Inhibition of YB-1 phosphorylation enhances cisplatin activity and disrupts cell division in pleural mesothelioma

Abstract

Background: The cold-shock domain protein YB-1 is overexpressed in pleural mesothelioma (PM) and was shown to contribute to increased cell migration and platinum resistance.

Methods: Phosphorylation of YB-1 at position serine 102 was analysed by immunohistochemistry, immunofluorescence and immunoblotting in PM tissue specimens and cell lines. Intracellular localisation experiments involved immunoblotting, transfection of fluorescent protein-tagged YB-1 and confocal imaging. YB-1 phosphorylation was inhibited with the RSK inhibitors BI-D1870 and LJH685. Effects of inhibition alone and in combination with radiation or cisplatin treatment were analysed by cell viability assays, clonogenic assays and videomicroscopy-based migration and cell fate map analyses.

Results: YB-1 phosphorylated at serine 102 is present in PM cell lines and tissue. Inhibition of phosphorylation with BI-D1870 reduced YB-1 localisation in the cell nucleus and led to reduced cell viability, clonogenicity, migration and disrupted cell division. Moreover, exposure to BI-D1870 increased the effect of radiation and cisplatin treatment with additive to synergistic effects in PM cell lines and primary cultures.

Conclusions: The serine 102 phosphorylated form of YB-1 contributes to the malignant phenotype of PM. Inhibition of YB-1 phosphorylation warrants further exploration as part of treatment strategies for this devastating disease.

Fig. 1. YB-1 is present in its serine 102 phosphorylated form in mesothelioma tissue and cell compartments.

a YB-1 and phospho-YB1 (pYB-1) stainings in PM and normal pleura (PL5) tissues, scored according to staining intensities: 0 = negative, 1 = weak, 2 = medium, 3 = strong. Representative images of PM tissue stained with YB-1 and phospho-YB-1 for the scores 2 and 3. Scale bar = 50 µm. b Representative pictures of PM cell lines. Phospho-YB-1 (pYB-1) is shown in green. Nuclei are stained with DAPI (blue) and actin is shown in red. Scale bar = 15 µm.

Fig. 2. BI-D1870 inhibits YB-1 S102 phosphorylation and reduces its nuclear localisation.

Representative Western blot pictures and densitometric quantification of pYB-1 in a total protein extracts and b YB-1 in nuclear and cytoplasmic fractions after the indicated times of treatment with 15 µM BI-D1870 (BI) or DMSO (Co). Beta-actin was used as loading control for total and cytoplasmic proteins, lamin b for nuclear fractions. Data are shown as mean + SEM of 3–5 replicates. *p < 0.05, **p < 0.005, ***p < 0.001. c Quantification of intracellular localisation and representative pictures of MSTO-211H cells transiently transfected with YB-1^EBFP2 (green) after 4 h treatment with 15 µM BI-D1870. The nucleus and cytoplasm were counterstained with Nuc-Red (blue) and a cell tracker (red), respectively. Scale bar = 20 µm. Quantification of the nuc/cyt ration was performed automatically using the Definiens software. The data are shown as single cells and mean + SEM. ***p < 0.001.

Fig. 3. BI-D1870 reduces PM cell viability.

a Viability of PM cells treated with BI-D1870 at the indicated concentrations for 72 h, determined by a SYBR green-based growth assay. b IC₅₀ values of BI-D1870 after 72 h, calculated from dose-response curves in correlation with cell viability 96 h after transfection with 5 nM of YB-1-specific siRNA compared to control siRNA (left), and YB-1 expression levels normalised to the immortalised mesothelial cell line Met-5A assessed by immunoblot. Each dot represents one cell line and is the mean of at least 3 biological replicates. Pearson correlation, *p < 0.05, **p < 0.005. c Representative images and d quantification of colony formation assays treated with DMSO (Co) or 5 µM and 15 µM BI-D1870 (BI) stained with crystal violet after 7–14 days. The data are shown as mean + SEM. ***p < 0.001. Scale bar = 1 cm.

Fig. 4. BI-D1870 (BI) changes cell morphology.

a Representative high magnification images from the colony formation assays and b quantification of cell areas assessed with ImageJ. Data are shown as mean + SEM of 20–50 cells. ***p < 0.001. Scale bar = 20 µm. c Automated quantification of cell area, aspect ratio, nuclei per cell and nucleus area in MSTO-211H cells using the Olympus CellSens software. ***p < 0.001.

Fig. 5. BI-D1870 induces cell cycle aberrations and reduces PM cell migration.

a Cell fate maps of SPC212 cells, created from live-cell videos. Each bar represents one cell over 96 h treated with DMSO control or 10 µM BI-D1870, a shorter bar indicates cell death. Interphases are shown in grey, nuclear divisions in blue and cytokinesis events in red. Cellular fusions, fissions and abnormal cell divisions into more than 2 daughter cells are indicated by respective symbols. b Quantification of doubling time, M-phase length, % cell death and % cytokinesis failure in DMSO (Co) and BI-D1870 (BI)-treated cells, derived from the cell fate maps. Data are shown as mean. c Migrated distance of SPC212 cells treated with DMSO or 10 µM BI-D1870 over 72 h, derived from live-cell videomicroscopy. Each dot represents one single cell and the coloured line indicates the mean. ***p < 0.001. Origin plots of representative SPC212 cells were generated using the DiPer migration tool. d Average speed of SPC212 cells, assessed by DiPer. Data are shown as mean + SEM. ***p < 0.001.

Fig. 6. BI-D1870 positively influences the effects of cisplatin and radiation treatment.

a Dose-response curves of cells treated with 2.5, 5 or 10 µM BI-D1870 in combination with 2 Gy or 0 Gy radiation. The red line represents the predicted value (PV) of additive effects (arithmetic product of the % viability of each treatment alone). Data are shown as mean + SEM. b PM cell lines and c primary cultures established from PM patients were treated with BI-D1870 (BI) or cisplatin (Cis) alone and in combination for 72 h and synergy maps derived from dose-response curves were created using the Combenefit software.