Abstract 3970: EGF has stimulatory and survival effects in osteosarcoma in vitro and pan-ERBB kinase inhibition causes cell growth inhibition in vitro and reduces lung metastases in vivo
Authors: Nelson Laura D. , Lynch Tiffany N. , Yang Yanwen , Wang Wei-Lien , Liu Diane , Thomas Dafydd G. , Hughes Dennis P. M. 
Published: May 2015

Osteosarcoma is the most common primary bone tumor in children and adolescents, with recurring disease or lung metastases being the primary cause of death in patients. Expression of EGFR, Her-2, and Her-4 (ERBB1, 2 and 4) have been reported in human primary osteosarcoma, suggesting that they may be contributing to osteosarcoma pathogenesis, but the correlation of expression levels with patient outcomes has been controversial. We have shown that a pan-ERBB small molecule kinase inhibitor causes growth inhibition and apoptosis of osteosarcoma cell in vitro, suggesting a potential therapeutic benefit of using this drug in vivo. In the present study, we show that protein expression levels of EGFR, Her-2 and Her-4 are higher in osteosarcoma lung metastases than in primary tumors by TMA, and that these levels are significantly higher for Her-2 and Her-4. We created eight new osteosarcoma cell lines from primary patient tissues and found that all eight, in addition to six commonly used osteosarcoma cell lines, express detectable levels of EGFR and Her-2, and several express detectable Her-4. EGF ligand stimulation in vitro produces robust phosphorylation of EGFR, Her-2 and Her-4 in most cell lines and moderate activation in others. EGF stimulation also produces phosphorylation of Akt, MAPK and p70S6K in most cell lines and of STAT5 in six cell lines, suggesting that EGF has growth and survival stimulation effects. When adding a pan-ERBB kinase inhibitor, Pf-299804, to cells pre-stimulated with EGF, phosphorylation of EGFR, Her-2, Her-4, Akt, etc. are inhibited in a dose-dependent manner. Reverse phase protein array also shows an increase in pro-apoptotic proteins Bim and cleaved PARP with Pf-299804 treatment, confirmed by Western blots. Mouse xenograft experiments, where cells injected in the tibia spontaneously form lung metastases, showed a significant decrease in three categories of Her-4-positive lung metastases in drug-treated mice based on size: 1-4 cell oligomets (p=0.0055); 5 cell-200 micron micromets (p=0.0276); and greater than 200 micron macromets (p=0.0055). These data suggest that EGF produces mitogenic and survival stimulation in osteosarcoma cells and that pan ERBB-family kinase inhibition causes growth inhibition and apoptosis in vitro and reduction of lung metastases in a mouse model in vivo. This provides evidence that pan-ERBB kinase inhibition has therapeutic potential for osteosarcoma lung metastases.

Citation Format: Laura D. Nelson, Tiffany N. Lynch, Yanwen Yang, Wei-Lien Wang, Diane Liu, Dafydd G. Thomas, Dennis P. M. Hughes. EGF has stimulatory and survival effects in osteosarcoma in vitro and pan-ERBB kinase inhibition causes cell growth inhibition in vitro and reduces lung metastases in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3970. doi:10.1158/1538-7445.AM2014-3970