Src-Beta Catenin-Snai1 Axis Is Critical For The Stemness And Bone Metastasis Of E-Cadherin-Lacking Prostate Cancer Cells

Bone metastasis of prostate cancer (PCA) cells is the main cause of high mortality and morbidity in patients. Recent studies have suggested that cancer stem cells not only sustain the growth and heterogeneity of primary tumors but also play a role in metastasis. Therefore, a better molecular understanding of metastatic cancer stem cells could be exploited towards targeting bone metastasis. Here, we analyzed the role of E-cadherin and Src-beta catenin-SNAI1 axis in regulating the stemness and bone metastasis of PCA cells. Human PCA PC3 cells were transfected with shRNA specific for E-cadherin or random shRNA, and selected for stable knock-down of E-cadherin (ShEC-PC3) or respective control cells (Sh-PC3). MTT, clonogenic and xenograft assays were performed to compare the in vitro and in vivo proliferation potential of Sh-PC3 and ShEC-PC3 cells. Stem cell population was characterized by FACS and prostasphere formation. Orthotopic xenograft model and optical imaging tools were employed to study metastasis. Immunoblotting and immunohistochemistry (IHC) methods were used to analyze proliferation, stemness, EMT, and osteomimicry biomarkers. Our results showed that ShEC-PC3 cells possessed mesenchymal phenotype, enhanced invasiveness and clonogenicity as well as higher growth rate both in cell culture and xenografts. Importantly, compared to Sh-PC3 cells, ShEC-PC3 cells formed significantly higher number and bigger sized prostaspheres suggesting an increase in the stem cell population with E-cadherin knock-down. Also, the ShEC-PC3 prostaspheres disintegration in the presence of serum generated bigger mass of attached proliferating cells compared to Sh-PC3. Cell sorting for established PCA stem cell phenotypic markers (CD44highCD24low) confirmed a two-fold increase in stem cell population along with a nine fold higher CD44high/CD44low population in ShEC-PC3 cells compared to Sh-PC3 cells. Immunoblotting and IHC analyses showed that E-cadherin knock-down increased the expression of stemness markers (CD44, Notch1 and Egr-1) as well as EMT and invasivness markers (Vimentin, pSrc-tyr416, beta catenin, NF-kappaB, integrin alpha 5 and beta 3) in both cell culture and xenograft tissues. Next, to define the role of E-cadherin in PCA metastasis, we orthotopically injected Sh-PC3 or ShEC-PC3 cells in the prostate of nude mice and after 4 weeks, imaged the mice using IR-dye (EGF optical probe). Mice injected with ShEC-PC3 cells showed remarkable metastasis to distant organs mainly to the bones, while mice injected with Sh-PC3 cells showed only prostate localized growth. We also observed increased expression of several osteomimicry molecules namely CXCR4, uPA, RANKL and Runx2 in ShEC-PC3 cells in cell culture as well as xenograft tissues. Importantly, we observed a remarkable increase in SNAI1 expression in both cytoplasmic and nuclear fractions from prostaspheres and xenograft tissues of ShEC-PC3 cells. Furthermore, the SNAI1 knock-down by specific siRNA completely inhibited the prostasphere formation, clonogenicity and invasiveness of ShEC-PC3 cells. The SNAI1 inhibition also decreased the level of pSrc-tyr416 and beta catenin in ShEC-PC3 cells suggesting SNAI1 role in regulating the expression of these molecules. Interestingly, inhibition of Src (by dasatinib) or beta catenin (by FH535) also decreased the SNAI1 expression. These results clearly showed that E-cadherin knock-down in PCA cells promotes the formation of Src-beta catenin-SNAI1 axis which then controls the stemness, invasiveness, and metastasis of PCA cells. Therefore, E-cadherin activators and/or Src-beta catenin-SNAI1 repressors could be useful in targeting the PCA stemness and bone metastasis, and lowering the PCA burden. Citation Format: Gagan Deep, Anil K. Jain, Samiha Mateen, Kavitha C. Vijendra, Subhash C. Gangar, Chapla Agarwal, Rajesh Agarwal. Src-beta catenin-SNAI1 axis is critical for the stemness and bone metastasis of E-cadherin-lacking prostate cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C73.