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[Cancer Research 65, 4762-4768, June 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

CCAAT/Enhancer-Binding Protein {delta}: A Molecular Target of 1,25-Dihydroxyvitamin D3 in Androgen-Responsive Prostate Cancer LNCaP Cells

Takayuki Ikezoe1,2, Sigal Gery1, Dong Yin1, James O'Kelly1, Lise Binderup3, Nathan Lemp1, Hirokuni Taguchi2 and H. Phillip Koeffler1

1 Division of Hematology/Oncology, Cedars-Sinai Medical Center, University of California at Los Angeles School of Medicine, Los Angeles, California; 2 Department of Internal Medicine, Kochi Medical School, Kochi, Japan; and 3 Leo Pharmaceuticals, Ballerup, Denmark

Requests for reprints: Takayuki Ikezoe, Department of Internal Medicine, Kochi Medical School, Nankoku, Kochi 783-8505, Japan. Phone: 81-88-880-2345; Fax: 81-88-880-2348; E-mail: ikezoet{at}med.kochi-ms.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D3, inhibits the proliferation of prostate cancer cells. However, the molecular mechanisms by which 1,25(OH)2D3 inhibits the proliferation of these cells remain to be fully elucidated. In this study, we used microarray technology to identify target genes of 1,25(OH)2D3 in androgen-responsive prostate cancer LNCaP cells. 1,25(OH)2D3 up-regulated CCAAT/enhancer-binding protein {delta} (C/EBP{delta}) by ~5-fold in these cells. Knockdown of C/EBP{delta} expression by RNA interference showed that C/EBP{delta} is essential for the significant growth inhibition of LNCaP cells in response to 1,25(OH)2D3 treatment. Moreover, we found that 1,25(OH)2D3 induced C/EBP{delta} in other cancer cells, including the estrogen receptor (ER)–expressing MCF-7 and T47D breast cancer cells that are sensitive to the growth inhibitory effects of 1,25(OH)2D3. On the other hand, 1,25(OH)2D3 was not able to induce C/EBP{delta} in either androgen receptor–negative PC-3 and DU145 or ER-negative breast cancer MDA-MB-231 cells that were relatively resistant to growth inhibition by 1,25(OH)2D3. Furthermore, forced expression of C/EBP{delta} in prostate cancer LNCaP as well as breast cancer MCF-7 and T47D cells dramatically reduced their clonal growth. Taken together, forced expression of C/EBP{delta} in cancer cells may be a promising therapeutic strategy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is a member of the seco-steroid hormone family, which controls calcium homeostasis (1). The effects of 1,25(OH)2D3 are mediated mainly via interaction with a specific nuclear vitamin D3 receptor (VDR), which forms heterodimers with retinoid X receptor and binds to the VDR response element, resulting in activation of target genes (1). 1,25(OH)2D3 induces growth arrest, differentiation, and apoptosis of a wide variety of cancer types, including those from prostate (2–5), breast (6, 7), as well as leukemia (8, 9). For example, 1,25(OH)2D3 induced G0-G1 cell cycle arrest in association with up-regulation of cyclin-dependent kinase inhibitor (CDKI) p21waf1 in androgen-responsive prostate cancer LNCaP cells (10). In addition, it induced apoptosis of LNCaP cells with down-regulation of the antiapoptotic protein Bcl-2 and Bcl-XL (11).

