ANXA3 Human

What is ANXA3 and what is its fundamental role in human cells?

ANXA3 (Annexin A3) is a member of the annexin family of proteins with calcium (Ca²⁺) and lipid-binding properties. It exists in two isoforms (36-kDa and 33-kDa) and can be found at various intra- and extracellular locations, including human cumulus oophorus cells . The annexin family proteins generally participate in membrane organization, calcium signaling, and cellular trafficking processes. In normal physiology, ANXA3 is expressed in various tissues including bone marrow and testis .

Structurally, ANXA3 contains a core domain with calcium-binding sites and an N-terminal domain that confers functional specificity. The protein's calcium-dependent phospholipid binding ability is central to its biological functions. Under normal conditions, ANXA3 participates in cellular processes like membrane trafficking and organization, though its precise physiological role requires further characterization in specific tissue contexts.

What are the recommended methods for detecting and quantifying ANXA3 expression?

Several complementary methods should be employed for comprehensive ANXA3 characterization:

Table 1: Methods for ANXA3 detection and functional analysis

In gastric cancer research, investigators employed RT-PCR and Western blot for initial ANXA3 characterization, followed by immunohistochemistry on 183 paraffin-embedded gastric cancer tissue samples for clinical correlation studies . For therapeutic studies, antibody-based detection is critical for confirming target engagement, as demonstrated in the development of ANXA3 degraders for triple-negative breast cancer .

How is ANXA3 expression regulated at the molecular level?

ANXA3 expression is regulated through multiple mechanisms:

• Hypoxia-induced regulation: ANXA3 expression is upregulated by HIF-1α (Hypoxia-inducible factor 1-alpha) in colon cancer in response to hypoxic stress . This represents an important microenvironmental regulation mechanism particularly relevant in solid tumors where hypoxia is common.

• Post-translational regulation: ANXA3 protein stability is regulated through the ubiquitin-proteasome system. Research shows that compounds like (R)-SL18 can promote ANXA3 ubiquitination, leading to its selective degradation . This mechanism has been exploited for therapeutic targeting of ANXA3.

• Pathway-mediated regulation: ANXA3 interacts with multiple signaling pathways including NFκB and Wnt/β-catenin . These interactions suggest regulatory feedback loops may exist where pathway activation influences ANXA3 expression or activity.

Researchers investigating ANXA3 regulation should consider these mechanisms when designing experiments, particularly the microenvironmental context (e.g., hypoxia) that appears to significantly influence ANXA3 expression levels.

In which cancer types has ANXA3 dysregulation been most conclusively demonstrated?

ANXA3 dysregulation has been documented in several cancer types with varying degrees of evidence:

• Breast Cancer: ANXA3 is significantly upregulated in breast cancer tissues . In triple-negative breast cancer (TNBC), ANXA3 overexpression correlates with poor prognosis and drives tumor growth and metastasis . Functional studies have shown that ANXA3 knockdown inhibits the NFκB pathway resulting in mesenchymal-epithelial transition (MET) and alterations in breast cancer stem cells .

• Gastric Cancer: Comprehensive studies involving 183 gastric cancer patients demonstrated high ANXA3 expression at both mRNA and protein levels in tumor tissues . Functional studies confirmed ANXA3's role in promoting proliferation, clone formation, migration, and invasion in gastric cancer cells .

• Colon Cancer: ANXA3 is upregulated by HIF-1α in colon cancer in response to hypoxic stress and contributes to tumor growth . This suggests an important role in adapting to the hypoxic microenvironment common in solid tumors.

• Ovarian Cancer: ANXA3 gene expression appears related to tumor aggressiveness, drug resistance, and immune infiltration in ovarian serous carcinoma .

The most robust evidence exists for breast and gastric cancers, where comprehensive molecular and clinical studies have been conducted.

What is the prognostic significance of ANXA3 expression across different cancer types?

ANXA3 demonstrates notable prognostic value across multiple cancer types, though its prognostic significance varies:

Table 2: Association between ANXA3 expression and clinicopathological parameters in gastric cancer

These findings suggest ANXA3 has significant prognostic value, but its implications may be cancer-type specific, necessitating context-specific interpretation.

How do mechanistic studies of ANXA3 in cancer cells correlate with clinical observations?

