The E3 ligase and tumor suppressor FBW7 targets drivers of cell-cycle progression such as the oncogenic transcription factor c-MYC, for proteasomal degradation. Vitamin D signaling regulates c-MYC expression and turnover in vitro and in vivo, which is highly significant as epidemiologic data link vitamin D deficiency to increased cancer incidence. We hypothesized that FBW7 and the vitamin D receptor (VDR) controlled each other's function as regulators of protein turnover and gene transcription, respectively. We found that hormonal 1,25-dihydroxyvitamin D3 (1,25D) rapidly enhanced the interaction of FBW7 with VDR and with c-MYC, whereas it blocked FBW7 binding to c-MYC antagonist MXD1. 1,25D stimulated the recruitment of FBW7, SCF complex subunits, and ubiquitin to DNA-bound c-MYC, consistent with 1,25D-regulated c-MYC degradation on DNA. 1,25D also accelerated the turnover of other FBW7 target proteins such as Cyclin E, c-JUN, MCL1, and AIB1, and, importantly, FBW7 depletion attenuated the 1,25D-induced cell-cycle arrest. Although the VDR contains a consensus FBW7 recognition motif in a VDR-specific insertion domain, its mutation did not affect FBW7–VDR interactions, and FBW7 ablation did not stabilize the VDR. Remarkably, however, FBW7 is essential for optimal VDR gene expression. In addition, the FBW7 and SCF complex subunits are recruited to 1,25D-induced genes and FBW7 depletion inhibited the 1,25D-dependent transactivation. Collectively, these data show that the VDR and FBW7 are mutual cofactors, and provide a mechanistic basis for the cancer-preventive actions of vitamin D.

Implications:

The key findings show that the VDR and the E3 ligase FBW7 regulate each other's functions in transcriptional regulation and control of protein turnover, respectively, and provide a molecular basis for cancer-preventive actions of vitamin D.

Visual Overview:http://mcr.aacrjournals.org/content/17/3/709/F1.large.jpg.

Components of the ubiquitin proteasome system (UPS) are targeted frequently by oncogenic mutations (1–3). SCFs (Skp1, Cullin-1, F box protein) are a class of E3 ligases of the UPS in which Cullin-1 is a scaffold and the F-box protein is the substrate recognition subunit. The F-box protein FBW7 (FBXW7/Sel-10/Ago/hCDC4) is one of the most commonly dysregulated UPS proteins in cancer and is a tumor suppressor (3–5). It targets several proteins that drive cell-cycle progression through recognition of “phospho-degron” motifs (consensus T/S-P-X-X-S/T/E), which are phosphorylated by GSK3β (6, 7). It is expressed in multiple isoforms of which α and γ are nuclear (8). Multiple FBW7 target proteins are oncogenes, including cyclin E, the transcriptional coactivator AIB1 (amplified in breast cancer 1; a.k.a. SRC3, ACTR), c-JUN, and the apoptosis regulator MCL1 (myeloid cell leukemia 1; refs. 9, 10). The best characterized FBW7 substrate is the transcription factor c-MYC, whose expression is dysregulated in approximately 50% of all cancers (11). Downregulation of FBW7 results in accumulation of cellular and chromatin-bound c-MYC (12). c-MYC activity can be antagonized by MXD1 (13), which, like c-MYC, binds cognate E-boxes on DNA as a heterodimer with MAX (13–15).

We have been investigating the mechanisms of cell-cycle arrest by hormonal vitamin D, 1,25-dihydroxyvitamin D (1,25D), and previously found that 1,25D treatment dramatically reduces expression of c-MYC protein partly by reducing its gene expression approximately 2-fold and partly by accelerating its proteasomal turnover (16). Loss of c-MYC expression in 1,25D-treated human cells was very robust, occurring in multiple cell lines and in primary cultures, and the effect of 1,25D on c-MYC turnover was strongly attenuated by ablation of FBW7. Conversely, the effects of 1,25D signaling on MXD1 expression were diametrically opposite, with a modest increase in MXD1 gene expression in 1,25D-treated cells and a reduction in FBW7-dependent proteasomal turnover (16). Collectively, these effects led to dramatic changes in the ratio of c-MYC/MXD1 in 1,25D-treated cells in vitro. Moreover, as predicted from in vitro studies, c-MYC expression was elevated in vivo in multiple tissues in Vdr −/− mice, notably in skin and intestine, and expression of MXD1 was diminished (16). These findings are intriguing because numerous studies have linked vitamin D deficiency, which is widespread (17, 18), to the increased incidence of cancers (19–21). Notably, vitamin D sufficiency was linked in a large prospective study with reduced incidence and mortality of several malignancies, in particular digestive cancers, including head and neck squamous cell carcinomas (HNSCC; ref. 19). Our previous work has demonstrated the efficacy of vitamin D analogues in arresting HNSCC cell proliferation in vitro and tumor growth in vivo (22, 23).

