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. 2009 Jan 22;150(5):2046–2054. doi: 10.1210/en.2008-1395

1α,25-Dihydroxyvitamin D3 Reduces c-Myc Expression, Inhibiting Proliferation and Causing G1 Accumulation in C4-2 Prostate Cancer Cells

JoyAnn N Phillips Rohan 1, Nancy L Weigel 1
PMCID: PMC2671895  PMID: 19164469

Abstract

There is an inverse correlation between exposure to sunlight (the major source of vitamin D) and the risk for prostate cancer, the most common noncutaneous cancer and second most common cause of death from cancer in American men. The active metabolite of vitamin D, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] acting through the vitamin D receptor decreases prostate cancer cell growth and invasiveness. The precise mechanisms by which 1,25(OH)2D3 inhibits growth in prostate cancer have not been fully elucidated. Treatment with 1,25(OH)2D3 causes an accumulation in the G0/G1 phase of the cell cycle in several prostate cancer cell lines. One potential target known to regulate the G0/G1 to S phase transition is c-Myc, a transcription factor whose overexpression is associated with a number of cancers including prostate cancer. We find that 1,25(OH)2D3 reduces c-Myc expression in multiple prostate epithelial cell lines, including C4-2 cells, an androgen-independent prostate cancer cell line. Reducing c-Myc expression to the levels observed after 1,25(OH)2D3 treatment resulted in a comparable decrease in proliferation and G1 accumulation demonstrating that down-regulation of c-Myc is a major component in the growth-inhibitory actions of 1,25(OH)2D3. Treatment with 1,25(OH)2D3 resulted in a 50% decrease in c-Myc mRNA but a much more extensive reduction in c-Myc protein. Treatment with 1,25(OH)2D3 decreased c-Myc stability by increasing the proportion of c-Myc phosphorylated on T58, a glycogen synthase kinase-3β site that serves as a signal for ubiquitin-mediated proteolysis. Thus, 1,25(OH)2D3 reduces both c-Myc mRNA levels and c-Myc protein stability to inhibit growth of prostate cancer cells.

1α,25(OH)2D3 reduces c-Myc expression through decreases in RNA expression and decreased protein stability; equivalent reductions in c-Myc using siRNA are sufficient to inhibit proliferation and cause comparable G1 accumulation.

There is increasing interest in the role of vitamin D metabolites in reducing the risk for a variety of cancers (1,2). Some studies have found inverse correlations between the risk for prostate cancer and exposure to sunlight (our major source of vitamin D) or the serum levels of 25-hydroxyvitamin D3 (3,4,5), whereas others have found no correlation or even an increase in risk (6). 1α,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active form of vitamin D, is the ligand for the vitamin D receptor (VDR), a member of the ligand activated nuclear receptor family of transcription factors (7). There are a number of reports demonstrating that 1,25(OH)2D3 and less calcemic analogs inhibit growth of prostate cancer cells in vitro (8,9) as well as in mouse xenograft models (10). In addition, there is some evidence from clinical trials of beneficial effects of combination treatments with calcitriol [1,25(OH)2D33] and dexamethasone or docetaxel (11). However, the precise mechanisms by which 1,25(OH)2D3 causes growth inhibition in prostate cancer are still unclear.

The actions of 1,25(OH)2D3 include cell cycle arrest (12) and, less commonly, induction of apoptosis (8,13). In many cells 1,25(OH)2D3 treatment induces G0/G1 accumulation (12,14,15,16). Progression to S phase requires phosphorylation and inactivation of the retinoblastoma (Rb) protein releasing the transcription factor E2F. Several studies have shown that 1,25(OH)2D3 treatment can reduce cyclin-dependent kinase (Cdk)-2 activity and induce the expression of cyclin-dependent kinase inhibitors p21 and p27 in prostate cancer cell lines (8,17,18). Our laboratory has shown that a consequence of 1,25(OH)2D3 treatment in LNCaP cells, an androgen-dependent prostate cancer cell line, is the reduction of c-Myc expression (19). c-Myc is a protooncogene important for progression through the early portion of the cell cycle; c-Myc induces the expression of cyclin D1 and cyclin D2 as well as Cdc25A, a phosphatase that dephosphorylates an inhibitory site in Cdk2, facilitating activation of the kinase and phosphorylation of Rb (20). Thus, 1,25(OH)2D3 may play a role in inhibiting cell cycle progression before the increases in cyclin-dependent kinase inhibitors reported previously.

Overexpression of c-Myc is observed in prostate cancer and many other cancers (21,22,23). Moreover, artificial overexpression of c-Myc is sufficient to induce prostate cancer in rodent models (24,25,26,27). Thus, c-Myc plays an important role in prostate cancer, and treatments that reduce its expression are of great interest. Regulation of c-Myc expression is complex. Transcription of c-Myc is induced both by growth factor signaling (28,29) and the wnt signaling pathway (30). β-Catenin/T-cell factor (TCF) responsive elements have been identified in the c-Myc promoter (31). In addition to transcriptional regulation, regulation of transcriptional elongation of c-Myc mRNA has been reported (32). Finally, c-Myc protein has a relatively short half-life (33). Posttranslational modifications and degradation processes mediated by E3 ubiquitin-ligases also regulate c-Myc protein levels (34).

