title: 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.
authors.affiliation: Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, San Diego, USA. Gagan.deep@ucdenver.edu.
authors.forename: Gagan
authors.initials: G
authors.lastname: Deep
authors.forename: Anil K
authors.initials: AK
authors.lastname: Jain
authors.forename: Anand
authors.initials: A
authors.lastname: Ramteke
authors.forename: Harold
authors.initials: H
authors.lastname: Ting
authors.forename: Kavitha C
authors.initials: KC
authors.lastname: Vijendra
authors.forename: Subhash C
authors.initials: SC
authors.lastname: Gangar
authors.forename: Chapla
authors.initials: C
authors.lastname: Agarwal
authors.forename: Rajesh
authors.initials: R
authors.lastname: Agarwal
mesh_terms: D000818:Animals; D015153:Blotting, Western; D015820:Cadherins / Q000378:metabolism*; D045744:Cell Line, Tumor; D058750:Epithelial-Mesenchymal Transition / Q000502:physiology*; D055785:Gene Knockdown Techniques; D064593:Heterografts; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008819:Mice, Nude; D018613:Microscopy, Confocal; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology*; D034741:RNA, Small Interfering; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
chemical_list: D015820:Cadherins; D034741:RNA, Small Interfering; C000604407:SNAI1 protein, human; C000604429:Snai1 protein, mouse; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors