. 2010 May 20;31(8):1434–1441. doi: 10.1093/carcin/bgq098

Apc^MIN modulation of vitamin D secosteroid growth control

Haibo Xu ³, Gary H Posner ¹, Michael Stevenson ², Frederick C Campbell ^1,^*

PMCID: PMC2915628 PMID: 20488884

Abstract

A central paradox of vitamin D biology is that 1α,25-(OH)₂ D₃ exposure inversely relates to colorectal cancer (CRC) risk despite a capacity for activation of both pro- and anti-oncogenic mediators including osteopontin (OPN)/CD44 and E-cadherin, respectively. Most sporadic CRCs arise from adenomatous polyposis coli (APC) gene mutation but understanding of its effects on vitamin D growth control is limited. Here we investigate effects of the Apc^Min/+ genotype on 1α,25-(OH)₂ D₃ regulation of OPN/CD44/E-cadherin signalling and intestinal tumourigenesis, in vivo. In untreated Apc^Min/+ versus Apc^+/+ intestines, expression levels of OPN and its CD44 receptor were increased, whereas E-cadherin tumour suppressor signalling was attenuated. Treatment by 1α,25-(OH)₂ D₃ or rationally designed analogues (QW or BTW) enhanced OPN but inhibited expression of CD44, the OPN receptor implicated in cell growth. These treatments also enhanced E-cadherin tumour suppressor activity, characterized by inhibition of β-catenin nuclear localization, T-cell factor 1 and c-myelocytomatosis protein expression in Apc^Min/+ intestine. All secosteroids suppressed Apc^Min/+-driven tumourigenesis although QW and BTW had lower calcium-related toxicity. Taken together, these data indicate that the Apc^Min/+ genotype modulates vitamin D secosteroid actions to promote functional predominance of E-cadherin tumour suppressor activity within antagonistic molecular networks. APC heterozygosity may promote favourable tissue- or tumour-specific conditions for growth control by vitamin D secosteroid treatment.

Introduction

Human vitamin D exposure, assessed by serum 25-OHD levels (1) is inversely associated with risk of colorectal adenomas (2), colorectal cancer (CRC) (3,4) and CRC mortality (5). However, direct associations have also been shown between serum 25-OHD levels and cancers of other organs (6,7). Although 1α,25-(OH)₂ D₃ target genes may represent potentially modifiable cancer risk factors, mechanisms responsible for the tissue or tumour specificity of 1α,25-(OH)₂ D₃ growth control remain unclear.

Expression profiling has identified diverse 1α,25-(OH)₂ D₃ molecular targets including G-coupled receptors, intercellular and intracellular signalling genes, cell cycle regulators and adhesion molecules (8). Within these signalling cascades, osteopontin (OPN) and E-cadherin orchestrate growth responses to 1α,25-(OH)₂ D₃ (9,10). OPN is a predominantly secreted glycoprotein (11) that is regulated by 1α,25-(OH)₂ D₃ transcription-dependent (12) and -independent mechanisms (13). OPN biological effects are primarily mediated through interactions between secreted OPN and its integrin or CD44 receptors on target cells (14). While integrin binding promotes cell adhesion (15), OPN binding to CD44 activates the phosphatidylinositol 3-kinase-Akt pathway, suppresses apoptosis (16), promotes foci formation, invasion and tumourigenesis (17). Conversely, antisense suppression of CD44 inhibits colorectal tumour growth and metastasis, partly by suppression of OPN–CD44 binding (18). OPN is a central effector of vitamin D-mediated anchorage-independent growth (19) and is a lead marker of CRC progression (20).

E-cadherin is induced by 1α,25-(OH)₂ D₃ transcription-independent rapid actions (13) and suppresses colorectal tumourigenesis (21), partly by inhibition of β-catenin transcriptional activity (22). β-catenin is sequestered by E-cadherin, rapidly phosphorylated by glycogen synthase kinase 3β, then ubiquitinated and degraded. Impaired E-cadherin function is a hallmark feature of CRC (23) and enables β-catenin accumulation particularly in the nucleus where it modulates the expression of T-cell factor (Tcf)/Lef-1-target genes (24). E-cadherin loss also activates other signalling pathways (25), including upregulation of Twist (26), a transcriptional coactivator of CD44 (27). Taken together, the above seminal studies have shown that 1α,25-(OH)₂ D₃ modulates a functionally antagonistic growth regulatory network, which may raise important risk-benefit questions.

Previous investigators have sought to dissociate some toxicities and benefits of 1α,25-(OH)₂ D₃ treatment by development of related molecules with different structural configurations (28). 1α,25-(OH)₂ D₃ and analogues may differ in their stabilization of vitamin D receptor (VDR), exposure of functional interfaces (29) and interactions with transcriptional coactivators. Posner et al. (30) developed new vitamin D analogues with a modified A ring that have high growth regulatory potential. QW-1624F2-2 1-alpha-hydroxymethyl-16-ene-24,24-difluoro-25-hydroxy-26,27-bishomovitamin D3 (QW) is a low-calcaemic hybrid molecule that has transactivating capacity (31), although its effects on OPN transcription is lower than that of 1α,25-(OH)₂ D₃ (32). BTW is a 2,2-dimethylated 19-nor analogue that has similar antiproliferative activity to 1α,25-(OH)₂ D₃ in vitro but has ∼75-fold lower binding affinity to VDR (33).

