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Endocrinology. Mar 2010; 151(3): 896–908.
Published online Feb 10, 2010. doi:  10.1210/en.2009-1116
PMCID: PMC2840684

Nuclear Targeting of Cyclin-Dependent Kinase 2 Reveals Essential Roles of Cyclin-Dependent Kinase 2 Localization and Cyclin E in Vitamin D-Mediated Growth Inhibition

Abstract

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), inhibits proliferation of a variety of cell types including adenocarcinoma of the prostate. We have previously shown that 1,25-(OH)2D3 increases the stability of the cyclin-dependent kinase inhibitor p27KIP1, decreases cyclin-dependent kinase 2 (CDK2) activity, and promotes G1 phase accumulation in human prostate cancer cells. These effects correlate with cytoplasmic relocalization of CDK2. In this study, we investigated the role of CDK2 cytoplasmic relocalization in the antiproliferative effects of 1,25-(OH)2D3. CDK2 was found to be necessary for prostate cancer cell proliferation. Although induced by 1,25-(OH)2D3, the cyclin-dependent kinase inhibitor p27KIP1 was dispensable for 1,25-(OH)2D3-mediated growth inhibition. Reduction in CDK2 activity by 1,25-(OH)2D3 was associated with decreased T160 phosphorylation, a residue whose phosphorylation in the nucleus is essential for CDK2 activity. Ectopic expression of cyclin E was sufficient to overcome 1,25-(OH)2D3-mediated cytoplasmic mislocalization of CDK2 and all antiproliferative effects of 1,25-(OH)2D3, yet endogenous levels of cyclin E or binding to CDK2 were not affected by 1,25-(OH)2D3. Similarly, knockdown of the CDK2 substrate retinoblastoma, which causes cyclin E up-regulation, resulted in resistance to 1,25-(OH)2D3-mediated growth inhibition. Human prostate cancer cells resistant to growth inhibition by 1,25-(OH)2D3 but retaining fully functional vitamin D receptors were developed. These cells did not exhibit 1,25-(OH)2D3-mediated cytoplasmic relocalization of CDK2. Targeting CDK2 to the nucleus of 1,25-(OH)2D3-sensitive cancer cells blocked G1 accumulation and growth inhibition by 1,25-(OH)2D3. These data establish central roles for CDK2 nuclear-cytoplasmic trafficking and cyclin E in the mechanism of 1,25-(OH)2D3-mediated growth inhibition in prostate cancer cells.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the most active metabolite of vitamin D, is best recognized for its pivotal role in regulating calcium and phosphorous homeostasis and skeletal mineralization (reviewed in Refs. 1 and 2). In addition to maintaining mineral balance, 1,25-(OH)2D3 promotes cell differentiation and inhibits proliferation of various normal and cancer cells (3,4). Epidemiologic studies have identified an inverse correlation between prostate cancer mortality and sunlight or UV B light exposure, which stimulates 1,25-(OH)2D3 synthesis (5,6). Furthermore, low serum 25-hydroxy vitamin D3 levels (commonly used to define overall vitamin D status) are associated with increased risk of prostate, breast, and colon cancers (5,6). 1,25-(OH)2D3 physiological functions are mediated by binding to the vitamin D receptor (VDR), a ligand-activated transcription factor, which forms heterodimers with retinoid X receptor (7,8). VDR/retinoid X receptor heterodimers bind preferentially to cis-acting DNA sequences known as vitamin D response elements. 1,25-(OH)2D3 binding to VDR results in the displacement of corepressor proteins such as SMRT and NCoR and the recruitment of chromatin-modifying coactivators to chromatin-bound VDR/retinoid X receptor resulting in modulation of gene transcription (8,9). 1,25-(OH)2D3 also elicits rapid signaling events that are termed nongenomic (reviewed in Ref. 10).

The most common mechanism for the antiproliferative effects of 1,25-(OH)2D3 is inhibition of cell cycle progression, particularly at the G1 to S phase transition (11). The cyclin-dependent kinase inhibitor (CKI) p21CIP1/WAF1/SDI1 is one vitamin D target in the growth inhibitory actions of 1,25-(OH)2D3 (12,13). However, 1,25-(OH)2D3 also has cell-specific effects on other G1 regulatory proteins including cyclin D1 (14), cyclin D3 (15), and the CKI p27KIP1 (13). In addition, 1,25-(OH)2D3 regulates the expression of a variety of signaling pathway modulators that impact cellular proliferation including cyclooxygenase-2 and 15-prostaglandin dehydrogenase (regulators of prostaglandin synthesis), as well as transforming growth factor β and IGF binding protein-3 (4,16,17,18). A more thorough understanding of the antiproliferative effects of 1,25-(OH)2D3 is imperative to employ vitamin D-based strategies most effectively for therapy and prevention.

Our group and others have demonstrated that 1,25-(OH)2D3 causes G1 accumulation in select human prostate carcinoma cells (13,19,20,21). Whereas VDR is necessary for the antiproliferative effects of 1,25-(OH)2D3, adequate levels of transcriptionally active VDR are not sufficient. In the human prostate cancer cell line, LNCaP, 1,25-(OH)2D3-mediated inhibition of cell proliferation correlates with relocalization of cyclin-dependent kinase 2 (CDK2) from nuclear to cytoplasmic compartments and a significant decrease in CDK2 activity (13,21). 1,25-(OH)2D3 treatment also increases the stability of the CKI p27 consistent with the observed decreased phosphorylation at Thr187. p27 stabilization by 1,25-(OH)2D3 may result from decreased CDK2-mediated phosphorylation, which results in impaired SKP2-mediated degradation of this CKI (13). Treatment of LNCaP, Hep-G2, and AT-84 cells with 1,25-(OH)2D3 or a vitamin D analog is associated with decreased levels of SKP2 (13,22,23). This effect may be secondary to G1 accumulation, which leads to rapid SKP2 degradation.

