Autoregulation of RANK ligand in oral squamous cell carcinoma tumor cells.

Oral squamous cell carcinoma (OSCC) is the most common malignancy among oral cancers and shows potent activity for local bone invasion. Receptor activator of nuclear factor κB (RANK) ligand (RANKL) is critical for bone-resorbing osteoclast formation. We previously demonstrated that OSCC tumor cells express high levels of RANKL. In this study, confocal microscopy demonstrated RANKL specific receptor, RANK expression in OSCC tumor cell lines (SCC1, SCC12, and SCC14a). We also confirmed the expression of RANK and RANKL in primary human OSCC tumor specimens. However, regulatory mechanisms of RANKL expression and a functional role in OSCC tumor progression are unclear. Interestingly, we identified that RANKL expression is autoregulated in OSCC tumor cells. The RANKL specific inhibitor osteoprotegerin (OPG) treatment to OSCC cells inhibits autoregulation of RANKL expression. Further, we showed conditioned media from RANKL CRISPR-Cas9 knockout OSCC cells significantly decreased osteoclast formation and bone resorption activity. In addition, RANKL increases OSCC tumor cell proliferation. RANKL treatment to OSCC cells demonstrated a dose-dependent increase in RANK intracellular adaptor protein, TRAF6 expression, and activation of IKK and IκB signaling molecules. We further identified that transcription factor NFATc2 mediates autoregulation of RANKL expression in OSCC cells. Thus, our results implicate RANKL autoregulation as a novel mechanism that facilitates OSCC tumor cell growth and osteoclast differentiation/bone destruction.