EMMPRIN regulates cytoskeleton reorganization and cell adhesion in prostate cancer

Abstract

Background: Proteins on cell surface play important roles during cancer progression and metastasis via their ability to mediate cell-to-cell interactions and navigate the communication between cells and the microenvironment.

Methods: In this study a targeted proteomic analysis was conducted to identify the differential expression of cell surface proteins in human benign (BPH-1) versus malignant (LNCaP and PC-3) prostate epithelial cells. We identified EMMPRIN (extracellular matrix metalloproteinase inducer) as a key candidate and shRNA functional approaches were subsequently applied to determine the role of EMMPRIN in prostate cancer cell adhesion, migration, invasion as well as cytoskeleton organization.

Results: EMMPRIN was found to be highly expressed on the surface of prostate cancer cells compared to BPH-1 cells, consistent with a correlation between elevated EMMPRIN and metastasis found in other tumors. No significant changes in cell proliferation, cell cycle progression, or apoptosis were detected in EMMPRIN knockdown cells compared to the scramble controls. Furthermore, EMMPRIN silencing markedly decreased the ability of PC-3 cells to form filopodia, a critical feature of invasive behavior, while it increased expression of cell-cell adhesion and gap junction proteins.

Conclusions: Our results suggest that EMMPRIN regulates cell adhesion, invasion, and cytoskeleton reorganization in prostate cancer cells. This study identifies a new function for EMMPRIN as a contributor to prostate cancer cell-cell communication and cytoskeleton changes towards metastatic spread, and suggests its potential value as a marker of prostate cancer progression to metastasis.

Figure 1. EMMPRIN expression and alternative splicing in prostate cancer cells

Panel A, EMMPRIN expression levels in total cell lysates of BPH-1, LNCaP and PC-3 cells. Bands of different motilities are likely due to glycosylation. Both LNCaP and PC-3 cell lines exhibited elevated levels of highly glycosylated EMMPRIN compared to BPH-1 cells. Panel B. LNCaP and PC-3 prostate cancer cells exhibited a significantly higher amount of membrane anchored EMMPRIN compared to BPH-1 cells. Membrane fractions (30µg protein) were subjected to Western blotting. (C). EMMPRIN transcripts were analyzed by RT-PCR and electrophoresis. Splicing variants 2, 3 and 4 were confirmed by DNA sequencing as Band #3, #1 and #2, respectively.

Figure 2. Suppression of EMMPRIN expression in transient and stable shRNA prostate cancer transfected cells

PC-3 cells were transfected with three EMMPRIN shRNA plasmids or scrambled control and subjected to Western blot analysis to establish EMPPPRIN protein expression levels (major band detected at 50 kDa). Panel A, after transient transfection a considerable reduction in EMMPRIN expression was detected in GFP-positive cells transfected with three different shRNA plasmids, while no changes in EMMPRIN levels were observed in the GFP negative control cells. Panel B; stably transfected cells with GFP marker were obtained after selection with G418 (300µg/ml). Substantial reduction in EMMPRIN levels was demonstrated in the individual stable shRNA clones.

Figure 3. Consequences of EMMPRIN silencing on prostate cancer cell adhesion to ECM, migration and invasion

EMMPRIN shRNA transfectant PC-3 cells were seeded on fibronectin-coated (panel A) or laminin (panel B) plates for 30mins and attached cells were fixed and counted. Panel C, Cell migration was assessed by wounding the cell monolayer and determining the number of cells migrating to the wounded area after 24hrs. Panel D, The effect of EMMPRIN silencing on prostate cancer cell invasion was determined using the matrigel assay as described in “Materials and Methods”. The average values from three independent experiments performed in triplicate are shown. Numerical values are expressed as percentage of control Sh scramble cells. Statistical significance is reached at p<0.01.

Figure 4. EMMPRIN loss reduces filopodia formation in prostate cancer cells

Panel A. Cells were plated on fibronectin-coated glass coverslips and after spreading (24hrs), they were exposed to F-actin staining, and subsequently visualized under confocal microscopy, Arrow heads indicate individual filopdia. The insert represents a zoom-in of region indicated in doted boxed areas. Panel B, The average number of filopodia in each cell was quantified in scramble control and the three shEMMPRIN clones, 98i, 277i and 776i cells. Filopodia from at least 20 cells were counted and representative average values are shown. Approximately 20 cells/field and 10 random fields were examined for each cell line; error bars indicate average values from these measurements (mean) ± standard error of mean (SEM) gathered from three independent experiments. Statistical difference is considered significant at p<0.01.

Figure 5. Effect of EMMPRIN on cell aggregation and tight junction proteins

Panel A. Cell aggregation increased with reduced EMMPRIN expression in prostate cancer cell EMMPRIN Sh clones. Panel B, Western blotting indicating expression of gap junction proteins in EMMPRIN silenced PC-3 cells. Panel C, Potential role of EMMPRIN in prostate cancer metastasis. EMMPRIN can stimulate production of MMPs, leading to reduced cell adhesion to ECM. Alternatively, EMMPRIN may directly promote the metastatic potential of prostate cancer cells by enhancing migration and invasion through cytoskeleton reorganization and impairing cell-microenvironment interactions.