Advances in the Study of IL-34 and DAD1 in Gastric Cancer

Congcong MA, Hairu JI, Jiemin QI

Department of Pathology, Chengde Medical College, Chengde 067000, China

Abstract Interleukin-34 (IL-34), an extremely important pro-inflammatory cytokine, participates in the regulation of related signal pathways in tumors, thereby mediating the proliferation and migration of tumor cells and affecting the prognosis of patients. DAD1, an anti-apoptotic factor, is highly expressed in a variety of tumors and is expected to become a tumor marker. The research on the function and mechanism of IL-34 and DAD1 is of great significance for the exploration of molecular targeted therapy. In this study, the regulatory functions of IL-34 and DAD1 in malignant tumors, the mechanisms of IL-34 and DAD1 involved in tumor invasion and immunity, and their expression in several tumors are briefly reviewed.

Key words IL-34, DAD1, Tumor, Mechanism

1 Introduction

Gastric cancer, one of the most common malignant tumors, seriously threatens human health. According to investigations and studies, gastric cancer has a high incidence and mortality rate in the world. China is a country with a high incidence of gastric cancer[1]. The occurrence and development of gastric cancer is a complex process. A large number of basic research and clinical data indicate that there is a close relationship between inflammatory factors, apoptotic factors and tumors. Linetal.studied the secreted protein and receptor system of cell signal transduction, and discovered a new inflammatory factor interleukin-34 (IL-34) that can promote the differentiation, proliferation and survival of monocytes and the formation of macrophage colonies during the culture of bone marrow cells[2]. Nakashimaetal.cloned an anti-apoptotic factor (defender against cell death 1, DAD1) from a mutant strain of a kidney-derived cell line (tsDN7), and it was named as an anti-apoptotic factor because of its function of inhibiting apoptosis[3]. Both of these factors play an important role in the occurrence and development of tumors. In this paper, the characteristics and signaling pathways of IL-34 and DAD1 and their roles in many tumors will be analyzed, aiming to better explain and understand the relationship between the expression of the two factors and cancer, and to provide new ideas for exploring new tumor markers and potential therapeutic targets.

2 IL-34 and tumors

2.1 Features of IL-34Interleukin-34 (IL-34) is a secreted dimeric protein composed of 241 amino acids, with a molecular weight of 39 kD. The amino acid sequence of human IL-34 has 99.6%, 72% and 71% homology with chimpanzee, rat and mouse IL-34, respectively[2]. Located on human chromosome 16 q22.1, IL-34 is highly conserved in biological evolution, and is secreted by the mononuclear macrophage system, epithelial cells, fibroblasts,etc[4]. IL-34 and macrophage colony stimulating factor (M-CSF, also known as CSF-1) are the ligands of colony stimulating factor 1 receptor (CSF-1R, also known as FMS), and each binds to CSF-1R. IL-34 binds to the N-terminal D1-D3 domain of the extracellular ligand domain of CSF-1R[5]. The combination of the two factors connects CSF-1R and promotes the autophosphorylation of tyrosine residues in the cytoplasm, recruits effector proteins, and promotes the function of mononuclear macrophages[6].

2.2 Signal pathways of IL-34 in tumorsIL-34 can promote the occurrence, development, angiogenesis and immunosuppression of tumors through both autocrine and paracrine. In the autocrine process, IL-34 and CSF-1R in tumor cells combine to activate signal pathways to stimulate the growth and spread of cancer cells and enhance their ability to resist chemotherapy drugs[7]. Macrophages are the key driver of tumor inflammation. The macrophages in tumors are named tumor-associated macrophages (TAMs). A large number of experimental and clinical evidences show that TAMs promote tumor growth, angiogenesis, immunosuppression, tumor invasion and metastasis in tumors, and adversely affect the prognosis of patients[8]. In the paracrine process, IL-34 produced by tumor cells and/or immune cells triggers the CSF-1R signaling pathway in TAMs, thereby promoting the recruitment of macrophages to the tumor area, promoting the formation of new blood vessels and the extravasation of immune inflammatory cells, and it plays an important role in the development of inflammatory diseases and tumors[9].

IL-34 can be transduced through the following signal pathways: (i) IL-34, as a specific and independent ligand of CSF-1 receptor, induces phosphorylation of ERK 1/2 and Akt to promote cell differentiation and survival by activating CSF-1R[10]; (ii) IL-34 promotes the adhesion and proliferation of osteoclast progenitor cells through the JAK2/STAT3 pathway, and stimulates the receptor activator (rankl) of NF-κB ligand to induce the formation and differentiation of osteoclasts[11]; (iii) TNF-α and IL-1β promote the production of IL-34 in synovial fibroblasts through NF-κB and JNK[12], and IL-34 can also regulate the production of inflammatory factors IL-6 and IL-8 in human lung fibroblasts through MAPK, PI3K-Akt, JAK and NF-κB signaling pathways[13].

