PAS1 Antibody

What is PAS1 and what is its role in cancer biology?

PAS1 (PHACTR2-AS1) is a long non-coding RNA that functions as a tumor suppressor in breast cancer. It forms a tripartite complex with the RNA-binding protein vigilin and histone methyltransferase SUV39H1 to suppress PH20 expression. This suppression inhibits breast cancer growth and metastasis, as PH20 is a member of the human hyaluronidase family that degrades hyaluronan in the extracellular matrix and contributes to tumor progression . The interaction between PAS1 and vigilin maintains the stability of PAS1, while PAS1 recruits SUV39H1 to trigger H3K9 methylation of PH20, leading to its silencing .

How is PAS1 expression regulated?

PAS1 expression is negatively regulated by DNA methyltransferase 1 (DNMT1). Studies have shown that DNMT1 knockdown results in upregulation of PAS1 expression, while overexpression of DNMT1 leads to reduction in PAS1 RNA levels . This regulation occurs through the direct binding of DNMT1 to the PAS1 promoter . Additionally, research identified 13 genes that could suppress PAS1 transcription, primarily involved in the negative regulation of transcription from RNA polymerase II promoter .

What methods are available for detecting PAS1 in tissue samples?

PAS1 can be detected in tissue samples using RNA in situ hybridization, which allows visualization of the RNA directly in tissues. This technique can be complemented with immunohistochemical staining for proteins like DNMT1 to establish expression correlations . For experimental studies, reverse transcription-PCR can be used to detect and quantify PAS1 expression levels in cell lines and tissue samples. RNA immunoprecipitation (RIP) can also be employed to study interactions between PAS1 and proteins like vigilin and SUV39H1 .

How does PAS1 stability affect its function in cancer cells?

The stability of PAS1 is critical for its tumor-suppressive function and is maintained through its interaction with vigilin. Research has shown that overexpression of vigilin leads to increased levels of PAS1 in both the nucleus and cytoplasm . When treated with the transcriptional inhibitor actinomycin D, the half-life of PAS1 was found to be approximately 4 hours in control cells, but this was extended to 8 hours with vigilin overexpression . This increased stability allows PAS1 to more effectively suppress its downstream targets, including PH20.

What is the mechanism of the tripartite complex formation between PAS1, vigilin, and SUV39H1?

The tripartite complex formation between PAS1, vigilin, and SUV39H1 involves specific molecular interactions. RNA pull-down experiments combined with mass spectrometry identified vigilin as a specific PAS1-interacting protein . Further investigation using PAS1-truncation mutants revealed that the C terminus from nucleotides 1301 to 2134 was sufficient for vigilin binding . The interaction between PAS1 and SUV39H1 is facilitated by vigilin, which is known to interact with both molecules. RNA immunoprecipitation (RIP) and Co-IP assays confirmed that both PAS1 and vigilin could be immunoprecipitated by SUV39H1, and biotin-labeled PAS1 RNA could bind to both SUV39H1 and vigilin . This complex is crucial for targeting and silencing PH20 through H3K9 methylation.

How do epigenetic modifications influence the PAS1-mediated suppression of PH20?

The epigenetic regulation of PH20 by PAS1 involves histone methylation, specifically H3K9 dimethylation and trimethylation (H3K9me2 and H3K9me3). PAS1 recruits SUV39H1, a histone methyltransferase, to the PH20 promoter, leading to increased H3K9me2 and H3K9me3 at this site . These histone modifications are known to mediate gene silencing. Experimental evidence shows that SUV39H1 knockdown enhances the expression of PH20, confirming the role of this epigenetic mechanism in PAS1-mediated PH20 suppression . This epigenetic control adds another layer to the transcriptional regulation of PH20 and provides insights into how lncRNAs can modulate gene expression through chromatin modifications.

What are the technical challenges in developing specific antibodies against PAS1-associated proteins?

Developing specific antibodies against PAS1-associated proteins like vigilin and SUV39H1 presents several technical challenges. First, these proteins may exist in multiple isoforms or have post-translational modifications that affect epitope accessibility. Second, the proteins might form complexes with other cellular components, potentially masking antibody binding sites. For antibody development, researchers must carefully consider epitope selection to ensure specificity and avoid cross-reactivity with related proteins. Validation of antibodies should include multiple techniques such as western blot, immunoprecipitation, and immunofluorescence to confirm specificity . Additionally, using knockout or knockdown controls is essential to verify antibody specificity in the context of PAS1-associated protein detection.

What are the optimal protocols for detecting PAS1 expression in different tissue types?

Optimal detection of PAS1 expression in different tissue types requires a combination of techniques tailored to the specific research question. For fixed tissue samples, RNA in situ hybridization with PAS1-specific probes provides spatial information about expression patterns . For fresh or frozen tissues, quantitative RT-PCR offers sensitive detection of PAS1 expression levels. The protocol should include careful RNA extraction using RNase-free conditions, DNase treatment to remove genomic DNA contamination, and reverse transcription with random hexamers or oligo(dT) primers. For qPCR, PAS1-specific primers should be designed to span exon junctions where possible, and multiple reference genes should be used for normalization. In cell lines, subcellular fractionation followed by RT-PCR can determine the nuclear versus cytoplasmic distribution of PAS1, which is important for understanding its function in forming the tripartite complex with vigilin and SUV39H1 .

