ERK2-mediated phosphorylation of ZEB1 at S322 enhances PD-L1 expression and EMT, leading to pancreatic cancer progression.

Background: Pancreatic cancer is the fourth leading cause of cancer-related deaths. Epithelial-mesenchymal transition (EMT) drives aggressive behaviour and unfavourable outcomes in this disease. The zinc finger E-box-binding homeobox 1 (ZEB1) transcription factor is pivotal in orchestrating EMT, promoting tumor cell mobility, metastasis, and immune evasion through phosphorylation events. However, the precise mechanisms underlying individual phosphorylation sites and their relationship with ZEB1's functions in vivo remain inadequately understood.

Methods: We assessed EMT using various techniques, including reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunoblotting, microscopy, migration, and invasion assays. ZEB1 knockdown was achieved via short hairpin RNA (shRNA), while plasmid transfection facilitated the overexpression of ZEB1, extracellular signal-regulated kinase 1 (ERK1), and extracellular signal-regulated kinase 2 (ERK2). Co-immunoprecipitation and kinase assays were used to examine the interaction between ZEB1 and ERK1/2. PANC-1 and HPAC cells were transplanted in an orthotopic mouse model for in vivo analysis.

Results: Sphingosylphosphorylcholine (SPC) induced EMT in PANC-1 and HPAC cells in a dose- and time-dependent manner through the phosphorylation and nuclear translocation of ZEB1. Notably, ERK2 interacted with ZEB1 and catalysed the phosphorylation of serine 322 (S322) within the ZEB1 molecule. Disrupting S322 phosphorylation hindered ZEB1's nuclear translocation, leading to reduced programmed death-ligand 1 (PD-L1) expression and suppressed migration and invasion of pancreatic cancer cells. Furthermore, in an orthotopic mouse model, implantation of S322 phosphorylation-deficient (shZEB1/S322A) pancreatic cancer cells suppressed tumour formation and metastasis. We developed a phosphoS322 detection antibody, which validated ZEB1 phosphorylation in pancreatic cancer cells and tissue samples from patients with pancreatic cancer.

Conclusion: SPC induces ZEB1 phosphorylation, with ERK2, rather than ERK1, targeting the S322 site. Inhibiting S322 phosphorylation mitigates EMT, PD-L1 expression, and progression of pancreatic cancer. The phosphoS322 detection antibody might be a valuable tool for predicting pancreatic cancer prognosis. These findings implicate ERK2 as a potential therapeutic target for pancreatic cancer and highlight phosphoZEB1 as a valuable prognostic marker.