Legumain Induces Oral Cancer Pain by Biased Agonism of Protease-Activated Receptor-2

Oral squamous cell carcinoma (OSCC) is one of the most painful cancers, which interferes with orofacial function including talking and eating. We report that legumain (Lgmn) cleaves protease-activated receptor-2 (PAR(2)) in the acidic OSCC microenvironment to cause pain. Lgmn is a cysteine protease of late endosomes and lysosomes that can be secreted; it exhibits maximal activity in acidic environments. The role of Lgmn in PAR(2)-dependent cancer pain is unknown. We studied Lgmn activation in human oral cancers and oral cancer mouse models. Lgmn was activated in OSCC patient tumors, compared with matched normal oral tissue. After intraplantar, facial or lingual injection, Lgmn evoked nociception in wild-type (WT) female mice but not in female mice lacking PAR(2) in Na(v)1.8-positive neurons (Par(2)Na(v)1.8), nor in female mice treated with a Lgmn inhibitor, LI-1. Inoculation of an OSCC cell line caused mechanical and thermal hyperalgesia that was reversed by LI-1. Par(2)Na(v)1.8 and Lgmn deletion attenuated mechanical allodynia in female mice with carcinogen-induced OSCC. Lgmn caused PAR(2)-dependent hyperexcitability of trigeminal neurons from WT female mice. Pare deletion, LI-1, and inhibitors of adenylyl cydase or protein kinase A (PICA) prevented the effects of Lgmn. Under acidified conditions, Lgmn deaved within the extracellular N terminus of PAR(2) at Asn(30)down arrow Arg(31), proximal to the canonical trypsin activation site. Lgmn activated PAR(2) by biased mechanisms in HEK293 cells to induce Ca2+ mobilization, cAMP formation, and PKA/protein kinase D (PKD) activation, but not beta-arrestin recruitment or PAR(2) endocytosis. Thus, in the acidified OSCC microenvironment, Lgmn activates PAR(2) by biased mechanisms that evoke cancer pain.

Automated summary

Oral squamous cell carcinoma (OSCC) is a type of cancer that causes severe pain, and it has been found that legumain (Lgmn) cleaves protease-activated receptor-2 (PAR(2)) in the acidic environment to trigger pain.