Theranostic application of miR-429 in HER2+ breast cancer

Abstract

Human epidermal growth factor receptor 2 (HER2) is overexpressed/amplified in one third of breast cancers (BCs), and is associated with the poorer prognosis and the higher metastatic potential in BC. Emerging evidences highlight the role of microRNAs (miRNAs) in the regulation of several cellular processes, including BC.

Methods: Here we identified, by in silico approach, a group of three miRNAs with central biological role (high degree centrality) in HER2+ BC. We validated their dysregulation in HER2+ BC and we analysed their functional role by in vitro approaches on selected cell lines and by in vivo experiments in an animal model.

Results: We found that their expression is dysregulated in both HER2+ BC cell lines and human samples. Focusing our study on the only upregulated miRNA, miR-429, we discovered that it acts as an oncogene and its upregulation is required for HER2+ cell proliferation. It controls the metastatic potential of HER2+ BC subtype by regulating migration and invasion of the cell.

Conclusions: In HER2+ BC oncogenic miR-429 is able to regulate HIF1α pathway by directly targeting VHL mRNA, a molecule important for the degradation of HIF1α. The overexpression of miR-429, observed in HER2+ BC, causes increased proliferation and migration of the BC cells. More important, silencing miR-429 succeeds in delaying tumor growth, thus miR-429 could be proposed as a therapeutic probe in HER2+ BC tumors.

Keywords: HER2+ breast cancer; Theranostics; diagnosis; microRNAs; therapeutic tool.

Figure 1

Workflow of the proposed computational approach.

Figure 2

Computational approach results Frequency of pairwise pathways in the top 10 for all 50 bootstraps (A). Pathways achieving the best performance in all 50 bootstraps (B) Boxplot representation of the AUC values in the training and testing dataset (C).

Figure 3

Pathway cross-talk networks in HER2 BC and their regulatory miRNAs. In red are indicated miRNAs and their connections, in blue are the functional pathways and their connections with the corresponding p-values.

Figure 4

miR-429 is overexpressed, while miR-190 and miR-584 are downregulated in HER2+ human BC. RT-PCR analysis of the fold change expression levels of miR-190 (A), miR-429 (B) and miR-584 (C) in HER2+ BC human samples compared to their corresponding healthy mammary tissue. The smaller panels in plot 4A and 4C represent an enlargement of the starting plot. The average value is indicated in red (n=10 samples, T test, p value<0.05, *).

Figure 5

miR-429 controls HER2+ BC cell proliferation , migration and invasion. MTT assay was performed on SKBR-3 cell at 0, 24, 48 h after 200nM Scramble or As miR-429 oligonucleotide treatment. The results are the average of three independent experiments in triplicate (t test compared to scramble treated cells, p value <0.05, *; <0.01, **)(A). 200nM 72h treatment with As miR-429 reduced the expression of CDK4 significantly (t test n=2 experiments in triplicate, p value <0.01**)(B). Wound healing test was performed on SKBR-3 treated with 200nM scramble oligonucleotide or As miR-429 in culture for 9 days. In each sample, the wound area was quantified by Image J following (t test compared to scramble-treated cells; n=3 experiments in triplicate, p value <0.01, **) (C). Boyden's chamber test was performed on SKBR-3 in the presence of 150nM scramble or As miR-429 in culture for 24h. The count of the cells was performed on 10 images from each sample (t test compared to scramble-treated cells; n=3 experiments, p value<0.01, **) (D).

Figure 6

As miR-429 counteracts the growth of HER2+ BC tumors xenografted in mouse. Tumors formed by SKBR3 in NOD/SCID mouse were treated with As miR-429 or scramble S oligonucleotide in the presence of atelogene reagent (vehicle). The volume of each tumor (height x length x depth) was measured at different time points and the curves represent the average volume in 15 days of measurements (A). The weights of the five Scramble S- or As miR-429-treated tumors are represented (t test p value<0.01, **, n=5) (B).

Figure 7

VHL is the direct target of miR-429. Luciferase assay: HER2+ cells were transfected with the Luciferase+ 3'UTR VHL (pGL3-VHL) construct alone, with 100nM 48h S Scramble or S miR-142 or As miR-429. The luciferase expression (LUC) was normalized on alkaline phosphatase content (AP). The chart represents the percentage of LUC/AP ratio (T test, p value<0.05, *; n=2 independent experiment)(A). RT-PCR analysis revealed that miR-429 modulation, obtained with 50nM As miR-429 for 72h (B), caused a significant increase of VHL expression, both at mRNA (C) and protein levels (D) (T test, p value<0.05, *; n=3 independent experiment). RT-PCR analysis on human HER2+ BC samples revealed that miR-429 is downregulated, compared to healthy tissue (E).

Figure 8

miR-429 is induced by hypoxia. RT-PCR analysis on SKBR3 revealed that 24h of hypoxia is able to induce miR-429 increased expression (A). The hypoxia induction has been verified by the RT-PCR analysis of HIF1α and its target VEGF mRNA induction (B).