Mechanistic studies of ANXA3 align closely with clinical observations, providing biological explanations for clinical phenotypes:

• Invasion and Metastasis: Laboratory studies showing ANXA3 promotes cell migration and invasion correlate with clinical observations that high ANXA3 expression associates with advanced T stage, lymph node metastasis, and distant metastasis in gastric cancer patients . The molecular mechanism involves ANXA3's role in epithelial-mesenchymal transition (EMT), which facilitates cancer cell invasion .

• Tumor Growth: Functional studies demonstrating ANXA3's role in promoting cell proliferation align with clinical observations of larger tumors in patients with high ANXA3 expression (P=0.006) . In vivo studies confirm that ANXA3 knockdown results in significantly smaller tumors .

• Drug Resistance: Mechanistic studies showing ANXA3 knockdown increases sensitivity to doxorubicin by enhancing drug uptake may explain why patients with high ANXA3 expression respond poorly to chemotherapy, contributing to worse survival outcomes.

• Pathway Dysregulation: ANXA3's involvement in NFκB and Wnt/β-catenin pathways provides molecular mechanisms underlying its clinical effects, as these pathways regulate crucial cancer hallmarks including proliferation, survival, and metastasis.

This strong correlation between laboratory findings and clinical observations validates ANXA3 as a mechanistically relevant biomarker and therapeutic target.

Which signaling pathways are modulated by ANXA3 in cancer progression?

ANXA3 interacts with several key signaling pathways that drive cancer progression:

• NFκB Pathway: ANXA3 positively regulates the NFκB pathway, as evidenced by studies showing that "ANXA3 knockdown inhibited the NFκB pathway via upregulating IκBα" . This inhibition results in mesenchymal-epithelial transition (MET) and changes in breast cancer stem cells, suggesting that ANXA3 normally promotes epithelial-mesenchymal transition (EMT) through NFκB activation .

• Wnt/β-catenin Pathway: ANXA3 appears to stabilize β-catenin levels, as the ANXA3 degrader (R)-SL18 "could reduce the β-catenin level, and accordingly inhibit the Wnt/β-catenin signaling pathway in TNBC cells" . This inhibition affected downstream genes (BIRC5, CCND1, MMP7, and MMP9) , all of which are involved in proliferation, survival, and invasion.

• Hypoxia Response Pathway: ANXA3 is upregulated by HIF-1α in colon cancer under hypoxic conditions , suggesting it may participate in cellular adaptation to hypoxia, a common feature of solid tumors that promotes aggressive behavior.

These pathway interactions position ANXA3 as a signaling node that integrates and influences multiple cancer-promoting cascades, making it a potentially valuable therapeutic target that could simultaneously affect several oncogenic mechanisms.

How does ANXA3 contribute to epithelial-mesenchymal transition in cancer metastasis?

ANXA3 promotes epithelial-mesenchymal transition (EMT) through multiple mechanisms, facilitating cancer metastasis:

• NFκB Pathway Activation: ANXA3 knockdown inhibits the NFκB pathway by upregulating IκBα (an NFκB inhibitor), resulting in mesenchymal-epithelial transition (MET) . This suggests ANXA3 normally suppresses IκBα, allowing NFκB activation which promotes EMT, a critical step in metastasis.

• EMT Marker Regulation: Studies directly demonstrate that "ANXA3 expression is associated with the epithelial-mesenchymal transition" . EMT involves loss of epithelial markers like E-cadherin and gain of mesenchymal markers like Vimentin, altering cellular adhesion and enhancing invasive properties.

• Matrix Metalloproteinase Expression: ANXA3 appears to regulate matrix metalloproteinases, as ANXA3 degradation leads to inhibition of MMP7 and MMP9 expression . These enzymes degrade extracellular matrix components, facilitating tumor cell invasion and metastasis.

• Enhanced Cellular Invasion: Experimental evidence confirms that "exogenous ANXA3 transduction promoted proliferation, clone formation, migration, and invasion" , while ANXA3 silencing inhibited these processes. These cellular capabilities are essential for the metastatic cascade.

The relationship between ANXA3 and EMT explains its clinical association with invasion depth, lymph node metastasis, and distant metastasis observed in gastric cancer patients .

What is the relationship between ANXA3 and cancer stem cell regulation?

ANXA3 appears to play a significant role in cancer stem cell biology, particularly in breast cancer:

Research indicates that "ANXA3 knockdown inhibited the NFκB pathway via upregulating IκBα, resulting in mesenchymal-epithelial transition (MET) and a heterogeneity change of breast cancer stem cells (BCSCs)" . This finding suggests that ANXA3 maintains BCSC properties through:

• NFκB Pathway Activation: The NFκB pathway is known to promote stemness in multiple cancer types. ANXA3's ability to activate this pathway likely contributes to BCSC maintenance.

