PDLIM2 Expression Is Driven by Vitamin D and Is Involved in the Pro-Adhesion, and Anti-Migration and -Invasion Activity of Vitamin D

ORIGINAL ARTICLE PDLIM2 expression is driven by vitamin D and is involved in the pro-adhesion, and anti-migration and -invasion activity of vitamin D

E Vanoirbeek1, G Eelen1,2, L Verlinden1, G Carmeliet1, C Mathieu1, R Bouillon1, R O’Connor 3, G Xiao4 and A Verstuyf1

1Alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active form of vitamin D3, is a pleiotropic hormone that exerts its effects on a wide range of tissues, resulting in different biological responses such as anticancer activity. It is the ligand of the vitamin D receptor (VDR), a nuclear receptor with transactivating capacity. We demonstrated in this study that 1,25(OH)2D3 induces PDZ-LIM domain-containing protein 2 (PDLIM2) expression. PDLIM2 is an adaptor molecule that links different components of the cytoskeleton, and was recently shown to be repressed in human breast cancer cells by hypermethylation of regulatory promoter regions, leading to enhanced tumorigenicity. We demonstrated that PDLIM2 was a direct target gene of 1,25(OH)2D3; its upregulation was VDR-dependent and a functional VDRE in the promoter was identiﬁed. Moreover, 1,25(OH)2D3 induced demethylation of the PDLIM2 promoter, leading to enhanced transcription. Finally, PDLIM2 was found to be crucial for 1,25(OH)2D3- induced cell adhesion and for mediating the ability of 1,25(OH)2D3 to suppress cancer cell migration and invasion. This study provides mechanistic insights into the anticancer activities of 1,25(OH)2D3 in human breast cancer cells.

Oncogene (2014) 33, 1904–1911; doi:10.1038/onc.2013.123; published online 15 April 2013 Keywords: PDLIM2; breast cancer; vitamin D; metastasis; DNA methylation

INTRODUCTION mediated by promoter hypermethylation, and treatment of cells Breast cancer accounts for 23% of all cancers and is the leading with a hypomethylating agent restores PDLIM2 expression and 10 cause of cancer death among women, for most of whom suppresses the tumorigenicity of the cells. Overexpression metastases are the major cause of death.1 Several preclinical of PDLIM2 in MCF7 cells results in suppression of anchorage- studies demonstrate the anticancer activities of vitamin D.2 It has independent growth and enhanced cell attachment to been shown that 1,25(OH)2D3, the biologically most active form of extracellular matrix (ECM) proteins. On the contrary, silencing of vitamin D, is able to inhibit the proliferation of cancer cells in vitro PDLIM2 expression in MCF7 cells results in impaired attachment and slow the progression of tumor growth in animal studies.3,4 and migration.9 PDLIM2 localizes at the actin cytoskeleton, The molecular pathways behind the antiproliferative action have where it serves as an adaptor that brings other proteins to the been extensively studied and many important effectors, such as cytoskeleton.11 PDLIM2 binds not only to a-actinin, but also cell cycle regulators and E2F transcription factors, have been to several other actin-binding proteins, such as myosin heavy 5,6 identified. Moreover, 1,25(OH)2D3 is able to induce cell adhesion chain IIA (Myh9), involved in cell functions such as motility and and to inhibit cancer cell migration and matrix invasion, thereby formation of focal adhesions,12 and Myosin VI, involved in reducing the malignancy of cancer cells. Nevertheless, little is membrane trafficking and cell movement.13 These data indicate known about the underlying molecular mechanisms. 1,25(OH)2D3 that PDLIM2 has an important role in breast cancer pathology. is a pleiotropic hormone that exerts its effects via the nuclear In this study we investigated whether PDLIM2 is a direct target vitamin D receptor (VDR). Binding of 1,25(OH)2D3 to its receptor gene of 1,25(OH)2D3 and we further explored its role in the induces the formation of a VDR-retinoid X receptor (RXR) anticancer activities of 1,25(OH)2D3. heterodimer, which binds to vitamin D response elements (VDREs) in the promoter of target genes, which leads to altered gene transcription and induction of various biological responses.7 RESULTS We previously identified PDLIM2 as a member of a gene cluster Expression of PDLIM2 is upregulated by 1,25(OH)2D3 that is quickly upregulated in MCF7 cells treated with 1,25(OH)2D3. Previously, we performed a microarray study in MCF7 cells treated PDLIM2 was recently identified as a tumor suppressor both in with 1,25(OH) D and identified a cluster of upregulated genes.14 8,9 2 3 in vitro cancer cell lines and in in vivo xenograft tumorigenicity PDLIM2 is a member of this gene cluster and its upregulation assays in mouse models.10 Its expression was shown to be was confirmed with QRT–PCR. ER-positive MCF7 cells were treated À 7 suppressed in various cancer cell lines compared with normal with 1,25(OH)2D3 (10 M) and the expression of PDLIM2 was epithelial cells. In both estrogen receptor (ER)-positive (MCF7) and determined at different time points, up to 24 h after treatment. ER-negative (MDA-MB-231) breast cancer cells this repression is Transcript levels were increased after 12 h, with a 3.7-fold

