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Precision-engineered reporter cell lines reveal ABCG2 regulation in live lung cancer cells
Daniella Kovacsics1, Anna Brózik1, Borbála Tihanyi1
1Research Centre for Natural Sciences, Institute of Enzymology, Budapest, Hungary.
Abstract:
Expression of the ABCG2 multidrug transporter is a marker of cancer stem cells and a predictor of recurrent malignant disease. Understanding how human ABCG2 expression is modulated by pharmacotherapy is crucial in guiding therapeutic recommendations and may aid rational drug development. Genome edited reporter cells are useful in investigating gene regulation and visualizing protein activity in live cells but require precise targeting to preserve native regulatory regions. Here, we describe a fluorescent reporter assay that allows the noninvasive assessment of ABCG2 regulation in human lung adenocarcinoma cells. Using CRISPR-Cas9 gene editing coupled with homology-directed repair, we targeted an EGFP coding sequence to the translational start site of ABCG2, generating ABCG2 knock-out and in situ tagged ABCG2 reporter cells. Using the engineered cell lines, we show that ABCG2 is upregulated by a number of anti-cancer medications, HDAC inhibitors, hypoxia-mimicking agents and glucocorticoids, supporting a model in which ABCG2 is under the control of a general stress response. To our knowledge, this is the first description of a fluorescent reporter assay system designed to follow the endogenous regulation of a human ABC transporter in live cells. The information gained may guide therapy recommendations and aid rational drug design.
Insights
Researchers developed a new fluorescent reporter assay to track ABCG2 transporter regulation in live lung cancer cells. This tool reveals how drugs and stress impact ABCG2 expression, aiding therapy and drug design.
Area of Science:
- Molecular Biology
- Cancer Research
- Pharmacology
Background:
- ABCG2 multidrug transporter expression is linked to cancer stem cells and disease recurrence.
- Understanding ABCG2 regulation by drugs is vital for cancer treatment and drug development.
- Existing methods for studying gene regulation in live cells require precise targeting.
Purpose of the Study:
- To develop a noninvasive fluorescent reporter assay for assessing ABCG2 regulation in human lung adenocarcinoma cells.
- To investigate the modulation of endogenous ABCG2 expression by various therapeutic agents and stress conditions.
Main Methods:
- Utilized CRISPR-Cas9 gene editing and homology-directed repair to tag the ABCG2 translational start site with EGFP.
- Generated ABCG2 knock-out and in situ tagged ABCG2 reporter cell lines.
- Employed the engineered cell lines to monitor ABCG2 expression changes in response to different treatments.
Main Results:
- Demonstrated upregulation of ABCG2 by anti-cancer drugs, HDAC inhibitors, hypoxia-mimicking agents, and glucocorticoids.
- Supported a model where ABCG2 is regulated by a general stress response pathway.
- Successfully established a live-cell assay for endogenous ABC transporter regulation.
Conclusions:
- The developed fluorescent reporter assay allows real-time monitoring of endogenous ABCG2 regulation in live cells.
- Findings suggest ABCG2 is part of a broader cellular stress response.
- This system can guide therapeutic recommendations and facilitate rational drug design for cancer treatment.
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