Carcinogenesis vol.31 no.8 pp.1434–1441, 2010
doi:10.1093/carcin/bgq098
Advance Access publication May 20, 2010
ApcMIN modulation of vitamin D secosteroid growth control
Haibo Xu3, Gary H.Posner1, Michael Stevenson2 and
Frederick C.Campbell
Centre for Cancer Research and Cell Biology, Queen’s University of Belfast,
Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK, 1Department of
Chemistry, The Johns Hopkins University, Baltimore, MD 21218, USA and
2
Centre for Public Health, Queen’s University of Belfast, Lisburn Road,
Belfast BT97BL, Northern Ireland, UK
3
Present address: Department of Physiology and Pharmacology, University
of Bristol, Bristol, BS8 1TD, UK
To whom correspondence should be addressed. Tel: þ44 28 90 972759;
Fax: þ44 28 90972776;
Email: [email protected]
A central paradox of vitamin D biology is that 1a,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 ApcMin/1 genotype on 1a,25-(OH)2 D3 regulation
of OPN/CD44/E-cadherin signalling and intestinal tumourigenesis, in vivo. In untreated ApcMin/1 versus Apc1/1 intestines,
expression levels of OPN and its CD44 receptor were increased,
whereas E-cadherin tumour suppressor signalling was attenuated. Treatment by 1a,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 b-catenin nuclear localization,
T-cell factor 1 and c-myelocytomatosis protein expression in
ApcMin/1 intestine. All secosteroids suppressed ApcMin/1-driven
tumourigenesis although QW and BTW had lower calciumrelated toxicity. Taken together, these data indicate that the
ApcMin/1 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 tumourspecific 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 1a,25-(OH)2 D3 target genes may
represent potentially modifiable cancer risk factors, mechanisms
responsible for the tissue or tumour specificity of 1a,25-(OH)2 D3
growth control remain unclear.
Expression profiling has identified diverse 1a,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 1a,25-(OH)2 D3 (9,10).
OPN is a predominantly secreted glycoprotein (11) that is regulated
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.
by 1a,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 1a,25-(OH)2 D3 transcription-independent
rapid actions (13) and suppresses colorectal tumourigenesis (21),
partly by inhibition of b-catenin transcriptional activity (22). b-catenin
is sequestered by E-cadherin, rapidly phosphorylated by glycogen
synthase kinase 3b, then ubiquitinated and degraded. Impaired
E-cadherin function is a hallmark feature of CRC (23) and enables
b-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
1a,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 1a,25-(OH)2 D3 treatment by development of related
molecules with different structural configurations (28). 1a,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 lowcalcaemic hybrid molecule that has transactivating capacity (31),
although its effects on OPN transcription is lower than that of
1a,25-(OH)2 D3 (32). BTW is a 2,2-dimethylated 19-nor analogue
that has similar antiproliferative activity to 1a,25-(OH)2 D3 in vitro
but has 75-fold lower binding affinity to VDR (33).
Here, we report in vehicle-only (VO)-treated ApcMin/þ versus
Apcþ/þ 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 1a,25-(OH)2 D3 or analogues enhanced
OPN but suppressed CD44. Treatment also enhanced E-cadherin
tumour suppressor activity characterized by inhibition of b-catenin
nuclear localization, Tcf1 and c-myelocytomatosis (c-Myc) protein
expression and inhibited adenoma formation in ApcMin/þ intestine.
Taken together, these data suggest that the molecular signature activated by the ApcMin/þ genotype may create favourable tissue- or
tumour-specific conditions for vitamin D growth control.
