Zamaporvint

In vitro studies revealed a downregulation of Wnt/β-catenin cascade by active vitamin D and TX 527 analog in a Kaposi’s sarcoma cellular model

Cinthya Tapia, Alejandra Suares, Pablo De Genaro, Verónica González-Pardo.
 
Abstract

The Kaposi’s sarcoma-associated herpesvirus G-protein-coupled receptor (vGPCR) is a key molecule in the pathogenesis of Kaposi’s sarcoma. We have previously demonstrated that 1α,25(OH)2D3 or its less calcemic analog TX 527 exerts antiproliferative effects in endothelial cells stable expressing vGPCR. Since it is well documented that vGPCR activates the canonical Wnt/β-catenin signaling pathway, the aim of this study was to evaluate if Wnt/β-catenin cascade is target of 1α,25(OH)2D3 or TX 527 as part of their antineoplastic mechanism. Firstly, Western blot studies showed an increase in β-catenin protein levels in a dose and time dependent manner; and when VDR was knockdown, β-catenin protein levels were significantly decreased. Secondly, β-catenin localization, investigated by immunofluorescence and subcellular fractionation techniques, was found increased in the nucleus and plasma membrane after 1α,25(OH)2D3 treatment. VE-cadherin protein levels were also increased in the plasma membrane fraction. Furthermore, β-catenin interaction with VDR was observed by co-immunoprecipitation and mRNA expression of β-catenin target genes was found decreased. Finally, Dkk-1, the extracellular inhibitor of Wnt/β-catenin pathway, showed an initial upregulation of mRNA expression. Altogether, the results obtained by different techniques revealed a downregulation of Wnt/β-catenin cascade after 1α,25(OH)2D3 or TX 527 treatment, showing the foundation for a potential chemotherapeutic agent.

Keywords: vGPCR, active vitamin D3, Wnt/β-catenin, neoplasia

1. Introduction

The Kaposi’s sarcoma-associated herpesvirus G protein-coupled receptor (vGPCR) is a key molecule in the pathogenesis of Kaposi’s sarcoma, which is the most common cancer in HIV-infected untreated individuals (Martin and Gutkind, 2009; Mesri et al., 2010). vGPCR induces in vivo the development of angioproliferative lesions similar to those observed in human KS lesions, showing powerful angiogenic and tumor properties (Montaner et al., 2003). As a result, to investigate vGPCR signaling pathway is of great interest for KS treatment.
The most active vitamin D form, 1α,25(OH)2D3, is a pleiotropic hormone that performs a main role in calcium homeostasis. Most of its actions depend on VDR, the vitamin D nuclear receptor. Besides its classical effects on intestinal calcium absorption and bone biology, 1α,25(OH)2D3 plays antiproliferative, pro-apoptotic and pro-differentiating actions on neoplastic cells and exerts antiinflammatory properties through the inhibition of pro-inflammatory cytokines and NF-κB signaling (Krishnan and Feldman, 2011). Unfortunately, calcemic effects like hypercalcemia, hypercalciuria and increased bone resorption have restricted the use for therapeutic purposes (Eelen et al., 2007). Therefore, analogs with less calcemic activity like TX 527, have presented diminished calcemic effects and enhanced antiproliferative and pro-differentiating capacities on malignant cells (Verlinden et al., 2000). Numerous investigations have described a family of 19 proteins called the human Wnts that participates controlling proliferation, survival, migration, differentiation, and lineage decisions in many cell types during development and adult life (Willert and Nusse, 2012). β-catenin is a multifunctional protein required for regulation of gene expression and cell-cell adhesion in response to Wnt. The Wnt/β-catenin pathway stabilizes β-catenin intracellular levels through the inactivation of a cytoplasmic protein complex (Axin, APC, GSK3β and CK1) after Dishevelled (Dsh) recruitment, which promotes β-catenin phosphorylation, ubiquitination and degradation by the proteasome when Wnt ligands bind to membrane heterodimeric receptors (Frizzled and LRP5/6). Consequently, un-phosphorylated β-catenin molecules are released in the cytosol; able to migrate to the nucleus to behave as a co-activator of TCF/LEF transcription factors (Cadigan and Waterman, 2012). Aberrant activation of this pathway triggers the accumulation of nuclear β-catenin and the consequent induction of excessive cell proliferation, critical for the initiation and progression of most types of cancers (Polakis, 2012). In a cellular model of viral oncogenesis, Angelova and collaborators have reported that the stable expression of vGPCR activates the canonical Wnt/β-catenin signaling and induces cyclin D1, VEGFA and MMP-9 mRNA expression in human endothelial cells (Angelova et al., 2014). The effect of 1α,25(OH)2D3 on the regulation of Wnt/βcatenin cascade has been studied in normal and pathological conditions. 1α,25(OH)2D3 has showed to antagonize the Wnt/β-catenin signaling in ron-mediated breast tumorigenesis (Johnson et al., 2015), to promote cardiac differentiation (Hlaing et al., 2014) and osteoblasts di erentiation and survival (Xiong et al., 2017). In addition, a direct VDR/β-catenin interaction triggered by 1α,25(OH)2D3 has revealed a decrease of β-catenin/TCF complexes resulting in a nuclear β-catenin non-transcriptional accumulation in colon cancer (Larriba et al., 2013, 2011; Pálmer et al., 2001). Furthermore, it has been demonstrated that 1α,25(OH)2D3 treatment, upregulated E-cadherin in a rat mammary tumor-derived cell line (Rama 37 cells) (Xu et al., 2009) and in colon carcinoma cells (Pálmer et al., 2001), promoting a phenotypic change of malignant cells towards a normal epithelial type. What is more, the Dickkopf (Dkk) gene family encodes secreted proteins, which bind to LRP5/6, in the presence of Kremen co-receptors, working as extracellular inhibitors of Wnt/β-catenin pathway (Mao et al., 2002). It has been demonstrated that 1α,25(OH)2D3 increases DKK-1 mRNA and protein levels in colon cancer cells associated to the induction of an epithelial adhesive phenotype (Aguilera et al., 2007).We have previously demonstrated that 1α,25(OH)2D3 or TX 527 has antiproliferative effects in vitro and in vivo models of KS. Moreover, both VDR agonists, similar to the proteasome inhibitor bortezomib, inhibit NF-κB pathway (González Pardo et al., 2013, 2012) and induce apoptosis in endothelial cells that express vGPCR by a VDR dependent mechanism (González Pardo et al., 2010). Since Wnt/β-catenin is activated in endothelial cells due to vGPCR expression, we investigated if this pathway is regulated by 1α,25(OH)2D3 or TX 527 as part of their mechanism of action.

