John C. Dolan

Nerve growth factor links oral cancer progression, pain, and cachexia.

Cancers often cause excruciating pain and rapid weight loss, severely reducing quality of life in cancer patients. Cancer-induced pain and cachexia are often studied and treated independently, although both symptoms are strongly linked with chronic inflammation and sustained production of proinflammatory cytokines. Because nerve growth factor (NGF) plays a cardinal role in inflammation and pain, and because it interacts with multiple proinflammatory cytokines, we hypothesized that NGF acts as a key endogenous molecule involved in the orchestration of cancer-related inflammation. NGF might be a molecule common to the mechanisms responsible for clinically distinctive cancer symptoms such as pain and cachexia as well as cancer progression. Here we reported that NGF was highly elevated in human oral squamous cell carcinoma tumors and cell cultures. Using two validated mouse cancer models, we further showed that NGF blockade decreased tumor proliferation, nociception, and weight loss by orchestrating proinflammatory cytokines and leptin production. NGF blockade also decreased expression levels of nociceptive receptors TRPV1, TRPA1, and PAR-2. Together, these results identified NGF as a common link among proliferation, pain, and cachexia in oral cancer. Anti-NGF could be an important mechanism-based therapy for oral cancer and its related symptoms. Mol Cancer Ther; 10(9); 1667–76. ©2011 AACR.

Peripheral endothelin A receptor antagonism attenuates carcinoma-induced pain.

In this study we investigated the role of endothelin‐1 (ET‐1) and its peripheral receptor (ET‐A) in carcinoma‐induced pain in a mouse cancer pain model. Tumors were induced in the hind paw of female mice by local injection of cells derived from a human oral squamous cell carcinoma (SCC). Significant pain, as indicated by reduction in withdrawal thresholds in response to mechanical stimulation, began at four days after SCC inoculation and lasted to 28 days, the last day of measurement. Intra‐tumor expression of both ET‐1 mRNA and ET‐1 protein were significantly upregulated compared to normal tissue, and local administration of the ET‐A receptor selective antagonist, BQ‐123 (100μM) significantly elevated withdrawal thresholds, indicating the induction of an antinociceptive effect. These findings support the suggestion that ET‐1 and ET‐A receptors contribute to the severity of carcinoma‐induced soft tissue cancer pain.

Novel Animal Models of Acute and Chronic Cancer Pain: A Pivotal Role for PAR2

Targeted therapy to prevent the progression from acute to chronic pain in cancer patients remains elusive. We developed three novel cancer models in mice that together recapitulate the anatomical, temporal, and functional characteristics of acute and chronic head and neck cancer pain in humans. Using pharmacologic and genetic approaches in these novel cancer models, we identified the interaction between protease-activated receptor 2 (PAR2) and serine proteases to be of central importance. We show that serine proteases such as trypsin induce acute cancer pain in a PAR2-dependent manner. Chronic cancer pain is associated with elevated serine proteases in the cancer microenvironment and PAR2 upregulation in peripheral nerves. Serine protease inhibition greatly reduces the severity of persistent cancer pain in wild-type mice, but most strikingly, the development of chronic cancer pain is prevented in PAR2-deficient mice. Our results demonstrate a direct role for PAR2 in acute cancer pain and suggest that PAR2 upregulation may favor the development and maintenance of chronic cancer pain. Targeting the PAR2–serine protease interaction is a promising approach to the treatment of acute cancer pain and prevention of chronic cancer pain.

The dolognawmeter: a novel instrument and assay to quantify nociception in rodent models of orofacial pain.

Rodent pain models play an important role in understanding the mechanisms of nociception and have accelerated the search for new treatment approaches for pain. Creating an objective metric for orofacial nociception in these models presents significant technical obstacles. No animal assay accurately measures pain-induced orofacial dysfunction that is directly comparable to human orofacial dysfunction. We developed and validated a high throughput, objective, operant, nociceptive animal assay, and an instrument to perform the assay termed the dolognawmeter, for evaluation of conditions known to elicit orofacial pain in humans. Using the device our assay quantifies gnawing function in the mouse. We quantified a behavioral index of nociception and demonstrated blockade of nociception in three models of orofacial pain: (1) TMJ inflammation, (2) masticatory myositis, and (3) head and neck cancer. This assay will be useful in the study of nociceptive mediators involved in the development and progression of orofacial pain conditions and it will also provide a unique tool for development and assessment of new therapeutic approaches.

