Abstract P6-05-07: The effects of microRNA modulation of polo-like kinase 1 in breast cancer cell lines
Authors: Zalles N , Gasparini P , Croce CM 
Published: May 2019

BACKGROUND: Elucidating a more universal therapeutic target is key to effectively combat the heterogeneity of breast cancer (BrCa). MicroRNAs (miRs) are small noncoding RNAs that are an abundant class of endogenous regulatory molecules, which act by targeting mRNAs for cleavage and/or translational repression. Continuing studies into BrCa genetics show that the various subtypes of BrCa are also affected by miR activity from the regulation of various cancer-related genes. MiR expression can even influence drug response in multiple cancer types. This prompted our investigation into the nuances of how miRs may influence BrCa behavior and response to treatment with targeted therapies. The Polo-like Kinase 1 (PLK1) enzyme plays an important role in the cell cycle, and is considered to be a proto-oncogene. Inhibiting PLK1 in has shown promise in reducing tumor volume and promoting tumor cell death in various cancers. Understanding how miRs regulate PLK1 in BrCa will improve our understanding of the PLK1 pathway, and whether this miR-directed regulation affects anti-PLK1 therapy. This knowledge could inform future anti-PLK1 BrCa treatment regimens, resulting in better outcomes for patients.

METHODS: The effects of an anti-PLK1 drug, NMS-P937, were tested on triple-negative (TN) (MDA-MB-231, BT549) and luminal (ZR-75-1, CAMA-1) BrCa cell lines. From miRNA target prediction sites (TargetScan, MiRTarBase), a panel of miRs predicted to bind to PLK1 was selected and then assessed using dual-reporter luciferase assays. MiR expression was transiently induced in TN and luminal BrCa cell lines, and the effects were verified using western blotting. Apoptosis was assessed by Annexin V staining in BrCa cell lines transiently transfected with the validated miR and treated with different concentration of NMS-P937 for 48 hrs. To validate the contribution of the miR-PLK1 axis for NMS-P937-induced apoptosis, BrCa cells were transfected with PLK1 siRNA, and the Annexin V staining experiment was repeated.

RESULTS: From the panel of miRs tested, miR-183-5p was the only one to demonstrate binding to the PLK1 gene. Western blotting showed that PLK1 expression was downregulated when miR-183-5p is overexpressed. Annexin V staining suggests that miR-183-5p induces apoptosis through PLK1 targeting in BrCa cell lines, and this effect seems to amplify NMS-P937-induced apoptosis in both cell line subtypes. Apoptotic markers assessed by western blot confirm the over-activation of apoptotic signals by miR-183-5p. Future work will continue to focus on determining the effects of miR-183-5p targeting on pathways downstream of PLK1, and utilizing tumor xenografts to elucidate the effects of miR-183-5p on NMS-P937 response in an in-vivo model.

CONCLUSIONS: MiR-183-5p appears to directly target PLK1 and reduce its level of expression. Additionally, miR-183-5p increases the number of apoptotic BrCa cells when used in combination with NMS-P937, and it is the inhibition of PLK1 specifically that contributes to the increase in the apoptosis of BrCa cells treated with NMS-P937. Taken together, our findings identify a new mechanism of PLK1 modulation through miR. Our next steps will focus on studying these modulatory effects in vivo, in an effort to move forward for clinical applications.

Citation Format: Zalles N, Gasparini P, Croce CM. The effects of microRNA modulation of polo-like kinase 1 in breast cancer cell lines [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-05-07.