Human Epididymis Protein 4 Protein Research Articles

Three-Dimensional Structure of Human Epididymis Protein 4 (HE4): A Protein Modelling of an Ovarian Cancer Biomarker Through In Silico Approach

The Human Epididymis Protein 4 (HE4) biomarker has been extensively investigated for its potential in diagnosing ovarian cancer (OC). For the application of diagnostic techniques and drug delivery, it is crucial to understand the protein tertiary structure. However, the Protein Data Bank (PDB) does not currently contain the three-dimensional (3D) structure of HE4. Therefore, an in silico analysis was conducted to model the HE4 protein using AlphaFold, I-TASSER, and Robetta servers, with the sequence retrieved from UniProt (ID: Q14508). These three servers employed deep learning algorithms, threading templates, and de novo methods, respectively. Subsequently, Molecular Dynamics (MD) simulation using the GROMACS software package improved each 3D structure model, resulting in optimized and refined structures: RF1, RF2, and RF3. PROCHECK and ERRAT programs were employed to assess the structure quality. The Ramachandran plots from PROCHECK indicated that 100% of residues were within the allowed regions for all servers except for I-TASSER. For the refined structures, RF1 and RF3, all residues were concentrated within the allowed regions. According to the ERRAT program, the RF1 model exhibited the highest overall quality factor of 97.701, followed by RF3 and AlphaFold models with scores of 94.643 and 93.750, respectively. After these validations, RF1 emerged as the most accurately predicted 3D structure of HE4 and has one tunnel identified by CAVER 3.0 tool that facilitates the transportation of small particles to the active site, supported by FTsite and PrankWeb binding site predictions. This model holds potential for various computational studies, including the development of OC diagnostic kits. It will enhance our comprehension of the interactions between the protein and other biomolecules.

Effect of human epididymis protein 4 on hyperoxia-induced bronchial dysplasia in newborn rats

Objective The study aimed to elucidate the role and the underlying mechanism of human epididymis protein 4 (HE4) in the pathogenesis of hyperoxia-induced bronchial dysplasia in newborn rats. Methods Forty neonatal Sprague–Dawley (SD) rats were separated into two groups: a normal control group (20.8% oxygen concentration) and a hyperoxia-induced group (85% oxygen concentration). Three time intervals of 24 h, 3 days and 7 days were chosen for each group. Haematoxylin–eosin staining was used to identify the pathological alterations in the lung tissue of the SD rats. Enzyme-linked immunosorbent assay was used to evaluate plasma protein levels. Real-time reverse transcription polymerase chain reaction was used to determine messenger RNA (mRNA) expression. Results In newborn SD rats, hyperoxia intervention within 7 days may result in acute lung damage. In the plasma and tissue of newborn SD rats, hyperoxia induction may raise levels of HE4, matrix metalloproteinases (MMP) 9 and tissue inhibitors of metalloproteinases (TIMP) 1. We discovered that the HE4 protein activates the phosphorylation of extracellular regulated protein kinases (ERK) and p65, activates the downstream MMP9 signalling pathway, inhibits MMP9 mRNA expression, inhibits protein activity, reduces type I collagen degradation, increases collagen secretion and promotes matrix remodelling and fibrosis in neonatal rat primary alveolar type II epithelial cells by overexpressing and silencing the HE4 gene. Conclusion Through the ERK, MMP9 and TIMP1 signalling pathways, HE4 mediates the pathophysiological process of hyperoxia-induced lung damage in rats. Lung damage and lung basal remodelling are mediated by HE4 overexpression.

Diagnostic value of cerebrospinal fluid human epididymis protein 4 for leptomeningeal metastasis in lung adenocarcinoma.