Here, we report in vehicle-only (VO)-treated Apc^Min/+ versus Apc^+/+ mice, maintained on a vitamin D-deficient diet, that expression levels of OPN and its CD44 receptor were increased, whereas E-cadherin tumour suppressor signalling was attenuated in the intestine and colon. Treatment by 1α,25-(OH)₂ D₃ or analogues enhanced OPN but suppressed CD44. Treatment also enhanced E-cadherin tumour suppressor activity characterized by inhibition of β-catenin nuclear localization, Tcf1 and c-myelocytomatosis (c-Myc) protein expression and inhibited adenoma formation in Apc^Min/+ intestine. Taken together, these data suggest that the molecular signature activated by the Apc^Min/+ genotype may create favourable tissue- or tumour-specific conditions for vitamin D growth control.

Materials and methods

Chemicals and reagents

1α,25-Dihydroxyvitamin D₃ [1α,25-(OH)₂ D₃], C₂₇H₄₄O₃, molecular weight: 416.64 was purchased from Sigma–Aldrich UK (Dorset, UK) (Cat Number: D1530). The 1α,25-(OH)₂ D₃ analogues QW (molecular weight: 492) and BTW (molecular weight: 432) were synthesized as described previously (33). 1α,25-(OH)₂ D₃ and analogues were dissolved in absolute ethanol at 10⁻³mol/l, stored in aliquots at −20°C and diluted with 5% ethanol in normal saline, prior to injection. All other reagents were commercially available.

Animals and treatment regimes

All animal experiments were carried out in strict accordance with the UK Animals (Scientific Procedures) Act 1986 and covered by the appropriate Home Office project licence (PPL2499). All animal research procedures were approved by Queen's University Research Ethics committee. Apc^Min/+ heterozygous C57BL/6J mice and wild-type (wt) C57BL/6J-Apc^+/+ mice were obtained from Clare Hall Laboratories, London Research Institute, Cancer Research UK, South Mimms, Hertfordshire, UK and the Apc^Min/+ pedigree was maintained by crossing C57BL/6J-Apc^Min/+ males to C57BL/6J-Apc^+/+ females. Offspring were genotyped for the Apc allele with polymerase chain reaction analysis of genomic DNA from samples of ear tissue using Min-specific primers (forward primer: 5′-TCTCGTTCTGAGAAAGACAGAAGCT-3′ and reverse primer: 5′-TGATACTTCTTCCAAAGCTTTGGCTAT-3′).

Four-week-old female wt C57BL/6J Apc^+/+ and Apc^Min/+ mice were weaned and arranged in five groups of eight mice. Mice were housed at four per cage in a ventilated, temperature- and humidity-controlled animal facility with 12 h light–12 h dark cycle and fed with vitamin D-deficient diet containing 0.47% calcium and 0.3% phosphorus (Harlan Teklad, Madison, WI; Cat No: TD.89123; supplementary Table is available at Carcinogenesis Online) and distilled water ad libitum. The absence of cholecalciferol from this dietary formula has previously been confirmed by high-performance liquid chromatography analysis (34). Apc^Min/+ mice received intraperitoneal injections of 1α,25-(OH)₂ D₃, QW and BTW each in doses of 0.33 μg/kg. Dose selection for 1α,25-(OH)₂ D₃ was based on previous in vivo studies of antiproliferative properties and toxicity (35). QW has comparable VDR transactivating capacity with 1α,25-(OH)₂ D₃ (32). Although BTW has lower VDR affinity than 1α,25-(OH)₂ D₃ (36), it has similar antiproliferative activity in vitro (33). Hence, iso-osmolar doses of all three compounds were used in the study. To ensure, uniform administration, treatments were given by thrice weekly injection in a total volume of 100 μl or equal volumes of VO(5% ethanol–95% saline) for 12 weeks. Wt C57BL/6J Apc^+/+ mice received VO injections. Body weights were assessed weekly. From each mouse, 30 μl of tail blood was collected for serum calcium estimation at weeks 0, 4, 8 and 12. All mice were then killed by CO₂ inhalation at week 12 for tissue retrieval.

Determination of serum calcium concentration

The Arsenazo III method of serum calcium estimation (36) was conducted with kit reagents from Randox Laboratories, Co. Antrim, UK according to manufacturer's instructions. Blood samples were centrifuged at 3000 r.p.m. for 5 min. Ten microliters of serum per mouse was aspirated from the supernatant and applied to the colorimetric determination of calcium concentration at 650 nm of wavelength, using R1 Arsenazo reagent and the Calibration Serum Level 3 (Randox Laboratories Ltd; Cat No: CA2390, CAL2351), according to the manufacturer's instructions. Calcium concentrations were calculated relative to the calibration serum standards and expressed in milligrams per deciliters.

