Conceived and designed the experiments: DK EH MC FHS. Performed the experiments: DK YL SA SS OH. Analyzed the data: DK WC LH YL FHS. Contributed reagents/materials/analysis tools: EH WC MC IP SS OH CH NG DC WAS MM FHS. Wrote the paper: DK WC LH FHS.
The authors have declared that no competing interests exist.
The emergence of castrate-resistant prostate cancer (CRPC) contributes to the high mortality of patients diagnosed with prostate cancer (PCa), which in part could be attributed to the existence and the emergence of cancer stem cells (CSCs). Recent studies have shown that deregulated expression of microRNAs (miRNAs) contributes to the initiation and progression of PCa. Among several known miRNAs, let-7 family appears to play a key role in the recurrence and progression of PCa by regulating CSCs; however, the mechanism by which let-7 family contributes to PCa aggressiveness is unclear. Enhancer of Zeste homolog 2 (EZH2), a putative target of let-7 family, was demonstrated to control stem cell function. In this study, we found loss of let-7 family with corresponding over-expression of EZH2 in human PCa tissue specimens, especially in higher Gleason grade tumors. Overexpression of let-7 by transfection of let-7 precursors decreased EZH2 expression and repressed clonogenic ability and sphere-forming capacity of PCa cells, which was consistent with inhibition of EZH2 3′UTR luciferase activity. We also found that the treatment of PCa cells with BR-DIM (formulated DIM: 3,3′-diindolylmethane by Bio Response, Boulder, CO, abbreviated as BR-DIM) up-regulated let-7 and down-regulated EZH2 expression, consistent with inhibition of self-renewal and clonogenic capacity. Moreover, BR-DIM intervention in our on-going phase II clinical trial in patients prior to radical prostatectomy showed upregulation of let-7 consistent with down-regulation of EZH2 expression in PCa tissue specimens after BR-DIM intervention. These results suggest that the loss of let-7 mediated increased expression of EZH2 contributes to PCa aggressiveness, which could be attenuated by BR-DIM treatment, and thus BR-DIM is likely to have clinical impact.
Prostate cancer (PCa) is the second leading cause of cancer death in men in the United States killing over 32,050 men in 2010
Radiation therapy and systemic therapy especially androgen deprivation therapy (ADT) are considered as reasonable adjuvant therapeutic options. Patients in the high-risk category have a recurrence rate of greater than 50% within their lifetime, which is unacceptable. However, one of the concerns in offering adjuvant therapy to all patients in the high risk group is that although greater than 50% of patients do recur, there are 38–50% of patients who do not
Since tumor recurrence and metastasis contribute to the high mortality, studies have suggested that the aggressiveness of PCa could be tightly linked with the acquisition of cancer stem cells (CSC) or cancer stem-like cells (CSLCs) characteristics. Emerging evidence suggests that deregulated expression of many microRNAs (miRNAs) including the let-7 family contributes to cancer progression and recurrence
Interestingly, let-7 family members have been demonstrated to regulate the self-renewal capacity of breast cancer cells
Enhancer of Zeste homolog 2 (EZH2) is one of the targets of the let-7 family of miRNAs, and that the expression of EZH2 is strongly associated with molecular features of both normal stem cells and CSCs or CSLCs. EZH2 is a histone-lysine N-methyltransferase, a subunit of polycomb repressive complex 2 (PRC2). The polycomb group of proteins are known to be involved in the regulation of gene repression through chromatin modifications, which is essential for the maintenance of the embryonic and adult stem cells
Previous pre-clinical studies from our laboratory have shown that indole-3-carbinol (I3C) found in cruciferous vegetables and its
LNCaP, C4-2B, and CWR22RV1 were maintained in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 10 µmol/L Hepes, 50 units/ml Penicillin and 50 µg/ml Streptomycin. Stable cell lines over-expressing PDGF-D and empty vector pcDNA3 were generated by transfection of PC3 cells with the pcDNA3-PDGF-D:His or corresponding empty vector pcDNA3 Neo as previously described and referred to as PC3 Neo, or PC3 PDGF-D cells
Antibody against EZH2 was purchased from BD Biosciences (Bedford, MA). Antibody to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was purchased from Affinity BioReagents (Golden, CO). Goat anti-mouse IgG (H+L)-HRP conjugate were obtained from Bio-Rad (Reinach, BL). BR-DIM, a formulated DIM with higher bioavailability, was kindly provided by Dr. Michael Zeligs (abbreviated as BR-DIM or B-DIM; Bio Response, Boulder, CO) and was dissolved in DMSO to make 50 mmol/L stock solutions and stored at −20°C in multiple aliquots for