1Laboratory of Clinical and Experimental Endocrinology, KU Leuven, Leuven, Belgium; 2Laboratory of Angiogenesis & Neurovascular link, Vesalius Research Center, VIB/KU Leuven, Leuven, Belgium; 3Department of Biochemistry, University College, Cork, Ireland and 4Hillman Cancer Center Research Pavilion, University of Pittsburgh, Pittsburgh, PA, USA. Correspondence: Professor A Verstuyf, Afdeling Experimentele Geneeskunde - Endocrinologie, UZ Herestraat 49 - bus 902 –O&NI, 3000 Leuven, Belgium. E-mail: [email protected] Received 26 June 2012; revised 29 January 2013; accepted 13 February 2013; published online 15 April 2013 Vitamin D-induced PDLIM2 reduces tumorigenicity E Vanoirbeek et al 1905 induction after 24 h (Figure 1a). The protein levels were induced (position À 2113 to À 2314 relative to the TSS of PDLIM2) that from 24 h on after treatment, and this induction was maintained contained the putative VDRE was cloned into a pGL3 basic throughout the time and became maximal at 72 h (2.8-fold, luciferase reporter plasmid to study the transactivating capacity of Figure 1a). PDLIM2 expression was also induced by 1,25(OH)2D3 1,25(OH)2D3 in transient transfection experiments. Relative to À 7 À 7 (10 M) in the ER-negative breast cancer cell line MDA-MB-231, at vehicle-treated cells, incubation with 10 M 1,25(OH)2D3 resulted both the mRNA and protein level, albeit at later time points in a 6.2-fold increase in luciferase activity in COS-1 cells co- (Figure 1b). At mRNA level, PDLIM2 was significantly induced after transfected with the PDLIM2 ER6-type VDRE-containing reporter 48 h of treatment with 1,25(OH)2D3 and induction was maximal construct and expression plasmids for VDR and RXR (Figure 2e), À 8 after 72 h (2.7-fold). Induction at protein level reached a maximum whereas treatment with 10 M 1,25(OH)2D3 induced luciferase of 2.3-fold after 72 h of treatment (Figure 1b). activity 3.8-fold. To confirm that the transactivation was driven by the putative VDRE, we made point mutations within the ER6-type VDRE (Figure 2e). In one mutant, the 50 hexamer sequence PDLIM2 is a direct target gene of 1,25(OH)2D3 TGACCC was mutated to TAGCCC (MutER6.1), whereas in the We tested if the induction of PDLIM2 by 1,25(OH)2D3 was 0 VDR-dependent. Therefore, we knocked down VDR expression in second, also the 3 hexamer was altered from GGTTCA to GCTTAA MCF7 cells with siRNA (Figure 2a). As a control, cells transfected (MutER6.2). As shown in Figure 2e, mutation of the VDRE within with non-targeting siRNA were used. We then determined PDLIM2 the PDLIM2 promoter construct completely abolished the transcript levels in VDR knockdown and control cells, treated with transactivation by 1,25(OH)2D3. À 7 1,25(OH)2D3 (10 M) for 72 h. 1,25(OH)2D3 clearly induced PDLIM2 expression in control MCF7 cells (Figure 2b), but not in VDR 1,25(OH)2D3 treatment results in demethylation of the PDLIM2 knockdown MCF7 cells (Figure 2b). promoter To examine if the VDR binds the human PDLIM2 promoter To determine whether 1,25(OH)2D3 mediates epigenetic directly, we tested the functionality of a putative VDRE that was regulation of PDLIM2, we investigated the effect of 1,25(OH)2D3 previously identified by in silico screening.15 The VDRE is an ER6- on PDLIM2 promoter methylation. By bisulfite genomic sequen- type binding motif, formed by everted repeats of PuG(G/T)TCA cing, a PDLIM2 promoter fragment, located À 1084 to À 800 separated by 6 bp. The sequence of the investigated ER6 motif is relative to the TSS, was previously shown to be hypermethylated as follows: TGACCCAGCAGGGGTTCA (half sites are underlined) in breast cancer cells and to repress PDLIM2 expression.10 After and is located at 2205 bp upstream of the TSS. ChIP experiments confirming the demethylation effect of 5-aza-dC (5 mM for 5 days, on MCF7 cells confirmed the actual association of the VDR with Figure 3), a well-known DNA methyltransferase inhibitor, we À 7 this PDLIM2 promoter fragment (Figure 2c). A fragment of the investigated the effect of 1,25(OH)2D3 (10 M for 5 days) on the PDLIM2 gene that contained no consensus VDR-binding site was methylation of this promoter fragment. On average, we observed included as a negative control. As a positive control, a promoter a decrease of 9% of methylated CpGs after treatment with fragment of 24-hydroxylase (CYP24) was used (Figure 2d). CYP24 is 1,25(OH)2D3, compared with vehicle-treated cells. 5-aza-dC one of the most responsive direct target genes of 1,25(OH)2D3, decreased the average methylation status with 31% (Figure 3). and this promoter fragment contains previously identified VDR- binding sites.16 Also here, a genomic fragment without consensus Methylation of the PDLIM2 promoter abolishes the transactivation VDRE sites was included as a negative control. This assay capacity of 1,25(OH)2D3 demonstrated that the VDR bound to the PDLIM2 promoter in We confirmed the findings from a previous study10 that treatment MCF7 cells. Next, a 202-bp fragment of the PDLIM2 promoter with 5-aza-dC (1 mM, 72 h) increased PDLIM2 expression in MCF7 cells (Figure 4a). Moreover, pre-treatment of the cells with 5-aza-dC (1 mM, 72 h) followed by a 24-h treatment with À 7 MCF7 1,25(OH)2D3 (10 M) significantly augmented the transcription 25 of PDLIM2 in comparison to treatment with 1,25(OH)2D3 alone. Vehicle * 24h 48h 72h 96h 20 1.25(OH) D These data suggest that promoter demethylation increased the 2 3 1.25(OH) D -++ -+-+- 2 3 transcriptional activity of 1,25(OH)2D3 (Figure 4a). To confirm the 15 Pdlim2 role of PDLIM2 promoter methylation in the transactivation 10 * capacity of 1,25(OH)2D3, we used SssI methylase to hypermethy- 5 Actin late the promoter fragment of PDLIM2 that contained the functional VDRE described above. Hypermethylation was con- 0

