Activation of the Hippo Pathway by ATN Suppresses Cancer Stem-Like Properties and EGFR-Associated Signaling in Castration-Resistant Prostate Cancer Cells in vitro

Background: Castration-resistant prostate cancer (CRPC) represents a lethal stage of prostate cancer for which effective therapeutic options remain limited. Aberrant activation of the Hippo pathway effector Yes-associated protein (YAP) and its crosstalk with epidermal growth factor receptor (EGFR) signaling have been implicated in tumor progression, cancer stemness, and metastasis. This study aimed to investigate the therapeutic potential and underlying mechanisms of ATN, a 1,2,4-oxadiazole-based YAP inhibitor, in CRPC cells. Methods: The anti-cancer activity of ATN was evaluated in CRPC cell lines, PC3 and DU145, using cell viability, colony formation, apoptosis, tumorsphere, and transwell migration assays. Western blotting and immunofluorescence analyses were performed to assess Hippo-YAP and EGFR signaling pathways. Functional rescue experiments were conducted using YAP overexpression and constitutively active Akt (CA-Akt). Results: cancer stem cell (CSC) population in CRPC cells. In addition, ATN inhibited cell migration and reduced EMT-related proteins, including N-cadherin, Twist, YB-1, and Snail. ATN also decreased amphiregulin (AREG) expression, leading to suppression of EGFR activation and downstream MEK/ERK and Akt/mTOR signaling pathways. Importantly, constitutive activation of Akt partially rescued ATN-induced cytotoxicity (~78% recovery), confirming the involvement of YAP-associated EGFR/Akt signaling. Conclusion: ATN exerts potent anti-tumor effects against CRPC cells by activating the Hippo pathway, promoting YAP degradation, and disrupting the YAP-associated EGFR signaling network. These findings highlight ATN as a promising therapeutic candidate for targeting YAP-driven oncogenic signaling in CRPC.