EMP3: A promising biomarker for tumor prognosis and targeted cancer therapy

1. Introduction

The peripheral myelin protein 22 kDa (PMP22) gene family encompasses several members, including epithelial membrane protein1, 2, and 3 (EMP1, EMP2, EMP3), PMP22, p53 apoptosis effect related to PMP22 (PERP), lens fiber cell protein MP20 (also named LIM2), and brain cell membrane protein [1]. Genes within this family are implicated in cellular proliferation, migration, and invasion and have active roles in tumor development [2]. Members of the PMP22 family share significant structural similarities and homology, suggesting potential overlap in function and involvement in cancer. PMP22 proteins are hydrophobic and approximately 160 amino acids long, containing two extracellular domains, four transmembrane domains, and small intracellular regions [3]. The amino acid sequence homology of EMP3 with PMP22, EMP1, EMP2 and MP20 is 41%, 33%, 38% and 23%, respectively. EMP3, PMP22, and EMP2 share the same C-terminal residue, suggesting that this motif may have functional importance as it closely resembles the retrieval signal of endoplasmic reticulum proteins [4]. Within the EMP protein family, all members feature N-glycosylation sites and share a highly conserved transmembrane domain [3]. There are notable structural differences between EMPs. EMP3 lacks a functional site for phosphorylation by casein kinase 2, whereas EMP1 and EMP2 contain the target site. EMP2 and EMP3 share similar molecular structures, both featuring an N-terminal glycosylation site in their first hydrophilic domain and a protein kinase C (PKC) phosphorylation site, both of which are absent in EMP1. EMP2 does not contain an N-myristoylation region. Moreover, expression of PMP22 proteins varies across organs and tissues. EMP1 mRNA levels are high in the esophagus, followed by adipose tissue, and the gallbladder, while EMP1 protein levels are high in the stomach but low elsewhere. EMP2 is most commonly expressed in the lung, skin, and esophagus and least commonly expressed in the pancreas and brain. A higher transcript level of EMP2 is found in the fetal lung and kidney but not in the adult thymus or peripheral leukocytes. In contrast to EMP2, EMP3 mRNA levels are high in peripheral leukocytes of adults and relatively high in fetal lungs, liver, and kidneys [4].

Many studies have confirmed the relationship between PMP22 family genes and cancer. Cañellas et al. [5] used EMP1 as a marker for highly recurrent cells in colon cancer. The group tracked and selectively eliminated this cell population and found that eliminating cells with high EMP1 expression could prevent metastasis and recurrence of colorectal cancer. In prostate cancer, EMP1 is downregulated and believed to negatively regulate tumor cells through modulation of caspase-9 and vascular endothelial growth factor (VEGF) levels [6]. A reduction in EMP1 expression is also observed in other cancers, such as laryngeal cancer, oral squamous cell carcinoma, nasopharyngeal carcinoma, and gastric cancer. EMP1 overexpression was found to inhibit tumor cell proliferation in esophageal cancer. On the other hand, EMP1 levels are higher in non-small-cell lung carcinoma than in tissues from benign controls, possibly due to the aberrant activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway [7, 8, 9, 10, 11]. EMP2 has been identified as an oncogene in human endometrial and ovarian cancers, promoting tumor formation through focal adhesion kinase (FAK) and steroid receptor coactivator (SRC) activation [1]. A study quantifying EMP2 expression levels in breast cancer cell lines found a 2.56-fold higher expression in MDA-MB-231 cells than in MCF7 cells. Importantly, EMP2 outperformed epithelial cell adhesion molecule in identifying breast cancer cells in patient blood samples, suggesting its potential as a novel biomarker and target [12]. While patients with normal brain tissue typically have low and undetectable levels of EMP2 expression, these levels are significantly elevated in 95% of patients with glioblastoma (GBM). EMP2 enhances glioma tumor growth in vivo by promoting cell migration and invasion. EMP2 upregulates α v β 3 integrin surface expression and enhances focal activation [13]. EMP2 is one of the upregulated genes of isoflavones, and its overexpression inhibits lesion formation, in vitro apposition-dependent growth, and tumorigenicity in uroepithelial carcinoma in severely combined immunodeficient mice. Furthermore, EMP2 is an oncogene of uroepithelial carcinoma [14]. Another notable member of the PMP22 gene family is the PMP22 protein, also known as growth arrest-specific gene 3. Initially isolated from growth-inhibited fibroblast cell line (NIH3T3) cells, this protein is abundantly expressed in Schwann cells. In the context of breast cancer, patients with high expression of the PMP22 gene face a 3.47-fold higher risk of death and a 1.77-fold higher rate of recurrence. PMP22 can serve as an independent prognostic indicator for breast cancer [15]. In addition, PMP22 is frequently amplified and overexpressed in osteosarcoma, suggesting its potential designation as an oncogene [16].