The diagnosis of lung adenocarcinoma (LUAD) leptomeningeal metastasis (LM) remains a clinical challenge. Human epididymis protein 4 (HE4) functions as a novel tumor biomarker for cancers. This study aimed to assess the diagnostic value of cerebrospinal fluid (CSF) HE4, and combined with CEACAM6, for LUAD LM. The CSF HE4 protein level was measured in two independent cohorts by electrochemiluminescence. Test cohort included 58 LUAD LM patients, 22 LUAD patients without LM (Wiot-LM), and 68 normal controls. Validation cohort enrolled 50 LUAD LM patients and 40 normal controls, in parallel with Wiot-LM patients without brain metastases (19 Wiot-LM/BrM patients) or with BrM (26 BrM patients). The CSF level of CEA, CA125, CA153, CA199, CA724, NSE and ProGRP of these samples was measured by electrochemiluminescence, whereas the CSF CEACAM6 level was detected by enzyme-linked immunosorbent assay (ELISA). In addition, the serum level of these biomarkers was detected by same method as CSF. The level of HE4 or CEACAM6 in CSF samples from LUAD LM patients was significantly higher than those from normal controls and Wiot-LM patients. The HE4 or CEACAM6 level in CSF was higher than that in serum of LM patient. The CSF HE4 or CEACAM6 level for distinguished LM from Wiot-LM showed good performance by receiver-operating characteristic analysis. The better discriminative power for LM was achieved when HE4 was combined with CEACAM6. In addition, the CSF HE4 and CEACAM6 level showed little or no difference between Wiot-LM/BrM and BrM patients, the BrM would not significantly influence the HE4 or CEACAM6 level in CSF. The diagnostic power of CSF CA125, CA153, CA199, CA724, NSE and ProGRP for LUAD LM were not ideal. The combination with HE4 and CEACAM6 has the promising application for the diagnosis of LUAD LM.

Identification and Characterization of Aptamers Targeting Ovarian Cancer Biomarker Human Epididymis Protein 4 for the Application in Urine.

Ovarian cancer is the deadliest gynecological cancer. With non-specific symptoms of the disease and the lack of effective diagnostic methods, late diagnosis remains the crucial hurdle of the poor prognosis. Therefore, development of novel diagnostic approaches are needed. The purpose of this study is to develop DNA-based aptamers as potential diagnostic probes to detect ovarian cancer biomarker Human epididymis protein 4 (HE4) in urine. HE4 is a protein overexpressed in ovarian cancer, but not in healthy or benign conditions. With high stability and diagnostic value for detection of ovarian cancer, urine HE4 appears as an attractive non-invasive biomarker. The high-affinity anti-HE4 DNA aptamers were selected through 10 cycles of High Fidelity Systematic Evolution of Ligands by EXponential enrichment (Hi-Fi SELEX), a method for aptamer selection based on digital droplet PCR. The anti-HE4 aptamers were identified using DNA sequencing and bioinformatics analysis. The candidate aptamer probes were characterized in urine for binding to HE4 protein using thermofluorimetry. Two anti-HE4 aptamers, AHE1 and AHE3, displayed binding to HE4 protein in urine, with a constant of dissociation in the nanomolar range, with Kd (AHE1) = 87 ± 9 nM and Kd (AHE3) aptamer of 127 ± 28 nM. Therefore, these aptamers could be promising tools for application in diagnostics and future development of urine tests or biosensors for ovarian cancer.

Exploring the relationship between preeclampsia and human epididymis protein 4

Background/Aim: The level of human epididymis protein 4 (HE4), a glycoprotein and protease inhibitor, increases under many malignancies and inflammatory conditions. HE4 is also associated with cell invasion, migration, and adhesion. In this study, we compared the HE4 protein levels in pregnant patients with preeclampsia to healthy pregnant and non-pregnant individuals with the aim of finding a biomarker that can be used to recognize preeclampsia. Methods: Our study is a prospective case control study and included 20 pregnant women with preeclampsia, 20 pregnant women without preeclampsia, and 20 healthy non-pregnant women (the control). The participants’ serum HE4 levels were analyzed statistically. Results: Data analysis revealed that the mean HE4 levels were significantly lower in the preeclampsia group than in the other two groups (P = 0.002). Mean HE4 protein levels were also lower in the non-pregnant women than in the pregnant women without preeclampsia; however, this difference was not significant. Conclusion: It is difficult to predict preeclampsia, and there is not any sensitive or specific biomarker for determining the condition. This study may support that HE4 protein may be useful and significant in predicting preeclampsia. The results we achieved provide proof that HE4 levels could be a potential biomarker for preeclampsia. Many more comprehensive studies are needed to support the association between HE4 protein and preeclampsia.

