Role of RNF144B in triple negative breast cancer proliferation

Abstract

Abstract Background Breast cancer (BC) is one of the leading causes of deaths in females worldwide. BC is classified into three distinct groups: hormone receptor positive (ER+/PR+), hormone negative/HER2 positive and triple negative breast cancer (TNBC). Patients with hormone positive (HR+) and HER2 positive(HER2+) cancers show a better prognosis and survival rate. However, due to a lack of biomarkers TNBC has the worst prognosis and approximately 70% of women with metastatic TNBC die within 5years. The results of recent published work from our lab showed that the oncogenic role of RNF144B protein in endometrial cancer is mediated only in the absence of oestrogen-mediated growth signalling. In this study we aim to investigate the role of RNF144B in TNBC where proliferation is independent of hormonal activity. Methods Cell culture: All cells were purchased from American Type Culture Collection (ATCC). Western blotting: Cells were lysed with 2x lysis buffer. Equal amount of proteins were electrophoretically separated by 10% SDS-PAGE gel. Then, proteins were subjected to electrotransfer into nitrocellulose membrane. Membranes were incubated for overnight with primary antibody, and then with the corresponding secondary antibody for an hour. Blots were developed with chemiluminescent substrates from BIO-RAD. siRNA transfection: Transfection was performed using Lipofectamine 2000 (Invitrogen) as per manufacturer’s instructions. Proliferation Assay: 5000cells/well were seeded into wells of a 24-well plate in triplicate and incubated overnight to adhere. Then, cells were fixed in 50/50% acetone and methanol. Fixed cells were stained with 4’,6-diamidino-2-phenylindole from (Sigma) for 10 minutes in the dark.Cells numbers were calculated using The Varioskan LUX multimode reader from Thermo. The data were normalised using Microsoft Excel. Results We found out that the E3 ubiquitin ligase RNF144B, is exclusively expressed in TNBC cell lines but not in HR+ or HER2+ breast cancer cell lines. By using MDA-MB-468 as a model for TNBC, we found that silencing RNF144B significantly decreased cell proliferation. Conclusions RNF144B May represent a novel biomarker for TNBC targeted therapy. Currently, further investigation is under way to identify the downstream targets of RNF144B. Legal entity responsible for the study Lamaia Altarjami. Funding The Saudi embassy in London. Disclosure The author has declared no conflicts of interest.