Abstract 5140: Phenotypic plasticity and cellular decision-making: role of SLIT2 in epithelial-to-mesenchymal transition

Abstract Background: Epithelial-to-mesenchymal transition (EMT), a key driver of cancer progression, is influenced by cell-specific factors. SLIT2, a ligand for ROBO1, regulates tumor growth and migration through a non-canonical ROBO1/EPHA2 pathway in lung and head and neck cancers. Modeling of the SLIT2-EPHA2 EMT circuit revealed SLIT2 stabilizes the epithelial phenotype, while EPHA2 coupling diminishes this suppression, inducing a tri-stable system with epithelial, hybrid, and mesenchymal states. In vitro, recombinant SLIT2 increased E-cadherin and decreased SNAIL in NSCLC and HNSCC cells. SLIT2 knockdown elevated mesenchymal markers, and recombinant SLIT2 suppressed migration in zebrafish xenografts, highlighting its anti-metastatic potential. These findings demonstrate SLIT2’s critical role in EMT regulation and its therapeutic promise for controlling metastasis. Introduction: Phenotypic plasticity, including epithelial-to-mesenchymal transition (EMT), drives cancer progression and resistance. SLIT2, signaling through ROBO1, has dual roles in cancer. In lung and head and neck cancers, SLIT2 inhibits tumor growth via a non-canonical ROBO1/EPHA2 pathway, though its role in EMT remains unclear. This study models SLIT2’s effect on EMT and confirms its potential to inhibit hybrid/mesenchymal transitions, offering therapeutic promise. Methods: SLIT2’s role in EMT was assessed in lung and head and neck cancers. A mathematical EMT model incorporating SLIT2 analyzed epithelial, hybrid, and mesenchymal states. Recombinant SLIT2 was tested on NSCLC and HNSCC cells, with EMT markers analyzed. SLIT2 knockdown was used to assess mesenchymal markers. Zebrafish xenografts quantified migration post-SLIT2 treatment. Results: SLIT2 expression was inversely correlated with EMT scores in LUSC and HNSCC. Epithelial subtypes had higher SLIT2 levels, while mesenchymal subtypes had lower levels. SLIT2 suppressed SNAIL expression, maintaining epithelial characteristics and reducing mesenchymal markers. Recombinant SLIT2 treatment decreased cell proliferation, migration, and spheroid growth. Zebrafish xenografts showed reduced tumor growth and metastasis. Patient tumor analysis indicated that high SLIT2 expression correlated with better survival. Conclusion: SLIT2 inhibits EMT in lung cancer cells, suppressing mesenchymal markers and maintaining the epithelial phenotype, thus limiting metastasis. Future studies will validate the therapeutic potential of the SLIT2 D2 fragment as an anti-metastatic treatment. Citation Format: Sravani Ramisetty, Ayalur Raghu Subbalakshmi, Madhu krishna B, Atish Mohanty, Aditya Agarwal, Mathias C Emery, Andrew Mc Carthy, Dana Do, Mari Ida, Tamara Mirzapoiazova, Siddhika Pareek, Deric L Wheeler, Sharad S Singhal, Prakash Kulkarni, Ravi Salgia. Phenotypic plasticity and cellular decision-making: role of SLIT2 in epithelial-to-mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5140.