Abstract P4-07-22: Evaluation of human epididymis protein 4 serum and tissue expression in ductal carcinoma in situ of the breast

Abstract Background: Human epididymis protein 4 (HE4), also known as WFDC2, is a member of whey acidic protein (WAP) family with a presumptive role in natural immunity. HE4 expression has been increased in many cancer types, particularly in gynecologic and pulmonary cancers. HE4 has been identified as an important serum biomarker in the diagnosis and monitoring of epithelial ovarian cancer. Recent researches have demonstrated that HE4 levels in serum and tissues are elevated in patients with breast cancer and HE4 is thought be involved in breast cancer progression. In contrast, expression of HE4 in ductal carcinoma in situ (DCIS) has not been well characterized yet. The present work was undertaken to evaluate serum and tissue levels of HE4 in DCIS and their correlations with clinicopathological features of DCIS. Materials and methods: Preoperative serum HE4 levels in 59 DCIS patients was analyzed using the ARCHITECT assay. Tissue microarray containing DCIS and adjacent normal tissues from the same cohort were tested for HE4 mRNA and protein by RNAscope in situ hybridization (ISH) and immunohistochemistry analysis, respectively. Tissue microarray containing only DCS tissues from 41 patients were also tested for HE4 mRNA and protein. To validate prognostic potential of HE4 in breast cancer patients, the BreastMark database was used. Results: HE4 levels in 59 DCIS patients ranged from 23.5 to 86.3 pmol/L (mean ± SD: 39.4 ± 11.9). Based on the cutoff level, there were no abnormal cases for HE4. Serum HE4 levels did not differ according to clinicopathological parameters except for menopause status. RNAscope ISH and immunohistochemistry confirmed an increase in HE4 in DCIS tissues compared with their corresponding normal tissues. Spearman’s correlation analyses revealed no significant correlation between serum and tissue levels of HE4. Among 100 DCIS tissues, there was a positive correlation between HE4 mRNA ISH scores and HE4 immunohistochemical staining scores (r = 0.771, P < 0.001). High mRNA and protein expression of HE4 were observed in 25 of 99 (25.3%) and 34 of 99 (34.3%) cases, respectively. High mRNA HE4 expression was significantly associated with low stromal tumor infiltrating lymphocyte (TIL) density scores, ER positivity, HER2 negativity, and HR+/HER2- subtype. High protein HE4 expression was also significantly associated with absence of comedo-type necrosis, low stromal TIL density scores, ER positivity, HER2 negativity, and HR+/HER2- subtype. HE4 mRNA and protein expression did not correlate with recurrence of DCIS. In breast cancer patients, high HE4 expression was significantly associated with good survival in the overall group (HR = 0.838, P = 0.003, n = 2,652) and lymph node negative group (HR = 0.791, P = 0.029, n = 1,183). Conclusions: Our study revealed that serum HE4 is not elevated in patients with DCIS. High expression of HE4 mRNA and protein in DCIS tissues are associated with good clinicopathological characteristics, which warrant. further investigations. Citation Format: Ji Shin Leee, Nah Ihm Kim, Min Ho Park. Evaluation of human epididymis protein 4 serum and tissue expression in ductal carcinoma in situ of the breast [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-07-22.

Targeting and neutralizing human epididymis protein 4 by novel nanobodies to suppress ovarian cancer cells and attenuate cisplatin resistance

Human epididymis protein 4 (HE4) is a glycoprotein secreted by epithelial ovarian cancer (EOC) cells and is a novel and specific biomarker for diagnosing and prognosing EOC. Previous studies have shown that overexpression of HE4 is correlated with EOC tumorigenesis and chemoresistance. However, less has been reported regarding the direct effect of the secreted HE4 protein as an autocrine factor in EOC cells. Here, we investigated the molecular mechanism of the secretory form of HE4 on the growth of EOC cells by applying nanobodies with a targeted interaction of free HE4. Three anti-HE4 nanobodies were selected from an immune library by phage display. HE4 secreted by serum-free cultured OVCAR3 cells increased and was effectively neutralized by anti-HE4 nanobodies, which inhibited cell viability. Treatment with the anti-HE4 nanobody 1G8 decreased Bcl-2 expression and increased BAX, cleaved PARP, and p53 levels, resulting in apoptosis of OVCAR3 cells. Moreover, 1G8 significantly improved the cisplatin response of OVCAR3 cells. Our data suggest that secretory HE4 played a novel pro-survival autocrine role and was a target of the anti-HE4 nanobody to improve the therapeutic effects of cisplatin-based chemotherapy.