A number of 1,25(OH)2D3 targets with potential to contribute to the growth-inhibitory actions of 1,25(OH)2D3 have been identified. These include IGF binding protein-3 (18), CCAAT/enhancer binding protein-Δ (35), and inhibition of targets in the prostaglandin pathway (36). Our goal was to determine how effective the reduction in c-Myc is in reproducing the growth inhibition induced by 1,25(OH)2D3 in C4-2 androgen-independent prostate cancer cells and identify the mechanisms by which 1,25(OH)2D3 reduces c-Myc expression. Our data show that the reduction in c-Myc is a major factor in 1,25(OH)2D3-mediated cell cycle accumulation and that 1,25(OH)2D3 reduces c-Myc expression both by reducing mRNA levels and by reducing protein stability.

Materials and Methods

1,25(OH)2D3 was obtained from Solvay Pharmaceuticals (Weesp, The Netherlands). 1,25(OH)2D3 stock solutions were prepared in ethanol and stored in the dark at −20 C. Isoton II and zapoglobulin II were purchased from Beckman Coulter Inc. (Fullerton, CA). Dulbecco’s PBS, TriZOL reagent, and Lipofectamine reagent were obtained from Invitrogen (Carlsbad, CA). Enhanced chemiluminescence (ECL) and ECL+ reagents were purchased from Amersham Pharmacia Biotech Inc. (Piscataway, NJ). SuperSignal West Femto maximum sensitivity substrate was purchased from Thermo Scientific (Rockford, IL). Cycloheximide was purchased from Sigma Chemicals (St. Louis, MO). (2′Z,3′E)-6-Bromoindirubin-3-oxime (BIO) was purchased from EMD Chemicals, Inc. (Gibbstown, NJ). Glycogen synthase kinase (GSK)-3β inhibitor I (I) was purchased from Calbiochem (San Diego, CA). Tissue culture supplies were obtained from Fisher Scientific (Pittsburgh, PA). All other chemicals were reagent grade unless otherwise stated.

Cell culture

The human prostate cancer cell line LNCaP and the normal prostate epithelial cell line RWPE-1 were obtained from American Type Culture Collection (Manassas, VA); the C4-2 prostate cancer cell line was obtained from Urocor Inc. (Oklahoma City, OK). The LNCaP cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). The RWPE-1 cells were grown in keratinocyte serum-free medium (Invitrogen) supplemented with 5 ng/ml human recombinant epithelial growth factor and 0.05 mg/ml bovine pituitary extract (Invitrogen). The C4-2 cells were grown in T medium (37) supplemented with 5% heat-inactivated FBS (56 C for 30 min). All cells were grown in a 37 C humidified incubator with an atmosphere of 5% CO2.

Cell growth assays

C4-2 cells were plated in six-well plates at a density of 100,000 cells/well. The medium was changed and fresh vehicle or 1,25(OH)2D3 added every 3 d. Cells were washed with PBS, harvested by scraping in PBS, and counted using a Coulter Particle Counter Z1 (Hialeah, FL).

Western blot analysis

Cells were washed, scraped into PBS, and pelleted by centrifugation. Cell pellets were resuspended in radioimmunoprecipitation assay buffer (Upstate, Temecula, CA) or buffer (pH 7.4) of 10 mm Tris, 1 mm EDTA, and 12 mm monothioglycerol containing 0.4 m NaCl plus 1× protease inhibitor mix and 1× phosphatase inhibitor mix (Pierce, Rockford, IL). Protein was extracted with three freeze/thaw cycles and the extract clarified by centrifugation. Protein concentrations were determined by Bradford analysis. Equal amounts of proteins were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membranes. The nitrocellulose membrane was blocked with 1% milk Tris-buffered saline and Tween 20 [TBST; 10 mm Tris (pH 7.5), 0.15 m NaCl, 1% Tween 20] for at least an hour at room temperature. Primary mouse monoclonal antibody against c-Myc (9E10 clone; Santa Cruz Biotechnology, Santa Cruz, CA) was diluted 1:200 in 1% milk-TBST. Primary rabbit monoclonal antibody (Y69 clone, Abcam, Cambridge, MA) against c-Myc was diluted 1:5000 in 1% milk-TBST. Primary rabbit polyclonal antibody against β-catenin (H-102; Santa Cruz) was diluted 1:400 in 1% milk-TBST. Primary mouse monoclonal antibody against phospho-tyrosine (clone 4G10; Millipore, Temecula, CA) was diluted 1:1000 in 1% milk-TBST. Primary rabbit polyclonal (Thr58/Ser62; Cell Signaling, Danvers, MA) against phospho-c-Myc was diluted 1:200 in 1% BSA-TBST. Primary mouse monoclonal antibody against tubulin (clone AA2; Millipore) was diluted 1:10,000 in 1% milk-TBST. Primary mouse monoclonal antibody against actin (Millipore) was diluted 1:10,000 in 1% milk-TBST.

