ASPH Human

What is ASPH and what is its normal expression pattern in human tissues?

Aspartate β-hydroxylase (ASPH) is a transmembrane protein that catalyzes the hydroxylation of aspartate and asparagine residues in epidermal growth factor-like domains (EGFDs) of various substrates . In normal adult tissues, ASPH is rarely expressed, with the notable exception of placental trophoblastic cells . This limited expression pattern in healthy adult tissues makes ASPH particularly interesting as a potential biomarker and therapeutic target in pathological conditions where its expression becomes dysregulated.

The protein's normal function appears to be developmentally regulated, with higher expression during embryogenesis followed by significant downregulation in most adult tissues. This expression pattern suggests ASPH plays important roles in cellular differentiation and tissue development that are largely unnecessary in fully differentiated adult tissues.

How is ASPH expression regulated in human cells?

ASPH expression is regulated through several signaling cascades, with insulin/insulin-like growth factor 1 (IGF-1) signaling pathways playing a central role. Research has demonstrated that stimulation with insulin and IGF-1 increases ASPH mRNA and protein expression in human cell lines . This upregulation is mediated primarily through:

• The ERK/MAPK (extracellular signal-regulated kinase/mitogen-activated protein kinase) pathway

• The PI3K/Akt (phosphatidylinositol 3-kinase/protein kinase B) pathway

Specifically, insulin receptor substrate 1 (IRS1)-mediated signaling has been identified as an upstream regulator of ASPH expression . These findings highlight how growth factor signaling can drive ASPH expression, which becomes particularly relevant in understanding its pathological overexpression in cancer contexts.

What is the relationship between ASPH and human malignancies?

ASPH overexpression has been documented in numerous human malignancies, occurring in approximately 70-90% of human tumors . Specifically, elevated ASPH expression has been observed in:

• Hepatocellular carcinoma (HCC)

• Cholangiocarcinoma

• Pancreatic cancer

• Lung cancer

• Breast cancer

• Colon cancer

• Neoplasms of the nervous system

In hepatocellular carcinoma patients, ASPH overexpression significantly correlates with higher recurrence rates and lower survival rates following surgical intervention . This association is particularly pronounced in early-stage HCC patients, suggesting ASPH may serve as a prognostic biomarker. In pancreatic cancer, ASPH overexpression promotes proliferation, migration, invasion, and malignant transformation through multiple signaling pathways .

What techniques are currently used to measure ASPH activity in research settings?

Recent methodological advances have significantly improved our ability to assess ASPH activity in research settings. A notable development is the solid-phase extraction mass spectrometry-based high-throughput ASPH inhibition assay . This technique employs:

• A stable synthetic thioether mimic of ASPH substrates

• Solid-phase extraction methodology

• Mass spectrometry detection

This assay demonstrates excellent robustness with high Z'-factors and good signal-to-noise/background ratios . The approach allows for efficient screening of potential ASPH inhibitors and has been successfully applied to screen approximately 1,500 bioactive small molecules, including natural products and active pharmaceutical ingredients .

Prior to this development, researchers faced significant challenges in efficiently monitoring the activity of isolated ASPH, which limited progress in identifying small-molecule ASPH inhibitors.

How does ASPH mechanistically affect the Notch signaling pathway in human cancer progression?

ASPH has been demonstrated to activate the Notch signaling pathway, which represents a key mechanism driving malignant transformation, particularly in pancreatic cancer cells . The Notch signaling cascade is a highly conserved pathway critical for cell-cell signaling and cell fate determination during embryogenesis.

The mechanistic relationship between ASPH and Notch activation in pancreatic ductal adenocarcinoma (PDAC) involves:

• Overexpression of activated Notch1 and hairy and enhancer of split-1 (HES1, a Notch-responsive gene) in PDAC cells compared to adjacent normal tissues

• ASPH-mediated hydroxylation of EGF-like repeats in Notch receptors, enhancing their interactions with ligands

• Subsequent activation of downstream transcriptional programs that promote cellular proliferation, survival, and invasiveness

While Notch receptors and ligands are typically downregulated in normal adult pancreatic tissue, they become activated during pancreatic tumorigenesis. Notch activation promotes epithelial-to-mesenchymal transition (EMT), facilitating invasion and metastasis of pancreatic cancer cells . This ASPH-Notch signaling axis represents a critical pathway that can be targeted therapeutically to potentially inhibit cancer progression.

What is the role of ASPH in mitochondrial DNA integrity and how does this contribute to oncogenesis?

