Abstract P4-07-03: Dynamic regulation of a microRNA-mRNA network during breast cancer metastasis reveals an essential tumor-promoting role for miR-203

Abstract

Abstract BACKGROUND: Metastasis represents a critical turning point from curable to incurable breast cancer. Alterations in gene expression underlie aspects of this transition and are coordinated both genetically and epigenetically. HYPOTHESIS: Epigenetically controlled microRNA-mRNA regulatory networks underlie the ability of breast cancer cells to metastasize and acquire treatment resistance. METHODS: Human MDA-MB-231 and MDA-MB-436 breast cancer xenograft primary tumors and corresponding lymph node, liver, lung and diaphragm metastases were generated by inoculation of cell lines into the mammary fat pad of NSG mice. Human-specific gene expression profiling was performed using microarrays with qPCR validation. MicroRNA targets were validated by 3'UTR mutagenesis-luciferase assays. Lentiviral shRNA and miRNA mimic constructs were stably transduced into the same cell lines and growth properties were assessed in vitro and in vivo. Mice were euthanized when tumors achieved 10% of body weight or at day 100, whichever came first. RESULTS: A common set of 18 mRNAs was differentially regulated at all 4 metastatic sites relative to primary tumors. Of these, 17 were downregulated, and 13 of these were strikingly enriched for microRNA binding sites. In the same tissues, we identified 21 microRNAs that underwent downregulation during primary tumor growth relative to parental cell cultures, but were highly upregulated at all 4 metastatic sites. 18 of these had at least one target among the downregulated genes, and 6 had multiple targets in the same 13 mRNA 3' UTRs. One, miR-203, previously described as a tumor suppressor, directly targeted two of the repressed genes, TWF1 and APBB2. MiR-203 levels positively correlated with primary tumor size in METABRIC (p=0.0096). MiR-203 overexpression blunted in vitro tumorigenic properties, modestly reduced primary tumor growth rates, and prevented metastasis formation. Remarkably, knockdown of miR-203 also reversed multiple tumorigenic properties of both cell lines, including proliferation, migration, and growth in soft agar, and conferred a fibroblastic morphology on MDA-MB-231 cells. In addition, primary miR-203KD xenograft tumors grew extremely slowly and underwent partial involution between days 80-100. Residual miR-203KD tumors and metastases at day 100 demonstrated re-gain of miR-203 expression through loss of the shRNA silencing vector. CONCLUSIONS: A small group of microRNAs displays biphasic expression during cancer growth. At least one of these, microRNA-203, is required for metastatic growth, but also inhibits metastasis formation when expressed at high levels. Our findings suggest opposing roles for the same microRNA at different stages of breast cancer progression, and support the existence of dynamic, context-sensitive epigenetic mechanisms that adapt breast cancer cells to thrive at remote sites. These mechanisms may serve as targets for intervention during the evolution of metastatic disease. Citation Format: Bishopric NH, Speransky S, Kajan D, Laderian B, Iorns E, Clarke J, Lippman ME. Dynamic regulation of a microRNA-mRNA network during breast cancer metastasis reveals an essential tumor-promoting role for miR-203 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-07-03.