CCAAT/enhancer binding proteins (C/EBP) are a highly conserved family of leucine zipper type (bZIP) DNA-binding proteins which are composed of six isoforms including C/EBP-{alpha}, C/EBP-ß, C/EBP-{gamma}, C/EBP-{delta}, C/EBP-{varepsilon} as well as C/EBP homologous protein (12). All C/EBPs share conserved COOH-terminal regions that contain leucine zipper dimerization motifs adjacent to basic DNA-binding domains. Their NH2-terminal regions are more diverse and contain transcriptional activation domains (12). C/EBPs form homodimers and heterodimers with other C/EBP family members as well as other transcription factors such as Fos, cyclic AMP–responsive element binding protein/activating transcription factor, and nuclear factor {kappa}B (NF-{kappa}B; refs. 10, 13–15). C/EBPs are implicated in the regulation of growth and differentiation of a wide variety of cells such as hepatocytes, adipocytes, pneumocytes, as well as hematopoietic cells. For example, C/EBP{varepsilon} plays an important role in the differentiation of hematopoietic progenitor cells toward the granulocytic lineage; indeed, forced expression of C/EBP{varepsilon} in U937 leukemic myeloblasts can induce these cells to differentiate towards granulocytes (16). In addition, C/EBP{varepsilon} deletional mice have incomplete maturation of their granulocytes, and these cells lack specific granule proteins (17). Furthermore, we have recently found that C/EBP{varepsilon} interacted with cell cycle regulatory molecules, retinoblastoma and E2F during granulocytic differentiation in human and murine myeloid precursor cells (18). C/EBP{alpha} is also pivotal in terminal differentiation of granulocytes as well as pneumocytes. Targeted inactivation of C/EBP{alpha} in mice showed hyperproliferation of type II pneumocytes and abnormal alveolar structure, and histopathology of the liver displayed a structure resembling regenerative changes or hepatocellular carcinoma (19). Others and ourselves have identified mutations in the C/EBP{alpha} gene in individuals with acute myeloid leukemia, myelodysplastic syndrome and non–small cell lung cancer (NSCLC); and these mutations disrupt the normal function of C/EBP{alpha} (20, 21). Recently, expression of C/EBP{alpha} was shown down-regulated in NSCLC cells compared with normal lung tissues, and forced expression of the p42 isoform of C/EBP{alpha} reduced proliferation of NSCLC cells (22).

C/EBP{delta} is induced in murine mammary epithelial cells as their cell growth slows by either withdrawal of serum or exposure to oncostatin M, an interleukin 6–type cytokine (23–25). In addition, we have found that forced expression of C/EBP{delta} in KCL-22 myeloid blast cells (cell line derived from chronic myeloid leukemia in myeloid blast crisis) induced G0-G1 cell cycle arrest and granulocytic differentiation of these cells in association with up-regulation of p27KIP1 protein (26).

In this study, we used microarray technology to identify the 1,25(OH)2D3 target genes in androgen-responsive LNCaP cells. 1,25(OH)2D3 increased the level of C/EBP{delta} in LNCaP cells; and forced expression of C/EBP{delta} in these cells inhibited their clonal growth.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines. Cell lines established from prostate cancer (LNCaP, PC-3, and DU145) and breast cancer (MCF-7, T47D, and MDA-MB-231) were obtained from American Type Culture Collection (Rockville, MD). LNCaP, PC-3, T47D, and MDA-MB-231 cells were cultured in RPMI 1640 (Life Technologies, Grand Islands, NY) with 10% FCS. DU145 and MCF-7 cells were maintained in DMEM with 10% FCS.

Chemicals. 1,25(OH)2D3 was obtained from Hoffmann-La Roche (Nutley, NJ) and was dissolved in absolute ethanol at 10–3 mol/L as a stock solution, stored at –20°C, and protected from light. All-trans retinoic acid (ATRA) was obtained from Sigma (St. Louis, MO), dissolved in absolute ethanol as a stock concentration of 10–2 mol/L, stored at –20°C, and protected from light. Cycloheximide was obtained from Sigma, dissolved as a stock concentration of 10 mg/mL, and stored at –20°C.

Soft agar colony assay. Cells were cultured in a two-layer soft agar system for 14 days as previously described (9). Washed, single-cell suspension of cells were enumerated and plated into 24-well flat-bottomed plates with a total of 500 cells per well in a volume of 400 µL per well. The feeder layer was prepared with agar that had been equilibrated at 42°C. Before this step, 1,25(OH)2D3 was pipetted into the wells. After incubation, colonies were counted. Experiments were done thrice using triplicate plates per experimental point.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Cells (104/mL) were incubated with a variety of concentrations of 1,25(OH)2D3 (10–9 to 10–6 mol/L) for 6 days in 96-well plates (Flow Laboratories, Irvine, CA). After culture, cell number and viability were evaluated by measuring the mitochondrial-dependent conversion of the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma), to a colored formazan product. MTT (0.5 mg/mL in PBS) was added to each well and incubated for 4 hours at 37°C. The medium was then carefully aspirated, and DMSO (Burdick & Jackson, Muskegon, MI) was added to solubilize the colored formazan product. Absorbance was read at 540 nm on a scanning multiwell spectrophotometer (Bio-Rad Laboratories, Hercules, CA) after agitating the plates for 5 minutes on a shaker.

