PRMT5-Regulated Splicing of DNA Repair Genes Drives Chemoresistance in Breast Cancer Stem Cells

Academic Background

Breast Cancer Stem Cells (BCSCs) are a rare subpopulation of cells within breast cancer that possess the ability to self-renew, initiate tumors, and metastasize. Despite their critical role in tumor initiation and progression, the mechanisms by which BCSCs resist chemotherapy and radiation remain poorly understood. Chemotherapy and radiation typically induce DNA damage to kill cancer cells, and BCSCs can resist these treatments by enhancing their DNA repair capabilities. Therefore, understanding how BCSCs resist chemotherapy through DNA repair mechanisms is crucial for developing new therapeutic strategies.

PRMT5 (Protein Arginine Methyltransferase 5) is an arginine methyltransferase involved in various cellular processes, including RNA splicing, DNA repair, and gene expression regulation. PRMT5 is upregulated in multiple cancers and is associated with tumor progression and poor prognosis. Previous studies have shown that PRMT5 is highly expressed in BCSCs and is closely related to their survival and self-renewal. However, how PRMT5 regulates the splicing of DNA repair genes to influence chemoresistance in BCSCs remains an unresolved question.

Source of the Paper

This paper was co-authored by Matthew S. Gillespie, Kelly Chiang, Gemma L. Regan-Mochrie, and other researchers from the Department of Cancer and Genomic Sciences at the University of Birmingham, UK. The paper was published in 2024 in the journal Oncogene, with the DOI: 10.1038/s41388-024-03264-1.

Research Process and Results

1. BCSCs Resist DNA-Damaging Agents Through Enhanced DNA Repair

The study first isolated BCSCs from the MCF7 breast cancer cell line using flow cytometry and anoikis-resistance (AR) methods. Experiments showed that BCSCs exhibited greater resistance to cisplatin and ionizing radiation (IR). By analyzing the clearance rate of 53BP1 foci, researchers found that BCSCs could repair cisplatin-induced DNA double-strand breaks (DSBs) more rapidly, indicating enhanced DNA damage response (DDR) capabilities in BCSCs.

2. PRMT5 Promotes DNA Repair and Chemoresistance in BCSCs

The high expression of PRMT5 in BCSCs is closely related to their chemoresistance. Through shRNA-mediated knockdown of PRMT5, researchers found that the loss of PRMT5 significantly increased the sensitivity of BCSCs to cisplatin and IR. Additionally, treatment with the PRMT5 inhibitor GSK3203591 (GSK591) also significantly increased the chemosensitivity of BCSCs. Further experiments demonstrated that the methyltransferase activity of PRMT5 is a key factor in the chemoresistance of BCSCs.

3. PRMT5 Regulates the Splicing of DNA Repair Genes

PRMT5 not only regulates DNA repair through the methylation of DNA repair proteins but also influences the expression of DNA repair genes by regulating RNA splicing. Through RNA sequencing (RNA-seq) analysis, researchers identified a large number of differential splicing events (DSEs) in BCSCs treated with the PRMT5 inhibitor GSK591, particularly in genes related to DNA repair. These splicing events primarily involved skipped exons (SE) and retained introns (RI), leading to the production of non-canonical splice isoforms that affect the function of DNA repair proteins.

4. Relationship Between Splicing Changes and Gene Expression Changes Induced by PRMT5 Inhibition

Although PRMT5 inhibition caused extensive splicing changes, these changes did not significantly affect overall gene expression levels. Researchers found that PRMT5 inhibition primarily led to the nuclear retention of intron-containing transcripts and the production of non-canonical splice isoforms. These results suggest that PRMT5 affects the function of DNA repair proteins through splicing regulation rather than by altering gene expression levels.

5. Differences in Splicing Regulation Between BCSCs and Bulk Tumor Cells

Researchers also compared the splicing regulation of PRMT5 inhibition in BCSCs and bulk tumor cells. Although both exhibited splicing changes in DNA repair genes, the splicing events in BCSCs were more specific, and BCSCs were less sensitive to PRMT5 inhibition. This indicates that BCSCs enhance their DNA repair capabilities and chemoresistance through high levels of PRMT5 expression and splicing regulation.

Conclusions and Significance

This study reveals the critical role of PRMT5 in the chemoresistance of BCSCs, particularly through the regulation of DNA repair gene splicing to enhance DNA repair capabilities. PRMT5 inhibition significantly increases the sensitivity of BCSCs to chemotherapeutic agents, especially when combined with DNA-damaging agents, effectively promoting BCSC apoptosis. This discovery provides important theoretical support for the development of new breast cancer treatment strategies, particularly combination therapies targeting PRMT5 inhibitors and DNA-damaging agents.

Research Highlights

1. PRMT5 Regulates DNA Repair Gene Splicing: PRMT5 enhances the DNA repair capabilities of BCSCs by regulating the splicing of DNA repair genes, leading to chemoresistance.
2. PRMT5 Inhibition Enhances Chemosensitivity: PRMT5 inhibition significantly increases the sensitivity of BCSCs to cisplatin and IR, especially when combined with DNA-damaging agents, effectively killing BCSCs.
3. Separation of Splicing Changes and Gene Expression Changes: Splicing changes induced by PRMT5 inhibition do not significantly affect overall gene expression levels but instead affect DNA repair protein function through the production of non-canonical splice isoforms.
4. Differences in Splicing Regulation Between BCSCs and Bulk Tumor Cells: BCSCs are less sensitive to PRMT5 inhibition, indicating that they enhance their DNA repair capabilities and chemoresistance through high levels of PRMT5 expression and splicing regulation.

Research Value

This study not only reveals the critical role of PRMT5 in the chemoresistance of BCSCs but also provides important theoretical support for the development of new breast cancer treatment strategies. By combining PRMT5 inhibitors with DNA-damaging agents, it may be possible to effectively kill chemoresistant BCSCs, thereby improving the long-term survival rates of breast cancer patients. Additionally, this study offers new research directions for further exploring the role of PRMT5 in other cancer stem cells.