CDK2 is a member of the cyclin-dependent kinase family, which regulates G1 to S phase progression of the cell cycle. Activation of CDK2 requires binding to the regulatory subunit cyclin E and nuclear import of the CDK2/cyclin E complexes via direct interactions between cyclin E and the importin α/β system (24,25). Cyclin E binding and nuclear translocation of CDK2/cyclin E are necessary for CDK2 enzymatic activation and substrate targeting because the CDK2 activating enzyme (CAK) and the majority of CDK2 targets reside in the nucleus (26,27,28). CDK2 activity is regulated through three phosphorylation sites: T14, Y15, and T160 (29). Phosphorylation of CDK2 T160 by CAK, a nuclear protein, improves substrate recognition and binding (30). Dephosphorylation of Thr14 and Tyr15 by the phosphatase CDC25A also facilitates CDK2 activation (31). Active CDK2/cyclin E complexes phosphorylate several cellular proteins including retinoblastoma (Rb) (32). Phosphorylation of Rb by CDK2/cyclin E results in the release and activation of E2F transcription factors, which promote the transcription of genes that encode proteins required for G1 to S phase progression (33,34). Whereas the necessity of CDK2 for G1 to S phase transition was challenged by the discovery that CDK2 knockout mice are viable with no signs of developmental abnormalities except for meiotic failure (35), this finding does not preclude an important role of CDK2 for cell cycle progression in adults or in pathological conditions such as cancer. Consistent with the critical nature of CDK2 in cell cycle regulation, inhibition of CDK2/cyclin activity blocks cancer cell proliferation in many but not all cancer cell types (36,37,38,39). Furthermore, because CDK2 activity is frequently deregulated in cancer, this kinase continues to be a compelling therapeutic target (40,41).

In this study, we examined the role of CDK2 subcellular localization and activity in 1,25-(OH)2D3-mediated inhibition of cell proliferation and cell cycle progression in vitamin D-sensitive and resistant human prostate cancer cells. We found that CDK2 is essential for prostate cancer cell proliferation and that 1,25-(OH)2D3-mediated cytoplasmic relocalization of CDK2 is an indispensable component of the antiproliferative effects of 1,25-(OH)2D3. Surprisingly, although p27 is up-regulated by 1,25-(OH)2D3, this key cell cycle inhibitor was not required for prostate cancer cell growth inhibition by 1,25-(OH)2D3. In addition, we identified an important role for cyclin E in 1,25-(OH)2D3-mediated growth inhibition. Our data establish a central function for CDK2/cyclin E nuclear-cytoplasmic translocation in 1,25-(OH)2D3-mediated G1 phase accumulation and decreased cell proliferation.

Materials and Methods

Materials

1,25-(OH)2D3 was purchased from BioMol Research Laboratories (Plymouth Meeting, PA) and ethanol-based stock solutions were stored at −20 C. Cell culture media (RPMI 1640 and DMEM-high glucose) were obtained from Mediatech (Manassas, VA), and fetal bovine serum was from Hyclone (Logan, UT). CalPhos transfection kit and anti-myc antibody were obtained from Clonetech (Mountain View, CA). Trizol and Prolong antifade reagent were purchased from Invitrogen (Carlsbad, CA). Antihuman p27, Cdk2, protein A-agarose beads, antirabbit IgG, antigoat IgG, and antimouse IgG antibodies with horseradish peroxidase conjugate were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Flag antibody was purchased from Sigma-Aldrich (St. Louis, MO). Antihuman phosphor T160CDK2 was obtained from Cell Signaling Technology (Danvers, MA). Fluorescence-conjugated secondary antibodies, Nup358, and mAb414 antibodies were generously provided by Dr. Beatriz Fontoura (University of Texas Southwestern Medical Center, Dallas, TX).

Cell culture

The human prostate cancer cell line LNCaP (American Type Culture Collection, Manassas, VA) and the LNCaP Rb knockdown cells (42) were passaged and maintained in RPMI medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100 g/ml streptomycin, and 100 g/ml l-glutamine. The rodent oral cancer cell line AT-84 was obtained from Dr. Edward Shillitoe (Upstate Medical University, Syracuse, NY) and was passaged and maintained as described above for LNCaP cells. All of the cultures were maintained at 37 C in a humidified atmosphere of 5% CO2.

Western blot analysis

Western blots were done as previously described (13). Briefly, protein concentrations were determined by the Bio-Rad Dc Protein Assay (Hercules, CA) according to the manufacturer’s instructions. Fifty micrograms of cell extract proteins were subjected to standard SDS-PAGE and transferred to nitrocellulose membrane filters. The filters were processed for Western blotting using standard procedures. Incubation with the primary antibody was done overnight in milk. For phospho-specific antibodies, the filters were incubated overnight in 5% BSA, 0.1% Tween in PBS, and primary antibody. After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibody, and the proteins were visualized using the ECL system (Amersham Biosciences, Piscataway, NJ) following the supplier’s instructions. All quantification was done using Image J software (National Institutes of Health, Bethesda, MD).