2. Materials and Methods

2.1. Chemicals and reagents

1α,25(OH)2D3, and the antibiotic G418 were from Sigma-Aldrich (St. Louis, MO, USA). The vitamin D analogue TX 527 [19-nor-14, 20-bisepi-23-yne-1α,25(OH)2D3], originally synthesized by M. Vandewalle and P. De Clercq (University of Ghent, Ghent, Belgium), was provided by Théramex (Monaco). Puromycin was provided by Invivogen (San Diego, CA, USA). The antibodies used were rat monoclonal anti-VDR (Affinity Bioreagents, Golden, CO, USA); rabbit monoclonal anti-β-catenin (Cell Signaling Technology, Migliore Laclaustra, Buenos Aires, AR); mouse monoclonal anti-Dkk-1, antitubulin and anti-VE-cadherin; rabbit polyclonal anti-VDR, and anti-rabbit horseradish peroxidase– conjugated secondary antibody (Santa Cruz, CA, USA). Protein A/G agarose, anti-αMEK, anti-Lamin B and anti-αQ11 were from Santa Cruz Biotechnology. Roche Applied Science (Indianapolis, IN, USA) provided high Pure RNA Isolation Kit. Immobilon P (polyvinylidene difluoride; PVDF) membranes were from Thermo Fisher; PCR primers for mouse Gapdh, Dkk-1, β-catenin,c-myc, MMP-9 and cyclin D1 were synthetized by Invitrogen (Thermo Fisher Scientific Inc., Rockford, IL, USA). For most applications, 1α,25(OH)2D3 and TX 527 were used at 10 nmol L-1 since this concentration consistently shows anti-proliferative effects in many tumor cell types.

2.2. Cell lines and transfections

As an experimental model of Kaposi’s sarcoma, SV-40 immortalized murine endothelial cells that stably express vGPCR full length receptor (vGPCR), were utilized as previously described (González Pardo et al., 2010). Stable overexpression of vGPCR promotes tumor formation in immune-suppressed mice and induces angiogenic lesions similar to those developed in Kaposi’s sarcoma (Montaner et al., 2003). vGPCR expression was routinely verified in cell cultures by qRT-PCR. Transfected cells were selected with 500 μg mL-1 G418. Stable vGPCR endothelial cells targeted with small hairpin RNA against mouse VDR (vGPCR-shVDR) or control shRNA (vGPCR-shctrl) were obtained by transduction of lentiviral particles and selected with 2 μg mL-1 of puromycin (González Pardo et al., 2013). Medium was freshly changed every other day and VDR knockdown was monitored by Western blot analysis.

2.3. SDS-PAGE and Western blot

Protein content from whole cell lysates were determined by the Bradford procedure (González Pardo et al., 2006). Proteins were resolved (with SDS–PAGE) and transferred to PVDF membranes followed by Western blot analyses that were effected as reported before (González Pardo et al., 2006). Antibodies used include monoclonal rabbit anti-β catenin (1:1000), anti-VDR (1:6000) and anti-Tubulin (1:1000), mouse anti-Dkk-1 (1:500) and anti-VE-cadherin (1:500); polyclonal goat anti-Lamin B (1:1000), rabbit anti-αQ11 (1:500) and anti-αMEK (1:20000). These were combined with anti-rabbit (1:5000) or antimouse (1:5000) or anti-goat (1:20000) horseradish peroxidase-conjugated secondary antibodies.

2.4. Confocal microscopy

vGPCR cells were cultured on cover slips and fixed in 2% paraformaldehyde with 5% of sucrose for 10 min. After two washes with PBS they were permeabilized in 0.1% Triton for 15 min and treated with 5% BSA in PBS for 30 min. Afterwards, cells were incubated with rabbit anti-β-catenin (1:50) overnight and after 3 washes with PBS (5 min) with secondary antibody anti-rabbit and TOPRO. Images were taken with a Leica DM IRB2 microscope with a confocal spectral module SP2 equipped with Ar laser (458, 476, 488 and 514 nm) and HeNe laser (633 nm). The observation was carried out with 63x 1.2 NA water-immersion objective. Nuclear Fluorescence intensity (N.F.I) was obtained by quantification with ImageJ platform of cellular nucleus for control and treated conditions in two different experiments. Means each five cellular nucleus, which is equivalent to 16 values per experiment for approximately 80 cells assayed (mean ± SEM), were used to graph fluorescence intensity and statistic. (Schneider et al., 2012; Uranga et al., 2017)

2.5. Subcellular fractionation

vGPCR cells were scrapped in ice-cold TES buffer (50 mmol L-1 Tris-HCl pH 7.4, 1 mmol L-1 EDTA, 250 mmol L-1 sucrose, 1 mmol L-1 DTT) containing proteases inhibitors (0.5 mmol L-1 PMSF, 20 µg mL-1 aprotinin and 20 µg mL-1 leupeptin). Lysates were passed through a 25 G needle 10 times using a 1 mL syringe and left on ice for 20 min. Then, homogenate was centrifuged at 100 g for 5 min to eliminate debris; supernatant was further centrifuged at 1500 g for 20 min to sediment the nuclear fraction. The supernatant was further centrifuged at 14000 g for 20 min to pellet mitochondria. The remaining supernatant was collected as cytosol fraction (González Pardo et al., 2008). Protein concentration from each fraction was estimated by the Bradford method. Goat anti-Lamin B and rabbit anti-αMEK antibodies were employed for the immunodetection of the nuclear protein marker Lamin B and the cytosol protein marker αMEK, in the different fractions. To obtain microsomes cytosol was further centrifuged at 100000 g for 75 min.