2.3 Expression and significance of IL-34 in tumors

2.3.1Tumor-promoting effect of IL-34. IL-34 can affect a variety of cells such as mononuclear macrophages, fibroblasts, and bone cells. IL-34 can participate in the occurrence of a variety of tumors, and exhibits a tumor-promoting effect. Through qRT-PCR and immunohistochemical staining, Baghdadietal.[14]confirmed that the expression of IL-34 in primary lung cancer tissues was higher than that in normal lung tissues; by Western blot, it was found that IL-34 mediated AKT signal through CSF-1R and enhanced the survival of chemotherapy-resistant cells and the immunosuppressive function of TAMs, thereby weakening the anti-tumor effect of chemotherapy, so that the high expression of IL-34 was related to poor prognosis of lung cancer; IL-34 can be used as a biomarker to monitor the progress of chemotherapy resistance in lung cancer patients undergoing chemotherapy. Through qRT-PCR and Western blot experiments, Franzèetal.[15]found that the expression levels of IL-34 mRNA and protein in colorectal cancer samples were higher than normal tissues, and IL-34 induced cancer cell proliferation through the ERK1/2 mechanism. Through immunohistochemical staining, Kobayashietal.[16]found that IL-34 was expressed in colorectal cancer tissues but almost undetectable in normal colonic epithelium. In addition, it was found that high expression of IL-34 was poorly associated with the survival of patients with colorectal cancer; the expression of IL-34 in colorectal cancer may be a good therapeutic target, and can be used to assist the treatment of colorectal cancer patients. Through qRT-PCR analysis, Endo[17]showed that the expression level of IL-34 mRNA in ovarian cancer tissue was higher than that in normal fallopian tube tissues and normal ovarian epithelial tissues. In addition, immunohistochemical staining of clinical samples revealed the level of IL-34 expression in ovarian cancer tissues. In a mouse ovarian cancer model, it was found that the tumor growth of mice vaccinated with the knockout IL-34 cell line was inhibited, and the survival rate of the mice was improved. By flow cytometry, it was found that IL-34 from ovarian cancer cells can regulate the number of immune cells at the tumor site and contribute to the formation of a tumor-promoting environment. Segalinyetal.[18]found that the overexpression of IL-34 in the mouse osteosarcoma model was related to the progression of osteosarcoma (tumor growth and lung metastasis) and the increase of new blood vessels; IL-34 had the function of promoting angiogenesisinvivoandinvitro; IL-34 can regulate the FAK, Src, Akt and ERK1/2 signaling pathways in endothelial cells, stimulate the proliferation of endothelial cells, and recruit M2 type TAMs into the tumor site to promote the growth of osteosarcoma; IL-34 played an important role in the occurrence and development of osteosarcoma, providing new evidence for IL-34 as an oncogene of osteosarcoma. Zhangetal.[19]found that IL-34 expression was significantly increased in serum and tissue specimens of patients with papillary thyroid carcinoma, which was significantly related to tumor size, stage and lymph node metastasis; IL-34 can promote the invasion and metastasis of tumors by activating the epithelial-mesenchymal transition of cells; these findings suggest that IL-34 may be used as a biomarker and therapeutic target for the diagnosis of papillary thyroid carcinoma. Zhou Hongjuetal.[20]used enzyme-linked immunosorbent assay to detect the serum of patients, and found that the expression level of IL-34 in the gastric cancer group was significantly higher than that in the healthy control group, and IL-34 promoted the occurrence and development of gastric cancer.

2.3.2Anti-tumor effect of IL-34. Recent studies have found that in some tumors, IL-34 can induce tumor cell apoptosis and exhibit an anti-tumor effect. By qPT-PCR detection, Zinsetal.[21]found that compared with normal breast cancer cell lines and normal breast tissue samples, IL-34 expression was down-regulated in tumor cell lines and primary tumor tissues; from Kaplan-Meier analysis of patients, it was found that patients with relatively high expression of IL-34 mRNA showed a higher survival rate and better prognosis. This shows that IL-34 can inhibit the progression of breast cancer tumors in this experiment. Similarly, Liuetal.[22]showed that the expression of IL-34 in gastric cancer was 40% lower than that in normal gastric tissue adjacent to the cancer by immunohistochemical staining; at the same time, the decrease of IL-34 in gastric cancer was negatively correlated with the tumor differentiation, age and female gender of gastric cancer patients, and the malignancy of female gastric cancer patients was more serious than that of older male patients; the high survival rate of gastric cancer patients was closely related to the high expression of IL-34, so IL-34 played an anti-tumor effect in gastric cancer.

3 DAD1 and tumors

3.1 Features of DAD1The human DAD1 gene is located on chromosome 14q11-q12, has 3 exons and 2 introns, and encodes a total of 113 amino acids, with a molecular weight of 12.5 kD. The gene database shows that DAD1 is widely expressed in human tissues, organs and cells, and does not have any sequence similarity with other apoptosis-related genes; it is conservative and stable during biological evolution. DAD1 plays a role in many tumors and is increasingly used as one of the landmark molecules related to apoptosis[23]. Protein N-glycosylation is a basic modification that occurs in all eukaryotes, and is catalyzed by oligosaccharyltransferase (OST) in the endoplasmic reticulum. DAD1, located in the endoplasmic reticulum of the cell, is the core subunit of OST and participates in aspartate-mediated N-glycosylation modification[24].