Assessment of small intestinal polyps

Small intestinal tumour formation was assessed as described previously (37,38). Briefly, small intestines (SIs) were divided into three equal proximal, middle and distal segments that were opened longitudinally and gently flushed with ice-cold phosphate-buffered saline. Intestinal segments were flattened on filter paper. Polyp number and diameter (mean of two largest diameters) were evaluated with a Leica dissecting microscope incorporating a calibrated eyepiece graticule at ×20 magnification by an experienced assessor, blinded to treatment categories. Polyp load was derived as the summation of polyp mean diameters in each intestinal segment.

Assay of colonic aberrant crypt foci

Aberrant crypt foci (ACF) were assessed as we have described previously (39). Briefly, colons were opened longitudinally, flushed with ice-cold phosphate-buffered saline, carefully pinned flat on a cork mat, painted with 0.2% methylene blue and left at room temperature for 10 min. Assay of ACF was performed using a Leica MZ 12.5 dissecting microscope at ×40 magnification. ACF number and crypt multiplicity were evaluated by an experienced assessor blinded to treatment categories.

Quantitative real-time polymerase chain reaction of VDR messenger RNA in SI and colon

Following assays of polyp or ACF formation, 30 mg segments of normal appearing tissue judged to be macroscopically tumour-free were excised from proximal, middle, distal SI and colon, snap frozen in liquid nitrogen and manually homogenized with glass mortar and pestle in 600 μl RNeasy Lysis Buffer containing 6 μl β-mercaptoethanol. VDR is a key regulator of 1α,25-(OH)₂ D₃ biological responses and was assayed at both transcript and protein level. The RNeasy Mini Kit (QIAGEN UK, West Sussex, UK; Cat No: 74104) was used for isolation of total RNA according to manufacturer's instructions, as we have described previously (40). Briefly, 500 ng total RNA was reverse transcribed in the presence of random hexamers (final concentration 2.5 μM), using MuLV reverse transcriptase (2.5 U/μl), 2 μl genomic DNA wipeout buffer and RNase-free water to a total volume of 20 μl per sample, using the QuantiTect Reverse Transcription Kit (QIAGEN UK; Cat No: 205311). This mixture was incubated at 42°C for 2 min to eliminate genomic DNA then incubated with 1 μl reverse transcriptase, RNase inhibitor, 4 μl of 5× reverse transcriptase buffer and 1 μl reverse transcriptase primer mix at 42°C for 15 min, followed by incubation at 95°C for 3 min to inactivate the transcriptase. The resulting complementary DNAs were then amplified using QuantiTect SYBR Green PCR Kit (QIAGEN UK; Cat No: 204143) on DNA Engine OPTICON 2 continuous fluorescence detector. Briefly, 500 ng complementary DNA per sample was quantified by NanoDrop 1000, using 0.75 μl of 10 μM forward primer, 0.75 μl of 10 μM reverse primer, 12.5 μl of 2× QuantiTect SYBR green PCR master mix and RNase-free water, made to a total volume of 25 μl. Primers were as follows: VDR forward 5′-ATCTGCATTGTCTCCCCAGACCGA-3′ and reverse 5′-GATCATCTTGGCGTAGAGCTGGTG-3′ and β-actin control forward 5′-GATCAAGATCATTGCTCCTCCTGAGC-3′ and reverse 5′-GACTCATCGTACTCCTGCTTGCTGA-3′. Final products of 147 bp (VDR) and 117 bp (β-actin) sequences were obtained.

Western blotting

Western blots were conducted in pooled samples containing macroscopically uninvolved mucosa from each respective intestinal region, as we have described previously (38). Western blot assays were conducted relating to VDR and 1α,25-(OH)₂ D₃ target genes that may be implicated in growth control, including E-cadherin, OPN, β-catenin, Tcf-1, c-Myc and CD44 (10,32). Briefly, tissue homogenates were incubated on ice in 1 ml of RIPA buffer (Sigma–Aldrich UK; Cat No: R0278-50ML), containing 150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate and 50 mM Tris, pH 8.0, with added 1.5 μl of protease inhibitor cocktail (Sigma–Aldrich UK; Cat No: P8340-5ML), containing 104 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride, 0.08 mM aprotinin, 2 mM leupeptin, 4 mM bestatin, 1.5 mM pepstatin A and 1.4 mM E-64. Following centrifugation at 14 000g for 10 minutes at 4°C, the supernatant was aspirated and stored at −80°C. For nuclear fractionation, freshly excised intestinal or colonic tissue was homogenized on ice with 0.5 ml buffer A, containing 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol and 0.05% IGEPAL CA-630, pH 7.9, with added 1.5 μl of protease inhibitor cocktail, followed by centrifugation at 3000 r.p.m. at 4°C for 10 min. The resulting pellet was resuspended with 374 μl buffer B, containing 5 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 1.5 mM MgCl₂, 0.2 mM ethylenediaminetetraacetic acid, 0.5 mM dithiothreitol and 26% glycerol (vol/vol), pH 7.9, with added 26 μl of 4.6 M NaCl, followed by homogenization on ice. The lysate was then left on ice for 30 min, centrifuged at 24 000g at 4°C for 20 min. The supernatant was collected and stored at −80°C.

