Robert Ungard

Inhibition of breast cancer-cell glutamate release with sulfasalazine limits cancer-induced bone pain.

Summary Inhibition of glutamate release via the system xC− cystine/glutamate antiporter with sulfasalazine reduces metastatic breast cancer‐induced bone pain in a novel animal behavioural model. ABSTRACT Cancer in bone is frequently a result of metastases from distant sites, particularly from the breast, lung, and prostate. Pain is a common and often severe pathological feature of cancers in bone, and is a significant impediment to the maintenance of quality of life of patients living with bone metastases. Cancer cell lines have been demonstrated to release significant amounts of the neurotransmitter and cell‐signalling molecule l‐glutamate via the system xC− cystine/glutamate antiporter. We have developed a novel mouse model of breast cancer bone metastases to investigate the impact of inhibiting cancer cell glutamate transporters on nociceptive behaviour. Immunodeficient mice were inoculated intrafemorally with the human breast adenocarcinoma cell line MDA‐MB‐231, then treated 14 days later via mini‐osmotic pumps inserted intraperitoneally with sulfasalazine, (S)‐4‐carboxyphenylglycine, or vehicle. Both sulfasalazine and (S)‐4‐carboxyphenylglycine attenuated in vitro cancer cell glutamate release in a dose‐dependent manner via the system xC− transporter. Animals treated with sulfasalazine displayed reduced nociceptive behaviours and an extended time until the onset of behavioural evidence of pain. Animals treated with a lower dose of (S)‐4‐carboxyphenylglycine did not display this reduction in nociceptive behaviour. These results suggest that a reduction in glutamate secretion from cancers in bone with the system xC− inhibitor sulfasalazine may provide some benefit for treating the often severe and intractable pain associated with bone metastases.

Inhibitors of glutamate release from breast cancer cells; new targets for cancer-induced bone-pain

Glutamate is an important signaling molecule in a wide variety of tissues. Aberrant glutamatergic signaling disrupts normal tissue homeostasis and induces several disruptive pathological conditions including pain. Breast cancer cells secrete high levels of glutamate and often metastasize to bone. Exogenous glutamate can disrupt normal bone turnover and may be responsible for cancer-induced bone pain (CIBP). CIBP is a significant co-morbidity that affects quality of life for many advanced-stage breast cancer patients. Current treatment options are commonly accompanied by serious side-effects that negatively impact patient care. Identifying small molecule inhibitors of glutamate release from aggressive breast cancer cells advances a novel, mechanistic approach to targeting CIBP that could advance treatment for several pathological conditions. Using high-throughput screening, we investigated the ability of approximately 30,000 compounds from the Canadian Compound Collection to reduce glutamate release from MDA-MB-231 breast cancer cells. This line is known to secrete high levels of glutamate and has been demonstrated to induce CIBP by this mechanism. Positive chemical hits were based on the potency of each molecule relative to a known pharmacological inhibitor of glutamate release, sulfasalazine. Efficacy was confirmed and drug-like molecules were identified as potent inhibitors of glutamate secretion from MDA-MB-231, MCF-7 and Mat-Ly-Lu cells.

Oxidative stress and cancer pain.

Breast cancers are the most common source of metastases to bone, of which cancer-induced bone pain is a frequent pathological feature. Cancer-induced bone pain is a unique pain state with multiple determinants that remains to be well understood and managed. Current standard treatments are limited by dose-dependent side effects that can reduce the quality of life of patients. Glutamate is a neurotransmitter and bone cell-signalling molecule that is released via the system x(c)(-) cystine/glutamate antiporter from cancer cell types that frequently metastasize to bone, including breast cancers. In cancer cells, glutamate release is understood to be a side effect of the cellular response to oxidative stress that upregulates the expression and activity of system x(c)(-) to promote the increased import of cystine. Attenuation of glutamate release from cancer cells has been demonstrated to result in reductions in associated cancer-induced bone pain in animal models. This review examines the clinical implications of attenuating cystine uptake and glutamate release in the treatment of cancer-induced bone pain.