Studies looking into EMP3 have been relatively limited, yet the protein’s unique characteristics warrant further investigation. The EMP3 gene is located on chromosome 19q13.3, a genomic locus characterized by high gene density. This region carries mediator29 (MED29), formerly known as IXL, polymerase-associated factor1 (PAF1), dual specificity tyrosine phosphorylation regulated kinase1B (DYRK1B), p21-activated kinase4 (PAK4), cyclin E3, which have undergone functional validation and are suggested to be putative amplicon targets [17, 18, 19]. Alaminos et al. [20] compared the expression profiles of 89 neuroblastoma tumors with that of benign ganglioneuromas through microarray analysis. The group found EMP3 was the only gene whose expression is down-regulated by more than threefold and contains CpG islands. While the exact biological function of EMP3 remains incompletely understood, some studies have employed EMP3 as a marker for tumor proliferation. EMP3 is associated with various cellular activities, including cell migration, antiviral and antitumor responses, and inflammatory processes, thereby influencing disease onset and progression [21, 22]. Notably, EMP3 is considered a tumor suppressor gene in certain tumor types and has also emerged as a potential target for cancer therapy [23, 24]. Therefore, a comprehensive investigation into the role of EMP3 in the context of tumorigenesis is timely.

OPEN IN VIEWER

The EMP3 gene is located on human chromosome 19q13.3 and mouse chromosome 7. The protein comprises 163 amino acids, encoding a hydrophobic integral membrane protein with an approximate molecular weight of 18 kDa. Its primary structural components comprise four transmembrane topological domains, two extracellular loops, and a short intracellular loop [25]. The two extracellular loops are 18 and 41 amino acids long, and the intracellular loop is composed of 13 amino acids. The N- and C-terminal domains are located within the cell [24, 26]. EMP3 is categorized as a tetraspanin membrane protein, known for its association with integrins and role in regulating integrin-mediated functions such as proliferation, survival, migration, and tumor invasion [27]. As the small size of the intracellular loop makes it unlikely to be involved in signaling pathways, the extracellular loops are free to interact with proteins and molecules, potentially holding functional significance. Post-translational modifications, including glycosylation and myristoylation, frequently occur on amino acid residues on the cell surface. Myristoylation, a lipid modification crucial for membrane targeting and signal transduction in various human tumors, is catalyzed by the enzyme N-myristoyltransferase, thereby playing a crucial role in signal transduction across different tumors [1] (Fig. 1). EMP3 contains a PKC phosphorylation site. It is well established that abnormal PKC activity is associated with cancer, as this kinase plays critical roles in cell proliferation, survival, migration, and apoptosis. The expression of the three PKC isoforms is regulated by various oncogenes such as rat sarcoma virus oncogene (RAS) and myelocytomatosis viral oncogene homolog (MYC). Different PKC isoforms mediate cell transformation, revealing its participation in cancer signaling pathways [28, 29]. Consequently, EMP3 may exert influence over the initiation and progression of tumors through its interactions with the aforementioned genes.

3. Expression of EMP3

EMP3 is expressed as both a 1.0-kb and a 1.4-kb mRNA in most tissues, with higher expression detected in the bone marrow, smooth muscle, uterus, bladder, lung, and ovary. There is lowerr expression found in tissues such as the thyroid, parathyroid gland, testis, and cerebral cortex [3]. In adult peripheral nerves, EMP3 can be detected in the axon core but not in the myelin sheath. It is also commonly expressed in Langerhans cells, monocytes, macrophages, granulocytes, and melanocytes [30, 31]. EMP3 protein is present on the surface of red blood cells and essential for the expression of the MAM blood group antigen. Individuals lacking the MAM antigen, known as MAM-negative individuals, carry inactivating mutations in the EMP3 gene. Experimental knockouts of the EMP3 gene in a human erythroid cell line resulted in the elimination of MAM antigen expression. MAM-negative erythrocytes show increased proliferation in vitro, revealing a regulatory role in erythropoiesis and cell production. Additionally, EMP3 is known to interact with and stabilize the receptor CD44 on the plasma membrane of erythrocytes. This association enables CD44 incorporation into signaling complexes such as lipid rafts [24].

