EPHA2 Human

What is the molecular structure of human EPHA2?

Human EPHA2 is a transmembrane glycoprotein comprising 976 amino acids with a molecular weight of approximately 130 kDa. It belongs to the ephrin receptor subfamily of protein-tyrosine kinase receptors. The protein structure includes a single domain of kinase and an extracellular region containing a cysteine-rich domain along with two repeats of fibronectin type III. The human EPHA2 gene demonstrates high conservation with its mouse counterpart, including adjacent gene orders and similar regulatory elements .

Which signaling pathways does EPHA2 participate in?

EPHA2 participates in signaling pathways primarily regulating cellular repulsion or adhesion. In cancer contexts, EPHA2 enhances tumorigenesis by modulating cell-cell interactions, proliferation, and cancer cell motility through complex signaling pathways including KRAS activation and ERK inhibition. During embryonic development, Eph receptor signaling (including EPHA2) is crucial in tissue organization, neuronal direction, and vascular morphogenesis . In pluripotent stem cells, EPHA2 appears to have roles in inhibiting MAPK or GSK3 signaling pathways, independent of LIF signaling .

How is EPHA2 expression regulated in human tissues?

EPHA2 expression is tightly regulated during development and in mature tissues. In human pluripotent stem cells, EPHA2 expression is regulated by core pluripotency factors. Analysis of genomic and epigenetic data revealed that in the human EPHA2 locus, active enhancer regions in iPSCs harbor multiple binding sites for OCT4 and NANOG, suggesting direct transcriptional regulation by these pluripotency factors. EPHA2 expression markedly declines following spontaneous differentiation of human iPSCs, mirroring the trend observed in mouse ESCs, indicating developmental regulation .

How does EPHA2 contribute to pluripotency maintenance?

EPHA2 plays a crucial role in maintaining pluripotency in stem cells. Experimental evidence shows that knock-down of Epha2 leads to spontaneous differentiation of mouse ESCs, even in the presence of LIF (Leukemia Inhibitory Factor, which normally helps maintain stemness). This differentiation is characterized by the loss of round-shaped mouse ES colonies and diminished alkaline phosphatase (AP) activity, both indicators of the undifferentiated state. The overexpression of human EPHA2 in Epha2-knocked-down mouse ESCs restored cell morphology and endogenous Oct4 expression levels, confirming EPHA2's direct role in pluripotency maintenance .

What is the relationship between EPHA2 and core pluripotency transcription factors?

EPHA2 shows a strong correlation with core pluripotency factors, particularly OCT4 and NANOG. In human iPSCs, EPHA2+ cells exhibit heightened OCT4 and NANOG expression compared to EPHA2- cells, though interestingly, SOX2 expression does not show the same correlation. During differentiation of both mouse and human PSCs, EPHA2 expression closely tracks with OCT4 expression. The human EPHA2 locus contains active enhancer regions with binding sites for OCT4 and NANOG, suggesting direct transcriptional regulation by these factors .

How can EPHA2 be used to identify undifferentiated cells in differentiated cell populations?

EPHA2 serves as an effective cell surface marker for identifying undifferentiated cells in heterogeneous populations. Using antibody-based magnetic-activated cell sorting (MACS) with anti-EPHA2 antibodies, researchers can separate EPHA2+ cells from differentiated populations. Studies have shown that depletion of EPHA2+ cells from mouse ESC-derived hepatic lineage cells reduces tumor formation after transplantation into immune-deficient mice. In human iPSC differentiation experiments, although not every EPHA2+ cell was OCT4+ in later stages of differentiation, the majority of OCT4+ cells did express EPHA2, making it a reliable marker for identifying remaining pluripotent cells .

What role does EPHA2 play in tumorigenesis?

EPHA2 serves as one of the primary functional receptors among EPH type A members and is implicated in enhancing tumorigenesis through multiple mechanisms. It modulates cell-cell interactions, proliferation, and cancer cell motility. These cellular actions are mediated by intricate signaling pathways such as KRAS activation and ERK inhibition. EPHA2's ability to regulate processes like cell proliferation and migration makes it a significant factor in cancer development and progression. Due to these functions, EPHA2 is considered an important cell surface marker for identifying and tracking the progression of aggressive and malignant tumors .