3.2 Signal transduction pathways of DAD1 in tumorsDAD1 is considered to be a negative regulator of apoptosis. DAD1 binds to the C-terminal region of Mcl-1, a member of the bcl-2 protein family containing BH2, anchors Mcl-1 in the endoplasmic reticulum localized by DAD1, and inhibits cell apoptosis[25]. Under normal circumstances, NF-κB dimer-binding inhibitor of κB (IκB) exists in the cell nucleus in an inactive form. When cells are stimulated, IκB protein is first released from NF-κB through IκBa phosphorylation and ubiquitin degradation, causing NF-κB nuclear ectopic and IκBa and other gene transcription activation[26]. DAD1 has been proven to be the downstream target and inducible gene of NF-κB, and IκBa inhibitor can reduce the expression of DAD1 in cells[27].

3.3 Expression and significance of DAD1 in tumorsThe occurrence and development of tumors are closely related to apoptosis. The infinite growth of tumor cells is the result of the inhibition of tumor cell apoptosis. Therefore, the obstruction of apoptosis can lead to the occurrence and development of tumors. As an important factor against apoptosis, DAD1 can inhibit apoptosis and is highly expressed in a variety of cancers. Through co-expression network analysis, Wangetal.[28]found that DAD1 is an important molecule in high-grade urothelial bladder cancer, and the expression of DAD1 was also found in invasive bladder cancer. Trueetal.[29]detected 131 benign prostate tissues and 306 prostate cancer tissues by immunohistochemical staining, and found that DAD1 was highly expressed in cancerous epithelium, and the expression level of DAD1 was significantly correlated with the clinicopathological grading of prostate cancer, so it can be used as a diagnostic marker of prostate cancer. By qRT-PCR and immunohistochemical staining, Nilsson[30]found that DAD1 was expressed in ileal carcinoids, and can be used as a candidate gene for targeted therapy of ileal carcinoids. Kulkeetal.[31]also adopted immunohistochemical staining to evaluate the expression of DAD1 protein in small bowel carcinoid tumors, and found that the degree of DAD1 staining in tumor cells was always higher than that of normal small intestinal mucosa and adjacent stroma. Sézary syndrome is a T-cell lymphoma that originates in the skin. Prasad[32]found that DAD1 may be involved in the pathogenesis of the disease in Sézary syndrome; DAD1 is an endogenous inhibitor of apoptosis protein; somatic cell loss at 14q11.2 site would trigger cell apoptosis. Through qRT-PCR and Western blot detection, Liu Bo[33]found that DAD1 was highly expressed in renal cancer cells, and knockdown of DAD1 inhibited the growth and migration of kidney cancer cells, while overexpression of DAD1 promoted the proliferation of cancer cells, confirming that DAD1 promoted the proliferation and invasion of kidney cancer cells. Jiang Qinghuaetal.[34]analyzed the data of 371 cases of gastric cancer using the Cox model, and found that low expression of DAD1 and gastric cancer radiotherapy sensitivity were statistically significant, and the survival rate of patients with low expression of DAD11 improved after receiving radiotherapy; DAD1 can be used as a potentially effective molecular marker for precise radiotherapy of gastric cancer.

4 Conclusions and outlook

With the deepening of IL-34 in disease research, it is found that IL-34 can be used as a tumor marker, which may be related to tumor invasion and metastasis; it can also be used as an independent prognostic evaluation factor, and plays an important role in tumors. The research of IL-34 has provided new directions for many tumor diseases. Similarly, DAD1 inhibits cell apoptosis, promotes cell proliferation, and plays an important role in the study of tumor pathogenesis, targeted therapy, and prognosis evaluation.

From a large number of relevant literatures, it is found that IL-34 has only been subjected to serum enzyme-linked immunosorbent assay and tissue immunohistochemical staining in gastric cancer. The research method is single, and the sample size is small; the mechanism of IL-34 in gastric cancer has not been studied at the cellular level and tissue level. For DAD1, the relationship with gastric cancer radiotherapy sensitivity in gastric cancer has only studied, cell-level and tissue-level research in gastric cancer have not been conducted. In gastric cancer, whether the influence of the inflammatory factor IL-34 on tumors is related to the regulatory apoptotic factor DAD1 has not been reported, and whether it induces the expression of DAD1 is still unclear. However, IL-34 can regulate multiple signaling pathways and the production of IL-6 and IL-8 in human lung fibroblasts through NF-κB. DAD1 has been proven to be the downstream target of NF-κB, and IkBa inhibitor reduces the expression of DAD1 in cells. In-depth study of the relationship between the inflammatory factor IL-34 and the apoptosis factor DAD1 in gastric cancer is of great significance. It is possible to find the connection point between the IL-34-mediated signaling pathway and DAD1 in gastric cancer and the new mechanism of IL-34 leading to the occurrence and development of gastric cancer. It can provide a theoretical basis for the early diagnosis of gastric cancer and open up a new way of gastric cancer treatment.