Molecular Mechanism Analysis Of PTPN1 In Promoting Invasion And Migration Of Esophageal Cancer Cells

As a member of PTPs family, PTPNl can dephosphorylate the tyrosine residues of its substrates to regulate several pathways involved in a wide array of cellular processes. Recently it has been reported that PTPN1 overexpressed in various cancers and promoted tumorigenesis via different mechanisms. In our preliminary work, PTPN1 was observed with obvious overexpression in 38%(37/98) of esophageal squamous cell carcinomas (ESCC) and demonstrated to be a key downstream molecule of CRT in promoting the lung metastasis of ESCC.To probe into the role of PTPN1 in the process of metastasis, we knocked down PTPN1 in ESCC cells and found that cell motility and invasion were both inhibited accompanied with a decrease of EGFR expression and Erkl/2 activity. We confirmed that inactivation of EGFR-Erkl/2 pathway could also suppress the motility and invasion of ESCC cells. Rescue experiment showed that PTPN1-induced the motility and invasion of ESCC cells mainly depended on EGFR-Erkl/2 pathway. Further investigation clarified that PTPN1 promoted a cytoplasm-to-nucleus translocation of the transcription factor Sp1, which enhanced the expression of EGFR at the mRNA Level.In order to shed new light on the mechanism how PTPn1 facilitated cell motility and invasion via EGFR-Erkl/2 pathway, GST-pulldown combined with LC/MS/MS was applied, and MYH9 was discovered to interact with PTPNl. MYH9 is a cytoskeletal protein, which is reported to regulate the motility of cells and the translocation of some proteins including Spl. By immunofluorescent localization and co-immunoprecipitation (CoIP), we confirmed that MYH9 interacted with PTPN1 in ESCC cells.Then we knocked down MYH9 in ESCC cells and found that the expression of EGFR decreased at both mRNA and protein levels and that the motility and invasion of ESCC cells were also restrained, suggesting that MYH9 may be a key molecule between PTPN1 and EGFR in the regulation of cell motility and invasion. But the ATPase inhibitor of MYH9, Blebbistatin, had no effect on the cell motility and invasion as well as the expression of EGFR. Further rescue experiment showed that the rod tail but cytoskeleton function of MYH9 was essential for the regulation of EGFR.Subsequently, Phos-tag and IP assays showed that PTPN1 could dephosphorylate MYH9. Using the catalytic-activity impaired mutant PTPN1, we demonstrated that phosphatase activity of PTPN1 was responsible for the regulation of cell motility and invasion as well as the expression of EGFR. By analysis of substrate-trap mutant PTPN1 and the non-phosphorylation mutant of MYH9, we identified that PTPN1 could directly dephosphorylate MYH9 on Tyr 1408. Rescue experiment confirmed that the non-phosphorylated Tyr 1408 in the rod tail of MYH9 was critical for PTPN1 in promoting the expression of EGFR.Taken together, these findings reveal that PTPN1 enhances the expression of EGFR via dephosphorylating of Tyr 1408 in MYH9, which promotes the invasion and migration of esophageal cancer cells. Our data provide new insights into the role of PTPN1 in ESCC.