Oral cancer induced TRPV1 sensitization is mediated by PAR2 signaling in primary afferent neurons innervating the cancer microenvironment.

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.

TNFα promotes oral cancer growth, pain, and Schwann cell activation.

Oral cancer is very painful and impairs a patient's ability to eat, talk, and drink. Mediators secreted from oral cancer can excite and sensitize sensory neurons inducing pain. Cancer mediators can also activate Schwann cells, the peripheral glia that regulates neuronal function and repair. The contribution of Schwann cells to oral cancer pain is unclear. We hypothesize that the oral cancer mediator TNFα activates Schwann cells, which further promotes cancer progression and pain. We demonstrate that TNFα is overexpressed in human oral cancer tissues and correlates with increased self-reported pain in patients. Antagonizing TNFα reduces oral cancer proliferation, cytokine production, and nociception in mice with oral cancer. Oral cancer or TNFα alone increases Schwann cell activation (measured by Schwann cell proliferation, migration, and activation markers), which can be inhibited by neutralizing TNFα. Cancer- or TNFα-activated Schwann cells release pro-nociceptive mediators such as TNFα and nerve growth factor (NGF). Activated Schwann cells induce nociceptive behaviors in mice, which is alleviated by blocking TNFα. Our study suggests that TNFα promotes cancer proliferation, progression, and nociception at least partially by activating Schwann cells. Inhibiting TNFα or Schwann cell activation might serve as therapeutic approaches for the treatment of oral cancer and associated pain.

Na+ /K+ -ATPase coupled to endothelin receptor type B stimulates peripheral nerve regeneration via lactate signalling.

We have recently demonstrated that endothelin (ET) is functionally coupled to Nax , a Na+ concentration-sensitive Na+ channel for lactate release via ET receptor type B (ETB R) and is involved in peripheral nerve regeneration in a sciatic nerve transection-regeneration mouse model. Nax is known to interact directly with Na+ /K+ -ATPase, leading to lactate production in the brain. To investigate the role of Na+ /K+ -ATPase in peripheral nerve regeneration, in this study, we applied ouabain, a Na+ /K+ -ATPase inhibitor, to the cut site for 4 weeks with an osmotic pump. While functional recovery and nerve reinnervation to the toe started at 5 weeks after axotomy and were completed by 7 weeks, ouabain delayed them by 2 weeks. The delay by ouabain was improved by lactate, and its effect was blocked by α-cyano-4-hydroxy-cinnamic acid (CIN), a broad monocarboxylate transporter (MCT) inhibitor. In primary cultures of dorsal root ganglia, neurite outgrowth of neurons and lactate release into the culture medium was inhibited by ouabain. Conversely, lactate enhanced the neurite outgrowth, which was blocked by CIN, but not by AR-C155858, a MCT1/2-selective inhibitor. ET-1 and ET-3 increased neurite outgrowth of neurons, which was attenuated by an ETB R antagonist, ouabain and 2 protein kinase C inhibitors. Taken together with the finding that ETB R was expressed in Schwann cells, these results demonstrate that ET enhanced neurite outgrowth of neurons mediated by Na+ /K+ -ATPase via ETB R in Schwann cells. This study suggests that Na+ /K+ -ATPase coupled to the ET-ETB R system plays a critical role in peripheral nerve regeneration via lactate signalling.

Role of c-Jun N-terminal kinase in late nerve regeneration monitored by in vivo imaging of thy1-yellow fluorescent protein transgenic mice.

The restoration of function to injured peripheral nerves separated by a gap requires regeneration across it and reinnervation to target organs. To elucidate these processes, we have established an in vivo monitoring system of nerve regeneration in thy1-yellow fluorescent protein transgenic mice expressing a fluorescent protein in their nervous system. Here we demonstrated that motor and sensory nerves were regenerated in a coordinated fashion across the gap and that the functional recovery of the response to mechanical stimuli correlated well with sensory innervation to the foot. Among the mitogen-activated protein kinase inhibitors examined, only the c-Jun N-terminal kinase (JNK) inhibitors delayed functional recovery. Although it did not affect the reinnervation of the muscle, the JNK inhibitor delayed sensory nerve innervation to the skin for over 8 weeks and increased the expression of activatng transcription factor 3 (ATF3), a neuronal injury marker, in the dorsal root ganglion over the same time period. Antibodies against nerve growth factor, glia-derived neurotrophic factor, and brain-derived neurotrophic factor applied to the transection site delayed the functional recovery in this order of potency. These neurotrophic factors enhanced neurite outgrowth from cultured dorsal root ganglion neurons, and the JNK inhibitor reversed their stimulatory effects. These results suggest that JNK played roles in nerve regeneration at both early and late phases. Taken together, the present study demonstrated that neurotrophic factors released from the distal nerve may accelerate motor and sensory nerve regeneration across the gap in a coordinated fashion and reinnervation of the target organs independently. The model characterized here has the advantage of in vivo monitoring of the evaluation of morphological and functional recovery in the same mice for a long period of time.

Involvement of Nax sodium channel in peripheral nerve regeneration via lactate signaling.

Na(x), a sodium concentration-sensitive sodium channel, is expressed in non-myelinating Schwann cells of the adult peripheral nervous system, but the pathophysiological role remains unclear. We found that functional recovery of the hind paw responses from the sciatic nerve transection was delayed in Na(x) knockout (Na(x)⁻/⁻) mice. Histological analyses showed a decrease in the number of regenerated myelinated axons in (Na(x)⁻/⁻) sciatic nerves. The delay in the recovery in Na(x)⁻/⁻ mice was improved by lactate and inhibited by a monocarboxylate transporter inhibitor. In vitro experiments using cultured Schwann cells showed that lactate release was enhanced by endothelin (ET)-1 and blocked by an ET receptor type B antagonist. Here, it is conceivable that Na(x) was activated by ET-1. The amount of lactate release by ET-1 was lower in Na(x)⁻/⁻ mice than in wild-type mice. These results indicated that Na(x) is functionally coupled to ET for lactate release via ET receptor type B and is involved in peripheral nerve regeneration.