EACR25-1777

Functional and molecular changes implicated in doxorubicin resistance in breast cancer

L. Trnkova¹, V. Buocikova¹, A. Soltysova^2,3, S. Miklikova¹, B. Tichy⁴, V. Bystry⁵, A. Cumova¹, M. Burikova¹, M. Cihova¹, B. Smolkova¹

¹Biomedical Research Center of the Slovak Academy of Sciences, Department of Molecular Oncology, Cancer Research Institute, Bratislava, Slovakia
²Comenius University in Bratislava, Department of Molecular Biology, Faculty of Natural Sciences, Bratislava, Slovakia
³Biomedical Research Center of the Slovak Academy of Sciences, Department of Human Genetics, Institute for Clinical and Translational Research, Bratislava, Slovakia
⁴CEITEC – Central European Institute of Technology, Masaryk University, Genomics Core Facility, Brno, Czech Republic
⁵CEITEC – Central European Institute of Technology, Masaryk University, Bioinformatics Core Facility, Brno, Czech Republic

Introduction:

Breast cancer (BC) is a prevalent oncological disease among women worldwide. Although therapeutic options for BC have improved significantly in recent years, the emergence of therapeutic resistance substantially decreases treatment efficacy. Doxorubicin (DOX), a chemotherapeutic agent classified as a topoisomerase II inhibitor, is commonly used in the treatment of triple-negative BC and is also employed in advanced stages of hormone receptor-positive or HER2-positive BC. This work describes the derivation of a cell line model to explore the mechanisms contributing to acquired DOX resistance in BC.

Material and method:

DOX-resistant cancer cells were derived from three BC cell lines (MDA-MB-231, JIMT-1, and T-47D) through continuous exposure to increasing DOX concentrations. A luminescent cell viability assay was used to assess the sensitivity of BC cells to chemotherapy. Functional assays included cell proliferation and migration analysis using the IncuCyte ZOOM system, in vivo tumorigenicity tests in SCID/bg immunodeficient mice, and β-galactosidase staining. Molecular and genomic changes in the resistant cancer cells were assessed using the RT2 Profiler PCR array, qRT-PCR, western blotting, array-based comparative genomic hybridization, and whole exome sequencing.

Result and discussion:

The derived BC cells exhibited a 5 to 8-fold increase in IC50 values upon the treatment with DOX, confirming their resistant phenotype. Cell proliferation and tumor growth were decreased in DOX-resistant cancer cells, but no statistically significant changes in cell migration were observed. Additionally, MDA-MB-231 and T-47D DOX-resistant cells displayed reduced β-galactosidase activity, a marker of cellular senescence. Molecular analysis revealed the deregulation of genes involved in drug resistance mechanisms, including those encoding ABC-transporters, genes involved in apoptotic signaling, drug metabolism, and genes and proteins implicated in DNA methylation processes. These changes partially corresponded with the observed copy number alterations in drug-resistant cells. Finally, single-nucleotide variants were prevalent mutations in the resistant cancer cells, affecting multiple genes, including the target enzyme of DOX.

Conclusion:

The acquisition of DOX resistance in BC is a complex process involving diverse changes at genomic, molecular, and functional levels. These changes include well-described multi-drug resistance mechanisms, alterations specific to DOX resistance, and newly observed changes in drug-resistant BC cells that warrant further investigation. This work was supported by the EraCoSysmed project RESCUER and VEGA 2/0067/22. We acknowledge the Genomics Core Facility and Bioinformatics Core Facility of CEITEC Masaryk University of A4L_ACTIONS, supported by the European Union’s Horizon 2020 under grant agreement No. 964997.