Cell proliferation assays

LNCaP and AT-84 cells (selected for expression of indicated constructs or uninfected) were plated at an initial density of 40,000 or 25,000 cells per well in 6- or 12-well dishes, respectively (unless otherwise stated). The following day, cells were treated with ethanol vehicle or 50 nm 1,25-(OH)2D3 (unless otherwise stated). After indicated treatment times, cells were trypsinized and viable cells were counted using a hemocytometer. Trypan blue was used to exclude dead cells. All experiments were performed in triplicate.

In vitro Cdk2 kinase assay

Kinase activity assays were done as previously described (13). Briefly, LNCaP cells were treated with either ethanol vehicle or 1,25-(OH)2D3 for the indicated periods. The protein concentrations of the lysates were determined and 200 μg of proteins were then incubated with 3 μg of rabbit antihuman Cdk2 or rabbit antihuman cyclin E or IgG control antibodies for 1 h at 4 C. After this incubation, samples were incubated with 40 μl of antirabbit IgG-agarose beads (Sigma-Aldrich) for 1 h at 4 C. Immune complexes were incubated in 30 μl of kinase buffer containing 1 μg of histone H1, 25 μm ATP, and 10 μCi of [γ-32P]ATP for 30 min at 30 C. The reactions were stopped by the addition of 4× Laemmli buffer. Phosphorylated histone H1 was visualized by autoradiography. After the autoradiography, the membrane was subjected to Western blotting for Cdk2 and cyclin E.

RNA extraction and quantitative RT-PCR

Total RNA was collected using Trizol according to the manufacturer’s protocol (Invitrogen). Five hundred nanograms of total RNA were used for reverse transcription using cDNA Archive Kit as per the manufacturer’s protocol (Applied Biosystems, Foster City, CA). One hundred nanograms of cDNA were used for quantitative PCR of CYP24A1, IGFBP3, and TRPV6, and 1 ng for 18S rRNA. Taqman probes for CYP24A1 and 18S were used to perform real-time PCR using ABI Prism 7700 (Applied Biosystems). The comparative threshold cycle method was used to determine the relative mRNA expression level.

Flow cytometry

LNCaP cells (selected for expression of indicated constructs or uninfected) were treated with ethanol vehicle or 50 nm 1,25-(OH)2D3 for 72 h. Cells were then incubated with 10 μg/ml BrdU (BD Biosciences, San Jose, CA) for 3 h, trypsinized, and collected. Cells (~1 × 106) were fixed at 4 C in 70% ethanol and processed following the manufacturer’s protocol using anti-BrdU antibody (BD Biosciences). Cells were counterstained with 50 ng/ml propidium iodide and RNA destroyed using 1 mg/ml ribonuclease (Roche Laboratories, Indianapolis, IN). To determine DNA content, BrdU- and propidium iodide-stained cells were analyzed by flow cytometry using the FACScan (Becton Dickinson, San Jose, CA). Analysis was done on 10,000 total gated cells.

Constructs and lentiviral production

Short hairpin RNA (shRNA) retroviral construct targeting CDK2 (shCDK2) contained in pLL3.7 as well as the Δ8.9 and Ampho vectors were generously provided by Dr. B. Amati (European Institute of Oncology, Milan, Italy). shRNA-targeting p27 (shp27) was created using the pLL3.7 backbone and sequence targeting bp 141–159 of human p27 mRNA (5′-gcactgcagagacatggaa-3′) (43). CDK2-nuclear localization signal (NLS)-myc cDNA was generously provided by Dr. J. J. Baldassare (Saint Louis University Medical School, Saint Louis, MO). Cyclin E wild-type and Cyclin E kinase-deficient (KD) cells were generously given by Dr. B. E. Clurman (Fred Hutchinson Cancer Research Center, Seattle, WA). Gene expression cDNAs for green fluorescent protein (GFP), CDK2, CDK2-myc, T160ACDK2-NLS-myc, CDK2-NLS-myc, Cyclin E, Cyclin E KD, Cyclin A, Cyclin B1, and Cyclin D1 were cloned into pQCXIN from BD Biosciences by PCR and verified by sequencing. For viral production, GP2–293 cells at 60–80% confluency in 100-mm dishes were transfected with 7.5 μg VSV-G and 12.5 μg pQCXIN (containing appropriate cDNA) using a CalPhos kit (Clontech, Palo Alto, CA). For viral production of shRNA, constructs of HEK-293T cells at 60–80% confluency in 100-mm dishes were then transfected with 7.5 μg Ampho and Δ8.9 and 12.5 μg pLL3.7 (containing appropriate shRNA) using CalPhos kit (Clontech). Forty-eight hours after transfection, media containing viral particles were collected and filtered through a 0.45-μm cellulose acetate filter and stored at −80 C. For selection, cells were infected with appropriate constructs 24 h after seeding and cultured in 1 mg/ml G418 48 h after infection for 5–8 d.

