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A Gene Expression Biomarker Identifies Activation of XBP-1 by Chemical Inducers of the Unfolded Protein Response (UPR) in a Gene Expression Compendium
Citation:
Ledbetter, V., J. Liu, Jessica Hanson, B. Chorley, AND Jon Corton. A Gene Expression Biomarker Identifies Activation of XBP-1 by Chemical Inducers of the Unfolded Protein Response (UPR) in a Gene Expression Compendium. SOT, Salt Lake City, UT, March 11 - 14, 2024. https://doi.org/10.23645/epacomptox.25534159
Impact/Purpose:
The US EPA has developed a tiered testing strategy, including high-throughput transcriptomics (HTTr) testing, to identify molecular targets of thousands of environmental chemicals. These findings can be linked to adverse outcomes and can be used in Integrated Approaches to Testing and Assessment (IATA). Exposure of human cells lines to environmental chemicals can lead to stress responses including the unfolded protein response (UPR). One of the three major arms of the UPR is controlled through activation of the endoplasmic reticulum (ER)-resident protein kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1), leading to activation of X-box binding protein 1 (XBP1). Here, we describe a method that uses a gene expression biomarker to identify UPR inducers that activate XBP1 in a microarray compendium after chemical exposure.
Description:
Background/Purpose The US EPA has developed a tiered testing strategy, including high-throughput transcriptomics (HTTr) testing, to identify molecular targets of thousands of environmental chemicals. These findings can be linked to adverse outcomes and can be used in Integrated Approaches to Testing and Assessment (IATA). Exposure of human cells lines to environmental chemicals can lead to stress responses including the unfolded protein response (UPR). One of the three major arms of the UPR is controlled through activation of the endoplasmic reticulum (ER)-resident protein kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1), leading to activation of X-box binding protein 1 (XBP1). Here, we describe a method that uses a gene expression biomarker to identify UPR inducers that activate XBP1 in a microarray compendium after chemical exposure. Methods Transcript profiles were generated using full genome Tempo-Seq from wild-type MCF-7 cells and those nullizygous for IRE1 treated for 6 hrs with 4 UPR inducers (AA147, brefeldin A, thapsigargin, tunicamycin). Genes were identified that were similarly regulated in wild-type cells across the 4 treatments but not in the nullizygous cells. The resultant gene list containing 83 genes and associated fold-changes averaged across the treatments in wild-type cells were imported into the Illumina BaseSpace Correlation Engine (BSCE) for comparison to other gene lists. Using the Running Fisher test, the resultant list of genes and fold-changes were compared to annotated lists of genes in BSCE from a variety of chemical treatment studies in human cell lines in vitro. Characterized gene expression biomarkers with known accuracy were used to provide additional insights into integrated stress responses and included those that predict modulation of NRF2, HIF1a, HSF1, MTF1, and NFKB. Additional biomarkers used in the analysis include those that predict histone deacetylase suppression (TGx-HDACi) and DNA damage (TGx-DDI). Results Using the upstream analysis function in Ingenuity Pathways Analysis, the 83 gene biomarker was most closely correlated to genes regulated by XBP1 and thus the biomarker was called the XBP1 biomarker. Examination of microarray datasets in BSCE from cells treated with known UPR inducers identified a 6-24 hr window in which XBP1 was maximally induced. Using treatment times of known inducers that were 6-24 hrs long (true positives) and chemical treatments 96 hrs or longer in which the UPR response was not expected to be induced, the balanced accuracy was 92%. A microarray compendium consisting of 12,092 comparisons from human cells exposed to 2,670 individual chemicals was then screened using our approach; 102 and 15 chemicals were identified as putative XBP1 activators or suppressors, respectively, and many appear to be novel modulators. The XBP1 biomarker was compared to profiles from MCF-7 cells treated with up to 10 concentrations of bisphenol A (BPA) and 16 BPA alternatives. XBP1, along with many stress factors, were activated with increasing chemical concentration in parallel with decreases in TGx-HDACi. Conclusions The approach using the XBP1 biomarker described here could be used to identify environmentally-relevant XBP1 activators in HTTr datasets. These findings along with those of other characterized biomarkers could be integrated into AOP frameworks as part of IATA. This abstract does not reflect EPA policy.
URLs/Downloads:
DOI: A Gene Expression Biomarker Identifies Activation of XBP-1 by Chemical Inducers of the Unfolded Protein Response (UPR) in a Gene Expression Compendium
XBP1POSTER_SOT_03_06_24_FINAL.PDF (PDF, NA pp, 1570.995 KB, about PDF)