Enhanced Expression of Human Epididymis Protein 4 (HE4) Reflecting Pro-Inflammatory Status Is Regulated by CFTR in Cystic Fibrosis Bronchial Epithelial Cells

Decreased human epididymis protein 4 (HE4) plasma levels were reported in cystic fibrosis (CF) patients under CFTR potentiator ivacaftor therapy, which inversely correlated with lung function improvement. In this study, we investigated whether HE4 expression was affected via modulation of CFTR function in CF bronchial epithelial (CFBE) cells in vitro. HE4 protein levels were measured in the supernatants of CFBE 41o− cells expressing F508del-CFTR or wild-type CFTR (wt-CFTR) after administration of lumacaftor/ivacaftor or tezacaftor/ivacaftor, while HE4 expression in CFBE 41o− cells were also analyzed following application of adenylate cyclase activators Forskolin/IBMX or CFTRinh172. The effect of all of these compounds on CFTR function was monitored by the whole-cell patch-clamp technique. Induced HE4 expression was studied with interleukin-6 (IL-6) in F508del-CFTR CFBE 41o− cells under TNF-α stimulation for 1 h up to 1 week in duration. In parallel, plasma HE4 was determined in CF subjects homozygous for p.Phe508del-CFTR mutation receiving lumacaftor/ivacaftor (Orkambi®) therapy. NF-κB-mediated signaling was observed via the nuclear translocation of p65 subunit by fluorescence microscopy together with the analysis of IL-6 expression by an immunoassay. In addition, HE4 expression was examined after NF-κB pathway inhibitor BAY 11-7082 treatment with or without CFTR modulators. CFTR modulators partially restored the activity of F508del-CFTR and reduced HE4 concentration was found in F508del-CFTR CFBE 41o− cells that was close to what we observed in CFBE 41o− cells with wt-CFTR. These data were in agreement with decreased plasma HE4 concentrations in CF patients treated with Orkambi®. Furthermore, CFTR inhibitor induced elevated HE4 levels, while CFTR activator Forskolin/IBMX downregulated HE4 in the cell cultures and these effects were more pronounced in the presence of CFTR modulators. Higher activation level of baseline and TNF-α stimulated NF-κB pathway was detected in F508del-CFTR vs. wt-CFTR CFBE 41o− cells that was substantially reduced by CFTR modulators based on lower p65 nuclear positivity and IL-6 levels. Finally, HE4 expression was upregulated by TNF-α with elevated IL-6, and both protein levels were suppressed by combined administration of NF-κB pathway inhibitor and CFTR modulators in CFBE 41o− cells. In conclusion, CFTR dysfunction contributes to abnormal HE4 expression via NF-κB in CF.

Human epididymis protein4 promotes P‑glycoprotein‑mediated chemoresistance in ovarian cancer cells through interactions with Annexin II.

The aim of the present study was to investigate the effects of human epididymis protein 4 (HE4) on drug resistance and its underlying mechanisms. The associations among proteins were detected by immunoprecipitation and immunofluorescence assays. Then, stably transfected cell lines CAOV3-HE4-L and CAOV3-A2-L expressing HE4 short hairpin (sh)RNAs and ANXA2 shRNAs, respectively, were constructed. MTT assay, immunocytochemistry, western blotting, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and flow cytometry were employed to examine drug sensitivity, as well as the expression and activity of P-glycoprotein (P-gp). HE4 and P-gp in epithelial ovarian cancer tissues were assessed via immunohistochemistry. MicroRNAs that targeted the P-gp gene, ABCB1, were predicted using bioinformatics methods, and their expression was evaluated by RT-qPCR. The common signaling pathways shared by HE4, ANXA2 and P-gp were selected by Gene Set Enrichment Analysis (GSEA). The interaction of HE4, ANXA2 and P-gp were confirmed. P-gp expression was positively associated with HE4 and ANXA2 expression, respectively. Moreover, it was observed that there was no significant rescue of P-gp expression in CAOV3-A2-L cells following the administration of active HE4 protein. In addition, the expression of HE4 and P-gp in ovarian cancer tissues of drug-resistant patients were higher compared with that of the drug-sensitive group (P<0.05). Furthermore, the results revealed that hsa-miR-129-5p was significantly increased accompanied by decreased HE4 or ANXA2 expression and P-gp expression in CAOV3-HE4-L and CAOV3-A2-L cells. GSEA analyses disclosed that HE4, ANXA2 and P-gp genes were commonly enriched in the signaling pathway involved in regulating the actin cytoskeleton. These results indicated that HE4 promotes P-gp-mediated drug resistance in ovarian cancer cells through the interactions with ANXA2, and the underlying mechanism may be associated with decreased expression of hsa-miR-129-5p and dysregulation of the actin cytoskeleton signaling pathway.