Immunofluorescence staining

Immunofluorescence staining was done as previously described (13). Briefly, after treatment, cells were fixed with 4% paraformaldehyde and permeablized in 0.2% Triton X-100. The cells were then incubated for 1 h with a different combination of antibodies: rabbit anti-CDK2, mouse anti-myc antibody, and rabbit anti-FLAG antibody, respectively. Cells were incubated for 1 h with Cy3-conjugated and fluorescein isothiocyanate-conjugated antibodies. Coverslips were mounted on glass slides in ProLong Gold antifade reagent supplemented with 4,6-diamidino-2-phenylindole (Invitrogen). The slides were visualized and images were acquired using LSM confocal microscopy (Carl Zeiss International, Jena, Germany). The optimal exposure settings were determined by deducting the background staining from the control slides stained with only the secondary antibodies for each experiment.

Coimmunoprecipitation

After treatment with ethanol vehicle or 50 nm 1,25-(OH)2D3, cells were lysed in immunoprecipitation buffer containing 50 mm HEPES (pH 7.5), 40 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, 5% glycerol, 10 mm sodium pyrophosphate, 10 mm β-glycerolphosphate, and 50 mm NaF. Five hundred micrograms of protein extracts were first precleared by incubating with 3 μg of rabbit-IgG and 20 μl of 50% protein agarose beads in 500 μl IP buffer at 4 C with agitation for 1 h. Three micrograms of rabbit-anti-CDK2 antibody were conjugated to protein A-agarose beads by incubating for 1 h at 4 C with agitation. The precleared protein extract was mixed with rabbit-anti-CDK2 conjugated to protein A-agarose beads and incubated with agitation for 2 h at 4 C. After five washes with the immunoprecipitation buffer, 40 μl 2× Laemmli gel loading buffer was added to each sample. After boiling at 95 C for 10 min, the samples were subjected to SDS-PAGE and the protein levels were evaluated by Western blot analysis.

Results

CDK2 is necessary for maximal prostate cancer cell proliferation

The findings that CDK2 knockout mice exhibit no deficiencies in cell proliferation have brought into question the necessity of CDK2 for cell cycle progression. To evaluate the importance of CDK2 for prostate cancer cell proliferation, we knocked down CDK2 using an shRNA retroviral construct targeting CDK2 (shCDK2) and measured the growth of LNCaP human prostate cancer cells. LNCaP cells infected with an shRNA construct targeting the luciferase gene (shLuc) were used as control (Fig. 1A1A).). Quantification of Western blots showed that CDK2 levels were decreased approximately 40% in cells expressing shCDK2 compared with control shLuc (Fig. 1A1A).). We found a significant decrease in the proliferation of cells infected with shCDK2 compared with shLuc-infected control cells suggesting that prostate cancer cells are highly dependent on CDK2 for maximal proliferation (Fig. 1B1B).

Figure 1
CDK2 is necessary for maximal LNCaP cellular proliferation. LNCaP cells were infected with either shCDK2 or control shLuciferase. A, Cellular lysates were harvested and CDK2 and actin were detected by Western blotting. This is representative of four experiments. ...

p27 is not required for 1,25-(OH)2D3-mediated growth inhibition

We have previously shown that 1,25-(OH)2D3 increases levels of the cyclin-dependent kinase inhibitor p27 (Fig. 2A2A)) through protein stabilization (13). To determine whether p27 plays a prominent role in the antiproliferative effects of 1,25-(OH)2D3, cells were infected with a retrovirus containing a shp27 or shLuc (Fig. 2B2B).). Whereas depletion of p27 by 74% resulted in increased cell proliferation, 1,25-(OH)2D3 continued to inhibit cellular proliferation to the same extent in p27 knockdown cells (33 ± 5.0%) compared with controls (37 ± 0.5%) (Fig. 2C2C).). These data show that p27 up-regulation is not essential for 1,25-(OH)2D3-mediated growth inhibition.

Figure 2
p27 is not required for 1,25-(OH)2D3-mediated growth inhibition. A, LNCaP cells were treated with either 50 nm 1,25-(OH)2D3 or EtOH vehicle control for 72 h. Cellular lysates were harvested and p27 and actin were detected by Western blotting. B, LNCaP ...

1,25-(OH)2D3 reduces CDK2 phosphorylation at T160, a key residue for activation

We previously reported that 1,25-(OH)2D3 inhibits the growth of LNCaP human prostate cancer cells by promoting G1 accumulation, which is associated with decreased CDK2 activity but not reduced CDK2 levels (13,21). We further showed that 1,25-(OH)2D3 exposure resulted in relocalization of CDK2 to the cytoplasm (13). Because phosphorylation of CDK2 at T160 by CAK, a constitutively active and nuclear protein, improves CDK2/cyclin E substrate recognition and binding (30,44), we examined the effect of 1,25-(OH)2D3 on phosphorylation of this residue. Vitamin D treatment of LNCaP cells resulted in decreased CDK2 activity as we previously observed (Fig. 3A3A).). Treatment with 1,25-(OH)2D3 also caused a significant decrease in T160-phospho-CDK2 whereas no change in total CDK2 protein levels was observed (Fig. 3B3B).). These results are consistent with the possibility that phosphorylation of CDK2 by nuclear CAK is impeded by the cytoplasmic relocalization of CDK2 after 1,25-(OH)2D3 treatment.

Figure 3
1,25-(OH)2D3 reduces CDK2 activity and CDK2T160 phosphorylation. LNCaP cells were treated with either 50 nm 1,25-(OH)2D3 or EtOH vehicle control for 72 h. A, Cellular lysates were harvested and in vitro CDK2 kinase assays were performed using rabbit anti-CDK2 ...

