Background & Aims

Oral cancer pain is one of the most intense among all cancers. Proteases released by the cancer [1] activate the protease-activated receptor 2 (PAR2, encoded by F2RL1) on trigeminal neurons. Canonical activation by trypsin promotes PAR2 endocytosis to elicit pain signaling from endosomes in colitis and irritable bowel syndrome [2,3]. Trypsin is not highly expressed in oral cancers. Proteases previously reported to mediate oral cancer pain via PAR2 do not promote PAR2 endocytosis [4,5], raising the question whether oral cancer pain involves PAR2 endosomal signaling. We studied PAR2 activation induced by three proteases overexpressed in human oral cancers and conditioned media (CM) from oral cancer cells, which evokes nociceptive behavior in preclinical models to identify (1) intracellular pools of PAR2 that drive oral cancer pain and (2) the associated signaling events. Understanding PAR2 spatiotemporal signaling could guide the development of pain relief treatments.

Methods

The elicited PAR2 spatiotemporal activation was studied by examining receptor trafficking, recruitment of signaling partners (G alpha proteins, G? and beta arrestins, ?arr), and the increase of second messengers (cAMP, Ca2+ and ERK). PAR2 was challenged with individual proteases (matrix metalloprotease (MMP1), serine protease 23 (PRSS23), matriptase (ST14) and cathepsin S (CTSS) or CM from oral cancer cells. Trafficking was examined by Bioluminescence Resonance Energy Transfer (BRET) assays using HEK293 cells transfected with human PAR2 and resident proteins from the plasma membrane (Kras), endosomes (Rab5 and Rab7), and the Golgi (Giantin and TGN38). Recruitment of G? and ?arr was studied using G?q-, Gs-, G?i- and G?12/13-Venus and ?arr1- and ?arr2-YFP. Second messengers were measured using the cAMP BRET sensor (CAMYEL), the ERK FRET sensor (EKAR) and the Ca2+ sensitive dye FURA-2 AM. Nociception was assessed in vivo by von Frey filaments after protease or CM injection.

Results

MMP1 and ST14 trigger PAR2 internalization. CTSS and PRSS23 do not. Upon ST14 addition, PAR2 redistributes to early endosomes (Rab5) and trans-Golgi (TGN38), whereas MMP1 promotes PAR2 redistribution to cis-Golgi (Giantin). CM from oral cancer cells, containing a mixture of proteases, induces PAR2 internalization to early endosomes and the cis- and trans-Golgi. PAR2 coupling of G? proteins is observed by ST14 (G?s and G?q) and CTSS (G?s), but not by MMP1 and PRSS23. ?arr recruitment by PAR2 is promoted by ST14 (?arr1 and ?arr2) and is absent after activation with CTSS, MMP1 and PRSS23. Proteases elicit differential signaling of PAR2. ST14 promotes Ca2+ influx and ERK. MMP1 induces ERK and cAMP. PRSS23 increases cAMP. MMP1 and PRSS23 signaling appears to be mediated by G?- and ?arr-independent mechanisms. Intraplantar administration of MMP1 and CM from oral cancer cells elicited nociceptive responses assessed with von Frey filaments.

Conclusions

Oral cancer proteases induce PAR2 trafficking to multiple intracellular locations from which signaling was observed. CTSS signals from the cell surface [4,6]. MMP1 redistributes PAR2 to the cis-Golgi and increases cAMP and ERK. ST14, which cleaves PAR2 at the same site as trypsin, induces PAR2 canonical trafficking and signaling. We could not find PAR2 on endosomes or the Golgi after PRSS23 stimulation, but we observed a rise in cAMP. Further studies exploring other intracellular compartments involved in PAR2 signaling are needed. The nucleus is a candidate, reported as a nociceptive signaling platform for the metabotropic glutamate receptor 5 [7]. CM from oral cancer cells triggers PAR2 trafficking to endosomes and the Golgi, consistent with our observations of PAR2 distribution elicited by MMP1 and ST14. Injection of MMP1 or CM elicits nociceptive behavior. Whether nociception is mediated by intracellular pools of PAR2 is yet to be determined.