What genetic alterations of EPHA2 are common in human cancers?

EPHA2 undergoes various genetic modifications in human cancers, including mutations and copy number alterations (CNAs). The Cancer Genome Atlas (TCGA) data allows investigation of modification frequencies, types of mutations, and CNAs across different cancer types. These genetic alterations can affect EPHA2 function and potentially contribute to cancer development or progression. Analysis of such modifications helps in understanding the role of EPHA2 in different cancer contexts and may inform therapeutic strategies targeting this receptor .

What techniques can be used to modulate EPHA2 expression in experimental models?

Several techniques can effectively modulate EPHA2 expression in experimental models:

• RNA interference (RNAi): Short hairpin RNA (shRNA) can be designed to target EPHA2 mRNA. These can be cloned into retroviral vectors like pSINsi-DK I and delivered to cells for stable knockdown.

• Overexpression systems: Human EPHA2 cDNA can be amplified by PCR and inserted into retroviral vectors like pMYs-IRES-puro for stable overexpression studies.

• Viral delivery: Retroviruses containing the constructs can be produced in packaging cells (like Plat-E cells), concentrated, and used to infect target cells with polybrene to enhance infection efficiency.

• Selection of stable cell lines: Transformed cells can be selected using appropriate antibiotics (G418 for knockdown vectors, puromycin for overexpression vectors) to establish stable cell lines with altered EPHA2 expression .

How can EPHA2-positive cells be isolated from heterogeneous cell populations?

EPHA2-positive cells can be isolated from heterogeneous populations using the following methods:

• Magnetic-activated cell sorting (MACS): Using anti-EPHA2 antibody-bound magnetic beads, EPHA2+ cells can be selectively captured from mixed populations. This technique was effectively used to deplete EPHA2+ cells from differentiated embryoid bodies.

• Fluorescence-activated cell sorting (FACS): Flow cytometry with fluorescently-labeled anti-EPHA2 antibodies enables the separation of EPHA2+ and EPHA2- cell populations based on surface expression levels. This approach allows for more precise quantification and isolation compared to MACS.

Both techniques can be combined with markers for other proteins (like OCT4-EGFP reporter systems) to further refine the isolation of specific cell subpopulations .

What markers should be co-analyzed with EPHA2 in stem cell differentiation experiments?

When analyzing EPHA2 in stem cell differentiation experiments, several categories of markers should be co-analyzed:

• Core pluripotency markers: OCT4, NANOG, SOX2 - These help establish the relationship between EPHA2 and the pluripotent state.

• Germ layer-specific markers: For comprehensive analysis of differentiation, markers for all three germ layers should be assessed:

  • Ectoderm: SOX1, PAX6

  • Mesoderm: BRACHYURY, MIXL1

  • Endoderm: SOX17, FOXA2

• Lineage-specific markers: Depending on the differentiation protocol, specific lineage markers should be included (e.g., AFP and ALB for hepatic differentiation).

• Other surface markers: Co-analysis with established surface markers like TRA-1-60, TRA-1-81, and SSEAs can provide comparative insights into EPHA2's specificity and sensitivity .

How does the heterogeneous expression of EPHA2 in human PSCs relate to developmental states?

The EPHA2+ subpopulation exhibits elevated OCT4 and NANOG expression, suggesting they may represent a transient state characterized by enhanced pluripotency factor expression. When isolated and cultured, EPHA2+ cells rapidly regenerate EPHA2- cell populations, and the elevated expression of OCT4 dissipates, indicating a dynamic equilibrium between these states. These observations suggest EPHA2+ cells may reflect a specific transitional state of PSCs, possibly related to the human embryo implantation period, though distinct from primitive endoderm .

