Background & Aims

Oral cancer patients suffer severe pain while eating and swallowing. The underlying mechanisms of oral cancer pain are unclear. Clinical data demonstrate a correlation between oral cancer pain severity and cathepsin S (CTSS) activity in the cancer (1). CTSS is a lysosomal cysteine protease expressed in oral cancer and immune cells that activates PAR2 (encoded by F2RL1) on sensory neurons to signal pain (1). Preclinical data suggest that CTSS expression is regulated by TRPV4 , a mechanosensitive ion channel that responds to shear stress, in central nervous system microglia (2, 3). The TRPV4 contribution of Schwann cells (SCs), the peripheral glia that ensheathe axons of sensory neurons, to oral cancer pain has not been studied. The aims of this research were to determine if: (i) oral cancer patients who report pain express functional TRPV4 on SCs in nerve fibers that innervate the cancer and (ii) TRPV4 activation on SCs regulates CTSS expression and extracellular activity.

Methods

Five new biopsy proven tongue cancer patients were enrolled into the study through the NYU Oral Cancer Center and consented for completion of the University of California San Francisco Oral Cancer Pain Questionnaire (UCSFOCPQ), mechanosensitivity testing and collection of their tissues. Lingual nerve fibers surgically removed during cancer resection were procured from patients who reported pain on the UCSFOCPQ and perceived von Frey testing as painful. Nerve fibers were used to isolate patient-specific SCs. The functional expression of TRPV4 on SCs was studied with whole-cell patch clamp and the selective TRPV4 agonist (GSK101) and TRPV4 antagonist (HC067). Human SC cultures were treated with vehicle, TRPV4 agonist alone or with the antagonist for 2 hours and supernatants (+/- extracellular Ca+2) were collected to measure CTSS activity levels using a fluorogenic probe selectively cleaved by CTSS. SC cultures were lysed after drug treatments to measure CTSS protein levels by ELISA.

Results

The mean score on the UCSFOCPQ for functional pain was 32.6 ± 13.6 (scale 0-100). Four out of five patients reported von Frey testing as painful. In patch clamp experiments, the TRPV4 agonist (GSK101, 0.1 ?M) increased the amplitude of SC currents which was inhibited by the TRPV4 antagonist, HC067 (1 ?M) (p<0.05, agonist vs. vehicle and antagonist; 1-way ANOVA, n=3 cells). In CTSS activity assays, the pre-treatment of SC cultures with GSK101 (0.1 ?M) vs. vehicle induced ~ 3-fold increase in the CTSS activity in supernatants and the effect was inhibited by HC067, 1 ?M. Removal of extracellular Ca+2 from the SC medium partially reduced the CTSS activity (p<0.05 vs control, 1-way ANOVA). Pre-treatment with a CTSS inhibitor blocked CTSS activity in supernatants. By contrast, GSK101 (0.1 ?M) pre-treatment had no significant effect on CTSS protein expression levels as measured by ELISA in SC lysates and supernatants.

Conclusions

TRPV4 is functionally expressed on SCs in nerve fibers that innervate the oral cancer in patients who report pain. TRPV4 activation on SCs increased the extracellular activity of CTSS. Our findings suggest a potential mechanism for oral cancer pain that depends on TRPV4 activation on SCs and CTSS extracellular activity.

References

1.N. H. Tu, K. Inoue, E. Chen, B. M. Anderson, C. M. Sawicki, N. N. Scheff, H. D. Tran, D. H. Kim, R. G. Alemu, L. Yang, J. C. Dolan, C. Z. Liu, M. N. Janal, R. Latorre, D. D. Jensen, N. W. Bunnett, L. E. Edgington-Mitchell, B. L. Schmidt, Cathepsin S Evokes PAR2-Dependent Pain in Oral Squamous Cell Carcinoma Patients and Preclinical Mouse Models. Cancers (Basel) 13, (2021).
2.X. Hu, L. Du, S. Liu, Z. Lan, K. Zang, J. Feng, Y. Zhao, X. Yang, Z. Xie, P. L. Wang, A. M. Ver Heul, L. Chen, V. K. Samineni, Y. Q. Wang, K. J. Lavine, R. W. t. Gereau, G. F. Wu, H. Hu, A TRPV4-dependent neuroimmune axis in the spinal cord promotes neuropathic pain. J Clin Invest 133, (2023).
3.B. D. Matthews, C. K. Thodeti, J. D. Tytell, A. Mammoto, D. R. Overby, D. E. Ingber, Ultra-rapid activation of TRPV4 ion channels by mechanical forces applied to cell surface beta1 integrins. Integr Biol (Camb) 2, 435-442 (2010).

Presenting Author

Yatendra Mulpuri

Poster Authors

Yatendra Mulpuri

PhD

New York University

Lead Author

E-mail

Samuel Nicholson

BS

New York University

Lead Author

E-mail

Kenji Inoue

New York University College of Dentistry

Lead Author

E-mail

Grace Harden

BS

Translational Research Center, NYU College of Dentistry

Lead Author

E-mail

Brooke Benton

RDH

Translational Research Center, NYU College of Dentistry

Lead Author

E-mail

Donna G. Albertson

PhD

Translational Research Center, NYU College of Dentistry

Lead Author

E-mail

Brian Schmidt

NYU Dentistry, Translational Research Center

Lead Author

E-mail

Topics

  • Specific Pain Conditions/Pain in Specific Populations: Cancer Pain & Palliative Care