Faculty Bibliography

BACKGROUND:Oral cancer causes intense pain at the primary site, and such pain can impair oral functions. However, the underlying mechanisms for oral cancer pain are still not fully understood. In the present study, it is investigated whether programmed cell death protein 1 (PD-1) is involved in the development of oral cancer pain. METHODS:RMP1-14, a specific anti-PD-1 antibody, was injected into spinal trigeminal nucleus caudalis (Sp5C) and measured pain behaviors using von Frey filaments and dolognawmeter. Western blotting and immunofluorescence staining were performed to analyze the expression of PD-1 and tumor necrosis factor alpha (TNFα) in the Sp5C. RESULTS:It was observed that the PD-1 antibody significantly inhibited mechanical hypersensitivity and functional allodynia in our oral cancer pain mouse model. Moreover, we found that TNFα was highly upregulated in the Sp5C following the induction of oral cancer pain and that intra-Sp5C injection of the PD-1 antibody diminished the upregulation of TNFα. It was found that genetic deletion of TNFα or its receptor antagonism synergized the analgesic effect of PD-1 antibody on oral cancer pain. CONCLUSION/CONCLUSIONS:Our results suggest that PD-1 in the Sp5C contributes to oral cancer pain by altering TNFα signaling in the trigeminal nociceptive system, and PD-1 could be targeted to develop a novel approach for oral cancer pain management.

Oral cancer is notoriously painful. Activation of protease-activated receptor 2 (PAR₂, encoded by F2RL1) by proteases in the cancer microenvironment is implicated in oral cancer pain. PAR₂ is a G protein-coupled receptor (GPCR) expressed on neurons and cells in the cancer microenvironment. Sustained signaling of PAR₂ from endosomes of neurons mediates sensitization and nociception. We focused on the differential contribution of PAR₂ on oral cancer cells and neurons to oral cancer pain and whether encapsulation of a PAR₂ inhibitor, AZ3451 in nanoparticles (NP) more effectively reverses PAR₂ activation. We report that F2RL1 was overexpressed in human oral cancers and cancer cell lines. Deletion of F2RL1 on cancer cells reduced cancer-associated mechanical allodynia. A third-generation polyamidoamine dendrimer, functionalized with cholesterol was self-assembled into NPs encapsulating AZ3451. NP encapsulated AZ3451 (PAMAM-Chol-AZ NPs) more effectively reversed activation of PAR₂ at the plasma membrane and early endosomes than free drug. The PAMAM-Chol-AZ NPs showed greater efficacy in reversing nociception than free drug, with respect to both level and duration, in three preclinical mouse models of oral cancer pain. The antinociceptive efficacy was confirmed with an operant orofacial assay. Genetic deletion of F2RL1 on cancer cells or F2rl1 on neurons each partially reversed mechanical cancer allodynia. The remaining nociception could be effectively reversed by PAMAM-Chol-AZ NPs. These findings suggest that PAR₂ on oral cancer cells and neurons contribute to oral cancer nociception and NPs loaded with a PAR₂ antagonist provide increased antinociception and improved oral function compared to free drug.

Head and neck squamous cell carcinoma (HNSCC) causes more severe pain and psychological stress than other types of cancer. Despite clinical evidence linking pain, stress, and cancer progression, the underlying relationship between pain and sympathetic neurotransmission in oral cancer is unknown. We found that human HNSCC tumors and mouse tumor tissue are innervated by peripheral sympathetic and sensory nerves. Moreover, [beta]-adrenergic 1 and 2 receptors ([beta]-AR) are overexpressed in human oral cancer cell lines, and norepinephrine treatment increased [beta]-AR2 protein expression as well as cancer cell proliferation in vitro. We have recently demonstrated that inhibition of tumor necrosis factor alpha (TNF[alpha]) signaling reduces oral cancer-induced nociceptive behavior. Norepinephrine-treated cancer cell lines secrete more TNF[alpha] which, when applied to tongue-innervating trigeminal neurons, evoked a larger Ca2+ transient; TNF-TNFR inhibitor blocked the increase in the evoked Ca2+ transient. Using an orthotopic xenograft oral cancer model, we found that mice demonstrated significantly less orofacial cancer-induced nociceptive behavior during systemic [beta]-adrenergic inhibitory treatment with propranolol. Furthermore, chemical sympathectomy via guanethidine led to a significant reduction in tumor size and nociceptive behavior. We infer from these results that sympathetic signaling modulates oral cancer pain via TNF[alpha] secretion and tumorigenesis. Further investigation of the role of neuro-cancer communication in cancer progression and pain is warranted.

Oral cancer patients report sensitivity to spicy foods and liquids. The mechanism responsible for chemosensitivity induced by oral cancer is not known. We simulate oral cancer-induced chemosensitivity in a xenograft oral cancer mouse model using two-bottle choice drinking and conditioned place aversion assays. An anatomic basis of chemosensitivity is shown in increased expression of TRPV1 in anatomically relevant trigeminal ganglion (TG) neurons in both the xenograft and a carcinogen (4-nitroquinoline 1-oxide)-induced oral cancer mouse models. The percent of retrograde labeled TG neurons that respond to TRPV1 agonist, capsaicin, is increased along with the magnitude of response as measured by calcium influx, in neurons from the cancer models. To address the possible mechanism of TRPV1 sensitivity in tongue afferents, we study the role of PAR₂, which can sensitize the TRPV1 channel. We show co-expression of TRPV1 and PAR₂ on tongue afferents and using a conditioned place aversion assay, demonstrate that PAR₂ mediates oral cancer-induced, TRPV1-evoked sensitivity in an oral cancer mouse model. The findings provide insight into oral cancer-mediated chemosensitivity.