All primary incubations were performed overnight at 4 C. All membranes were washed three times with 1% milk-TBST at room temperature. Membranes that were incubated with mouse primary antibodies were next incubated with rabbit antimouse IgG (Zymed Laboratories, South San Francisco, CA) diluted to 1:10,000 in 1% milk- or BSA-TBST for 1 h at room temperature. All membranes were washed with 1% milk- or BSA-TBST and incubated in horseradish peroxidase-conjugated donkey antirabbit IgG (Amersham Pharmacia Biotech) diluted 1:30,000 in TBST for 1 h at room temperature. Membranes were washed three times with TBST followed by two Tris-buffered saline [10 mm Tris (pH 7.5), 0.15 m NaCl] washes. Proteins were detected using ECL, ECL+, or Femto reagents according to manufacturer’s recommendations. Films were scanned using the Molecular Dynamics Personal Densitometer SI (Amersham Pharmacia Biotech) or GeneSnap 7.04 (SynGene, Cambridge, UK) and quantified used Image Quant 5.2 (Amersham Pharmacia Biotech) or GeneTools 4.00 (SynGene).

Small interfering RNA (siRNA)

C4-2 cells, plated in six-well plates at a density of 100,000 cells per well, were transfected with the indicated amounts of control or target-specific siRNA using Lipofectamine (Invitrogen) following the manufacturer’s recommendations in serum-free medium (OPTI-MEM; Invitrogen). c-Myc (siRNA no. 1, catalog no. 4250) and silencer negative control siRNA (catalog no. 4611) were purchased from Ambion (Austin, TX). c-Myc (siRNA no. 2; catalog no. M-003282-04-0005) and nontargeting control siRNA (catalog no. D-001210-01-05) were purchased from Dharmacon (Lafayette, CO). β-Catenin (also known as CTNNB1; catalog no. M-003482) siRNA was purchased from Millipore. Six hours after transfection, medium was removed and replaced with fresh full medium (T medium supplemented with 5% heat inactivated FBS) and cells treated with vehicle or 100 nm 1,25(OH)2D3 for 48 h.

Quantitative RT-PCR (qPCR)

RNA was isolated using TriZOL reagent (Invitrogen) following the manufacturer’s recommendations, quantified, and diluted to 100 ng/μl for preparation of cDNA using SuperScript II reverse transcriptase (Invitrogen) as recommended by the manufacturer. Primer and probe sets for 18S (4319413E-040211), c-Myc (Hs99999003_m1; Applied Biosystems, Foster City, CA), cdc25A (5′-TCATTGATTCTAGAGAGACCTGTTTCAG-3′ and 5′-AGTGAAGCCGTGATGGTAAGGA-3′), E2F (5′-TCCAAGAACCACATCCAGTG-3′ and 5′-CTGGGTCAACCCCTCAAG-3′), and c-Myc (5-CGTCTCCACACATCAGCACAA-3′ and 5-CTCTTGGCAGCAGGATAGTCCTT-3′) were used to measure cDNA using the 7500 real-time PCR system (Applied Biosystems).

[3H]thymidine incorporation

Cells were incubated with 2 μCi/ml [3H]thymidine (MP Biomedicals, Solon, OH) for 3 h. Incorporated [3H]thymidine was extracted and counted as previously described (37).

Cell cycle analysis

C4-2 cells, plated in 6-well plates at a density of 100,000 cells/well, were transfected with siRNA as described above and treated with vehicle or 1,25(OH)2D3 for 2 d. Cells were washed with PBS, fixed in ethanol, and prepared for fluorescence activated cell sorting analysis as described previously (38), analyzed using the Coulter EPICS XL-MCL, and quantified by flow cytometry using FlowJo version 8.8.2 (for Mac; Tree Star, Inc. Ashland, OR).

c-Myc stability

C4-2 cells, plated in 10-cm plates at a density of 500,000 cells/well, were pretreated with vehicle or 1,25(OH)2D3 for 3 d and then incubated with 10 μg/ml cycloheximide (CHX) for the indicated times. Cell extracts were prepared and c-Myc detected using the Y69 antibody as described above.

GSK-3β inhibitors

C4-2 cells, plated in six-well plates at a density of 100,000 cells/well, were treated with 2 μm BIO or 10 μm GSK-3β I for 24 h (alone) or 6 h [when cells were pretreated with vehicle or 1,25(OH)2D3]. c-Myc and phospho-c-Myc were detected as described above.

Statistics

The SigmaStat program (Jandel Scientific, San Rafael, CA) was used to perform Student’s unpaired t test to compare the means of two groups and one-way ANOVA was used to compare multiple means to control group or an all pair-wise multiple comparison procedure was used (Holm-Sidak method). Values are indicated as means and the error bars indicate sem. P < 0.05 was considered statistically significant. All experiments were performed a minimum of three times and a representative experiment is shown.

