CtBP1 gene expression data were procured from cDNA microarray analysis [20
]. To measure the CtBP1 transcript levels, total RNA was isolated from prostate cell lines and prostate tissue samples using the RNeasy Mini Kit (Qiagen, Valencia, CA). Quantitative polymerase chain reaction (qPCR) was performed as described [21
]. All primers were synthesized by Integrated DNA Technologies, Coralville, IA. PCR reactions were performed in triplicates. Primer sequences used in the present study include CtBP1: F, TCACAGGCCGGATCCCAGACAG and R, GGTACCTATAGGCAGCCCCATTGAGC and F, CCGTCAAGCAGATGAGACAA and R, GGCTAAAGCTGAAGGGTTCC; E cadherin: F, GGAGGAGAGCGGTGGTCAAA and R, TGTGCAGCTGGCTCAAGTCAA; ARHGDIB: F, ACAGGACTGGGGTGAAAGTG and R, GAGCCTCCTCAACTGGAGTG; LCN2: F, CAAGGAGCTGACTTCGGAAC and R, TACACTGGTCGATTGGGACA.
For immunoblot analysis, 10 µg of normal and prostate cancer tissues as well as prostate cancer cell line lysates was boiled in sample buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride membrane (GE Healthcare, Piscataway, NJ). The membrane was incubated for 1 hour in blocking buffer (TBS, 0.1% Tween, 5% nonfat dry milk) and incubated overnight at 4°C with respective primary antibodies, and signals were visualized after incubating with secondary antibody conjugated with HRP. Densitometric scan of the immunoblot was performed using ImageJ. The following antibodies and dilutions were used for the immunoblots: anti-CtBP1 (1:2000 in blocking buffer, BD Biosciences [San Jose, CA], Cat. No. 612042,), anti-LCN2 (1:10,000, R&D Systems [Minneapolis, MN], Cat. No. AF1757), phospho-H2AX (1:2000, Millipore [Billerica, MA], Cat. No. 16-202A), anti-β-actin mouse monoclonal antibody (1:20,000, Sigma [St Louis, MO], Cat. No. A5316-500ul), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1:5000, Abcam [Cambridge, MA], Cat. No. ab8245), and β-tubulin (1:2000, Santa Cruz Biotechnology [Santa Cruz, CA], sc-9104).
Benign and prostate cancer tissues were obtained from the radical prostatectomy series at the University of Michigan and from the Rapid Autopsy Program, both part of the Michigan Prostate SPORE Tissue Core. Institutional Review Board approval was obtained to procure and analyze the tissues used in this study. Immunohistochemistry was carried out using standard biotin-avidin complex to evaluate CtBP1 expression using mouse monoclonal antibody against CtBP1 (BD Biosciences) as well as a rabbit polyclonal antibody [22
Small interfering RNA (siRNA) duplexes for RNA interference of CtBP1 was purchased from Dharmacon, Lafayette, CO (Thermo Scientific, Cat. No. LQ-008609-00-0002). Short hairpin RNA (shRNA) constructs were generated using pGreen-puro vector for two of the most efficient siRNA duplexes by SBI (System Biosciences, Mountain View, CA). Lentivirus for the stable knockdown of CtBP1 was generated by the University of Michigan Vector Core. To perform the siRNA knockdown, we plated prostate cancer cell lines DU145, PC3, and LNCaP at 2 x 105 cells per well in a 6-well plate for immunoblot analysis and cell proliferation analysis and at 1.5 x 103 cells per well in a 96-well plate for Cell Titer-Glo (Promega, Madison, WI) proliferation assays. Twelve hours after plating, the cells were transfected with siRNA duplex, using Oligofectamine (Invitrogen, Carlsbad, CA). A second identical transfection was performed 24 hours later. Sixty-four hours after the first transfection, the cells were harvested for RNA isolation or lysed for immunoblot analysis. For knockdown of LCN2 (NGAL), specific siRNA (Dharmacon, Cat. Nos J-003679-07 and J-003679-09) were used in DU145 and PC3 stable CtBP1 knockdown cells.
Gene Expression Analysis of CtBP1 Knockdown Cells
RNA isolated from shRNA knockdown DU145, PC3, and LNCaP as well as nontarget control cells were used for gene expression profiling. Expression profiling was performed using the Agilent Whole Human Genome Oligo Microarray (Agilent, Santa Clara, CA) according to the manufacturer's protocol. Statistical analysis of gene expression array was performed. Microarray probes were identified as differential on CtBP1 knockdown if the mean log2
(Cy5/Cy3) ratio across cell lines was significantly different from zero as measured by one-sample two-sided Student's t
tests, using a P
-value cutoff of .05. The list of differentially expressed genes was additionally filtered such that the mean log2
(Cy5/Cy3) ratio exceeded log2
(2.5) in absolute value. The resulting list of 155 genes are shown in as a heat map and listed in Table W1
. Statistical analysis was performed using R (www.r-project.org
), version 2.15.0.
Figure 3 CtBP1 knockdown reactivate tumor suppressors in prostate cancer. (A) Heat map of genes that are significantly altered by CtBP1 stable knockdown in LNCaP, PC3, and DU145 cells. Log2(Cy5/Cy3) ratios are shown for each expression array. Red and green represent (more ...)
