EBAG9 Human

What is EBAG9 and what is its basic molecular classification?

EBAG9, also known as Receptor-binding cancer antigen expressed on SiSo cells, is a protein encoded by the EBAG9 gene in humans. It was initially identified as an estrogen-responsive gene in MCF-7 breast cancer cells using CpG genomic binding-site cloning technique . EBAG9 is regulated transcriptionally by estrogen through the estrogen receptor, which binds to an estrogen-responsive element (ERE) in the 5'-flanking region of this gene . Two transcript variants differing in the 5' UTR but encoding the same protein have been identified for this gene . As a tumor-associated antigen, EBAG9 is expressed at high frequency across various cancers and functions prominently in immune modulation and cancer progression.

What is the expression pattern of EBAG9 across normal human tissues?

EBAG9 shows widespread expression across human tissues with relatively low brain regional specificity . According to protein atlas data, EBAG9 expression can be detected in multiple organs and tissue types including the hippocampal formation, amygdala, basal ganglia, midbrain, spinal cord, cerebral cortex, cerebellum, hypothalamus, thyroid gland, adrenal gland, lung, gastrointestinal tract, reproductive organs, and lymphoid tissues . This broad expression pattern suggests that EBAG9 likely serves fundamental biological functions beyond its role in cancer, though these functions are less well characterized than its pathological roles.

How does EBAG9 contribute to cancer immune evasion mechanisms?

EBAG9 functions as a novel inhibitor of cytotoxic immune responses through multiple mechanisms:

• Suppression of secretory lysosomes: EBAG9 inhibits the production of secretory lysosomes through negative regulation of adaptor proteins involved in intracellular vesicle transfer . These lysosomes contain cell lysis effector molecules that would normally be released into the immune synapse formed between cytotoxic immune cells and their target tumor cells.

• Inhibition of cytotoxic enzyme release: EBAG9 inhibits the release of cytotoxic enzymes from immune cells, which slows the desired immune response against cancer cells .

• Extracellular vesicle-mediated immune suppression: EBAG9 can be transferred from cancer cells to surrounding T cells via extracellular vesicles (EVs), inhibiting T-cell cytotoxicity and modulating immune-related gene expression in these cells .

• Induction of immune cell apoptosis: Secreted EBAG9 can function as a ligand for a putative receptor present on T-lymphocytes, B-lymphocytes, and natural killer cells, inducing their apoptosis and death, which enables cancer cells to evade immune surveillance .

These mechanisms collectively position EBAG9 as a significant immune checkpoint molecule that contributes to tumor immune escape.

What evidence supports EBAG9 as a cancer biomarker?

EBAG9 has been established as a relevant cancer biomarker across multiple tumor types:

• Elevated tissue expression: EBAG9 protein expression is often upregulated in various malignant tumors, including cancers of the gastrointestinal tract, hepatocellular carcinoma, non-small cell lung cancers, and breast, ovarian, endometrial, and cervical cancers .

• Prognostic significance: The tissue expression of EBAG9 is negatively correlated with prognosis in patients bearing these malignancies, suggesting its potential utility as a prognostic biomarker .

• Detection in patient serum: EBAG9 is a secretory protein that can be detected in the sera of cancer patients, making it potentially valuable for liquid biopsy approaches .

• Association with advanced disease: In ovarian cancer, EBAG9 immunoreactivity was detected in 51.1% of epithelial ovarian carcinomas, with expression significantly higher in serous histology and advanced disease stages .

These findings collectively support the potential of EBAG9 as both a tissue and serum biomarker for cancer diagnosis, prognosis, and potentially treatment response monitoring.

What genetic models have been developed to study EBAG9 function?

Researchers have developed several genetic models to investigate EBAG9 function, with knockout mouse models being particularly informative:

• Ebag9-knockout mice: These have been generated through homologous recombination in embryonic stem cells followed by crossing with mice expressing Cre recombinase to remove the neomycin-resistance cassette and exon 2 of the Ebag9 gene . These models have revealed:

  • Substantially reduced tumor formation and metastasis to the lung when challenged with implanted cancer cells

  • Enhanced infiltration of CD8+, CD3+, and CD4+ T cells into generated tumors

  • Upregulation of immunity- and chemoattraction-related genes in CD8+ T cells isolated from tumors

  • Enhanced degranulation and increased cytolytic activity of CD8+ T cells

• TRAMP/Ebag9-knockout model: By crossing Ebag9 knockout mice with transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, researchers have shown that spontaneous development of prostate cancer was repressed in the absence of Ebag9 .

These genetic models have been instrumental in establishing the role of EBAG9 in immune suppression and cancer progression.

What methodologies are used to detect and quantify EBAG9 in research and clinical settings?

Several methodologies have been employed to detect and quantify EBAG9:

• Immunohistochemistry (IHC): Used to detect EBAG9 protein in tissue sections. In ovarian cancer studies, EBAG9 immunoreactivity was detected in the surface and cytoplasm of carcinoma cells .

