survival analysis indicated that PLKl+/β-catenin+was a poor-prognostic factor in ESCC.It has been reported that activation ofβ-catenin/TCF pathway protected cells from anoikis and we also confirmed this phenomenon in ESCC cells. We then investigated whether PLK1 was also involved in anoikis resistance. We found that detachment of esophageal tumor cells triggered upregulation of both PLK1 mRNA and protein. Enforced overexpression of PLKl delayed ESCC cell anoikis by activating MEK-ERK pathway. EMSA, CHIP and gene reporter assay indicated that NF-kappaB p65 bound directly to PLKl promoter and positively regulated PLK1 mRNA expression during cell suspension. Inhibition of NF-kappaB pathway caused a noticeable downregulation of PLKl and an increase of cell apoptosis after detachment. We conclude that PLKl is an important regulator of the detachment-triggered life signals in ESCC cells.Taken together, our findings showed that PLKl played an important role in regulating both (3-catenin protein degradation andβ-catenin/TCF signaling pathway. Detachment-induced upregulation of PLK1 was driven by NF-kappaB at the transcription level. These mechanisms were involved in anoikis resistance of esophageal squamous carcinoma cells.