After quantification with BCA protein assay kit (Thermo Scientific Pierce, Thermo Fisher Scientific, Northumberland, UK; Cat No: 23225), 20 μg of protein per lane were separated on sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane with 0.45 μm of pore size (Sigma–Aldrich UK; Cat No: N8392). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% tween-20 (TBST) for 45 min and incubated with polyclonal rabbit anti-VDR antibody (Santa Cruz Biotechnology, Insight Bio, Wembley, UK; Cat No: sc-9164, 51 kDa), monoclonal mouse anti-OPN antibody (The Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, IA; Cat No: MPIIIB10, 75 kDa), polyclonal goat anti-β-catenin antibody (Santa Cruz Biotechnology; Cat No: sc-1496, 92 kDa), polyclonal goat anti-Tcf1 antibody (Santa Cruz Biotechnology; Cat No: sc-8589, 48 kDa), polyclonal rabbit anti-CD44 antibody (Abcam, Cambridge, UK; Cat No: ab24504, 82 kDa), monoclonal mouse anti-c-Myc antibody (Santa Cruz Biotechnology; Cat No: sc-40, 67 kDa), monoclonal mouse anti-E-cadherin antibody (BD Biosciences; Cat No: 610181, 120 kDa), TATA-binding protein antibody (loading control for nuclear β-catenin; Abcam; Cat No: 1TBP18, 37 kDa), monoclonal mouse anti-β-actin antibody (Sigma–Aldrich UK; Cat No: A5441, 42 kDa) at 1:5000 in 1% bovine serum albumin–TBST overnight. Following incubation with horseradish peroxidase-conjugated secondary antibody (DAKO UK, Cambridge, UK) at dilution of 1:2000 for 90 min, the immunoreactivities were detected with western blotting luminol reagent (Santa Cruz Biotechnology; Cat No: sc-2048) and Amersham high-performance autoradiography film (GE Healthcare, Bucks, UK; Cat No: 28906844). Bands were quantified by densitometry, using a CS-9000 dual wavelength Flying Spot scanner (Shimadzu, Tokyo, Japan).

Data analysis

Effects of genotype and treatment on expression of specific genes in intestine or colon were assayed by one- or two-way analysis of variance (ANOVA) with the Tukey's post hoc test. Treatment effects on serum calcium levels, small intestinal polyp formation and colonic ACF were assessed by one- or two-way ANOVA. To assess regional variations in treatment effectiveness against polyp number or load, post-treatment values were expressed as a ratio to their pretreatment counterparts. Effects of treatment and small intestinal region on these ratios were assessed by two-way ANOVA. Large datasets of repeated or related variables were evaluated by principal component analysis (PCA) for data reduction and preparation of smaller coherent data subsets. Effects of genotype and treatment on weight gain were assessed by PCA and then by one-way ANOVA for mice in each genotype grouping. Descriptive statistics included the mean ± SE. Statistical assessment was conducted using SPSS version 17 (SPSS, Chicago, IL).

Results

Gene expression in Apc^Min/+ versus Apc^+/+ intestine or colon

1α,25-(OH)₂ D₃ exerts biological activity in part, by interaction with the VDR which acts as a ligand-dependent transcription factor. VDR messenger RNA levels, assessed by quantitative real-time polymerase chain reaction and densitometry, were greater in wt Apc^+/+ versus Apc^Min/+ SI and colon after VO treatment (Apc^+/+ versus Apc^Min/+ proximal, mid and distal SI = 3.26−3.19 ± 0.25−0.29 versus 1.04−1.16 ± 0.1−0.14 arbitrary densitometry units (ADU) P < 0.001; Apc^+/+ versus Apc^Min/+ colon = 3.43 ± 0.30 versus 1.19 ± 0.23 ADU; Figure 1a). VDR protein expression was also greater in VO-treated Apc^+/+ versus Apc^Min/+ SI and colon (SI 6.34 ± 0.41 versus 1.0 ± 0.12; colon 5.6 ± 0.45 versus 1.0 ± 0.14; Apc^+/+ versus Apc^Min/+, P < 0.001 ANOVA (Figure 1b). Apc^Min/+ mice expressed lower E-cadherin protein but increased OPN, CD44, Tcf1, c-Myc, total β-catenin and nuclear β-catenin in SI and colon in comparison with Apc^+/+ mice (Figure 1b and c). No differences of TATA-binding protein (nuclear β-catenin loading control) or β-actin levels were observed (Figure 1b and c).