Chronic Inhibition of STAT3/STAT5 in Treatment-Resistant Human Breast Cancer Cell Subtypes: Convergence on the ROS/SUMO Pathway and Its Effects on xCT Expression and System xc- Activity

Pharmacologically targeting activated STAT3 and/or STAT5 has been an active area of cancer research. The cystine/glutamate antiporter, system xc-, contributes to redox balance and export of intracellularly produced glutamate in response to up-regulated glutaminolysis in cancer cells. We have previously shown that blocking STAT3/5 using the small molecule inhibitor, SH-4-54, which targets the SH2 domains of both proteins, increases xCT expression, thereby increasing system xc- activity in human breast cancer cells. The current investigation demonstrates that chronic SH-4-54 administration, followed by clonal selection of treatment-resistant MDA-MB-231 and T47D breast cancer cells, elicits distinct subtype-dependent effects. xCT mRNA and protein levels, glutamate release, and cystine uptake are decreased relative to untreated passage-matched controls in triple-negative MDA-MB-231 cells, with the inverse occurring in estrogen-responsive T47D cells. This “ying-yang” effect is linked with a shifted balance between the phosphorylation status of STAT3 and STAT5, intracellular ROS levels, and STAT5 SUMOylation/de-SUMOylation. STAT5 emerged as a definitive negative regulator of xCT at the transcriptional level, while STAT3 activation is coupled with increased system xc- activity. We propose that careful classification of a patient’s breast cancer subtype is central to effectively targeting STAT3/5 as a therapeutic means of treating breast cancer, particularly given that xCT is emerging as an important biomarker of aggressive cancers.

Differences in electrophysiological properties of functionally identified nociceptive sensory neurons in an animal model of cancer-induced bone pain

Background Bone cancer pain is often severe, yet little is known about mechanisms generating this type of chronic pain. While previous studies have identified functional alterations in peripheral sensory neurons that correlate with bone tumours, none has provided direct evidence correlating behavioural nociceptive responses with properties of sensory neurons in an intact bone cancer model. Results In a rat model of prostate cancer-induced bone pain, we confirmed tactile hypersensitivity using the von Frey test. Subsequently, we recorded intracellularly from dorsal root ganglion neurons in vivo in anesthetized animals. Neurons remained connected to their peripheral receptive terminals and were classified on the basis of action potential properties, responses to dorsal root stimulation, and to mechanical stimulation of the respective peripheral receptive fields. Neurons included C-, Aδ-, and Aβ-fibre nociceptors, identified by their expression of substance P. We suggest that bone tumour may induce phenotypic changes in peripheral nociceptors and that these could contribute to bone cancer pain. Conclusions This work represents a significant technical and conceptual advance in the study of peripheral nociceptor functions in the development of cancer-induced bone pain. This is the first study to report that changes in sensitivity and excitability of dorsal root ganglion primary afferents directly correspond to mechanical allodynia and hyperalgesia behaviours following prostate cancer cell injection into the femur of rats. Furthermore, our unique combination of techniques has allowed us to follow, in a single neuron, mechanical pain-related behaviours, electrophysiological changes in action potential properties, and dorsal root substance P expression. These data provide a more complete understanding of this unique pain state at the cellular level that may allow for future development of mechanism-based treatments for cancer-induced bone pain.