1,25D signaling is mediated by the vitamin D receptor (VDR), a nuclear receptor and ligand-regulated transcription factor. The α-helical ligand-binding domain (LBD) controls the transcriptional regulation by the VDR (24, 25). The LBD also contains a unique 23-residue “insertion” domain (26). In studies presented below, we also find that, although the VDR insertion domain contains a motif corresponding to a consensus phospho-degron, the sequence does not appear to control interaction of the VDR with FBW7, nor does its mutation affect the stability of the VDR, indicating that the VDR is not a typical FBW7 target protein.

In this study, our working hypothesis was that the VDR and FBW7 interact functionally in a hormone-dependent manner, leading to the 1,25D-dependent turnover of multiple FBW7 target proteins, and that these interactions are an important component of the capacity of 1,25D to control cell proliferation. We found that the VDR and FBW7 are mutual cofactors in control of proteasomal turnover of FBW7 target proteins, and, unexpectedly, in 1,25D-dependent transcriptional regulation by the DNA-bound VDR. Collectively, our results strongly suggest that the functional interaction of the VDR with FBW7 is a fundamental component of the anticancer properties of 1,25D signaling.

Cell culture

SCC25 cells were obtained from the ATCC and cultured in DMEM/F12 (319-085-CL, Wisent Bioproducts) supplemented with 10% FBS. HaCaT cells (gift from the laboratory of Dr. Jean-Jacques Lebrun, McGill University Montreal, Quebec, Canada), which are immortalized keratinocyte cells from adult human skin, were cultured in DMEM (319-005-CL, Wisent Bioproducts) supplemented with 10% FBS. SCC25 cells with stable expression for tagged-FBW7 were generated using LeGO-iG2-Flag-FBW7, LeGO-iG2-HA-FBW7, and pLVX-IRES-Hyg-V5-FBW7 vectors. HEK 293-T17 (gift from the laboratory of Dr. Jerry Pelletier, McGill University, Montreal, Quebec, Canada) were cultured in DMEM (319-005-CL, Wisent Bioproducts) supplemented with 10% FBS. Primary Human keratinocytes (HEK-a) were obtained from ScienCell (#2100) and cultured in keratinocytes basal medium (#2101, ScienCell) supplemented with keratinocyte growth supplement (# 2152, ScienCell). On the basis of the company protocol, plates were coated with poly-l-Lysine (1 mg/mL; #0403, ScienCell).

Reagents

1α,25-Dihydroxyvitamin D3 (BML-DM200) was purchased from Enzo Life Sciences. Cycloheximide (C7698) and MG-132 (M7449) were purchased from Sigma. Bortezomib (504314) was from EMD Millipore.

Plasmids

In this study, the following constructs were generated: pcDNA 3.1-MXD1, pcDNA 3.1-MYC, pGEX-4T3-VDR, LeGO-iG2-Flag-FBW7, LeGO-iG2-HA-FBW7, pLVX-IRES-Hyg-V5-FBW7, pLVX-IRES-Hyg-HA-VDR, and pLVX-IRES-Hyg-HA-mVDR (TPSFS > AAAAA).

FBW7 knockdown

SCC25 cells were transfected with SMARTpool: ON-TARGETplus FBXW7 siRNA L-004264-00-0005 or scrambled siRNA (Dharmacon) for 24 hours, using Pepmute transfection reagent (SL100566) from SignaGen. The final concentration of siRNAs was 10 nmol/L.

Immunoprecipitation and Western blot analysis

Cells were lysed with a lysis buffer (20 mmol/L Tris, pH 7.5, 100 mmol/L NaCl, 0.5% Nonidet P-40, 0.5 mmol/L EDTA, 0.5 mmol/L phenylmethylsulfonyl fluoride). For coIP assays, 4 ng of antibody were prebound for 2 hours to Dynabeads protein A, then was washed with lysis buffer, and added to the lysate, followed by immunoprecipitation overnight. Beads were then washed five times with washing buffer (20 mmol/L Tris, pH 7.5, 200 mmol/L NaCl, 1% Nonidet P-40, 0.5 mmol/L EDTA, 0.5 mmol/L phenylmethylsulfonyl fluoride) and processed for Western blotting, performed with standard protocols. The following antibodies were used for Western blot analysis and were diluted into 2.5% milk as follows: VDR (D-6, Santa Cruz Biotechnology) 1/500; MXD1(c-19, Santa Cruz Biotechnology) 1/1,000; HA (Y-11, Santa Cruz Biotechnology) 1/500; c-Jun (H-79, Santa Cruz Biotechnology) 1/1,000; Cyclin E (M-20, Santa Cruz Biotechnology) 1/500; Lamin-A (H-102, Santa Cruz Biotechnology) 1/1,000; AIB1 (5E11, Cell Signaling Technology) 1/1,000; c-MYC (D84C12, Cell Signaling Technology) 1/1,000; and Flag (M2, Sigma) 1/500.