Fig. 1. — Effects of genotype and/or treatment on gene expression. (a) VDR messenger RNA in SI or colon was lower in Apc^Min/+ versus Apc^+/+ SI or colon after VO treatment. Expression was enhanced in Apc^Min/+ tissues by 1α,25-(OH)₂ D₃ or analogue treatment. (b and c) E-cadherin protein expression and signalling were attenuated in Apc^Min/+ versus Apc^+/+ intestine or colon after VO treatment, whereas OPN and CD44 protein expression were increased. Treatment by 1α,25-(OH)₂ D₃ or analogues enhanced E-cadherin signalling, upregulated OPN expression but suppressed CD44 protein, in Apc^Min/+ SI or colon (b and c). Total β-catenin, (TATA-binding protein, nuclear β-catenin loading control) and β-actin protein levels were unaffected by treatment.

Effects of treatment on expression of vitamin D target genes

Treatment by 1α,25-(OH)₂ D₃, QW and BTW increased VDR messenger RNA [P < 0.001 ANOVA (Figure 1a)] and VDR protein expression in Apc^Min/+ SI and colon [Apc^Min/+ SI VDR protein = 1.0 ± 0.12 VO versus 5.3 ± 0.59 versus 3.1 ± 0.37 versus 2.5 ± 0.36 ADU after 1α,25-(OH)₂ D₃ versus QW versus BTW treatment; P < 0.001; ANOVA (Figure 1b)]. OPN is a central 1α,25-(OH)₂ D₃ target gene and a key effector of anchorage-independent growth (41). Although OPN levels were enhanced by 1α,25-(OH)₂ D₃, QW or BTW treatment in Apc^Min/+ SI or colon, expression of its CD44 receptor was suppressed (Figure 1b and c). These treatments also enhanced expression of E-cadherin protein but suppressed nuclear β-catenin, Tcf1 and c-Myc in comparison with VO-treated Apc^Min/+ mice controls. Treatment by 1α,25-(OH)₂ D₃ appeared more effective than QW or BTW for selective upregulation or suppression of these genes, respectively. Total β-catenin, TATA-binding protein and β-actin levels were unaffected by treatment.

Treatment effects on small intestinal polyp formation in Apc^Min/+ mice

In VO-treated Apc^Min/+ mice, fewer polyps were found in the proximal than mid or distal regions of the SI (proximal = 21.75 ± 2.18, mid = 24.75 ± 1.90, distal = 23.6 ± 1.86; P = 0.017, ANOVA; Figure 2a and b). Polyp diameter was greater in the mid SI than other SI regions (1.98 ± 0.05 mid versus 1.81± 0.1 proximal versus 1.52 ± 0.7 mm distal SI; P < 0.001, ANOVA). Treatment by 1α,25-(OH)₂ D₃ or analogues significantly inhibited polyp number and polyp load in all regions of the SI (P < 0.001, ANOVA; Table I). 1α,25-(OH)₂ D₃ had a significantly greater inhibitory effect on polyp number than QW or BTW at all SI regions (P < 0.01, ANOVA). There were significant interactive effects of treatment and small intestinal region on polyp number (P < 0.001, two-way ANOVA). 1α,25-(OH)₂ D₃ and BTW had maximal inhibitory effects on polyp number (P = 0.002, 1α,25-(OH)₂ D₃; P < 0.001 BTW; ANOVA) and polyp load (P < 0.001, 1α,25-(OH)₂ D₃ and BTW) in the mid SI. Treatment by QW showed maximal inhibition of polyp load only in the mid SI (P = 0.016, ANOVA; Figure 2b and c).

Fig. 2. — SI polyp formation. (a) Small intestinal polyp (marker −200 μM). (b and c) Polyp number was greater in mid or distal than proximal Apc^Min/+ SI (P = 0.017). Polyp load was greater in mid than in proximal or distal Apc^Min/+ SI (P < 0.001). Treatment by 1α,25-(OH)₂ D₃ or analogues significantly inhibited polyp number and polyp load in all Apc^Min/+ regions (P < 0.001). 1α,25-(OH)₂ D₃ had a significantly greater inhibitory effect on polyp number than QW or BTW at all SI regions (P < 0.01). Statistically significant interactive effects of treatment and small intestinal region were observed for polyp number (P < 0.001). 1α,25-(OH)₂ D₃ and BTW had maximal inhibitory effects on polyp number and polyp load in the mid SI, whereas QW showed maximal inhibition of polyp load only in the mid SI.

Table I.

Effects of treatment on APC^Min/+ SI tumourigenesis

Treatment	n	Proximal	Middle	Distal
Polyp number
VO	8	21.75 ± 2.18	24.75 ± 1.90	23.6 ± 1.86
Vit D₃	8	11.62 ± 1.30	9.5 ± 1.50	11.12 ± 1.2
QW	8	13.13± 1.36	15.25 ± 1.16	13.75 ± 1.38
BTW	8	15.25± 1.28	12.12 ± 1.35	16.12 ± 1.35
Total polyp load
VO	8	38.84 ± 0.82	48.84 ± 0.82	35.71 ± 0.89
Vit D₃	8	20.55 ± 0.52	17.55 ± 0.62	18.05 ± 0.58
QW	8	17.20 ± 0.53	21.20 ±0.70	17.94 ± 0.60
BTW	8	21.75 ± 0.64	20.75 ± 0.64	18.38 ± 0.66

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P = 0.017 for polyp number in proximal versus mid or distal SI in VO-treated APC^Min/+ mice. P < 0.001 for polyp load in mid SI versus proximal or distal SI, in VO-treated APC^Min/+ mice. P < 0.001 for effects of 1α,25-(OH)₂ D₃, (Vit D₃), QW or BTW versus VO control on polyp number and polyp load in APC^Min/+ mice.

