1153 - Molecular Mechanisms Underlying Liver Metastasis in Hypofractionated Radiotherapy-Resistant Colorectal Cancer

W. Wen¹, X. Zhang², J. Wang², F. Sun², D. Chen³, and J. Yu⁴; ¹Department of Radiation Oncology and Shandong Provincial Key Laboratory of Radiation Oncology, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China, ²Department of Radiation Oncology and Shandong Provincial Key Laboratory of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shangdong, China, ³Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China, ⁴Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China

Purpose/Objective(s): Colorectal cancer (CRC) remains a global health concern, with liver metastasis presenting a significant treatment challenge and mortality risk. Approximately 20-30% of CRC patients develop liver metastasis following resistance to radiotherapy. This study aimed to elucidate the molecular mechanisms underlying HFRT-resistant CRC and its impact on liver metastasis. Materials/

Methods: HFRT-resistant CT26 cell line obtained through in vivo screening served as models for in vitro and in vivo investigations. RNA-Seq analysis identified differentially expressed genes and enriched pathways in HFRT-resistant CT26 cell line. Liver metastasis models were established via splenic injection of luciferase-expressing HFRT-resistant CT26 cell line in BALB/c mice. Flow cytometry was utilized to examine the immune microenvironment in the liver. Mechanistic investigations employed techniques such as immunoprecipitation-mass spectrometry (IP-MS), co-immunoprecipitation (CO-IP), Chromatin Immunoprecipitation (ChIP), in vitro kinase assays, Western blotting, QPCR, immunofluorescence staining, wound healing, and transwell assays. Clinical relevance was explored through multi-color immunofluorescence staining.
Results: HFRT-resistant CRC cells exhibited increased propensity for liver metastasis, characterized by shorter tumor latency, larger metastatic foci, and reduced mouse survival. RNA-seq revealed upregulation of RGS14 in HFRT-resistant CRC cells. CRISPR-Cas9-mediated knockout of RGS14 significantly inhibited liver metastasis in HFRT-resistant CRC cells. Flow cytometry showed decreased M2-polarized tumor-associated macrophages (TAMs) and enhanced cytotoxicity of tumor-infiltrating CD8⁺ T cells upon RGS14 knockout. Mechanistically, RGS14 interacted with the N-terminal domain of ß-catenin, competitively inhibiting GSK-3ß-mediated phosphorylation of ß-catenin, leading to ß-catenin activation and nuclear translocation to drive downstream gene transcription, resulting in an increase in the expression of snail, slug, vimentin, and N-cadherin, enhancing tumor epithelial-mesenchymal transition (EMT), and upregulated IL-6 expression, fostering TAM recruitment and polarization towards a pro-tumorigenic M2 phenotype, thus modulating pre-metastatic niche and facilitating liver metastasis. Multi-color immunofluorescence on CRC tissue microarrays linked high RGS14 expression to poor prognosis and decreased CD8⁺ T cell infiltration.
Conclusion: RGS14 promotes EMT and orchestrates pre-metastatic niche formation to enhance liver metastasis in HFRT-resistant CRC via ß-catenin activation. Understanding these mechanisms could inform novel therapeutic strategies to counter HFRT resistance and prevent liver metastasis in CRC patients.