ChIP and re-ChIP assays

Chromatin immunoprecipitation (ChIP) assays were performed as described previously (16). DNA fragments were purified with a PCR purification Kit (Qiagen) and were analyzed by SsoFast-EvaGreen RT-PCR. For re-ChIP assays, the ChIPed immunocomplexes were eluted by adding 40 μL 10 mmol/L DTT for 30 minutes at 37°C. Supernatants were diluted 1:40 in dilution buffer (150 mmol/L NaCl, 1% Triton X-100, 2 mmol/L EDTA and 50 mmol/L Tris-HCl, pH 8), and were used for the second round of ChIP assay. Four nanograms of antibodies HA (Y-11), MXD1 (c-19), and VDR (D-6) from Santa Cruz Biotechnology; V5 (R960-25) from Thermo Fisher Scientific; c-MYC (9402) from Cell Signaling Technology; RBX1 (ab133565) and SKP1 (ab76502) from Abcam; UB (FK2) from Enzo Life Sciences were used in ChIP and reChIP assays.

GST pull-down assay

GST pull-down assays were performed using the MagneGST Pull-Down System (Promega) according manufacturer's instruction. BL21 bacteria were transformed with appropriate pGEX4T3 constructs and induced to express GST or GST fusion proteins. The total lysates of bacteria were used to pull-down in vitro translated proteins. The pulled down proteins were analyzed by Western blot assay.

Proliferation assay

Proliferation assays were performed by using Click-iT EdU Alexa Fluor 647 High-Throughput Imaging Assay Kit (Invitrogen) according to the manufacturer's protocol. SCC25 cells were transfected with FBW7 or scrambled siRNAs and treated with 1,25D for 24 hours. Cells were incubated with Alexa-EdU for 1 hour and fixed with 3.7% formaldehyde. DAPI was used to stain the nucleus. Images were captured by high-content screening microscope using ImageXpressMicro program and analyzed with MetaXpress.

qRT-PCR

qRT-PCR was performed with BrightGreen Express 2X qPCR MasterMix-ROX (Abmgood). Expression was normalized to the expression of Actin. Primers for mRNA expression are provided in Supplementary Table S1.

Statistical analysis

All experiments are representative of 3–5 biological replicates. Statistical analysis (Student t test) was conducted using R (version 3.2.3) statistical software package; P values are as follows: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

1,25D-Dependent interactions of the VDR, c-MYC, and FBW7

C-MYC expression is elevated in skin in Vdr-null mice (16), and we confirmed that FBW7 ablation enhanced the c-MYC expression and attenuated 1,25D-induced turnover in well-differentiated SCC25 squamous carcinoma cells and in primary human keratinocytes (Supplementary Fig. S1A and S1B). We hypothesized that the hormone-bound VDR may act as an FBW7 cofactor, at least partly, by regulating access of FBW7 to c-MYC. In control experiments, the VDR was predominantly nuclear and treatment with 1,25D led to a rapid transfer of residual protein to the nucleus. C-MYC was essentially nuclear and short-term 1,25D treatment diminished the c-MYC protein levels, as expected, in the absence of any substantial nucleo-cytoplasmic shift (Supplementary Fig. S2A and S2B). Coimmunoprecipitation (co-IP) experiments revealed partially hormone-dependent association of the VDR and c-MYC (Fig. 1A). Similarly, GST pull-down assays showed partially hormone-dependent binding of c-MYC to GST-VDR (Fig. 1B). Importantly, co-IP of lentivirally transduced Flag-FBW7 with c-MYC was enhanced by 1,25D (Fig. 1C; note that commercial antibodies do not reliably recognize endogenous FBW7). Turnover of c-MYC antagonist MXD1 is also regulated by FBW7. However, 1,25D treatment stabilizes MXD1 protein expression, and enhances its DNA binding (16). Under the conditions where MXD1 levels were increasing in the presence of 1,25D (Supplementary Fig. S3A), its association with the VDR over 8 hours was largely unchanged but declined at 24 hours (Supplementary Fig. S3B). In the absence of 1,25D, MXD1 expression levels are low (Supplementary Fig. S3A–S3C). However, we were able to detect co-IP of Flag-FBW7 with MXD1 in vehicle-treated cells, which was disrupted by addition of 1,25D (Supplementary Fig. S3C) under the conditions of increasing MXD1 expression. These effects of 1,25D on the association of FBW7 with c-MYC and MXD1 are consistent with the destabilization of c-MYC and stabilization of MXD1. To probe the interaction of tagged FBW7 with the endogenous VDR, we performed co-IPs of transduced SCC25 cells, which revealed a rapid 1,25D-dependent association of the two proteins (Fig. 1D and E).

Figure 1.

