DOI: 10.25345/c5d795p3j
title: MassIVE MSV000096551 - Inducible re-epithelialization of cancer cells increases autophagy and DNA damage: implications for breast cancer dormancy
title: Project description: Epithelial lineage differentiation is pivotal to mammary gland development and it can pause breast cancer (BC) metastasis by inducing tumor dormancy. To simulate this, we expressed epithelial genes in mesenchymal BC cells. Inducible expression of the epithelial OVOL genes in metastatic BC cells suppressed proliferation and migration. We found that C1ORF116, a novel OVOLs target, is susceptible to genetic and epigenetic aberrations in BC. It is regulated by steroids and functions as a putative autophagy receptor that inhibits antioxidants like thioredoxin. Accordingly, boosting epithelialization lowered glutathione, elevated reactive oxygen species and increased both DNA oxidation and double strand breaks. Epithelialization also associated with redistribution of NRF2 and an altered interplay among p38, ATM, and the other kinases regulating the DNA damage response. Hence, hormonal regulation of OVOLs and chronic stress might permit epithelial differentiation and retard exit from dormancy, while altering redox homeostasis and permitting DNA damage accumulation, which may eventually awaken dormant tumors. Sample processing protocol: Using 55 ug protein as starting material, reduction, alkylation and tryptic digestion was performed using automated SP3, adapted from Mueller et al., 2020 (https://www.embopress.org/doi/full/10.15252/msb.20199111). Proteins were digested with Trypsin in a protease:protein ratio of 1:25 for 16 hours at 37 degrees Celsius. Peptides were vacuum centrifuged to dryness and stored at -20 degrees Celsius. For LC-MS/MS analysis, peptides from 1 ug protein starting material were dissolved in ULC/MS grade water containing 0.1% trifluoracetic acid (TFA) and 2.5% 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP), followed by sonication for 5 minutes. The samples were transferred to autosampler vials and placed in the autosampler module of the Ultimate 3000 (Thermo Fisher Scientific) liquid chromatography (LC) system. The LC was operated at a flow of 300 nl/min and the column was heated to 35 degrees celsius. Peptides were first loaded onto a trapping column (Thermo Fisher Scientific, 160454) in the presence of 98% loading buffer A (0.1% TFA in water) and 2% loading buffer B (0.1% TFA in acetonitrile). Peptides were then transferred to the analytical column (Waters, 186008795, BEH C18 130A 1.7 um 75x250 mm), on which they were separated by hydrophobicity with a linear gradient of 4-30% acetonitrile. Separated peptides were ionized by electrospray ionization (ESI) with an applied voltage of 2200 V and subjected to MS/MS analysis (Exploris 480, Thermo Fisher Scientific). In DIA mode, MS1 scans were acquired at a resolution of 120K covering the range from 350 to 1400 m/z. Maximum injection time was 45 ms and the automated gain control (AGC) target was set to 3e6. MS2 scans were acquired in 47 precursor isolation windows of variable width and 1 m/z overlap that covered the range from 400 to 1000 m/z. The orbitrap was operated at a resolution of 30K and a normalized collision energy of 28% was applied. Maximum injection time was 54 ms and the AGC target was set to 1e6. Data processing protocol: Peptide and protein identification and quantification from DIA raw data was performed with Spectronaut software (version 17, Biognosys, Switzerland) in directDIA+ mode against the human proteome database with isoforms (79,052 entries).