ARL4C in oral cancer cells promotes stromal RANKL expression via a RelA-dependent inflammatory axis in stromal fibroblasts

Abstract

Jawbone destruction in oral squamous cell carcinoma (OSCC) is driven by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-mediated osteoclastogenesis. Although the cellular sources of RANKL and its regulatory mechanisms within the OSCC microenvironment remain elusive, this study aimed to elucidate how ADP-ribosylation factor-like 4 C (ARL4C), a key oncogene in oral cancer, promotes the pro-osteoclastogenic microenvironment by identifying the specific signaling axis responsible for RANKL induction in stromal fibroblasts. Stromal cells were cultured with OSCC-conditioned medium (CM), followed by comprehensive RNA sequencing and pathway enrichment analysis. Identified signaling pathways were validated using siRNA and pharmacological inhibitors. Spatial localization in human clinical specimens was evaluated by immunohistochemistry. Transcriptomic profiling revealed significant enrichment of the NF-κB and Stat3 pathways in stromal cells exposed to OSCC-CM. Inhibition of RelA subunit (a major subunit of NF-κB) significantly reduced tumor-induced RANKL expression. We found that ARL4C expression in cancer cells promoted the secretion of proinflammatory cytokines, establishing a sustained RelA-mediated inflammatory response in stromal fibroblasts. Immunohistochemical analysis of human OSCC specimens confirmed that phosphorylated RelA and RANKL were co-expressed in α-SMA-positive stromal fibroblasts at the bone-invasive front, even in areas distant from tumor nests. This study identifies a tumor–stromal signaling axis where ARL4C in OSCC cells triggers a RelA-mediated inflammatory response in stromal fibroblasts. These fibroblasts act as a critical, non-canonical RANKL source driving jawbone destruction. Targeting the ARL4C–RelA axis might represent a promising therapeutic strategy for suppressing stromal RANKL expression and improving quality of life for OSCC patients.