Abstract
Resistance to trastuzumab, which specifically target HER2-positive breast and gastric cancer, can develop ultimately in cancer patients. However, the underlying mechanisms of resistance in gastric cancer have not been fully elucidated. Here, we established trastuzumab-resistant MKN45 and NCI N87 gastric cancer sublines from their parental cells. The resistant cells exhibited characteristics of epithelial-mesenchymal transition (EMT) and acquired higher migratory and invasive capacities. To exploit the activated pathways and develop new strategies to overcome trastuzumab resistance, we investigated MKN45 and MKN45/R cells via label-free quantitative proteomics, and found pathways that were altered significantly in MKN45/R cells, with the Wnt/β-catenin pathway being the most significant. We further confirmed the activation of this pathway by detecting its key molecules in MKN45/R and NCI N87/R cells via Western blot, in which Wnt3A, FZD6, and CTNNB1 increased, whereas GSK-3β decreased, manifesting the activation of the Wnt/β-catenin pathway. Correspondingly, inhibition of Wnt/β-catenin pathway by ICG-001, a specific Wnt/β-catenin inhibitor, preferentially reduced proliferation and invasion of trastuzumab-resistant cells and reversed EMT. Concurringly, CTNNB1 knockdown in stable cell lines potently sensitized cells to trastuzumab and induced more apoptosis. Taken together, our study demonstrates that the Wnt/β-catenin pathway mediates trastuzumab resistance, and the combination of Wnt/β-catenin inhibitors with trastuzumab may be an effective treatment option.
Highlights
Gastric carcinoma, which is the fourth leading cause of cancer death worldwide, has half of its incidences detected in East Asian countries, with mortality rates being higher than other countries [1,2]
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/Human epidermal growth factor receptor-2 (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
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 and NCI N87 cells (Figure S1a)
Summary
Gastric carcinoma, which is the fourth leading cause of cancer death worldwide, has half of its incidences detected in East Asian countries, with mortality rates being higher than other countries [1,2]. 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]. 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. As opposed to breast cancer, gastric cancer still lacks extensive research in signaling pathways which mediate acquired trastuzumab resistance. Knockdown of β-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
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