May human epididymis 4 protein play a role in the etiopathogenesis of hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome?

Hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome is an extremely advanced form of preeclampsia. Currently, there is no parameter or marker to predict this syndrome; however, it is emphasized that vascular endothelial damage and abnormal immune responses can be the possible etiologies of HELLP syndrome. It is known that human epididymis protein 4 (HE4) is a protease inhibitor and previous studies have shown that HE4 protein levels are increased in many malignancies and inflammatory conditions. Considering that metalloproteinases may also play a role in endothelial damage, which is thought to be involved in the etiopathogenesis of HELLP syndrome, we thought that HE4 protein, which is a protease inhibitor, may be associated with vascular damage. We aimed to investigate the relationship between HELLP syndrome and HE4 protein and to identify a biomarker that can be utilized in the diagnosis of HELLP syndrome. In this study, 40 patients with HELLP syndrome and 40 healthy pregnant women with similar characteristics without HELLP syndrome were compared. When the results were evaluated, no statistically significant difference was found between serum HE4 protein levels in patients with HELLP syndrome and patients without HELLP syndrome in this study (p: 0.29). HE4 protein has no field of use in obstetrics yet. In our study, we aimed to find a new biomarker to identify patients with HELLP syndrome. However, we could not find any statistically significant difference in terms of HE4 protein levels between patients with and without HELLP syndrome. Our study is an important study as it is the first study aiming to use HE4 protein in obstetrics.

Evaluation of serum human epididymis protein 4 in patients with relapsing-remitting multiple sclerosis

So far, little is known about the properties of human epididymis protein 4 (HE4) in multiple sclerosis (MS). This type 4 glycoprotein belongs to a family of genes encoding proteins whose expression is associated with the process of spermatogenesis in the epididymis. The biological function of HE4 is not fully understood. Overexpression of HE4 has been found in several malignant tumors, particularly in ovarian cancer, as well as in mesothelioma, lung, endometrial, breast, and kidney cancers. To evaluate serum HE4 in patients with relapsing-remitting multiple sclerosis (RRMS) as compared to healthy controls. Fifty patients with RRMS undergoing first-line immunomodulatory treatment were enrolled in the prospective study. We analyzed correlations between serum HE4 levels and gender, age, disease duration, the Expanded Disability Status Scale (EDSS), annualized relapse rate (ARR), and magnetic resonance imaging (MRI) lesions. The patients from the study group had higher concentrations of HE4 than the subjects from the control group. Patients with EDSS > 2 had significantly higher concentrations of HE4. Positive correlations were found between HE4 concentrations and age as well as between HE4 concentrations and disease duration. No significant correlations were found between HE4 concentrations and EDSS or between HE4 concentrations and ARR. The results of the study indicate a novel aspect of the HE4 protein in the pathomechanisms of MS.

Human Epididymis Protein 4 (HE4) Reference Limits in Polish Population of Healthy Women, Pregnant Women, and Women with Benign Ovarian Tumors.

Objectives Defining precisely the normal range of HE4 protein is crucial for the proper interpretation of tumor marker results and for a more efficient diagnosis of ovarian malignancy. The aim of our study was to evaluate a reference limit of HE4 protein in a population to promote and facilitate the common use of HE4 protein assays. We also tried to identify potential association of HE4 levels with other conditions such as smoking, age, BMI, and creatinine levels. Methods Blood samples were collected from 617 patients divided into three groups: healthy, pregnant, and with benign ovarian tumors. Serum HE4 concentrations were measured following a standard procedure. HE4 reference ranges for each group and association of HE4 levels with BMI, creatinine, and smoking were investigated. Results HE4 reference limit for healthy patients equals 85 pmol/l, which becomes 73 pmol/l and 93 pmol/l for pre and postmenopausal subgroups, respectively. There is a statistically significant correlation between HE4 serum level and smoking (p = 0.000001) and there is no correlation with creatinine. But if we take into account age and smoking, in multivariate analysis, there is a correlation. For pregnant, the upper limit values of normal HE4 levels are 55 pmol/l (median = 40 pmol/l), 80 pmol/l (median = 43 pmol/l), and 106 pmol/l (median = 53 pmol/l) for the first, second, and third trimesters, respectively. Conclusions HE4 protein value strongly depends on the patient's age and smoking. The serum concentration of HE4 marker increases with the duration of pregnancy. Understanding the normal range of HE4 protein enables the correct interpretation of marker measurements. This may result in an earlier and more effective diagnosis of ovarian cancer.

