Abstract 4690: Inhibition of AKT signaling by targeting both VEGF-R and ErbB-2 in breast cancer cell lines

Abstract

Abstract Deregulation of PI3K/AKT signaling occurs commonly in breast cancer and is associated with HER2 amplification as well as activating PI3K mutations. Trastuzumab (Herceptin), a recombinant humanized anti-ErbB-2 monoclonal antibody, has been found to have potent antiproliferative effects in ErbB-2 overexpressing human breast tumors, and to inhibit PI3K/AKT pathways. Vascular endothelial growth factor receptor (VEGF-R) is overexpressed in human breast tumors and cell lines, and specifically targeted knockdown of VEGF-R1 expression by siRNA has been shown to downregulate AKT phosphorylation and decrease the survival of breast cancer cells. The purpose of this study was to evaluate the antiproliferative effect of the novel combination of trastuzumab and a specific VEGF-R tyrosine kinase inhibitor in human breast cancer cell lines, and to assess their effect on AKT activation and apoptosis. Increasing doses of trastuzumab (0.125-2 nM) and a VEGF-R tyrosine kinase inhibitor (1.25-20 μM) were tested alone and in combination. Three breast carcinoma cell lines were studied: two ErbB-2 overexpressing lines (SKBR3 and BT474) and one cell line that expressed low levels of the receptor (MDA-MB231). Inhibition of growth was assessed after 5 and 7 days of treatment by the MTT colorimetric assay. Inactivation of pAKT (Ser 473) was assessed by Western Blot analysis, while apoptosis was determined by quantitating Caspase-3, 8 and 9 levels. A time and dose-dependent growth inhibition was demonstrated in all three cell lines tested with the VEGF-R inhibitor, while trastuzumab was only effective in the ErbB-2 positive cells. After 5 days of treatment, trastuzumab (0.5 nM) inhibited cell growth by 37.5% and 33.8%, and the VEGF-R inhibitor (5 μM) by 41.3% and 20.3% in the SKBR3 and BT474 cell lines, respectively. The combination of trastuzumab and the VEGF-R inhibitor, at these doses, significantly inhibited growth in SKBR3 and BT474 cells by 57.9% and 54.4 % (p < 0.01) compared to either agent alone. In the ErbB-2 negative cell line (MDA-MB231) no increase in inhibition was seen with the combination compared to VEGF-R inhibition alone. A significant reduction in pAKT levels was demonstrated with trastuzumab in the ErbB-2 positive cell lines, which was enhanced by VEGF-R inhibition in the BT474 cells, which interestingly also harbors a PI3K mutation. Caspase-3 activity increased 2.6 to 9.3-fold using the trastuzumab and VEGF-R inhibitor combination in these cell lines. Trastuzumab plus VEGF-R inhibition decreased proliferation and increased apoptosis via inactivation of AKT in ErbB-2 overexpressing breast cancer cell lines. This combination may have important clinical therapeutic implications, and further investigation is warranted. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4690.