Results

1,25(OH)2D3-mediated growth inhibition is accompanied by down-regulation of c-Myc

To determine whether down-regulation of c-Myc might be responsible for 1,25(OH)2D3-mediated growth inhibition, we first compared the time course of growth inhibition with the time required to down-regulate c-Myc. Treatment of androgen-independent C4-2 cells grown in medium containing FBS with 100 nm 1,25(OH)2D3 inhibits cell growth with initial differences in cell number detected at 2 d of 1,25(OH)2D3 treatment; the differences in cell number and percent growth inhibition increase with time (Fig. 1A). The treated cells essentially stop growing, although there is no net loss in cell number. Previous publications from our laboratory showed that treatment with growth-inhibitory concentrations of 1,25(OH)2D3 reduces c-Myc protein levels in the LNCaP human androgen-dependent prostate cancer cell line (19,39). 1,25(OH)2D3 treatment also strongly reduces c-Myc expression in C4-2 cells and to a lesser extent in the nontransformed human prostate epithelial cell line RWPE-1 (Fig. 1B). Consistent with the down-regulation contributing to 1,25(OH)2D3 mediated growth inhibition, c-Myc proteins levels begin to decrease before the changes in cell number and expression is strongly inhibited by 1,25(OH)2D3 treatment (Fig. 1C).

Figure 1.

Figure 1

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1,25(OH)2D3 treatment reduces cell growth in C4-2 cells and c-Myc expression in normal and malignant prostate epithelial cell lines. A, C4-2 cells were treated with vehicle or 100 nm 1,25(OH)2D3 (1,25D) for the indicated times, harvested, and counted using a Coulter counter. Data are expressed as the mean ± sem. *, Significance at P < 0.05 with respect to vehicle-treated cells within the same time frame; **, significance at P < 0.005 with respect to vehicle-treated cells within the same time frame. B, LNCaP, C4-2, and RWPE-1 cells were treated with vehicle (−) or 100 nm 1,25(OH)2D3 (1,25D; +) for 3 d. Cells were harvested and c-Myc and actin protein levels were detected by Western blotting. C, C4-2 cells were treated with vehicle (−) or 100 nm 1,25(OH)2D3 (1,25D; +) for the indicated times. Cells were harvested, extracts run on an SDS-PAGE gel, and proteins (c-Myc and actin) detected by Western blotting.

Reducing c-Myc expression is sufficient to inhibit proliferation and induce G1 accumulation in C4-2 cells

To investigate the role of reduced c-Myc levels in 1,25(OH)2D3-mediated growth inhibition in C4-2 cells, c-Myc expression was reduced using siRNA. The reduction in c-Myc protein by c-Myc targeted siRNA (siRNA no. 1) compared with control siRNA was similar to the reduction in c-Myc achieved by treatment with 1,25(OH)2D3 (Fig. 2A). Reducing c-Myc expression led to a significant reduction in proliferation, as measured by [3H]thymidine incorporation and the reduction was similar to that caused by 1,25(OH)2D3 treatment (Fig. 2B). Similar results were obtained with a second independent c-Myc-targeted siRNA (siRNA no. 2) (Fig. 2, C and D). Because reducing c-Myc expression with siRNA similar to levels achieved by 1,25(OH)2D3 treatment is sufficient to inhibit proliferation, we asked whether reducing c-Myc expression would cause G1 accumulation as has been reported for 1,25(OH)2D3 treatment. Cells were transfected with control or c-Myc siRNA no. 2 and treated with vehicle or 100 nm 1,25(OH)2D3 as indicated for 48 h and the percentages of cells in G0/G1, S, and G2/M phases of the cell cycle were determined by flow cytometry using the FlowJo 8.8.2 (Mac; Tree Star) program (Fig. 3A). The cell cycle distribution of 1,25(OH)2D3-treated and c-Myc siRNA-treated cells show remarkably similar increases in G1 (from 77% in control to ∼87–91% in treated) with corresponding reductions in S and G2/M and are statistically different from the control (Fig. 3A). Thus, reducing c-Myc expression is sufficient to induce G1 accumulation. To determine whether reducing c-Myc or treating with 1,25(OH)2D3 induces changes in gene expression consistent with reduced c-Myc activity, we measured expression of Cdc25A, E2F1, p21, and p27 mRNA. Both Cdc25A and E2F1 expression were reduced when cells were treated with c-Myc siRNA or 1,25(OH)2D3 (Fig. 3B). However, we did not detect changes in p21 or p27 in either case (data not shown).

Figure 2.

Figure 2

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Proliferation in the C4-2 prostate cancer cell line is reduced by c-Myc siRNA or 1,25(OH)2D3 treatment. A, C4-2 cells were treated with vehicle or 100 nm 1,25(OH)2D3 (1,25D) or transiently transfected with 75 pm of control or c-Myc siRNA no. 1 for 2 d. Cells were harvested, extracts run on an SDS-PAGE gel, and c-Myc and actin protein levels detected by Western blotting. Representative westerns corresponding to the samples in B are shown. B, C4-2 cells were treated with vehicle or 100 nm 1,25(OH)2D3 (1,25D) or transiently transfected with 75 pm of control or c-Myc siRNA no. 1 for 2 d. Proliferation was measured by [3H]thymidine incorporation. Data are expressed as the mean ± sem. *, Significance at P < 0.005 with respect to vehicle or control siRNA. C, C4-2 cells were transiently transfected with 150 pm control or c-Myc siRNA no. 2. C4-2 cells transfected with control siRNA were further treated with vehicle or 100 nm 1,25(OH)2D3 (1,25D) for 2 d. c-Myc mRNA was measured by qPCR and normalized with 18S RNA. Results from two separate experiments, each performed in triplicate, were pooled and analyzed. Data are expressed as the mean normalized to control ± sem. *, Significance at P ≤ 0.001 with respect to control siRNA plus vehicle. D, C4-2 cells were transiently transfected with 150 pm control or c-Myc siRNA no. 2. C4-2 cells transfected with control siRNA were further treated with vehicle or 100 nm 1,25(OH)2D3 (1,25D) for 2 d. Proliferation was measured by [3H]thymidine incorporation. Data are expressed as the mean ± sem. Results from two separate experiments, each performed in triplicate, were pooled and analyzed. *, Significance at P ≤ 0.001 with respect to control siRNA plus vehicle. 1,25(OH)2D3, 1,25D.