HDAC3 positively regulates HE4 expression to promote ovarian carcinoma progression

ObjectiveTo identify the relationship between Histone deacetylase 3 (HDAC3) and Human epididymis protein 4 (HE4) and to explore the mechanisms underlying their effects on the malignant behaviors of ovarian carcinoma cells. MethodsThe expression levels of HDAC3 in ovarian carcinoma tissues were identified by immunohistochemistry, Western blot and real-time PCR. A wound healing assay, a Transwell assay and a CCK8 proliferation assay were used to assess the proliferation, invasion and metastatic capacities of ovarian carcinoma cells before and after transfection and HDAC3 protein treatment. HDAC3 and HE4 protein expression level in epithelial ovarian tissues were detected by immunohistochemistry, and the relationship between them was examined. ResultsHE4 was identified as an HDAC3-interacting protein. HDAC3 promotes ovarian carcinoma cell proliferation, invasion and migration by increasing the expression of HE4. HE4 and HDAC3 expression levels were significantly higher in malignant epithelial ovarian tissues than they were in benign and normal epithelial ovarian tissues. HDAC3 gene interference downregulated the expression of the PI3K/AKT signaling pathway-associated molecules P-PI3K/PI3K and P-AKT/AKT. ConclusionHDAC3 expression is higher in ovarian carcinoma and promotes ovarian carcinoma cell proliferation, invasion and migration. HDAC3 and HE4 binding activates the PI3K/AKT signaling pathway, enhances ovarian carcinoma and promotes ovarian carcinoma cell proliferation, invasion and migration. Therefore, inhibiting the relationship between HDAC3 and HE4 may therefore have potential therapeutic value in patients with ovarian carcinoma.

Diagnostic and prognostic role of HE4 expression in multiple carcinomas: A protocol for systematic review and meta-analysis.

Background:Human epididymis protein 4 (HE4) protein has garnered a great degree of interest as a complementary biomarker to carbohydrate antigen 125 (CA125), or even as an independent biomarker for monitoring, diagnosis, and prognostication of ovarian cancer. Its use is currently limited to ovarian cancer. Recent studies have suggested that it could also be used in other types of cancers.Methods:The Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) guidelines was used to design this meta-analysis protocol. The final study will also be conducted under the PRISMA guidelines for systematic reviews and meta-analyses. The core bibliographic database search will be carried out by 2 reviewers working individually, with each conducting an initial screening based on titles and abstracts. The shortlisted articles will be selected for review and statistical analysis based on predefined inclusion and exclusion criteria. Study characteristics, relevant clinicopathological characteristics and statistical data required for meta-analysis (hazard ratios [HRs] and 95% confidence interval [CIs) will be extracted and compiled into a MS Excel datasheet. Meta-analysis will be performed, using a random-effects model, and the results (pooled HR and 95% CI) will be presented in the form of a forest plot. Publication bias will also be assessed by use of Egger bias indicator test and funnel plot symmetry. If data are insufficient, a narrative line of review will be pursued.Discussion:HE4 protein has been shown to have great potential for clinical use as a diagnostic and prognostic marker in epithelial ovarian cancer (EOC). However, HE4 is not only limited to expression in ovarian cancer, but is also overexpressed in lung and endometrial cancers. The effectiveness of HE4 as a biomarker in cancers (other than EOC) has not yet been studied in the form of a comprehensive systematic review and meta-analysis. The results of this study should allow for expanded use of HE4 as a multiutility biomarker in multiple cancer types, thereby, elevating HE4's value as a cancer biomarker.PROSPERO registration:CRD42019120326.

Electrochemical quantum dots-based magneto-immunoassay for detection of HE4 protein on metal film-modified screen-printed carbon electrodes

A novel enzyme-free electrochemical immunosensor was developed for highly sensitive detection and quantification of human epididymis protein 4 (HE4) in human serum. For the first time, core/shell CdSe/ZnS quantum dots were conjugated with anti-HE4 IgG antibodies for subsequent sandwich-type immunosensing with superparamagnetic microparticles functionalized with anti-HE4 IgG antibodies, which allow rapid and efficient HE4 capture from the sample. Electrochemical detection of anti-HE4 IgG – HE4 – anti-HE4 IgGCdSe/ZnS immunocomplex was performed by recording the current response of Cd(II) ions, released from dissolved quantum dots at screen-printed carbon electrode (SPCE), modified with mercury or bismuth film. The linear range of the detection was from 20 pM to 40 nM with limit of detection of 12 pM using three times the standard deviation of blank criterion at mercury-film SPCE and from 100 pM to 2 nM with limit of detection of 89 pM at bismuth-film SPCE. Proposed electrochemical immunosensor meets the requirements for fast and sensitive quantification of HE4 biomarker in early stage of ovarian cancer and due to the proper sensitivity and specificity presents a promising alternative to enzyme-based probes used routinely in clinical diagnostics.