Treatment effects on colonic ACF

ACF were more numerous (15.6 ± 1.4 versus 1.63 ± 0.74) and had greater crypt multiplicity (6.49 ± 0.90 versus 1.39 ± 0.44 crypts per focus in VO-treated Apc^Min/+ versus wt mouse colon (P < 0.001; independent samples Student's t-test). Treatment of Apc^Min/+ mice by 1α,25-(OH)₂ D₃, QW or BTW significantly inhibited ACF number [1α,25-(OH)₂ D₃, 6.75 ± 1.28; QW, 7.37± 1.30; BTW, 8.37± 1.30 versus VO, 15.62 ± 1.41;P < 0.001) and ACF crypt multiplicity (1α,25-(OH)₂ D₃, 3.90 ± 0.878; QW, 4.46 ± 0.71; BTW, 4.55± 0.81 versus VO, 6.49 ± 0.90;P < 0.001, ANOVA; Figure 3a and b).

Fig. 3. — Colonic ACF formation. (a and b) Treatment of Apc^Min/+ mice by 1α,25-(OH)₂ D₃, QW or BTW significantly inhibited ACF number [(a) P < 0.001] and size [(b) P < 0.001].

Treatment effects on serum calcium in APC^Min/+ mice

No significant differences were observed in serum calcium levels between VO-treated Apc^+/+ and Apc^Min/+ mice at 0, 4, 8 or 12 weeks (wt 9.09–9.14 ± 0.77–0.62 versus APC^Min/+ 9.29–8.38 ± 0.91–0.80; P = not significant; two-way ANOVA). Treatment by 1α,25-(OH)₂ D₃, QW or BTW was associated with significantly higher serum calcium levels than VO-treated Apc^Min/+ mice (1α,25, 15.1 ± 1.05; QW, 10.43 ± 0.84, BTW, 11.57 ± 0.91 versus VO, 8.38 ± 0.8, at 12 weeks (P < 0.001; two-way ANOVA). Levels were significantly lower with QW or BTW than 1α,25-(OH)₂ D₃ (serum Ca⁺⁺ at 8 and 12 weeks, QW, 10.2 ± 0.9; 10.45 ± 0.84; BTW, 11.07± 0.77; 11.57 ± 0.9, 1α,25-(OH)₂ D₃, 13.76 ± 0.96; 15.1 ± 1.05, P < 0.001; two-way ANOVA), whereas differences between QW and BTW treatment approached significance (P = 0.058; Figure 4; all serum Ca²⁺ values expressed in milligrams per deciliters).

Fig. 4. — VO-treated Apc^Min/+ mice (solid line) developed lower mean serum calcium levels than that of VO-treated Apc^+/+ littermates (short fragment broken line) by 12 weeks although differences were not statistically significant. All secosteroid treatments increased serum calcium levels in Apc^Min/+ mice (P < 0.001). 1α,25-(OH)₂ D₃ induced maximal hypercalcaemia (broken line, wide grouped fragments) that was significantly greater than that induced by BTW (next in magnitude, broken line, close grouped fragments) or QW (long fragment broken line) treatment at 8 and 12 weeks (P < 0.001; all serum Ca²⁺ values mg/dl).

Effects of genotype and treatment on weight gain

The 13 related body weight measurements during the study interval were subjected to PCA. PCA revealed a dichotomy between weeks 3 and 4 suggesting that the optimal approach to the data was to average the weights over the first 3 weeks and then over the final 9 weeks. Weight gain was assessed as the difference between these averages. VO-treated wt mice gained significantly more weight (5.4 ± 1.3 g) than VO-treated Apc^Min/+ mice (1.7 ± 1.4 g; P < 0.001, ANOVA; Figure 5). Apc^Min/+ mice receiving QW or BTW but not 1α,25-(OH)₂ D₃ had significantly greater weight gain than VO-treated Apc^Min/+ mice (QW, 4.02 ± 1.03 gversus VO, 1.7 ± 1.36 g, P = 0.004; BTW, 3.65 ± 1.18 g; P = 0.019 versus VO, 1α,25-(OH)₂ D₃, 2.46 ± 1.32 P = not significant versus VO ANOVA; Figure 5).