Relative Pdlim2 expression 1h 3h 6h 12h 24h ﬁrmed by digestion with McrBC, a methylation-dependent Time points (hours) restriction enzyme, and was compared to mock-treated fragments (no SssI methylase enzyme) (Figure 4b). Subsequently, the MDA-MB-231 transcriptional activity of the hypermethylated and mock-treated 3 * Vehicle reporter constructs was tested by co-transfection with VDR 24h 48h 72h 96h 1.25(OH) D 2 3 1.25(OH)2D3 - + - + - + - + and RXR expression vectors in COS-1 cells. In cells transfected 2 with a mock-treated reporter construct, luciferase activity Pdlim2 * increased 1.8-fold, relative to vehicle-treated cells, after incubation À 7 1 with 1,25(OH)2D3 (10 M) for 48 h. In cells transfected with a Actin hypermethylated promoter construct, the induction of the 0 luciferase activity by 1,25(OH)2D3 was completely abolished Relative Pdlim2 expression 1h 3h 6h 12h 24h 48h 72h (Figure 4c). Time points (hours)

Figure 1. 1,25(OH)2D3-induced expression of PDLIM2. PDLIM2 mRNA PDLIM2 mediates the effect of 1,25(OH) D on cell migration, and protein levels were determined at different time points after 2 3 À 7 invasion and adhesion stimulation with 10 M 1,25(OH)2D3 in MCF7 (a) and MDA-MB-231 (b) cells and normalized to b-actin levels. RT–PCR data represent the Malignancy-associated cell traits such as cell migration and matrix mean±s.e.m. of three independent experiments. The upregulation invasion are suppressed by 1,25(OH)2D3. To investigate the role of of PDLIM2 was signiﬁcant according to two-way ANOVA and PDLIM2 in the anti-migration and -invasion effect, and the Bonferroni post hoc test for multiple comparisons (*Po0.05). pro-adhesion activity of 1,25(OH)2D3 on breast cancer cells,

& 2014 Macmillan Publishers Limited Oncogene (2014) 1904 – 1911 Vitamin D-induced PDLIM2 reduces tumorigenicity E Vanoirbeek et al 1906

