School of Medicine and Health Sciences Poster Presentations

Title

Exosomal miRNAs may modulate chemoresistance in triple negative breast cancer

Document Type

Poster

Abstract Category

Cancer/Oncology

Keywords

exosomal miRNA, chemoresistance, Annexin 6, triple negative breast cancer

Publication Date

Spring 5-1-2019

Abstract

Triple-negative breast cancer (TNBC) is one of the most aggressive breast cancer subtypes. Chemoresistance leads to high mortality, and its underlying mechanisms are poorly understood. Extracellular vesicles (EVs), including exosomes, are nanoscale particles surrounded by phospholipid bilayers, act as efficient carriers of RNAs (including miRNAs), proteins and other bioactive molecules and also known to play a key role in cell-cell communication between cancer cells and adjacent microenvironments, accelerating pathological processes including immune suppression, angiogenesis, cell migration, tumor invasion, and drug resistance. The primary purpose of this study was to characterize the potential role of exosomal miRNAs in TNBC chemoresistance and to analyze their underlying mechanisms. Exosomes were isolated from drug-resistant TNBC cell lines (MDA-MB-231/Epirubicin, Paclitaxel, Cisplatin) and their parental cell line using the Total Exosome Isolation kit (Invitrogen). Exosome RNAs and proteins were isolated using the Total Exosome RNA and Protein isolation kit. Quantitative real-time reverse transcription-PCR (qRT-PCR) assays were used to detect the expression of several known miRNAs that are associated with drug-resistance or tumor invasions, such as miR-19b, miR-141, miR-181a, miR-181c, miR-196b, miR-197, miR-200b, miR-203, miR-222, miR-375, miR-638 and miR-671-5p in both cellular RNA and exosomal RNA. The expression of miR-181c, miR-203, miR-375, miR-638, and miR-671-5p was downregulated in most drug-resistant cell lines compared to the parental cell line. In particular, miR-671-5p was downregulated in all three drug-resistant cell lines in both cellular and exosomal RNA. Furthermore, target predictions using the miRanda algorithm (microRNA.org) showed that miR-181c, miR-203, and miR-671-5p collectively shared the same target gene, Annexin-6 (AXNA6), which is a calcium-dependent membrane and phospholipid binding protein implicated in membrane-related events along exocytotic and endocytotic pathways. Recently, EV-associated AXNA6 was found to be linked to breast cancer metastasis after treatment with neoadjuvant chemotherapy in mouse models and human breast cancer cell lines. Given that EV-associated AXNA6 may confer chemoresistance, we explored the expression of EV-AXNA6 in chemoresistant breast cancer cell lines via qRT-PCR. Primers for ANXA6 were designed using the Primer-BLAST. The expression of AXNA6 in both cellular and exosomal RNA was assayed by qRT-PCR. The expression of the ANXA6 gene in drug-resistant cell lines was upregulated in both cellular and exosomal RNA compared to that in the parental cell line. These data suggest that the downregulation of miR-181c, miR-203, and miR-671-5p in exosomes may result in ANXA6 overexpression, leading to chemoresistance in TNBC. Targeting such miRNAs may be a new avenue to tackle drug resistance.

Open Access

1

Comments

Presented at Research Days 2019.

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Exosomal miRNAs may modulate chemoresistance in triple negative breast cancer

Triple-negative breast cancer (TNBC) is one of the most aggressive breast cancer subtypes. Chemoresistance leads to high mortality, and its underlying mechanisms are poorly understood. Extracellular vesicles (EVs), including exosomes, are nanoscale particles surrounded by phospholipid bilayers, act as efficient carriers of RNAs (including miRNAs), proteins and other bioactive molecules and also known to play a key role in cell-cell communication between cancer cells and adjacent microenvironments, accelerating pathological processes including immune suppression, angiogenesis, cell migration, tumor invasion, and drug resistance. The primary purpose of this study was to characterize the potential role of exosomal miRNAs in TNBC chemoresistance and to analyze their underlying mechanisms. Exosomes were isolated from drug-resistant TNBC cell lines (MDA-MB-231/Epirubicin, Paclitaxel, Cisplatin) and their parental cell line using the Total Exosome Isolation kit (Invitrogen). Exosome RNAs and proteins were isolated using the Total Exosome RNA and Protein isolation kit. Quantitative real-time reverse transcription-PCR (qRT-PCR) assays were used to detect the expression of several known miRNAs that are associated with drug-resistance or tumor invasions, such as miR-19b, miR-141, miR-181a, miR-181c, miR-196b, miR-197, miR-200b, miR-203, miR-222, miR-375, miR-638 and miR-671-5p in both cellular RNA and exosomal RNA. The expression of miR-181c, miR-203, miR-375, miR-638, and miR-671-5p was downregulated in most drug-resistant cell lines compared to the parental cell line. In particular, miR-671-5p was downregulated in all three drug-resistant cell lines in both cellular and exosomal RNA. Furthermore, target predictions using the miRanda algorithm (microRNA.org) showed that miR-181c, miR-203, and miR-671-5p collectively shared the same target gene, Annexin-6 (AXNA6), which is a calcium-dependent membrane and phospholipid binding protein implicated in membrane-related events along exocytotic and endocytotic pathways. Recently, EV-associated AXNA6 was found to be linked to breast cancer metastasis after treatment with neoadjuvant chemotherapy in mouse models and human breast cancer cell lines. Given that EV-associated AXNA6 may confer chemoresistance, we explored the expression of EV-AXNA6 in chemoresistant breast cancer cell lines via qRT-PCR. Primers for ANXA6 were designed using the Primer-BLAST. The expression of AXNA6 in both cellular and exosomal RNA was assayed by qRT-PCR. The expression of the ANXA6 gene in drug-resistant cell lines was upregulated in both cellular and exosomal RNA compared to that in the parental cell line. These data suggest that the downregulation of miR-181c, miR-203, and miR-671-5p in exosomes may result in ANXA6 overexpression, leading to chemoresistance in TNBC. Targeting such miRNAs may be a new avenue to tackle drug resistance.