Preparation and characterization of a high-affinity monoclonal antibody against human epididymis protein-4

Human epididymis protein-4 (HE4) may serve as a putative biomarker for the early diagnosis, therapy and especially prognosis of ovarian, lung and breast cancer. Detection and targeting of HE4 using the anti-HE4 antibody could be one of the effective strategies for the cancer diagnosis and treatment in clinical practice. In this study, a high-efficiency expression system was established to purify recombinant HE4. We obtained high purity HE4 in 400 mg quantity from 1 L culture supernatant of HEK293F cells. CCK-8 and cell cycle assays indicated that the purified recombinant HE4 protein could promote SKOV3 cell cycle and proliferation at the concentration of 0.1 mg/L. Furthermore, an anti-HE4 high-affinity monoclonal antibody 9C3 (ka = 8.1 × 106 1/MS, kd = 4.4 × 10−5 1/S, KD = 5.5 × 10−12 M) was prepared using hybridoma technique and analyzed by surface plasmon resonance technology using this HE4 protein. Differential Scanning calorimeter (DSC) analysis showed that 9C3 had a commendable thermal stability with the Tm value of 73 °C. Analyses of western blot, immunohistochemistry and immunofluorescence showed that the 9C3 was highly specific to HE4 in human cancer cells and tissues. In conclusion, our study designed a method to prepare human recombinant HE4 with high yield and generated a high-affinity anti-HE4 monoclonal antibody that might have potential for basic research and clinical application.

Detection of Human Epididymis Protein 4 (HE4) in Human Serum Samples Using a Specific Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assay (ELISA).

The human epididymis protein 4 (HE4) may have high specificity in the detection of malignant diseases, making the development of an immunoassay for HE4 essential. In our study, a fusion gene was constructed encoded with the HE4 protein. This protein was then produced in the bacterial cells (Escherichia coli) and used to immunize mice in order to eventually generate hybridomas specific to HE4. The hybridoma supernatants were then screened, and four positive anti-HE4 cell lines were selected. These cell lines produce monoclonal antibodies against HE4 epitopes, as demonstrated in the Western blot as well as by direct enzyme-linked immunosorbent assay (ELISA). Using the developed antibodies, we successfully identified several good antibody pairs from the hybridomas, which allowed for the development of a sandwich ELISA to measure HE4 levels. By using the HE4 ELISA, we measured HE4 levels of 60 clinical human serum samples. Compared with the Food and Drug Administration (FDA) approved kit (Roche), our results showed a strong positive correlation to those of the FDA-approved kit. In summary, highly sensitive antibody pairs were screened against HE4, and a sandwich ELISA was developed as an accurate analytical tool for the detection of HE4 in human serum, which could be especially valuable for diagnosing ovarian carcinomas.

Expression and clinical significance of annexin A2 and human epididymis protein 4 in endometrial carcinoma.

BackgroundIt is well-known that the treatment and monitoring methods are limited for advanced stage of endometrial carcinoma. Biological molecules with expression changes during tumor progression become potential therapeutic targets for advanced stage endometrial carcinoma. Annexin A2 (ANXA2) has been reported to be overexpressed in recurrent endometrial carcinoma, and the expression of human epididymis protein 4 (HE4) is upregulated in endometrial carcinoma. What’s more, ANXA2 and HE4 interacted in ovarian cancer and promoted the malignant biological behavior. We speculated that their interaction may exist in endometrial carcinoma as well. We evaluated the expression and the correlation relationship of ANXA2 and HE4 in endometrial carcinoma.MethodsThe expression of ANXA2 and HE4 protein in 84 endometrial carcinoma, 30 endometrial atypical hyperplasia, and 18 normal endometrial tissue samples were then measured using an immunohistochemical assay in paraffin embedded endometrial tissues. The structural relationship between ANXA2 and HE4 was explored by immunoprecipitation and double immunofluorescent staining.ResultsANXA2 and HE4 co-localized in both endometrial tissues and endometrial carcinoma cells. ANXA2 and HE4 were expressed in 95.2 % and 85.7 % of the the endometrial carcinoma, respectively, which were significantly higher than normal endometrium (55.6 % and 16.7 %, both p < 0.05). The expression of ANXA2 and HE4 was significantly correlated with FIGO stage, degree of differentiation, myometrial invasion, and lymph node metastasis. ANXA2 was an independent risk factor for the prognosis of endometrial carcinoma (p < 0.05, hazard ratio [HR] = 8.004). The expression of ANXA2 and HE4 was positively correlated (Spearman correlation coefficient = 0.228, p < 0.05). HE4 was an independent factor for ANXA2 in multivariate linear regression model (p < 0.05).ConclusionWe revealed the co-localization of ANXA2 and HE4 in endometrial carcinoma. Expression levels of ANXA2 and HE4 were closely related to the malignant biological behavior of endometrial carcinoma, and ANXA2 was an independent risk factor for poor prognosis. The expression of ANXA2 and HE4 can affect each other.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0208-8) contains supplementary material, which is available to authorized users.

