These data indicate that prolonged treatment of trastuzumab induces the EMT process, which is involved in trastuzumab resistance of gastric cancer cells. Mounting evidence suggests that Wnt/-catenin pathway plays an important role in EMT regulation of various cancers. and induced more apoptosis. Taken together, our study demonstrates that the Wnt/-catenin pathway mediates trastuzumab resistance, and the combination of Wnt/-catenin inhibitors with trastuzumab may be an effective treatment option. gene located on chromosome 17q21 [3,4]. A positive correlation exists, as inferred from numerous studies, between HER-2 over-expression and cancer cell proliferation, malignancy, metastasis, and poor outcomes [5,6,7]. HER-2 over-expression and/or gene amplification (20% of gastric cancer cases) represents a negative predictor of response to chemotherapy and a positive factor to anti-HER2 agents [4]. Previous studies have confirmed that HER-2 activation can be perceived as a trigger of multiple cell signal transduction pathways, which promotes aberrant cell proliferation and drug resistance [8,9]. As a result of rapid advancement in the field of tumor biology, attention has been focused on the new modality of molecular targeted therapy for advanced cancer [10,11]. Molecular-targeted drugs such as trastuzumab (Herceptin?), a humanized monoclonal antibody interfering with the extracellular domain of HER2/neu receptor, has been proved to be beneficial in patients with HER2-positive advanced gastric and breast cancer in clinical treatment [12,13]. Unfortunately, the acquired resistance could hinder the effectiveness of trastuzumab [14,15]. In clinical practice, acquired resistance can be a major barrier for antineoplastic agents. Some potential mechanisms of trastuzumab resistance include mutational activation of the phosphatidylinositide 3-kinase (PI3K)/AKT pathway [16], up-regulation of insulin-like growth factor receptor (IGFR) and hetero-dimerization of IGFR/HER-2 [17,18], loss of phosphatase and tensin homolog gene (PTEN) function [19], and accumulation of truncated HER-2 receptor (p95HER-2) [20], all of which have been verified as principal pathways in breast cancer. Although gastric cancer does possess some of these pathway modulations, there are some gastric cancer-specific mechanisms too. LY 3200882 For instance, Srebf1 over-expression of miR-223 in miR-223/FBXW7 pathway [21], up-regulation of fibroblast growth factor receptor 3 (FGFR3)/AKT axis [22], activation of 2-adrenergic receptor (2-AR) signaling, and loss of HER-2 [23,24] are some of the mechanisms. As opposed to breast cancer, gastric cancer still lacks extensive research in signaling pathways which mediate acquired trastuzumab resistance. Mass spectrometry-based proteomics has emerged as a powerful tool for large-scale protein analysis in biological research [25,26]. Ding et al. have developed a novel technique in recent years named label-free quantification workflow (Fast-quan) for protein quantification, in which 7000 proteins can be detected and quantified within 12 h of mass spectrometry running time [27]. Here, the trastuzumab-resistant sublines, MKN45/R and NCI N87/R, were obtained by continuous exposure to increasing doses of trastuzumab up to 80 g/mL. We proved that there is an association between acquirement of trastuzumab resistance and EMT. We also performed label-free proteome profiling of MKN45 and MKN45/R, analyzed differential proteins and explored the corresponding pathways using bioinformatics techniques. In addition, a series of biological validation were conducted and the activation of canonical Wnt/-catenin pathway in both MKN45/R and NCI N87/R cells was confirmed. Suppression of Wnt/-catenin signaling by ICG-001 decreased viability and induced apoptosis of trastuzumab resistant cells in a dose-dependent manner and reversed EMT. Also, knockdown of -catenin suppressed cell proliferation and enhanced sensitivity to trastuzumab of resistant cells, implying this pathway to be a possible treatment target for trastuzumab-resistant gastric carcinoma. 2. Results 2.1. Establishment of Trastuzumab-Resistant Gastric Cancer Cell Lines We employed Western blot to detect the expression of HER-2 in all six gastric cancer cell lines, including NCI N87, MKN45, MKN28, BGC823, MGC803, and SGC7901, with a relatively high level being observed in MKN45 LY 3200882 and NCI N87 cells (Figure S1a). To simulate the in vivo mode of LY 3200882 resistance, we treated MKN 45 and NCI N87 cell lines with increasing doses of trastuzumab for five months. Once the drug concentration level reached up to 80 g/mL, trastuzumab-resistant sublines MKN45/R and NCI N87/R were then harvested. The IC50 values of MKN45 and MKN45/R cells were 56.48 and 414.52 g/mL,.