Abstract
Purpose
Dysregulation of HER2 signaling pathway in breast cancer is well documented. Our bioinformatics analysis predicted hsa-miR-512-3p (miR-512-3p) as a bona fide regulator of HER2 as well as HER3, PIK3R2, and AKT1 genes. Then, we intended to examine the effect of miR-512-3p on the predicted target genes that are involved in HER2 signaling pathway.
Methods and results
RT-qPCR results indicated lower expression of miR-512-3p in breast cancer specimens, compared to their normal pairs. Overexpression of miR-512-3p resulted in HER2, HER3, PIK3R2, and AKT1 gene downregulation, detected by RT-qPCR and the result was confirmed by western analysis and ELIZA test against p-AKT, BAX, FADD, and HER2 proteins in SKBR3 cells, respectively. Then, dual-luciferase assay supported the direct interaction of miR-512-3p with 3′UTR sequences of HER2, HER3, PIK3R2, and AKT1 target genes. When miR-512-3p was overexpressed, BAX/BCL2 expression ratio and proportion of sub-G1 cell population were increased in transfected SKBR3 cells, detected by RT-qPCR and flow cytometry, respectively. These results were consistent with the decreased viability of transfected cells, documented by MTT assay. In addition, results were consistent with the upregulation of BAX, BAK, BOK, PTEN, P53, and P21 genes and downregulation of CCND1 gene in SKBR3 cells. Although the overexpression of miR-512 resulted in cell cycle arrest at Sub-G1 stage in MDA-MB-231 cells, this effect seemed independent of targeting HER2, HER3, PIK3R2, and AKT1 target genes.
Conclusion
Overall, results indicated that miR-512-3p acts as a cell-type-specific tumor suppressor, through targeting HER2, HER3, PIK3R2, and AKT1 transcripts. These results suggest miR-512-3p as a potential candidate marker for breast cancer diagnosis.
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References
Avgeris M, Mavridis K, Scorilas A (2012) Kallikrein-related peptidases in prostate, breast, and ovarian cancers: from pathobiology to clinical relevance. Biol Chem 393(5):301–317
Eroles P et al (2012) Molecular biology in breast cancer: intrinsic subtypes and signaling pathways. Cancer Treat Rev 38(6):698–707
Zaczek A, Brandt B, Bielawski K (2005) The diverse signaling network of EGFR, HER2, HER3 and HER4 tyrosine kinase receptors and the consequences for therapeutic approaches. Histol Histopathol 20(3):1005–1015
Wang S-C, Hung M-C (2009) Nuclear translocation of the epidermal growth factor receptor family membrane tyrosine kinase receptors. Clin Cancer Res 15(21):6484–6489
Krishnamurti U, Silverman JF (2014) HER2 in breast cancer: a review and update. Adv Anat Pathol 21(2):100–107
English DP, Roque DM, Santin AD (2013) HER2 expression beyond breast cancer: therapeutic implications for gynecologic malignancies. Mol Diagn Ther 17(2):85–99
Wu S, Karger B, Dai S (2010) Characterization of ErbB2 phosphorylation and their dynamic changes upon EGF stimulation in human breast cancer cells. J Biomol Tech JBT 21(3 Suppl):S60
Garay C et al (2015) Epidermal growth factor–stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis. Mol Biol Cell 26(19):3504–3519
Tamaskovic R et al (2016) Intermolecular biparatopic trapping of ErbB2 prevents compensatory activation of PI3K/AKT via RAS–p110 crosstalk. Nat Commun 7:11672
Bertotti A et al (2011) A molecularly annotated platform of patient-derived xenografts (‘xenopatients’) identifies HER2 as an effective therapeutic target in cetuximab-resistant colorectal cancer. Cancer Discov 1(6):508–523
Mitri Z, Constantine T, O’Regan R (2012) The HER2 receptor in breast cancer: pathophysiology, clinical use, and new advances in therapy. Chemother Res Pract 2012
Josse C et al (2013) Identification of a microRNA landscape targeting the PI3K/Akt signaling pathway in inflammation-induced colorectal carcinogenesis. Am J Physiol Gastrointest Liver Physiol 306(3):G229–G243