ASPH has been implicated in disrupting mitochondrial DNA (mtDNA) integrity, which contributes to oncogenesis through multiple mechanisms. Somatic mtDNA mutations have been detected in various tumor types, including pancreatic cancer . Research in hepatocellular carcinoma has demonstrated that ASPH overexpression significantly correlates with:

• Decreased copy numbers of the displacement loop (D-loop) region of mtDNA

• Reduced NADH dehydrogenase subunit 1

• Increased somatic mutations in the D-loop region

The mechanistic basis for these effects involves ASPH disruption of the H2AX-mtTFA signaling pathway:

• Under normal conditions, H2AX (H2A histone family member X) functions as a shuttle protein transporter that carries mitochondrial transcription factor A (mtTFA) from the cytoplasm to mitochondria

• mtTFA plays a crucial role in mtDNA replication, transcription, and repair

• Overexpressed ASPH competes with mtTFA for binding to H2AX, blocking mtTFA translocation into mitochondria

• This competition reduces mtTFA binding to the D-loop, disrupting mtDNA maintenance

These alterations ultimately lead to:

• Increased mutations in mtDNA

• Reduced mtDNA copy number

• Decreased expression of mitochondrial respiratory chain enzymes

• Aberrant mitochondrial membrane potential

• Decreased ATP production

• Increased reactive oxygen species (ROS)

This cascade of mitochondrial dysfunction contributes to the metabolic reprogramming characteristic of cancer cells and potentially enhances their invasive capabilities.

What therapeutic approaches targeting ASPH are under development for human cancers?

ASPH has emerged as a promising therapeutic target due to its overexpression in 70-90% of human tumors and its cell surface localization in cancer cells . Several therapeutic approaches are being investigated:

Immunotherapeutic Approaches

ASPH-based immunotherapy leverages the protein's translocation from the endoplasmic reticulum to the plasma membrane in cancer cells, where it is exposed to the extracellular environment and can function as a tumor-associated antigen (TAA) .

Research has demonstrated that:

• ASPH-loaded dendritic cells (DCs) exhibit substantial anti-tumor effects in hepatocellular carcinoma models

• Both CD4+ T cells and CD8+ cytotoxic T cells contribute to these anti-tumor effects

• ASPH protein-loaded DCs can activate CD4+ T cells and CD8+ CTLs via ASPH-derived HLA class I- and class II-restricted peptides

Similar effects have been observed in cholangiocarcinoma models, where ASPH-loaded DCs exhibited cytotoxic effects in vitro, suppressed intrahepatic tumor growth and metastasis in rats, and increased infiltration of CD3+ lymphocytes into tumors .

Small Molecule Inhibitors

Several small molecule inhibitors targeting ASPH have shown promise:

• MO-I-1100: This β-hydroxylase inhibitor reduced ASPH activity by 80% and inhibited ASPH-induced proliferation, migration, invasion, and colony formation in pancreatic cancer models, while suppressing Notch signaling

• MO-I-1151: Demonstrated significant reduction in viability and directional motility of glioblastoma multiforme cells

• Vadadustat: Originally developed as a hypoxia-inducible factor prolyl-hydroxylase inhibitor, this compound was found to inhibit ASPH with moderate potency

Antibody-Based Approaches

Monoclonal antibodies targeting ASPH show potential for both imaging and therapeutic applications:

• Radiolabeled human monoclonal antibody PAN-622 targeting cell surface ASPH shows promise for imaging and potentially treating metastatic breast cancer

• Monoclonal antibodies against the ASPH C-terminal (ASPH-C) increase antibody-dependent cellular cytotoxicity of NK cells against various cancer cell lines

Nucleic Acid-Based Therapies

RNA interference approaches have demonstrated efficacy in preclinical models:

• Antisense oligodeoxynucleotide inhibition of ASPH expression reduced motility of cholangiocarcinoma cells

• Small interfering RNAs (siRNAs) targeting exon 2 of the ASPH gene inhibited ASPH expression and reduced directional motility in HCC cells

What methodological advances have enabled high-throughput screening for ASPH inhibitors?

Recent methodological advances have overcome previous limitations in ASPH inhibitor screening. A key breakthrough came with the development of a stable synthetic thioether mimic of ASPH substrates that can be employed in solid-phase extraction mass spectrometry-based high-throughput ASPH inhibition assays .

The development process involved:

• Understanding that ASPH substrates have a non-canonical EGFD disulfide pattern

• Creating a stable synthetic thioether mimic that retains appropriate substrate properties

• Incorporating this mimic into a solid-phase extraction methodology

• Coupling with mass spectrometry detection for high-throughput analysis

This assay system demonstrates:

• Excellent robustness (high Z'-factors)

• Good signal-to-noise and signal-to-background ratios

• Suitability for screening large compound libraries

Using this methodology, researchers successfully screened approximately 1,500 bioactive small molecules, including natural products and active pharmaceutical ingredients of approved human therapeutics . This screening identified potent ASPH inhibitors from both compound classes, demonstrating the utility of the approach for drug discovery efforts.

How does ASPH expression correlate with specific cancer subtypes and patient outcomes?

ASPH expression patterns vary across cancer subtypes and correlate with specific clinical outcomes. In hepatocellular carcinoma, Wang et al. demonstrated a significant association between ASPH overexpression and higher recurrence rates as well as lower survival rates following surgical intervention . This correlation is particularly pronounced in early-stage HCC patients, suggesting ASPH may serve as an early prognostic biomarker.

Research has established correlations between ASPH expression and:

These correlations highlight the potential utility of ASPH as both a prognostic biomarker and a therapeutic target.