RNA extraction. LNCaP (5 x 105/mL) cells were plated on 100-mm plates. On the following day, the medium was replaced with fresh RPMI 1640 containing 10% FCS with either 1,25(OH)2D3 (10–7 mol/L) or control diluent. After 18 hours of incubation, total RNA was extracted as previously described using TRIzol (Life Technologies; ref. 27) followed by a purification step using the RNeasy cleanup kit (QIAGEN, Inc., Valencia, CA) according to the manufacturer's protocol. The RNA quality was confirmed on an agarose gel.

Oligonucleotide microarrays. The HuGeneFL Array (Affymetrix, Inc., Santa Clara, CA) provides gene expression data for 5,600 full-length human sequences. The set of matched samples [LNCaP cells treated either with or without 1,25(OH)2D3] was studied independently twice. The preparation and microarray processing was done per standard Affymetrix protocol as previously published (28). The raw data images were analyzed using the GeneChip Analysis Suite (Affymetrix), and the data for each microarray were normalized by global scaling to a target value of 2,500 as we have previously reported (27, 28). The average background noise was in the range of 500.

Real-time reverse transcription-PCR. One microgram of DNase I–treated RNA was reverse transcribed by using Moloney murine leukemia virus reverse transcriptase (Life Technologies), and 50 ng of the resulting cDNAs were used as templates for PCR. Real-time PCR was done using specific primers (sequences will be provided upon request), HotMaster Taq DNA Polymerase (Eppendorf, Hamburg, Germany) and SYBRGreen I (Molecular Probes, Eugene, OR). PCR conditions were as follows: 2 minutes at 94°C followed by 45 cycles of 94°C for 20 seconds, 60°C for 10 seconds, 65°C for 25 seconds, and fluorescence determination at the melting temperature of the product for 20 seconds. Specificity of PCR products was checked on agarose gel. All reactions were done in triplicates in an iCycler iQ system (Bio-Rad Laboratories). For each sample, the amount of the target gene and ß-actin (for internal control) was determined from a standard curve. The results are expressed in arbitrary units as a ratio of the target gene transcripts/ß-actin transcripts (each value represent the mean of three measurements of the sample).

Western blot analysis. Lysates were made by standard methods as previously described (29). Protein concentrations were quantitated using a Bio-Rad assay (Bio-Rad Laboratories). Proteins were resolved on a 4% to 15% SDS polyacrylamide gel, transferred to an immobilon polyvinylidene difuride membrane (Amersham Corp., Arlington Heights, IL), and probed sequentially with antibodies. Anti-C/EBP{delta} (Santa Cruz Biotechnology, Santa Cruz, CA), anti–ß-actin (Santa Cruz Biotechnology), and anti–glyceraldehyde-3-phosphate dehydrogenase (Research Diagnostics, Flanders, NJ) antibodies were used.

Small interference RNA. Primers were designed using the RNA interference oligo retriever web site (http://katahdin.cshl.org:9331/RNAi/html/rnai.html). The following small interfering (siRNA) sequence was used: 5'-GCTGTCGGCTGAGAACGAGAAGCTGCACC. Scrambled siRNA was designed by the same method for control. The siRNA primers together with the U6 promoter were cloned into pCR2.1 and confirmed by sequencing. LNCaP cells were cotransfected with either C/EBP{delta} siRNA or control siRNA along with pMSCVpuro vector (Clontech, Palo Alto, CA) and selected with puromycin. Equal numbers of surviving cells (3 x 103 cell per well) were plated in 96-well plates and 24 hours later, treated either with control diluent or with 1,25(OH)2D3 (10–7 mol/L). Cell proliferation was measured after 5 days using MTT assays (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol.

