Deciphering the Molecular Machinery-Influence of sE-Cadherin on Tumorigenic Traits of Prostate Cancer Cells

Abstract

The serum level of soluble (s)E-cadherin is elevated in several malignancies, including prostate cancer (PCa). This study was designed to investigate the effects of sE-cadherin on the behavior of PCa cells in vitro, with the aim of identifying a potential therapeutic target. Growth as well as adhesive and motile behavior were evaluated in PC3, DU-145, and LNCaP cells. Flow cytometry was used to assess cell cycle phases and the surface expression of CD44 variants as well as α and β integrins. Confocal microscopy was utilized to visualize the distribution of CD44 variants within the cells. Western blot was applied to investigate expression of α3 and β1 integrins as well as cytoskeletal and adhesion proteins. Cell growth was significantly inhibited after exposure to 5 µg/mL sE-cadherin and was accompanied by a G0/G1-phase arrest. Adhesion of cells to collagen and fibronectin was mitigated, while motility was augmented. CD44v4, v5, and v7 expression was elevated while α3 and β1 integrins were attenuated. Blocking integrin α3 reduced cell growth and adhesion to collagen but increased motility. sE-cadherin therefore appears to foster invasive tumor cell behavior, and targeting it might serve as a novel and innovative concept to treat advanced PCa.

Keywords: cell growth; metastasis; migration; prostate cancer; sE-cadherin.

Figure 1

(A): Influence of sE-cadherin on cell growth of PC3, DU145, and LNCaP cells. Cell number evaluated after 24 (100%), 48, and 72 h by MTT assay. Error bars indicate standard deviation, * = p ≤ 0.05, n = 6. (B): Influence of 5 µg/mL sE-cadherin on proportionate G0/G1, S, and G2/M-phases of the cell cycle in PC3, DU145, and LNCaP cells over the course of 24 h (n = 3; * indicates significant difference to untreated controls). (C): Influence of sE-cadherin (5.0 µg/mL) on cell growth of BPH-1 and PNT-2 cells. Cell number evaluated after 24 (100%) and 72 h by MTT assay. Error bars indicate standard deviation, * = p ≤ 0.05, n = 3.

Figure 2

(A): Influence of sE-cadherin on adhesion of PC3, DU145, and LNCaP cells to plastic dishes, collagen- or fibronectin-coated plates. Five separate fields of 0.25 mm² were counted at 200 × magnification (means ± SD, n = 6); * indicates significant difference to untreated controls. (B): Influence of sE-cadherin on chemotaxis of PC3 or DU145 towards a serum gradient after 24 h. 5 separate fields of 0.25 mm² were counted at 200 × magnification (means ± SD, n = 6); * indicates significant difference to untreated controls. (C): Influence of sE-cadherin on chemotaxis of BPH-1 and PNT-2 cells towards a serum gradient after 24 h (means ± SD, n = 3); * indicates significant difference to untreated controls.

Figure 2

(A): Influence of sE-cadherin on adhesion of PC3, DU145, and LNCaP cells to plastic dishes, collagen- or fibronectin-coated plates. Five separate fields of 0.25 mm² were counted at 200 × magnification (means ± SD, n = 6); * indicates significant difference to untreated controls. (B): Influence of sE-cadherin on chemotaxis of PC3 or DU145 towards a serum gradient after 24 h. 5 separate fields of 0.25 mm² were counted at 200 × magnification (means ± SD, n = 6); * indicates significant difference to untreated controls. (C): Influence of sE-cadherin on chemotaxis of BPH-1 and PNT-2 cells towards a serum gradient after 24 h (means ± SD, n = 3); * indicates significant difference to untreated controls.

Figure 3

Wound closure analyzed after sE-cadherin exposure and compared to untreated controls. One representative figure and the mean wound closure of treated versus untreated PC3 or DU145 are shown (n = 3). Scale bar indicates 600 µm. * indicates significant difference to the untreated controls.

Figure 4

(A): CD44 variants v4, v5, and v7 on PC3, DU145, and LNCaP cells recorded by flow cytometry. Single representative of three separate experiments. Solid line: specific fluorescence; dashed line: isotype IgG1-APC. (B): CD44 variants v4, v5, and v7 on PC3 cells following sE-cadherin exposure for 24, 48 and 72 h. Means related to untreated controls (100%); MFU = mean fluorescence units; error bars indicate SD; * and ** = significant difference to corresponding control with p < 0.05 and p < 0.01, respectively, n = 3. (C): Distribution of CD44v4, v5, and v7 on PC3 cells after 72h sE-cadherin exposure. Pictures were taken by CLSM with a Plan-Neofluar × 63/1.3 oil immersion objective.

Figure 5

(A): Surface expression of α and β integrins on PC3 cells. Solid line: specific fluorescence, dashed line: IgG1-PE or IgG2a-PE. The abscissa shows the relative logarithmic distribution of the relative fluorescence intensity of α2, α3, α5, α6, β1, and β4. The ordinate shows cell number. 10,000 cells were counted. The figure is representative for n = 6. Scatter plots are shown in Supplement S1. (B): Influence of sE-cadherin (5 µg/mL) on the integrin expression profile of PC3 cells. The untreated control is set to 100%. Values are means ± SD, n = 4; * indicates significant difference to controls. (C): Western blot of α and β integrins, ILK, FAK, pFAK, and pAkt in PC3 depending on the influence of sE-cadherin (5 µg/mL). Protein levels were measured 24 h after treatment. All bands are representative of n = 3. β-actin served as loading control and is representatively shown once. 50 µg were used per sample. (D): Adhesion to collagen and chemotaxis of PC3 cells after blockade of integrins α3 or β1. The untreated control is set to 100%. 5 separate fields of 0.25 mm² were counted at 200× magnification (means ± SD, n = 3); * indicates significant difference to controls. (E): PC3 cell growth after blockade of integrins α3 or β1. Cell number evaluated after 24, 48, and 72 h by MTT assay. Error bars indicate standard deviation, * = p ≤ 0.05. (F): Western blot of cytoskeleton-related proteins in PC3 following sE-cadherin exposure (0.5, 5 µg/mL). Protein levels were measured after 24 h of treatment. All bands are representative of n = 3. β-actin served as loading control and is representatively shown once. 50 µg were used per sample.

Figure 6

(A): Influence of sE-cadherin on cell growth of PC3 and DU145 cells treated with the sE-cadherin specific antibody HECD-1 (versus untreated). Cell number evaluated after 24 (100%) and 72 h by MTT assay. Error bars indicate standard deviation, * = p ≤ 0.05, n = 3. (B): Influence of sE-cadherin on chemotaxis of PC3 and DU145 cells treated with the sE-cadherin specific antibody HECD-1 (versus untreated). Endpoints after 24 h (means ± SD, n = 3); * indicates significant difference to controls. Ctrl: treatment with cell culture medium, HECD-1: treatment with the antibody alone, HECD-1+sE-Cad: treatment with HECD-1 and sE-cadherin (5 µg/mL).