Deep Gagan, Jain Anil K, Ramteke Anand, Ting Harold, Vijendra Kavitha C, Gangar Subhash C, Agarwal Chapla, Agarwal Rajesh
Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, San Diego, USA.
Mol Cancer. 2014 Feb 24;13:37. doi: 10.1186/1476-4598-13-37.
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.
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.
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.
Together, these results suggest that E-cadherin loss promotes SNAI1 expression that controls the aggressiveness of PCA cells.
为了设计针对前列腺癌（PCA）的新型靶向预防和治疗策略，有必要对前列腺癌发生进行更好的分子层面理解，前列腺癌是男性死亡的主要原因之一。在此，我们研究了两种上皮-间质转化（EMT）调节因子，即黏着连接蛋白E-钙黏蛋白及其转录抑制因子SNAI1，在调节PCA细胞侵袭性中的作用。
在细胞培养的MTT和克隆形成试验以及体内裸鼠异种移植模型中，比较了稳定敲低E-钙黏蛋白的人前列腺癌PC3细胞（ShEC-PC3）和相应对照细胞（Sh-PC3）的生长速率。通过前列腺球形成试验分析ShEC-PC3和Sh-PC3细胞的干性。采用蛋白质免疫印迹法和免疫组织化学（IHC）研究E-钙黏蛋白和SNAI1敲低后的蛋白质表达变化。运用小干扰RNA（siRNA）技术敲低ShEC-PC3细胞中SNAI1蛋白的表达。
与Sh-PC3细胞相比，ShEC-PC3细胞在细胞培养和无胸腺裸鼠中均表现出更高的增殖速率。ShEC-PC3细胞还形成了更大且数量显著更多的前列腺球，表明随着E-钙黏蛋白的敲低，干细胞样群体增加。此外，与Sh-PC3前列腺球相比，在有血清和贴壁的情况下，ShEC-PC3前列腺球解体产生了更大的增殖细胞团。免疫印迹/免疫组化分析表明，在细胞培养和异种移植组织中，E-钙黏蛋白敲低会增加干性调节因子/生物标志物（CD44、切割的Notch1和Egr-1）以及EMT相关因子（波形蛋白、pSrc-tyr416、整合素β3、β-连环蛋白和NF-κB）的表达。在ShEC-PC3细胞中，几种与骨转移相关分子即CXCR4、uPA、RANKL和RunX2的表达也增加了。重要的是，我们观察到ShEC-PC3细胞的细胞质和细胞核组分、前列腺球和异种移植组织中SNAI1表达显著增加。此外，通过特异性siRNA敲低SNAI1可强烈抑制ShEC-PC3细胞的前列腺球形成、克隆形成能力和侵袭性，并降低pSrc-tyr416、总Src和CD44的水平。对RWPE-1、WPE1-NA22、WPE1-NB14和DU-145细胞的特性分析进一步证实，低E-钙黏蛋白与更高的SNAI1表达和前列腺球形成相关。
总之，这些结果表明E-钙黏蛋白的缺失促进了SNAI1的表达，而SNAI1控制着PCA细胞的侵袭性。