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Figure 2. PDLIM2 is a direct target gene of 1,25(OH)2D3. VDR expression in knockdown in MCF7 cells using siRNA (a). As a control, a non-targeting siRNA is used. PDLIM2 transcript levels were determined in control and VDR-knockdown MCF7 cells after treatment for 72 h À 7 with 10 M 1,25(OH)2D3 (b). ChIP assays with anti-VDR antibody were performed on MCF7 cells. The VDREs in the PDLIM2 promoter (c)orin the CYP24A1 promoter (positive control) (d) were ampliﬁed by PCR. Non-VDRE promoter regions were included as negative controls for both genes. Ratios represent fold enrichment above background of 1,25(OH)2D3/vehicle-treated cells. COS-1 cells were transfected with pGL3 basic luciferase reporter containing a wild-type or mutated VDRE, or with empty pGL3 basic luciferase reporter vector as control and were treated with different concentrations of 1,25(OH)2D3 for 48 h. Luciferase activity was measured and normalized to b-galactosidase activity (e). All experiments were performed in triplicate and found to be signiﬁcant according two-way ANOVA (a, b and e) or one-way ANOVA (c and d). (*Po0.05). Point mutations (one half site MutER6.1, both half sites MutER6.2) were introduced within the ER6-type VDRE (wildtype) (f).

PDLIM2 knockdown cell lines were generated. For both MCF7 and capacity of 1,25(OH)2D3 was completely lost (Figures 6a and b). MDA-MB-231, two independently established cell lines were Subsequently, we tested whether PDLIM2 interferes with the generated, each transduced with a different PDLIM2 shRNAmiR anti-migration and -invasion effects of 1,25(OH)2D3. In control sequence (PDLIM2 knockdown (1) and (2)). As a control, cells MDA-MB-231 cells, 1,25(OH)2D3 significantly reduced cell migra- transduced with non-silencing shRNAmiR particles were used tion (71%) and invasion (69%), while in PDLIM2 knockdown (control cells). Cell adhesion was examined in both MCF7 and MDA-MB-231 cells, 1,25(OH)2D3 could only reduce cell migration MDA-MB-231 cells, but migration and invasion were studied only and invasion to non-significant levels (Figures 6c and d). in MDA-MB-231 breast cancer cells, since this ER-negative cell line has a high potential to migrate and to invade the ECM. Actual knockdown was confirmed at mRNA and protein levels (Figures 5a DISCUSSION and b). To determine whether loss of PDLIM2 expression would 1,25(OH)2D3 inhibits proliferation and induces differentiation of a affect the anti-proliferative action of 1,25(OH)2D3, we performed wide variety of cells, including cancer cells. Therefore, the use of 3 [ H]thymidine incorporation assays on MCF7 cells (because 1,25(OH)2D3 may be an alternative strategy in the treatment of 1,25(OH)2D3 has a strong antiproliferative effect on MCF7 cells, cancer. Consequently, many studies investigated the molecular but not on MDA-MB-231 cells). We found no difference in mechanism behind these antiproliferative and pro-differentiation 1,25(OH)2D3-induced antiproliferative action between knockdown effects. Nevertheless, insights into other anticancer effects of and control cell lines (Figure 5c). On the other hand, PDLIM2 1,25(OH)2D3, such as interference with cell adhesion and inhibition knockdown interfered with the capacity of 1,25(OH)2D3 to induce of cell migration, are more limited. It was shown that adhesive cell adhesion to collagen I-coated wells. Treatment with interactions have a critical role in tumor cell dissemination À 7 1,25(OH)2D3 (10 M) for 48 h significantly enhanced the adhesion and subsequent migration and metastasis. Cancer cell adhesion of both MCF7 (35%, Figure 6a) and MDA-MB-231 (34%, Figure 6b) can both positively and negatively modulate metastasis. cells. However, in PDLIM2 knockdown cells, the pro-adhesive Some adhesive molecules induce interactions between cancer

Oncogene (2014) 1904 – 1911 & 2014 Macmillan Publishers Limited Vitamin D-induced PDLIM2 reduces tumorigenicity E Vanoirbeek et al 1907 1bp 31 bp 14 bp 0 bp 19 bp 27 bp 29 bp 11 bp 63 bp 5 bp 22 bp