Lee JJ, Loh K, Yap Y-S (2015) PI3K/Akt/mTOR inhibitors in breast cancer. Cancer Biol Med 12(4):342
Guo H et al (2010) Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466(7308):835
Cheng CJ et al (2015) MicroRNA silencing for cancer therapy targeted to the tumour microenvironment. Nature 518(7537):107
Smith B, Agarwal P, Bhowmick NA (2017) MicroRNA applications for prostate, ovarian and breast cancer in the era of precision medicine. Endocr Relat Cancer 24(5):R157–R172
Ruchi Sharma V et al (2017) Pi3k/akt/mtor intracellular pathway and breast cancer: factors, mechanism and regulation. Curr Pharm Des 23(11):1633–1638
Li G et al (2016) CCAR1 5′ UTR as a natural miRancer of miR-1254 overrides tamoxifen resistance. Cell Res 26(6):655
Lesurf R et al (2016) Molecular features of subtype-specific progression from ductal carcinoma in situ to invasive breast cancer. Cell Rep 16(4):1166–1179
Menard S et al (2004) Role of HER2/neu in tumor progression and therapy. Cell Mol Life Sci 61(23):2965–2978
Li X et al (2018) Posttranscriptional upregulation of HER3 by HER2 mRNA induces trastuzumab resistance in breast cancer. Mol Cancer 17(1):113
Li X et al (2015) Cell membrane gp96 facilitates HER 2 dimerization and serves as a novel target in breast cancer. Int J Cancer 137(3):512–524
Manning BD, Toker A (2017) AKT/PKB signaling: navigating the network. Cell 169(3):381–405
Saxton RA, Sabatini DM (2017) mTOR signaling in growth, metabolism, and disease. Cell 168(6):960–976
Martinou J-C, Youle RJ (2011) Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics. Dev Cell 21(1):92–101
Del Principe MI et al (2016) Clinical significance of bax/bcl-2 ratio in chronic lymphocytic leukemia. Haematologica 101(1):77–85
Chène P (2003) Inhibiting the p53–MDM2 interaction: an important target for cancer therapy. Nat Rev Cancer 3(2):102
Ogawara Y et al (2002) Akt enhances Mdm2-mediated ubiquitination and degradation of p53. J Biol Chem 277(24):21843–21850
Abraham AG, O’Neill E (2014) PI3K/Akt-mediated regulation of p53 in cancer. Biochemical Soc Trans 42(4):798–803
Moll UM, Petrenko O (2003) The MDM2–p53 interaction. Mol Cancer Res 1(14):1001–1008
Mende N et al (2015) CCND1–CDK4–mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo. J Exp Med 212(8):1171–1183
Liang T, Wang P (2013) MiR-512-3p expression pattern and function in breast cancer. Chin J Clin Oncol 19:1145–1149
Kominami K et al (1823) (2012) The molecular mechanism of apoptosis upon caspase-8 activation: quantitative experimental validation of a mathematical model. Biochimica et Biophysica Acta (BBA) Mol Cell Res 10:1825–1840
Acknowledgements
We wish to thank 4402 Lab members at Tarbiat Modares University and Motamed Cancer Institute (MCI) for their supports throughout the project.
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This study was funded by INSF (Grant Number 96012144).
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ZM and BMS conceived and designed the study, analyzed the results, and wrote the paper. ZM performed the experiments. FM provided cancer tissue samples with preparation of pathological features. All authors reviewed the results and approved the final version of the manuscript.
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This study was approved by the Institutional ethics committee at TMU. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
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Mohamadzade, Z., Mahjoubi, F. & Soltani, B.M. Introduction of hsa-miR-512-3p as a new regulator of HER2 signaling pathway in breast cancer. Breast Cancer Res Treat 185, 95–106 (2021). https://doi.org/10.1007/s10549-020-05937-3
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DOI: https://doi.org/10.1007/s10549-020-05937-3