• EMT Promotion: EMT and cancer stemness are closely linked, with the mesenchymal state often associated with increased stemness characteristics. By promoting EMT, ANXA3 may enhance cancer stem cell properties.

• BCSC Heterogeneity Regulation: The observation of "heterogeneity change" in BCSCs following ANXA3 knockdown suggests ANXA3 may influence the composition or phenotypic diversity of the cancer stem cell population, which has implications for tumor behavior and treatment resistance.

The study explicitly describes "the role and mechanisms of ANXA3 in regulating BCSCs and breast cancer growth and metastasis" , highlighting the importance of this relationship. Since cancer stem cells drive tumor initiation, metastasis, and therapeutic resistance, ANXA3's role in their regulation represents a significant mechanism through which it influences cancer progression.

What are the optimal methods for ANXA3 genetic manipulation in cellular models?

Several effective approaches for ANXA3 genetic manipulation have been validated in cancer research:

• CRISPR/Cas9 Knockout: CRISPR/Cas9 technology has successfully established ANXA3-knockout cell lines, as demonstrated in MDA-MB-231 triple-negative breast cancer cells . This approach provides complete elimination of ANXA3 expression, confirmed by Western blot analysis. Functional studies showed that "ANXA3 knockout markedly weakened the proliferation of MDA-MB-231 cells" , validating the system's efficacy.

• siRNA/shRNA Knockdown: RNA interference offers flexibility for both transient and stable ANXA3 suppression. In gastric cancer research, "SGC7901 and MKN45 cells with high ANXA3 expression were infected with ANXA3 siRNA and negative control" , resulting in 77-79% reduction in ANXA3 protein levels. This approach effectively inhibited proliferation, colony formation, migration, and invasion .

• Lentiviral Overexpression: For gain-of-function studies, lentiviral vectors carrying ANXA3 cDNA have been used: "MPC803 and HGC27 cells with low ANXA3 expression were infected with ANXA3 overexpression Lentivirus and control" . This method demonstrated that "ANXA3 overexpression accelerated the growth of MPC803 and HGC27 cells" .

For in vivo applications, stable cell lines are preferable. In xenograft studies, "SGC7901 subline with stably knockdown ANXA3 and a control cell line were injected subcutaneously into the flanks of nude mice" , demonstrating that tumor volumes and weights were significantly reduced in the ANXA3 knockdown group.

The choice of approach should be guided by experimental goals: CRISPR for complete gene elimination, siRNA for transient effects, and shRNA for stable long-term suppression.

What methodological approaches are recommended for validating ANXA3-targeting compounds?

A comprehensive validation framework for ANXA3-targeting compounds includes:

Table 3: Validation framework for ANXA3-targeting compounds

This multi-dimensional validation approach, exemplified in the development of (R)-SL18, ensures target specificity, mechanism clarification, and therapeutic potential assessment. The use of genetic knockout/knockdown models is particularly important for confirming on-target effects, as demonstrated by comparing compound efficacy in control versus ANXA3-depleted cells .

What in vivo models best represent ANXA3's role in cancer progression?

Several in vivo models have demonstrated utility for studying ANXA3 in cancer:

• Cell Line Xenograft Models: These provide direct assessment of ANXA3's role in tumor growth. Studies in gastric cancer used "SGC7901 subline with stably knockdown ANXA3 and a control cell line were injected subcutaneously into the flanks of nude mice" . Results showed significantly smaller tumor volumes and weights in the ANXA3 knockdown group, confirming ANXA3's role in promoting tumor growth.

• Patient-Derived Xenograft (PDX) Models: These maintain tumor heterogeneity and better recapitulate clinical scenarios. The ANXA3 degrader (R)-SL18 was evaluated in "a TNBC patient-derived xenograft (PDX) model with high ANXA3 expression" , demonstrating its ability to reduce tumor growth in a clinically relevant model.

• Metastasis Models: While not explicitly described in the search results, models examining metastatic spread would be valuable given ANXA3's role in invasion and metastasis. Tail vein injection or orthotopic implantation models would allow assessment of ANXA3's impact on metastatic potential.