Figure 3.

Figure 3

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c-Myc siRNA or 1,25(OH)2D3 induces G0/G1 accumulation. A, C4-2 cells were transiently transfected with 150 pm control or c-Myc siRNA. Control siRNA-transfected cells were treated with vehicle (+Veh) or 100 nm 1,25(OH)2D3 (+1,25D) for 2 d. Cells were then stained with propidium iodide (PI) for total DNA content and analyzed by flow cytometry using FlowJo. G0/G1, S, and G2/M phases of a representative experiment performed in triplicate are indicated. Data are expressed as the mean ± sem for each treatment group. *, Significance at P ≤ 0.05 with respect to vehicle or control siRNA. B, cdc25A, E2F, and c-Myc mRNA were measured by qPCR and normalized with 18S RNA. Results from a representative experiment performed in triplicate are shown. Data are expressed as the mean ± sem. *, Significance at P ≤ 0.05 with respect to vehicle or control siRNA.

Wnt signaling and the regulation of c-Myc in C4-2 cells

Expression of c-Myc is often induced by signaling pathways such as the canonical wnt signaling pathway and 1,25(OH)2D3 inhibits wnt signaling under some conditions (40). To assess the overall contribution of canonical wnt signaling through β-catenin-dependent transcription to c-Myc expression in C4-2 cells, β-catenin expression was reduced using β-catenin-specific siRNA and expression of β-catenin and c-Myc measured. Expression of c-Myc is diminished when β-catenin expression is reduced verifying that wnt signaling contributes to c-Myc expression in C4-2 cells (Fig. 4A). However, the reduction is not as extensive as that observed with 1,25(OH)2D3 treatment (Fig. 1) nor was reducing β-catenin expression sufficient to inhibit proliferation in C4-2 cells (Fig. 4B). Moreover, we were unable to reproducibly detect a 1,25(OH)2D3-dependent reduction in β-catenin/TCF activity measured using a TOP Flash reporter (Millipore, Billerica, MA; data not shown). Thus, 1,25(OH)2D3 must regulate c-Myc expression through multiple pathways. Tyrosine kinase signaling also has been implicated in inducing expression of c-Myc mRNA (28,41,42,43). Our laboratory has shown previously that 1,25(OH)2D3 reduces the tyrosine phosphorylation of a subset of proteins in LNCaP and C81 LN (an androgen independent derivative of LNCaP) prostate cancer cell lines including HER2 in C81 LN cells (39). To test whether tyrosine kinase signaling was reduced by 1,25(OH)2D3 treatment in C4-2 cells, we performed Western blot analysis using an antibody against phosphotyrosine residues. Similar to our results in other cell lines (39), we found that 1,25(OH)2D3 treatment reduces tyrosine phosphorylation of a subset of proteins in C4-2 cells (Fig. 5A).

Figure 4.

Figure 4

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Reducing β-catenin modestly reduces c-Myc protein. A, C4-2 cells were transiently transfected with 80 pm control siRNA or β-catenin siRNA. Two days after transfection, C4-2 cells were harvested for protein analysis and extracts run on an SDS-PAGE gel. β-Catenin, c-Myc, and tubulin protein levels were detected by Western blotting. B, C4-2 cells were transiently transfected with 80 pm control siRNA or β-catenin siRNA. Three days after transfection, C4-2 cells were harvested for protein analysis and extracts run on an SDS-PAGE gel. β-Catenin protein levels were detected by Western blotting. In parallel, proliferation was measured after 3 d of treatment with siRNA by [3H]thymidine incorporation. Data are expressed as the mean ± sem.

Figure 5.

Figure 5

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1,25(OH)2D3 reduces c-Myc mRNA and protein. A, C4-2 cells were treated with vehicle or 100 nm 1,25(OH)2D3 (1,25D) for 3 d. Cells were harvested for protein analysis, extracts run on an SDS-PAGE gel, and phosphotyrosine residues in proteins detected by Western blotting. Arrows indicate reduction in phosphotyrosine residues after 1,25D treatment in a subset of proteins. B, C4-2 cells were treated with vehicle or 100 nm 1,25(OH)2D3 (1,25D) for 3 d. c-Myc mRNA was measured by qPCR and normalized with 18S. Data are expressed as the mean ± sem. B, C4-2 cells were treated with vehicle (−) or 100 nm 1,25(OH)2D3 (1,25D; +) for 3 d. Cells were harvested for protein analysis, extracts run on an SDS-PAGE gel, and proteins (c-Myc and actin) detected by Western blotting. Intensity of signals was measured with a densitometer. Densitometric values represent c-Myc/actin normalized to vehicle (−).

