SNAI1 is critical for the aggressiveness of prostate cancer cells with low E-cadherin

Abstract

Background: A better molecular understanding of prostate carcinogenesis is warranted to devise novel targeted preventive and therapeutic strategies against prostate cancer (PCA), a major cause of mortality among men. Here, we examined the role of two epithelial-to-mesenchymal transition (EMT) regulators, the adherens junction protein E-cadherin and its transcriptional repressor SNAI1, in regulating the aggressiveness of PCA cells.

Methods: The growth rate of human prostate carcinoma PC3 cells with stable knock-down of E-cadherin (ShEC-PC3) and respective control cells (Sh-PC3) was compared in MTT and clonogenic assays in cell culture and in nude mouse xenograft model in vivo. Stemness of ShEC-PC3 and Sh-PC3 cells was analyzed in prostasphere assay. Western blotting and immunohistochemistry (IHC) were used to study protein expression changes following E-cadherin and SNAI1 knock-down. Small interfering RNA (siRNA) technique was employed to knock- down SNAI1 protein expression in ShEC-PC3 cells.

Results: ShEC-PC3 cells exerted higher proliferation rate both in cell culture and in athymic nude mice compared to Sh-PC3 cells. ShEC-PC3 cells also formed larger and a significantly higher number of prostaspheres suggesting an increase in the stem cell-like population with E-cadherin knock-down. Also, ShEC-PC3 prostaspheres disintegration, in the presence of serum and attachment, generated a bigger mass of proliferating cells as compared to Sh-PC3 prostaspheres. Immunoblotting/IHC analyses showed that E-cadherin knock-down increases the expression of regulators/biomarkers for stemness (CD44, cleaved Notch1 and Egr-1) and EMT (Vimentin, pSrc-tyr416, Integrin β3, β-catenin, and NF-κB) in cell culture and xenograft tissues. The expression of several bone metastasis related molecules namely CXCR4, uPA, RANKL and RunX2 was also increased in ShEC-PC3 cells. Importantly, we observed a remarkable increase in SNAI1 expression in cytoplasmic and nuclear fractions, prostaspheres and xenograft tissues of ShEC-PC3 cells. Furthermore, SNAI1 knock-down by specific siRNA strongly inhibited the prostasphere formation, clonogenicity and invasiveness, and decreased the level of pSrc-tyr416, total Src and CD44 in ShEC-PC3 cells. Characterization of RWPE-1, WPE1-NA22, WPE1-NB14 and DU-145 cells further confirmed that low E-cadherin is associated with higher SNAI1 expression and prostasphere formation.

Conclusions: Together, these results suggest that E-cadherin loss promotes SNAI1 expression that controls the aggressiveness of PCA cells.

Figure 1

E-cadherin knock-down increases the proliferation of human PCA PC3 cells. (A) Multiplication rate of Sh-PC3 and ShEC-PC3 cells was determined by MTT assay. Data shown is mean ± SD of 12 samples. (B) Clone formation by Sh-PC3 and ShEC-PC3 cells was examined in a clonogenic assay as detailed in the methods. Number of clones with more than 50 cells were counted and presented in a bar diagram. Data shown is mean ± SD of 6 samples. (C-D) Sh-PC3 and ShEC-PC3 cells were injected subcutaneously in athymic nude mice, and average tumor volume (mean ± SEM) as a function of time is presented. Tumor tissues were analyzed for proliferation biomarkers (PCNA and Ki-67) by IHC. Percentage of PCNA and Ki-67 positive cells was calculated by counting the number of positive stained cells (brown stained) and the total number of cells at five arbitrarily selected fields from each tumor at 400x magnification. The data shown in the bar diagrams is the mean±SEM of 7–10 samples. *, p ≤ 0.001; #, p ≤ 0.01; $, p ≤ 0.05.

Figure 2

E-cadherin knock-down enhances the stemness of human PCA PC3 cells. (A-B) Sh-PC3 or ShEC-PC3 cells were plated on 6 well Corning ultra-low attachment plates in DMEM/F-12(Ham) media containing supplements B27 and N2. Prostasphere formation was measured after 5 days. Thereafter, prostaspheres were collected and plated on normal cell culture plate with or without serum and monitored for 5 days. Representative pictures are shown for prostaspheres’ state at day 1, day 3 and day 5. Data shown is mean ± SD of 3–6 samples. * p ≤ 0.001.