For therapeutic studies, PDX models with confirmed high ANXA3 expression offer advantages in predicting clinical responses, as they maintain tumor heterogeneity and microenvironment interactions. For mechanistic studies, cell line xenografts with genetic manipulation of ANXA3 (overexpression, knockdown, or knockout) provide controlled systems to isolate ANXA3's specific contributions to tumor biology.

What ANXA3-targeting compounds have been developed and what are their mechanisms?

Currently, (R)-SL18 represents the most advanced ANXA3-targeting compound in the research literature:

Table 4: Properties of the first-in-class ANXA3 degrader (R)-SL18

(R)-SL18 represents a targeted protein degradation approach rather than conventional enzyme inhibition. This compound "directly bound to ANXA3 and increased its ubiquitination, thereby inducing ANXA3 degradation with moderate family selectivity" . The development of this compound provides proof-of-concept that ANXA3 can be successfully targeted with small molecules.

This targeted degradation strategy offers potential advantages over conventional inhibition, as it removes the protein entirely rather than just blocking its function, potentially providing more complete suppression of ANXA3-dependent signaling.

How does ANXA3 contribute to chemotherapy resistance and how might this be overcome?

ANXA3 contributes to chemotherapy resistance through multiple mechanisms:

• Reduced Drug Uptake: Research demonstrates that "ANXA3 knockdown increased the sensitivity of breast cancer cells to doxorubicin by increasing the drug uptake" . This suggests ANXA3 normally reduces intracellular drug accumulation, potentially by altering membrane properties or drug transport.

• Enhanced Tumor-Initiating Capacity: ANXA3's role in maintaining breast cancer stem cells (BCSCs) may contribute to therapy resistance, as cancer stem cells typically exhibit enhanced resistance to conventional therapies.

• Pro-Survival Signaling: ANXA3's activation of the NFκB pathway likely promotes cell survival mechanisms that protect cancer cells from chemotherapy-induced apoptosis.

These mechanisms can be therapeutically targeted through:

• Combination Therapy: "The combination of ANXA3 knockdown and doxorubicin treatment simultaneously inhibited tumor growth and metastasis in vivo" . This suggests that ANXA3 inhibition serves as a chemosensitizing strategy.

• ANXA3 Degraders: Compounds like (R)-SL18 that induce ANXA3 degradation could potentially reverse resistance by eliminating ANXA3-mediated protective mechanisms.

• Pathway Inhibitors: Since ANXA3 operates through NFκB and Wnt/β-catenin pathways, inhibitors of these pathways might circumvent ANXA3-mediated resistance.

These findings suggest that "downregulating ANXA3 together with chemotherapy might be a novel therapeutic strategy for treating breast cancer" , highlighting the potential for dual-targeting approaches to overcome resistance.

What challenges and opportunities exist in developing ANXA3-targeted therapies?

Developing ANXA3-targeted therapies presents both challenges and opportunities:

Challenges:

• Achieving Selectivity: The "moderate family selectivity" of current ANXA3 degraders highlights difficulties in selectively targeting ANXA3 versus other annexin family members, potentially leading to off-target effects.

• Normal Tissue Expression: ANXA3 is expressed in normal tissues including bone marrow and testis , raising concerns about potential toxicity in these tissues.

• Context-Dependent Functions: The contrasting prognostic implications of ANXA3 in different cancers (poor prognosis in gastric and breast cancers versus potentially favorable outcomes in ovarian cancer ) suggest tissue-specific functions that complicate therapeutic development.

Opportunities:

• Targeted Protein Degradation: The successful development of (R)-SL18, which "directly bound to ANXA3 and increased its ubiquitination, thereby inducing ANXA3 degradation" , demonstrates the feasibility of targeted ANXA3 degradation as a therapeutic strategy.

• Combination with Chemotherapy: The finding that "ANXA3 knockdown increased the sensitivity of breast cancer cells to doxorubicin" suggests potential for combination regimens that enhance the efficacy of standard treatments.

• Cancer Type Specificity: The strong evidence for ANXA3's role in breast cancer, particularly TNBC, provides a clear therapeutic rationale for these difficult-to-treat cancer types where new targets are urgently needed.

• Patient Selection Biomarkers: ANXA3 expression levels could serve as biomarkers for patient selection, as studies show particularly promising results in "high ANXA3-expressing TNBC patient-derived xenograft model" .

The development of (R)-SL18 represents a significant milestone, demonstrating that ANXA3 "possesses the potential to treat TNBC" , a cancer subtype with limited targeted therapy options.