1,25D-dependent interactions of the VDR, c-MYC, and FBW7. A, Western blot analysis of VDR co-IPed with IgG or c-MYC following treatment of SCC25 cells with 100 nmol/L 1,25D. B, Left, Western blot analysis of c-MYC pulled down by GST or GST-VDR −/+1,25D in the reaction; 1,25D was added to the pull-down reaction. Right, Western blot analysis of GST and GST-VDR as input. C, Western blot analysis of Flag-FBW7 Co-IPed with IgG or c-MYC, following treatment of SCC25 cells with 1,25D. Cells were transfected with empty vector or Flag-FBW7 and treated with 100 nmol/L of 1,25D for 6 hours. c-MYC was immunoprecipitated from SCC25 lysate. The total lysate as “input” and immunoprecipitations (IP) were probed for Flag. D (left) and E, Western blot analysis of Flag-FBW7 co-IPed with IgG or VDR, following treatment of SCC25 cells with 100 nmol/L of 1,25D, as indicated. The total lysate as “input” and immunoprecipitations were probed for Flag. The total lysate as “input” was probed for VDR. D, Quantification of Western blot analysis of Flag-FBW7 co-IPed with IgG or VDR (right).

Figure 1.

1,25D-dependent interactions of the VDR, c-MYC, and FBW7. A, Western blot analysis of VDR co-IPed with IgG or c-MYC following treatment of SCC25 cells with 100 nmol/L 1,25D. B, Left, Western blot analysis of c-MYC pulled down by GST or GST-VDR −/+1,25D in the reaction; 1,25D was added to the pull-down reaction. Right, Western blot analysis of GST and GST-VDR as input. C, Western blot analysis of Flag-FBW7 Co-IPed with IgG or c-MYC, following treatment of SCC25 cells with 1,25D. Cells were transfected with empty vector or Flag-FBW7 and treated with 100 nmol/L of 1,25D for 6 hours. c-MYC was immunoprecipitated from SCC25 lysate. The total lysate as “input” and immunoprecipitations (IP) were probed for Flag. D (left) and E, Western blot analysis of Flag-FBW7 co-IPed with IgG or VDR, following treatment of SCC25 cells with 100 nmol/L of 1,25D, as indicated. The total lysate as “input” and immunoprecipitations were probed for Flag. The total lysate as “input” was probed for VDR. D, Quantification of Western blot analysis of Flag-FBW7 co-IPed with IgG or VDR (right).

Close modal

FBW7 target proteins controlling cell-cycle progression are regulated by 1,25D

Results presented above show that the hormone-bound VDR enhances the association of FBW7 and c-MYC and blocks that with MXD1, and suggest that the VDR may alter the expression of other FBW7 target proteins. We examined the effect of 1,25D treatment on FBW7-regulated proteins, AIB1, Cyclin E, MCL1, and c-JUN (9, 10). In all cases, 1,25D treatment strongly reduced the protein expression (Fig. 2A and B). Under the same conditions, 1,25D treatment only modestly affected the corresponding mRNA levels (Fig. 2C). Significantly, the 1,25D-dependent loss in AIB1 and Cyclin E expression in SCC25 cells was blocked by ablation of FBW7 expression (Fig. 2D).

Figure 2.

Regulation of FBW7 target proteins by 1,25D treatment. A, Western blot analysis of AIB1, Cyclin E, MCL1, and c-JUN levels in SCC25 cells following treatment with vehicle or 100 nmol/L 1,25D. B, Western blot analysis of AIB1 in HaCaT cells following treatment with 100 nmol/L 1,25D for 24 hours. C, qRT-PCR analysis of FBW7 target genes, AIB1, CCNE1, MCL1, and JUN, transcription in SCC25 cells following treatment with 100 nmol/L 1,25D. D, Western blot analysis of AIB1 and Cyclin E levels in SCC25 cells transfected with scrambled or FBW7 siRNA and treated with vehicle or 100 nmol/L of 1,25D.

Figure 2.

Regulation of FBW7 target proteins by 1,25D treatment. A, Western blot analysis of AIB1, Cyclin E, MCL1, and c-JUN levels in SCC25 cells following treatment with vehicle or 100 nmol/L 1,25D. B, Western blot analysis of AIB1 in HaCaT cells following treatment with 100 nmol/L 1,25D for 24 hours. C, qRT-PCR analysis of FBW7 target genes, AIB1, CCNE1, MCL1, and JUN, transcription in SCC25 cells following treatment with 100 nmol/L 1,25D. D, Western blot analysis of AIB1 and Cyclin E levels in SCC25 cells transfected with scrambled or FBW7 siRNA and treated with vehicle or 100 nmol/L of 1,25D.

