<scp>EN2</scp> Regulates Pancreatic Cancer Initiation, Progression, and Epithelial‐Mesenchymal Transition Through the Notch Signalling Pathway

The incidence and mortality of pancreatic cancer are steadily increasing worldwide, and the disease is projected to become the second leading cause of cancer-related deaths by 2030. Pancreatic tumorigenesis is driven by multiple genetic alterations, underscoring the need to elucidate the molecular mechanisms underlying pancreatic carcinogenesis. In this context, the present study is the first to identify a novel oncogenic role for Engrailed 2 (EN2) in the initiation and progression of pancreatic cancer and to characterise its underlying molecular pathogenesis. Immunohistochemical analysis of a pancreatic cancer tissue microarray revealed significantly elevated EN2 expression in tumour tissues compared with adjacent normal tissues. Consistent with these findings, EN2 was markedly upregulated in human pancreatic cancer cell lines, but absent in normal pancreatic epithelial cells, and functional studies demonstrated that EN2 expression is oncogenic in pancreatic cancer. TCGA data further corroborated the significantly higher EN2 expression in pancreatic cancer tissues and showed that elevated EN2 levels are associated with poor overall survival. To define the biological significance of EN2, we investigated its role in promoting pancreatic cancer initiation and progression. Gain- and loss-of-function studies revealed that EN2 regulates key target genes involved in pluripotency, cell survival, and cell-cycle progression, drug resistance, and epithelial-mesenchymal transition. Moreover, lentiviral-mediated shRNA knockdown of EN2 suppressed pancreatic cancer cell proliferation, invasion, and metastasis in vitro and significantly inhibited tumour growth in a xenograft mouse model, in part by inhibiting Notch signalling. Taken together, these findings identify EN2 as a critical driver of pancreatic cancer initiation, progression, and metastasis, representing the first report of its oncogenic function in this malignancy.