How can researchers effectively modulate PAS1 expression in experimental models?

For PAS1 modulation, researchers have successfully used DNMT1 inhibitors like decitabine to increase PAS1 expression, as well as direct methods such as lentiviral delivery of PAS1 for overexpression studies . When studying the effects of PAS1 modulation, it's important to confirm expression changes using RT-PCR and to assess downstream effects on target genes like PH20. Additionally, using synthetic PAS1 fragments with modifications like 2′-O-methylation and 5′-Cholesterol can enhance RNA stability for in vivo studies .

What techniques can be used to study PAS1 interactions with protein partners?

Several techniques can be employed to study PAS1 interactions with protein partners such as vigilin and SUV39H1:

• RNA pull-down: Using biotinylated PAS1 RNA as bait to capture interacting proteins, followed by mass spectrometry or western blot analysis for identification .

• RNA Immunoprecipitation (RIP): Utilizing antibodies against potential protein partners to precipitate protein-RNA complexes, followed by RT-PCR to detect PAS1 .

• Cross-linking Immunoprecipitation (CLIP): Adding a cross-linking step to RIP to capture transient or weak interactions.

• Co-localization studies: Using RNA FISH for PAS1 combined with immunofluorescence staining for protein partners to visualize co-localization in cells .

• Proximity Ligation Assay (PLA): Detecting protein-RNA interactions in situ with high sensitivity.

• Truncation mutants: Creating deletion variants of PAS1 to map the specific regions required for protein interactions, as demonstrated with the C-terminus binding to vigilin .

These techniques should be used in combination to provide robust evidence of specific interactions and their functional significance.

How should researchers interpret changes in PAS1 expression in relation to DNMT1 and PH20 levels?

When interpreting changes in PAS1 expression in relation to DNMT1 and PH20 levels, researchers should consider the following principles:

• Inverse correlation analysis: A negative correlation between DNMT1 and PAS1 expression is expected based on the regulatory mechanism . Similarly, an inverse relationship between PAS1 and PH20 levels should be observed. Quantitative analysis of this correlation can be performed using Pearson or Spearman correlation coefficients.

• Temporal dynamics: Consider the time course of expression changes, as there may be delays between changes in DNMT1, subsequent PAS1 upregulation, and downstream effects on PH20 expression.

• Threshold effects: Determine whether there are threshold levels of PAS1 expression required for significant suppression of PH20, which could indicate non-linear relationships in the pathway.

• Cell type specificity: The DNMT1-PAS1-PH20 axis may function differently in various cell types or cancer subtypes, requiring context-specific interpretation of expression data.

• Confounding factors: Other regulatory mechanisms may influence any of these three components, potentially obscuring the expected correlations in some experimental contexts.

Statistical analysis should include multivariate approaches to account for these complex relationships when interpreting experimental data .

What controls are essential when validating antibodies against proteins in the PAS1 pathway?

When validating antibodies against proteins in the PAS1 pathway, several essential controls should be included:

• Positive controls: Use cell lines or tissues known to express the target protein at high levels, such as HepG2, Hepa 1-6, IMR-32, Neuro-2A, or C6 cell lines for related pathway proteins .

• Negative controls: Include samples where the target protein is absent or knocked down using siRNA, shRNA, or CRISPR-Cas9 to confirm antibody specificity.

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to demonstrate that binding is specifically blocked.

• Multiple techniques validation: Confirm antibody specificity across multiple applications (Western blot, immunoprecipitation, immunohistochemistry, immunofluorescence) as performance can vary between techniques .

• Cross-reactivity testing: Especially important for antibodies detecting proteins in the PAS1 pathway, test against related family members to ensure specificity.

• Loading controls: Use established housekeeping proteins (e.g., GAPDH, β-actin) to normalize protein loading in Western blots.

• Recombinant protein standards: Include purified recombinant proteins as reference standards for size verification and quantification .

Documentation of these controls is essential for ensuring reproducibility and reliability of results using antibodies against proteins in the PAS1 pathway.

How might targeting the DNMT1-PAS1-PH20 axis impact cancer treatment strategies?

Targeting the DNMT1-PAS1-PH20 axis presents several promising avenues for cancer treatment strategies:

• DNMT1 inhibitors: Drugs like decitabine can suppress PH20 expression by activating PAS1, potentially inhibiting tumor growth and metastasis . This approach leverages existing FDA-approved drugs, potentially expediting clinical translation.

• PAS1 RNA therapeutics: Synthetic PAS1 fragments with modifications for in vivo delivery (such as PAS1-30nt-RNA) have shown efficacy in preclinical models . These could be developed as novel RNA therapeutics to restore PAS1 function in cancers where it is downregulated.