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Figure 3. Epigenetic regulation of PDLIM2 promoter. Bisulfite sequencing of genomic DNA isolated from MCF7 cells treated with 1,25(OH)2D3, 5-aza-dC or vehicle. Each square represents a CpG site. Yellow squares represent methylated CpG dinucleotides whereas blue squares represent unmethylated CpG sites. If the methylation state of a CpG site could not be defined, it is represented as not present. The ratios of the filled area in the squares represent the methylation status in the CpG sites. The number of base pairs between each CpG dinucleotide is indicated at the top. One representative experiment out of three is shown. cells (homotypic adhesion) to confine them to the primary anchorage-independent growth and impairs cell adhesion, thus 9 tumor sites. 1,25(OH)2D3 enhances the homotypic adhesion by emphasizing its role in breast cancer pathology. Moreover, its inducing genes like E-cadherin.17,18 On the other hand, heterotypic expression is suppressed in several breast and colon cancer cell adhesion of cancer cells to vessel endothelial cells is required lines and re-expression reduces the tumorigenicity of the cancer for the progression to distal metastases and is hindered by cells.8,10 1,25(OH)2D3 due to the promotion of homotypic cell–cell Importantly, 1,25(OH)2D3 induced PDLIM2 expression in other aggregation.17 Finally, cancer cells adhere to the ECM, which is cell lines such as mouse mammary GR cells, MC3T3-E1 mouse pre- usually mediated by cell surface adhesion molecules that are osteoblasts and ST-2 mouse osteoblasts (unpublished results), connected to the cytoskeleton and that can induce intracellular indicating that the induction is not restricted to human breast signaling. The integrin-associated tetraspanin CD151, for example, cancer cells. However, its function in (pre-)osteoblasts needs to be promotes the interaction between tumor cells and the ECM and elucidated. PDLIM2-induced osteoblast adhesion to the ECM thereby prevents dissociation of individual cells from the tumor, might, in analogy to BMP2, enhance cytoskeletal organization.25 leading to reduced migration and invasion.19 Conversely, some of PDLIM2 expression also increases upon monocyte differentiation these ECM-linked adhesive molecules induce cell migration. For to adherent macrophages,26 further suggesting a general function instance, in thyroid cancer cells, CEACAM1 expression is reduced in adhesion associated with cellular differentiation. by 1,25(OH)2D3, thereby decreasing metastasis, probably due to Further, we demonstrated that the induction of PDLIM2 by 20 interference with the intracellular insulin/IGF-I signaling cascade. 1,25(OH)2D3 is VDR dependent, and we identified a VDRE in Cancer cell adhesion, migration and invasion are also affected by the PDLIM2 promoter that bound the VDR and consequently microRNAs (miRNAs), as miRNAs have been shown to affect target induced gene transcription. The identification of this functional genes that are important in the metastatic cascade.21 Recently it VDRE, located B2 kb upstream of the promoter, does not was shown that miR-23b/-27b suppresses cell invasion, migration however exclude the involvement of other regulatory elements and anchorage-independent survival of castration-resistant in the transcriptional regulation of PDLIM2 by 1,25(OH)2D3. prostate cancer cells via its effects on E-cadherin and the Previously, PDLIM2 was shown to be transcriptionally repressed 22 Rho-GTPase Rac1. Interestingly, 1,25(OH)2D3 can also modulate by DNA methylation in different cancer cell lines. As it is known the expression of several miRNAs regulating cell proliferation that 1,25(OH)2D3 can regulate transcription at the epigenetic in different cancer cell lines,23,24 and a similar mechanism may level,27 we investigated the PDLIM2 promoter and demonstrated control cell adhesion and migration. that 1,25(OH)2D3 indeed reduced its methylation status. The aim of this study was to gain more insight into the Hypermethylated promoters often recruit methyl-CpG binding molecular processes by which 1,25(OH)2D3 regulates cell migra- domain proteins, leading to a closed chromatin status that is tion and invasion. A previous microarray study in MCF7 cells was incapable of binding transcription factors such as the VDR.28 performed to identify genes that are involved in the anticancer Indeed, in PC3 prostate cancer cells the CYP24 promoter is 14 effects of 1,25(OH)2D3. Here PDLIM2 was present in a cluster of hypermethylated, and treatment with 5-aza-dC enhances upregulated genes. PDLIM2 potentially functions as an adaptor recruitment of the VDR to the promoter and induces the of cytoskeletal proteins that mediate cell adhesion, migration expression of CYP24.27 Here we showed that in vitro methylation and matrix invasion.11 Overexpression in MCF7 cells suppresses of the PDLIM2 promoter fragment that contained the

& 2014 Macmillan Publishers Limited Oncogene (2014) 1904 – 1911 Vitamin D-induced PDLIM2 reduces tumorigenicity E Vanoirbeek et al 1908 5 demonstrated effects of 1,25(OH)2D3 on the expression of * histone-modifying enzymes involved in epigenetic regulation.32 4 Therefore, further research is required to investigate the interplay 3 between histone modiﬁcations and DNA methylation, and DNA Ladder Mock SssI expression to decipher the mechanism that controls DNA demethylation of 2 1500 bp PDLIM2 by 1,25(OH)2D3. 1 Repression of PDLIM2 may be essential for malignant transfor- PDLIM2 600 bp ratio treatment/vehicle mation and cancer progression.10 Therefore its re-expression by 0 1,25(OH)2D3 is possibly important to reverse the malignant 5-aza-dC + - + 100 bp phenotype, and it was thus interesting to examine the 1.25 - ++ importance of PDLIM2 in 1,25(OH)2D3-induced anticancer effects. (OH)2D3 D 3 D 3 We showed that the 1,25(OH)2D3-induced enhanced adhesion of 2 2 cancer cells to the ECM was mediated by PDLIM2. This interaction 5-AZA-dC 1.25(OH) is important to prevent dissociation of individual tumor cells and is therefore important to inhibit metastasis.19 Indeed, we demonstrated that 1,25(OH)2D3 signiﬁcantly reduced the capacity 5-AZA-dC + 1.25(OH) of MDA-MB-231 cells to migrate towards chemoattractants that