Colony formation assay. Cells were split evenly into 6-well plates. After growing to ~60% confluence, cells were transfected with either pcDNA3.1-C/EBP{delta} (26) or pcDNA3.1 empty vector (both contain the Neo-resistant gene). After 48 hours, medium was replaced with fresh medium containing G418. Two weeks later, the cells were stained with 0.1% crystal violet to assess colony formation. Colonies containing >40 cells were counted. This study was done twice in triplicate.

Statistical analysis. Statistical analysis was done by Student's t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of 1,25(OH)2D3 on clonogenic growth of prostate and breast cancer cells. The LNCaP, PC-3, and DU145 prostate cancer cells and MCF-7, T47D, and MDA-MB231 breast cancer cells were cloned in soft agar in the presence of various concentrations (10–10 to 10–7 mol/L) of 1,25(OH)2D3. Dose-response curves were drawn, and the effective dose that inhibited 50% colony formation (ED50) was determined. 1,25(OH)2D3 inhibited clonal proliferation of LNCaP, PC-3, MCF-7, and T47D cells in a dose-dependent manner (Fig. 1A and B) with an ED50 of 6 x 10–10, 1 x 10–8, 7 x 10–9, and 8 x 10–9 mol/L, respectively (Table 1). On the other hand, both DU145 and MDA-MB-123 cells were resistant to growth inhibition by 1,25(OH)2D3 (Fig. 1A and B). We also assessed the ability of 1,25(OH)2D3 to inhibit the proliferation of LNCaP cells by MTT assay on day 6 of culture; the effective dose that inhibited growth of LNCaP cells by 50% compared with diluent-treated cells was ~10–6 mol/L (figure not shown).



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  		Figure 1. Dose-response effects of 1,25(OH)2D3 on clonal proliferation of prostate (A) and breast (B) cancer cells. Results are expressed as a mean percentage of control plates containing no 1,25(OH)2D3. Point, mean of three independent experiments with triplicate dishes; bars, ±SD.

 

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  		Table 1. Inhibition of clonal proliferation of tumor cells by 1,25(OH)2D3

 
Microarray analysis of 1,25(OH)2D3-inducible genes in LNCaP cells. To do oligonucleotide array analysis, LNCaP cells were cultured either without or with 1,25(OH)2D3 (10–7 mol/L, 18 hours). Total RNA was extracted and subjected to microarray analysis using HuGeneFL Array. A short exposure duration (18 hours) was chosen to enhance the identification of direct target genes that are responsible for growth inhibition and differentiation induced by ligand-activated VDR. By using GeneChip Analysis Suite (Affymetrix), we sorted the genes that were called "present" in control LNCaP cells and were either up-regulated or down-regulated by ≥2-fold after treatment with 1,25(OH)2D3 in two independent experiments (Table 2). Six genes were up-regulated and three were down-regulated by 1,25(OH)2D3 in both experiments. The induction/repression of the genes identified by the microarray analysis was confirmed by quantitative real-time reverse transcription-PCR (RT-PCR; Table 2). In eight of the nine genes examined, the real-time RT-PCR data were consistent with the chip data, demonstrating that our microarray analysis was reliable.


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  		Table 2. Genes either induced or repressed after exposure of LNCaP cells to 1,25(OH)2D3

 
1,25(OH)2D3 induces C/EBP{delta} in LNCaP cells. The gene showing the highest fold induction following 1,25(OH)2D3 treatment was C/EBP{delta} (4.7-fold). To confirm further that 1,25(OH)2D3 induces C/EBP{delta} expression in LNCaP, we did Western blot analysis. As shown in Fig. 2A, the level of C/EBP{delta} protein was negligible in untreated LNCaP cells and treatment with 1,25(OH)2D3 (10–9 to 10–7 mol/L, 18 hours) markedly up-regulated its expression in a dose-dependent manner. ATRA binds to retinoic acid receptor (RAR) and induces growth arrest and differentiation of LNCaP cells (30); however, ATRA (10–6 mol/L, 18 hours) was not able to induce expression of C/EBP{delta} in LNCaP cells under similar culture conditions (Fig. 2B), suggesting that induction of C/EBP{delta} seems specific to ligand-activated VDR not RAR.



