miR-503-5p induces doxorubicin resistance in triple-negative breast cancer.
Authors: Garrido-Cano Iris , Adam-Artigues Anna , Lameirinhas Ana , Pattanayak Birlipta , Tormo Eduardo , Miranda-Gonçalves Vera Mónica , Macedo-Silva Catarina , Rojo Federico , Zazo Sandra , Madoz-Gúrpide Juan , Burgues Octavio , Hernando Cristina , Martínez Maite , Moragón Santi , Bermejo De Las Heras Begoña , Lluch Ana , Jerónimo Carmen , Eroles Pilar , Cejalvo Juan Miguel 
Published: June 2021

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Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer (BC) subtype comprising approximately 15% of BC. Conventional cytotoxic chemotherapies continue to be the mainstay for treatment of this BC, which lacks targetable markers. In this context, microRNAs have been described to have an important role. The aim of this work was to elucidate the function of miR-503-5p in doxorubicin resistance in TNBC. Methods: miR-503-5p expression was evaluated in the TNBC cell line with acquired resistance to doxorubicin (MDA-MB-231R) and its parental cell line (MDA-MB-231), by qRT-PCR. Studies of gain/loss of function of miR-503-5p were carried out in MDA-MB-231 and MDA-MB-231R cells by transient transfection of mimics and inhibitors. Cells were treated with doxorubicin, and viability was measured by flow cytometry and MTT assay. The role of miR-503-5p was also evaluated in vivo by Chicken Chorioallantoic Membrane (CAM) assay. MDA-MB-231 cells transfected with miR-503-5p mimic or scramble miRNA were inoculated onto the CAM of fertilized chicken eggs. After 48 hours, tumours were treated with doxorubicin or supplemented media for 48 hours and tumour growth was measured. miR-503-5p expression was quantified by qRT-PCR in a retrospective cohort of 74 TNBC patients treated with anthracycline + taxane regimens. Overall survival analysis for miR-503-5p in TNBC patients from METABRIC dataset was evaluated by the KM plotter online tool. Results: miR-503-5p was significantly upregulated in the resistant MDA-MB-231R TNBC cell line when compared to its parental cell line MDA-MB-231 (̃3.5-fold; p< 0.0001). Then, gain/loss function assays showed that upregulation of miR-503-5p in MDA-MB-231 cells increased resistance to doxorubicin ( p< 0.0001) and its downregulation in MDA-MB-231R cells had the opposite effect ( p< 0.0001). Moreover, the role of miR-503-5p was also confirmed in the CAM assay in vivo model, where miR-503-5p overexpression inhibited the effect of doxorubicin. In our cohort of patients, miR-503-5p expression levels in core biopsies sampled before preoperative chemotherapy were associated with residual cancer burden (RCB). miR-503-5p expression was significantly higher in patients with poor response to chemotherapy (RCB II and III; median, 95% CI: 0.00055, 0.00024 - 0.00136) than in patients with good response (RCB 0 and I; median, 95% CI: 0.00018, 0.00011 - 0.00034; p = 0.036). Moreover, we confirmed that TNBC patients with high expression of miR-503-5p had worse overall survival than patients with low expression ( p= 0.016). Conclusions: We identified miR-503-5p as a modulator of doxorubicin resistance in TNBC. Our in vitro findings are supported by the clinical data of TNBC patients and in vivo assays. Hence, the inhibition of miR-503-5p may be a promising strategy to improve chemotherapeutic efficacy. Moreover, the expression levels of miR-503-5p may be used as a biomarker for therapy response in TNBC.