Abstract P3-07-21: Cytotoxic potential of the RG7388 MDM2-p53 binding antagonist and the GSK2830371 WIP1 inhibitor on MX-1 and MCF-7 human breast cancer cells
Authors: Manoharan V , Lunec J , Esfandiari A , Mahdi A , Wu C-E , Zanjirband M , Karunanayake EH , Tennekoon KH , De Silva S 
Published: May 2019

Background

The tumor suppressor p53 is a central hub in molecular signaling pathways that control the integrity of the human genome. The p53 protein functions as a transcription factor and increases the expression of many cellular genes which contribute to activation of cell cycle arrest, apoptosis and DNA repair. MDM2 is another important p53 target gene, and the MDM2 protein is capable of binding directly to p53 and directing it for degradation through the ubiquitin-dependent proteolytic pathway. Inhibition of MDM2 stabilizes p53 and MDM2 inhibitors are being explored clinically as therapies. Stabilization alone may not be enough to increase the activity of p53, and posttranslational modification of p53 by phosphorylation has been proposed to be an important contributory mechanism by which p53 becomes functionally active. Therefore maintaining the phosphorylated status of p53 in tumor cells may help to enhance its growth inhibitory and pro-apoptotic role. Wild type p53 – induced phosphatase (Wip1) is a serine – threonine phosphatase which dephosphorylates central players in the DNA damage response, including p53 and may be an additional target to enhance p53-dependent treatments. Therefore this work was focused on the effect of MDM2 (RG7388) and Wip1 (GSK2830371) inhibitors on MX-1 and MCF breast carcinoma cell lines. These two cell lines were recorded to have wild type TP53 status as well as high expression of Wip1.

Trial design

RG7388 and GSK2830371 were tested for growth inhibition on MX-1 and MCF-7 breast cancer cell lines using the sulforhodamine B (SRB) assay. The results were further confirmed and mechanism explored by western blotting using extracted protein from drug treated cell lines. Contradictory evidence regarding the TP53 mutation status of the MX-1 cell line was clarified by direct sequencing of MX-1 DNA.

Results

The MCF-7 cells responded to both RG7388 and GSK2830371 with GI50 value of 0.034 µM and 2.92 µM respectively. The MX-1 cells did not respond to either drug. The results of western blotting showed there was no expression of p53 in the MX-1 cell line. Failure to respond to RG7388and also no expression of p53 in western blotting made us suspicious about the TP53 status of the MX-1 cells. The direct sequencing results confirmed that there was a 5bp deletion in exon 4 of the TP53 gene of the MX-1 cells. The c.154_158delCAATG mutation creates a stop codon at the 54th aminoacid position and results in a truncated p53 protein (p.Gln52Valfs*3).

Conclusion

RG7388 and GSK2838371 showed cytotoxic effects on MCF-7 cells, whereas both RG7388 and GSK2838371 had no effect on the MX-1 cell line due to the truncated p53 and loss of p53 function. In conclusion, the potency of both drugs depends on the TP53 mutation status and they are likely to be mediated via p53-dependent growth inhibition and apoptosis. Further studies are needed to evaluate the combination effect of both drugs on TP53 wild type cell lines.

Citation Format: Manoharan V, Lunec J, Esfandiari A, Mahdi A, Wu C-E, Zanjirband M, Karunanayake EH, Tennekoon KH, De Silva S. Cytotoxic potential of the RG7388 MDM2-p53 binding antagonist and the GSK2830371 WIP1 inhibitor on MX-1 and MCF-7 human breast cancer cells [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 P3-07-21.