Targeting EphA2 with CRISPR/Cas9 Identifies a Novel EphA2-CDH1 Regulatory Axis in Migration of Castration-Resistant Prostate Cancer

Abstract

Objectives: Progression to castration-resistant prostate cancer (CRPC) and metastasis are the greatest challenges to effective treatment. Anticancer strategies targeting the key kinases associated with the development of CRPC may represent a breakthrough. The tyrosine kinase receptor Erythropoietin-producing hepatocellular (Eph) A2 receptor is highly expressed in CRPC cell lines and may be associated with tumor invasion and metastasis. However, the effects and exact mechanisms of EphA2 in CRPC are only partially understood. This study aimed to investigate the impact of EphA2 on CRPC cell behaviors and underlying molecular pathways Methods: CRISPR/Cas9-mediated gene editing induced EphA2-disrupted in human-derived PC3 and DU145 cells. Single-guide RNAs (sgRNAs) targeting EphA2 were designed, and editing efficiency was validated. Optimal sgRNA sequences were selected to generate EphA2-knockdown (KD) and -overexpressing (OE) cell lines. Cell migration, proliferation, and apoptosis were assessed via functional assays. Transcriptomic analysis, quantitative PCR, and Western blotting were performed to identify downstream effectors. Bioinformatics analyses were used to correlate EphA2 and CDH1 expression with clinical parameters in prostate cancer patients. Results: Editing efficiency was found to vary among different sgRNAs targeting the EphA2 gene. EphA2-KD significantly inhibited CRPC cell migration but did not affect cell proliferation or apoptosis. Conversely, EphA2-OE significantly enhanced the migration of DU145 cells. Molecular analyses revealed that the expression of CDH1 (an important marker of the epithelial-mesenchymal transition (EMT) in tumors) was significantly upregulated in PC3-EphA2-KD cells and downregulated in DU145-EphA2-OE cells, indicating that CDH1 is a downstream regulator of EphA2. Bioinformatic analysis revealed that higher EphA2 levels and lower CDH1 expression were both associated with an advanced tumor T stage, higher Gleason scores, and lymph node metastases in prostate cancer patients. More importantly, EphA2 was found to be an important predictor of lymph node metastasis, in addition to the Gleason score. Adding EphA2 to the Gleason score could significantly improve the detection of lymph node metastasis. Conclusion: CRISPR/Cas9-mediated EphA2-KD significantly suppressed the migration of CRPC cells through the inhibition of the EphA2-CDH1 axis. Strategies targeting the EphA2 gene may be promising for the treatment of CRPC.