Abstract 3426: E-cadherin regulates ‘stemness’ and ‘bone metastasis’ of prostate cancer cells
Authors: Deep Gagan , Vijendra Kavitha C. , Jain Anil K. , Gangar Subhash C. , Agarwal Chapla , Agarwal Rajesh 
Published: August 2012

Prostate cancer (PCA) is the most common non-cutaneous cancer and is the second leading cause of cancer-related deaths in American men. Bone metastasis of PCA cells is main cause of high mortality; therefore, a better understanding of the metastatic events might help to develop newer strategies that would lower the PCA burden. Recent studies suggest that cancer stem cell population not only sustains the growth and heterogeneity of the primary tumors but also metastasis. Here, we analyzed the role of E-cadherin, a transmembrane protein, in regulating the ‘stemness’ and ‘bone metastasis’ of PCA cells. First, we transfected human PCA PC3 cells with shRNA specific for E-cadherin or random shRNA, and selected cells with stable knock-down of E-cadherin (ShEC-PC3 cells) and respective control cells (Sh-PC3 cells). ShEC-PC3 cells showed mesenchymal phenotype, shorter doubling time, enhanced invasiveness and clonogenicity. Importantly, compared to Sh-PC3 cells, ShEC-PC3 cells also formed significantly higher number and bigger sized prostaspheres suggesting an increase in the stem cell population with E-cadherin knock-down. Cell sorting for established stem cell phenotypic markers (CD44hiCD24lo) confirmed a two-fold increase in stem cell population along with a nine fold higher CD44hi/CD44lo population in ShEC-PC3 cells compared to Sh-PC3 cells. Western blot analysis showed that E-cadherin knock-down increases the expression of stemness markers (CD44, Notch1 and Egr-1) and EMT markers (Vimentin, phospho-Src, beta-catenin and NF-kB). Importantly, we observed a remarkable increase in SNAI1 expression in cytoplasmic and nuclear fractions with E-cadherin knock-down. We also observed a remarkably higher SNAI1 expression in the prostaspheres formed by ShEC-PC3 cells, and SNAI1 knock-down by specific siRNA completely inhibited the prostaspheres formed by ShEC-PC3 cells, further validating the critical role of SNAI1 in regulating the self-renewal capability of stem cells in ShEC-PC3 cells. 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 molecules that are important in PCA bone metastasis namely CXCR4, uPA, RANKL and Runx2 with E-cadherin knock-down. Overall, these results suggest the important role of E-cadherin in regulating the stemness and bone metastasis of PCA cells, which have significant translational implications as E-cadherin is mostly silenced epigenetically in cancer cells. We conclude that it might be possible to prevent metastasis in PCA patients with localized disease through re-activating E-cadherin expression in PCA cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3426. doi:1538-7445.AM2012-3426