Abstract P2-09-06: The role of p53 family tumor suppressors in mediating response to aurora kinase inhibition in triple-negative breast cancer

Abstract

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype defined by the lack of expression of the estrogen and progesterone receptors and lack of HER2 over-expression. TNBC carries an increased risk of developing distant metastatic disease and cancer-related death. TNBC is heterogeneous in its underlying biology, however, mutations in p53 are found in approximately 80% of tumors. The purpose of this study was to utilize TNBC cell line models to investigate the role of p53 and p73 in mediating sensitivity to MLN8237, a selective Aurora kinase A inhibitor, in the setting of mutant or wildtype p53. Additionally, we used p53 and p73 knock-down models to determine the role of p53 and p73 in mediating induction of apoptosis and cellular senescence in response to Aurora kinase inhibition. Methods: Eighteen TNBC cell lines were exposed to MLN8237 and the effects on proliferation, apoptosis, and cell cycle distribution were evaluated. Proliferation was assessed using an SRB assay, apoptosis was analyzed using a caspase 3/7 assay and cell cycle was measured using flow cytometry. Proliferation was confirmed using a cell counting technique to control for an increase in cell size following MLN8237 exposure. The p53 wildtype TNBC cell line CAL51 was transduced with several clones of shRNA constructs targeting p53 and p73 with > 80% knockdown by RT-PCR. These clones were exposed to escalating doses of MLN8237 and the effect on proliferation was determined using an SRB assay and the effect on cellular senescence was determined using a senescence associated beta-galactosidase (SA b-gal) assay. Results: In vitro exposure to MLN8237 resulted in robust inhibition of proliferation in TNBC cell lines which was associated with dose-dependent G2/M cell cycle arrest and induction of caspase-dependent apoptosis in a subset of sensitive cell lines. Knock-down of p53 and p73 in the CAL51 cell line resulted in an increase in the absolute 50% inhibitory concentration (IC50) calculated from the SRB proliferation assay curves from 0.04 μM to > 2 μM, 1.8 μM, > 2 μM and 1.5 μM in the CAL51scr, CAL51p5310, CAL51p5312, CAL51p7326, CAL51p7355, respectively. The sensitive p53 mutated MDA-MB-468 cell line (IC50 40 nM) and the sensitive p53 wild-type CAL51 cell line (IC50 45 nM) were selected for further experimentation. Exposure to MLN8237 at concentrations of 50 nM and 100 nM for 7 days resulted in induction of cellular senescence as detected by the SA b-gal assay in the CAL51 p53 wild-type cell line, but not in the MDA-MB-468 cell line where induction of apoptosis at 48 hours was observed using the caspase 3/7 assay. In the CAL51 p73 knock-down clones, induction of cellular senescence was not observed following exposure to MLN8237. Conclusions: MLN8237 exhibited robust anticancer activity towards preclinical models of p53 mutated and p53 wild-type TNBC, supporting future clinical investigation of Aurora kinase inhibitors in TNBC. In p53 wild-type TNBC, both p53 and p73 mediate sensitivity to MLN8237 and p73 is essential for induction of cellular senescence following exposure to MLN8237. Biomarkers predictive of response to MLN8237 in p53 mutated TNBC are being developed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-06.