2.6. Co-immunoprecipitation

Co-immunoprecipitation assays were conducted under native conditions in order to preserve proteinprotein associations as previously described (Buitrago et al., 2002). Cells were lysed (45 min at 4°C) in co-immunoprecipitation buffer (Co-IP buffer) containing 50 mmol L-1 Tris-HCl pH 7.4, 150 mmol L-1 NaCl, 3 mmol L-1 KCl, 0.5 mmol L-1 EDTA, 1% Tween-20, 1 mmol L-1 sodium orthovanadate, 1 mmol L-1 PMSF, 1 mmol L-1 DTT, 1mmol L-1 β-glycerophospate and, 10 g mL-1 of each leupeptin and aprotinin. Lysates were clarified by centrifugation (14000 g, 10 min at 4°C). 500 µg of protein were incubated with 2 µl of rat anti-VDR antibody or 2 µl of rabbit anti-β catenin antibody (Fig. 5) plus buffer for 5 h in cold agitation, followed by incubation with protein A/G plus agarose (PAG) overnight. Whole cell lysate plus PAG was added as control. The precipitated immunocomplexes were washed 3 times with Co-IP buffer and subject to Western blot with anti-β catenin (rabbit) or anti-VDR (rabbit).

2.7. Quantitative real-time PCR

Total RNA for real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis was isolated using the High Pure RNA Isolation Kit (Roche). RNA (0.5-1µg) was reverse transcribed using the kit High Capacity cDNA RT (Applied biosystem) and qRT-PCR reactions were performed on the resulting cDNA (2 µl of cDNA; dilution 1/10) in an ABI 7500 Real Time PCR system (Applied Biosystems, CA, USA). Specific primers were used to detect Dkk-1, β-catenin, c-myc, Mmp-9 and cyclin D1 levels. The real time PCR data was analyzed by 2-delta delta Ct method using Gapdh as reference parameter (Suares et al., 2017). Reactions were carried out using the SYBR Green PCR Master Mix reagent (Applied biosystem). Primers sequences were as follows: Dkk-1 forward: 5’-TCTCTTTTCCTGACCTTTGCC-3’ Dkk-1 reverse: 5’-TGAGTTCAAGGTGGCACTG-3’, β-catenin (Ctnnb1) forward: 5’-ACAAGAAGCGGCTTTCAGTC-3’ β-catenin (Ctnnb1) reverse: 5’-CTGCAGTCTCATTCCAAGCC-3’. c-myc forward: 5’-GCCCAGTGAGGATATCTGGA-3’ c-myc reverse: 5’-ATCGCAGATGAAGCTCTGGT-3’ cyclin D1(Cnnd1) forward: 5’-TCCCAGACGTTCAGAACC-3’ cyclin D1 (Cnnd1) reverse: 5’-AGGGCATCTGTAAATACACT-3’ Mmp-9 forward: 5’-ACGACATAGACGGCATCCAGTATC-3’ Mmp-9 reverse: 5’-AGGTATAGTGGGACACATAGTGGG-3’

