Abstract
BackgroundHER2 gene amplification generates an enormous number of HER2 transcripts, but the global effects on endogenous miRNA targets including HER family members in breast cancer are unexplored.MethodsWe generated a HER2–3’UTR expressing vector to test the tumor-promoting properties in HER2 low expressing T47D and MCF7 cells. Through microarray analysis and real-time PCR analysis we identified genes that were regulated by HER2–3’UTR. Positive and negative manipulation of miRNA expression, response element mutational studies and transcript reporter assays were performed to explore the mechanism of competitive sequestration of miR125a/miRNA125b by HER2 3’UTR.To investigate if trastuzumab-induced upregulation of HER3 is also mediated through miRNA de-repression, we used the CRISPR/cas9 to mutate the endogenous HER2 mRNA in HER2 over-expressing Au565 cells. Finally, we looked at cohorts of breast cancer samples of our own and the TCGA to show if HER2 and HER3 mRNAs correlate with each other.ResultsThe HER2 3’UTR pronouncedly promoted cell proliferation, colony formation, and breast tumor growth. High-throughput sequencing revealed a significant increase in HER3 mRNA and protein levels by the HER2 3’untranslated region (3’UTR). The HER2 3’UTR harboring a shared miR-125a/b response element induced miR-125a/b sequestration and thus resulted in HER3 mRNA derepression. Trastuzumab treatment upregulated HER3 via elevated HER2 mRNA expression, leading to trastuzumab resistance. Depletion of miR-125a/b enhanced the antitumor activity of trastuzumab. Microarray data from HER2-overexpressing primary breast cancer showed significant elevation of mRNAs for predicted miR-125a/b targets compared to non-targets.ConclusionsThese results suggest that HER2 3’UTR-mediated HER3 upregulation is involved in breast cell transformation, increased tumor growth, and resistance to anti-HER2 therapy. The combinatorial targeting of HER3 mRNA or miR-125a/b may offer an effective tool for breast cancer therapy.
Highlights
Human epidermal growth factor receptor 2 (HER2) gene amplification generates an enormous number of HER2 transcripts, but the global effects on endogenous miRNA targets including HER family members in breast cancer are unexplored
Similar to cells transfected with the HER2 coding sequence (CDS), cells transfected with the HER2 3’untranslated region (3’UTR) displayed increased cell proliferation compared to control vector-transfected cells (Fig. 1a)
The effect of the HER2 3’UTR on cell proliferation was largely abolished by miR-125a and miR-125b depletion (Fig. 3l). These results demonstrate that the miR-125a/b response element is essential for HER2 3’UTR regulation of Human epidermal growth factor receptor 3 (HER3) and cell malignancy
Summary
HER2 gene amplification generates an enormous number of HER2 transcripts, but the global effects on endogenous miRNA targets including HER family members in breast cancer are unexplored. The HER-2/neu oncogene is amplified two-fold to > 20-fold in approximately 25% of breast cancers. Overexpression or gene amplification of HER2 is associated with poor prognosis and an aggressive course of the disease, such as oncogenic transformation, tumorigenesis, and metastasis [1]. HER2 belongs to the Type I receptor tyrosine kinase family, which includes four family members: EGFR (HER1), HER2 (neu or ERBB2), HER3, and HER4 [2]. In spite of possessing no known ligand, HER2 is the preferred heterodimerization partner within the family and can form heterodimers with HER1 and HER3, leading to phosphorylation of tyrosine residues within the cytoplasmic domain [3, 4].
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