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

Abstract

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 (PAR₂) 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 PAR₂-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 PAR₂ in Na_V1.8-positive neurons (Par₂Na_v1.8), 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. Par₂Na_v1.8 and Lgmn deletion attenuated mechanical allodynia in female mice with carcinogen-induced OSCC. Lgmn caused PAR₂-dependent hyperexcitability of trigeminal neurons from WT female mice. Par₂ deletion, LI-1, and inhibitors of adenylyl cyclase or protein kinase A (PKA) prevented the effects of Lgmn. Under acidified conditions, Lgmn cleaved within the extracellular N terminus of PAR₂ at Asn³⁰↓Arg³¹, proximal to the canonical trypsin activation site. Lgmn activated PAR₂ by biased mechanisms in HEK293 cells to induce Ca²⁺ mobilization, cAMP formation, and PKA/protein kinase D (PKD) activation, but not β-arrestin recruitment or PAR₂ endocytosis. Thus, in the acidified OSCC microenvironment, Lgmn activates PAR₂ by biased mechanisms that evoke cancer pain.

SIGNIFICANCE STATEMENT Oral squamous cell carcinoma (OSCC) is one of the most painful cancers. We report that legumain (Lgmn), which exhibits maximal activity in acidic environments, cleaves protease-activated receptor-2 (PAR₂) on neurons to produce OSCC pain. Active Lgmn was elevated in OSCC patient tumors, compared with matched normal oral tissue. Lgmn evokes pain-like behavior through PAR₂. Exposure of pain-sensing neurons to Lgmn decreased the current required to generate an action potential through PAR₂. Inhibitors of adenylyl cyclase and protein kinase A (PKA) prevented the effects of Lgmn. Lgmn activated PAR₂ to induce calcium mobilization, cAMP formation, and activation of protein kinase D (PKD) and PKA, but not β-arrestin recruitment or PAR₂ endocytosis. Thus, Lgmn is a biased agonist of PAR₂ that evokes cancer pain.