Proteomic analysis of zoledronic-acid resistant prostate cancer cells unveils novel pathways characterizing an invasive phenotype

Abstract

Proteomic analysis identified differentially expressed proteins between zoledronic acid-resistant and aggressive DU145R80 prostate cancer (PCa) cells and their parental DU145 cells. Ingenuity Pathway Analysis (IPA) showed a strong relationship between the identified proteins within a network associated with cancer and with homogeneous cellular functions prevalently related with regulation of cell organization, movement and consistent with the smaller and reduced cell-cell contact morphology of DU145R80 cells. The identified proteins correlated in publically available human PCa genomic data with increased tumor expression and aggressiveness. DU145R80 exhibit also a clear increase of alpha-v-(αv) integrin, and of urokinase receptor (uPAR), both included within the same network of the identified proteins. Interestingly, the actin-rich structures localized at the cell periphery of DU145R80 cells are rich of Filamin A, one of the identified proteins and uPAR which, in turn, co-localizes with αv-integrin, in podosomes and/or invadopodia. Notably, the invasive feature of DU145R80 may be prevented by blocking anti-αv antibody. Overall, we unveil a signaling network that physically links the interior of the nucleus via the cytoskeleton to the extracellular matrix and that could dictate PCa aggressiveness suggesting novel potential prognostic markers and therapeutic targets for PCa patients.

Keywords: cytoskeleton organization; prostate cancer; urokinase receptor (uPAR); zoledronic acid; αv integrin.

Figure 1. PCA plot, spot map, functional distribution of identified protein and validation by 1-D and 2-D Western blot of ALDH7A1

(A) Unsupervised multivariate analysis of the 2-DE DIGE results shows that the eight spot maps clearly clustered into two groups corresponding to DU145 (circled in red) or DU145R80 (circled in black). The PCA plot also shows a good experimental reproducibility as demonstrated by the closely relation between the four biological replicates (DU145 red circles; DU145R80 black circles). To be included in the analysis, protein spots had to be found in 80% of the spot maps and display an expression variation of at least 1.4-fold with t-Student test (p < 0.05). (B) Representative DeepPurple^TM stained spot map image of DU145R80. All the detected differences existing between DU145 and DU145R80 cells are visualized by circles. For MS-identified protein spots, the spot numbers match those listed in Table 1. (C) Pie chart of functional distribution of proteins identified by 2-DE DIGE/MS. (D) The upper-left panels showed the 3D view of the 2-DE DIGE quantification for spot of interest (ALDH7A1). The corresponding between the log of standard abundance of the spot of interest (y-axis) for the two different cell lines (x-axis) on the four replicates are also shown in lower-left panel. Representative 2-DE DIGE gels images are shown on the upper-right panel. The spots of interest corresponding to the ALDH7A1 protein are circled and the spot number match those listed in Table1. The 2D and 1D Western blot results are shown below. Protein lysates from DU145R80 and DU145 cell lines, obtained after 48 h of cell culture, were immunostained with anti-ALDH7A1 antibody. For 1D Western blot CDK4 ensured equal loading of sample in each lane. The quantization of the bands was obtained using the software ImageQuantTL and the value relative to each band normalized on loading control is reported.

Figure 2. Validation by 1-D Western blot of protein identified as differentially expressed in the 2-DE DIGE/MS analysis

In the middle of each validation images 1D Western blot experiment was showed. Protein lysate from DU145R80 and DU145 were immunostained with anti-LAMIN A/C (A); anti-LAMIN B2 (B); anti-AXNA1 (C); anti-FLNA (D), anti-PSMA6 (E) and anti-eEF1γ antibodies (F). CDK4 or α-tubulin immunoblotting ensured equal loading of sample in each lane. The quantization of the bands was obtained using the software ImageQuantTL. Below each image the value relative to each band normalized on loading control is reported. On the left of each images the 2-DE DIGE quantification for spots of interest is reported in a 3D view that shows significant difference in protein expression. On the right the corresponding between log of standard abundance of the spot of interest (y-axis) for the two different cell lines (x-axis) on the four replicates are shown.

Figure 3. Morphology of DU145 and DU145R80 cells and cellular localization of FLMNA and F-actin

(A) Representative images of DU145 and DU14580 cells grown on glass slides to semi-confluence and then analysed by phase contrast microscopy; or (B) by confocal microscopy after double staining for FLMNA and F-actin. Original magnifications: 200x (A) or 630x (B). Scale bars: 50 μm (A) or 10 μm (B).

Figure 4. Involvement of αv integrin in cell invasion ability of DU145R80 cells expressing higher levels of uPAR and αv as compared to DU145 cells

(A) Western blot analysis of uPAR and αv integrin proteins evaluated after 48 h of cell culture; (B) representative images of DU145 and DU14580 cells grown on glass slides and analyzed by confocal microscopy after double staining for αv and uPAR; or (C) uPAR and F-actin. Original magnifications: 630x. Scale bars:10 μm. (D) Cell invasion of DU145 and DU145R80 cells toward 10% FBS, with/without 1:500 anti-αv or anti-Akt monoclonal antibodies, the last used as antibody negative control. The extent of invasion is expressed as a percentage of the basal cell invasion assessed in the absence of chemo-attractants, considered as 100% (None). Data represent the mean ± SD of three experiments in duplicate. *Statistical significance calculated against the positive control with p = 0.0003.

Figure 5. Visual representation of the principal network generated by Ingenuity Pathway Analysis (IPA)

The network included 6 of the 7 validated proteins with direct and indirect relations (ANXA1, FLMNA, PSMA6, ALDH7A1, LMNA, LMNB2) connected with both αv (ITGAV) and uPAR (PLAUR) proteins and localized to a specific cell compartment (all colored in orange). Network proteins are visualized by proper symbols, which specify the functional nature of protein. Each node represents a protein and its direct (represented by solid lines) and indirect (represented by dotted lines) association with other proteins. Proteins with no background color were undetected in the study but have been inserted by IPA to produce a highly connected network.