Endothelin-1: A Dual Role in Oral Cancer Pain

The role of ET-1 in oral cancer pain is complex. The nociceptive effects of ET-1 depend on the location of the two endothelin receptor subtypes and the action of those receptor subtypes upon ligand binding. ET-1 is a potent vasoactive peptide that produces nociceptive behavior upon injection in animals and humans (Hans et al., 2007). ET-1 also drives cancer pain (Davar, 2001). ET-1 binds to two G-protein-coupled receptors, the endothelin-A receptor (ETAR) and the endothelin-B receptor (ETBR). ETARs are distributed on peripheral sensory neurons; ETBRs are expressed on non-myelinating Schwann cells of the sciatic nerve and dorsal root ganglion satellite cells (Peters et al., 2004) as well as on keratinocytes, which are known to secrete opioids upon binding and activation.

Patients with oral SCC have high levels of ET-1 in the cancer microenvironment (Pickering et al., 2007; Schmidt et al., 2007) and report severe functional pain upon mechanical stimulation. To parallel the mechanical allodynia that is observed in human oral SCC patients, Pickering and colleagues established a mouse model of cancer pain by inoculating a human oral tongue SCC into the mouse hind paw (Pickering et al., 2008). The role of ET-1 in oral cancer pain has been confirmed and characterized in this mouse model, and the ET-1 concentration has been shown to be a more important factor than tumor volume in establishing cancer pain.

Proteases and Protease-activated Receptors

Proteolytic activity is critical to carcinogenesis, and the cancer microenvironment is replete with both proteases and proteolytic peptide products. Cancer-associated trypsin has been identified in cancers such as ovarian carcinoma, pancreatic cancer, hepatocellular and cholangiocarcinomas, lung neoplasms, colorectal cancers, fibrosarcoma, leukemia, gastric cancer, and oral cancer (Nyberg et al., 2006). Proteases activate cell-surface receptors on primary afferent nociceptors within the cancer microenvironment, either directly or via their peptide products.

Protease-activated receptors (PARs) belong to a family of G-protein-coupled receptors (PAR1 to PAR4) that are activated by proteolytic cleavage. Such cleavage can result from a number of different enzymes, including serine proteases, trypsin, and tryptase. Cleavage exposes a tethered ligand that binds the receptor and initiates signal transduction (Russo et al., 2009). PAR2 activates multiple second-messenger pathways, which sensitize TRPV1 and TRPV4 receptors on nociceptive afferents and result in TRPV1-dependent thermal hyperalgesia and TRPV4-dependent mechanical allodynia, respectively (Amadesi et al., 2006).

PAR2 has recently been implicated in cancer pain by pharmacologic, behavioral, biochemical, and genetic approaches (Lam and Schmidt, 2010). Proteases capable of activating PAR2 on sensory neurons are recovered in the supernatants of human oral SCC cells. Injection of supernatants alone without cancer cells causes marked and prolonged mechanical allodynia in mice. This nociceptive effect is abolished by serine protease inhibition, diminished by mast cell depletion, and absent in PAR2 knockout mice. Serine proteases, such as trypsin from cancer cells and tryptase from mast cells, both of which can activate PAR2, contribute to cancer pain. Fibroblasts in the surrounding stroma of oral carcinoma also produce trypsin. Chronic exposure to serine proteases secreted by human cancer up-regulates PAR2 levels in peripheral neurons (Lam and Schmidt, 2010). The continual release of serine proteases from cancer and non-malignant cells in the microenvironment could produce ongoing excitation of primary nociceptive afferents, leading to mechanical allodynia in oral cancer patients.

Nerve Growth Factor

In the microenvironment of many cancers, sensory neurons are chronically exposed to nerve growth factor (NGF), which is normally secreted to promote the local growth and survival of afferent sensory neurons. Acute peripheral administration of NGF leads to thermal hyperalgesia, whereas chronic administration produces mechanical allodynia. Similarly, a transgenic mouse engineered to overexpress NGF exhibits mechanical hypersensitivity (Davis et al., 1993). Signals from NGF are mediated via a high-affinity receptor tyrosine kinase (TrkA) and a low-affinity p75 receptor on the neuronal membrane (Friedman, 1999). NGF and its high-affinity TrkA receptor can also facilitate proliferation and invasion of multiple cancers, including breast, prostate, pancreatic, and oral cancers (Zhu et al., 1999). Expression of NGF by cancer and regulation of both high- and low-affinity receptors have also been extensively investigated (Kolokythas et al., 2010). NGF secretion by cancer cells into the microenvironment likely leads to a number of changes that contribute to pain. One possible mechanism of NGF-induced cancer pain is its association with perineural involvement, a pathologic term for the invasion and proliferation of a cancer within a nerve, associated with pain and recurrence following surgical resection. NGF is associated with perineural invasion in adenoid cystic carcinoma, a salivary gland malignancy known for its neurotropism, as well as in pancreatic and oral cancer (Ma et al., 2008). Both NGF mRNA and protein levels are also significantly higher in cancer tissues from oral cancer patients and in oral SCC culture (Ye et al., 2011). Anti-NGF with a monoclonal antibody reduces cancer pain and restores function in a bone sarcoma rat model (Sevcik et al., 2005). Furthermore, NGF blockade in two separate mouse oral cancer models decreases tumor proliferation, nociception, and weight loss through modulation of pro-inflammatory cytokines and leptin production. NGF blockade also decreased expression of TRPV1, TRPA1, and PAR-2 nociceptive receptors (Ye et al., 2011). These results unveil anti-NGF as a novel therapeutic for two of the most obstinate oral cancer symptoms, especially in its later stages, namely, pain and cachexia.

Opioid Receptors and Peptides

In 1973, opioid receptors were identified by three separate groups (Pert and Snyder, 1973; Simon et al., 1973; Terenius, 1973). Since then, the endogenous opioid system has become well-characterized. Molecular characterization and cloning of the mu-, delta-, and kappa-opioid receptors occurred in the 1990s (Chen et al., 1993). Opioid receptors are G-protein-coupled receptors belonging to the subfamily of rhodopsin. They are comprised of 7 alpha-helical transmembrane domains and an extracellular N-terminus with multiple glycosylation sites (Law et al., 2000). Mu-, delta-, and kappa-opioid receptors are highly homologous to each other but are diverse at the N- and C-termini and extracellular loops (Pil and Tytgat, 2003). Activation of opioid receptors causes inhibition of cAMP production and voltage-gated calcium-channels, and stimulation of inwardly rectifying potassium channels and the MAP kinase pathway. These combined effects result in inhibition of neuronal activity.

Endogenous opioid peptides are derived from either the pro-opiomelanocortin (POMC), pro-enkephalin (PENK), or pro-dynorphin (PDYN) family. These precursors form the final active peptides, beta-endorphin, met-enkephalin, leu-enkephalin, dynorphin, and neo-endorphin (Przewlocki and Przewlocka, 2001). Each endogenous opioid peptide exhibits different affinities for the 3 opioid receptors. Opioid receptors and endogenous opioid peptides are widely distributed in the central nervous system (CNS) and peripheral tissues (Niikura et al., 2010). They are also present in peripheral neurons and can contribute to anti-nociception.

Many studies have shown that endogenous opioids in the central nervous system are involved in the anti-nociceptive process. Beta-endorphin administration to the lateral brain ventricle and intrathecally produces a strong anti-nociceptive response that is blocked by naloxone (Przewlocki and Przewlocka, 2001). Interestingly, however, endogenous opioids are also expressed in non-neuronal, peripheral tissues, like immunocytes and keratinocytes, and could result in an anti-nociceptive effect (Khodorova et al., 2003). Keratinocytes have been identified as a source of beta-endorphin production. Activation of the endothelin B receptor (ETBR) and cannabinoid 2 receptor (CBr2) in keratinocytes induces secretion of beta-endorphin and elicits anti-nociception in vivo (Khodorova et al., 2003; Ibrahim et al., 2005). More importantly for cancer pain, oral SCC cells, which are malignant keratinocytes, also secrete endogenous opioids.

Endothelin Receptors and Opioid Release

Several studies have proposed harnessing endogenous opioids as pharmacologic solutions to opiate tolerance. Specifically, one study demonstrated that treatment with an ETBR agonist in a mouse oral SCC model results in mechanical anti-nociception through secretion of beta-endorphin (Quang and Schmidt, 2010b). Oral SCC consists of malignant keratinocytes that bear ETBRs and secrete opioids which can modulate the activity of the surrounding primary afferent nociceptors in skin. In addition, ET-1 activation of ETBRs on keratinocytes leads to analgesia that is reversed with naloxone, implicating the keratinocytes as a source of opioid released upon ETBR activation (Khodorova et al., 2002, 2003).

Surprisingly, in parallel with the role of ETBR activation, increased production of β-endorphin and leu-enkephalin occurs in oral SCC cell culture treated with an endothelin A receptor (ETAR) antagonist (Quang and Schmidt, 2010a). In the cancer mouse model, significant mechanical nociception begins at 4 days after inoculation of SCC cells and lasts up to 30 days when the mice are sacrificed. Local administration of naloxone methiodide significantly blocks the anti-nociceptive effect of the ETBR agonist or ETAR antagonist.

Modulation of ET-1 receptors in the management of cancer pain might have additional benefits. ETAR antagonism has been shown to prevent morphine tolerance (Bhalla et al., 2003). Theoretically, the combination of ETAR antagonism, which produces anti-nociception and simultaneously prevents morphine tolerance, and ETBR agonism, which leads to local opioid release, might hold promise for the treatment of oral cancer pain.

Cannabinoid Receptors and Endogenous Analgesia

Another endogenous analgesic mechanism that could be exploited for oral cancer pain treatment is the cannabinoid system. The two cannabinoid receptors, CBr1 and CBr2, both contribute to analgesia. CBr1 is expressed at central and peripheral nerve terminals and in keratinocytes after being synthesized in dorsal root ganglia (Munro et al., 1993). CBr2 is found on immune cells and keratinocytes (Ibrahim et al., 2006). CBr2 activation stimulates beta-endorphin release, leading to endogenous analgesia.

The link between cannabinoids and cancer pain was made by Kehl et al., who demonstrated that cannabinoids produce anti-nociception through CBr1 in a murine model of bone sarcoma pain (Kehl et al., 2003). The only soft-tissue carcinoma model exploring the analgesic effects of cannabinoids is by Guerrero et al., who created an oral cancer pain model by inoculating oral carcinoma cells into the hind paws of mice (Guerrero et al., 2008). These authors demonstrated that activation of CBr2 leads to the release of opioids from the carcinoma cells (Saghafi et al., 2011). Similar to the findings with the endothelin receptors, these findings target cannabinoid receptor activation as a possible endogenous approach to oral cancer pain treatment.