Cyclin E expression overcomes 1,25-(OH)2D3-mediated antiproliferative effects

Due to the importance of cyclin E in promoting CDK2 activity and nuclear localization, we investigated the possibility that 1,25-(OH)2D3 interferes with CDK2/cyclin E interaction. As we previously showed, 1,25-(OH)2D3 treatment did not affect cyclin E protein levels (Fig. 4A4A)) (21). Coimmunoprecipitation experiments indicated that there was no decrease in CDK2/cyclin E association after 1,25-(OH)2D3 treatment (Fig. 4B4B).). CDK2/cyclin E association was also analyzed by mammalian two-hybrid assays, which confirmed that 1,25-(OH)2D3 did not influence CDK2/ cyclin E interaction (data not shown).

Figure 4
Cyclin E levels and CDK2/cyclin E binding are not affected by 1,25-(OH)2D3. LNCaP cells were treated with either 50 nm 1,25-(OH)2D3 or EtOH vehicle for 72 h. A, Cellular lysates were harvested and cyclin E and actin were detected by Western blotting. ...

Because CDK2 translocates to the nucleus as a complex with cyclin E, we used immunofluorescence staining and microscopy to determine CDK2 localization after ectopic expression of wild-type cyclin E. As shown in Fig. 55,, A and B, whereas 1,25-(OH)2D3 induced cytoplasmic relocalization of CDK2 in cells expressing GFP, this was not observed in LNCaP infected with a wild-type cyclin E construct. The ability of wild-type cyclin E to overcome CDK2 cytoplasmic relocalization led us to question whether ectopic expression of cyclin E would also block 1,25-(OH)2D3-mediated growth inhibition. Interestingly, cells expressing wild-type cyclin E were not growth inhibited by 1,25-(OH)2D3 (Fig. 5C5C).). To verify that these results were specific to cyclin E and its kinase-dependent function, proliferation of cells expressing a kinase-deficient mutant of cyclin E (KD-E) or wild-type cyclins A, B1, and D1 were examined. 1,25-(OH)2D3-mediated growth inhibition in KD-E-, cyclin A-, cyclin B1-, and cyclin D1-expressing cells was similar to control GFP cells (Fig. 5C5C).). Ectopic expression of cyclin E also reversed 1,25-(OH)2D3-mediated G1 accumulation (Fig. 5D5D).). Although 1,25-(OH)2D3 caused an 8% decrease in S phase and a comparable increase in G2/M in the cyclin-E-expressing cells, these effects were quite modest when compared with changes in cell cycle distribution in 1,25-(OH)2D3-treated control cells or cells expressing KD-E. To determine whether these effects might occur in another cell line that is growth inhibited by 1,25-(OH)2D3 and analogs (45) similar to LNCaP cells, we introduced cyclin E into AT-84 cells, a murine oral cancer cell line. Expression of cyclin E but not KD-E or GFP in AT-84 cells blocked 1,25-(OH)2D3-mediated growth inhibition (data not shown). Cyclin E did not alter VDR-mediated gene transcription as shown by 1,25-(OH)2D3-mediated regulation of VDR target gene expression (Fig. 5E5E).). Thus, disruption of VDR activity was not the cause of the blockade by cyclin E of 1,25-(OH)2D3-mediated growth inhibition. Taken together, these data affirm the importance of 1,25-(OH)2D3-mediated cytoplasmic relocalization of CDK2 and inhibition of CDK2 activity in the antiproliferative effects of 1,25-(OH)2D3 and show that cyclin E is central to these effects.

Figure 5
Cyclin E1 expression overcomes 1,25-(OH)2D3-mediated antiproliferative effects. A, LNCaP cells were infected with GFP or cyclin E1–3XFLAG lentiviral constructs. Forty-eight hours postinfection, cells were treated with either 50 nm 1,25-(OH)2D ...

Rb is necessary for 1,25-(OH)2D3-mediated antiproliferative effects

Phosphorylation of the pocket protein Rb by CDK2/cyclin E promotes G1 to S phase cell cycle progression (33,34). Because Rb is a major substrate of CDK2/cyclin E and knockdown of Rb in LNCaP cells results in the deregulation of a subset of Rb target genes (most notably cyclin E) (42), we asked whether Rb depletion would be sufficient to overcome 1,25-(OH)2D3-mediated antiproliferative effects. LNCaP cells stably expressing an shRNA construct targeting Rb showed no significant growth inhibition when treated with 1,25-(OH)2D3 compared with control cells (Fig. 6A6A).). As 1,25-(OH)2D3 exerts antiproliferative effects through VDR, we examined whether the lack of 1,25-(OH)2D3-mediated growth inhibition in the Rb-depleted cells was due to deregulated VDR activity. VDR transcriptional activity was retained in the Rb knockdown cells as expression of CYP24A1 was comparable in short hairpin Rb-expressing cells and in control cells (Fig. 6B6B).). These data further validate the importance of Rb and cyclin E in the antiproliferative response of prostate cancer cells to 1,25-(OH)2D3.

Figure 6
Rb knockdown overcomes 1,25-(OH)2D3-mediated growth inhibition. A, LNCaP cells expressing short hairpin RB or EV control were treated with either 50 nm 1,25-(OH)2D3 or EtOH vehicle for 72 h, and cell numbers were determined. Experiments were performed ...