1,25(OH)2D3 treatment reduces the stability of c-Myc

Our initial studies (Figs. 1 and 2) suggested that c-Myc protein levels were reduced more than c-Myc mRNA levels. In a direct comparison within a single experiment, we found that treating C4-2 cells with 1,25(OH)2D3 for 3 d reduces c-Myc protein expression approximately 90% (Fig. 5C) with only an approximately 50% reduction in c-Myc mRNA (Fig. 5B), suggesting an additional posttranscriptional level of control. Densitometric scans of c-Myc protein in Fig. 1C yielded similar reductions in protein (∼80–85%). Because c-Myc expression is also regulated at the level of protein stability (34), we asked whether 1,25(OH)2D3 treatment reduced c-Myc protein stability. To address this, cells were pretreated with vehicle or 1,25(OH)2D3 for 3 d; CHX was then added to inhibit de novo protein synthesis, and cells were harvested at the indicated times. As previously established, 1,25(OH)2D3 decreases the total amount of c-Myc protein. Furthermore, c-Myc expression decreases more quickly in the presence of 1,25(OH)2D3 compared with control-treated cells (Fig. 6A). To determine half-life, expression of c-Myc was normalized to actin and levels of control or 1,25(OH)2D3-treated c-Myc expression at T = 0 for the CHX treatment were set as 100. In this experiment, 50% of maximal signal remains at about 35 min in vehicle-treated cells compared with about 20 min for the 1,25(OH)2D3-treated cells (Fig. 6B). Figure 6C shows the averages and means of the half-lives for c-Myc in vehicle-treated cells and 1,25(OH)2D3-treated cells from five independent experiments. The average reduction in half-life is about 50%.

Figure 6.

Figure 6

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1,25(OH)2D3 treatment reduces stability of c-Myc protein in C4-2 cells. A, C4-2 cells were pretreated with vehicle or 100 nm 1,25(OH)2D3 (1,25D) for 3 d. Before harvesting, cells were treated with CHX for 0–60 min. Cells were harvested for protein analysis at the indicated times, extracts run on an SDS-PAGE gel, and c-Myc and actin protein levels detected by Western blotting. A representative western is shown. B, Percent of time 0 was calculated from the Western blot after normalization to actin. Percentage reductions are graphed on a log scale. The line indicates 50% of maximal signal. C, Graph indicating mean values of 50% of maximum signal from five independent experiments for each treatment group. *, Significance at P ≤ 0.005 with respect to vehicle-treated cells. D, C4-2 cells were treated with vehicle, BIO, or GSK-3β I for 24 h. Cells were harvested for protein analysis, extracts run on an SDS-PAGE gel, and c-Myc and tubulin protein levels detected by Western blotting. E, Cells were pretreated with vehicle (−) or 100 nm 1,25(OH)2D3 (1,25D; +) for 3 d. Then cells were treated with vehicle (−) or BIO (+) for 6 h. Twice as much protein extract (2 times) compared with 1 time were run on an SDS-PAGE gel to enhance signal in selected cases. c-Myc and phosphorylated c-Myc (T58/S62) were detected by Western blotting. The signals were measured with a densitometer. Results are presented as ratios of phosphorylated c-Myc to total c-Myc normalized to the ratio for the control sample (arbitrarily set at 1).

1,25(OH)2D3 increases phosphorylation of T58 in c-Myc, a signal for degradation

Phosphorylation of T58 in c-Myc serves as a signal to induce degradation through binding of SCFFbw7 (E3 ubiquitin ligase complexes) (34,44). GSK-3β has been identified as the primary kinase responsible for T58 phosphorylation targeting c-Myc for degradation (34). Thus, we asked whether GSK-3β plays a role in regulating the basal levels of c-Myc protein in C4-2 cells. Treatment with either of two GSK-3β inhibitors for 24 h (I or BIO) increased levels of c-Myc protein (Fig. 6D). Because GSK-3β negatively regulates the expression of c-Myc protein in C4-2 cells, we asked whether 1,25(OH)2D3 increases phosphorylation of T58 facilitating c-Myc degradation. C4-2 cells were pretreated with vehicle or 100 nm 1,25(OH)2D3 for 3 d and then with vehicle or the GSK-3β inhibitor, BIO, for 6 h before extraction and measurement of total c-Myc and P-T58 c-Myc by Western blotting. The ratio of phospho to total c-Myc in 1,25(OH)2D3-treated cells is substantially increased compared with vehicle-treated C4-2 cells (fold increase over multiple experiments ranged from 3 to 6) (Fig. 6E), suggesting that 1,25(OH)2D3 enhances GSK-3β activity, leading to increased phosphorylation and degradation. Furthermore, the reduction in the ratio of phospho to total c-Myc in 1,25(OH)2D3-treated plus BIO cells compared with 1,25(OH)2D3-treated cells is greater than the reduction in control plus BIO-treated compared with control cells, suggesting that the 1,25(OH)2D3 stimulated phosphorylation is GSK-3β dependent (Fig. 6E). Whether the residual phosphorylation is due to incomplete inhibition of GSK-3β by BIO or the presence of another kinase that also phosphorylates T58 is not known, but collectively the data indicate that 1,25(OH)2D3 enhances c-Myc phosphorylation and that this phosphorylation event is reduced by the GSK-3β inhibitor.