Head and neck squamous cell carcinoma (HNSCC) patients report severe function-induced pain at the site of the primary tumor. The current hypothesis is that oral cancer pain is initiated and maintained in the cancer microenvironment due to secretion of algogenic mediators from tumor cells and surrounding immune cells that sensitize the primary sensory neurons innervating the tumor. Immunogenicity, which is the ability to induce an adaptive immune response, has been widely studied using cancer cell transplantation experiments. However, oral cancer pain studies have primarily used xenograft transplant models in which human-derived tumor cells are inoculated in an athymic mouse lacking an adaptive immune response; the role of inflammation in oral cancer-induced nociception is still unknown. Using syngeneic oral cancer mouse models, we investigated the impact of tumor cell immunogenicity and growth on orofacial nociceptive behavior and oral cancer-induced sensory neuron plasticity. We found that an aggressive, weakly immunogenic mouse oral cancer cell line, MOC2, induced rapid orofacial nociceptive behavior in both male and female C57Bl/6 mice. Additionally, MOC2 tumor growth invoked a substantial injury response in the trigeminal ganglia as defined by a significant upregulation of injury response marker ATF3 in tongue-innervating trigeminal neurons. In contrast, using a highly immunogenic mouse oral cancer cell line, MOC1, we found a much slower onset of orofacial nociceptive behavior in female C57Bl/6 mice only as well as sex-specific differences in the tumor-associated immune landscape and gene regulation in tongue innervating sensory neurons. Together, these data suggest that cancer-induced nociceptive behavior and sensory neuron plasticity can greatly depend on the immunogenic phenotype of the cancer cell line and the associated immune response.

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 Na_v1.8-positive neurons (Par₂Na_v1.8), 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.

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 to 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 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 protein kinase A/D 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 STATEMENTOral 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 to 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 prevented the effects of Lgmn. Lgmn activated PAR₂ to induce calcium mobilization, cAMP formation and activation of protein kinase D and A, but not β-arrestin recruitment or PAR₂ endocytosis. Thus, Lgmn is a biased agonist of PAR₂ that evokes cancer pain.

Cancer invading into nerves, termed perineural invasion (PNI), is associated with pain. Here we show that oral cancer patients with PNI report greater spontaneous pain and mechanical allodynia compared with patients without PNI, suggesting unique mechanisms drive PNI-induced pain. We studied the impact of PNI on peripheral nerve physiology and anatomy using a murine sciatic nerve PNI model. Mice with PNI exhibited spontaneous nociception and mechanical allodynia. PNI induced afterdischarge in A high threshold mechanoreceptors (AHTMRs), mechanical sensitization (i.e., decreased mechanical thresholds) in both A and C HTMRs, and mechanical desensitization in low threshold mechanoreceptors (LTMRs). PNI resulted in nerve damage, including axon loss, myelin damage, and axon degeneration. Electrophysiological evidence of nerve injury included decreased conduction velocity, and increased percentage of both mechanically-insensitive and electrically-unexcitable neurons. We conclude that PNI-induced pain is driven by nerve injury and peripheral sensitization in HTMRs.

Oral cancer patients often have severe, chronic, and mechanically induced pain at the site of the primary cancer. Oral cancer pain is initiated and maintained in the cancer microenvironment and attributed to release of mediators that sensitize primary sensory nerves. This study was designed to investigate the histopathology associated with painful oral cancers in a preclinical model. The relationship of pain scores with pathologic variables was also investigated in a cohort of 72 oral cancer patients. Wild-type mice were exposed to the carcinogen, 4-nitroquinoline 1-oxide (4NQO). Nociceptive (pain) behavior was measured with the dolognawmeter, an operant device and assay for measuring functional and mechanical allodynia. Lesions developed on the tongues and esophagi of the 4NQO-treated animals and included hyperkeratoses, papillomas, dysplasias, and cancers. Papillomas included lesions with benign and dysplastic pathological features. Two histologic subtypes of squamous cell carcinomas (SCCs) were identified-SCCs with exophytic and invasive components associated with papillary lesions (pSCCs) and invasive SCCs without exophytic histology (iSCCs). Only the pSCC subtype of tongue cancer was associated with nociceptive behavior. Increased tumor size was associated with greater nociceptive behavior in the mouse model and more pain experienced by oral cancer patients. In addition, depth of invasion was associated with patient-reported pain. The pSCC histology identifies 4NQO-induced tongue cancers that are expected to be enriched for expression and release of nociceptive mediators.

Migraine is commonly reported among patients with temporomandibular disorders (TMDs), especially myogenic TMD. The pathophysiologic mechanisms related to the comorbidity of the two conditions remain elusive. In the present study, we combined masseter muscle tendon ligation (MMTL)-produced myogenic TMD with systemic injection of nitroglycerin (NTG)-induced migraine-like hypersensitivity in mice. Facial mechanical allodynia, functional allodynia, and light-aversive behavior were evaluated. Sumatriptan, an FDA-approved medication for migraine, was used to validate migraine-like hypersensitivity. Additionally, we examined the protein level of calcitonin gene-related peptide (CGRP) in the spinal trigeminal nucleus caudalis using immunohistochemistry. We observed that mice with MMTL pretreatment have a prolonged NTG-induced migraine-like hypersensitivity, and MMTL also enabled a non-sensitizing dose of NTG to trigger migraine-like hypersensitivity. Systemic injection of sumatriptan inhibited the MMTL-enhanced migraine-like hypersensitivity. MMTL pretreatment significantly upregulated the protein level of CGRP in the spinal trigeminal nucleus caudalis after NTG injection. Our results indicate that a pre-existing myogenic TMD can upregulate NTG-induced trigeminal CGRP and enhance migraine-like hypersensitivity.