Generation of a 1,25-(OH)2D3-resistant cell line

To investigate further the mechanisms of 1,25-(OH)2D3-mediated antiproliferative effects, we developed LNCaP cells resistant to the growth effects of 1,25-(OH)2D3 (LNCaP VitD.R). LNCaP VitD.R were obtained by continuous culture of LNCaP cells in medium supplemented with 10 nm 1,25-(OH)2D3. Control LNCaP cells (LNCaP Con.R) were cultured in parallel in vehicle. Whereas LNCaP VitD.R cells were not growth-inhibited by 50 nm 1,25-(OH)2D3 treatment, the same treatment led to over 67% growth inhibition of the control LNCaP Con.R cells (Fig. 7A7A).). Unlike LNCaP Con.R cells or parental LNCaP cells, LNCaP VitD.R cells showed no G1 accumulation after 50 nm 1,25-(OH)2D3 treatment (Fig. 7B7B).). As 1,25-(OH)2D3 exerts its antiproliferative effects through VDR, we examined whether this resistance was due to a decrease in VDR or deregulated VDR activity. We found that VDR was expressed at comparable levels in LNCaP VitD.R and LNCaP Con.R cells treated with 50 nm 1,25-(OH)2D3 (Fig. 7C7C).). VDR transcriptional activity was retained in VitD.R cells as 1,25-(OH)2D3 induced similar levels of CYP24A1 in the resistant and control Con.R cells (Fig. 7D7D).). Thus, the resistance of LNCaP VitD.R cells to growth inhibition by 1,25-(OH)2D3 does not appear to be due to down-regulation of VDR levels or VDR transcriptional activity. Although ectopic expression of cyclin E results in resistance to 1,25-(OH)2D3-mediated effects (Fig. 55),), the levels of cyclin E were comparable between LNCaP Con.R (sensitive) and LNCaP VitD.R (resistant) cells (data not shown). Thus, the resistance of LNCaP VitD.R cells is unlikely to be due to up-regulation of cyclin E.

Figure 7
Characterization of 1,25-(OH)2D3-resistant LNCaP cells. Cells sensitive (LNCaP Con.R) and insensitive (LNCaP VitD.R) to 1,25-(OH)2D3 were treated with either 50 nm 1,25-(OH)2D3 or EtOH vehicle for 5 d unless otherwise stated. A, Five days after treatment, ...

1,25-(OH)2D3-resistant cells do not exhibit 1,25-(OH)2D3-mediated CDK2 cytoplasmic relocalization or inhibition of CDK2 activity

Because 1,25 D-mediated growth inhibition is associated with CDK2 cytoplasmic relocalization and decreased CDK2 activity, we examined these events in LNCaP VitD.R cells. We found that 50 nm 1,25-(OH)2D3 treatment of LNCaP VitD.R cells did not affect the subcellular localization of CDK2, whereas significant cytoplasmic relocalization of CDK2 was seen in both LNCaP Con.R cells (Fig. 7E7E)) and LNCaP cells (data not shown). CDK2 activity in LNCaP VitD.R cells cultured in 1,25-(OH)2D3 was comparable to LNCaP Con.R cells treated with ethanol vehicle (data not shown). LNCaP VitD.R cells exhibited none of the previously observed 1,25-(OH)2D3 effects on CDK2 and this correlated with the resistance of these cells to 1,25-(OH)2D3 antiproliferative effects. These data support the contention that the antiproliferative effects of 1,25-(OH)2D3 occur through disruption of CDK2 localization and activity.

Nuclear targeting of CDK2 overcomes 1,25-(OH)2D3-mediated antiproliferative effects

To address more definitely the importance of CDK2 cytoplasmic relocalization in mediating 1,25-(OH)2D3 antiproliferative effects, a nuclear-targeted CDK2 construct containing full-length human CDK2 linked to three repeats of the simian virus 40 T-antigen nuclear localization signal and a myc tag (CDK2-NLS-myc) was used (Fig. 8A8A).). We first demonstrated that addition of the NLS repeats caused CDK2 protein to be expressed solely in nuclei even after 1,25-(OH)2D3 treatment (Fig. 8B8B).). We next examined the capacity of cells expressing CDK2-NLS-myc to overcome G1 attenuation and growth inhibition by 1,25-(OH)2D3. As expected in control GFP-infected cells, 1,25-(OH)2D3 treatment caused a significant increase in the percentage of cells in G1 phase of the cell cycle and a subsequent decrease of cells in S phase (Fig. 8C8C).). In contrast, cells expressing CDK2-NLS-myc protein showed similar cell cycle profiles in the presence and absence of 1,25-(OH)2D3. In addition, cells expressing CDK2-NLS-myc were virtually resistant to growth inhibition by 1,25-(OH)2D3 compared with control cells expressing GFP, CDK2-myc, or CDK2/T160A-NLS-myc, a mutant of CDK2 that cannot be activated (44) (Fig. 8D8D).). Similarly, expression of CDK2-NLS-myc in AT-84 cells significantly decreased 1,25-(OH)2D3-mediated growth inhibition (data not shown). Lastly, we confirmed that the activity of nuclear-targeted CDK2 was not inhibited by 1,25-(OH)2D3 treatment (Fig. 8E8E).). Collectively, these data suggest that the central mechanism by which 1,25-(OH)2D3 inhibits CDK2 activity and subsequent G1 accumulation and cell proliferation is by promoting CDK2 cytoplasmic relocalization.