Patient tissues were collected after receiving the approval from Wayne State University Institutional Review Board (IRB). This clinical protocol is classified as exempt #4 categories under the NIH policy because such studies are retrospective studies using archival sample. The IRB waived the need for consent for use of the archive samples, which was consistent with NIH guidelines, and the samples were analyzed anonymously. PCa tissue specimens from our on-going clinical trial of BR-DIM (B-DIM) intervention prior to radical prostatectomy were also obtained after receiving approval from Wayne State University Institutional Review Board (IRB) and informed consents were obtained from all study subjects. Because this study is a conventional clinical trial, the IRB process required written consenting from patients prior to accrual into the clinical trial, which is routinely coordinated through the clinical trial office (CTO). The approval through IRB implicates complying with consenting and accrual without which patients cannot be enrolled into the study, and thus our study fulfilled all the required compliance guidelines from the CTO and the IRB. The local clinical trial protocol number is 2007-128, which can be found in the NIH clinical trials.gov. (
After obtaining institutional review board approval, retrospective archival pre-treatment PCa tissues and matched adjacent normal tissues were obtained from Biospecimen Core of Karmanos Cancer Institute (KCI) collected from patients who underwent radical prostatectomy from 2004–2010 at KCI. We also obtained PCa tissue specimens from our on-going clinical trial of BR-DIM (B-DIM) intervention prior to radical prostatectomy of newly diagnosed PCa patients from 2009–2011 at KCI and Henry Ford Health System (HFHS), Detroit, Michigan. Formalin-fixed paraffin-embedded (FFPE) tissues were cut for miRNA and mRNA analysis. Patients' clinical characteristics were obtained from the hospital database including race as shown in
Characteristics | Patients who underwent RP (N = 129) | Patients who took BR-DIM prior to surgery (N = 11) |
Age (years), median (range) | 59 (45–78) | 59.5 (47–64) |
Gleason score | ||
6 | 44 (34.1%) | 3 (27.3%) |
7 | 52 (40.3%) | 6 (55.5%) |
≥8 | 33 (25.6%) | 2 (18.2%) |
PSA |
||
ng/ml, median (range) | 6.5 (1.1–153) | 8.15 (3.2–29.14) |
Race | ||
African American | 78 (60.5%) | 5 (45.5%) |
European American | 51 (39.5%) | 4 (36.4%) |
Other | 0 (0%) | 2 (18.1%) |
Pre-intervention PSA was available from 88 of the 129 patients who underwent RP and from 10 of the 11 patients who underwent BR-DIM intervention.
Data for Gleason score and PSA levels were reported previously (Kong
C4-2B, PC3 PDGF-D, and PC3 PDGF-D cells transfected with the let-7 family for 24 h were collected after trypsinization, and re-suspended in the complete medium. Single cell suspensions were plated in regular 10 cm in diameter Petri dishes at the colony density of 2,000 cells per dish. After 2–3 weeks of incubation with changing fresh media every 3–4 days, colonies were stained with crystal violet for an additional 10 min, and washed with 1× PBS. The colonies were photographed.
C4-2B cells were harvested and suspended in culture medium. Soft agar assay was performed as previously described by our laboratory
Single cell suspensions of C4-2B and PC3 PDGF-D cells were plated in ultra low adherent wells of 6-well plates (Corning, Lowell, MA) at 2000 cells/well in DMEM/F12 (Invitrogen) containing 1× B27 and N2 (Invitrogen). Single cell status was confirmed under microscope. Fresh medium with 10 or 25 µM was added every 3–4 days. After one week, numbers of prostaspheres were counted under a microscope and data were presented as sphere numbers per 1000 cells seeded. Prostaspheres were photographed (40×) under a phase contrast microscope.
Total cell lysates were obtained by lysing the cells in RIPA buffer. Protein concentration was determined using BCA protein assay (Pierce, Rockford, IL). Western blotting was performed as previously described
Cells were transfected with 40 nmol/L of let-7 precursors or miRNA precursors negative control#1 (Ambion, Austin, TX) using DharmaFECT3 transfection reagent (DHARMACON, Lafayette, CO). After 1 day of transfection, the cells were trypsonized, and re-suspended in the complete medium. Single cell suspensions were confirmed under a microscope for clonogenic assay. In addition, after 3 days of transfection, the cell lysates were prepared for Western blot analysis.
PC3 PDGF-D cells with lower levels of let-7 were seeded at a density of 6×103 cells/well in a 96-well plate and incubated for 24 h. The cells were co-transfected with EZH2 3′UTR luciferase plasmid (Origene, Rockville, MD) or Renilla luciferase plasmid and control miRNA, let-7a, let-7b, let-7c, and let-7d precursors using DharmaFECT duo transfection reagent (DHARMACON, Lafayette, CO). After 48 h of incubation, luciferase activity was assayed using Steady–Glo Luciferase Assay System (Promega). The Renilla luciferase activity was used as a control for transfection efficiency.