4. Effect of EMP3 on immune cells and signaling pathways

5. Effect of EMP3 on cell proliferation and apoptosis

EMP3 negatively correlates with a cluster of genes associated with the S phase of the cell cycle, including cyclin E2 (CCNE2), cyclin-dependent kinase 2 (CDK2), proliferating cell nuclear antigen (PCNA), replication factorC4 (RFC4), minichromosome maintenance 4 (MCM4), and GINS1 [22]. Studies suggest a significant relationship between EMP3 expression and the tumor cell cycle. Downregulation of EMP3 has been linked to decreased levels of phosphorylated Akt (p-Akt), phosphorylated epidermal growth factor receptor (p-EGFR), and p-ERK. Additionally, the absence of EMP3 expression decreases TGF- β , nuclear factor kappa-B(NF- κ B), and PI3K/Akt signaling, resulting in reduced cell proliferation and migration. Moreover, EMP3 has been found to sensitize cells to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and staurosporine [34]. Notably, EMP3 overexpression in urothelial cancer cells substantially elevates the proportion of cells in S phase while increasing the Ki67 index. Inhibiting EMP3 expression in liver cancer cells leads to growth inhibition in vivo, reduced cell proliferation in vitro, and cellular arrest in the G1 phase. Studies conducted in breast cancer have found that EMP3 knockdown upregulates cyclin-dependent kinase 2, and proliferating cell nuclear antigen while downregulating P21, a negative modulator of the S-phase. EMP3 knockdowns have been shown to increase the fraction of S-phase cells and EdU-positive cells, preventing breast cancer cells from entering the S-phase. In addition, EMP3 inhibits DNA damage repair and interferes with cancer stem cell properties in breast cancer. EMP3 regulates autophagy, stem cell-like properties, DNA replication, and DNA damage repair through inhibition of the PI3K/Akt pathway. Regulation of DNA replication and damage repair by EMP3 is associated with N6-methyladenosine reader YTHDC1 [22]. Furthermore, EMP3 exhibits rapid and sustained expression in peripheral nerves distal to injuries, whereas PMP22 expression is reversibly downregulated. During nerve regeneration, EMP3 participates in Schwann cell proliferation during the early stages and has been found to have a prolonged impact as well [47]. EMP3 also affects cell migration and apoptosis. EMP3 interacts with purinergic P2X₇ receptors at the cell membrane. This interaction has been shown to trigger apoptosis, whereas EMP3 overexpression alone can induce membrane blebbing. Studies have also indicated that EMP3 depletion leads to an upregulation of the apoptosis marker caspase-3 in tumor lysates. In gallbladder cancer, elevated EMP3 levels inhibit cancer cell migration and promote apoptosis by increasing caspase-3 and caspase-9 expression levels [3, 48].

6. EMP3 and cancers

EMP3 is strongly involved in cancer development and progression, playing different roles in promoting or inhibiting cancer growth across different diseases. While it may act as a tumor-suppressive protein in esophageal squamous cell carcinoma and gallbladder cancer, EMP3 functions as a tumor promotor in endometrial cancer and hepatocellular carcinoma. The function of EMP3 is still controversial in many cancers. In summary, EMP3 is involved in several pathways: methylation of dependent tumor suppressors, interference with the MAPK/ERK axis affects tumor progression, action as an oncogene by regulating the HER-2 pathway, activation of the PI3K/Akt signaling pathway; regulation of TGF- β /Smad2/3 signaling in tumor cell proliferation and survival; promotion of tumor progression by recruiting TAMs and inhibiting intra-tumor T cell infiltration, etc. We found that EMP3 seems to be closely related to the MAPK/ERK and the PI3K/Akt signaling pathways, suggesting a potentially greater impact on cancers associated with these signaling pathways. Furthermore, the extracellular domain of EMP3 contains myristoylation regions, which are essential for membrane targeting and signaling in several human tumors. Myristoylation, catalyzed by NMT on glycine residues exposed during co-translational, is upregulated in several human cancers, including colorectal, gallbladder, and brain cancers, as well as in oral SCC [83, 84]. Knocking down EMP3 reduced p-AKT, p-ERK, and p-EGFR levels and inhibited cell proliferation. It also made the cells more sensitive to apoptosis induced by TRAIL and astrosporin. Therefore, EMP3 may be involved in the regulation of receptor tyrosine kinase-mediated mitosis [34]. Regulation of EMP3 expression in tumors, alongside the use of other drugs, holds promise as a novel direction of therapy options.