Discussion

Our current and previous studies show that 1,25(OH)2D3 down-regulates c-Myc in a variety of prostate cancer cell lines as well as to a lesser extent in nontransformed RWPE-1 prostate epithelial cells. We found that down-regulation of c-Myc is achieved through regulation both at the RNA and protein levels. There is strong evidence that increased c-Myc expression is a major factor in prostate cancer. c-Myc is a protooncogene that is frequently deregulated either through gene translocation, gene amplification, or mutations in many human cancers including prostate cancer (21,23,45) Amplification of chromosome 8q24 (c-Myc locus) is associated with more aggressive prostate cancer (22). The most severe subclass in this analysis included a gain of 8q24 (MYC) combined with a loss at 10q23 (phosphatase and tensin homolog deleted on chromosome 10) among other chromosomal alterations (22). Loss or inactivation of this phosphatase results in higher Akt activity, leading to inactivation of GSK-3β and increased stability and thus expression of c-Myc. LNCaP cells and its androgen-independent derivative C4-2, which were used for this study, both lack functional phosphatase and tensin homolog deleted on chromosome 10 (46,47). Interestingly, recent studies have revealed that a substantial proportion of prostate cancers contain a translocation or deletion that fuses the androgen regulated TMPRSS2 promoter to the coding region of an E26 transformation specific (ETS) factor, most commonly ETS-related gene (ERG;48). Induction of c-Myc by this fusion has been identified as a major factor in regulating growth of VCaP prostate cancer cells (49).

There are numerous studies addressing the mechanisms by which 1,25(OH)2D3 inhibits cell growth and identifying candidates that contribute to the beneficial actions of 1,25(OH)2D3 (9,13,19,39,50,51,52). 1,25(OH)2D3 inhibits growth of both normal (53,54) and malignant (55) prostate cells and at least in some cases tends to induce a more differentiated phenotype. Understanding the mechanisms by which 1,25(OH)2D3 inhibits growth will aid both to identify tumors that are likely to respond to treatment as well as to ascertain which treatments might be enhanced when combined with a VDR agonist. Treatment with 1,25(OH)2D3 causes G0/G1 accumulation in many cell types including some of the prostate cancer cell lines (8,12,19). Transition from G1 to S phase requires the transcription factor E2F, which is sequestered by active Rb (56). Inactivation of Rb is accomplished by phosphorylation by the Cdks, Cdk2, Cdk4, and Cdk6 (57). Cdk activities depend on not only the levels of Cdk but also levels of their partner cyclins whose expression is regulated during cell cycle progression, levels of the Cdk inhibitors (p16, p21, and p27) (58) and the phosphorylation state of the kinases themselves. 1,25(OH)2D3 treatment results in a reduction in Cdk2 activity (8). In some cases, this reduction in activity has been attributed to increases in p21 and/or p27 (8,17,18). Treatment of LNCaP cells with 1,25(OH)2D3 also causes enhanced cytoplasmic localization of Cdk2 (17).

1,25(OH)2D3-induced down-regulation of c-Myc is observed in several types of cancer cells (19,59,60). Although there may be redundant mechanisms by which 1,25(OH)2D3 causes G1 accumulation, our data are the first to show that mimicking the down-regulation of c-Myc by 1,25(OH)2D3 by reducing c-Myc expression with siRNA is sufficient to cause both the growth inhibition and G1 accumulation observed in 1,25(OH)2D3-treated C4-2 cells. A previous report using an antisense oligonucleotide to reduce c-Myc expression in LNCaP cells that were serum starved overnight and then grown in reduced serum (2.5%) also showed growth inhibition (61). The change in growth was accompanied by an increase in a sub-G1 population, suggesting that the cells were becoming apoptotic under these conditions. In C4-2 cells in full serum, reducing c-Myc causes a G1 accumulation similar to 1,25(OH)2D3. Some of the previously reported actions of 1,25(OH)2D3 may be secondary to the down-regulation of c-Myc. For example, c-Myc induces expression of Cdc25A, and we have shown previously that 1,25(OH)2D3 treatment reduces Cdc25A expression in LNCaP cells (39). We have now demonstrated that 1,25(OH)2D3 treatment or depleting c-Myc using siRNA reduces Cdc25A expression in C4-2 cells (Fig. 3B). Cdc25A is required for the removal of an inhibitory phosphate on Cdk2 (62). Thus, reducing c-Myc expression results in reduced Cdk2 activity. Yang and Burnstein (17) reported that 1,25(OH)2D3 treatment of LNCaP cells resulted in increases in the stability of p27 through reduced Cdk2-dependent phosphorylation of p27. 1,25(OH)2D3 treatment reduces E2F activity in LNCaP cells (63). Recently c-Myc has been shown to play a role in inducing expression of E2F, promoting proliferation (64,65). We find that treatment with 1,25(OH)2D3 or depleting c-Myc mRNA reduces E2F1 mRNA (Fig. 3B). Thus, the observed reduction in activity likely is a combination of reduced E2F1 expression due to down-regulation of c-Myc and sequestration of E2F1 by activated Rb.

