Antitumor Activity Of Trastuzumab Plus Saracatinib In Gastric Cancer And Its Mechanism Of Action

Overexpression of ErbB2is found in many solid tumors. Trastuzumab is ahumanized monoclonal antibody directed against ErbB2. Recently, the FDA hasapproved trastuzumab for ErbB2-positive metastatic gastric and gastro-oesophagealjunction cancer. Despite the effectiveness of trastuzumab, the majority oftrastuzumab-responsive patients develop resistance within one year of treatmentinitiation. Thus, there is an urgent need to overcome trastuzumab resistance. In thisstudy, we investigated the antitumor effect of trastuzumab in combination with the SRCinhibitor saracatinib on gastric cancer cell lines and as well as its mechanism of action.To obtain trastuzumab-resistant NCI-N87R, NCI-N87cells were treated with10μg/ml of trastuzumab for nine months．ErbB receptor expression levels were determinedby indirect immunofluorescence assays．The cell proliferation of both NCI-N87andNCI-N87R cells which were incubated with increasing concentrations of trastuzumab,saracatinib, or trastuzumab and saracatinib in combination was detected by MTS assay.Combination index (CI) values were calculated using the Chou-Talalay method.NCI-N87or NCI-N87R cells were inoculated into the nude mice which were thentreated with trastuzumab, saracatinib, or trastuzumab plus saracatinib. Tumors weremeasured with digital calipers.Immunoblots were used to detect the impact oftrastuzumab plus saracatinib on cell signaling changes in NCI-N87andtrastuzumab-resistant NCI-N87R cells.Phosphorylation of AKT was also determined byimmunohistochemistry in NCI-N87and NCI-N87R xenografts treated with trastuzumab,saracatinib, or trastuzumab plus saracatinib. Trastuzumab-resistant NCI-N87R was significantly more resistant to trastuzumabthan NCI-N87both in vitro and in vivo. We found a substantial downregulation ofEGFR, ERBB2and ErbB3in the NCI-N87R compared with the NCI-N87by flowcytometry and western blotting. However, NCI-N87R cells showed an increase inEGFR and ErbB3phosphorylation. The phosphorylation of SRC,AKT and MAPK wasalso markedly enhanced in the NCI-N87R cell line. Next, the results showed thatsaracatinib plus trastuzumab exhibited a significantly greater antiproliferative activityagainst NCI-N87cells than either agent alone in vitro. Similar results were obtainedwith NCI-N87R cells. Data analyzed using the method of Chou and Talalay showed thatsaracatinib and trastuzumab synergistically inhibited the proliferation of both NCI-N87and NCI-N87R cell lines.In nude mice model, combinatorial treatment withtrastuzumab and saracatinib resulted in a significant benefit over either agent alone inboth NCI-N87and NCI-N87R xenograft models. In vitro, ErbB signaling pathwaysindicated that trastuzumab treatment caused a decrease in ErbB3and AKTphosphorylation in the NCI-N87cell line but not in the NCI-N87R cell line. We foundthat saracatinib inhibited the phosphorylation of SRC, EGFR, ErbB2, ErbB3, AKT andMAPK in both cell lines. Remarkably, the addition of trastuzumab to saracatinib furtherreduced the phosphorylation of ErbB3and AKT in both trastuzumab-sensitive and-resistant gastric cancer cell lines. The results in xenograft models also showed thatcombinatorial treatment decreased AKT phosphorylation much better than singletreatment alone in both NCI-N87and NCI-N87R xenograft tumors.Our results demonstrated that trastuzumab in combination with saracatinibsynergistically inhibited the in vitro growth and was significantly more effective thaneither agent alone in inhibiting tumor growth in both NCI-N87and NCI-N87Rxenograft models, suggesting that the combination therapy of trastuzumab andsaracatinib may provide a new effective strategy for the treatment of gastric cancer.