2.8. Statistical analysis

Data are shown as means ± SD. Results from qRT-PCR and Western blot were analyzed by one way ANOVA followed by Bonferroni test or two-tailed t-test to evaluate differences between control (vehicle) and treated conditions (1α,25(OH)2D3 or TX 527) at each time (*p<0.05 or **p<0.01). 3. Results 3.1. 1α,25(OH)2D3 and TX 527 increase β-catenin expression As outlined in the introduction, stable expression of vGPCR in endothelial cells activates the canonical Wnt/β-catenin pathway (Angelova et al., 2014). Therefore, we first investigated whether VDR agonists regulate β-catenin protein levels. vGPCR cells were plated and starved for 24 h, afterwards cultures were incubated with 1α,25(OH)2D3 or TX 527 in DMEM 2% FBS for 12-48 h or vehicle (0.01% ethanol) or during 48 h at different doses. Cell lysates were prepared and subject adequately for immunoblot analysis. According to Fig. 1A and B, β-catenin protein levels significantly increased in time and dosedependent manner. To determine the nature of β-catenin protein accumulation, we next blocked transcription with Actinomycin D (Act-D, 10 µg L-1) alone or in combination with 1α,25(OH)2D3 (10 nmol L-1) for 24 h (Fig 1 C). Under these conditions, β-catenin protein levels slightly decreased in presence of Act-D, whereas in combination with 1α,25(OH)2D3 significantly increased and this effect was comparable to 1α,25(OH)2D3 treatment alone. 3.2. β-catenin protein levels are VDR dependent Previously, we demonstrated that 1α,25(OH)2D3 or TX 527 treatment induced cell cycle arrest and apoptosis of vGPCR cells by a mechanism that involved VDR (González Pardo et al., 2014, 2010). Consequently, VDR participation on β-catenin protein levels was examined using the stable VDR knockdown cell line vGPCR-shVDR. To that end, vGPCR-shctrl and vGPCR-shVDR cells were plated and incubated with 1α,25(OH)2D3 or TX 527 or vehicle in DMEM 2% FBS for 48 h. Cell lysates were prepared and subject to immunoblot analysis with VDR, β-catenin and Tubulin antibodies. First, VDR knockdown was monitored as we have previously reported (data not shown) (González Pardo et al., 2014, 2010). The results in Fig. 2 indicate that β-catenin protein levels significantly increased during 1α,25(OH)2D3 or TX 527 treatment in control cells (vGPCR-shctrl) and when VDR was knocked down in vGPCR-shVDR, even though treated conditions showed a lesser increase compared to control, these effects were significantly decreased when comparing vGPCR-shctrl/shVDR treated conditions (**p<0.01). This suggests β-catenin increase depends on VDR expression. 3.3. Nuclear and membrane accumulation of β-catenin is increased by 1α,25(OH)2D3 treatment β-catenin localization was studied in vGPCR cells treated with 1α,25(OH)2D3 or vehicle for 48 h by two different approaches. First, β-catenin immunostaining was investigated with confocal microscopy as described in methods. As indicated in Fig. 3A left panel, β-catenin was mostly observed in the nucleus, cytosol and less in plasma membrane in control cells (vehicle); whereas β-catenin was more concentrated in nucleus and plasma membrane after 1α,25(OH)2D3 treatment as is indicated by arrows in the images in Fig. 3A right panel. To corroborate nuclear β-catenin accumulation, nuclear fluorescence intensity was quantified (Fig. 3B). for 24 h. Confocal microscopy samples were obtained as described in Methods. Images are representative of two independent experiments. Arrows mark β-catenin localization in the plasma membrane and nucleus. Total magnification 630x, bar = 50 µm. (B) Nuclear Fluorescence Intensity (N.F.I) from two independent experiments (mean ± SEM) was quantified as described in Methods and analyzed using Student’s test (**p < 0.01). Secondly, β-catenin protein was quantified in each compartment by Western blot analysis in enriched nucleus, cytosol and microsomal fractions. As described in methods, cells were collected in TES buffer and subject to differential centrifugation to obtain enriched subcellular fractions. Western blot analysis with anti-β-catenin antibody was performed, and with Lamin B, α-MEK and αQ11 antibodies as nuclear, cytosol and plasma membrane markers respectively. The results shown in Fig. 4 confirmed that 1α,25(OH)2D3 significantly increased β-catenin in the nucleus (Fig. 4A) and in plasma membrane in TES buffer and subject to differential centrifugation to obtain enriched nuclear (N), cytosolic (C) and microsomal fractions (M). Western blot analyses were performed with anti-β-catenin, VE-cadherin, Lamin B, α-MEK and αQ11 antibodies. Representative blots of five (A) and three (B) independent experiments were quantified by Image J and data were then represented in bar graphs. (C) Nucleus: Cytosol (N:C) and Membrane: Cytosol (M:C) ratios are shown. The statistical significance was evaluated using Student’s t-test (**p< 0.01, *p< 0.05). 3.4. 1α,25(OH)2D3 increases VE-cadherin in the plasma membrane Since E-cadherin induction allows a phenotypic change from malignancy towards a normal epithelial type (Pálmer et al., 2001), we also investigated whether β-catenin raise by 1α,25(OH)2D3 in plasma membrane correlates with VE-cadherin protein expression. Hence, vGPCR cells were treated with 1α,25(OH)2D3 or vehicle for 48 h in DMEM 2% FBS. As mentioned previously in methods, cells were collected in TES buffer and subject to differential centrifugation to obtain enriched membrane fraction. Western blot analyses with anti-β-catenin and anti-VE-cadherin antibodies were performed, αQ11 antibody was used as membrane marker. The results shown in Fig. 4B indicate that VE-cadherin protein is presence in the membrane fraction and increased significantly upon 1α,25(OH)2D3 treatment as well as β- catenin. 