0.5 * are present in the serum and to invade the ECM. These capacities Vehicle were lost or severely reduced in the absence of PDLIM2 1.25(OH) D 0.4 2 3 expression, suggesting its role in mediating these functions.9 0.3 Future research should conﬁrm the importance of PDLIM2 in the anticancer activities of 1,25(OH)2D3 in vivo. Conﬁning metastasis is 0.2 an important objective of cancer treatment, as the invasion and 0.1 the growth of tumor cells in distant organs is responsible for the majority of the cancer deaths. In this study we showed that

Relative luciferase activity Relative luciferase 0.0 administration of 1,25(OH)2D3 to human breast cancer cells

SssI Mock reinduced PDLIM2 expression, providing a mechanism to inhibit tumor metastasis. Figure 4. PDLIM2 promoter methylation reduces the transcriptional inducibility of 1,25(OH)2D3. Effect of pre-treatment with 5-aza-dC on induction of PDLIM2 expression by 1,25(OH)2D3 for 24 h (a). Ratios of MATERIALS AND METHODS PDLIM2 transcript levels in treated cells versus vehicle-treated cells are represented. The figure represents the mean±s.e.m. of three Cell culture independent experiments. PDLIM2 expression levels were tested to MCF7- and COS-1 cells (ATCC, Manassas, VA, USA) were maintained in be significantly changed by one-way ANOVA (*Po0.05). In vitro DMEM with 2 mmol/l glutaMAX-I containing 10% heat-inactivated fetal methylation of human PDLIM2 promoter (VDRE region). Promoter bovine serum (FBS, Biochrom, Berlin, Germany), 100 units/ml penicillin, and methylation was carried out by SssI methylase. A digestion with 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA). MDA-MB-231 cells McrBC was performed to confirm the methylation status of the (ATCC) were maintained in RPMI 1640 with the same additives. PDLIM2 promoter construct (b). A luciferase activity assay of methylated and mock-treated PDLIM2 promoter constructs trans- Retroviral transduction and generation of stable PDLIM2 fected in COS-1 cells was performed (c). Cells were treated with knockdown cell lines 1,25(OH)2D3 for 48 h and luciferase activity was measured and Stable cell lines (MCF7 and MDA-MB-231) were generated by transducing normalized to b-galactosidase activity. One representative experi- the cells with GIPZ lentiviral shRNAmiR particles (Open Biosystems, ment out of three is shown. PDLIM2 expression levels were found Lafayette, CO, USA) targeting PDLIM2 (PDLIM2 knock-down cells (1) and to be significantly different by two-way ANOVA and subsequent (2) or a non-silencing control sequence (control cells)). Approximately Bonferonni post hoc test for multiple comparisons (*Po0.05). 4 Â 106 total transducing units (TU)/ml were used to infect the cells. A stable cell culture was obtained by growing the cells in the presence of 0.5 mg/ml puromycin (Invitrogen). newly identified VDRE repressed its promoter activity. The transactivating capacity of 1,25(OH)2D3 on this fragment was Cell proliferation assay completely lost. As a measure of cell proliferation, [3H]thymidine incorporation in breast Hypermethylation of CpG islands in the promoter region of cancer MCF7 cells was determined after a 72-h incubation with various 33 genes is an established mechanism to inactivate tumor suppres- concentrations of 1,25(OH)2D3 as described previously. sors and is seen in almost every tumor type.29 The reversibility of this epigenetic mark stimulated the development of epigenetic Cell adhesion assay altering drugs. Therefore, the ability of 1,25(OH)2D3 to reactivate a Culture plates were coated with 40 mg/ml Collagen I (Sigma, Saint-Louis, selection of hypermethylated genes is of great therapeutic MI, USA) and were incubated at 4 1C overnight. The coating solution was advantage. Different mechanisms and factors can be involved in then removed, replaced with 1% BSA (Applichem, Darmstadt, Germany) À 7 1 M the DNA demethylation events induced by 1,25(OH)2D3 at the and kept at 4 C for 4 h. Cells were pre-treated with 1,25(OH)2D3 (10 ) for 48 h and serum starved for 4 h before being harvested and seeded in PDLIM2 promoter. It has been demonstrated that alterations in 4 DNA methylation of the VDR target gene CDKN1A, the gene the Collagen I-coated plates at a concentration of 2 Â 10 cells/well in SFM with 0.01% BSA. Cells were allowed to attach at 37 1C for 30 min, and coding for the cyclin dependent kinase inhibitor p21, can occur 30 floating cells were removed by gentle washing with DMEM/BSA. Attached through recruitment of the corepressor NCOR1. In the prostate cells were fixed with 4% paraformaldehyde (Merck, Darmstadt, Germany) cancer cell line PC3 the transrepression of CDKN1A was associated and stained with 0.1% crystal violet, which was subsequently extracted with increased NCOR1 binding on the CDNK1A promoter in with 50 ml of 0.5% TX-100 (Sigma) at room temperature and measured by response to 1,25(OH)2D3. The inappropriate recruitment of NCOR1 reading its absorbance at A595. was accompanied by elevated expression levels of H3K9 methyltransferases, which can sustain gene-repressive histone Cell migration and invasion test À 7 modifications like H3K3me2, and this in turn can attract the MDA-MB-231 knockdown and control cells were treated with 10 M 31 DNA methylation machinery. Other studies as well have 1,25(OH)2D3 for 48 h. Cells were then serum starved overnight and