Close modal

Loss of FBW7 expression affects VDR gene expression but not protein stability

The VDR ligand–binding domain contains a linker, or insertion domain, between helices 1 and 3 of 72 to 81 amino acids that is much longer than comparable regions of other nuclear receptors (15–25 a.a.; ref. 27). Remarkably, analysis of the human VDR amino acid sequence revealed a consensus FBW7 phospho-degron in this region (Fig. 3A), which is conserved in primates, whereas a near-consensus sequence exists in mice and rats (Supplementary Fig. S4A). The presence of the motif raises the possibility that, although 1,25D influences FBW7 association with its targets, VDR stability may be regulated in a negative-feedback loop by FBW7. However, mutation of the motif had no effect on the 1,25D-dependent interactions of the VDR and FBW7 (Fig. 3B). Intriguingly, the depletion of FBW7 (e.g., see Fig. 3D) led to a substantial reduction in VDR protein, an effect partially alleviated by 1,25D (Fig. 3C). VDR turnover in cycloheximide-treated cells was unaffected or modestly accelerated by FBW7 depletion (Supplementary Fig. S4B and S4C). 1,25D may stabilize the VDR in FBW-depleted cells, although the effect was not statistically significant (Supplementary Fig. S4D). These observations are not consistent with FBW7 recognizing the phospho-degron motif or with the VDR acting as a typical substrate. It is not clear from structural studies what the conformation of the VDR-specific insertion region is; however, our data suggest that the phospho-degron is sequestered and that FBW7 interacts with other site(s) on the VDR LBD. Significantly, FBW7 ablation did lead to an approximately 2-fold reduction in VDR transcript levels (Fig. 3D). To confirm this finding in the primary cells, FBW7 was depleted in human keratinocytes, with very similar effect of VDR mRNA and protein expression (Supplementary Fig. S5A and S5B). These results suggest that FBW7 may control the turnover of factor(s) that suppress VDR mRNA expression, and that diminished VDR mRNA strongly contributes to the loss of VDR protein in the absence of FBW7.

Figure 3.

The VDR is not a typical FBW7 substrate. A, Schematic representation of the VDR and location of the putative phospho-degron motif TPSFS (left). Right, tertiary structure of VDR/RXR showing the phospho-degron in the insertion domain. B, Western blot analysis of Flag-FBW7 co-IPed with IgG, VDR, or mutated VDR (TPSFS > AAAAA), following treatment of SCC25 cells with 100 nmol/L of 1,25D for 4 hours. Total lysate was probed for Flag or VDR as input. C, Western blot analysis of VDR in FBW7-ablated SCC25 cells (left). Cells were transfected for scrambled or FBW7 siRNA and treated with 100 nmol/L 1,25D. Actin was used as an internal control. Right, quantification of Western blot analysis of VDR. D, qRT-PCR assay of FBW7 and VDR mRNA expression after knockdown of FBW7 in SCC25 cells and treatment with 100 nmol/L of 1,25D (**, P ≤ 0.01; ***, P ≤ 0.001).

Figure 3.

The VDR is not a typical FBW7 substrate. A, Schematic representation of the VDR and location of the putative phospho-degron motif TPSFS (left). Right, tertiary structure of VDR/RXR showing the phospho-degron in the insertion domain. B, Western blot analysis of Flag-FBW7 co-IPed with IgG, VDR, or mutated VDR (TPSFS > AAAAA), following treatment of SCC25 cells with 100 nmol/L of 1,25D for 4 hours. Total lysate was probed for Flag or VDR as input. C, Western blot analysis of VDR in FBW7-ablated SCC25 cells (left). Cells were transfected for scrambled or FBW7 siRNA and treated with 100 nmol/L 1,25D. Actin was used as an internal control. Right, quantification of Western blot analysis of VDR. D, qRT-PCR assay of FBW7 and VDR mRNA expression after knockdown of FBW7 in SCC25 cells and treatment with 100 nmol/L of 1,25D (**, P ≤ 0.01; ***, P ≤ 0.001).

Close modal

FBW7 as a cofactor on VDR target genes

Given the above findings, we examined the potential role of FBW7 in VDR-dependent transactivation. FBW7 depletion compromised 1,25D-dependent induction of direct VDR target genes over 6 hours (Fig. 4A), and VDR DNA binding was substantially diminished (Supplementary Fig. S6A). We also observed 1,25D-dependent recruitment of tagged FBW7 and endogenous SCF FBW7 complex subunits, SKP1 and RBX1, to VDR target genes by ChIP (Fig. 4B–D). This suggests that in addition to maintaining expression of the VDR, FBW7 and SCF complex subunits function as cofactors in VDR-dependent transactivation. Indeed, we found that proteasome inhibitors MG132 and bortezomib inhibited the 1,25D-dependent transactivation of CYP24A1 and CAMP (Fig. 4E and F). Thus, E3 ligase complex recruitment and function is an integral part of the VDR-dependent transactivation.

Figure 4.

FBW7 as a cofactor on VDR target genes. A, qRT-PCR of VDR target gene transcription in SCC25 cells transfected with scrambled or FBW7 siRNA and treated with vehicle or 100 nmol/L 1,25D. B, Analysis of V5-FBW7 recruitment to VDREs by ChIP-qPCR in V5-FBW7 SCC25 cells treated with vehicle or 100 nmol/L of 1,25D (1 and 2 in CYP24 ChIP-qPCR refers to two different VDREs in the promoter of CYP24). C and D, Analysis of recruitment of SKP1 and RBX1 to VDREs by ChIP-qPCR in SCC25 cells treated with vehicle or 100 nmol/L 1,25D. E and F, qRT-PCR analysis of VDR target genes transcription in SCC25 cells in the absence or presence of proteasome inhibitors, 10 μmol/L of MG-132 (E) or 5 nmol/L of bortezomib (F; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Figure 4.