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  		Figure 2. 1,25(OH)2D3 induces C/EBP{delta} in LNCaP cells. A, Western blot analysis of C/EBP{delta} expression in LNCaP cells treated either with control diluent or 1,25(OH)2D3 for 18 hours at the indicated concentrations. B, Western blot analysis of C/EBP{delta} expression in LNCaP cells treated with either control diluent, 1,25(OH)2D3 (10–7 mol/L), or ATRA (10–6 mol/L) for 18 hours. The membranes were probed sequentially with anti-C/EBP{delta} and either ß-actin (A) or GAPDH (B) antibodies. C, real-time PCR analysis of RNA from LNCaP cells treated either with control diluent or 1,25(OH)2D3 (10–7 mol/L) for 1, 4, and 6 hours either in the absence (–CHX) or presence (+CHX) of cycloheximide (10 µg/mL). The results are expressed in arbitrary units as a ratio of C/EBP{delta} transcripts/ß-actin transcripts. Columns, means of triplicate samples; bars, ±SD.

 
Experiments were done to determine if the 1,25(OH)2D3-dependent increase in C/EBP{delta} mRNA levels is a primary response or requires ongoing protein synthesis. LNCaP cells were treated with cycloheximide for 30 minutes followed by 1,25(OH)2D3 (10–7 mol/L) for 1, 4, and 6 hours. Real-time RT-PCR showed that the expression profile of C/EBP{delta} following 1,25(OH)2D3 treatment was not perturbed by pretreating the cells with cycloheximide, indicating that de novo protein synthesis was not required for 1,25(OH)2D3 to exert its effect (Fig. 2C).

Suppression of C/EBP{delta} reduces 1,25(OH)2D3-induced growth inhibition in LNCaP cells. To evaluate the role of C/EBP{delta} in 1,25(OH)2D3-mediated growth inhibition, we applied siRNA to silence C/EBP{delta} expression. Inhibition of C/EBP{delta} expression by the C/EBP{delta} siRNA construct was shown in 293T cells cotransfected with a C/EBP{delta} expression vector (pcDNA3.1-C/EBP{delta}) and either control siRNA (scrambled siRNA) or C/EBP{delta} siRNA (Fig. 3A). The C/EBP{delta} siRNA also inhibited 1,25(OH)2D3-dependent induction of C/EBP{delta} protein in LNCaP cells (Fig. 3B). To test the effect of C/EBP{delta} suppression on 1,25(OH)2D3-induced growth inhibition, LNCaP cells were transfected with either control siRNA or C/EBP{delta} siRNA and selected for 2 days with puromycin. Equal numbers of surviving cells were grown either in the presence or absence of 1,25(OH)2D3 (10–7 mol/L) for 5 days and their growth rate was determine by MTT assays. As expected, 1,25(OH)2D3 treatment resulted in a marked inhibition (64%) in cell proliferation in the control-transfected cells (Fig. 3C). On the other hand, the responsiveness to 1,25(OH)2D3, although not completely extinguished, was significantly reduced (34%) in LNCaP cells transfected with the C/EBP{delta} siRNA (Fig. 3C). These results indicate that C/EBP{delta} plays a major role in 1,25(OH)2D3-mediated growth inhibition of LNCaP cells.



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  		Figure 3. Suppression of C/EBP{delta} by siRNA reduces the antiproliferative effects of 1,25(OH)2D3 in LNCaP cells. Inhibition of C/EBP{delta} by siRNA in 293T (A) and LNCaP (B) cells. 293T cells were cotransfected with pcDNA3.1-C/EBP{delta} and either control (scrambled) siRNA or C/EBP{delta} siRNA. LNCaP cells were transfected with either control siRNA or C/EBP{delta} siRNA and treated with either control diluent or 1,25(OH)2D3 (10–7 mol/L, 18 hours). C/EBP{delta} expression was examined by Western blotting. GAPDH was used to control for equal loading and siRNA specificity. C, cell proliferation. LNCaP cells were cotransfected with either control siRNA or C/EBP{delta} siRNA, along with a vector containing the puromycin resistance gene (pMSCVpuro) and selected with puromycin for 2 days. Equal numbers of transfected cells were plated and 24 hours later were treated with either control diluent or 1,25(OH)2D3 (10–7 mol/L). After 5 days, cell proliferation was determined by MTT assays. Columns, means of quadruplicate samples; bars, ±SD. sic, control siRNA; si{delta}, C/EBP{delta} siRNA; –, 1,25(OH)2D3 absent; +, 1,25(OH)2D3 present.

