Abstract 2532: Wild-type p53 represses MELK expression by blocking the recruitment of FOXM1 to MELK promoter
Authors: Bollu Lakshmi Reddy , Shepherd Jonathan , Zhao Dekuang , Ma Yanxia , Mazumdar Abhijit , Brown Powel H. 
Published: August 2018

Background: Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer with poor prognosis due to frequent distant metastasis and lack of successful targeted therapies. To identify potential druggable targets, previously we performed a gene expression analysis and identified protein kinases that are highly expressed in TNBCs. In this study, we investigated the molecular mechanism through which wild-type p53 represses MELK expression in TNBC cells. Through these studies, we discovered that the expression of Maternal Embryonic Leucine zipper Kinase (MELK) is highly elevated in TNBC when wild-type p53 is lost or mutated. We also show that overexpression of wild-type p53 represses MELK expression without binding to MELK promoter.

Experimental Design and Methods: To identify the p53-responsive region in MELK promoter, we have made multiple promoter deletions using PCR deletion mutagenesis. The p53-responsive region was determined by measuring the activity of these promoter constructs in the presence of wild-type p53 using luciferase assays. TransFac software was used to identify potential transcription factors in the p53-responsive region. The effect of overexpression of p53 and FOXM1 on MELK expression was examined through Western blotting analysis. Using co-immuno precipitation (Co-IP) studies, we determined the protein-protein interactions between wild-type p53 and FOXM1. The effect of wild-type on FOXM1 recruitment was determined using chromatin immuno precipitation (ChIP) assays.

Results: Through promoter deletion analysis, we identified that deletion of the region between -1.69kb to -0.69kb in MELK promoter significantly reduced p53-dependent suppressive effect on MELK promoter activity. Screening this p53-responsive region using TransFac software, we identified a binding site for FOXM1, a previously reported MELK transcription factor. Similar to the regulation of MELK by wild-type p53, overexpression of wild-type p53 repressed FOXM1 and knockdown of wild-type p53 increased FOXM1 expression. Overexpression of FOXM1 increased MELK expression in p53-null and p53-mutant cells but not in p53 wild-type cells. Co-IP studies revealed that wild-type p53 interacts with FOXM1. ChIP studies revealed that overexpression of wild-type p53 significantly reduced the recruitment of FOXM1 to MELK promoter in TNBC cells.

Conclusion: These studies elucidated the regulation of MELK expression by wild-type p53 and demonstrated that wild-type p53 represses MELK expression by inhibiting expression and recruitment of FOXM1 to the MELK promoter. In this study, we identified a novel mechanism for upregulation of MELK expression in TNBC, which is associated with poor prognosis of breast cancer patients.

This work was supported by Susan G Komen Promise Grant (PB, SH, GM), SAB Komen grant (PB) and Young Foundation grant (PB).

Citation Format: Lakshmi Reddy Bollu, Jonathan Shepherd, Dekuang Zhao, Yanxia Ma, Abhijit Mazumdar, Powel H. Brown. Wild-type p53 represses MELK expression by blocking the recruitment of FOXM1 to MELK promoter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2532.