AZGP1 Human, HEK

What is the molecular structure and basic function of human AZGP1?

AZGP1 is a 34.5kDa single, non-glycosylated polypeptide chain when expressed in E. coli, containing 301 amino acids (specifically amino acids 21-298 of the full sequence). It stimulates lipid degradation in adipocytes and causes fat losses associated with advanced cancers. The protein may also bind polyunsaturated fatty acids, suggesting multiple metabolic functions . Structurally, recombinant AZGP1 is often produced with an N-terminal His-tag to facilitate purification, resulting in a fusion protein with proprietary chromatographic properties .

What expression systems are most effective for producing human AZGP1?

While E. coli has been successfully used to produce human AZGP1, mammalian expression systems like HEK293 cells offer advantages for proper post-translational modifications. For bacterial expression systems, recombinant AZGP1 can be produced as a non-glycosylated protein with a molecular mass of approximately 34.5kDa . Purification typically employs proprietary chromatographic techniques following fusion with affinity tags such as an N-terminal His-tag. Optimal storage conditions include 20mM Tris-HCl (pH8.0) buffer with 10% glycerol at 4°C for short-term use or -20°C with a carrier protein (0.1% HSA or BSA) for long-term storage .

How is AZGP1 regulated in normal tissues versus pathological conditions?

AZGP1 shows differential expression between normal and cancerous tissues. In hepatocellular carcinoma (HCC), AZGP1 expression is significantly downregulated in approximately 80% of tumorous tissues compared to adjacent non-tumorous liver tissues . Quantitative RT-PCR analyses have demonstrated that AZGP1 mRNA levels in HCC tissues can be on average 15.5-fold lower than in corresponding adjacent liver tissue . Similar downregulation has been observed in soft tissue sarcomas, where reduced AZGP1 expression correlates with metastatic potential .

What are the optimal methods for detecting AZGP1 expression in tissue samples?

Multiple techniques have proven effective for AZGP1 detection:

• Quantitative RT-PCR: Highly sensitive for measuring AZGP1 mRNA levels in fresh tissues. This method has successfully detected significant differences between tumorous and non-tumorous tissues in HCC and soft tissue sarcomas .

• Immunohistochemistry (IHC): Particularly effective for tissue microarray (TMA) analysis, allowing visualization of AZGP1 expression patterns within tissue architecture. Researchers typically classify AZGP1 expression into categories (negative, weak, medium, and strong) based on staining intensity .

• Western Blotting: Useful for semi-quantitative protein analysis, with published studies showing approximately 1.55-fold lower AZGP1 expression in tumorous tissues compared to paired non-tumorous tissues .

How can researchers effectively modulate AZGP1 expression in cellular models?

For mechanistic studies, researchers have successfully manipulated AZGP1 expression using the following approaches:

• Lentiviral overexpression: Particularly effective in cell lines with low endogenous AZGP1 expression, such as RD rhabdomyosarcoma cells .

• Small hairpin RNA (shRNA) knockdown: Successfully employed in HT1080 fibrosarcoma cells to reduce AZGP1 expression by 55-80% compared to controls with scramble oligonucleotides .

• Cell line selection: Research shows that RD cells have lower endogenous AZGP1 levels compared to HT1080 and SW982 cells, making RD cells suitable for overexpression studies and HT1080 cells appropriate for knockdown experiments .

What functional assays are most informative for studying AZGP1's biological effects?

Several validated assays have been used to evaluate AZGP1's functional impact:

• Wound healing assay: Measures cell spreading capability following AZGP1 modulation. This assay has demonstrated decreased cell spreading in RD cells overexpressing AZGP1 .

• Transwell migration and invasion assays: Quantitatively assess cell motility and invasive potential. In RD cells, AZGP1 overexpression decreased migration by 62% and invasion by 81% compared to control cells . Conversely, AZGP1 knockdown in HT1080 cells increased migration 3.1-fold and invasion 5.2-fold .

• Protein-protein interaction studies: Important for elucidating AZGP1's molecular partners and downstream signaling pathways.

How does AZGP1 expression correlate with clinical outcomes in different cancer types?

AZGP1 expression shows significant associations with patient prognosis across multiple cancer types:

What molecular mechanisms explain AZGP1's role in tumor progression and metastasis?

Experimental evidence suggests AZGP1 functions as a metastasis suppressor through several mechanisms:

• Inhibition of cellular migration: In vitro studies show AZGP1 overexpression significantly decreased RD cell migration by 64%, while AZGP1 knockdown in HT1080 cells increased migration 3.1-fold .

• Suppression of invasion: AZGP1 overexpression reduced RD cell invasion by 78%, whereas AZGP1 knockdown enhanced HT1080 cell invasion by 5.2-fold .

• Effects on cell spreading: Wound healing assays demonstrated decreased cell spreading in AZGP1-overexpressing RD cells compared to controls .

