Abstract P2-08-03: An essential role of GRB7 in promoting the growth of therapy resistant HER-2 positive human breast cancer cells in culture and animal models

Abstract

Abstract Background- GRB7 gene encodes a multi-domain signal transduction molecule and is part of the core of the HER-2 amplicon. GRB7 is commonly co-amplified and over-expressed with HER-2 in human breast cancer. Earlier studies found a functional role of GRB7 in breast cancer. The role of GRB7 in HER-2 positive human breast cancer resistant to HER-2 targeted therapy remains unexplored however. Materials and Methods- HCC-1954, 21MT1 and JimT1 are human HER-2 positive breast cancer cell lines that are resistant to trastuzumab and lapatinib treatment. Transient knock down of GRB7 protein expression was achieved with siRNA transfection and stable knock down with lentiviral vector mediated shRNA over-expression. Cell lines transfected with non-targeting siRNA or shRNA serve as negative controls. Knock down of GRB7 protein expression is verified by Western blotting. The growth of human breast cancer cell lines after GRB7 knock down in vitro is measured with the CellTiter Glo assay as well as the Incucyte live cell imaging. Activation status of specific signaling pathways was examined with phospho-specific antibody by immune-blotting and immune-precipitation. To assess the growth promoting function of GRB7 in human breast cancer cell lines in vivo, polyclonal HCC-1954, 21MT1 and JimT1 cells, with GRB7 knock down or their corresponding negative control, were orthotopically injected into the mammary fat pads of female immune-deficient NSG mice. The growth rates of these tumors, measured serially with caliper, and final tumor weights were compared between GRB7 knock down and the negative control. The proliferation rate and apoptosis of these tumors were studied with ki-67 staining and Tunel assay.The effects of GRB7 knock down on signaling were investigated with a proteome profiler receptor tyrosine kinase kit (R&D). The role of signaling molecules differentially activated in the growth of breast cancer cells by GRB7 knock down was examined utilizing siRNA mediated knock down, and antibody and small molecule inhibitors. Results- GRB7 knock down decreased the growth of HCC-1954, 21MT1 and JimT1 cells in vitro and the growth of tumor xenograft these cells formed in animal models. When assayed by ki67 staining and Tunel assay, the mechanism of reduced tumor xenograft growth appeared to be distinct. Reduced proliferation and increased apoptosis were seen in 21MT1 cells, while reduced proliferation was seen in HCC-1954 cells and increased apoptosis in JimT1 cells. Protein profiling found that tyrosine phosphorylation of candidate signaling molecules was reduced with GRB7 knock down in JimT1 cells. Immuno-blotting and immuno-precipitation experiments were performed to evaluate these effects in other cell lines. The effect of targeting these molecules in breast cancer cell growth by siRNA and inhibitors is being examined. Discussion- GRB7 has essential growth promoting function in therapy resistant HER-2 positive human breast cancer cells. GRB7 knock down has pleiotropic effects on signaling in various cellular contexts. The potential of targeting GRB7 signaling in treating therapy resistant HER-2 positive breast cancer merits further study. Citation Format: Luoh S-W, Wagoner W, Lai X, Hu Z, Chin K, Ramsey B. An essential role of GRB7 in promoting the growth of therapy resistant HER-2 positive human breast cancer cells in culture and animal models [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-08-03.