FBW7 as a cofactor on VDR target genes. A, qRT-PCR of VDR target gene transcription in SCC25 cells transfected with scrambled or FBW7 siRNA and treated with vehicle or 100 nmol/L 1,25D. B, Analysis of V5-FBW7 recruitment to VDREs by ChIP-qPCR in V5-FBW7 SCC25 cells treated with vehicle or 100 nmol/L of 1,25D (1 and 2 in CYP24 ChIP-qPCR refers to two different VDREs in the promoter of CYP24). C and D, Analysis of recruitment of SKP1 and RBX1 to VDREs by ChIP-qPCR in SCC25 cells treated with vehicle or 100 nmol/L 1,25D. E and F, qRT-PCR analysis of VDR target genes transcription in SCC25 cells in the absence or presence of proteasome inhibitors, 10 μmol/L of MG-132 (E) or 5 nmol/L of bortezomib (F; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Close modal

VDR and FBW7 are corecruited to E-box motifs of c-MYC target genes

Our previous studies detected association of the VDR with promoters of c-MYC target genes (16). Because FBW7 is recruited to and is required for 1,25D-stimulated expression of VDRE-containing genes by VDR, we analyzed the hormone-dependent recruitment of FBW7 to c-MYC target genes, whose expression is repressed by 1,25D. Short-term treatment with 1,25D reduced the c-MYC recruitment to the CDC25A and CDK4 promoters (Fig. 5A) and increased that of MXD1 (Fig. 5B). Under the same conditions, association of tagged FBW7 with the same loci was detected by ChIP, with little apparent effect of 1,25D (Fig. 5C). ChIP assays also revealed an association of SCF complex subunits SKP1 and RBX1 with the CDC25A and CDK4 loci (Supplementary Fig. S7A and S7B) similar to total FBW7 (Fig. 5C). Significantly, SKP1 recruitment was diminished by the depletion of endogenous FBW7, consistent with its association with c-MYC target genes being dependent on recruitment of FBW7 (Supplementary Fig. S7C).

Figure 5.

VDR and FBW7 are corecruited to E-box motifs of c-MYC target genes. Analysis of c-MYC (A) and MXD1 (B) recruitment to E-boxes of target genes by ChIP-qPCR in SCC25 cells treated with vehicle or 100 nmol/L 1,25D for 6 hours. C, Analysis of HA-FBW7 recruitment to E-box motifs by ChIP-qPCR in HA-FBW7 SCC25 cells treated with 100 nmol/L 1,25D for 6 hours. D, Corecruitment of HA-FBW7 and c-MYC or MXD1 to E-box motif of CDC25A by re-ChIP in SCC25 cells after 6-hour 1,25D treatment. The first round of ChIP was for HA-FBW7, followed by second round of immunoprecipitation for c-MYC or MXD1. E, Analysis of the corecruitment of HA-FBW7 and VDR to E-box motifs of CDC25A and CDK4 by re-ChIP assay in SCC25 cells. The first round of ChIP was for HA-FBW7, followed by second round of immunoprecipitation for VDR. F, Recruitment of ubiquitin by ChIP assay (left) and corecruitment of ubiquitin and c-MYC by re-ChIP assay to E-box motif of CDC25A promoter after 6 hours of 1,25D treatment in SCC25 cells (right; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Figure 5.

VDR and FBW7 are corecruited to E-box motifs of c-MYC target genes. Analysis of c-MYC (A) and MXD1 (B) recruitment to E-boxes of target genes by ChIP-qPCR in SCC25 cells treated with vehicle or 100 nmol/L 1,25D for 6 hours. C, Analysis of HA-FBW7 recruitment to E-box motifs by ChIP-qPCR in HA-FBW7 SCC25 cells treated with 100 nmol/L 1,25D for 6 hours. D, Corecruitment of HA-FBW7 and c-MYC or MXD1 to E-box motif of CDC25A by re-ChIP in SCC25 cells after 6-hour 1,25D treatment. The first round of ChIP was for HA-FBW7, followed by second round of immunoprecipitation for c-MYC or MXD1. E, Analysis of the corecruitment of HA-FBW7 and VDR to E-box motifs of CDC25A and CDK4 by re-ChIP assay in SCC25 cells. The first round of ChIP was for HA-FBW7, followed by second round of immunoprecipitation for VDR. F, Recruitment of ubiquitin by ChIP assay (left) and corecruitment of ubiquitin and c-MYC by re-ChIP assay to E-box motif of CDC25A promoter after 6 hours of 1,25D treatment in SCC25 cells (right; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Close modal

