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Mass spectroscopy‑based proteomics and metabolomics analysis of triple‑positive breast cancer cells treated with tamoxifen and trastuzumab

ABSTRACT: HER2-enriched breast cancer with high levels of hormone receptor expression, known as triple positive breast cancer, may represent a new entity with a relatively favourable prognosis against which the combination of chemotherapy, HER-2 inhibition, and endocrine treatment may be considered overtreatment. We explored the effect of the anticancer drugs tamoxifen and trastuzumab, both separately and in combination, on the integrated proteomic and metabolic profile of triple positive breast cancer cells (BT-474). Method We employed ultra-high-performance liquid chromatography-quadrupole time of flight mass spectrometry using a Bruker timsTOF to investigate changes in BT-474 cell line treated with either tamoxifen, trastuzumab or a combination. Differentially abundant metabolites were identified using the Bruker Human Metabolome Database metabolite library and proteins using the Uniprot proteome for Homo sapiens using MetaboScape and MaxQuant, respectively, for identification and quantitation. Results A total of 77 proteins and 85 metabolites were found to significantly differ in abundance in BT-474 treated cells with tamoxifen 5 μM/and or trastuzumab 2.5 μM. Findings suggest that by targeting important cellular signalling pathways which regulate cell growth, apoptosis, proliferation, and chemoresistance, these medicines have a considerable anti-growth effect in BT-474 cells. Pathways enriched for dysregulation include RNA splicing, neutrophil degranulation and activation, cellular redox homeostasis, mitochondrial transmembrane transport, ferroptosis and necroptosis, ABC transporters and central carbon metabolism. Conclusion Our findings in protein and metabolite level research revealed that anti-cancer drug therapy had a significant impact on the key signalling pathways and molecular processes in triple positive BT-474 cell lines.

ORGANISM(S): Human Homo Sapiens

TISSUE(S): Breast Cancer Cells

DISEASE(S): Cancer

SUBMITTER: Nelson Soares

PROVIDER: ST002319 | MetabolomicsWorkbench | Tue Oct 18 00:00:00 BST 2022

REPOSITORIES: MetabolomicsWorkbench

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Project description:Tamoxifen, an antagonist to estrogen receptor (ER), is a first line drug used in breast cancer treatment. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying the resistance to tamoxifen, we established a tamoxifen-resistant cell line by treating the MCF7 breast cancer cell line with tamoxifen for over 6 months. We showed that this cell line exhibited resistance to tamoxifen both in vitro and in vivo. In order to quantify the phosphorylation alterations associated with tamoxifen resistance, we performed SILAC-based quantitative phosphoproteomic profiling on the resistant and vehicle-treated sensitive cell lines where we identified >5,600 unique phosphopeptides. We found phosphorylation levels of 1,529 peptides were increased (>2 fold) and 409 peptides were decreased (<0.5-fold) in tamoxifen resistant cells compared to tamoxifen sensitive cells. Gene set enrichment analysis revealed that focal adhesion pathway was the top enriched signaling pathway activated in tamoxifen resistant cells. We observed hyperphosphorylation of the focal adhesion kinases FAK1 and FAK2 in the tamoxifen resistant cells. Of note, FAK2 was not only hyperphosphorylated but also transcriptionally upregulated in tamoxifen resistant cells. Suppression of FAK2 by specific siRNA knockdown could sensitize the resistant cells to the treatment of tamoxifen. We further showed that inhibiting FAK activity using the small molecule inhibitor PF562271 repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 significantly associated with short metastasis-free survival of ER-positive breast cancer patients treated with tamoxifen-based hormone therapy. Our studies suggest that FAK2 is a great potential target for the development of therapy for the treatment of hormone refractory breast cancers.

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Mass spectroscopy‑based proteomics and metabolomics analysis of triple‑positive breast cancer cells treated with tamoxifen and trastuzumab

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