3.5. 1α,25(OH)2D3 induces β-catenin-VDR nuclear association As reported, 1,α25(OH)2D3 promotes a direct VDR-β-catenin interaction, proving a nuclear β-catenin non-transcriptional accumulation (Larriba et al., 2011; Pálmer et al., 2001). However, the existence of this interaction is unknown in other neoplastic cell types. To elucidate in more depth the inhibitory effect of 1α,25(OH)2D3 in Wnt/β-catenin pathway, we investigated the possible association of VDR with βcatenin in vGPCR cells. To that end, the cells were treated as in Fig. 5 and then collected in coimmunoprecipitation buffer (Co-IP) followed by immunoprecipitation of β-catenin or VDR as described in Methods. VDR protein was analyzed in β-catenin immunoprecipitates or reverse by Western blot. As shown in Fig. 5, VDR was detected in β-catenin immunoprecipitates; when reverse immunoprecipitation was carried out with VDR antibody, β-catenin was revealed in VDR immunoprecipitates indicating that the association between VDR and β-catenin was effectively induced by 1α,25(OH)2D3 treatment. 3.6. Dkk-1 extracellular inhibitor is early upregulated by 1α,25(OH)2D3 According to existing data, 1α,25(OH)2D3 increases DKK-1 mRNA and protein levels in colon cancer cells associated to the induction of an epithelial adhesive phenotype (Aguilera et al., 2007). Therefore, changes in Dkk-1 and β-catenin mRNA were analyzed. vGPCR cells were incubated with 1α,25(OH)2D3 or vehicle in presence of DMEM 2% FBS for 0.3-1 h. Total RNA from control (vehicle) and treated conditions was isolated. The RNA was reverse transcribed followed by qRT-PCR using specific primers to detect Dkk-1 and β-catenin mRNA. To normalize gene expression Gapdh mRNA was used. The results shown in Fig. 6A revealed that 1α,25(OH)2D3 significantly increased Dkk-1 mRNA levels before an hour of treatment (3.5 fold at 40 min), whereas β-catenin increase was minor (0.25 fold, Fig. 6B). independent experiments was evaluated using Student’s t-test (**p< 0.01, *p< 0.05). For (C) cell lysates were subject to Western blot analysis with anti-β-catenin and anti-tubulin antibodies. Blots were quantified and represented in bar graphs. Data from at least two independent experiments were analyzed using Student’s t-test (**p< 0.01, *p< 0.05). Next, to evaluate whether β-catenin variations in mRNA levels were reflected in protein expression, cells were incubated with 1α,25(OH)2D3, TX 527 or vehicle for 0.5-3 h. Cell lysates were prepared and subject to immunoblot. As shown in Fig. 6C β-catenin protein levels significantly decreased at the beginning (0.5-1.5 h) of both treatments. 3.7. 1α,25(OH)2D3 downregulates β-catenin dependent transcription of c-myc, Mmp-9 and cyclin D1 There is evidence indicating that VDR/β-catenin interaction reduces the quantity of β-catenin bound to TCF, inhibiting the expression β-catenin/TCF target genes like c-MYC in human cancer cells (Larriba et al., 2011, 2007; Pálmer et al., 2001). c-MYC regulates cell cycle progression and its expression is often elevated or deregulated in human cancer (Morrish et al., 2009). Moreover, it has been reported a significant increase in expression of β-catenin target genes cyclin D1, MMP-9 and VEGFA by vGPCR in human endothelial cells (HUVEC) (Angelova et al., 2014) that have been implicated in angiogenesis and KS development (Arasteh and Hannah, 2000; Hong et al., 2004; Qian et al., 2007). Consequently, cmyc, Mmp-9 and cyclin D1 gene expression were analyzed in vGPCR cells treated with 1α,25(OH)2D3 or vehicle in presence of DMEM 2% FBS for 48 h. Total RNA from control (vehicle) or treated conditions was then isolated and RNA was reverse transcribed followed by qRT-PCR using specific primers to detect c-myc, Mmp-9 and cyclin D1 mRNA. To normalize gene expression Gapdh mRNA was used. As can be seen in Fig. 7, c-myc, Mmp-9 and cyclin D1 mRNA expression was effectively diminished after 1α,25(OH)2D3 treatment, suggesting the downregulation of β-catenin activity as a transcription factor. 4. Discussion In many types of tissues 1α,25(OH)2D3 has immunomodulatory activity, affecting both innate and adaptive immune responses. Clinical data suggested its potential application in cancer prevention and treatment, due to 1α,25(OH)2D3 antiproliferative effects in neoplastic processes (Beer and Myrthue, 2004). Nevertheless, its calcemic effects such as hypercalcemia, hypercalciuria, and increased bone resorption, have limited the use of the hormone with therapeutic purposes (Eelen et al., 2007). Varieties of synthetic analogs indicate dissociation between unwanted calcemic and beneficial antiproliferative effects. Some of them have a stronger antiproliferative action (Bouillon et al., 2005; Eelen et al., 2007, 2005). TX 527 [19-nor-14,20-bisepi-23-yne1,25(OH)2D3] biological activity has been reported showing its capacity to inhibit human breast cancer in vivo and in vitro (Eelen et al., 2005; Verlinden et al., 2000). Previously, we have demonstrated that the antiproliferative effects of 1α,25(OH)2D3 and its analog TX 527 in vGPCR occur in part by negative modulation of the NF-κB pathway, a family of evolutionarily conserved transcription factors critical in mediating the pro-inflammatory response (González Pardo et al., 2013, 2012). NF-κB role is fundamental in the generation and maintenance of malignancies (Nishikori et al., 2005). However, the molecular mechanism of action of VDR agonists in inflammatory gene alteration and tumor development has not been completely elucidated yet. There are several data of cross-talk between