Materials and methods
Chemicals and reagents
1a,25-Dihydroxyvitamin D3 [1a,25-(OH)2 D3], C27H44O3, molecular weight:
416.64 was purchased from Sigma–Aldrich UK (Dorset, UK) (Cat Number:
D1530). The 1a,25-(OH)2 D3 analogues QW (molecular weight: 492) and
BTW (molecular weight: 432) were synthesized as described previously
(33). 1a,25-(OH)2 D3 and analogues were dissolved in absolute ethanol at
103mol/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. ApcMin/þ heterozygous
Ó The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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ApcMin modulation of vitamin D growth signalling
C57BL/6J mice and wild-type (wt) C57BL/6J-Apcþ/þ mice were obtained from
Clare Hall Laboratories, London Research Institute, Cancer Research UK,
South Mimms, Hertfordshire, UK and the ApcMin/þ pedigree was maintained
by crossing C57BL/6J-ApcMin/þ males to C57BL/6J-Apcþ/þ 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 Apcþ/þ and ApcMin/þ 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). ApcMin/þ mice received intraperitoneal injections of 1a,25-(OH)2 D3, QW and BTW each in doses of 0.33 lg/kg. Dose
selection for 1a,25-(OH)2 D3 was based on previous in vivo studies of antiproliferative properties and toxicity (35). QW has comparable VDR transactivating capacity with 1a,25-(OH)2 D3 (32). Although BTW has lower VDR
affinity than 1a,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 ll or equal volumes of VO(5%
ethanol–95% saline) for 12 weeks. Wt C57BL/6J Apcþ/þ mice received VO
injections. Body weights were assessed weekly. From each mouse, 30 ll 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 phosphatebuffered 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 ll RNeasy Lysis
Buffer containing 6 ll b-mercaptoethanol. VDR is a key regulator of 1a,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 lM),
using MuLV reverse transcriptase (2.5 U/ll), 2 ll genomic DNA wipeout
buffer and RNase-free water to a total volume of 20 ll 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 ll reverse transcriptase, RNase inhibitor, 4 ll of 5 reverse
transcriptase buffer and 1 ll 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 ll of 10 lM
forward primer, 0.75 ll of 10 lM reverse primer, 12.5 ll of 2 QuantiTect
SYBR green PCR master mix and RNase-free water, made to a total volume of
25 ll. Primers were as follows: VDR forward 5#-ATCTGCATTGTCTCCCCAGACCGA-3# and reverse 5#-GATCATCTTGGCGTAGAGCTGGTG-3#
and b-actin control forward 5#-GATCAAGATCATTGCTCCTCCTGAGC-3#
and reverse 5#-GACTCATCGTACTCCTGCTTGCTGA-3#. Final products of
147 bp (VDR) and 117 bp (b-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 1a,25(OH)2 D3 target genes that may be implicated in growth control, including
E-cadherin, OPN, b-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 ll 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-2hydroxyethylpiperazine-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 ll 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 ll 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 ll 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 lg of
protein per lane were separated on sodium dodecyl sulphate–polyacrylamide
gel electrophoresis and transferred to nitrocellulose membrane with 0.45 lm 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-b-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
b-catenin; Abcam; Cat No: 1TBP18, 37 kDa), monoclonal mouse antib-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: sc2048) 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
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H.Xu et al.
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 ApcMin/þ versus Apcþ/þ intestine or colon
1a,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 Apcþ/þ
versus ApcMin/þ SI and colon after VO treatment (Apcþ/þ versus
ApcMin/þ proximal, mid and distal SI 5 3.263.19 ± 0.250.29
versus 1.041.16 ± 0.10.14 arbitrary densitometry units (ADU)
P , 0.001; Apcþ/þ versus ApcMin/þ colon 5 3.43 ± 0.30 versus
1.19 ± 0.23 ADU; Figure 1a). VDR protein expression was also
greater in VO-treated Apcþ/þ versus ApcMin/þ SI and colon (SI
6.34 ± 0.41 versus 1.0 ± 0.12; colon 5.6 ± 0.45 versus 1.0 ± 0.14;
Apcþ/þ versus ApcMin/þ, P , 0.001 ANOVA (Figure 1b). ApcMin/þ
mice expressed lower E-cadherin protein but increased OPN, CD44,
Tcf1, c-Myc, total b-catenin and nuclear b-catenin in SI and colon
in comparison with Apcþ/þ mice (Figure 1b and c). No differences
of TATA-binding protein (nuclear b-catenin loading control) or
b-actin levels were observed (Figure 1b and c).
Effects of treatment on expression of vitamin D target genes
Treatment by 1a,25-(OH)2 D3, QW and BTW increased VDR
messenger RNA [P , 0.001 ANOVA (Figure 1a)] and VDR protein
expression in ApcMin/þ SI and colon [ApcMin/þ SI VDR
protein 5 1.0 ± 0.12 VO versus 5.3 ± 0.59 versus 3.1 ± 0.37 versus
Fig. 1. Effects of genotype and/or treatment on gene expression. (a) VDR messenger RNA in SI or colon was lower in ApcMin/þ versus Apcþ/þ SI or colon after
VO treatment. Expression was enhanced in ApcMin/þ tissues by 1a,25-(OH)2 D3 or analogue treatment. (b and c) E-cadherin protein expression and signalling
were attenuated in ApcMin/þ versus Apcþ/þ intestine or colon after VO treatment, whereas OPN and CD44 protein expression were increased. Treatment by 1a,25(OH)2 D3 or analogues enhanced E-cadherin signalling, upregulated OPN expression but suppressed CD44 protein, in ApcMin/þ SI or colon (b and c). Total
b-catenin, (TATA-binding protein, nuclear b-catenin loading control) and b-actin protein levels were unaffected by treatment.
