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Published on: June 13, 2020
LRRC8A Inhibition Overcomes Chemoresistance by Downregulating MRP3 and CYP3A4 in the 3D Spheroid Model of Human
Ryo Otsuka1, Junko Kajikuri1, Miki Matsui1
1Department of Pharmacology, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, Japan.
Abstract:
Leucine-rich repeat-containing 8A (LRRC8A; also known as SWELL1), the essential subunit of volume-regulated anion channels (VRACs), is amplified in multiple malignancies and has been implicated in tumor progression and therapeutic resistance. Three-dimensional (3D) cancer spheroids have been well-established as in vitro models that recapitulate characteristics of tumor stemness and intrinsic drug resistance. In the present study, spheroid formation in human breast cancer cell lines, YMB-1 and MDA-MB-468, conferred resistance to multiple anticancer drugs, including doxorubicin (DOX), gemcitabine (GEM), and 5-fluorouracil (5-FU), thereby mimicking the characteristic properties of breast cancer stem-like cells. LRRC8A expression was upregulated in 3D spheroids compared with adherent 2D monolayers, and its pharmacological inhibition induced membrane hyperpolarization accompanied by intracellular Cl- accumulation. Inhibition of LRRC8A significantly sensitized spheroids to DOX, GEM, and 5-FU. Spheroid formation increased the expression of multidrug resistance-related protein 3 (MRP3) and the drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4), whereas LRRC8A inhibition suppressed their expression. The transcriptional upregulation of MRP3 and CYP3A4 was mediated through the NRF2-CEBPB/D transcriptional axis. Collectively, these findings suggest that LRRC8A inhibition may represent a therapeutic strategy to overcome chemoresistance by repressing MRP3 and/or CYP3A4 expression in breast cancer stem cells.
Insights
Inhibiting Leucine-rich repeat-containing 8A (LRRC8A) in 3D breast cancer spheroids overcomes chemoresistance. This approach reduces drug efflux and metabolism, sensitizing cancer stem-like cells to chemotherapy.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Leucine-rich repeat-containing 8A (LRRC8A) is crucial for volume-regulated anion channels (VRACs) and is amplified in cancers, contributing to tumor progression and drug resistance.
- Three-dimensional (3D) cancer spheroids model tumor stemness and intrinsic drug resistance, making them valuable for studying chemoresistance mechanisms.
Purpose of the Study:
- To investigate the role of LRRC8A in chemoresistance within 3D breast cancer spheroids.
- To explore LRRC8A inhibition as a therapeutic strategy to overcome drug resistance in breast cancer stem-like cells.
Main Methods:
- Human breast cancer cell lines (YMB-1, MDA-MB-468) were cultured as 2D monolayers and 3D spheroids.
- LRRC8A expression and function were assessed in both culture models.
- Pharmacological inhibition of LRRC8A was performed, followed by drug sensitivity assays (doxorubicin, gemcitabine, 5-fluorouracil).
- Expression of multidrug resistance-related protein 3 (MRP3) and cytochrome P450 3A4 (CYP3A4) was analyzed, along with their transcriptional regulation via the NRF2-CEBPB/D axis.
Main Results:
- Spheroid formation enhanced resistance to doxorubicin, gemcitabine, and 5-fluorouracil, mimicking breast cancer stem-like cell properties.
- LRRC8A expression was upregulated in 3D spheroids compared to 2D cultures.
- LRRC8A inhibition sensitized spheroids to chemotherapy and suppressed the expression of MRP3 and CYP3A4.
- The NRF2-CEBPB/D transcriptional axis mediated the upregulation of MRP3 and CYP3A4 in spheroids.
Conclusions:
- LRRC8A plays a significant role in mediating chemoresistance in breast cancer stem-like cells within 3D spheroid models.
- Inhibiting LRRC8A can re-sensitize resistant breast cancer cells to chemotherapy.
- Targeting LRRC8A may be a viable strategy to overcome chemoresistance by suppressing MRP3 and CYP3A4 expression.