The necessity for tightly regulating the expression of c-Myc in normal cells has resulted in the evolution of a complex series of regulatory mechanisms. The rates of transcriptional initiation and elongation are both regulated (32,66,67). The protein has a relatively short half-life (<1 h), and its stability is regulated by a complex series of phosphorylation and dephosphorylation (68) followed by proteasome mediated degradation (34). We found that 1,25(OH)2D3 modestly reduces c-Myc mRNA levels. The expression of c-Myc mRNA is often regulated by wnt signaling through β-catenin/TCF/lymphoid enhancer binding factor (31). We found that reducing β-catenin expression modestly reduces c-Myc expression; however, reducing β-catenin expression was insufficient to inhibit proliferation, suggesting that there are additional mechanisms regulating c-Myc expression in C4-2 cells. Consistent with the observation that tyrosine kinase activity has been implicated in the induction of c-Myc mRNA in some cells (41,42,43), we observed decreases in tyrosine phosphorylated proteins after 1,25(OH)2D3 treatment potentially contributing to the reduction of c-Myc mRNA. One of the mechanisms by which growth factor signaling increases c-Myc mRNA is through p42/p44 MAPK-dependent activation (in some cases induced by HER2) of ETS factors, such as ETV1/ER81 (28,48,69), an ETS factor that is overexpressed in the LNCaP lineage (48). In some cells, 1,25(OH)2D3 induces homeobox gene 4, a protein that inhibits transcriptional elongation of c-Myc mRNA (32). However, we did not see evidence of homeobox gene 4 induction on 1,25(OH)2D3 treatment (data not shown).

The discrepancy between the steady-state levels of c-Myc mRNA and steady-state protein levels in cells treated with 1,25(OH)2D3 suggested regulation at the level of protein expression. We found that 1,25(OH)2D3 treatment reduced the half-life of c-Myc protein by about 50%. Phosphorylation of c-Myc at T58 serves as a signal for proteasome-mediated degradation (34). T58 is phosphorylated by GSK-3β, which requires a prior phosphorylation at S62 to phosphorylate T58 (70). S62 is then dephosphorylated and the protein ubiquitinated and degraded (71). Consistent with GSK-3β playing a role in regulating c-Myc levels in C4-2 cells, we showed that treatment with a GSK-3β inhibitor increases c-Myc expression. Moreover, the increased ratio of T58 P c-Myc/total c-Myc is consistent with increased activity of GSK-3β in 1,25(OH)2D3-treated cells. GSK-3β activity can be inhibited through wnt signaling (72,73) and through growth factor-mediated activation of Akt (70,74). Thus, 1,25D may contribute to activation of GSK-3β through either or both of these pathways. The importance of phosphorylation of T58 in regulating the expression of c-Myc is highlighted by the frequency of T58 mutations in human Burkitt’s lymphoma (75,76).

Other VDR target genes have been suggested as contributing to 1,25(OH)2D3-mediated growth inhibition in prostate cancer cells. These include IGF binding protein-3 (18), CCAAT/enhancer binding protein-Δ (35), and inhibition of the prostaglandin pathway (36). Whether these pathways are redundant with down-regulation of c-Myc or cooperate in producing the down-regulation remains to be determined.

Our findings that down-regulation of c-Myc mimics the growth inhibition and G1 accumulation induced by 1,25(OH)2D3 suggests that this is a major factor in the beneficial actions of 1,25(OH)2D3. Moreover, the ability of 1,25(OH)2D3 to reduce both c-Myc mRNA and c-Myc protein stability indicates that 1,25(OH)2D3 treatment can reduce overexpression caused by a variety of genetic alterations.

Acknowledgments

The authors thank William E. Bingman III for technical expertise and Dr. Carolyn Smith’s and Dr. Austin Cooney’s laboratories for reagents.

Footnotes

This work was supported by National Institutes of Health Grant 5R01CA107691 (to N.L.W.), Department of Defense Prostate Cancer Research Program Grant W81-04-1-0192 (to N.L.W.), and Training Grant 5T32HD07165 (to J.N.P.R.).

Disclosure Summary: The authors have nothing to disclose.

First Published Online January 22, 2009

Abbreviations: BIO, 2′Z,3′E)-6-Bromoindirubin-3-oxime; Cdk, cyclin-dependent kinase; CHX, cycloheximide; ECL, enhanced chemiluminescence; FBS, fetal bovine serum; GSK, glycogen synthase kinase; I, inhibitor I; 1,25(OH)2D3, 1α,25-dihydroxyvitamin D3; qPCR, quantitative RT-PCR; Rb, retinoblastoma; siRNA, small interfering RNA; TBST, Tris-buffered saline and Tween 20; TCF, T-cell factor; VDR, vitamin D receptor.

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