What are the mechanistic interactions between EPHA2 and the MAPK/GSK3 signaling pathways in pluripotency?

The exact molecular mechanisms remain to be fully elucidated, but EPHA2 likely interfaces with these signaling pathways through its tyrosine kinase activity. In the context of pluripotency maintenance, EPHA2 may phosphorylate downstream targets that negatively regulate MAPK pathway components or positively regulate inhibitors of the pathway. Alternatively, EPHA2 could influence GSK3 activity, affecting β-catenin stability and consequently modulating Wnt signaling, which is important for pluripotency maintenance in certain contexts.

Further investigations using phosphoproteomic approaches and targeted manipulation of pathway components would help clarify these interactions and potentially reveal novel regulatory mechanisms in pluripotency maintenance .

What are the implications of EPHA2 as a marker for eliminating tumorigenic cells in regenerative medicine applications?

The identification of EPHA2 as a marker for tumorigenic undifferentiated cells has significant implications for regenerative medicine. The major challenge in translating pluripotent stem cell-derived therapies to clinical applications is the risk of teratoma formation from residual undifferentiated cells. EPHA2's strong correlation with OCT4 expression during differentiation of both mouse and human PSCs positions it as a valuable tool for addressing this challenge.

Experimental evidence demonstrated that removing EPHA2+ cells from mouse ESC-derived hepatic lineage reduced tumor formation after transplanting them into immune-deficient mice. This suggests practical applications:

• Quality control: EPHA2-based sorting could be implemented as a final purification step in therapeutic cell production to remove tumorigenic cells.

• Safety enhancement: Combining EPHA2 with other markers might create more robust safety protocols for cell therapy products.

• Monitoring methods: Developing quick, sensitive assays for EPHA2+ cells could provide real-time quality assessment during differentiation processes.

• Targeted elimination strategies: Beyond sorting, developing EPHA2-targeted cytotoxic agents could selectively eliminate undifferentiated cells within heterogeneous populations.

These approaches could substantially reduce the risk of tumor formation in stem cell-based therapies, potentially accelerating the clinical translation of regenerative medicine applications .

How does EPHA2 influence cellular behaviors beyond pluripotency?

EPHA2's influence extends well beyond pluripotency to diverse cellular behaviors. As a receptor tyrosine kinase, it is activated during early development and pathogenesis, playing crucial roles in managing various biological functions. In cancer contexts, EPHA2 modulates cell-cell interactions, proliferation, and cancer cell motility. These functions are mediated through complex signaling cascades, including KRAS activation and ERK inhibition.

During embryonic development, Eph receptor signaling (including EPHA2) contributes to tissue organization, neuronal direction, and vascular morphogenesis. In differentiated cells, EPHA2 continues to influence cellular behaviors, particularly those related to migration, adhesion, and tissue boundary formation. The widespread expression and multifunctional nature of EPHA2 suggest it serves as a key regulator of cellular behavior across various contexts, making it both an interesting research target and a potential therapeutic intervention point .

What phenotypic changes result from EPHA2 manipulation in different cell types?

Phenotypic changes resulting from EPHA2 manipulation vary significantly depending on cell type and context:

• In mouse ESCs: Knockdown of Epha2 leads to:

  • Loss of round-shaped colony morphology

  • Reduced alkaline phosphatase activity

  • Suppression of undifferentiated state-specific markers

  • Spontaneous differentiation into all three germ layers

  • Enhanced induction of differentiation markers during embryoid body formation

• In human iPSCs:

  • EPHA2+ cells show elevated OCT4 and NANOG expression

  • After passage, EPHA2+ cells rapidly regenerate EPHA2- populations

  • The elevated expression of OCT4 dissipates over time

• In cancer cells (from broader literature):

  • EPHA2 overexpression often correlates with enhanced proliferation, migration, and invasion

  • Manipulation of EPHA2 can alter tumor growth, metastatic potential, and response to therapies

These diverse phenotypic changes highlight the context-dependent nature of EPHA2 function and its ability to regulate fundamental cellular processes across different cell types .