References

1. Lam DK, Schmidt BL. Serine proteases and protease-activated receptor 2-dependent allodynia: a novel cancer pain pathway. Pain. 2010;149(2):263–72.

2. Jimenez-Vargas NN, Pattison LA, Zhao P, Lieu T, Latorre R, Jensen DD, Castro J, Aurelio L, Le GT, Flynn B, Herenbrink CK, Yeatman HR, Edgington-Mitchell L, Porter CJH, Halls ML, Canals M, Veldhuis NA, Poole DP, McLean P, Hicks GA, Scheff N, Chen E, Bhattacharya A, Schmidt BL, Brierley SM, Vanner SJ, Bunnett NW. Protease-activated receptor-2 in endosomes signals persistent pain of irritable bowel syndrome. Proc National Acad Sci. 2018;115(31):E7438–47.

3. Latorre R, Hegron A, Peach CJ, Teng S, Tonello R, Retamal JS, Klein-Cloud R, Bok D, Jensen DD, Gottesman-Katz L, Rientjes J, Veldhuis NA, Poole DP, Schmidt BL, Pothoulakis CH, Rankin C, Xie Y, Koon HW, Bunnett NW. Mice expressing fluorescent PAR2 reveal that endocytosis mediates colonic inflammation and pain. Proc National Acad Sci. 2022;119(6).

4. Tu NH, Inoue K, Chen E, Anderson BM, Sawicki CM, Scheff NN, Tran HD, Kim DH, Alemu RG, Yang L, Dolan JC, Liu CZ, Janal MN, Latorre R, Jensen DD, Bunnett NW, Edgington-Mitchell LE, Schmidt BL. Cathepsin S Evokes PAR2-Dependent Pain in Oral Squamous Cell Carcinoma Patients and Preclinical Mouse Models. Cancers. 2021;13(18):4697.

5. Tu NH, Jensen DD, Anderson BM, Chen E, Jimenez-Vargas NN, Scheff NN, Inoue K, Tran HD, Dolan JC, Meek TA, Hollenberg MD, Liu CZ, Vanner SJ, Janal MN, Bunnett NW, Edgington-Mitchell LE, Schmidt BL. Legumain Induces Oral Cancer Pain by Biased Agonism of Protease-Activated Receptor-2. J Neurosci. 2021;41(1):193–210.

6. Zhao P, Lieu T, Barlow N, Metcalf M, Veldhuis NA, Jensen DD, Kocan M, Sostegni S, Haerteis S, Baraznenok V, Henderson I, Lindström E, Guerrero-Alba R, Valdez-Morales EE, Liedtke W, McIntyre P, Vanner SJ, Korbmacher C, Bunnett NW. Cathepsin S causes inflammatory pain via biased agonism of PAR2 and TRPV4. J Biol Chem. 2014;289(39):27215–34.

7. Vincent K, Cornea VM, Jong YI, Laferriere A, Kumar N, Mickeviciute A, Fung JST, Bandegi P, Ribeiro-da-Silva A, O’Malley KL, Coderre TJ. Intracellular mGluR5 plays a critical role in neuropathic pain. Nat Commun. 2016;7(1):10604.

Presenting Author

Paulina Ramirez Garcia

Poster Authors

Paulina Ramirez Garcia

BSc

New York University

Lead Author

E-mail

Zinaida Dubeykovskaya

New York University

Lead Author

E-mail

Leticia Arbex

New York University

Lead Author

E-mail

Rocco Latorre

NYU-Pain Research Center

Lead Author

E-mail

Tu Huu Nguyen

NYU

Lead Author

E-mail

Brian Schmidt

NYU Dentistry, Translational Research Center

Lead Author

E-mail

Topics

  • Mechanisms: Biological-Molecular and Cell Biology