There was a lack of apparent dependence of the recruitment of FBW7 and SCF complex subunits on 1,25D treatment. However, such results likely represent a composite of dynamic, 1,25D-dependent interactions of FBW7 with changing levels of c-MYC, MXD1, and the VDR at these sites. To probe this further, reChIP assays were performed with an anti-HA antibody against FBW7. These experiments revealed an increased association of FBW7 with DNA-bound c-MYC after 6 hours of 1,25D treatment (Fig. 5D), even though DNA-bound c-MYC was in decline (Fig. 5A). Conversely, we observed a markedly decreased colocalization of FBW7 with MXD1 under the same conditions (Fig. 5D), as well as a decline in reChIP of the VDR with FBW7 associated with these sites (Fig. 5E). This is to be expected given that these sites are predominately occupied by MXD1 after extended 1,25D treatment, which would be refractory to FBW7 binding in the presence of 1,25D. Importantly, reChIP assays also revealed a 1,25D-dependent increase in ubiquitin associated with DNA-bound c-MYC (Fig. 5F), consistent with 1,25D inducing c-MYC turnover on DNA. Finally, the depletion of FBW7 strongly reduced association of the VDR with the CDC25A E box (Supplementary Fig. S6B), consistent with its effects on VDR gene expression and binding of the receptor to VDRE-containing genes.

Loss of FBW7 attenuates 1,25D-dependent cell-cycle arrest

Taken together, our results show that FBW7 and the VDR regulate each other's function in control of protein turnover and regulation of gene transcription, and that FBW7 is a key contributor to signal transduction by the VDR. To test for possible functional consequences of the role of FBW7 in 1,25D-dependent signaling, we examined the effect of FBW7 ablation on the antiproliferative effects of 1,25D in SCC25 cells. These experiments revealed that loss of FBW7 enhanced SCC25 cell proliferation as measured by EdU incorporation and strongly attenuated the capacity of 1,25D to induce cell-cycle arrest (Fig. 6A and B). In conclusion, the hormone-bound VDR regulates tumor suppressor FBW7 and the turnover of several of its target proteins. Moreover, FBW7 is required for optimal VDR expression and function as a transcriptional regulator, revealing that the two proteins act as mutual cofactors.

Figure 6.

Loss of FBW7 attenuates 1,25D-dependent cell-cycle arrest. A, qRT-PCR analysis of FBW7 transcription in SCC25 cells transfected with scrambled or FBW7 siRNA. B, Left, EdU cell proliferation assay in SCC25 cells transfected with scrambled or FBW7 siRNA and treated with vehicle or 100 nmol/L of 1,25D. Right, quantification of EdU cell proliferation assay (**, P ≤ 0.01).

Figure 6.

Loss of FBW7 attenuates 1,25D-dependent cell-cycle arrest. A, qRT-PCR analysis of FBW7 transcription in SCC25 cells transfected with scrambled or FBW7 siRNA. B, Left, EdU cell proliferation assay in SCC25 cells transfected with scrambled or FBW7 siRNA and treated with vehicle or 100 nmol/L of 1,25D. Right, quantification of EdU cell proliferation assay (**, P ≤ 0.01).

Close modal

Epidemiologic studies, supported by preclinical data, link vitamin D deficiency to increased cancer risk (19–21, 28–34); for example, a large prospective analysis linked vitamin D sufficiency to the reduced total incidence and mortality from digestive cancers and leukemias (19). Vitamin D deficiency represents a potentially widespread problem as, in the absence of adequate supplementation, most diets are vitamin D deficient. In addition, climate, extended vitamin D winters (inadequate solar UVB) in major population centers, conservative dress, and sun avoidance also contribute to vitamin D deficiency worldwide (17, 18). However, although clinical and preclinical data point to the cancer-preventive effects of vitamin D signaling, much remains to be learned about the underlying molecular mechanisms of its action.

Data presented above strongly suggest that the hormone-bound VDR is a key cofactor of FBW7, and show that 1,25D regulates the association of FBW7 with two of its substrates, c-MYC and MXD1. We show that the VDR interacts with FBW7 and c-MYC, and that 1,25D enhanced the association of FBW7 with c-MYC as assessed by co-IP. Treatment with 1,25D also induced the association of FBW7 and ubiquitin with DNA-bound c-MYC. These results, summarized in Fig. 7, are consistent with the hormone-bound VDR accelerating the proteasomal turnover of DNA-bound c-MYC, providing a mechanism for eliminating the protein and clearing E boxes for binding of MXD1, whose expression and DNA binding increase strongly in 1,25D-treated cells. Other data presented above provide evidence that the effects of 1,25D on FBW7 function as an E3 ligase are not limited to c-MYC and MXD1, as 1,25D treatment reduced protein, but not mRNA expression, of FBW7 targets AIB1, MCL1, c-JUN, and Cyclin E1, and the ablation of FBW7 attenuated loss of AIB1 expression in 1,25D-treated cells. Taken together, these findings bridge genomic and nongenomic actions of the VDR; our data provide evidence that induced c-MYC degradation and suppression of c-MYC target gene regulation takes place on the DNA, which would represent a genomic action, whereas the accelerated turnover of proteins such as Cyclin E and MCL1 would take place off DNA, thus representing a nongenomic action.

Figure 7.

Schematic representation of 1,25D-regulated proteasomal turnover of c-MYC and MXD1. A, Schematic representation of VDR-regulated proteasomal degradation of DNA-bound c-MYC. B, Schematic representation of the association of the VDR with MXD1 blocking binding of FBW7.

Figure 7.