Fig. 5. — Body weight increased over 12 weeks in all mice. Weight gain was greatest in VO-treated Apc^+/+ mice (broken line short fragments) from 15.25 ± 1.04 to 24.2 ± 1.39 g. VO-treated Apc^Min/+ mice (solid line) had least weight gain, increasing from 15.13 ± 1.0 to 18.13 ± 1.3 g (P < 0.001 versus Apc^+/+ mice). Apc^Min/+ mice receiving QW (broken line, single long fragments) or BTW (broken line, closely spaced grouped fragments) but not 1α,25-(OH)₂ D₃ (broken line, widely spaced grouped fragments) had significantly greater weight gain than VO-treated Apc^Min/+ mice (QW P = 0.004 versus VO; BTW P = 0.019 versus VO).

Discussion

Germ line Apc heterozygosity alters the gene expression and proteome profiles of normal-appearing intestinal or colonic epithelium (42) and preferentially promotes tumourigenesis in these tissues (43). Mutational inactivation of APC leads to β-catenin stabilization, nuclear β-catenin accumulation, enhanced β-catenin/Tcf binding and activity (44). β-Catenin/Tcf target genes include Twist, (45) a coactivator of CD44 (27). The present study shows that VO-treated Apc^Min/+ mice maintained on a vitamin D-deficient diet, expressed greater β-catenin (total and nuclear), Tcf1, c-Myc, CD44 and OPN but lower E-cadherin and VDR in tumour-free SI and colon than Apc^+/+ wt controls. Although the molecular basis of these expression differences have not been fully elucidated, the Apc^Min/+ genotype also upregulates specific histone deacetylases in flat mucosa (46) that may enhance expression of OPN (47), synergize with β-catenin/Tcf signalling (48) and corepress both E-cadherin (49) and VDR (50).

The heighted intestinal or colonic β-catenin/Tcf activity reported here contrasts with a previous study showing unclear effects of Apc^1638N+/− heterozygosity, on Wnt signalling in murine colonic mucosa (51). However, the specific location of APC mutation within the coding sequence influences signalling and phenotype (52). Most APC mutations (mAPCs) underlying human CRC occur in the mutation cluster region within the first half of the coding sequence (53). These mAPCs produce truncated APC (trAPC) proteins that lack a variable number of 20 amino acid repeats associated with β-catenin binding (53). trAPCs that retain the first 171 amino acids may homodimerize with the wt allele (54) and act as a dominant-negative APC (55). The germ line Apc^Min truncating mutation at Apc codon 850 retains these characteristics, acts as a dominant-negative APC, promotes β-catenin accumulation (55) and a severe intestinal tumourigenesis phenotype (56). Conversely, mutations of the extreme 5′ end of APC may not produce trAPCs (57). The Apc^1638N mutation raised by insertion of a neomycin cassette at Apc codon 1638 produces very low amounts (1–2%) of trApcs (52). The Apc^1638N protein product neither homodimerize with the wt allele nor act as a dominant-negative APC (52). The Apc^1638N+/− genotype is not associated with intestinal intracellular β-catenin accumulation and produces only an attenuated intestinal tumourigenesis phenotype (55). However, Apc^1638N+/− heterozygosity activates a geneset in murine colonic mucosa that partially overlaps with that induced by a vitamin D and calcium-deficient (new Western-style) diet (51).

Previous studies have shown that vitamin D and specific analogues suppress β-catenin and its growth promoting target genes in vitro (10) and inhibit tumour load in Apc^Min/+ mice (58). Derivatized vitamin D analogues can have important functional differences from the natural 1α,25(OH)₂D₃ hormone. Analogues locked in cis- or trans- conformations can preferentially elicit rapid non-genomic and/or genomic responses and may be useful for dissecting the relative importance of vitamin D secosteroid genomic versus non-genomic signalling (59). OPN is regulated by vitamin D secosteroid genomic (12) and non-genomic mechanisms (13,60), whereas E-cadherin is regulated by 1a,25-(OH)2 D3 rapid non-genomic actions (13). The present study compared analogues QW and BTW against 1α,25-(OH)₂ D₃. QW retains retains transactivating capacity (31), whereas BTW has lower VDR binding affinity than 1α,25-(OH)₂ D₃ (33). All treatments increased E-cadherin but inhibited nuclear β-catenin localization as well as Tcf1, c-Myc and CD44 expression in Apc^Min/+ SI and colon. Our findings that QW and BTW were weaker in these effects than 1α,25-(OH)₂ D₃, suggests that the efficacy of vitamin D secosteroids for upregulation of E-cadherin may be proportional to their VDR binding affinity. In this context, our findings support those of Eelen et al. (61) who demonstrated that analog effects on E-cadherin transcription and growth suppression in vitro may be proportional to their VDR transactivating potency. In addition to their effects of upregulation of E-cadherin tumour suppressor activity, all treatments in the present study also upregulated pro-oncogenic OPN.