nuclear hormone receptors and Wnt/β-catenin pathway, particularly VDR-β-catenin interaction (Beildeck et al., 2011; Pálmer et al., 2001; Shah et al., 2006), which is complex and not fully understood. Evidence shows that Wnt/β-catenin signaling is abnormally activated in most colorectal cancers and 1α,25(OH)2D3 antagonizes this cascade. VDR-β-catenin association lows the amount of β-catenin bound to TCF, which impedes its transcriptional activity in target genes such as c-MYC, TCF1, LEF1 and AXIN2 in human colon cancer cells (Larriba et al., 2011, 2007). These antagonism mechanisms are VDR dependent, as they are absent in VDR-negative human colon cancer cells (SW480-R and SW620) or in VDR-positive cells (SW480-ADH), where VDR expression has been repressed by SNAIL1 or SNAIL2 overexpression or by shRNA technology (Larriba et al., 2009; Pálmer et al., 2004). Many studies have reported the high expression of TCF/β-catenin target genes such as c-myc, MMP-9 and cyclin D1 in different types of tumors (Burandt et al., 2016; Cai et al., 2017; Hessmann et al., 2016). In addition, E-cadherin is a protein linker between cells being part of the adherens junctions. It plays the main role in the adhesive and polarized phenotype maintenance of epithelial cells (Fagotto and Gumbiner, 1996). The loss of E-cadherin expression involves the obtainment of an invasive phenotype (Christofori and Semb, 1999). There is evidence that 1α,25(OH)2D3 increases E-cadherin protein levels leading to the relocation of β-catenin to the adherens junctions contributing to the hormone antiangiogenic effect in colon carcinoma cells (Pálmer et al., 2001). Apart from that, another event at the plasma membrane happens through the extracellular inhibitor DKK-1, blocking the formation of Wnt/βcatenin signaling complex of destruction (González-Sancho et al., 2005). Numerous data demonstrate coordination between DKK-1 regulation and Wnt signaling inhibition in different cell types (Kim et al., 2015). DKK-1 adenoviral expression inhibits Wnt target gene expression, losing the proliferative crypts and eventual inflammation (Hoffman et al., 2004). Also in colon cancer DKK-1 is downregulated, but the treatment with 1α,25(OH)2D3 induces DKK-1 gene expression associated to the differentiation of human colon cancer cells (Aguilera et al., 2007; González-Sancho et al., 2005). Recently, Rubin and collaborators have presented DKK-1 increased mRNA levels in a human adrenocortical carcinoma cell line after 1α,25(OH)2D3 treatment in combination with mitotane as part of the molecular mechanism of cellular growth inhibition (Rubin et al., 2019). In this study we found β-catenin protein levels increased in a time (Fig. 1A) and dose-dependent (Fig. 1B) manner, during 1α,25(OH)2D3 or TX 527 treatment in vGPCR cells. This mechanism depends on VDR expression (Fig. 2). What is more, we observed β-catenin nuclear and plasma membrane localization after 1α,25(OH)2D3 treatment (Fig. 3 and 4) and VDR-β-catenin association (Fig. 5) which could be preventing β-catenin to act as a transcription factor of c-myc, Mmp-9 and cyclin D1 (Fig. 7), key result to relate the antiproliferative effect of VDR agonists previously reported (González Pardo et al., 2010) with the inhibition of Wnt/β-catenin pathway. Our results also suggest an anti-angiogenic action through VE-cadherin elevated expression after 1α,25(OH)2D3 treatment leading to β-catenin relocation at the plasma membrane (Fig. 3 and 4B). Finally, 1α,25(OH)2D3 treatment for less than 3 hours indicated a Dkk-1 mRNA increment which correlates with a β-catenin protein low expression (Fig. 6). These results could be related to an initial attempt to diminish β-catenin levels during 1α,25(OH)2D3 short times treatment due to the extracellular inhibitor increase. However, after longer periods, 1α,25(OH)2D3 treatment indicated β-catenin non-transcriptional nuclear accumulation in vGPCR cells, depending on VDR (Fig. 8). In conclusion, despite further investigations are needed to elucidate the mechanism involved in the antiproliferative action of 1α,25(OH) D , our study provides evidence of Wnt/β-catenin signaling References Aguilera, O., Peña, C., García, J.M., Larriba, M.J., Ordóñez-Morán, P., Navarro, D., Barbáchano, A., López de Silanes, I., Ballestar, E., Fraga, M.F., Esteller, M., Gamallo, C., Bonilla, F., GonzálezSancho, J.M., Muñoz, A., 2007. The Wnt antagonist DICKKOPF-1 gene is induced by 1alpha,25dihydroxyvitamin D3 associated to the differentiation of human colon cancer cells. Carcinogenesis 28, 1877–84. https://doi.org/10.1093/carcin/bgm094 Angelova, M., Ferris, M., Swan, K.F., McFerrin, H.E., Pridjian, G., Morris, C.A., Sullivan, D.E., 2014. Kaposi’s sarcoma-associated herpesvirus G-protein coupled receptor activates the canonical Wnt/βcatenin signaling pathway. Virol. J. 11, 218. https://doi.org/10.1186/s12985-014-0218-8 Arasteh, K., Hannah, A., 2000. The Role of Vascular Endothelial Growth Factor in Restenosis. Oncologist 5, 28–31. https://doi.org/https://doi.org/10.1634/theoncologist.5-suppl_1-28 Beer, T.M., Myrthue, A., 2004. Calcitriol in cancer treatment: from the lab to the clinic. Mol. Cancer Ther. 3, 373–81. https://doi.org/10.1023/A:1026203418976 Beildeck, M.E., Gelmann, E.P., Byers, S.W., 2011. Cross-regulation of signaling pathways: an example of nuclear hormone receptors and the canonical Wnt pathway. Cell 316, 1763–1772. https://doi.org/10.1016/j.yexcr.2010.02.001.Cross-regulation Bouillon, R., Verlinden, L., Eelen, G., De Clercq, P., Vandewalle, M., Mathieu, C., Verstuyf, A., 2005. Mechanisms for the selective action of Vitamin D analogs. J. Steroid Biochem. Mol. Biol. 97, 21–30. https://doi.org/10.1016/j.jsbmb.2005.06.013 Buitrago, C., González Pardo, V., de Boland, A.R., 2002. Nongenomic action of 1 alpha,25(OH)(2)vitamin D3. Activation of muscle cell PLC gamma through the tyrosine kinase c-Src and PtdIns 3kinase. Eur. J. Biochem. 