SILAC-Based Proteomic Profiling of the Human MDA-MB-231 Metastatic Breast Cancer Cell Line in Response to the Two Antitumoral Lactoferrin Isoforms: The Secreted Lactoferrin and the Intracellular Delta-Lactoferrin

Abstract

BackgroundLactoferrins exhibit antitumoral activities either as a secretory lactoferrin or an intracellular delta-lactoferrin isoform. These activities involve processes such as regulation of the cell cycle and apoptosis. While lactoferrin has been shown to exert its function by activating different transduction pathways, delta-lactoferrin has been proven to act as a transcription factor. Like many tumor suppressors, these two proteins are under-expressed in several types of cancer, particularly in breast cancer.Methodology/Principal FindingsIn order to compare the differential effects of the re-introduction of lactoferrin isoforms in breast cancer cells we chose the cancerous mammary gland MDA-MB-231 cell line as a model. We produced a cell line stably expressing delta-lactoferrin. We also treated these cells with fresh purified human breast lactoferrin. We performed two quantitative proteomic studies in parallel using SILAC coupled to mass spectrometry in order to compare the effects of different doses of the two lactoferrin isoforms. The proteome of untreated, delta-lactoferrin expressing and human lactoferrin treated MDA-MB-231 cells were compared. Overall, around 5300 proteins were identified and quantified using the in-house developed MFPaQ software. Among these, expression was increased by 1.5-fold or more for around 300 proteins in delta-lactoferrin expressing cells and 190 proteins in lactoferrin treated cells. At the same time, about 200 and 40 proteins were found to be downregulated (0-0.7-fold) in response to delta-lactoferrin and lactoferrin, respectively.Conclusions/SignificanceRe-introduction of delta-lactoferrin and lactoferrin expression in MDA-MB-231 mainly leads to modifications of protein profiles involved in processes such as proliferation, apoptosis, oxidative stress, the ubiquitin pathway, translation and mRNA quality control. Moreover, this study identified new target genes of delta-lactoferrin transcriptional activity such as SelH, GTF2F2 and UBE2E1.

The roles of ribosomal protein S19 C-terminus in a shortened neutrophil lifespan through delta lactoferrin

“…To examine other roles of ␦Lf, in silico analyses of the same promoter sequence, AGCACTTTGG, were performed, and we found expression of the ubiquitin-conjugating enzyme E2 E1 in the cancerous mammary gland MDA-MB-231 cells (Hoedt et al, 2014), suggesting the participation of ␦Lf in the maintenance of cell homeostasis. We identified the same promoter against ␦Lf at −468 bp upstream of the ATG start codon in the RGS3 gene (NG 029512.1) in this study.…”

Selenoproteins and Metastasis

“…There is evidence using the MDA-MB-231 metastatic breast cancer cell line that SELENOH is transcriptionally upregulated by the anti-proliferation and anti-migration protein, delta-lactoferrin (Hoedt et al, 2014). These different studies do not clarify how SELENOH may affect metastasis, but redox directed transcription regulated by SELENOH is likely to both influence and be influenced by the stage of cancer cell progression into aggressive metastatic tumors.…”

Modification by SUMOylation Controls Both the Transcriptional Activity and the Stability of Delta-Lactoferrin

“…ΔLf mainly exerts its anti-proliferative and pro-apoptotic activities via its role as a transcription factor. Indeed, ΔLf transactivates different target genes such as Skp1 , DcpS , Bax , SelH , GTF2F2 and UBE2E1 [ 9 , 12 – 14 ]. A genome-wide pathway analysis and our quantitative proteomic analysis showed that the re-introduction of Lf isoforms in cancerous cells modified essential genes and/or signaling networks responsible mainly for cell survival, apoptosis and RNA processing [ 14 , 15 ].…”