 
Forced expression of C/EBP{delta} inhibits the growth of LNCaP cells. Further studies explored the biological function of C/EBP{delta} in cancer cells. We transiently transfected the C/EBP{delta} expression vector (pcDNA3.1-C/EBP{delta}) into LNCaP cells. Colony formation assays showed that LNCaP cells transfected with pcDNA3.1-C/EBP{delta} formed about 90% fewer colonies [mean 38 ± 19 (SD) colonies per well] compared with the number of colonies developing from control LNCaP cells transfected with the same amount of pcDNA3.1 empty vector (379 ± 23 colonies per well; Fig. 4A). Western blot analysis showed that level of C/EBP{delta} in the transiently transfected LNCaP cells, was comparable with levels in the 1,25(OH)2D3-treated LNCaP cells (Fig. 4B).



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  		Figure 4. C/EBP{delta} inhibits the clonal proliferation of LNCaP cells. LNCaP cells were transfected with either pcDNA3.1 empty vector or pcDNA3.1-C/EBP{delta} expression vector. A, colony formation assay. Transfected cells were treated for 2 weeks with G418, fixed, stained, and photographed. Colonies with >40 cells were counted. Columns, means of three experiments done in triplicate plates; bars, ±SD. B, Western blot analysis. Transfected cells were harvested 2 days after transfection and analyzed for C/EBP{delta} expression. C/EBP{delta} expression in LNCaP cells treated with either control diluent or 1,25(OH)2D3 (10–7 mol/L, 18 hours) is shown for comparison. GAPDH was used to control for equal loading.

 
1,25(OH)2D3 up-regulates the expression of C/EBP{delta} in both androgen receptor– and estrogen receptor–positive cancer cells. Among the prostate cancer lines, 1,25(OH)2D3 significantly inhibited growth of androgen receptor (AR)–positive LNCaP cells, had an intermediate effect on AR-negative PC-3 cells, and had minimally inhibitory activity on AR-negative DU145 cells (Fig. 1A). We explored whether 1,25(OH)2D3 induced C/EBP{delta} in these AR-negative cells. Real-time RT-PCR analysis showed that in contrast to LNCaP cells, 1,25(OH)2D3 (10–7 mol/L, 18 hours) was unable to increase the expression of C/EBP{delta} in the AR-negative cells (Fig. 5).



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  		Figure 5. 1,25(OH)2D3 up-regulates the expression of C/EBP{delta} in both AR- and ER-positive cancer cells. The cancer cells were cultured with either control diluent or 1,25(OH)2D3 (10–7 mol/L). After 18 hours, RNA was extracted and levels of C/EBP{delta} transcripts were measured by real-time RT-PCR. The results are expressed in arbitrary units as a ratio of C/EBP{delta} transcripts/ß-actin transcripts. Columns, means of triplicate samples; bars, ±SD. –, 1,25(OH)2D3 absent; +, 1,25(OH)2D3 present.

 
Some breast cancer cell lines, including MCF-7 and T47D cells, express estrogen receptor (ER) whose signaling plays an important role in carcinogenesis and progression of breast cancer cells (31). These ER-expressing breast cancer cells are growth inhibited when exposed to vitamin D3 (Fig. 1B). We therefore explored whether 1,25(OH)2D3 induced C/EBP{delta} in these cells. Real-time RT-PCR analysis showed that 1,25(OH)2D3 (10–7 mol/L, 18 hours) increased the levels of C/EBP{delta} by ~5.7- and 3.5-fold, in MCF-7 and T47D, respectively (Fig. 5). On the other hand, 1,25(OH)2D3 failed to increase the level of C/EBP{delta} in ER-negative MDA-MB231 cells (Fig. 5) that are resistant to growth inhibition by 1,25(OH)2D3 (Fig. 1B).

