Inhibition of GAS6/AXL improves efficacy of HER2 inhibitor trastuzumab in uterine serous cancer

Abstract

<h3>Objectives:</h3> To determine whether inhibition of GAS6/AXL can improve efficacy to the humanized anti-HER2 monoclonal antibody trastuzumab by decreasing uterine serous cancer cell invasion and cell survival <i>in vitro</i> and tumor burden <i>in vivo.</i> <h3>Methods:</h3> We treated AXL-expressing and HER2-expressing uterine serous cancer cell lines (ARK1, ARK2) with trastuzumab, AVB-500, or trastuzumab plus AVB-500. Clonogenic cell survival assays were performed. Matrigel invasion assays were used to assess the invasive capacity of ARK2 cells after 24 hours of treatment with vehicle, AVB-500, trastuzumab, or trastuzumab plus AVB-500. A proximity ligation assay was used to examine colocalization of HER2/neu and AXL. Western blots were performed to assess the effects of the individual drugs and combination on phosphorylation of HER2 and AXL. An intraperitoneal tumor model with 10 million ARK1 cells was treated with vehicle, AVB-500, trastuzumab, or trastuzumab plus AVB-500 for 35 days. The number, size, and volume of tumor nodules were measured. GraphPad Prism was used for statistical analysis. <h3>Results:</h3> We found decreased cell survival by clonogenic assays in ARK1 and ARK2 cells treated with trastuzumab plus AVB-500 compared to trastuzumab alone (relative absorbance 0.155 nm vs 0.202 nm, P=0.015). There was significantly less invasion from treatment with trastuzumab plus AVB-500 than with trastuzumab alone (3.2 vs 17.8 invading tumor cells/hpf, <i>P</i>=0.0004). Western blot showed that cells treated with trastuzumab plus AVB-500 had reduced phospho-AXL compared to cells treated with AVB-500 or trastuzumab alone. Furthermore, the proximity ligation assay demonstrated co-localization of the AXL and HER2 receptors in both ARK1 and ARK2 cells indicating a physical closeness that could account for this co-regulation. We found that mice treated with trastuzumab plus AVB-500 developed significantly less tumor burden than mice treated with trastuzumab alone (0.03205 g vs 0.08316 g, P=0.024), AVB-500 alone (0.0320 g vs 0.1638 g, P<0.0001), and vehicle alone (0.03205 g vs 0.1154 g, P=0.0395). <h3>Conclusions:</h3> The GAS6/AXL inhibitor AVB-500 potentiated the effect of trastuzumab to decrease uterine serous cancer cell proliferation and invasion <i>in vitro</i> and tumor burden <i>in vivo.</i> There was co-localization of AXL and HER2 receptors seen in both uterine serous cancer cell lines.