Figure 8
Nuclear targeting of CDK2 overcomes 1,25-(OH)2D3-mediated antiproliferative effects. A, Schematic illustration of CDK2-NLS-myc and CDK2-myc constructs. B, G418 selected LNCaP/CDK2-NLS-myc and LNCaP-GFP cells were plated on coverslips for 24 h, 72 h after ...

Discussion

In this study, we show that 1,25-(OH)2D3-mediated regulation of CDK2 localization and activity were the critical triggers for the antiproliferative actions of this hormone in prostate cancer cells. Given the finding that compensatory mechanisms participate in G1 to S phase progression in CDK2 knockout mice and in some cancer cell lines depleted of CDK2, we first established the importance of CDK2 for LNCaP cell proliferation. Even relatively modest depletion of CDK2 in LNCaP cells had a pronounced growth repressive effect.

We previously demonstrated that 1,25-(OH)2D3 treatment of LNCaP cells resulted in increased levels of p27 but decreased p27 phosphosphorylation of Thr187. Because CDK2 mediates phosphorylation of p27-Thr187 leading to p27 degradation, we also demonstrated that increased p27 levels in response to 1,25-(OH)2D3 are due to p27 stabilization (13). In the present study, we examined whether elevated p27 was required for vitamin D-mediated growth inhibition. As expected, depletion of p27 leads to more rapid LNCaP cell proliferation; however, the cells retained full sensitivity to growth inhibition by 1,25-(OH)2D3. Based on these data and our previous findings, we conclude that increased p27 levels may be a result of CDK2 inhibition by 1,25-(OH)2D3, but this effect is not essential for growth inhibition by vitamin D. Clinically, decreased p27 levels in prostate tumors are associated with cancer recurrence and aggressiveness as well as decreased survival (46,47,48,49). One implication of our finding is that p27 status may not be informative with respect to sensitivity to the antitumor actions of vitamin D.

CDK2/cyclin E complexes phosphorylate and inactivate Rb thereby promoting G1 to S phase transition. 1,25-(OH)2D3 treatment inhibits CDK2 activity and results in hypophosphorylation of Rb and G1 accumulation (13). In the present study, we showed that Rb depletion, which causes a selective increase in cyclin E levels in LNCaP cells, results in resistance to growth inhibition by 1,25-(OH)2D3. These data support the critical importance of Rb and cyclin E in enforcing 1,25-(OH)2D3-mediated cell cycle inhibition.

Given the importance of CDK2 for prostate cancer cell proliferation and our previous finding that 1,25-(OH)2D3 decreased CDK2 activity and promoted relocalization of CDK2 from the nucleus to cytoplasm, we focused on the effects of vitamin D on CDK2 activation. Phosphorylation of CDK2-T160 by the nuclear complex CAK is essential for CDK2 activation. We found that 1,25-(OH)2D3 causes decreased phosphorylation of CDK2-T160 but no change in CDK2 levels. 1,25-(OH)2D3 inhibition of CAK levels or activity could explain the decrease in the activating phosphorylation of CDK2. However, CAK also activates CDK1 and CDK4/6 and we failed to see a substantial inhibition of the activation of these kinases by 1,25-(OH)2D3 (data not shown and Ref. 21). However, these results could be explained by decreased accessibility of CDK2 to the nuclear CAK as a result of CDK2 cytoplasmic relocalization in vitamin D-treated cells.

The intracellular trafficking of CDK2 is not well understood; however, studies suggest that cyclins may play a role in the translocation of the cyclin/CDK complex via the classical importin α/β pathway (24,25). Importin α binds to the nuclear localization signal on cyclins whereas importin β binds to nuclear transport machinery proteins as well as to cyclin-bound importin α. The cyclin/CDK/importin α/β complex is then translocated into the nucleus via an energy-dependent process. If this complex is disrupted directly or indirectly as a result of 1,25-(OH)2D3 treatment, it is possible that CDK2 nuclear transport would be diminished and CDK2 would accumulate in the cytoplasm. However, we did not detect disruption of the association between CDK2 and cyclin E with 1,25-(OH)2D3 treatment. Ectopic expression of wild-type cyclin E blocked 1,25-(OH)2D3-mediated growth inhibition, CDK2 cytoplasmic relocalization, and inhibition of CDK2 activity. The ability to overcome these effects of 1,25-(OH)2D3 was specific to cyclin E as overexpression of other cyclins did not block 1,25-(OH)2D3 mediated growth inhibition.

It is unclear whether 1,25-(OH)2D3 treatment reduces CDK2 nuclear import or accelerates CDK2 export. Because cyclin E protein levels and CDK2-cyclin E binding are unaffected by 1,25-(OH)2D3 treatment, other proteins are likely to be involved in CDK2 nuclear-cytoplasmic translocation. One protein that inhibits the activity of CDK2 as well as affects the subcellular localization of CDK2 and inhibits the nuclear translocation of c-ABL is 14-3-3σ (50,51). Levels of 14-3-3σ were not affected by 1,25-(OH)2D3 in prostate cancer cells (data not shown) although there is still the possibility that an isoform of 14-3-3 that may play a role in 1,25-(OH)2D3-mediated effects on CDK2.