Analgesia targeting IB4-positive neurons in cancer-induced mechanical hypersensitivity

UNLABELLED Cancer patients often suffer from pain and most will be prescribed μ-opioids. μ-opioids are not satisfactory in treating cancer pain and are associated with multiple debilitating side effects. Recent studies show that μ and δ opioid receptors are separately expressed on IB4 (-) and IB4 (+) neurons, which control thermal and mechanical pain, respectively. In this study we investigated IB4 (+) and IB4 (-) neurons in mechanical and thermal hypersensitivity in an orthotopic mouse oral cancer model. We used a δ opioid receptor agonist and a P2X(3) antagonist to target IB4 (+) neurons and to demonstrate that this subset plays a key role in cancer-induced mechanical allodynia, but not in thermal hyperalgesia. Moreover, selective removal of IB4 (+) neurons using IB4-saporin impacts cancer-induced mechanical but not thermal hypersensitivity. Our results demonstrate that peripherally administered pharmacological agents targeting IB4 (+) neurons, such as a selective δ-opioid receptor agonist or P2X(3) antagonist, might be useful in treating oral cancer pain. PERSPECTIVE To clarify the mechanisms of oral cancer pain, we examined the differential role of IB4 (+) and IB4 (-) neurons. Characterization of these 2 subsets of putative nociceptors is important for further development of effective clinical cancer pain relief.

Adenosine triphosphate drives head and neck cancer pain through P2X2/3 heterotrimers

IntroductionCancer pain creates a poor quality of life and decreases survival. The basic neurobiology of cancer pain is poorly understood. Adenosine triphosphate (ATP) and the ATP ionotropic receptor subunits, P2X2 and P2X3, mediate cancer pain in animal models; however, it is unknown whether this mechanism operates in human, and if so, what the relative contribution of P2X2- and P2X3-containing trimeric channels to cancer pain is. Here, we studied head and neck squamous cell carcinoma (HNSCC), which causes the highest level of function-induced pain relative to other types of cancer.ResultsWe show that the human HNSCC tissues contain significantly increased levels of ATP compared to the matched normal tissues. The high levels of ATP are secreted by the cancer and positively correlate with self-reported function-induced pain in patients. The human HNSCC microenvironment is densely innervated by nerve fibers expressing both P2X2 and P2X3 subunits. In animal models of HNSCC we showed that ATP in the cancer microenvironment likely heightens pain perception through the P2X2/3 trimeric receptors. Nerve growth factor (NGF), another cancer-derived pain mediator found in both human and mouse HNSCC, induces P2X2 and P2X3 hypersensitivity and increases subunit expression in murine trigeminal ganglion (TG) neurons.ConclusionsThese data identify a key peripheral mechanism in cancer pain and highlight the clinical potential of specifically targeting nociceptors expressing both P2X2 and P2X3 subunits (e.g., P2X2/3 heterotrimers) to alleviate cancer pain.

Surveying proteolytic processes in human cancer microenvironments by microdialysis and activity-based mass spectrometry

Purpose: We present a strategy to survey proteolytic processes that occur in human cancer microenvironments.

Conditional TNF-α Overexpression in the Tooth and Alveolar Bone Results in Painful Pulpitis and Osteitis.