Oncogene (2014) 1904 – 1911 & 2014 Macmillan Publishers Limited Vitamin D-induced PDLIM2 reduces tumorigenicity E Vanoirbeek et al 1909 2.0 MCF7 2.0 MDA-MB-231

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MCF7 control cells MCF7 knock-down (1) H]Thymidine incorporation (%) H]Thymidine 3 [ MCF7 knock-down (2) 40 10-10 10-9 10-8 10-7 10-6 10-5 Concentration (M) Figure 5. Generation of stable PDLIM2-knockdown MCF7 and MDA-MB-231 cell lines. PDLIM2-knockdown was confimed at mRNA level and at protein level in MCF7 (a) and MDA-MB-231 (b) cells. RT–PCR experiments represent the mean±s.e.m. of 3 independent experiments. PDLIM2 down-regulation was tested to be significant by one-way ANOVA (*Po0.05). The arrow indicates PDLIM2 protein. Proliferation of the control MCF7 cells was compared with the knockdown cell lines by [3H]thymidine incorporation (c). The figure represents the mean±s.e.m. of two independent experiments. One-way ANOVA showed no overall differences between the cell lines (P40.05).

35 Â 103 cells were reseeded in the upper chamber of a modified Boyden anti-PDLIM2 (previously described9 and purchased from Abcam two-chamber system (transwell, polycarbonate, pore size 8 mm; Corning, (Cambridge, UK); ab68220) and anti-b-actin (A5441; Sigma). Newark, NY, USA). Serum (10%) was added in the lower chamber as chemoattractant. After 4 h the remaining cells at the top surface of the membrane were removed. Cells at the lower surface were fixed Luciferase reporter assay 4 with 4% paraformaldehyde, stained with a 0.5% crystal violet solution 2 Â 10 COS-1 cells were transfected with 40 ng pSG5-VDR, 40 ng pSG5- (Acros Organics, Morris Plains, NJ, USA) in 25% methanol, and counted. RXR and 200 ng pGL3 basic luciferase reporter (Promega, Madison, WI, À 7 For invasion tests, the cells were treated for 72 h with 10 M USA) and 15 ng b-galactosidase expression construct. A putative VDRE from the human PDLIM2 promoter (NM_021630.4), located at 2205 bp 1,25(OH)2D3, the membranes were coated with 100 ml matrigel (BD Biosciences, Franklin Lakes, NJ, USA) diluted in PBS (300 mg/ml) and upstream of the transcription start site (TSS), was cloned in the pGL3 basic the cells were allowed to migrate for 12 h. vector. This fragment is present on the human chromosome 8 between nucleotides 22 434 438 and 22 434 455. All the human genome data are retrieved from the UCSC genome browser and based on the GrCh37/hg19 Quantitative real-time PCR (RT-PCR) and western blotting assembly. To test the functionality of this response element, point Quantitative RT–PCR analyses were performed as previously described.5,34 mutations were introduced in either one or both half sites of this VDRE. b-Actin levels were used to normalize gene expression. The sequence of The cells were treated with the indicated doses of 1,25(OH)2D3 for 48 h. PCR primers and fluorogenic probes are available on request. Luciferase activity was measured and corrected for b-galactosidase activity. Preparation of whole-cell extracts and subsequent western blotting Specific methylation of the insert was performed as described were performed as previously described.35 Antibodies used were previously.27 Briefly, the cloned fragment was excised from pGL3 basic

