Exploring miRNA‑target gene profiles associated with drug resistance in patients with breast cancer receiving neoadjuvant chemotherapy

Abstract

Exosomal microRNAs (miRNAs) are closely related to drug resistance in patients with breast cancer (BC); however, only a few roles of the exosomal miRNA-target gene networks have been clinically implicated in drug resistance in BC. Therefore, the present study aimed to identify the differential expression of exosomal miRNAs associated with drug resistance and their target mRNAs. In vitro microarray analysis was used to verify differentially expressed miRNAs (DEMs) in drug-resistant BC. Next, tumor-derived exosomes (TDEs) were isolated. Furthermore, it was determined whether the candidate drug-resistant miRNAs were also significant in TDEs, and then putative miRNAs in TDEs were validated in plasma samples from 35 patients with BC (20 patients with BC showing no response and 15 patients with BC showing a complete response). It was confirmed that the combination of five exosomal miRNAs, including miR-125b-5p, miR-146a-5p, miR-484, miR-1246-5p and miR-1260b, was effective for predicting therapeutic response to neoadjuvant chemotherapy, with an area under the curve value of 0.95, sensitivity of 75%, and specificity of 95%. Public datasets were analyzed to identify differentially expressed genes (DEGs) related to drug resistance and it was revealed that BAK1, NOVA1, PTGER4, RTKN2, AGO1, CAP1, and ETS1 were the target genes of exosomal miRNAs. Networks between DEMs and DEGs were highly correlated with mitosis, metabolism, drug transport, and immune responses. Consequently, these targets could be used as predictive markers and therapeutic targets for clinical applications to enhance treatment outcomes for patients with BC.

Keywords: breast cancer; drug resistance; exosomes; gene profile; miRNA; neoadjuvant chemotherapy.

Figure 1.

Schematic of the research design for determining the associations between exosomal miRNAs and their target genes. DEGs, differentially expressed genes; pCR, pathological complete response; BC, breast cancer; miRNA, microRNA.

Figure 2.

(A) Flow chart illustrating the steps for miRNA expression microarray analysis using AffymetrixGeneChip^®. (B) Heat maps with hierarchical clustering showing the differential expression of miRNAs in MM231_wild-type, MM231_{resistant-type}, MM468_wild-type, MM468_{resistant-type}, HCC1395_wild-type, and HCC1395_{resistant-type} BC cell lines. (C) Venn diagrams illustrating the number of all upregulated and downregulated DEMs in three pairs of BC cell lines. The intersection in the center represents the common DEMs among the three groups. miRNA or miR, microRNA; BC, breast cancer; DEMs, differentially expressed miRNAs.

Figure 3.

(A) Venn diagram showing candidate exosomal miRNAs related to drug resistance. Bar charts showing fold-change in exosomal miRNAs from wild-type and resistant-type cells. A total of 12 selected candidate exosomal miRNAs were analyzed in the three different breast cancer cell lines (B) MM231, (C) MM468, and (D) HCC1395, using quantitative PCR. Significant miRNAs are indicated in bold text. miRNA or miR, microRNA.

Figure 4.

(A) Radar plots of miRNA signature profiles from quantitative PCR in BC cells, including MDA-MB-231 (MM231), MDA-MB-468 (MM468), and HCC1395. The relative expression levels of miRNAs in wild-type BC cells, drug-resistant exosome 24 h-treated (educated-type) BC cells, and resistant-type BC cells were compared. The line labels represent the drug-resistant miRNAs, and the ring labels represent the fold change calculated from each group/wild-type difference. (B) The cell viabilities of wild-type, educated-type, and resistant-type BC cells were compared after 48 h of incubation with Adriamycin and Taxotere. miRNA or miR, microRNA; BC, breast cancer; ADR, Adriamycin; TAX, Taxotere.

Figure 5.

(A) Characterization of tumor-derived exosomes isolated from plasma of patients with BC via nanoparticle tracking analysis. (B) Confocal and SEM images of exosomes bound to microbead surfaces after immuno-affinity capture. Microbeads were functionalized with antibodies against the BC-targeting markers ITGAV, ITGA2 and EpCAM. Scale bars represent 500 and 200 nm, respectively. The size distribution and exosome imaging were obtained from representative patient 1. (C) Exosomal miRNAs related to drug resistance were validated in plasma samples from 35 patients with BC. Statistical analyses were performed using an unpaired Student's t-test between two groups. *P<0.05 and **P<0.01. The statistically significant miRNA candidates are indicated using blue dots and non-significant miRNA candidates are indicated using red dots. BC, breast cancer; SEM, scanning electron microscopy; miRNA or miR, microRNA.

Figure 6.

Discriminatory effect of candidate exosomal miRNAs that were significantly upregulated in patients with breast cancer exhibiting no pathological complete response assessed using receiver operating characteristic curves and area under the curve values. (A) Five exosomal miRNAs were validated as targets with high discriminatory ability and (B) four exosomal miRNAs were validated as targets with poor discriminatory ability. miRNA or miR, microRNA.

Figure 7.

Transcriptome analysis in drug-resistant and drug-sensitive breast cancer tissues. (A) Volcano plot of DEGs in drug-resistant tissue samples compared with those in drug-sensitive tissue samples as identified by GSE25066 and GSE41988 public data analyses. (B) GO cluster map of significantly upregulated and downregulated DEGs in drug-resistant tissue samples compared with those in drug-sensitive tissue samples. Top-ranked terms consisted of interesting GO clusters that were selectively labeled. DEGs, differentially expressed genes; GO, gene ontology; pCR, pathological complete response.

Figure 8.

(A) GO analysis of target genes of the candidate drug-resistant exosomal miRNAs. (B) miRNA-mRNA interaction network. The network illustrates the putative miRNAs and their predicted target mRNAs associated with drug resistance in BC after neoadjuvant chemotherapy. The candidate miRNAs were classified into two groups of drug resistance: Highly-related miRNAs (miRNA^{high R}) and moderately-related miRNAs (miRNA^{moderate R}) based on their discriminatory power. (C) The survival analysis in BC patients with target genes of miRNA^{high R} and (D) miRNA^{moderate R} using public databases. GO, gene ontology; miRNA or miR, microRNA; BC, breast cancer.