Behavioural Effects of Using Sulfasalazine to Inhibit Glutamate Released by Cancer Cells: A Novel target for Cancer-Induced Depression

Despite the lack of robust evidence of effectiveness, current treatment options for cancer-induced depression (CID) are limited to those developed for non-cancer related depression. Here, anhedonia-like and coping behaviours were assessed in female BALB/c mice inoculated with 4T1 mammary carcinoma cells. The behavioural effects of orally administered sulfasalazine (SSZ), a system xc− inhibitor, were compared with fluoxetine (FLX). FLX and SSZ prevented the development of anhedonia-like behaviour on the sucrose preference test (SPT) and passive coping behaviour on the forced swim test (FST). The SSZ metabolites 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP) exerted an effect on the SPT but not on the FST. Although 5-ASA is a known anti-inflammatory agent, neither treatment with SSZ nor 5-ASA/SP prevented tumour-induced increases in serum levels of interleukin-1β (IL-1β) and IL-6, which are indicated in depressive disorders. Thus, the observed antidepressant-like effect of SSZ may primarily be attributable to the intact form of the drug, which inhibits system xc−. This study represents the first attempt at targeting cancer cells as a therapeutic strategy for CID, rather than targeting downstream effects of tumour burden on the central nervous system. In doing so, we have also begun to characterize the molecular pathways of CID.

Functional effects of TrkA inhibition on system xC−-mediated glutamate release and cancer-induced bone pain:

Breast cancer cells release the signalling molecule glutamate via the system xC− antiporter, which is upregulated to exchange extracellular cystine for intracellular glutamate to protect against oxidative stress. Here, we demonstrate that this antiporter is functionally influenced by the actions of the neurotrophin nerve growth factor on its cognate receptor tyrosine kinase, TrkA, and that inhibiting this complex may reduce cancer-induced bone pain via its downstream actions on xCT, the functional subunit of system xC−. We have characterized the effects of the selective TrkA inhibitor AG879 on system xC− activity in murine 4T1 and human MDA-MB-231 mammary carcinoma cells, as well as its effects on nociception in our validated immunocompetent mouse model of cancer-induced bone pain, in which BALB/c mice are intrafemorally inoculated with 4T1 murine carcinoma cells. AG879 decreased functional system xC− activity, as measured by cystine uptake and glutamate release, and inhibited nociceptive and physiologically relevant responses in tumour-bearing animals. Cumulatively, these data suggest that the activation of TrkA by nerve growth factor may have functional implications on system xC−-mediated cancer pain. System xC−-mediated TrkA activation therefore presents a promising target for therapeutic intervention in cancer pain treatment.

Oxidative Stress and Glutamate Release in Glioma

Glioma are a family of glial cell tumours of the central nervous system (CNS) wellcharacterized as aggressive cancers with dismally limited treatment options. The relatively recent discoveries of mechanisms surrounding glioma cell antioxidant protection and neuronal and glial cell destruction have opened the gates to a new therapeutic avenue whose implications to the field of cancer biology extend far beyond the treatment of glioma alone. Stemming from the discovery of significant glutamate release and glutathione production by glioma cells, the mechanisms through which glioma mediate oxidative stress and influence their extracellular microenvironment are now being unravelled. This chapter will discuss the upregulation of the cystine/glutamate antiporter, system xc-, in glioma and the far ranging consequences that stem from this compensatory action. Specifically, a characteristic shift of cancer cell metabolism away from the tricarboxylic acid (TCA) cycle and towards increased rates of glycolysis, a process termed the Warburg effect, produces a high amount of reactive oxygen species (ROS) that would prove cytotoxic without adequate cellular antioxidant defences. In response to this metabolic abnormality, glioma have demonstrated increased synthesis of the primary cellular antioxidant glutathione and an increased circulation of the cystine/cysteine redox cycle. These antioxidant increases are driven by an upregulation of system xc-, which supplies the cell with the rate limiting substrate for glutathione synthesis, cysteine, and acts as one half of the transport machinery for the cystine/cysteine cycle. The increased tolerance to oxidative stress that is conferred by these mechanisms allows glioma survival and growth advantages and mediate chemoand radiation-resistance to treatment. The corollary effect of cystine import via system xcis the export of the neurotransmitter and ubiquitous cell-signalling molecule, glutamate. This release has destructive consequences for the peritumoral brain. Glutamate induces neuronal and glial excitotoxic cell death, and acts in an autocrine and paracrine signalling manner to stimulate glioma cell growth and migration. Treatments based on these mechanisms are currently under development and some have progressed as far as clinical trials. Glutamate receptor antagonists and system xcinhibitors are as of yet the primary avenues of investigation. The potential treatment benefits of targeting these pathways are great, and the discovery of system xcprevalence in other cancers beyond glioma suggests that study of this pathway may produce wide-ranging cancer treatment options.

