An integrative approach to identify YB-1-interacting proteins required for cisplatin resistance in MCF7 and MDA-MB-231 breast cancer cells

Abstract

The Y-box binding protein 1 (YB-1) is a multifunctional protein that affects transcription, splicing, and translation. Overexpression of YB-1 in breast cancers causes cisplatin resistance. The exact mechanism by which YB-1 confers cisplatin resistance is unknown. The aim of the present study was to identify, using mass spectrometry, proteins that interact with YB-1 that are important for cisplatin resistance in two breast cancer cell lines, namely MCF7 and MDA-MB-231. A tagged YB-1 construct was used to identify proteins interacting directly with YB-1 in breast cancer cells. We then focused on proteins that are potentially involved in breast cancer progression based on the ONCOMINE public microarray database. Genes encoding for these YB-1-interacting proteins were examined in the public NCBI comparative genomic hybridization database to determine whether they are localized to regions of chromosomes that are rearranged in breast cancer tissues. From these analyses, we generated a list of proteins potentially involved in cisplatin resistance. Cisplatin dose-response curves were constructed in MCF7 and MDA-MB-231 transfected with four siRNA corresponding to each of these YB-1 interactors to identify proteins significantly affecting cisplatin sensitivity upon gene silencing. Depletion of only the X-linked ribosomal protein S4 (RPS4X) resulted in consistent resistance to cisplatin in both cell lines with at least three different siRNA sequences against RPS4X. Further analyses indicated that the knock down of RPS4X decreased DNA synthesis, induced cisplatin resistance, and is equivalent to the overexpression of YB-1 in both MCF7 and MDA-MB-231 cells. These results suggest that the RPS4X/YB-1 complex is a significant potential target to counteract cisplatin resistance in breast cancer.

Figure 1

Validation of an interaction between tandem affinity purification (TAP)‐tagged Y box‐binding protein 1 (YB‐1) and 11 proteins identified from liquid chromatography–tandem mass spectrometry (LC‐MS/MS). (a) MCF7 clones expressing the construct. Clone A was used for LC‐MS/MS analyses. (b) Interaction between TAP‐YB‐1 and the C1QBP, DAP3, HNRPK, HNRPL, PABPC1, RALYL, RIBC2, RPL31, RPS7, RPS4X, and SARG proteins. Proteins from TAP‐ and TAP‐YB‐1‐expressing MCF7 cells were eluted from the streptavidin beads and analyzed by SDS‐PAGE with antibodies against the proteins indicated. Proteins were revealed using an ECL Plus kit (GE Healthcare Bio‐Sciences Corp., Piscataway, NJ, USA). WCE, whole‐cell lysate; TAP eluate, protein eluted from the streptavidin beads. (c) Nuclear expression of C1QBP, DAP3, HNRPK, HNRPL, PABPC1, RALYL, RIBC2, RPL31, RPS7, RPS4X, SARG, and lamin B1 (protein control) in TAP cells and the TAP‐YB‐1 clone A.

Figure 2

Histograms showing changes in cisplatin EC₅₀ values for each siRNA sequence as a ratio of the EC₅₀ in cells transfected with an siRNA against GFP (control) in (a) MCF7 and (b) MDA‐MB‐231 transfected cells. The EC₅₀ values were calculated from cisplatin concentration–response curves obtained after transfection of individual siRNA sequences a, b, c, and d targeting the proteins indicated. Data are the mean ± SEM (n = 4). *P < 0.05, ^† P = 0.0719 compared with control (unpaired Student’s t‐test).

Figure 3

Knock down of RPS4X with specific siRNA molecules in (a,c) MCF7 and (b,d) MDA‐MB‐231 cells. Cells were transfected with the indicated siRNA molecules and, 96 h later, proteins were extracted for western blot analyses. (a,b) Representative blots following transfection of MCF7 (a) and MDA‐MB‐231 (b) cells with siRNAs against RPS4X. siControl, non‐specific siRNA molecules. In every blot, β‐actin and KU86 were used as loading controls. (c,d) Effect of RPS4X depletions on bromodeoxyuridine (BrdU) incorporation in MCF7 (c) and MDA‐MB‐231 (d) cells transfected with the indicated siRNA sequences, as determined by ELISA. Cells were transfected with the indicated siRNA sequences for 72 h and then incubated with medium containing BrdU for an additional 24 h before the ELISA. Data are the mean ± SEM (n = 4). *P < 0.03 (unpaired Student’s t‐test).

Figure 4

Effect of Y box‐binding protein 1 (YB‐1) overexpression on bromodeoxyuridine (BrdU) incorporation in MCF7 and MDA‐MB‐231 cells. (a) Incorporation of BrdU, as determined by ELISA, in MCF7 and MDA‐MB‐231 cells. Cells were transfected with a YB‐1 expression vector or a control empty vector and 24 h later were incubated with medium containing BrdU for an additional 24 h before the ELISA. Indicated P‐values were determined using an unpaired Student’s t‐test. (b) Representative western blots showing increased expression of YB‐1 in MCF7 and MDA‐MB‐231 cells transfected with a YB‐1 expression vector compared with an empty control vector. Cells were transfected and 24 h later proteins were extracted for western blot analysis. (c) Percentage of MCF7 and MDA‐MB‐231 transfected cells in each phase of the cell cycle. Cells were transfected with the indicated constructs and subjected to FACS analysis 24 h later. Data are the mean ± SEM (n = 4).

Figure 5

Cell growth of Y box‐binding protein 1 (YB‐1)‐overexpressing cells. (a) Growth curves for tandem affinity purification (TAP) and TAP‐YB‐1‐expressing MCF7 clones. (b) Growth curves for MDA‐MB‐231 cells transfected with control or YB‐1 expression vectors. Fifty thousand cells were plated on 60‐mm Petri dishes 24 h after transfection in duplicate. Cells were counted every other day with a hemocytometer. Data are the mean ± SEM. MCF7 and MDA‐MB‐231 cells reached confluence on Days 9 and 11, respectively.

Figure 6

Resistance of Y box‐binding protein 1 (YB‐1)‐overexpressing cells to cisplatin after RPS4X depletion. (a) MDA‐MB‐231 cells were transfected with either siControl (non‐specific siRNA molecules), siRPS4X, empty or YB‐1 expression vectors and treated with the indicated cisplatin concentrations. Experiments were performed in duplicate. Cells were treated with cisplatin for 18 h, after which they were plated (1000 cells per plate) in 100‐mm Petri dishes and cultured for 10 days without drug to allow colony formation. The y‐axis is in logarithmic scale. (b) Tandem affinity purification (TAP) or TAP‐YB‐1 MCF7 cells were transfected with siControl or siRPS4X molecules as indicated and treated with cisplatin for 18 h. The percentage of live cells was counted with a hemocytometer the next day. Experiments were performed in triplicate. Data are the mean ± SEM.