New findings in people, mice, and flies points to a role for epidermal growth factor receptor (EGFR) in chronic pain. The work, led by Jeffrey Mogil and Luda Diatchenko, both from McGill University, Montreal, Canada, demonstrates that genetic variation in EGFR and in one of its ligands, epiregulin (EREG), is associated with chronic orofacial pain in people, and that existing EGFR inhibitors reduce inflammatory and neuropathic pain in mice.
The work, the first to systematically assess the role of EGFR in pain, also demonstrates that signaling through the mRNA translation regulator, mechanistic target of rapamycin (mTOR), and the downstream effector, matrix metallopeptidase 9 (MMP-9), is responsible for EGFR’s analgesic effects.
“This is a remarkably interesting and really impressive paper. It is a very comprehensive study that uses human genetics, mouse models, and even Drosophila to show quite convincingly that EGFR plays a role in nociception,” said Theodore Price, University of Texas at Dallas, US, who was not involved in the study.
The findings were published online August 7 in the Journal of Clinical Investigation.
EGFR is a member of the ErbB family of receptor tyrosine kinases that play a role in a number of important cellular processes, including cell growth, proliferation, and survival. EGFR has been widely studied in connection with cancer, and EGFR inhibitors are the preferred treatment for non-small cell lung cancer.
“Case reports and cancer clinical trials have reported that people who are treated with EGFR inhibitors report less pain and a significant improvement in quality of life, a finding that has largely been attributed to tumor reduction,” said Diatchenko. However, recent analyses have demonstrated that these effects on pain and quality of life are independent of tumor shrinkage, providing a clue that EGFR inhibitors may provide analgesia through cancer-independent mechanisms, she said.
From humans …
The current project began when genetic findings in chronic pain patients provided the research team with another clue that EGFR signaling promotes pain. The researchers analyzed genetic variants associated with the orofacial pain condition temporomandibular disorder (TMD) in a sample of 127 females from the Orofacial Pain: Prospective Evaluation and Risk Assessment (OPPERA) study (see PRF related news stories here and here) and 231 matched controls. On a panel of 358 pain-relevant candidate genes, EGFR and one of its ligands, EREG, showed the highest association with TMD. In addition, nearly all of the cohort’s single nucleotide polymorphisms (SNPs) significantly associated with TMD were located in these two genes. The researchers also replicated their findings in two additional female TMD cohorts.
The team then used quantitative PCR (qPCR) to measure EREG mRNA levels in leukocytes from TMD patients; EGFR levels could not be examined due to low expression. Major allele carriers of the EREG SNP with the strongest association with TMD (rs1563826) had increased levels of EREG mRNA compared to minor allele carriers.
… to mice and then flies
The researchers then examined pain behavior in mice treated with one of three EGFR inhibitors (the preclinical compound tyrphostin AG 1478, and two clinically available drugs, gefitinib and lapatinib), finding a very robust analgesic effect in nearly all pain models tested, Diatchenko said. “All three inhibitors really worked very well, with efficacies that were comparable to morphine,” she added.
While the drugs had no effect on acute noxious thermal or mechanical pain, they were effective in two models of acute inflammatory pain. They blocked tonic inflammatory pain in the formalin test and completely reversed thermal hypersensitivity induced by the inflammatory mediator, λ-carrageenan. When the researchers examined chronic pain models, they found that the EGFR inhibitors completely and dose-dependently reversed allodynia in three models: the complete Freund’s adjuvant (CFA) model of inflammatory pain, the spared nerve injury (SNI) model of neuropathic pain, and the chronic constriction injury (CCI) model of neuropathic pain.
Consistent with the human data, only intrathecal injection of EREG, but not four other EGFR ligands tested, increased nociceptive responses, as measured by licking behavior, in mice treated with formalin. In addition, similar to the human chronic pain patients, CFA and SNI exposure produced an upregulation of blood EREG levels in mice.
In a partial loss-of-function EGFR mutant mouse model in which EGFR is constitutively active but unable to bind to ligands, EREG-induced hypersensitivity following formalin exposure was abolished, as expected. Basal sensitivity of these animals to formalin was increased, a finding the researchers attributed to the receptor’s constitutive activation.
To see if the role of EGFR was conserved across species, the research team then turned to fruit flies. They knocked down the Drosophila orthologue of EGFR specifically in peripheral neurons expressing the nociceptive marker pickpocket (ppk), and found prolonged withdrawal latencies in response to a heated probe. Reintroduction of the Egfr gene in these neurons rescued this effect.
“We started from human data and then asked if mice have a similar involvement of EGFR and EREG in pain. The answer was yes. Then we went further down the evolutionary tree to Drosophila and again the answer was yes. EREG signaling through EGFR is not only a very significant pain pathway, but it’s also highly conserved,” said Diatchenko.
Toward a mechanism
The researchers then turned their focus to the mechanism underlying the EGFR-EREG pathway’s role in pain. Based on the increased EREG levels in chronic pain patients, the team hypothesized that EGFR would have higher levels of activation in the mouse models of chronic pain. Indeed, activity of the receptor, measured by phosphorylation levels of a specific tyrosine residue in EGFR, was increased in dorsal root ganglion (DRG) lysate after CFA and SNI.
Because of prior findings that signaling through EGFR increases the activity of the mRNA translation master regulator, mTOR, the researchers then investigated whether mTOR signaling played a role in the hypersensitivity induced by EREG administration. Using pharmacological inhibitors and knockout mice, they systematically assessed the contributions of various members of the mTOR signal transduction pathway to hypersensitivity during the formalin test.
They found that EREG promoted pain through mTOR signaling via effects on the downstream eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and the eukaryotic translation initiation factor 4F (eIF4F) complex, ultimately resulting in increased levels of MMP-9.
“This is a nice confirmation that translation regulation, especially translation of MMP-9, is an important contributor to chronic pain,” said Price. “In addition, this paper reports a novel regulatory mechanism of MMP-9, which we know is involved in lots of different chronic pain states.”
“Our findings have uncovered a whole new pain pathway that deepens our understanding of the pathophysiology of chronic pain. EGFR-mediated pain occurs through a signaling pathway involving mTOR, the eIF4F translation initiation complex, and MMP-9,” said Diatchenko.
New targets for pain
Overall, EGFR is a very promising analgesic target, one for which drugs already exist, Diatchenko said. EGFR inhibitors don’t carry the risk of dependence, constipation, and respiratory depression that opioids do, and while a proportion of people using EGFR inhibitors develop a rash, that is easily treatable. Repurposing existing drugs for new indications would be much faster than developing new ones from scratch, she added.
The researchers are planning a clinical trial of EGFR inhibitors for pain, likely starting with cancer patients who are already taking these drugs, Diatchenko said. The team will examine if patients’ pain reports are related to the genetic markers identified in the current study and levels of EREG in the blood.
“Now is a wonderful time to be finding new pain targets, given the major emphasis on the non-opioid analgesics that are badly needed,” said Price. And, “cancer therapeutics that are existing EGFR inhibitors or yet to be discovered programmed cell death protein 1 [PD-1; see PRF related news story] modulators that also have analgesic effects could open up an exciting new area of drug targets that could have a combined effect on cancer and cancer-induced pain,” he said.
Allison Marin, PhD, is a neuroscientist-turned-science writer who resides in Pittsburgh, US.
Image credit: Eikuch/Wikimedia Commons.