For determining mRNA levels, the total RNA from cells was isolated using the RNeasy Mini Kit (Qiagen) and the DNA was removed using an RNase-free NDase kit (Qiagen). One microgram of RNA was reverse transcribed into cDNA using a High Capacity RNA-to-cDNA Kit (Applied biosystems, Fostor, CA) according to the manufacturer's instruction. PCR was performed as previously described
miRNA microarray as well as data analysis were conducted following procedures as previously described by Kong et al
All data is MIAME compliant and that the raw data has been deposited in a MIAME compliant database. GEO accession number is GSE34310.
Experiments presented in the figures for cell line studies are representative of three or more independent repetitions. The data are presented as the mean and standard deviation (SD) in the bar charts. The miRNA and mRNA data from patient tissue specimen was log transformed before analysis. Comparisons of the continuous variables between two independent groups were made using the Wilcoxon rank sum test. For paired related groups (i.e. Normal tissues and paired G6 tumor tissues), the Wilcoxon signed rank test was used. Spearman correlations were used to describe the strength of linear relationship between two variables. Statistical test of significance of the correlation was based on approximated
To determine the levels of the let-7 family in PCa tissue specimens, we collected pre-treatment PCa tissues and matched adjacent normal tissue specimens (used as control for comparison with tumor tissues). The results from miRNA expression by mircroarray expression profiling showed that all the members of the let-7 family (miR-98 was undetectable) decreased in the PCa tissues compared to adjacent normal tissue specimens (
(A) Microarray profiling was done for assessing the expression of miRNA using total RNA extracted from three PCa tissue specimens and matched adjacent normal prostate tissues. The results showed that let-7a, let-7b, let-7c and let-7d was highly expressed in prostate tissues and their expression was lost in human PCa tissue specimens (* p<0.05, ** p<0.01). (B) The results from real time-RT-PCR confirmed that the levels of let-7b, let-7c and let-7d were significantly down-regulated in tumors with higher Gleason grade. (7a: let-7a; N: normal; TG6: tumor tissues from patients with Gleason grade 6. n = 39 for normal, n = 44 for TG6, n = 52 for TG7, n = 33 for TG8, 9). (C) A significant up-regulation of the expression of EZH2 was observed in PCa tissue specimens with Gleason grade 7 (n = 46) and higher (n = 33), but not in PCa specimens with Gleason grade 6 (n = 39). (D) EZH2 levels were inversely correlated with let-7b and let-7c expressions in tumor specimens with Gleason grade 7.
Since the expression of the let-7 family was lost in PCa tissues of patients with higher Gleason grade, it raises a question of how the let-7 family could regulate PCa aggressiveness. We have searched targets of the let-7 family using TargetScan software and we found that EZH2 could be regulated by the let-7 family because there is a specific binding site in the 3′UTR of EZH2 mRNA. Thus, we have assessed the expression of EZH2 mRNA in PCa tissues. Our results showed that EZH2 expression was increased in PCa tissue specimens with Gleason grade 7 and higher compared with normal tissue control (
We examined the expression of EZH2 in PCa cell lines and immortalized prostate epithelial cell lines, and found that EZH2 was expressed in prostate cancer cells at relatively higher levels compared to immortalized prostate epithelial cell lines: PZ-HPV-7 and RWPE-1 cells (
(A) Expression of EZH2 was found to be higher in PCa cell lines compared with immortalized prostate epithelial cell lines: PZ-HPV-7 and RWPE-1 (upper panel) and transfection of let-7 precursors inhibited EZH2 expression in PC3 and PC3 PDGF-D cells 3 days after transfection (middle and lower panel). (B) let-7 family members repressed EZH2 3′UTR luciferase activity in PC3 PDGF-D cells (lower levels of let-7 family in these cells) co-transfected with let-7 and EZH2 3′UTR luciferase plasmid. (C) let-7 binding sites in the 3′UTR of EZH2 mRNA were shown. (D) Transfection of let-7 precursors significantly reduced clonogenic growth capacity of PC3 PDGF-D cells (Con: control, 7a: pre-let-7a, ** p<0.01).
In the present study, we found that BR-DIM treatment increased the expression of let-7 family in several PCa cell lines including LNCaP, C4-2B and PC3 cells (
(A) Total RNA was isolated from LNCaP, C4-2B and PC3 cells treated with 25 µM BR-DIM for 24 h and the results from real time RT-PCR showing that the expression of let-7 was increased following BR-DIM treatment compared to untreated control (c: DMSO control). (B) Levels of EZH2 mRNA were repressed by BR-DIM treatment in a dose dependent meaner. (C and D) The cell lysates were prepared from cells treated with BR-DIM for 48 h and Western blot showing the protein levels of EZH2, which was down-regulated by BR-DIM treatment (PC3 PD: PC3 PDGF-D cells, *, p<0.05; **, p<0.01).
