P3-18-02: A Combination of Pathway-Targeted Inhibitor with DNA-Repair Inhibitor: Preclinical Efficacy of Zactima and Olaparib in Triple Negative Subset of Breast Cancer.
Authors: Dey N , Wu H , Sun Y , De P , Leyland-Jones B 
Published: June 2012

Abstract
Introduction: Pathway-targeted therapy has not been established for the treatment of Triple Negative (TN) subset of breast cancer (BC). Despite emerging data supporting the use of polyADP-ribose polymerase (PARP) inhibitors, complete and durable responses are rare in TNBC and exploration of additional pathway-targeted therapies is needed. The high frequency (72-75%) of EGFR expression in TNBCs suggests that loss of BRCA1 may be coupled, either directly or indirectly, with EGFR overexpression in breast cancer (Van der Groep et al., 2006), and a recent report shows that even a partial loss of BRCA1 leads to an overall increase in EGFR expression in mammary epithelial cells (Burga et al., 2011). We hypothesize that the combined inhibition of growth factor driven EGFR pathway (by Zactima, a small molecule kinase inhibitor of EGFR and VEGFR-2) and DNA-repair pathway (by Olaparib, PARP inhibitor) in the presence of DNA-damaging agent (carboplatin) will have anti-proliferative and anti-migratory effects on triple negative breast tumor (TNBT) cells.
Methods: The effects of Zactima, Olaparib (single or in combination) and carboplatin were studied on: (a) the cell survival/proliferation (MTT, SRB, & cell titer-GLO assay), (b) EGF-induced upregulation of downstream proliferation markers, (c) the cellular signals for apoptosis, (d) fibronectin-directed migration (scratch-assay), (e) the vascular mimicry, and (f) the clonogenic survival (3D-ON-TOP assay) in different TNBT cell lines.
Results: Our in vitro data show that, (1) Zactima (10 uM) blocked EGF-induced phosphorylation of AKT (S473 & T308), S6RP, 4EBP1 and ERK as early as 1 hr (after the treatment), (2) the range of EC50s for Zactima (96 hrs) varied from 5 uM −15 uM (HCC70, HCC1937, MDA-MB231, MDA-MB468, BT20), (3) EGFR over-expressing, PTEN-null, and ATM kinase mutated MDA-MB468 cell line exhibited lowest EC50 for Zactima as compared to other TNBT cell lines and was found to be the most sensitive cell line to Zactima (∼ 0.05 uM) when combined with 10 uM fixed dose of Olaparib, (4) a combination of Zactima with Olaparib plus carboplatin altered an adhesion-dependent cell-survival, increased caspase3 activity (expression of cleaved caspase3 and cleaved PARP) time dependently, inhibited vascular mimicry (at 6 and 24 hrs), blocked fibronectin-directed migration (scratch assay, 24 hrs), changed the organization of actin-cytoskeleton, and suppressed clonogenic growth in different TNBT cells lines.
Conclusion: Zactima alone showed anti-proliferative/pro-apoptotic, and anti-migratory effects in TNBT cells. The combination of Zactima with Olaparib plus carboplatin was most effective in blocking the clonogenic growth of TNBT cells. We are currently pursuing studies to, (1) delineate the relationship between the anti-proliferative effects (clonogenic assay) of Zactima (alone or in combination) and the status of the EGFR/PI3K pathway using PIK3CA-mutated, PTEN-null and EGFR overexpressed TNBT cell lines, and (2) determine the effect of Zactima on angiogenesis using HUVEC cells, the results of which will be presented in the meeting.
This work was generously supported by AVON foundation.
Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-18-02.