• Combination approaches: Combining DNMT1 inhibitors with PAS1-30nt-RNA has demonstrated superior efficacy compared to either treatment alone in inhibiting tumor growth and metastasis . This suggests that multi-targeted approaches may overcome resistance mechanisms and enhance therapeutic outcomes.

• Biomarker-guided therapy: Assessment of DNMT1, PAS1, and PH20 levels could potentially serve as biomarkers to identify patients most likely to benefit from these targeted therapies, enabling precision medicine approaches.

• Metastasis prevention: Given the role of PAS1 in suppressing cancer cell migration through PH20 inhibition, targeting this axis may be particularly valuable in preventing metastatic spread, addressing a major challenge in cancer treatment .

These strategies could potentially expand the therapeutic arsenal for breast cancer and possibly other malignancies where this regulatory axis is active.

What are the potential biomarkers for monitoring response to therapies targeting the PAS1 pathway?

Several potential biomarkers could be used to monitor response to therapies targeting the PAS1 pathway:

• PAS1 expression levels: Measured by RT-PCR in liquid biopsies (circulating tumor RNA) or in tumor tissue, increased PAS1 levels could indicate successful DNMT1 inhibition or effective PAS1 RNA therapy .

• PH20 expression: As a downstream target of PAS1, reduced PH20 levels in tumor tissue would suggest effective pathway modulation .

• H3K9 methylation status: Since PAS1 recruits SUV39H1 to trigger H3K9 methylation of PH20, assessing the methylation status at the PH20 promoter could serve as a mechanistic biomarker of pathway activity .

• Hyaluronan levels: As PH20 degrades hyaluronan in the extracellular matrix, changes in hyaluronan content in tumor tissue or circulation might reflect PH20 activity and indirectly indicate PAS1 pathway function .

• Vigilin-PAS1-SUV39H1 complex formation: Although technically challenging to assess in clinical samples, development of assays to detect this tripartite complex could provide direct evidence of functional PAS1 activity .

• Proliferation and apoptosis markers: Downstream effects such as decreased Ki67 expression (proliferation) and increased Bax expression (apoptosis) have been observed with PAS1 pathway modulation and could serve as pharmacodynamic markers .

Longitudinal monitoring of these biomarkers could help assess treatment efficacy and guide decisions about continuation or modification of therapy.

What are the unexplored aspects of PAS1 function beyond breast cancer?

While the role of PAS1 in breast cancer has been investigated, several unexplored aspects warrant further research:

• PAS1 in other cancer types: Given that hyaluronan degradation and extracellular matrix remodeling are relevant to multiple cancer types, PAS1 may play similar tumor-suppressive roles in other malignancies beyond breast cancer. Comprehensive expression profiling across cancer types could identify additional contexts where PAS1 dysregulation contributes to pathogenesis.

• Non-cancer functions: The physiological role of PAS1 in normal tissue development and homeostasis remains largely unexplored. PAS1 may have tissue-specific functions in regulating gene expression through its interaction with vigilin and SUV39H1.

• Regulatory networks: While DNMT1 has been identified as a negative regulator of PAS1, the complete transcriptional and post-transcriptional regulatory network controlling PAS1 expression requires further elucidation .

• Alternative binding partners: Beyond vigilin and SUV39H1, PAS1 may interact with other proteins in a context-dependent manner, potentially affecting diverse cellular processes beyond PH20 regulation .

• Role in therapy resistance: Whether alterations in the DNMT1-PAS1-PH20 axis contribute to resistance to conventional therapies remains to be determined and could provide insights into combination treatment strategies.

Investigating these aspects would enhance our understanding of PAS1 biology and potentially reveal new therapeutic opportunities.

How can high-throughput methods advance our understanding of the PAS1 interactome?

High-throughput methods offer powerful approaches to expand our understanding of the PAS1 interactome:

• RNA-protein interaction mapping: Techniques such as CLIP-seq or RAP-MS (RNA antisense purification with mass spectrometry) could comprehensively identify proteins that interact with PAS1 across different cellular contexts, potentially revealing context-specific binding partners beyond vigilin and SUV39H1 .

• Genomic binding site analysis: ChIRP-seq (Chromatin Isolation by RNA Purification followed by sequencing) could map the genomic binding sites of PAS1, potentially identifying additional genes beyond PH20 that are regulated by this lncRNA.

• Functional genomic screens: CRISPR-Cas9 or RNAi screens in the presence or absence of PAS1 could identify genes that synthetically interact with PAS1, revealing functional dependencies and pathway connections.

• Single-cell approaches: Single-cell RNA-seq combined with spatial transcriptomics could reveal cell type-specific expression patterns and functions of PAS1, providing insights into its role in heterogeneous tumor environments.

• Computational prediction and validation: Machine learning approaches could predict additional RNA-protein interactions based on known binding motifs and structural features, guiding targeted experimental validation.

These high-throughput approaches would provide a systems-level understanding of PAS1 function, potentially identifying new nodes for therapeutic intervention in the PAS1 pathway.