To assess the oncological significance of these molecular changes, we investigated their association with tumour formation. Total polyp numbers in VO-treated animals in this study were similar to that previously reported for Apc^Min/+ mice given a vitamin D-deficient diet (37). We found that the number and load of polyps in VO-treated Apc^Min/+ mice were greater in the mid SI than other regions. Treatment by 1α,25-(OH)₂ D₃ or analogues significantly inhibited polyp load, in agreement with Huerta et al. (58) but also suppressed polyp number. The experimental background of vitamin D deficiency in the present study could have influenced the greater effectiveness of 1α,25-(OH)₂ D₃ or analogue treatment against tumour formation. We also found differences in treatment effectiveness and site specificity. 1α,25-(OH)₂ D₃ showed significantly greater overall inhibitory effects on SI tumorigenesis than QW or BTW and showed maximal activity against polyp number in the mid SI. Conversely, QW showed similar polyp inhibition throughout all SI regions. 1α,25-(OH)₂ D₃, QW and BTW treatments also inhibited both the number and crypt multiplicity of colonic ACF. Such region-specific differences in polyp formation and 1α,25-(OH)₂ D₃ or analogue responsiveness are unexplained. Although these effects could not be attributed to any regional differences in VDR messenger RNA, unknown molecular regulators of intestinal region-specific functional specialization could be implicated.

A diet lacking in vitamin D but containing 0.47% calcium has previously been shown to be associated with normocalcaemia in Balb/c mice (34) and was used in the present study. Apc^Min/+ tumourigenesis could impede intestinal transit and/or calcium absorption. Although we found lower serum calcium levels in Apc^Min/+ versus Apc^+/+ mice after 12 weeks of the above diet, differences were not statistically significant (8.38 ± 0.8 versus 9.14 ± 0.62 mg/dl). Adverse effects of 1α,25-(OH)₂ D₃ treatment involve calcium homeostasis. 1α,25-(OH)₂ D₃ treatment induced higher serum calcium levels than QW or BTW in Apc^Min/+ mice, after 4 weeks. Despite the greater effects of 1α,25-(OH)₂ D₃ in activation of E-cadherin and inhibition of SI tumour formation, weight gain was significantly less in Apc^Min/+ animals treated by 1α,25-(OH)₂ D₃ than QW or BTW. Hence, the greater tumour suppressor effects of 1α,25-(OH)₂ D₃ are offset by its higher calcium related toxicity. All treatments induced a rise in serum calcium levels but were administered at potentially higher exposures than would be used in clinical studies (62).

The present study shows that β-catenin accumulation invoked by the Apc^Min/+ genotype (55) associates with increased expression CD44 and OPN but decreased E-cadherin. Other oncogenic signatures implicated in CRC progression can induce similar changes in the OPN-CD44 versus E-cadherin equilibrium. Oncogenic Ras drives progression from benign colonic adenoma to dysplastic adenocarcinoma (63,64), upregulates OPN (65) and CD44 (66) and suppresses E-cadherin by coactivation of Snail, through a Raf-MEK-ERK cascade (67). However, pathway-specific mechanisms responsible for deregulation of the OPN–CD44 versus E-cadherin equilibrium may invoke different growth responses to vitamin D. As shown previously (58) and in the present study, the Apc^Min/+ genotype is sensitive to vitamin D secosteroid-mediated suppression of tumourigenesis. However, Ras hyperactivity may confer resistance to vitamin D growth control (68) and Snail may abrogate vitamin D inhibition of β-catenin/Tcf signalling (69).

Taken together, the present study shows that the Apc^Min/+ genotype modulates vitamin D secosteroid actions, to promote functional predominance of E-cadherin tumour suppressor activity over OPN/CD44 pro-oncogenic signalling. These properties may assign tissue or tumour specificity to 1α,25-(OH)₂ D₃ growth control and provide a scientific rationale for vitamin D inhibition of CRC (4,5) that arises from mAPC (70) but incongruous effects on other cancers (6,7,71) that may be initiated by different oncogenic pathways. Furthermore, the chemopreventive efficacy of vitamin D secosteroids against CRC may be maximal at early stage tumourigenesis when mAPC is the principal driver of adenoma formation (72) before deregulation of OPN/CD44/E-cadherin by mutant K-Ras or other oncogenic signatures that may be resistant to vitamin D growth control.

Supplementary material

Supplementary Table can be found at http://carcin.oxfordjournals.org/

Funding

Wellcome Trust, UK (GR069313A1A); National Institues of Health, USA (CA 93547).

Supplementary Material

[Supplementary Data]

bgq098_index.html^{(669B, html)}

Acknowledgments

We would also like to express our gratitude to Ms Victoria Bingham for help in preparation of graphs and figures.

Conflict of Interest Statement: None declared.

Glossary

Abbreviations

ACF: aberrant crypt foci
APC: adenomatous polyposis coli
ANOVA: analysis of variance
c-Myc: c-myelocytomatosis
CRC: colorectal cancer
OPN: osteopontin
PCA: principal component analysis
SI: small intestine
tcf: T cell factor
trAPC: truncated APC
VDR: vitamin D receptor
VO: vehicle only
wt: wild-type

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PERMALINK

ApcMIN modulation of vitamin D secosteroid growth control

Haibo Xu

Gary H Posner

Michael Stevenson

Frederick C Campbell