269, 2506–15. https://doi.org/10.1046/j.1432-1033.2002.02915.x Burandt, E., Grünert, M., Lebeau, A., Choschzick, M., Quaas, A., Jänicke, F., Müller, V., Scholz, U., Bokemeyer, C., Petersen, C., Geist, S., Paluchowski, P., Wilke, C., Heilenkötter, U., Simon, R., Sauter, G., Wilczak, W., 2016. Cyclin D1 gene amplification is highly homogeneous in breast cancer. Breast Cancer 23, 111–119. https://doi.org/10.1007/s12282-014-0538-y Cadigan, K.M., Waterman, M.L., 2012. TCF/LEFs and Wnt signaling in the nucleus. Cold Spring Harb. Perspect. Biol. 4, 1–22. https://doi.org/10.1101/cshperspect.a007906 Cai, X., Zhu, H., Li, Y., 2017. PKCξ, MMP-2 and MMP-9 expression in lung adenocarcinoma and association with a metastatic phenotype. Mol. Med. Rep. 16, 8301–8306. https://doi.org/10.3892/mmr.2017.7634 Christofori, G., Semb, H., 1999. The role of the cell-adhesion molecule E-cadherin as a tumoursuppressor gene. Trends Biochem. Sci. 24, 73–6. https://doi.org/10.1016/S0968-0004(98)01343-7 Eelen, G., Gysemans, C., Verlinden, L., Vanoirbeek, E., De Clercq, P., Van Haver, D., Mathieu, C., Bouillon, R., Verstuyf, A., 2007. Mechanism and potential of the growth-inhibitory actions of vitamin D and ana-logs. Curr. Med. Chem. 14, 1893–910. https://doi.org/10.2174/092986707781058823 Eelen, G., Verlinden, L., Van Camp, M., Claessens, F., De Clercq, P., Vandewalle, M., Bouillon, R., Verstuyf, A., 2005. Altered Vitamin D receptor-coactivator interactions reflect superagonism of Vitamin D analogs. J. Steroid Biochem. Mol. Biol. 97, 65–8. https://doi.org/10.1016/j.jsbmb.2005.06.009 Fagotto, F., Gumbiner, B.M., 1996. Cell contact-dependent signaling. Dev. Biol. 180, 445–54. https://doi.org/10.1006/dbio.1996.0318 González-Sancho, J.M., Aguilera, O., García, J.M., Pendás-Franco, N., Peña, C., Cal, S., García de Herreros, A., Bonilla, F., Muñoz, A., 2005. The Wnt antagonist DICKKOPF-1 gene is a downstream target of beta-catenin/TCF and is downregulated in human colon cancer. Oncogene 24, 1098–103. https://doi.org/10.1038/sj.onc.1208303 González Pardo, V., Boland, R., de Boland, A.R., 2008. Vitamin D receptor levels and binding are reduced in aged rat intestinal subcellular fractions. Biogerontology 9, 109–18. https://doi.org/10.1007/s10522-007-9118-2 González Pardo, V., Boland, R., de Boland, A.R., 2006. 1alpha,25(OH)(2)-Vitamin D(3) stimulates intestinal cell p38 MAPK activity and increases c-Fos expression. Int. J. Biochem. Cell Biol. 38, 1181–90. https://doi.org/10.1016/j.biocel.2005.12.018 González Pardo, V., D’Elia, N., Verstuyf, A., Boland, R., Russo de Boland, A., 2012. NFκB pathway is down-regulated by 1α,25(OH)(2)-vitamin D(3) in endothelial cells transformed by Kaposi sarcomaassociated herpes virus G protein coupled receptor. Steroids 77, 1025–32. https://doi.org/10.1016/j.steroids.2012.05.006 González Pardo, V., Martin, D., Gutkind, J.S., Verstuyf, A., Bouillon, R., de Boland, A.R., Boland, R.L., 2010. 1α,25-dihydroxyvitamin D3 and its TX527 analog inhibit the growth of endothelial cells transformed by Kaposi sarcoma-associated herpes virus G protein-coupled receptor in vitro and in vivo. Endocrinology 151, 23–31. https://doi.org/10.1210/en.2009-0650 González Pardo, V., Suares, A., Verstuyf, A., De Clercq, P., Boland, R., de Boland, A.R., 2014. Cell cycle arrest and apoptosis induced by 1α,25(OH)2D3 and TX 527 in Kaposi sarcoma is VDR dependent. J. Steroid Biochem. Mol. Biol. 144 Pt A, 197–200. https://doi.org/10.1016/j.jsbmb.2013.11.014 González Pardo, V., Verstuyf, A., Boland, R., Russo de Boland, A., 2013. Vitamin D analogue TX 527 down-regulates the NF-κB pathway and controls the proliferation of endothelial cells transformed by Kaposi sarcoma herpesvirus. Br. J. Pharmacol. 169, 1635–45. https://doi.org/10.1111/bph.12219 Hessmann, E., Schneider, G., Ellenrieder, V., Siveke, J.T., 2016. MYC in pancreatic cancer: Novel mechanistic insights and their translation into therapeutic strategies. Oncogene 35, 1609–1618. https://doi.org/10.1038/onc.2015.216 Hlaing, S.M., Garcia, L.A., Contreras, J.R., Norris, K.C., Ferrini, M.G., Artaza, J.N., 2014. 1,25-vitamin D3 promotes cardiac differentiation through modulation of the WNT signaling pathway. J. Mol. Endocrinol. 53, 303–317. https://doi.org/10.1530/JME-14-0168 Hoffman, J., Kuhnert, F., Davis, C.R., Kuo, C.J., 2004. Wnts as essential growth factors for the adult small intestine and colon. Cell Cycle 3, 554–7. Hong, A., Davies, S., Stevens, G., Lee, S., 2004. Cyclin D1 Overexpression in AIDS-related and Classic Kaposi Sarcoma. Dermatological Cryosurgery and Cryotherapy 12, 26–30. https://doi.org/10.1007/978-1-4471-6765-5_133 Johnson, A.L., Zinser, G.M., Waltz, S.E., 2015. Vitamin D3-dependent VDR signaling delays ronmediated breast tumorigenesis through suppression of β-catenin activity. Oncotarget 6, 16304–20. https://doi.org/10.18632/oncotarget.4059 Kim, H.Y., Park, J.H., Won, H.Y., Lee, J.Y., Kong, G., 2015. CBX7 inhibits breast tumorigenicity through DKK-1-mediated suppression of the Wnt/β-catenin pathway. FASEB J. 29, 300–313. https://doi.org/10.1096/fj.14-253997 Krishnan, A. V., Feldman, D., 2011. Mechanisms of the anti-cancer and anti-inflammatory actions of vitamin D. Annu. Rev. Pharmacol. Toxicol. 51, 311–36. https://doi.org/10.1146/annurev-pharmtox010510-100611 Larriba, M.J., González-Sancho, J.M., Barbáchano, A., Niell, N., Ferrer-Mayorga, G., Muñoz, A., 2013. Vitamin D Is a Multilevel Repressor of Wnt/b-Catenin Signaling in Cancer Cells. Cancers (Basel). 