Figure 3

E-cadherin knock-down increases the expression of stemness, EMT, and bone metastasis biomarkers in PC3 cells. (A-B) Sh-PC3 or ShEC-PC3 cells were collected at similar confluency and total cell lysates were prepared and analyzed for the protein expression of E-cadherin, CD44, cleaved Notch-1, Egr-1, Vimentin, Integrin β3, N-cadherin, OB-cadherin, CXCR4, uPA, RANKL, and RunX2. Tubulin and β-actin were used as loading controls.

Figure 4

Expression of stemness, EMT, and bone metastasis biomarkers in Sh-PC3 and ShEC-PC3 xenograft tissues. Sh-PC3 and ShEC-PC3 xenograft tissues were analyzed for the expression of E-cadherin, CD44, Notch1, pSrc-tyr416, β-catenin, CXCR4 and RANKL by IHC as detailed in the methods. Immunoreactivity was analyzed in 5 random areas for each tumor tissue and was scored as 0+ (no staining), 1+ (weak staining), 2+ (moderate staining), 3+ (strong staining), 4+ (very strong staining). IHC scores (as mean ± SEM) are shown as bar diagram of 5–10 samples.

Figure 5

Effect of E-cadherin knock-down on the expression of SNAI1 and other transcriptional factors. (A) Sh-PC3 and ShEC-PC3 cells were collected at similar confluency and nuclear/cytoplasmic fractions were prepared and analyzed for SNAI1, β-catenin, and p65 expression by Western blotting. Tubulin and histone H1 were used as loading control for cytoplasmic and nuclear fractions respectively. (B) Sh-PC3 and ShEC-PC3 prostaspheres were collected following centrifugation and cell lysates were prepared and analyzed for SNAI1 expression by Western blotting. (C) Sh-PC3 and ShEC-PC3 xenograft tissues were analyzed for the expression of SNAI1 by IHC. Immunoreactivity score was analyzed in 5 random areas for each tumor tissue and was scored as 0+ (no staining), 1+ (weak staining), 2+ (moderate staining), 3+ (strong staining), 4+ (very strong staining). Percentage of SNAI1 positive cells was calculated by counting the number of positive stained cells (brown stained) and the total number of cells at five arbitrarily selected fields from each tumor at 400x magnification. The data shown in the bar diagrams is the mean±SEM of 7–10 samples. *, p ≤ 0.001.

Figure 6

SNAI1 knock-down inhibited the prostaspheres formation, clonogenicity, and invasiveness of ShEC-PC3 cells. (A-C) SNAI1 expression was knocked down using SNAI1 specific siRNA. Mock and si-SNAI1 transfected cells were collected and analyzed for prostasphere formation, clonogenicity and invasiveness. Representative photomicrographs are shown at 100x. (D) Total cell lysates were prepared from mock and si-SNAI1 transfected ShEC-PC3 cells, and analyzed for SNAI1, pSrc-tyr416, Src, and CD44 by Western blotting. Tubulin was used as loading control. *, p ≤ 0.001; #, p ≤ 0.01.

Figure 7

Low E-cadherin is associated with high SNAI1 and prostasphere formation. (A-B) E-cadherin and SNAI1 expression was analyzed in RWPE-1, WPE1-NA22, WPE1-NB14, and DU-145 cells via immunoblotting and confocal microscopy methods. Representative confocal pictures are shown (at 1500x) where Alexa Fluor 555-red is for E-cadherin, Alexa Fluor 488-green is for SNAI1, while DAPI-blue stains nuclei. (C) RWPE-1, WPE1-NA22, WPE1-NB14, and DU-145 cells were plated on 6 well Corning ultra-low attachment plates in DMEM/F-12(Ham) media containing supplements B27 and N2. Prostasphere formation was measured after 8 days. Representative prostasphere pictures are shown at 100x. Data shown is mean ± SEM of 3 samples. * p ≤ 0.001 (compared to RWPE-1 prostasphere number).