These findings suggest AZGP1 may inhibit key processes required for metastatic dissemination, possibly through regulation of cytoskeletal dynamics, cell adhesion, or extracellular matrix interactions.

How do AZGP1 expression patterns vary across different cancer subtypes?

AZGP1 expression shows distinct patterns across cancer types with significant clinical correlations:

• Hepatocellular carcinoma:

  • Decreased AZGP1 expression observed in 80.1% (197/246) of HCC patient tissues .

  • Low AZGP1 expression significantly associated with high serum AFP levels (P = 0.013), liver cirrhosis (P = 0.002), and poor tumor differentiation (P = 0.025) .

• Soft tissue sarcomas:

  • AZGP1 expression negatively correlated with metastasis and disease-specific mortality .

  • Immunohistochemical analysis showed lower AZGP1 expression in patients with metastasis compared to those without .

• Other cancers:

  • Research has documented altered AZGP1 expression in prostate, breast, lung, and colorectal carcinomas, suggesting broad relevance across diverse malignancies .

How might AZGP1 function as a biomarker in precision oncology approaches?

AZGP1 shows promise as both a prognostic and potentially predictive biomarker:

What technical challenges exist in measuring AZGP1 for clinical applications?

Several methodological considerations affect reliable AZGP1 assessment:

• Tissue preservation and processing:

  • Fresh versus formalin-fixed tissues yield different quantitative results for AZGP1 detection.

  • Standardization of tissue collection, processing, and storage protocols is essential for consistent measurements.

• Detection methodology:

  • Quantitative RT-PCR provides sensitive mRNA quantification but requires stringent quality control.

  • Immunohistochemistry allows spatial visualization but depends on antibody specificity and optimal staining protocols.

  • Western blotting offers protein-level confirmation but with lower throughput.

• Scoring systems:

  • For IHC, development of standardized scoring methods is critical for clinical application.

  • Current research employs various semi-quantitative approaches that require further validation and harmonization.

How does AZGP1 interact with the tumor microenvironment?

Emerging research suggests complex interactions between AZGP1 and the tumor microenvironment:

• Influence on stromal cells:

  • AZGP1's role in lipid metabolism may affect cancer-associated adipocytes and metabolic reprogramming within the tumor microenvironment.

  • Potential interactions with immune cells given AZGP1's structural similarity to MHC class I molecules.

• Matrix interactions:

  • AZGP1's impact on invasion suggests potential effects on extracellular matrix composition or turnover.

  • These interactions may help explain AZGP1's role in regulating cancer cell migration and invasion.

• Metabolic effects:

  • AZGP1-mediated lipid mobilization could alter nutrient availability within the tumor microenvironment.

  • This metabolic function may contribute to AZGP1's broader role in cancer progression beyond direct effects on tumor cells.

What are promising therapeutic strategies targeting AZGP1 or its pathways?

Several therapeutic approaches warrant investigation:

• AZGP1 restoration strategies:

  • Gene therapy approaches to restore AZGP1 expression in cancers showing downregulation.

  • Small molecules that induce endogenous AZGP1 expression.

• Pathway modulation:

  • Targeting downstream effectors of AZGP1 signaling.

  • Identification of compounds that mimic AZGP1's inhibitory effects on migration and invasion.

• Combination approaches:

  • Integration of AZGP1-targeting strategies with conventional chemotherapy or immunotherapy.

  • Exploiting AZGP1's potential role in modulating tumor metabolism alongside metabolic inhibitors.

How might single-cell analysis enhance our understanding of AZGP1 biology?

Single-cell technologies offer several advantages for AZGP1 research:

• Cellular heterogeneity:

  • Characterization of AZGP1 expression across distinct cell populations within tumors.

  • Identification of specific cell types responsible for AZGP1 production in the tumor microenvironment.

• Rare cell populations:

  • Detection of AZGP1-expressing circulating tumor cells and their relationship to metastasis.

  • Analysis of therapy-resistant cell subpopulations and their AZGP1 expression patterns.

• Spatial context:

  • Integration of AZGP1 expression data with spatial information about the tumor microenvironment.

  • Correlation between AZGP1 expression patterns and local invasion or metastatic potential.

What contradictions in current AZGP1 research require resolution?

Several knowledge gaps and contradictions require further investigation:

• Tissue-specific effects:

  • While AZGP1 downregulation correlates with poor outcomes in HCC and STS, its expression pattern and significance may differ in other cancer types.

  • Reconciling these differences requires comprehensive pan-cancer analysis of AZGP1 function.

• Mechanistic understanding:

  • The precise molecular pathways through which AZGP1 inhibits migration and invasion remain incompletely characterized.

  • Identification of direct AZGP1 binding partners and signaling mediators requires further investigation.

• Causality versus correlation:

  • Whether AZGP1 downregulation is a cause or consequence of cancer progression remains unclear in many contexts.

  • Rigorous mechanistic studies with appropriate animal models are needed to establish causality.