Cancer-related neuropathic pain (NeP) is a common cause of chronic pain, which is one of the most difficult clinical problems[1, 2]. Despite an increasing number of available diagnosis and therapies, adequately diagnosis of NeP is often difficult and satisfactory pain control is not always achieved[2, 6, 7]. Therefore, novel molecular network biomarkers or targets are needed for earlier detection or treatment.
In the present study, we collected malignant tumors of the spine data based on RNA sequencing from published datasets, and demonstrated ENPP6 was significantly down-regulated in with pain samples compared with no pain samples. There are few reports on the expression of ENPP6 in cancer, while Yano Y et al. demonstrated that ENPP3 which is the same family as ENPP6 was expressed in the tumor cells of bile duct malignancies and may play a role in tumor infiltration[32]. And ENPP2 was expressed in thyroid cancer cells and may associated with tumor cell motility and tumorigenic capacity[33]. Then, we analyzed the function of ENPP6 in malignant tumors of the spine using GSEA, and the results indicated that the low expression of ENPP6 in with pain tissue may mediate pain through indirect mechanisms such as a variety of cytokines and metabolic enzymes involved in the RAS and AKT pathway. There is evidence indicates that Ras-AKT signaling can promote the progression of glioma[34]. Further analysis using the HPA database showed high expression of ENPP6 in normal brain tissue and low expression in brain tumor tissue (glioma)[35], which consistent with the foregoing results. Above evidence suggested that ENPP6 may be related to cancer and was differentially expressed in cancer.
Subsequently, using GO and KEGG pathway analysis, we found that DEGs was mostly enriched in the immunoinflammation-related and regulation of trans-synaptic signaling GO terms, and Inflammation and cancer associated pathways. Accumulating evidence shows that inflammation involved in the development of NeP. It has been demonstrated that ENPP6 is expressed in oligodendrocytes which necessary for transmission of bioelectrical signals and the protection of the normal function of neurons[36]. In addition, ENPP2 have been revealed may play an important role in neural and vascular development, tumour progression and metastasis, as well as inflammation, neuropathic pain and fibrotic disease[37]. Xiao L et al have reported that ENPP6 may play a role in lipid metabolism during myelin sheath formation and might be required to initiate myelination rapidly in response to differentiation induced signals[36]. ENPP6, ENPP2 and ENPP7 shared recognition of phospholipids with choline. Evidence supported that ENPP7 is associated with anti-inflammatory and anti-tumorigenic activity by affecting the conversion of sphingomyelin to ceramide[38, 39]. Through these data, we found that ENPP6 may be involved in lipid metabolism, inflammation, cancer or nerve signaling, which prompted us to further analyze.
Afterward, the results of CIBERSORT analysis for the proportion of tumor-infiltrating immune cell (TIC) revealed that the expression of ENPP6 was positively correlated with immunophenotype and autophagy phenotype in spinal tumors patients. Then, we analyzed the expression differences of immune cells between the groups with high and low ENPP6 expression, and found that the activated NK cells were significantly highly expressed in the tissues with low ENPP6 expression. Natural Killer (NK) cells are lymphocytes with the capacity to target tumor cells via innate and adaptive responses[40, 41]. Activated NK cells can rapidly produce cytokines and activate other leukocytes, resulting increased complex fluctuations of cytokines observed in the blood and cerebrospinal fluid of NeP[42, 43]. Immune effector cells, especially NK cells, are associated with NeP[44]. Gao YH et al. reported that electroacupuncture improved NeP by affecting the activity and number of NK cells[45]. Morphine can inhibit the cytotoxic activity of NK cells through opioid receptors and Toll-like receptor-4 (TLR4), which is of great significance for the maintenance of immune function during pain[46]. Therefore, it is tempting to speculate that drugs targeted in activated NK cell may improve the proportion of immune cell distribution, and alleviate pain subsequently.
Furthermore, by analyzing the area under the ROC curve (AUC), we surprisingly found that ENPP6 had a good performance in diagnosing NeP. Recent reports indicated that choline plays a role in NeP and neuroinflammatory disorders[47, 48], and the role of ENPP6 in supplying choline to the cells have been well demonstrated[11]. Then, we analyzed the mutation status of the ENPP family, and the results showed that there were most of the mutations in ENPP4-7 were phosphorylation sites. Molecular and channel phosphorylation can affect the pathophysiological processes of NeP[49, 50]. García G et al reported that with mutations at PKC/PKA phosphorylation sites reversed tactile allodynia in neuropathic rats[51]. These suggested that the mutation of ENPP6 phosphorylation site may be one of the pathogenic links.
There are some limitations of our study. Firstly, the current study was only based on data analysis and additional experiments are needed to demonstrate the biological impact of ENPP6 in NeP. Secondly, the sample size of the data involved was small, and the study failed to cover different regions, which may affect the gene expression in tumors. Thirdly, because our study only focused on those genes that showed significant changes in the data set, some biological information might be ignored in our study. Therefore, further studies about direct mechanisms in NeP are needed.
In conclusion, this is the first study that uncovers ENPP6 may had a good performance in diagnosing NeP and may had an important role in the regulation of inflammatory and cancer pathways in malignant spinal tumors. The present study may offer new ideas for diagnosis and treatment of malignant spinal tumour patients with NeP.