• Reverse Transcription-Polymerase Chain Reaction (RT-PCR): Used to evaluate mRNA expression of EBAG9, as demonstrated in ovarian cancer cases .

• Immunoblotting (Western blot): Used to detect EBAG9 protein expression in cell lines and tissue samples .

• Flow cytometry: Can be used to detect cell surface and intracellular EBAG9 in immune cells and cancer cells.

• ELISA: Potentially useful for detecting secreted EBAG9 in patient serum samples.

The selection of detection method depends on the specific research question, sample type, and required sensitivity and specificity.

What approaches are being developed to target EBAG9 therapeutically?

Several approaches for therapeutic targeting of EBAG9 are being explored:

• Neutralizing antibodies: Research has demonstrated that a neutralizing antibody for EBAG9 could rescue extracellular vesicle-mediated immune suppression by recovering T-cell cytotoxicity . This approach could potentially enhance endogenous anti-tumor immune responses.

• Genetic silencing: Studies have successfully used microRNA to curb synthesis of the EBAG9 protein. This approach has been applied to CAR T cells, where "EBAG9-silenced" CAR T cells showed enhanced anti-tumor activity against human leukemia or lymphoma cells .

• Combined approaches with immunotherapy: Given EBAG9's role as an immune checkpoint, combining EBAG9 inhibition with existing immunotherapies could potentially enhance therapeutic efficacy .

These approaches position EBAG9 as a promising target for cancer immunotherapy, particularly for advanced diseases with EBAG9 overexpression.

How does EBAG9 silencing enhance CAR T cell efficacy?

EBAG9 silencing has been shown to significantly enhance the efficacy of CAR T cell therapy through several mechanisms:

• Enhanced cytotoxic substance release: Shutting down EBAG9 allows CAR T cells to release more cytotoxic substances, enabling more effective killing of tumor cells .

• Earlier and more radical tumor eradication: EBAG9 silencing allows the body to eradicate tumor cells earlier and more radically, potentially achieving longer-lasting therapeutic success and increasing chances of cure .

• Reduced relapse rates: In studies with human leukemia or lymphoma cells, EBAG9-silenced CAR T cells reduced tumor growth much more significantly, and relapses developed much later .

• Reduced risk of cytokine storms: Interestingly, despite enhanced cytotoxicity, EBAG9-silenced CAR T cells do not cause the strong cytokine storm typically associated with CAR therapy. In fact, the risk is minimized because fewer cells are needed to achieve therapeutic effects .

• Broad applicability: The EBAG9 silencing approach works across different CAR T cell types, regardless of which type of blood cancer they target .

These findings suggest that EBAG9 silencing could be a valuable strategy to enhance the efficacy and safety of CAR T cell therapies for various malignancies.

How does extracellular vesicle-mediated EBAG9 transfer impact the tumor microenvironment?

Extracellular vesicle (EV)-mediated EBAG9 transfer represents a novel mechanism by which cancer cells can influence the tumor microenvironment:

• Transfer of immunosuppressive signals: EVs from EBAG9-overexpressing prostate cancer cells can facilitate immune escape by inhibiting T-cell cytotoxicity and modulating immune-related gene expression in T cells .

• Establishment of an immunosuppressive gradient: Through EV-mediated transfer, cancer cells can potentially establish a gradient of EBAG9-mediated immunosuppression extending beyond the immediate tumor vicinity.

• Interference with antigen presentation: EBAG9 transferred via EVs might impact antigen-presenting cells within the tumor microenvironment, further compromising anti-tumor immunity.

• Epithelial-mesenchymal transition (EMT) regulation: EBAG9 has been identified to interact with TM9SF1, which regulates EMT in cancer cells . This interaction could influence tumor progression and metastatic potential.

This mechanism of intercellular communication adds another layer to understanding how EBAG9 contributes to tumor immune evasion and progression.

What is known about EBAG9's role in different immune cell populations beyond CD8+ T cells?

While much of the research on EBAG9 has focused on its effects on CD8+ T cells, its impact extends to other immune cell populations:

• CD4+ T cells: EBAG9 knockout mice show increased infiltration of not only CD8+ but also CD4+ T cells into tumors, suggesting EBAG9 may regulate helper T cell recruitment or retention .

• Natural killer (NK) cells: Secreted EBAG9 can function as a ligand for putative receptors on NK cells, potentially inducing their apoptosis and death, thereby contributing to immune evasion .

• B lymphocytes: Similar to its effects on T cells and NK cells, EBAG9 can induce apoptosis in B lymphocytes, further compromising anti-tumor immunity .

• Antigen-presenting cells: Given EBAG9's role in regulating secretory lysosomes, it may affect antigen processing and presentation by dendritic cells and other antigen-presenting cells, though this aspect requires further investigation.

Understanding EBAG9's effects across different immune cell populations provides a more comprehensive picture of its role in immune modulation and cancer progression.