5, 1242–60. https://doi.org/10.3390/cancers5041242 Larriba, M.J., Martín-Villar, E., García, J.M., Pereira, F., Peña, C., de Herreros, A.G., Bonilla, F., Muñoz, A., 2009. Snail2 cooperates with Snail1 in the repression of vitamin D receptor in colon cancer. Carcinogenesis 30, 1459–68. https://doi.org/10.1093/carcin/bgp140 Larriba, M.J., Ordóñez-Morán, P., Chicote, I., Martín-Fernández, G., Puig, I., Muñoz, A., Pálmer, H.G., 2011. Vitamin D receptor deficiency enhances Wnt/β-catenin signaling and tumor burden in colon cancer. PLoS One 6, e23524. https://doi.org/10.1371/journal.pone.0023524 Larriba, M.J., Valle, N., Pálmer, H.G., Ordóñez-Morán, P., Alvarez-Díaz, S., Becker, K.-F., Gamallo, C., de Herreros, A.G., González-Sancho, J.M., Muñoz, A., 2007. The inhibition of Wnt/betacatenin signalling by 1alpha,25-dihydroxyvitamin D3 is abrogated by Snail1 in human colon cancer cells. Endocr. Relat. Cancer 14, 141–51. https://doi.org/10.1677/ERC-06-0028 Mao, B., Wu, W., Davidson, G., Marhold, J., Li, M., Mechler, B.M., Delius, H., Hoppe, D., Stannek, P., Walter, C., Glinka, A., Niehrs, C., 2002. Kremen proteins are Dickkopf receptors that regulate Zamaporvint Wnt/beta-catenin signalling. Nature 417, 664–7. https://doi.org/10.1038/nature756
Martin, D., Gutkind, J.S., 2009. Kaposi’s sarcoma virally encoded, G-protein-coupled receptor: a paradigm for paracrine transformation. Methods Enzymol. 460, 125–50. https://doi.org/10.1016/S0076-6879(09)05206-9
Mesri, E.A., Cesarman, E., Boshoff, C., 2010. Kaposi’s sarcoma and its associated herpesvirus. Nat. Rev. Cancer 10, 707–719. https://doi.org/10.1038/nrc2888
Montaner, S., Sodhi, A., Molinolo, A., Bugge, T.H., Sawai, E.T., He, Y., Li, Y., Ray, P.E., Gutkind, J.S., 2003. Endothelial infection with KSHV genes in vivo reveals that vGPCR initiates Kaposi’s sarcomagenesis and can promote the tumorigenic potential of viral latent genes. Cancer Cell 3, 23–36. https://doi.org/10.1016/S1535-6108(02)00237-4
Morrish, F., Isern, N., Sadilek, M., Jeffrey, M., David, M., 2009. c-Myc activates multiple metabolic networks to generate substrates for cell cycle entry Hockenbery1. Oncogene 28, 2485–2491. https://doi.org/10.1038/onc.2009.112.c-Myc
Nishikori, M., Ohno, H., Haga, H., Uchiyama, T., 2005. Stimulation of CD30 in anaplastic large cell lymphoma leads to production of nuclear factor-kappaB p52, which is associated with hyperphosphorylated Bcl-3. Cancer Sci. 96, 487–97. https://doi.org/10.1111/j.1349-7006.2005.00078.x
Pálmer, H.G., González-Sancho, J.M., Espada, J., Berciano, M.T., Puig, I., Baulida, J., Quintanilla, M., Cano, A., de Herreros, A.G., Lafarga, M., Muñoz, A., 2001. Vitamin D(3) promotes the differentiation of colon carcinoma cells by the induction of E-cadherin and the inhibition of betacatenin signaling. J. Cell Biol. 154, 369–87. https://doi.org/10.1083/jcb.200102028
Pálmer, H.G., Larriba, M.J., García, J.M., Ordóñez-Morán, P., Peña, C., Peiró, S., Puig, I., Rodríguez, R., de la Fuente, R., Bernad, A., Pollán, M., Bonilla, F., Gamallo, C., de Herreros, A.G., Muñoz, A., 2004. The transcription factor SNAIL represses vitamin D receptor expression and responsiveness in human colon cancer. Nat. Med. 10, 917–9. https://doi.org/10.1038/nm1095
Polakis, P., 2012. Wnt signaling in cancer. Cold Spring Harb. Perspect. Biol. 4, 9. https://doi.org/10.1101/cshperspect.a008052
Qian, L.-W., Xie, J., Ye, F., Gao, S.-J., 2007. Kaposi’s Sarcoma-Associated Herpesvirus Infection Promotes Invasion of Primary Human Umbilical Vein Endothelial Cells by Inducing Matrix Metalloproteinases. J. Virol. 81, 7001–7010. https://doi.org/10.1128/jvi.00016-07
Rubin, B., Pilon, C., Pezzani, R., Rebellato, A., Fallo, F., 2019. The effects of mitotane and 1α,25dihydroxyvitamin D3 on Wnt/beta-catenin signaling in human adrenocortical carcinoma cells. J. Endocrinol. Invest. https://doi.org/10.1007/s40618-019-01127-1
Schneider, C.A., Rasband, W.S., Eliceiri, K.W., 2012. NIH Image to ImageJ: 25 years of Image Analysis. Fundam. Digit. Imaging Med. 9, 185–188. https://doi.org/10.1007/978-1-84882-087-6_9
Shah, S., Islam, M.N., Dakshanamurthy, S., Rizvi, I., Rao, M., Herrell, R., Zinser, G., Valrance, M., Aranda, A., Moras, D., Norman, A., Welsh, J.E., Byers, S.W., 2006. The molecular basis of vitamin D receptor and β-catenin crossregulation. Mol. Cell 21, 799–809. https://doi.org/10.1016/j.molcel.2006.01.037
Suares, A., Mori Sequeiros Garcia, M., Paz, C., González Pardo, V., 2017. Antiproliferative effects of Bortezomib in endothelial cells transformed by viral G protein-coupled receptor associated to Kaposi’s sarcoma. Cell. Signal. 32, 124–132. https://doi.org/10.1016/j.cellsig.2017.01.025
Uranga, R.M., Alza, N.P., Conde, M.A., Antollini, S.S., Salvador, G.A., 2017. Phosphoinositides: TwoPath Signaling in Neuronal Response to Oligomeric Amyloid β Peptide. Mol. Neurobiol. 54, 3236–3252. https://doi.org/10.1007/s12035-016-9885-3
Verlinden, L., Verstuyf, A., Van Camp, M., Marcelis, S., Sabbe, K., Zhao, X.Y., De Clercq, P., Vandewalle, M., Bouillon, R., 2000. Two novel 14-Epi-analogues of 1,25-dihydroxyvitamin D3 inhibit the growth of human breast cancer cells in vitro and in vivo. Cancer Res. 60, 2673–9.
Willert, K., Nusse, R., 2012. Wnt proteins. Cold Spring Harb. Perspect. Biol. 4, a007864. https://doi.org/10.1101/cshperspect.a007864
Xiong, Y., Zhang, Y., Xin, N., Yuan, Y., Zhang, Q., Gong, P., Wu, Y., 2017. 1α,25-Dihydroxyvitamin D3promotes osteogenesis by promoting Wnt signaling pathway. J. Steroid Biochem. Mol. Biol. 174, 153–160. https://doi.org/10.1016/j.jsbmb.2017.08.014