The role of schwann cell TRPA1 in cancer pain and perineural nerve invasion

Acute pain is a frequent and evolutionary conserved survival mechanism. Chronic pain, which can last for years, is linked to inflammatory disorders, peripheral and central neuropathies and cancer and it is a debilitating painful syndrome that affects 25% of European people. Pain, a common symptom of cancer that can reduce quality of life more than the disease itself, affects around 70% of cancer patients. It becomes more frequent and debilitating in the presence of bone metastases, which is a common consequence of many primary and metastatic tumors. Overall, the burden of cancer-related pain is enormous in consideration of suffering, disability, health care, and social and economic costs. The current medications for treating different kind of pain are frequently ineffective. Identifying the mechanisms in the peripheral and central neural systems that maintain chronic pain (allodynia/hyperalgesia) is a major challenge in pain research. Transient receptor potential ankyrin 1 (TRPA1), a proalgesic ion channel play a role in cellular transduction of mechanical, chemical and thermal stimuli. TRPA1 is highly expressed in nociceptive sensory fibers, where it transduces exogenous and endogenous stimuli into a painful signal, and it is uniquely sensitive to oxidative stress byproducts. Recently TRPA1 was found to be expressed in many non-neuronal cells, included Schwann cells (SCs). SCs are the major peripheral glial cells and recently a crucial contribution of SC TRPA1, in sustaining mechanical hypersensitivity, has been highlighted in different mouse models of pain, including peripheral nerve injury, ischemia/reperfusion, alcoholic neuropathy, and migraine. The primary goal of the three-year PhD project was to identify the cellular and molecular mechanisms that, from cancer growth and treatment result in a chronic pain condition. We firstly studied the mechanism underlying the pain symptoms associated with chemotherapeutic-induced peripheral neuropathy (CIPN) induced by thalidomide in its derivatives. Thalidomide causes a painful CIPN in patients via an unknown mechanism. TRPA1, TRP vanilloid 4 (TRPV4), vanilloid 1 (TRPV1) and oxidative stress have been implicated in several rodent models of CIPN-evoked allodynia. In the first part of the study, we revealed that a single systemic administration of thalidomide and its derivatives, lenalidomide and pomalidomide, elicits prolonged (35 days) mechanical and cold hypersensitivity in C57BL/6J mouse hind paw. Pharmacological antagonism or genetic deletion studies indicated that both TRPA1 and TRPV4, but not TRPV1, contribute to mechanical allodynia, whereas cold hypersensitivity was entirely due to TRPA1. Thalidomide per se did not stimulate recombinant and constitutive TRPA1 and TRPV4 channels, in vitro, which, however, were activated by the oxidative stress byproduct, hydrogen peroxide. Systemic treatment with an antioxidant attenuated mechanical and cold hypersensitivity, and the increase in oxidative stress in hind paw, sciatic nerve and lumbar spinal cord produced by thalidomide. Notably, central (intrathecal) or peripheral (intraplantar) treatments with channel antagonists or an antioxidant revealed that oxidative stress-dependent activation of peripheral TRPA1 mediates cold allodynia and part of mechanical allodynia. However, oxidative stress-induced activation of central TRPV4 mediated the residual TRPA1-resistant component of mechanical allodynia. Thus, targeting of peripheral TRPA1 and the central TRPV4 may be required to attenuate pain associated with CIPN elicited by thalidomide and related drugs. In the second part of the PhD project, we aimed at deciphering the contribution of macrophages (MΦ) in different model cancer pain due to primary and a metastatic cancer growth. Here we report that depletion of sciatic nerve resident MΦs (rMΦ) in mice attenuates mechanical/cold hypersensitivity and spontaneous pain evoked by intraplantar injection of melanoma or lung carcinoma cells. MΦ-colony stimulating factor (M-CSF) was upregulated in the sciatic nerve trunk and mediated cancer-evoked pain via rMΦ expansion, TRPA1 activation, and oxidative stress. Targeted deletion of Trpa1 revealed a key role for SC TRPA1 in sciatic nerve rMΦ expansion and pain-like behaviors. Collectively, we identified a feed-forward pathway involving M-CSF, rMΦ, oxidative stress and Schwann cell TRPA1 that operates throughout the nerve trunk to signal cancer-evoked pain. In line with this, we further investigated, in the last part of this 3-years project, the role of SC TRPA1 and oxidative stress in a mouse model of metastatic bone cancer pain (MBCP). Insulin growth factor-1 (IGF-1), an osteoclast-dependent osteolysis biomarker, contributes to MBCP, but the underlying mechanism is poorly understood. In mice, the femur metastasis caused by intramammary inoculation of breast cancer cells resulted in IGF-1 increase in femur and sciatic nerve. We hypothesized that osteoclast-dependent osteolysis mediators target SCs to elicit MBCP. To address this hypothesis, we used adeno-associated virus (AAV)-mediated cell-specific IGF-1R silencing in SCs or DRG neurons. AAV-based shRNA selective silencing of IGF-1 receptor (IGF-1R) in SCs, but not in DRG neurons, attenuated pain-like behaviors. Intraplantar IGF-1 evoked acute nociception and mechanical/cold allodynia, which were reduced by selective IGF-1R silencing in DRG neurons and Schwann cells, respectively. SCs IGF-1R intracellular signaling promoted an endothelial nitric oxide synthase-mediated TRPA1 activation and release of reactive oxygen species that, via M-CSF-dependent endoneurial macrophage expansion, sustained pain-like behaviors. Osteoclast derived IGF-1 initiates a Schwann cell-dependent neuroinflammatory response that sustains a proalgesic pathway that provides new options for MBCP treatment. Overall, we revealed that the targeting these cellular pathway may be a promising therapeutic method for the treatment of pain symptoms associated with cancer. TRPA1 and TRPV4 antagonists, as well as TRPA1/TRPV4-desensitizing medicines, may significantly improve pain therapy.