2.1 IL-6

IL-6 is a multifunctional cytokine mainly expressed in the basal cells of prostate epithelium and is also commonly present in glandular cells of advanced prostatic intraepithelial neoplasia and prostate cancer. In the tumor microenvironment, IL-6 affects the occurrence and development of tumors by regulating tumor cell proliferation, apoptosis, differentiation, metabolism, angiogenesis, and metastasis processes. Research [9] has shown that the expression level of IL-6 is elevated in CRPC patients with drug resistance and metastasis, which is associated with poor prognosis and reduced survival rate in prostate cancer patients. Downregulation of IL-6 expression level suggests a good prognosis. The method of obtaining NE like cell lines cultured in vitro can be obtained not only through the commonly used ADT therapy induction, but also through IL-6 overexpression stimulation. In the past, most researchers believed that the role of IL-6 in NEPC differentiation was mediated by its involvement in related signaling pathways such as Janus kinase/signal transducer and transcription factor 3 (JAK/STAT3) pathway, phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) pathway. The pathways of mitogen activated protein kinases/extracellular regulated protein kinases (MEK/ERK) and mitogen activated protein kinases/extracellular regulated protein kinases (MAPKs) have been studied, the results are correlated and show that IL-6 participates in the process of inducing NEPC. However, with further research, it has been found that in addition to the functions of these signaling pathways, IL-6 itself has the ability to induce NE like differentiation. Natani et al. [10] found that the AMP activated protein kinase/silencing information regulator of transcription 1 (AMPK/SIRT1) signaling pathway, mediated by p38 mitogen activated protein kinase (p38MAPK), can induce NE like differentiation of LNCaP (Lymph node-derived carcinoma of the prostate) prostate cancer cells under reduced androgen conditions. The relationship between IL-6 (interleukin-6 AMPK) and AMPK (adenylate activated protein kinase) is mainly reflected in their synergistic effect on metabolic regulation, especially in the interaction between skeletal muscle glucose metabolism and fat breakdown processes. Antiandrogenic drugs activate tumor macrophages during treatment, leading to increased secretion of IL-6 and inducing NEPC. IL-6 can also participate in the activation of the JAK-STAT3 pathway through autocrine or paracrine mechanisms, increasing its own expression to induce NEPC differentiation [11]. In the latest study, Ji et al. [12] found in vitro experiments on NEPC mouse models that the antihistamine drug ketotifen reverses lineage switching by targeting the IL-6/STAT3 pathway, effectively inhibits NE differentiation, and reduces tumor cell viability.

Research has found that in addition to the aforementioned stimulus induced mechanisms, IL-6 can also regulate transcription factors to achieve NE differentiation. Suppressoreelement-1-silencing transcription factor (REST) is a silencing transcription factor that can inhibit NE gene expression. Zhu et al. [13] found that IL-6 can inhibit the expression of REST and degrade and inactivate REST to eliminate factors that hinder NE cell differentiation. In addition, autophagy regulation, which has attracted much attention in prostate cancer ADT and chemotherapy resistance, has an impact on IL-6 induced NE differentiation. Chang et al. [14] found through cell counting and Western blot analysis that treating LNCaP cells with IL-6 not only induced NE like differentiation of tumor cells, but also activated a specific autophagy pathway, which was further silenced by Beclin1 and Atg5.

The MTT (Methylthiazolyldiphenyl-tetrazolium bromide) colorimetric assay confirmed that the autophagy pathway reduces apoptosis in NE-like cells. If the autophagy pathway is inhibited, the number of apoptotic NE-like cells significantly increases, indicating that autophagy is the fundamental cause of chemotherapy resistance in NE-like cells. In summary, it further proves that IL-6 is one of the important cytokines that induce NE differentiation in prostate cancer cells.

2.2 IL-8

IL-8 is a cytokine secreted by Th1 cells, which can be highly expressed in various human tumor cells. It can act on different cells, promote inflammatory response, stimulate blood vessel formation, promote mitosis, regulate host immune function, etc. It is closely related to the occurrence and development of various inflammatory diseases, tumors, and immune diseases. In prostate cancer patients, serum IL-8 levels increase with disease progression [15]. Patel et al. [16] found that IL-8 was not expressed in the prostate cancer cell line LNCaP but was expressed in cell lines cultured in androgen deficient medium, suggesting a preliminary association between IL-8 and prostate cancer drug resistance. There are two types of epithelial cell populations in prostate cancer: (1) secretory tumor cells, which account for the majority of tumor cells; (2) a few scattered NE cells. After hormone therapy, the number of NE cells increases, especially when prostate cancer is in the androgen dependent phase. These NE tumor cells may contribute to the androgen independent growth of prostate cancer. In 2004, Lee et al. [17] further explored the expression and effects of IL-8 in LNCaP cells. Through cell migration and chromatin immunoprecipitation methods, they found that IL-8 is a promoter of androgen independent transformation in prostate cancer cells and may be related to IL-8-induced tyrosine kinase signaling and androgen receptor activation, but the specific mechanism is still unclear. The main binding receptor of IL-8 is CXC chemokine receptor 1 (CXCR1).