[EXPRESS] Cancer pain and neuropathic pain are associated with Aβ sensory neuronal plasticity in dorsal root ganglia and abnormal sprouting in lumbar spinal cord

Evidence suggests that there are both nociceptive and neuropathic components of cancer-induced pain. We have observed that changes in intrinsic membrane properties and excitability of normally non-nociceptive Aβ sensory neurons are consistent in rat models of peripheral neuropathic pain and cancer-induced pain. This has prompted a comparative investigation of the intracellular electrophysiological characteristics of sensory neurons and of the ultrastructural morphology of the dorsal horn in rat models of neuropathic pain and cancer-induced pain. Neuropathic pain model rats were induced with a polyethylene cuff implanted around a sciatic nerve. Cancer-induced pain model rats were induced with mammary rat metastasis tumour-1 rat breast cancer or MATLyLu rat prostate cancer cells implanted into the distal epiphysis of a femur. Behavioural evidence of nociception was detected using von Frey tactile assessment. Aβ-fibre low threshold mechanoreceptor neurons in both cancer-induced pain and neuropathic pain models exhibited slower dynamics of action potential genesis, including a wider action potential duration and lower action potential amplitude compared to those in control animals. Enhanced excitability of Aβ-fibre low threshold mechanoreceptor neurons was also observed in cancer-induced pain and neuropathic pain models. Furthermore, both cancer-induced pain and neuropathic pain models showed abundant abnormal axonal sprouting in bundles of myelinated axons in the ipsilateral spinal laminae IV and V. The patterns of changes show consistency between rat models of cancer-induced pain and neuropathic pain. These findings add to the body of evidence that animal models of cancer-induced pain and neuropathic pain share features that may contribute to the peripheral and central sensitization and tactile hypersensitivity in both pain states.

Cancer-Induced Pain

Most commonly, but not exclusively, cancer pain is a result of late-stage metastatic cancers and primary and metastatic cancers that grow in the bone. Cancer pain, like the disease itself, is widely diverse in its quality and extent, and can result from many different causative factors. Many factors have been implicated in the causation and maintenance of cancer pain. Neuropathic pain results from damaged peripheral or central neuronal tissue and from chronically altered neuronal signalling resulting from central and peripheral sensitization. Neuronal tissue can be damaged by direct invasion by tumour cells, as is the case of tumours of the central nervous system (CNS) or by invasion of peripheral neurons in peripheral host tissues. Cancer cells and associated cells also secrete a large number of chemical factors, some of which can directly damage or simulate neurons. Direct physical interaction between the tumour mass and the altered host tissues with neuronal tissue can also cause neuropathic damage through nerve disruption and destruction. Cancer cells and associated cells including stromal and immune cells also secrete a host of chemical signalling molecules that can directly and indirectly stimulate nociceptors. Thermal stimuli of sensory neurons can become pathological following peripheral and central sensitization, which decreases the threshold temperature at which thermally sensitive neurons will respond. Pain is also often a side effect of many treatments of cancer, although the mechanisms of these treatment-induced conditions are beyond the scope of this review. Treatment of cancer pain itself largely relies on analgesics and therapies directed against the cancers themselves, although specific treatments for cancer pain are more recently becoming available. It is often the case, however, that cancer pain conditions become intractable, or are poorly controlled. Breakthrough pain which is prevalent in cancer pain is defined by its relationship to treatment where it is an episodic painful event that occurs during a routine of normally effective pain control. Cancer pain is a serious and prevalent oncodynamic effect that arises from a highly variable array of stimuli. The study of cancer pain as a distinct phenomenon is still in its infancy.