Overexpression of C/EBP{delta} inhibits growth of MCF-7 and T47D cells and does not affect the growth of PC-3 cells. Whereas 1,25(OH)2D3 inhibited proliferation of PC-3, MCF-7, and T47D cells (Fig. 1), induction of C/EBP{delta} following 1,25(OH)2D3 treatment was only observed in the ER-positive MCF-7 and T47D breast cancer cells but not in the AR-negative PC-3 prostate cells (Fig. 4). Colony formation assays showed that in MCF-7 cells, forced expression of C/EBP{delta} (pcDNA3.1-C/EBP{delta}) resulted in a significant (68%) growth reduction (486 ± 28 colonies per well pcDNA3.1; 154 ± 14 colonies per well pcDNA3.1-C/EBP{delta}; Fig. 6). The growth of T47D cells was also inhibited by C/EBP{delta} although to a lesser extent (43%, 98 ± 9 colonies per well pcDNA3.1; 56 ± 6 colonies per well pcDNA3.1-C/EBP{delta}; Fig. 6). In contrast, C/EBP{delta} did not significantly inhibit the growth rate of PC-3 cells (47 ± 4 colonies per well pcDNA3.1; 42 ± 7 colonies per well pcDNA3.1-C/EBP{delta}; Fig. 6). These results suggest that 1,25(OH)2D3-mediated growth suppression in PC-3 cells occurs via a C/EBP{delta}-independent pathway.



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  		Figure 6. C/EBP{delta} inhibits growth of MCF-7 and T47D cells and does not affect the growth of PC-3 cells. Colony formation assays (A) and Western blot analysis (B) were done as described in Fig. 3 legend.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we used microarray analysis to identify potential 1,25(OH)2D3 target genes under conditions where 1,25(OH)2D3 causes growth inhibition in the AR-expressing prostate cancer cell line, LNCaP. Our study identified C/EBP{delta} as being the gene maximally induced by 1,25(OH)2D3. Knockdown of C/EBP{delta} expression by siRNA significantly reduced the ability of 1,25(OH)2D3 to inhibit growth in LNCaP cells. Furthermore, forced expression of C/EBP{delta} in LNCaP cells inhibited their clonal growth, suggesting that C/EBP{delta} behaves as a tumor suppressive molecule in these prostate cancer cells.

The ER-expressing breast cancer cell lines MCF-7 and T47D also exhibited growth inhibition in response to 1,25(OH)2D3. As we have shown for LNCaP cells, 1,25(OH)2D3 induced C/EBP{delta} in these cancer cells as well; and forced expression of C/EBP{delta} in MCF-7 and T47D cells inhibited their clonal growth. Among LNCaP MCF-7 and T47D cells, LNCaP was most sensitive to growth inhibition by 1,25(OH)2D3 followed by MCF-7 and then T47D. The degree of 1,25(OH)2D3-mediated growth inhibitory activity correlated with the induction levels of C/EBP{delta} by 1,25(OH)2D3 and with the growth inhibitory effects of C/EBP{delta} itself when overexpressed in these cell lines. These results further support our hypothesis that C/EBP{delta} is a key mediator of 1,25(OH)2D3 effects in AR-positive prostate and ER-positive breast cancer cells.

On the other hand, 1,25(OH)2D3 did not increase the levels of C/EBP{delta} in the AR- and ER-negative DU145 and MDA-MB-231 cells, respectively; likewise, these cells have been shown resistant to growth inhibition after treatment with 1,25(OH)2D3. These data suggest that the induction of C/EBP{delta} mediated by 1,25(OH)2D3 in prostate and breast cancer cells correlates with either a functionally intact AR or ER and the responsiveness of the cells to 1,25(OH)2D3.

Our results together with earlier studies suggest that AR- and ER-positive cancer cells are generally more sensitive to 1,25(OH)2D3. Still, 1,25(OH)2D3 exerts antiproliferative effects on AR- and ER-negative cells as well (5, 31). In the present study, we show that 1,25(OH)2D3 slows the growth of the AR-negative prostate cancer cells, PC-3, although the level of inhibition is modest compared with that observed in the AR-positive prostate (LNCaP) and ER-positive breast (MCF-7 and T47D) cancer cells. Interestingly, levels of C/EBP{delta} are not induced in PC-3 cells following 1,25(OH)2D3 treatment and forced expression of C/EBP{delta} does not lead to their growth inhibition. These results suggest that the signaling pathways of 1,25(OH)2D3 in AR- and ER-negative cells are, at least partly, different from the ones occurring in AR- and ER-positive cells. Previous studies done in other human cells including squamous carcinoma cells (32) and breast cancer cells (31) have also suggested that the molecular mechanisms underlying 1,25(OH)2D3 growth inhibitory effects are cell type specific.