Cell Proliferation Assays
For cell counts at 96 and 120 hours, the cells were treated with trypsin and replated in six-well dishes 64 hours after the first transfection. Stable knockdown of CtBP1 was performed using shRNA strategy using lentiviral construct with specific duplex sequences targeting CtBP1. DU145 and PC3 cell lines were used for stable CtBP1 knockdown. LCN2 and ARHGDIB were knocked down in stable CtBP1 knockdown PC3 and DU145 cells. Sequence information of all the siRNA used in this study has been given in the Supplementary materials. Cell proliferation was determined using ATPase assay kit (Promega) as described [23
]. Additionally, cell proliferation was measured by cell counting. For this, 10,000 cells/well (DU145 and PC3) were seeded in 24-well plates (n
= 3), and cells were harvested and counted at specified time points by Coulter counter (Beckman Coulter, Fullerton, CA).
Basement Membrane Matrix Invasion Assay
For invasion assays, control shRNA stable cells or CtBP1 stable knockdown cells as well as wild-type DU145 and PC3 cells were used. Equal numbers of the indicated cells were seeded onto the basement membrane matrix (BD Biosciences) present in the insert of a 24-well culture plate. RPMI medium supplemented with 10% FBS was added to the lower chamber as a chemoattractant. After 48 hours, non-invading cells and extracellular matrix were removed with a cotton swab. Invaded cells were stained with crystal violet and photographed. The inserts were treated with 10% acetic acid, and absorbance was measured at 560 nm.
Chromatin Immunoprecipitation Assay
The ChIP assays were performed as described [24
]. Briefly, DU145 cells at 60% confluency were cross-linked with 1% formaldehyde for 10 minutes, followed by quenching with 0.125 M glycine for 5 minutes at room temperature. Cells were lysed and sonicated to fragment the chromatin to an average size of 500 bp. This was followed by overnight incubation with the antibodies and protein A or G magnetic beads. Cross-links were reversed by incubating chromatin at 62°C for 2 hours, and DNA was isolated. Tri-Methyl-Histone H3 (Lys4) antibody was obtained from Cell Signaling Technology, Danvers, MA (Cat. No. 9751S). Rabbit IgG (Diagenode [Denville, NJ], Cat. No. kch-504-250) was used as a control. Purified DNA was analyzed by qPCR to determine fold enrichment relative to input DNA. The primer sequences for the promoters analyzed are provided as follows. Primers used for ChIP assay are LCN2: F, TGCAGAAATCTTGCCAAGTG and R, GGGATCTAGGGTGGGTTGAT; ARHGDIB: F, CCCAGGGTTTCCTCTTCAA and R, TCAGTGCTTCACGTCTCTGTC; GAPDH: F, TACTAGCGGTTTTACGGGCG and R, TCGAACAGGAGGAGCAGAGAGCGA.
Clonogenic Survival Assay
Clonogenic survival assays were performed using standard techniques [25
]. Cells were subcultured at clonal density immediately after irradiation. Cell survival curves were fitted using the linear quadratic equation, and the mean inactivation dose was calculated according to the method of Fertil and Malaise [26
Chicken Embryo Chorioallantoic Membrane Assay
Chicken embryo chorioallantoic membrane (CAM) assay was performed as described previously [27
]. To measure metastasis, we harvested lungs on day 18 of embryonic growth and analyzed for the presence of tumor cells by quantitative human Alu-specific PCR. Genomic DNA from lungs was prepared using the Puregene DNA purification system (Qiagen) and was quantified as previously described [28
]. For measuring tumor growth, embryos were sacrificed on day 18 and extraembryonic xenografts were excised and weighed.
Prostate Tumor Xenograft Model
All procedures involving mice were approved by the University Committee on Use and Care of Animals at the University of Michigan and conform to their relevant regulatory standards. To evaluate the role of CtBP1 in tumor formation, we propagated stable CtBP1 knockdown DU145 pools, single clone, and vector control cells and inoculated 5 x 106 cells subcutaneously into the dorsal flank of 5-week-old male nude athymic BALB/c nu/nu mice (n = 10 for each group; Charles River Laboratory, Wilmington, MA). Tumor size was measured weekly, and tumor volumes were calculated using the formula (π/6) (L x W2), where L = length of tumor and W = width.
Murine Tumor Metastasis Models and Bioluminescent Imaging
Experimental procedures were approved by the University Committee on Use and Care of Animals. Male CB17 severe combined immunodeficient mice (4–6 weeks of age) were bred in-house. CtBP1 knockdown PC3-Luc cell pools or nontargeting shRNA-transduced control cells were used for the metastasis model. Animals underwent intracardiac injections of 200,000 cells and were imaged once weekly by bioluminescent imaging (BLI) using a Xenogen IVIS 200 System at the University of Michigan's Center for Molecular Imaging as previously described [29
]. Mice were injected with luciferin (100 µl at 40 mg/ml) by intraperitoneal injections. Ventral images were acquired 13 minutes after injection under 1.5% isoflurane anesthesia. Tumor burden of each animal was determined with Living Image software using regions of interest encompassing the entire animal. Animals with no tumor take were defined as those with bioluminescent flux less than 1.151 x 106
p/s at week 8, and these animals were removed from subsequent analysis. Statistical significance was determined using one-sided two-sample t
tests. Three animals closest in bioluminescent flux to each group's mean reading at week 8 were selected as representative images.