RNA was obtained from BR-DIM intervention clinical trial samples where BR-DIM was given to patients for 2–4 weeks prior to surgery. RNA was also obtained from formalin-fixed paraffin-embedded (FFPE) tissue specimens of PCa patients with matched tumor Gleason grade, tumor stage and patient age as control group. The expression of miRNAs and mRNA was assessed using real time RT-PCR. Relative miRNA and mRNA levels were normalized to RNU1A1 and beta-actin, respectively. (A) BR-DIM intervention led to the increased trend in levels of Let-7a expression. (B and C) let-7b, let-7c and let-7d were significantly up-regulated by BR-DIM intervention. (D) EZH2 expression was down-regulated by BR-DIM intervention.
To test whether BR-DIM could regulate the self-renewal capacity and clonogenic ability of PCa cells, we performed sphere-forming assays and found that the treatment of C4-2B and PC3 PDGF-D cells with 10 or 25 µM BR-DIM markedly reduced the number and the size of prostaspheres compared to untreated control (DMSO control) (
(A) Single cell suspensions of C4-2B and PC3 PDGF-D cells were plated in ultra low adherent wells of 6-well plate at 2000 cells/well in DMEM/F12 supplemented with B27 and N2 and with 10, 25 µM BR-DIM and incubated for 6 days. Prostasphere numbers were reduced by BR-DIM treatment (upper panel). Prostaspheres were photographed and the results showed that 10 and 25 µM BR-DIM significant reduced the size of prostaspheres (lower panel). (B) C4-2B and PC3 PDGF-D cells were seeded in 100 mm dishes at 2000 cells/dish, after 24 h incubation, the cells were treated with 10 or 25 µM BR-DIM for 72 h and then the culture medium was changed with fresh media without BR-DIM for every 3 days. After 2 weeks, the colonies were stained and photographed. (C and D) Soft agar assay showed that BR-DIM treatment reduced the size (left panel) and the numbers of colonies of C4-2B cells (right panel; **, p<0.01).
Although it is known that the let-7 family is associated with maintenance of stem cell signature, which is believed to be strongly linked with cancer recurrence, the mechanism by which let-7 family regulates the stem cell signatures is unknown. In this study, we found that the expression of the let-7 family was lost in PCa tissue specimens with Gleason grade 7 or higher but not in patients with Gleason grade 6 tumors. These results were consistent with corresponding increased expression of EZH2, which appears to be a target of the let-7 family. Our results suggest that the loss of expression of let-7 with a consequent over-expression of EZH2 could be associated with PCa aggressiveness. Moreover, the results from 3′UTR of the EZH2 luciferase assay and Western blot analysis further confirmed that let-7 could repress EZH2 expression by binding to 3′UTR element of EZH2 mRNA.
Recent studies have shown that miR-101 negatively regulates the expression of EZH2
EZH2 is a component of the PRC2, which mediates chromatin-based gene silencing through trimethylation of lysine 27 on histone H3. PRC2 also plays a critical role in the maintenance of the embryonic and adult stem cells
In the current study, we found that the levels of EZH2 were increased in PCa tissue specimens, especially in patients with higher Gleason grade tumors and were also highly expressed in PCa cell lines, suggesting that the over-expression of EZH2 is associated with PCa aggressiveness. Therefore, finding novel approaches by which one could re-express the lost miRNAs such as let-7 family with consequent down-regulation of EZH2 could become a newer avenue for the prevention of PCa and/or treatment of aggressive PCa. To that end, we tested the effects of BR-DIM (B-DIM), which is a “natural agent”-derived compound (formulated DIM: 3,3′-diindolylmethane by BioResponse, Boulder, CO). The results of pharmacokinetic studies of BR-DIM in mice have been reported
In summary, we found that BR-DIM up-regulated the expression of the let-7 family and consequently down-regulated the expression of EZH2 not only in PCa cell lines but also in human PCa tissue specimens from our on-going phase II clinical trial. These results suggest that BR-DIM could serve as a novel agent for the inhibition of PCa progression and recurrence.
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Special thanks for Dr Gerold Bepler (President and CEO of Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan) and Dr. Anthony Shields (Professor & Associate Center Director for Clinical Research), Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan for supporting BR-DIM clinical trial. Special thanks to Dr. Hyeong-Reh Choi Kim who kindly provided the PC3 Neo and PC3 PDGF-D cell lines (Department of Pathology, Wayne State University School of Medicine, Detroit, MI).