miR-125a Induces HER2 Expression and Sensitivity to Trastuzumab in Triple-Negative Breast Cancer Lines

Abstract

The EGFR/HER2 signaling network is an effective therapeutic target for HER2-positive cancers, which are known for their aggressive biological course. Evidence indicates that the EGFR/HER2 network plays a role in the aggressive basal-like subtype as well. Here, we studied the potential role of miR-125a-3p as a modulator of the EGFR/HER2 pathway in basal-like breast cancer. Over-expression of miR-125a-3p reduced the migratory capability of MDA-MB-231 cells and led to an increase in the expression of ErbB2 transcript and protein. The induced ErbB2 responded to trastuzumab and underwent internalization and subsequent intra-lysosomal degradation. Trastuzumab treatment further reduced the migratory capability and induced the apoptosis of the cells. An in-vivo mouse model, which supported the in-vitro findings, showed a synergistic effect for miR-125a-3p and trastuzumab. Trastuzumab-treated miR-125a-3p-induced tumors were significantly smaller than control induced tumors. Our findings indicate that, in the basal-like subtype of breast cancer, miR-125a-3p may act as a tumor suppressor. miR-125a-3p induces an increase in the expression of ErbB2 that may render the cells suitable for treatment with anti-HER2 therapies.

Keywords: ErbB2; TNBC; apoptosis; epigenetics; miRNA; migration.

Figure 1

Characterization of breast cancer cell lines. (A–C) Three breast cancer cell lines were subjected to qPCR analysis with specific primers for (A) estrogen receptor, (B) ErbB2 calibrated with HPRT1, and (C) miR-125a-3p calibrated with U6 snRNA. Data were normalized to MDA-MB-231 cells. (D) Non-transfected MDA-MB-231 cells (naive cells) or cells transfected with either scrambled miRNA (control) or miR-125a were subjected, 48 h later, to qPCR analysis with specific primers for miR-125a-3p and for U6 snRNA as an endogenous control. All experiments were repeated three times and analyzed by a one-sample Student's t-test. Data are presented as mean ± SEM. *P < 0.05—significantly different from MDA-MB-231 cells (A–C), or naive cells (D).

Figure 2

miR-125a-3p regulates the migration capability of MDA-MB-231 cells. MDA-MB-231 cells, stably expressing miR-125a-3p (miR-125a-3p) or scrambled miRNA (scrambled), were subjected to the following analyses. (A) Cell migration, assayed by a Transwell system for 12 h. Upper panel—representative cell culture micrographs. Bar = 50 μm. Lower panel—summary of three experiments, analyzed using Image J software. (B) qPCR analysis with specific primers for Fyn, FAK, and Akt; HPRT1 served as an endogenous control. The expression of each gene was normalized to its expression in control cells (100%) and is shown as mean ± SEM. The experiment was repeated three times and analyzed by a Student's t-test. *P < 0.05; **P < 0.01.

Figure 3

miR-125a-3p increases the expression of ErbB2 in MDA-MB-231 cells. (A–D) MDA-MB-231 cells, stably overexpressing either miR-125a-3p (miR-125a-3p) or scrambled miRNA as a control (scrambled), were subjected to: (A) qPCR with specific primers for miR-125a-3p, miR-125a-5p, and U6 snRNA as an endogenous control; (B) qPCR with specific primers for ErbB2 and HPRT1 as an endogenous control; (C,D) Western blot (WB) using anti-ErbB2 antibody and anti-actin to normalize loading; (C) one representative blot from analysis of three independent WB assays (D,E) immunofluorescence after staining with anti-ErbB2 antibody (green) and Hoechst (blue) as a nuclear marker; SKBR3 cells were used as positive control. (F,G) MDA-MB-231 cells, transfected with miR-125a-3p mimic (miR-125a-3p) or scrambled RNA as a control (scrambled), were cultured for 48 h and subjected to (F) qPCR with specific primers for miR-125a-3p, miR-125a-5p, and U6 snRNA as an endogenous control or to (G) qPCR with specific primers for ErbB2 and HPRT1 as an endogenous control. All experiments were repeated three times. *P < 0.05, **P < 0.01.

Figure 4

miR-125a-3p increases the expression of ErbB2 in TNBC cell lines. BT-549 or Hs578T cells transfected with either miR-125a-3p (miR-125a-3p) or scrambled miRNA as a control (scrambled) were subjected after 48 h to (A) qPCR with specific primers for miR-125a-3p and U6 snRNA as an endogenous control; (B) qPCR with specific primers for ErbB2 and HPRT1 as an endogenous control. All experiments were performed three times. *P < 0.05.

Figure 5

Induced-ErbB2 responds to Trastuzumab. (A) MDA-MB-231 cells stably expressing miR-125a-3p (miR-125a-3p) or scrambled RNA (scrambled) were stained with FITC anti-human ErbB2 IgG1 (#324404, BioLegend) or FITC mouse IgG1 isotype control (#400109, BioLegend) and analyzed by FACS, confirming their membrane expression of ErbB2. (B,C) Cells were treated with 100 μg/ml trastuzumab for 6 h, washed and subjected to immunofluorescence assay to track (B) ErbB2 internalization, immediately after incubation with trastuzumab, and (C) lysosomal localization 6 h after trastuzumab addition. Cells were stained with anti-ErbB2 antibody (green; B,C), Hoechst (blue) as a nuclear marker, and a lysosome marker (Lysotracker; red; C). Arrows indicate co-localization of ErbB2 and lysotracker (yellow dots). (B,C) Bars = 50 μm. Similar results were obtained in three independent experiments.

Figure 6

miR-125a-3p and trastuzumab have a synergistic effect in inducing apoptosis and reducing the migration reduction of MDA-MB-231 cells. (A) MDA-MB-231 cells, stably expressing miR-125a-3p (miR-125a-3p) or scrambled RNA as a control (scrambled), were left untreated or treated with 100 μg/ml trastuzumab for 6 h and subjected to Transwell assay. Thick arrows indicate migrating cells. Thin arrows point at the membrane pores. Bars = 50 μm. The results were analyzed using Image J software. (B,C) MDA-MB-231 cells, stably expressing miR-125a-3p (miR-125a-3p) or scrambled RNA (scrambled), were left untreated or treated with 100 μg/ml trastuzumab for 3 weeks and subjected to FACS analysis of apoptosis using PI and annexin staining. (B) Plots showing a representative assay. (C) Graph summarizing three FACS analyses. Bars are mean ± SEM analyzed by one-sample t-test. *P < 0.05—significantly different from control value. (A,B) Similar results were obtained in three independent experiments.

Figure 7

miR-125a-3p and trastuzumab have a synergistic effect on tumor growth. Cells (1.5 × 10⁶), stably expressing miR-125a-3p (miR-125a-3p) or scrambled miRNA as a control (scrambled), were injected into the mammary fat pads of 6 week-old mice. Starting after 1 week, the mice were treated twice a week for 28 days with 10 mg/kg trastuzumab or with vehicle (control). Each group contained six mice. (A) Representative CT images taken after 16 days of trastuzumab treatment. (B) Graphical representation of tumor growth, shown as mean ± SEM. Mice were sacrificed on the 28th day, their tumors were excised, and the expression of ErbB2 and miR-125a-3p was analyzed by WB and by qPCR (C,D, respectively). Data were analyzed by a Student's t-test. *P < 0.05—significantly different from “scrambled.” *P < 0.05—significantly different from miR-125a-3p.