Schematic representation of 1,25D-regulated proteasomal turnover of c-MYC and MXD1. A, Schematic representation of VDR-regulated proteasomal degradation of DNA-bound c-MYC. B, Schematic representation of the association of the VDR with MXD1 blocking binding of FBW7.

Close modal

Consistent with the fact that many FBW7 target proteins are drivers of cell-cycle progression, ablation of FBW7 expression strongly compromised 1,25D-dependent cell-cycle arrest. Taken together, these data strongly suggest that the hormone-bound VDR has widespread effects on FBW7-stimulated proteasomal turnover, and that the regulation of FBW7, which controls the turnover of several drivers of cell division, is a key component of the capacity of 1,25D to arrest cell proliferation. There are examples of hormonal signaling regulating the expression of E3 ligases (35, 36). Our results provide an example of hormonal signaling directly regulating the protein–protein interactions of an E3 ligase and thus its substrate access.

Sequence analysis revealed that the VDR has a motif corresponding to a consensus phospho-degron in a conserved, VDR-specific insertion in the receptor LBD. However, mutational analysis and co-IP experiments revealed that the motif is not required for the 1,25D-dependent binding of FBW7 to the VDR. Moreover, FBW7 ablation did not have a significant effect on VDR stability in the absence or presence of 1,25D. Thus, our findings are not consistent with the VDR being a target of FBW7-regulated proteasomal turnover. It is not clear what the structure of the VDR-specific insertion region is, however, our data suggest that the phospho-degron motif is sequestered and that FBW7 interacts with other site(s) on the VDR LBD. The lack of stabilization of the VDR upon FBW7 depletion suggests that FBW7 associates with the VDR through region(s) independent of the substrate recognition domain. Unexpectedly, we found that the ablation of FBW7 reduces VDR gene expression, which appeared to be the major contributor to the reduce expression of VDR protein seen in the FBW7-depleted cells. This led to reduced receptor binding to cognate VDREs of target genes. In addition, the FBW7 expression and proteasome function were necessary for optimal VDR-dependent transactivation; we detected hormone-dependent recruitment of FBW7 and proteasome subunits to VDREs, and the depletion of FBW7 or addition of proteasome inhibitors attenuated the induction by 1,25D of multiple target genes.

Other studies have provided evidence for hormone-dependent recruitment of proteasome subunits to nuclear receptor target genes and their contribution to hormone-dependent transcriptional regulation. Estrogen receptor α (ERα) is degraded by the proteasome in a receptor-specific manner that is regulated by ligand binding, and proteasome inhibitors block estradiol-dependent transactivation (37, 38). Subsequent work provided evidence that ERα-dependent transactivation and proteasome activity are intrinsically linked, and that proteasomal degradation of ERα occurs on estrogen-responsive promoters (39). Analysis of androgen-regulated gene expression showed that recruitment of the MDM2 E3 ligase led to ubiquitination and degradation of the DNA-bound androgen receptor and that disruption of this process impaired hormone-dependent transactivation (40). Similarly, proteasome inhibitors block transactivation by PPARγ, and receptor ubiquitination is enhanced by ligand binding (41). Collectively, these studies provide strong evidence for a role of proteasomal degradation of nuclear receptors in the process of hormone-dependent transactivation, and suggest that receptor degradation may contribute to the putative turnover and cycling of transcription factors occurring during transactivation (42, 43). However, it is unlikely that a mechanism of FBW7-dependent receptor turnover is operative on genes transactivated by the VDR, as FBW7 does not appear to recognize the VDR as a typical substrate and does substantially regulate VDR stability. If it functions as an E3 ligase, FBW7 likely targets other components of the transcription apparatus recruited by the VDR. If a similar mechanism of proteasomal degradation of the VDR occurs during transactivation, it would be mediated by other E3 ligase(s).

In conclusion, our studies provide a molecular basis for the anticancer activities of vitamin D. FBW7 is a tumor suppressor because of its role in regulating cell division by controlling the proteasomal turnover of several drivers of cell proliferation. 1,25D treatment diminished the expression of several FBW7 target proteins, and consistent with these findings, FBW7 depletion attenuated the vitamin D–induced cell-cycle arrest. Unexpectedly, we also found that FBW7 is essential for normal VDR gene expression and function of the VDR as a transactivator. Thus, the two proteins stimulate each other's function and are mutual cofactors.

No potential conflicts of interest were disclosed.

Conception and design: R. Salehi-Tabar, J.H. White

Development of methodology: R. Salehi-Tabar, J.H. White

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R. Salehi-Tabar, B. Memari, H. Wong

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R. Salehi-Tabar, V. Dimitrov, J.H. White

Writing, review, and/or revision of the manuscript: R. Salehi-Tabar, J.H. White

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R. Salehi-Tabar, J.H. White

Study supervision: J.H. White

Other (provided structural data): N. Rochel

This work was supported by an operating grant from the Cancer Research Society (to J.H. White, R. Salehi-Tabar, and B. Memari). This work was also supported by McGill University Internal Studentships.

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.

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