Mechanistically, the co-operation between RAS activation and loss of PTEN led to the acquisition of stem/ progenitor subpopulation with mesenchymal characteristics, and these cells were highly metastatic upon orthotopic transplantation [21]. Low expression of ETS transcription factor ESE3/EHF was demonstrated to be associated with increased biochemical recurrence and reduced overall survival of PCa patients after radical prostatectomy, which was found to be consistent with induction of EMT and acquisition of CSLC signatures that led to tumor-initiating and metastatic properties of PCa cells [22]. Androgen-deprivation therapy (ADT) is commonly used for the treatment of metastatic PCa, and Jennbacken et al. showed that ADT could enhance N-cadherin expression, a hallmark for EMT, which was associated with increased of metastasis [23]. Increased expression of N-cadherin in non-metastatic, androgen-dependent PCa in animal model led to castration resistance, increased invasion and metastasis, which was consistent with findings showing elevated expression of N-cadherin in primary and metastatic tumors of patients with castration resistant PCa (CRPC) [24]. Moreover, a recent study has shown that ADT could induce EMT and leads to the acquisition of CSLC signatures in both normal mouse prostate tissue and human LuCaP35 prostate tumor explants. Similar changes in mesenchymal features were also observed in prostate tumors from patients treated with ADT [25]. These results suggest that the induction of EMT and CSLC signatures leads to therapeutic resistance and leads to prostate cancer progression. In the present study, we found that treatment of PCa cells with TSA or SAHA induced EMT phenotype mediated through upregulation of transcription factors and mesenchymal markers via promoting acetylation of histone 3 on proximal promoters of specific genes. Moreover, TSA treatment also led to further increase in the expression of stem cell markers in EMT phenotypic PCa cells. These results were consistent with increased cell motility of TSA or SAHA treated PCa cells. our results provide a mechanistic basis for the disappointing outcome of HDACIs in the treatment of solid tumors, and further suggest that reversal of EMT phenotype or CSLC characteristics by novel approach prior to HDACIs could become a rational strategy for the treatment of solid tumors especially PCa.
Research Reagents and Antibodies
Trichostatin A (TSA) was obtained from Sigma Aldrich (St. Louis, MO) and Suberoylanilide hydroxamic acid (SAHA) was purchased from Aton Pharma, Inc, (Lawrenceville, NJ). Antibodies against acety-histone H3 (Lys9), HDAC1, HDAC2, HDAC3, HDAC4, HDAC6, Slug, Sox2 and Nanog were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against Ncadherin and vimentin were purchased from BD Biosciences (Bedford, MA) and Abcam (Cambridge MA), respectively. Antibodies against ZEB1, Fibronectin and E-cadherin were obtained from Santa Cruz (Santa Cruz, CA). Goat anti-rabbit IgG (H+L)-HRP conjugate and goat anti-mouse IgG (H+L)-HRP conjugate were purchased from Bio-Rad (Reinach, BL). Antibody to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was obtained from Affinity BioReagents (Golden, CO). Alex Fluro 594 goat anti-rabbit IgG or Alex Fluro 594 goat anti-mouse IgG and Alexa Fluor 594 phalloidin for F-actin staining were purchased from Invitrogen (Carlsbad, CA).
Materials and Methods Cell Lines and Culture Condition
LNCaP and PC3 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 10 mmol/L Hepes, 50 units/ml Penicillin and 50 mg/ml Streptomycin. Stable cell lines overexpressing PDGF-D (referred to PC3 PDGF-D) was generated as previously described [19] and cultured in RPMI 1640 (Invitrogen) supplemented with 5% fetal bovine serum (FBS), 2 mmol/LFigure 1. HDACIs led to the induction of EMT phenotype. (A and B) PC3 cells treated with TSA and SAHA for 24 h at indicated doses. The photomicrographs of PC3 cells treated with TSA and SAHA exhibited a fibroblastic-type phenotype, while cells treated with DMAO control displayed rounded epithelial cell morphology (original magnification, 6100). (C) PC3 cells were seeded in the chamber. After 24 h incubation, cells were treated
with 400 nM TSA or 5 mM SAHA for another 24 h. The results from immuno-fluorescence staining for vimentin and ZEB1 (Red) indicated that treatment of PC3 cells with TSA and SAHA increased the expression of vimentin and ZEB1 (middle and right panel) compared to control cells (left panel). Phalloidin staining showed F-actin (Red) reorganization in TSA and SAHA treated PC3 cells. Nuclear DNA was stained with DAPI (Blue, original magnification, 6200).
Cell Migration Assay
Cell migration assay was performed using 24 well Transwell Permeable Supports with 8 mm pores (Corning, Lowell, MA) according to the instruction supplied by the manufacturer. Briefly, PC3 cells treated with TSA, SAHA or DMSO control for 6 hours were collected and suspended in serum free RPMI 1640 medium and 16105 cells in each well were seeded into the Transwell inserts. Bottom wells was filled with 0.5 ml of media containing 10% FBS. After 24 h of incubation, migrated cells were determined as previously described [9].
free NDase kit (Qiagen). High Capacity RNA-to-cDNA Kit (Applied biosystems, Fostor, CA) was used to reverse transcribed one microgram of RNA into cDNA according to the manufacturer’s instruction. Real time PCR was performed using specific primers to quantify gene expression by using SYBRH Green RTPCR Reagents (Applied biosystems). The relative amount of mRNA was normalized to the expression of GAPDH. Primer sequences are shown in the table 1.