Tumor necrosis factor–α (TNF-α) is a proalgesic cytokine that is commonly expressed following tissue injury. TNF-α expression not only promotes inflammation but can also lead to pain hypersensitivity in nociceptors. With the established link between TNF-α and inflammatory pain, we identified its increased expression in the teeth of patients affected with caries and pulpitis. We generated a transgenic mouse model (TNF-αglo) that could be used to conditionally overexpress TNF-α. These mice were bred with a dentin matrix protein 1 (DMP1)–Cre line for overexpression of TNF-α in both the tooth pulp and bone to study oral pain that would result from subsequent development of pulpitis and bone loss. The resulting DMP1/TNF-αglo mice show inflammation in the tooth pulp that resembles pulpitis while also displaying periodontal bone loss. Inflammatory infiltrates and enlarged blood vessels were observed in the tooth pulp. Pulpitis and osteitis affected the nociceptive neurons innervating the orofacial region by causing increased expression of inflammatory cytokines within the trigeminal ganglia. With this new mouse model morphologically mimicking pulpitis and osteitis, we tested it for signs of oral pain with an oral function assay (dolognawmeter). This assay/device records the time required by a mouse to complete a discrete gnawing task. The duration of gnawing required by the DMP1/TNF-αglo mice to complete the task was greater than that for the controls; extended gnaw time in a dolognawmeter indicates reduced orofacial function. With the DMP1/TNF-αglo mice, we have shown that TNF-α expression alone can produce inflammation similar to pulpitis and osteitis and that this mouse model can be used to study dental inflammatory pain.

Ex vivo nonviral gene delivery of μ-opioid receptor to attenuate cancer-induced pain.

Abstract Virus-mediated gene delivery shows promise for the treatment of chronic pain. However, viral vectors have cytotoxicity. To avoid toxicities and limitations of virus-mediated gene delivery, we developed a novel nonviral hybrid vector: HIV-1 Tat peptide sequence modified with histidine and cysteine residues combined with a cationic lipid. The vector has high transfection efficiency with little cytotoxicity in cancer cell lines including HSC-3 (human tongue squamous cell carcinoma) and exhibits differential expression in HSC-3 (∼45-fold) relative to HGF-1 (human gingival fibroblasts) cells. We used the nonviral vector to transfect cancer with OPRM1, the &mgr;-opioid receptor gene, as a novel method for treating cancer-induced pain. After HSC-3 cells were transfected with OPRM1, a cancer mouse model was created by inoculating the transfected HSC-3 cells into the hind paw or tongue of athymic mice to determine the analgesic potential of OPRM1 transfection. Mice with HSC-3 tumors expressing OPRM1 demonstrated significant antinociception compared with control mice. The effect was reversible with local naloxone administration. We quantified &bgr;-endorphin secretion from HSC-3 cells and showed that HSC-3 cells transfected with OPRM1 secreted significantly more &bgr;-endorphin than control HSC-3 cells. These findings indicate that nonviral delivery of the OPRM1 gene targeted to the cancer microenvironment has an analgesic effect in a preclinical cancer model, and nonviral gene delivery is a potential treatment for cancer pain.

Tumor necrosis factor alpha secreted from oral squamous cell carcinoma contributes to cancer pain and associated inflammation

Abstract Patients with oral cancer report severe pain during function. Inflammation plays a role in the oral cancer microenvironment; however, the role of immune cells and associated secretion of inflammatory mediators in oral cancer pain has not been well defined. In this study, we used 2 oral cancer mouse models: a cell line supernatant injection model and the 4-nitroquinoline-1-oxide (4NQO) chemical carcinogenesis model. We used the 2 models to study changes in immune cell infiltrate and orofacial nociception associated with oral squamous cell carcinoma (oSCC). Oral cancer cell line supernatant inoculation and 4NQO-induced oSCC resulted in functional allodynia and neuronal sensitization of trigeminal tongue afferent neurons. Although the infiltration of immune cells is a prominent component of both oral cancer models, our use of immune-deficient mice demonstrated that oral cancer–induced nociception was not dependent on the inflammatory component. Furthermore, the inflammatory cytokine, tumor necrosis factor alpha (TNF&agr;), was identified in high concentration in oral cancer cell line supernatant and in the tongue tissue of 4NQO-treated mice with oSCC. Inhibition of TNF&agr; signaling abolished oral cancer cell line supernatant-evoked functional allodynia and disrupted T-cell infiltration. With these data, we identified TNF&agr; as a prominent mediator in oral cancer–induced nociception and inflammation, highlighting the need for further investigation in neural–immune communication in cancer pain.