miR-489 Confines Uncontrolled Estrogen Signaling Through a Negative Feedback Mechanism and Regulates Tamoxifen Resistance in Breast Cancer

Abstract:

Background: Approximately 75% of diagnosed breast cancer tumors are estrogen-receptor (ER) positive tumors and are associated with better prognosis due to their response to hormonal therapies. However, around 40% of patients relapse after hormonal therapies. In the current study, we aim to evaluate miR-489 as a novel molecular target to combat tamoxifen resistance.

Methods: Genomic analysis of gene expression profiles in primary breast cancers and tamoxifen resistant cell lines unveiled the potential role of miR-489 in regulation of estrogen signaling and development of tamoxifen resistance. We manipulated miR-489 expression in breast cancer cell lines by transient transfection of a miR-489 mimic or establishment of knockout cell lines using the CRISPR/Cas9 system to study the reciprocal regulation of miR-489 and estrogen/ER signaling pathways. Cell proliferation assays, Sphere-formation assays and flow cytometry analysis were conducted to investigate the role of miR-489 in estrogen-induced cell proliferation, cancer stem cell expansion and development of tamoxifen resistance.

Results: miR-489 expression was significantly downregulated in tamoxifen-resistant cell lines. Low levels of miR-489 were associated with poor clinical outcomes in patients with hormone treatment. In vitro analysis showed that loss of miR-489 expression promoted tamoxifen resistance while overexpression of miR-489 in tamoxifen-resistant cells restored tamoxifen sensitivity. Mechanistically, we found that miR-489 is an estrogen regulated miRNA that negatively regulated estrogen receptor signaling by using at least the following two mechanisms: i) modulation of ER phosphorylation status by inhibiting MAPK and AKT kinase activities and downregulating SHP2 expression; ii) regulation of nucleus to cytosol translocation of estrogen receptor α (ERα) by decreasing p38 expression and consequently ER phosphorylation. In addition, miR-489 could break the positive feed-forward loop between the estrogen-ERα axis and p38 MAPK in breast cancer cells, which was necessary for its function as a transcription factor.

Conclusion: Our study unveiled the underlying molecular mechanism by which miR-489 regulates estrogen signaling pathway through a negative feedback loop and uncovered its role in both the development of and overcoming of tamoxifen resistance in breast cancers.