Label-Free Quantitative Proteomics Combined with Biological Validation Reveals Activation of Wnt/β-Catenin Pathway Contributing to Trastuzumab Resistance in Gastric Cancer

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.

Cisplatin resistance in gastric cancer cells is involved with GPR30‐mediated epithelial‐mesenchymal transition

“…EMT‐expressing cancer cells showed decreased expression of epithelial cell markers such as E‐cadherin and ZO‐1, while expression of mesenchymal cell markers such as vimentin and N‐cadherin increased. Although there is increasing evidence that EMT is closely related to the development of drug resistance in gastric cancer cells, its underlying mechanisms have not yet been fully elucidated …”

“…Although there is increasing evidence that EMT is closely related to the development of drug resistance in gastric cancer cells, its underlying mechanisms have not yet been fully elucidated. [14][15][16] To date, many studies have investigated the role of oestrogen receptor (ER) in gastric cancer, and the possible mechanisms of these effects and the clinical relevance of dysregulated ER in GC.…”

The Dynll1-Cox4i1 Complex Regulates Intracellular Pathogen Clearance via Release of Mitochondrial Reactive Oxygen Species

“…Protein concentration was determined using a Bradford protein assay kit (P0006; Beyotime, China). Samples (200 g each) containing extracted membrane proteins were digested with trypsin, as described previously (54). Tryptic peptides were preseparated on a Waters 2695 HPLC system equipped with a C 18 column (5 m, 4.6 ϫ 250 mm; Thermo Fisher) and eluted using a continuous acetonitrile gradient as follows: time ϭ 0 min, 100% A (water), 0% B (acetonitrile), 0.5 ml/min; time ϭ 35 min, 2% A (water), 98% B (acetonitrile), 0.5 ml/min; and time ϭ 40 min, 2% A (water), 98% B (acetonitrile), 0.5 ml/ min.…”

Global metabolomic profiling of trastuzumab resistant gastric cancer cells reveals major metabolic pathways and metabolic signatures based on UHPLC-Q exactive-MS/MS

“…Western blot was performed according to standard protocols as described previously. 13,14 To put it briey, cells were collected and lysed with RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, Beyotime) containing 1% protease inhibitor. Then lysates were centrifuged at 12 000g for 15 min at 4 C, and the supernatants were collected for further use.…”

“…8,12 Our previous proteomics demonstrated that acquired resistance to trastuzumab leads to distinct alterations of several signaling pathways in NCI N87/R and MKN45/R cells, out of which mTOR and Wnt/ b-catenin were conrmed by further studies. 13,14 Recently, we explored transcription factors related to trastuzumab resistance in NCI N87/R cells by liquid chromatography-mass spectrometry coupled with concatenated tandem array of transcription factor response elements (catTFRE) pull down proteomic technique. Our study indicated several transcription factors including TCF7L2, TCF7, FOXC1, JUN, MYC, FOS, ELK4, FOXC and DDIT3 had a crucial regulatory effect on epithelialmesenchymal transformation (EMT), Wnt/b-catenin and MAPK signaling pathways contributing to trastuzumab-resistance.…”