This study also found that 1,25(OH)2D3 increased the level of I{kappa}B{alpha} in LNCaP cells. I{kappa}B{alpha} forms a binding complex with NF-{kappa}B in the cytoplasm, preventing the latter from entering the nucleus and behaving as a progrowth transcription factor by activating target genes such as Bcl-2 and cyclin D (33). The level of I{kappa}B{alpha} protein is regulated by the ubiquitin-proteasome pathway. Another gene whose expression was up-regulated by 1,25(OH)2D3 is the small ubiquitin-related modifier 1 (SUMO 1; Table 2). This protein stabilizes I{kappa}B{alpha} and prevents 26S proteasome–mediated degradation of I{kappa}B{alpha} (34). Therefore, overexpression of SUMO 1 also inhibits the activity of NF-{kappa}B via up-regulation of I{kappa}B{alpha} (34). A gene that was down-regulated by 1,25(OH)2D3 was the paternally expressed gene 3 (PEG 3), which is also implicated in the regulation of NF-{kappa}B activity; PEG 3 activates NF-{kappa}B by aiding the dissociation of the I{kappa}B{alpha}/NF-{kappa}B complex (35). Thus, several genes revolving around NF-{kappa}B were modulated by 1,25(OH)2D3 in LNCaP cells. Further studies are warranted to explore the effects of 1,25(OH)2D3 on NF-{kappa}B activity in cancer cells.

The 1,25(OH)2D3 up-regulates the levels of CDKIs, including p21waf1 and p27kip1 in cancer cells (36, 37). These CDKIs are also substrates for degradation by the ubiquitin-proteasome pathway (38). Up-regulation of SUMO 1 in LNCaP cells mediated by vitamin D could protect CDKIs from degradation by the ubiquitin-proteasome pathway stabilizing these proteins, resulting in retardation of their proliferation.

The 1,25(OH)2D3 down-regulated by 60% the expression of neuronal cell adhesion molecule (NRCAM) in LNCaP cells compared with control cells. Neuroblastoma cells express NRCAM and their levels of this protein paralleled their growth rate (39). However, the relationship between prostate cancer and NRCAM has not been studied.

Recently, other investigators did microarray analysis to identify target genes of 1,25(OH)2D3 in adenocarcinoma cells from the prostate including LNCaP cells (40, 41). They used arrays carrying 20,000 genes and identified IGFBP-3 as an up-regulated gene. Expression of IGFBP-3 in LNCaP cells under the conditions that we used, was extremely low as measured by real-time PCR (data not shown); therefore, it was not identified as "present" in our microarray analysis. We note that with the exception of one gene (FK506-binding protein 5), the gene expression profiles generated by the two studies do not overlap. It is likely that the dissimilarities are the result of different experimental conditions as well as different criteria used to analyze the data and that each study complements the other.

Taken together, we have found that 1,25(OH)2D3 stimulated the expression of C/EBP{delta} in LNCaP cells, which contributed to their growth arrest mediated by 1,25(OH)2D3. Further studies are required to clarify the molecular mechanisms by which ligand-activated VDR enhances the expression of C/EBP{delta}.


   Acknowledgments
 
Grant support: NIH, AT00151, the University of California at Los Angeles Center for Dietary Supplements Research: Botanicals, Leo Pharmaceuticals, the Parker Hughes Fund, Aaron Eschman Fund, University of California-Los Angeles Jonsson Comprehensive Cancer Center and Molecular Biology Institute membership (H.P. Koeffler), and Mark Goodson Chair of Oncology Research at Cedars-Sinai Medical Center (H.P. Koeffler).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: T. Ikezoe and S. Gery contributed equally to the article.

Received 11/18/03. Revised 2/ 9/05. Accepted 3/24/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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