& 2014 Macmillan Publishers Limited Oncogene (2014) 1904 – 1911 Vitamin D-induced PDLIM2 reduces tumorigenicity E Vanoirbeek et al 1910 Adhesion MCF7 Adhesion MDA-MB-231

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Figure 6. PDLIM2 expression is required for enhanced cell adhesion and reduced cell migration and invasion, mediated by 1,25(OH)2D3. The ability of 1,25(OH)2D3 to increase adhesion of MCF7 (a) and MDA-MB-231 (b) control cells to Collagen I-coated cell culture wells compared with PDLIM2-knockdown cells. Cell migration (c) and invasion (d) was examined in MDA-MB-231 control and PDLIM2-knockdown cells treated À 7 with 10 M 1,25(OH)2D3 or vehicle for 48 h. In the ﬁgures the mean absorbance at 595 nm of 1,25(OH)2D3 or vehicle-treated cells±s.e.m. of three independent experiments are represented. Student’s t-tests were performed between vehicle- and 1,25(OH)2D3-treated cells to examine the effects of 1,25(OH)2D3 on cell adhesion, migration and invasion (*Po0.05).

luciferase reporter and methylated in vitro with SssI methylase harvested for QRT–PCR analysis. For bisulfite sequencing, MCF7 cells were À 7 (New England Biolabs, Ipswich, MA, USA), or no enzyme (mock), all in treated for 5 days with 1,25(OH)2D3 (10 M), 5-aza-dC (5 mM) or vehicle. the presence of the cofactor S-adenosyl methionine (New England Biolabs). Genomic DNA was isolated using PureLink Genomic DNA Purification Kit Methylation efficiency was confirmed using McrBC digests (New England (Invitrogen). Aliquots of the genomic DNA were treated with sodium Biolabs). Methylated or mock-treated fragments were purified by QIAquick bisulfite using the EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, PCR Purification kit (QIAGEN, Hilden, Germany) and ligated back into pGL3 CA, USA), followed by PCR to amplify PDLIM2 promoter using Hot-start Taq basic luciferase reporter using standard methods. 3 Â 103 COS-1cells were enzyme (Fermentas, St Leon-Rot, Germany). Primers were designed to transfected with 100 ng of the methylated or mock-treated pGL3 basic recognize the bisulfite-modified regions ( À 1084 to À 800 upstream of luciferase reporter constructs along with 10 ng of pSG5-VDR, 10 ng pSG5- TSS) of the PDLIM2 promoter (sequences are available on request). The PCR RXR and 5 ng b-galactosidase expression construct to normalize for products were used for DNA sequencing to determine the methylation À 7 transfection efficiency. Cells were treated with 10 M 1,25(OH)2D3 for 48 h, status of the CpG dinucleotides in the PDLIM2 promoter, as described after which luciferase activity was measured. in Qu et al.8

Chromatin immunoprecipitation assay (ChIP) Statistical analysis ChIP assays were performed as previously described.36 ChIP output DNA Statistical analysis was performed with the software program GraphPad was analyzed by QRT-PCR, using a primer set around the putative Prism5 (Graphpad Software, La Jolla, CA, USA). All results are expressed as VDRE and a primer set in a distant region (exon 10) of the human PDLIM2 means±s.e.m. ANOVA analyses followed by Bonferroni multiple-compar- gene. Fold enrichment above background (input) was calculated as ison tests and Student’s t-tests were carried out to detect significant described in the user manual ‘Epitect ChIP qPCR assay handbook differences, unless otherwise noted. Po0.05 was accepted as significant. (SABiosciences, Qiagen, Hilden, Germany) and take into account IgG controls. As a positive control, a primer set specific for the human CYP24A1 promoter was used. Sequences of primers are available on request. CONFLICT OF INTEREST Antibodies used for ChIP assays are anti-VDR (C-20: Sc-1800; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit (anti-mouse) The authors declare no conflict of interest. immunoglobulins (Santa Cruz Biotechnology). ACKNOWLEDGEMENTS 0 Treatment with 5-aza-2 -deoxycytidine (5-aza-dC) and bisulfite We thank Ine Beullens, Mark Van Camp, Suzanne Marcelis and Biauw Keng Tan for genomic DNA sequencing excellent technical assistance. This work was supported by grants from the University MCF7 cells were treated with 5-aza-dC (1 mM) for 3 days, followed by the of Leuven (GOA/04/10), Flanders Research Foundation (FWO G.0587.09 and À 7 addition of 1,25(OH)2D3 (10 M) for an additional 24 h. Cells were then G.0859.11) and Stichting tegen Kanker (212-2008).

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