SERPINE2 (Protease Nexin I) Is a Feedback Regulator of EGF/PKC/MAPK/EGR1 Signaling in Breast Cancer Cells Metastasis and Stemness

Background: Protease nexin-1 (PN-1) contributes to enhanced metastasis of cancer cells. In this study, we showed PN-1 levels were remarkably upregulated in breast cancer cells and tissues, we aimed to uncover an unidentified mechanism underlying the up-regulation of PN-1 in breast cancers.   Methods: The up-regulation of PN-1 in MCF-7 spheroid cells and breast cancer tissues was detected by RNA sequencing and qRT-PCR. The functions of PN-1 on breast cancer cells were investigated. The signaling pathway involved in epidermal growth factor (EGF) up-regulated PN-1 in breast cancer cells was identified by specific inhibitors or siRNAs of the candidate downstream genes. The feedback regulation of EGF/MAPK/EGR1 signaling by PN-1 was analyzed by Enzyme-linked immunosorbent assay (ELISA) and western blotting. Findings: The up-regulation of PN-1 in breast cancer mainly promoted cell migration, invasion and stemness. We uncovered that EGF induced the expression of PN-1 via its transcriptional factor, early growth response protein 1 (EGR1), through cascade activation of epidermal growth factor receptor (EGFR) to the activation of protein kinase Cδ (PKCδ), mitogenactivated protein kinase (MEK) and extracellular signal-related kinase (ERK). We further demonstrated EGF-induced PN-1 up-regulation could promote breast tumor cell metastasis in the mouse model for human breast cancer lung and liver metastasis. We also proved there was a positive feedback towards the activation of EGF signals by overexpression of PN-1. Interpretation: Our findings reveal a novel signaling axis that up-regulated PN-1 expression in breast cancer cells, which may provide new insights into identifying novel therapeutic targets for breast cancer.   Funding Statement: This project is funded by National Natural Science Foundation of China (Grant No. 81570696, No. 81702941). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All the patients provided written consent, and the experiments were approved by the Ethics Committee of China Pharmaceutical University (Nanjing, China).