Prolonged mild hypoxia induced alterations in the proteome of triple negative breast cancer cell lines MDA-MB-231 and MDA-MB-436

Objectives: Hypoxic areas are hallmark of most breast tumors. Adaptation of cancer cells to hypoxia is a driving force for the clonal selection of more aggressive phenotype. The aim of this exploratory study was to characterize alterations in the protein abundances in triple negative breast cancer (TNBC) cell lines MDA-MB-231 (M231) and MDA-MB-436 (M436) during prolonged adaptation to mild hypoxia.
Materials and Methods: Cell cultures were exposed to hypoxia (2% O2) for 4 subcultures (length of hypoxic exposure was ~70 days for M231 and ~90 days for M436); normoxic cultures (19.6 %O2) were used as controls; culture experiments were performed in triplicate. Two protein samples were obtained from control cultures and each culture after 1st (H1) and 4th hypoxic (H4) passage. Peptides obtained by filter-aided sample preparation (FASP) and tryptic digestion were analyzed by NanoUPLC-HDMSE (Waters). Three UPLC-MS/MS runs were performed for each
tryptic sample. Identification and label-free quantification of proteins was performed using Progenesis QI (Nonlinear Dynamics). Further stastistical analysis was performed using R, version 3.6. Significance of differences was determined by ROTS test.
Results: 1894 proteins were consistently quantified in both cell lines. Statistically significant (p≤0.05) at least 2-fold differences in hypoxic vs normoxic samples were detected for 293 proteins in m231_H1, 143 in m231_H4, 108 in m436_H1, and 73 in m436_H4. Only tropomyosin alpha 3 (TPM3) and ubiquitin carboxyl terminal hydrolase 5 (USP5) were significantly altered in all hypoxic samples. 60 and 10 proteins of M231 and M436, respectively, were significantly altered in both early and late hypoxia. Those proteins were associated with proteostasis
related processes (ribosomes, translation, nonsense-mediated decay), amino acid metabolism and extracellular vesicle formation in M231, and with NRF2 pathway and cellular senescence/necroptosis in M436.
Conclusions: Adaptation to hypoxia in TNBC cancer cells involves modifications in nitrogen metabolism and protein synthesis, as well as oxidative stress regulation and cell fate determination pathways.