Park Mi Kyung, Lee Hye Ja, Sung Jee Young, Byun Hyun Jung, Kim Hyun Ji, Kim Eun Ji, Nguyen Tuan Minh, Kang Gyeoung Jin, Oh Seung Hyun, Shim Jae Gal, Lee Ho, Nam Ki Taek, Kim Yong Yun, Rho Seung Bae, Kim Sang Gun, Lee Chang Hoon
Department of Biomedical Science, Hwasung Medi-Science University, Hwaseong-si, 18274, Republic of Korea.
BK21 FOUR Team and Integrated Research Institute for Drug Development, College of Pharmacy, Dongguk University, Seoul 100-715, Goyang, 10326, Republic of Korea.
Cell Commun Signal. 2025 Apr 28;23(1):204. doi: 10.1186/s12964-025-02182-3.
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.
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.
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.
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.
胰腺癌是癌症相关死亡的第四大主要原因。上皮-间质转化（EMT）驱动该疾病的侵袭性生物学行为并导致不良预后。锌指E盒结合同源框1（ZEB1）转录因子在协调EMT过程中起关键作用，通过磷酸化事件促进肿瘤细胞迁移、转移和免疫逃逸。然而，单个磷酸化位点背后的确切机制及其在体内与ZEB1功能的关系仍未得到充分了解。
我们使用多种技术评估EMT，包括逆转录定量聚合酶链反应（RT-qPCR）、免疫印迹、显微镜检查、迁移和侵袭实验。通过短发夹RNA（shRNA）实现ZEB1基因敲低，而质粒转染促进ZEB1、细胞外信号调节激酶1（ERK1）和细胞外信号调节激酶2（ERK2）的过表达。采用免疫共沉淀和激酶实验检测ZEB1与ERK1/2之间的相互作用。将PANC-1和HPAC细胞移植到原位小鼠模型中进行体内分析。
鞘氨醇磷酸胆碱（SPC）通过ZEB1的磷酸化和核转位，以剂量和时间依赖的方式诱导PANC-1和HPAC细胞发生EMT。值得注意的是，ERK2与ZEB1相互作用并催化ZEB1分子内丝氨酸322（S322）的磷酸化。破坏S322磷酸化会阻碍ZEB1的核转位，导致程序性死亡配体1（PD-L1）表达降低，并抑制胰腺癌细胞的迁移和侵袭。此外，在原位小鼠模型中，植入缺乏S322磷酸化的（shZEB1/S322A）胰腺癌细胞可抑制肿瘤形成和转移。我们开发了一种S322磷酸化检测抗体，该抗体验证了胰腺癌患者癌细胞和组织样本中ZEB1的磷酸化情况。
SPC诱导ZEB1磷酸化，其中ERK2而非ERK1靶向S322位点。抑制S322磷酸化可减轻EMT、PD-L1表达和胰腺癌进展。S322磷酸化检测抗体可能是预测胰腺癌预后的有价值工具。这些发现表明ERK2是胰腺癌的潜在治疗靶点，并突出了磷酸化ZEB1作为有价值的预后标志物。
