Proteomic analysis of radio-resistant breast cancer xenografts: Increased TGF-ß signaling and metabolism.

ABSTRACT

Our previous studies have shown that MCF-7 breast cancer cell line exposed to 6 Gy and allowed to recover for 7 days (D7-6G) developed radio-resistance. In this study, we have tested the ability of these cells to form tumors in severe combined immunodeficiency (SCID) mice and characterized these tumors by proteomic analyses. Untreated (MCF-C) and D7-6G cells (MCF-R) were injected s.c. in SCID mice and tumor growth monitored. On Day 18, the mice were killed and tumor tissues were fixed in formalin or RNA later. Expression of genes was assessed by reverse transcription-polymerase chain reaction and proteins by enzyme-linked immunosorbent assay/antibody labeling and flow cytometry. Label free proteomic analyses was carried out by liquid chromatography-mass spectrometry. Metabolic analysis was carried out using Seahorse analyzer. MCF-R cells had a shorter latency and formed larger tumors. These tumors were characterized by an increased expression of transforming growth factor ß (TGF-ß) isoforms; its downstream genes pSMAD3, Snail-1, Zeb-1, HMGA2; hybrid epithelial/mesenchymal phenotype; migration, enrichment of cancer stem cells and radioresistance following challenge dose of radiation. Proteomic analysis of MCF-7R tumors resulted in identification of a total of 649 differentially expressed proteins and pathway analyses using protein annotation through evolutionary relationship indicated enrichment of genes involved in metabolism. Data are available via ProteomeXchange with identifier PXD022506. Seahorse analyzer confirmed increased metabolism in these cells with increased oxidative phosphorylation as well as glycolysis. Increased uptake of 2-NBDG further confirmed increased glycolysis. In summary, we demonstrate that radioresistant breast cancer cells had an enrichment of TGF-ß signaling and increased metabolism.