Baldassare and colleagues identified a role for MAPK in CDK2 localization. In this study, inhibition of ERK1/2 kinase activity in fibroblasts results in the cytoplasmic mislocalization of CDK2, decreased phosphorylation of CDK2 Thr160, and decreased G1 to S phase progression (28,44). Moreover, the nuclear translocation of CDK2 is associated with the formation of molecular complexes containing active MAPK (26). Although 1,25-(OH)2D3 has been shown to inhibit ERK activation in certain cell types (52), we did not observe this effect in LNCaP cells (data not shown). Thus, it is unlikely that MAPK is involved in 1,25-(OH)2D3-mediated CDK2 cytoplasmic relocalization.

There have been a variety of proposed mechanisms through which 1,25-(OH)2D3 inhibits cell proliferation. 1,25-(OH)2D3 treatment has been shown to down-regulate c-myc mRNA and protein levels (53). Because c-myc promotes the transcription of cell cycle regulators such as CDC25A and cyclin D, lower c-myc protein levels would be expected to result in decreased levels of these G1 regulatory proteins. We saw no effect of 1,25-(OH)2D3 on cyclin D1 levels (21). CDC25A activates CDK2 by dephosphorylating CDK2 inhibitory sites Thr14 and Tyr15. We did not observe an increase in CDK2 Thr14 Tyr15 phosphorylation with 1,25-(OH)2D3 treatment (data not shown) indicating that decreased CDC25A levels are unlikely to explain 1,25-(OH)2D3 inhibition of CDK2 activity. A reduction in c-myc protein levels has also been shown to correlate with reduced E2F1 mRNA, which together with decreased CDK2 activity may contribute to decreased cell proliferation (53). The liberation of E2F family members from Rb by CDK2/cyclin E phosphorylation leads to transcription of E2F family members and c-myc genes in a positive feedback loop thus, it is possible that the reduction seen in both c-myc and E2F1 levels is the result and not the cause of reduced CDK2/cyclin E activity (54).

The establishment of 1,25-(OH)2D3-resistant LNCaP VitD.R cells provides an invaluable tool for understanding 1,25-(OH)2D3 mechanisms of action and supports the critical nature of CDK2 relocalization in the antiproliferative effects of 1,25-(OH)2D3. The resistance of LNCaP VitD.R cells to 1,25-(OH)2D3-mediated antiproliferative effects correlates with the ability of these cells to maintain CDK2 nuclear localization and activity. Furthermore, VDR is expressed and is transcriptionally active in these resistant cells. Interestingly, LNCaP VitD.R cells grow more slowly than control LNCaP Con.R even in the absence of 1,25-(OH)2D3. This difference in growth rate is maintained after prolonged culture of the resistant cells without 1,25-(OH)2D3 and the growth of VitD.R cells remains unaffected by 1,25-(OH)2D3 (data not shown). Thus, the growth rate of the resistant cells appears to be stably altered by the prolonged culture in 1,25-(OH)2D3.

Differential gene expression and epigenetic modifications may contribute to 1,25-(OH)2D3 resistance in VitD.R cells. CDK2 phosphorylation of histones has been shown to alter chromatin structure, gene transcription, and cell cycle progression (55). Furthermore, aberrant DNA methylation is often detected in cancer cell lines and tissues including prostate cancer (56). Understanding the mechanisms by which 1,25-(OH)2D3 exerts its antiproliferative effects in prostate cancer is essential for effective targeted therapy and identifying susceptibility to 1,25-(OH)2D3 effects. This knowledge may perhaps contribute to optimal use of vitamin D, which has been shown to increase survival in prostate cancer patients when combined with chemotherapeutic drugs such as docetaxel (57).

Acknowledgments

We thank Drs. B. Amati (European Institute of Oncology, Milan, Italy), J. J. Baldassare (St. Louis University Medical School, St. Louis, Missouri), B. E. Clurman (Fred Hutchinson Cancer Research Center, Seattle, Washington), E. Shillitoe (Upstate Medical University, Syracuse, New York), and B. Fontoura (University of Texas Southwestern Medical Center, Dallas, Texas) for generously providing reagents. We also thank Drs. Adena Rosenblatt, Shuyun Rao, Fayi Wu, and Ines Garcia, as well as Carol Maiorino for their advice and assistance (University of Miami Miller School of Medicine, Miami, Florida). We are grateful to Dr. Wayne Balkan for help with the figures (Department of Medicine, University of Miami Miller School of Medicine; GRECC and Research Services, Miami Veterans Affairs Medical Center, Miami, Florida).

Footnotes

This work was supported by National Institutes of Health Grant CA107705 (to K.L.B.) and the James and Esther King Biomedical Research Program. O.F. was supported by NIH training grant T32-HL007188.

Disclosure Summary: The authors have nothing to disclose.

First Published Online February 10, 2010

Abbreviations: CAK, CDK2 Activating enzyme; CDK2, cyclin-dependent kinase 2; CKI, cyclin-dependent kinase inhibitor; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; GFP, green fluorescent protein; KD, kinase-deficient; KD-E, kinase-deficient mutant of cyclin E; LNCaP Con.R, control LNCaP cell; NLS, nuclear localization signal; Rb, retinoblastoma; shCDK2, shRNA retroviral construct targeting CDK2; shLuc, shRNA construct targeting the luciferase gene; shp27, shRNA-targeting p27; shRNA, short hairpin RNA; VDR, vitamin D receptor.

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