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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. Par 2 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 PAR 2 at Asn 30 ↓Arg 31 , proximal to the canonical trypsin activation site. Lgmn activated PAR 2 by biased mechanisms in HEK293 cells to induce Ca 2+ mobilization, cAMP formation, and PKA/protein kinase D (PKD) activation, but not β-arrestin recruitment or PAR 2 endocytosis. Thus, in the acidified OSCC microenvironment, Lgmn activates PAR 2 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 (PAR 2 ) 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 PAR 2 Exposure of pain-sensing neurons to Lgmn decreased the current required to generate an action potential through PAR 2 Inhibitors of adenylyl cyclase and protein kinase A (PKA) prevented the effects of Lgmn. Lgmn activated PAR 2 to induce calcium mobilization, cAMP formation, and activation of protein kinase D (PKD) and PKA, but not β-arrestin recruitment or PAR 2 endocytosis. Thus, Lgmn is a biased agonist of PAR 2 that evokes cancer pain., (Copyright © 2021 the authors.)

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 NaV1.8-positive neurons (Par2Nav1.8), 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. Par2Nav1.8 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. [ABSTRACT FROM AUTHOR]

Simple Summary: Oral cancer is often deadly and severely painful. Because this form of cancer pain cannot be adequately treated with current medications including opiates, new treatment approaches are needed. Cathepsin S, a lysosomal cysteine protease may play a role in oral cancer pain through a protease-activated receptor-2 (PAR2)-dependent mechanism. We undertook a series of experiments to define the role of cathepsin S in oral cancer pain. We compared cathepsin S activity in human oral cancer tumors versus patient-matched normal tissue; a human oral cancer cell line versus a benign dysplastic oral keratinocyte cell line; and in an orthotopic xenograft tongue cancer mouse model versus normal controls in mice. We localized cathepsin S in macrophages and carcinoma cells in human oral cancers. The injection of cathepsin S caused nociception in a mouse model while the injection of oral cancer cells in which the gene for cathepsin S is deleted generated less nociception. Our findings will lay the foundations for clinical trials of cathepsin S inhibitors for treating oral cancer pain. Oral squamous cell carcinoma (SCC) pain is more prevalent and severe than pain generated by any other form of cancer. We previously showed that protease-activated receptor-2 (PAR2) contributes to oral SCC pain. Cathepsin S is a lysosomal cysteine protease released during injury and disease that can activate PAR2. We report here a role for cathepsin S in PAR2-dependent cancer pain. We report that cathepsin S was more active in human oral SCC than matched normal tissue, and in an orthotopic xenograft tongue cancer model than normal tongue. The multiplex immunolocalization of cathepsin S in human oral cancers suggests that carcinoma and macrophages generate cathepsin S in the oral cancer microenvironment. After cheek or paw injection, cathepsin S evoked nociception in wild-type mice but not in mice lacking PAR2 in Nav1.8-positive neurons (Par2Nav1.8), nor in mice treated with LY3000328 or an endogenous cathepsin S inhibitor (cystatin C). The human oral SCC cell line (HSC-3) with homozygous deletion of the gene for cathepsin S (CTSS) with CRISPR/Cas9 provoked significantly less mechanical allodynia and thermal hyperalgesia, as did those treated with LY3000328, compared to the control cancer mice. Our results indicate that cathepsin S is activated in oral SCC, and that cathepsin S contributes to cancer pain through PAR2 on neurons. [ABSTRACT FROM AUTHOR]