Correlation between eIF3a and HE4 expression and ovarian cancer

To investigate the correlation between eukaryotic translation initiation factor 3, subunit A (eIF3a) and human epididymis protein 4 (HE4) expression and ovarian cancer. RT-PCR or immunohistochemistry was used to examine eIF3a and HE4 mRNA or protein expression in ovarian tissues from patients with ovarian cancer (n=181) or benign ovarian tumors, or from the healthy women. There were significant differences in mRNA and protein expression of eIF3a and HE4 among normal ovarian tissues, benign ovarian tumor tissues, and ovarian cancer tissues (P< 0.05). There were significant differences in mRNA expression of eIF3a and HE4 between the normal tissues and the ovarian cancer tissues, or between the benign ovarian tumor tissues and the normal tissues (P< 0.001). The mRNA expression of eIF3a in the normal ovarian tissues was significantly higher than that in the benign ovarian tumor tissues or that in the ovarian cancer tissues. The mRNA expression of HE4 was gradually increased from the normal ovarian tissues, the benign ovarian tumor tissues to the ovarian cancer tissues. The mRNA expression of HE4 in the ovarian cancer tissues was significantly higher than that in the benign ovarian tumor tissues (P< 0.001). Positive expression rates for eIF3a or HE4 protein in normal, benign tumor, and cancer tissues were 0, 66.7%, and 81.0% or 0, 27.8%, and 56.2%, respectively. There were significant differences in positive expression rates of eIF3a protein and HE4 protein between the ovarian tumor tissues and benign ovarian tumor tissues, between the ovarian cancer tissues and the normal ovarian tissues, or between the benign ovarian tumor tissues and the normal ovarian tissues (P< 0.001). The eIF3a protein expression was positively correlated with HE4 protein expression (r=0.575, P< 0.05). The expressions of eIF3a and HE4 are associated with ovarian cancer, and extracellular regulated protein kinases may play a role in the interaction between eIF3a and HE4.

Human epididymis protein 4 in association with Annexin II promotes invasion and metastasis of ovarian cancer cells

BackgroundThe objective of the present study was to identify human epididymis protein 4 (HE4) interacting proteins and explore the mechanisms underlying their effect on ovarian cancer cell invasion and metastasis.MethodsHE4 interacting proteins were identified by mass spectrometry and validated by co-immunoprecipitation and pull-down assays. The scratch test, the Transwell assay and animal experiments were used to assess the invasive and metastatic abilities of ovarian cancer cells before and after transfection and HE4 protein treatment. HE4 and annexin II protein expression in epithelial ovarian tissues was detected by immunohistochemistry, and the relation between their expression levels was examined.ResultsAnnexin II was identified as an HE4 interacting protein. HE4 and annexin II binding interaction promoted ovarian cancer cell invasion and metastasis. HE4 and annexin II expression levels were significantly higher in malignant epithelial ovarian tissues than in benign and normal epithelial ovarian tissues, and they were higher in tissues with lymph node metastases than in those without. HE4 gene interference downregulated the expression of MAPK and the FOCAL adhesion signaling pathway-associated molecules MKNK2 and LAMB2, and HE4 protein supplementation reversed this effect.ConclusionThe binding interaction between HE4 and annexin II activates the MAPK and FOCAL adhesion signaling pathways, promoting ovarian cancer cell invasion and metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-243) contains supplementary material, which is available to authorized users.