# LRRC8A Inhibition Overcomes Chemoresistance by Downregulating MRP3 and CYP3A4 in the 3D Spheroid Model of Human Breast Cancer Cells

## TL;DR

Inhibiting LRRC8A in 3D breast cancer models reduces drug resistance by lowering MRP3 and CYP3A4 levels.

## Contribution

LRRC8A inhibition is shown to overcome chemoresistance via downregulation of MRP3 and CYP3A4 in 3D breast cancer spheroids.

## Key findings

- LRRC8A inhibition sensitized 3D spheroids to doxorubicin, gemcitabine, and 5-fluorouracil.
- Spheroid formation increased MRP3 and CYP3A4 expression, which was suppressed by LRRC8A inhibition.
- NRF2–CEBPB/D axis mediates the transcriptional upregulation of MRP3 and CYP3A4.

## 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.