There are limited reports on EMP3 and tumor therapy, however, some studies have noted EMP the relationship between EMP3 and drug resistance. EMP3 can act downstream of CD13 and regulate the PI3K/Akt pathway, hereby promoting EMT and cisplatin resistance in gastric cancer cells [71]. Similarly, in primary gastric cancer, EMP3 can function as a novel downstream effector target of TWIST1/2 and as a modifier gene involved in cisplatin resistance [70]. EMP3 modulates a variety of membrane receptor activities associated in IDH-WT GBM, including increasing GBM-associated receptor tyrosine kinase activity and promoting the expression, stability, or activity of several extracellular matrix receptors and matrix degraders. In addition, EMP3 interacts with purinergic receptor P2RX7, which has received increasing attention for its role in GBM. EMP3 regulates the trafficking and enhancing the stability of EGF, and EMP3 deletion renders tumor cells more sensitive to the pan-kinase inhibitor staurosporine and osimertinib, an EGFR inhibitor. The stabilizing effect of EMP3 provides tumor cells with an additional defense barrier against targeted kinase inhibition. Therefore, EMP3 may be an interesting target for therapeutic intervention [53]. In primary breast cancer, studies found the downregulation of EMP3 by miR-765 mimics or RNA-EMP3 resulted in inhibition of homologous recombination repair of DNA double-strand breaks through negative regulation of BRCA1 and RAD51 expression. Consequently, this led to a reduction in the resistance of breast cancer cells to DNA-damaging drug Adriamycin [22, 42]. As previously described, patients with overexpression of EMP3 in upper urinary tract urothelial carcinoma exhibited significantly poorer overall survival and metastasis-free survival [37]. This suggests that EMP3 serves as an important prognostic marker for patients who may benefit from more aggressive therapy. In addition, the co-expression of EMP3 and HER-2 is one of the most important indicators for progression-free and metastasis-free survival of patients, as there is a functional interaction between the two proteins. Therefore, EMP3 can be used as an innovative target for targeted therapy in cancer treatment.

The development of monoclonal antibodies targeting EMP family members is challenging due to the clinical and pathologic heterogeneity and biological complexity of cancers. Some progress has been made in the research and development of anti-EMP monoclonal antibodies. Research shows that anti-EMP2 diabody treatment demonstrated significant antiproliferative effects through enhancing caspase-3-mediated apoptosis in endometrial adenocarcinoma cell lines. It proved successful in reducing tumor size and promoting cell death in human endometrial cancer xenografts [85]. Anti-EMP2 IgG1 improves endometrial cancer survival through reduction in cancer stem cells [86]. In a model of transplanted ovarian cancer tumors, anti-EMP2 bivalent antibody therapy reduced the proliferation of ovarian cancer cells and promoted apoptosis [87]. In addition, treatment of breast cancer with anti-EMP2 IgG1 promoted tumor cell death and inhibited tumor cell invasion. In a mouse model of breast cancer, anti-EMP2 treatment reduced the number of spontaneous metastases from xenografts expressing high levels of EMP2 [88]. So far, there has been no report on the use of anti-EMP3 in clinical trials. EMP3 is of great significance in tumor drug resistance and evaluation of disease prognosis. Therefore, it is necessary to further study the specific role of EMP3 and its application value in the clinical setting.

8. Conclusion and future perspective

Rapid advancements in genomics research have catalyzed the emergence of molecular targeted therapy as a promising avenue for cancer treatment and facilitated the discovery of numerous novel tumor biomarkers. Among these proteins, EMP3, a member of the epithelial membrane protein family, has shown expression across various tissues and appears to regulate signaling pathways related to cell proliferation and cell–cell interactions. A notable advantage of using EMP3 as a potential drug target is its location on the cell membrane and its involvement in diverse signaling pathways, making it physically accessible for drug targeting. However, the precise biological function of EMP3 remains incompletely understood. Studies suggest that the protein acts as an oncogene in several tumors while suppressor function in others, highlighting the complex nature of EMP3-mediated pathways in tumorigenesis. Further research is necessary to understand the patterns of EMP3 expression and activity. Ultimately, EMP3 holds promise as a potential prognostic indicator across various cancers. There is a need to further elucidate the potential associations between EMP3 expression and tumor immunity. Therefore, it is crucial to further investigate the interplay between EMP3-mediated immune dysregulation and tumor progression, with a focus on understanding the underlying molecular mechanisms.