CXCR1 and CXC chemokine receptor 2 (CXCR2), two IL-8 receptors widely expressed in various types of prostate cancer cells, among which CXCR2 is expressed in Purkinje cells and various neurons in the human body, and plays an important role in promoting neuronal proliferation and migration when combined with IL-8. In 2005, Huang et al. [18] further demonstrated that NE cells are an independent source of IL-8 in prostate cancer. When combined with CXCR2, NE cells induce androgen independent proliferation of tumor cells through autocrine effects and stimulate more NE cell differentiation. This conclusion was once again validated in the study by Li et al. [19] in 2013. According to reports, activation of the MAPK/ERK pathway in the tyrosine kinase signaling pathway of LNCaP cells can induce NE differentiation and increase NSE expression. IL-8 is one of the upstream regulatory factors of this pathway [20], but there is still no direct evidence to suggest the specific role of IL-8 on the MAPK/ERK pathway in prostate cancer cells. Forkhead box A1 (FOXA1) is a transcription factor. In 2017, Kim et al. [21] found that FOXA1 can inhibit the MAPK/ERK pathway by reducing ERK phosphorylation, thereby preventing NE like differentiation of prostate cancer cells. At the same time, gene expression analysis and Western blot analysis showed that the binding of FOXA1 to the IL-8 promoter sequence knocked out the expression of FOXA1, which may be one of the mechanisms by which IL-8 participates in causing NEPC.

3.1 Epidermal growth factor (EGF)

EGF is one of the important mitogenic factors in prostate cells, which is overexpressed in tumor cells to promote proliferation, differentiation, and metastasis. Its specific binding receptors include not only EGFR (Epidermal Growth Factor Receptor), but also members of the ErbB receptor family, such as ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4). DiLorenzo et al. [22] observed increased expression of EGF and EGFR in prostate cancer cells. Research [23] found that overexpression of EGF increased the expression of NSE. This indicates that EGF may induce increased differentiation of NE-like cells in prostate cancer cells, but there are few studies reporting the direct mechanism of EGF action. Most of them induce NE differentiation by binding to receptors and enhancing their expression. Research [24] has shown that using the PI3K/Akt (Phosphatidylinositol 3-kinase/Protein Kinase B) inhibitor LY294002 to observe cell apoptosis in LNCaP cell lines through flow cytometry, the number of apoptotic cells decreased after the addition of EGF, and a sharp increase in ErbB2 levels was observed. After a period of time, LNCaP cells began to exhibit NE like differentiation, during which the levels of NE differentiation markers increased parallel to ErbB2 levels. This result indicates a clear positive correlation between EGF binding to ErbB2 overexpression and NE differentiation. Zhang et al. [25] also found that activating the EGFR/ERK signaling pathway can promote angiogenesis and gene mutation induced NEPC formation, increasing tumor metastasis and invasiveness.

3.2 Transforming growth factor beta (TGF β)

The TGF β superfamily contains a group of structurally related cytokines, including TGF β, growth and differentiation factors (GDF), and bone morphogenetic protein (BMP). TGF β can be detected in almost all tumor cells and is involved in regulating various cell growth processes, including cell proliferation, invasion, apoptosis, epithelial mesenchymal transition, tissue homeostasis, immune suppression, and extracellular matrix remodeling. It is overexpressed under conditions of fibrosis, inflammation, cancer, and excessive proliferation, especially in glioblastoma where it is highly secreted and may be related to neuronal differentiation. In prostate cancer cells, TGF β transmits signals through the TGF β R Ⅱ (type II receptor subunit) on the cell surface to mediate the phosphorylation of TGF β R Ⅰ (type I receptor subunit), activating the classical TGF β SMAD2 pathway to control cell growth by inhibiting proliferation, inducing apoptosis, promoting migration and invasion [26, 27], and also obtaining lineage transformation ability by inducing demethylation of CD133 (Prominin-1) promoter. Dicken et al. [28] proposed that the interaction between TGF β signaling and AR signaling axis induces epithelial mesenchymal transition, interferes with the homeostasis of epithelial cell lines, and provides a key site for epithelial cell differentiation into tumor cells. At the same time, it increases the invasiveness and metastasis of the derived tumor, supports the later acquisition of NE phenotype by prostate tumor cells, increases castration resistance, and reduces the efficacy of drug therapy. Previous reports have shown that under IL-6 stimulation, cells can release a large amount of TGF β. Sagara et al. [29] added IL-6 to androgen deficient culture medium to cultivate LNCaP cells and release TGF β. Proteomic data and bioinformatics analysis revealed that TGF β SMAD2 signaling can activate p38MAPK to promote NED, while targeted TGF β therapy can prevent the progression of NED in LNCaP cells, suggesting that TGF β targeted therapy may become a new direction for NEPC treatment, but the relevant mechanisms still need further exploration.