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ApcMin modulation of vitamin D growth signalling
2.5 ± 0.36 ADU after 1a,25-(OH)2 D3 versus QW versus BTW treatment; P , 0.001; ANOVA (Figure 1b)]. OPN is a central 1a,25(OH)2 D3 target gene and a key effector of anchorage-independent
growth (41). Although OPN levels were enhanced by 1a,25-(OH)2 D3,
QW or BTW treatment in ApcMin/þ 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 b-catenin, Tcf1 and c-Myc in comparison with VO-treated ApcMin/þ mice controls. Treatment by 1a,25-(OH) D appeared more
2
3
effective than QW or BTW for selective upregulation or suppression
of these genes, respectively. Total b-catenin, TATA-binding protein
and b-actin levels were unaffected by treatment.
Treatment effects on small intestinal polyp formation in ApcMin/þ mice
In VO-treated ApcMin/þ mice, fewer polyps were found in the proximal than mid or distal regions of the SI (proximal 5 21.75 ± 2.18,
mid 5 24.75 ± 1.90, distal 5 23.6 ± 1.86; P 5 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 1a,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). 1a,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). 1a,25-(OH)2 D3 and
BTW had maximal inhibitory effects on polyp number (P 5 0.002,
1a,25-(OH)2 D3; P , 0.001 BTW; ANOVA) and polyp load
(P , 0.001, 1a,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 5 0.016, ANOVA; Figure 2b and c).
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 ApcMin/þ versus wt mouse colon (P , 0.001;
independent samples Student’s t-test). Treatment of ApcMin/þ mice
by 1a,25-(OH)2 D3, QW or BTW significantly inhibited ACF number
[1a,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
(1a,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).
Treatment effects on serum calcium in APCMin/þ mice
No significant differences were observed in serum calcium levels
between VO-treated Apcþ/þ and ApcMin/þ mice at 0, 4, 8 or 12 weeks
(wt 9.09–9.14 ± 0.77–0.62 versus APCMin/þ 9.29–8.38 ± 0.91–0.80;
P 5 not significant; two-way ANOVA). Treatment by 1a,25-(OH)2
D3, QW or BTW was associated with significantly higher serum calcium levels than VO-treated ApcMin/þ mice (1a,25, 15.1 ± 1.05; QW,
Fig. 2. SI polyp formation. (a) Small intestinal polyp (marker 200 lM). (b and c) Polyp number was greater in mid or distal than proximal ApcMin/þ SI
(P 5 0.017). Polyp load was greater in mid than in proximal or distal ApcMin/þ SI (P , 0.001). Treatment by 1a,25-(OH)2 D3 or analogues significantly inhibited
polyp number and polyp load in all ApcMin/þ regions (P , 0.001). 1a,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).
1a,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.
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H.Xu et al.
Table I. Effects of treatment on APCMin/þ SI tumourigenesis
Treatment
n
Polyp number
VO
8
8
Vit D3
QW
8
BTW
8
Total polyp load
VO
8
8
Vit D3
QW
8
BTW
8
Proximal
Middle
Distal
21.75 ± 2.18
11.62 ± 1.30
13.13± 1.36
15.25± 1.28
24.75 ± 1.90
9.5 ± 1.50
15.25 ± 1.16
12.12 ± 1.35
23.6 ± 1.86
11.12 ± 1.2
13.75 ± 1.38
16.12 ± 1.35
38.84 ± 0.82
20.55 ± 0.52
17.20 ± 0.53
21.75 ± 0.64
48.84 ± 0.82
17.55 ± 0.62
21.20 ±0.70
20.75 ± 0.64
35.71 ± 0.89
18.05 ± 0.58
17.94 ± 0.60
18.38 ± 0.66
P 5 0.017 for polyp number in proximal versus mid or distal SI in VO-treated
APCMin/þ mice. P , 0.001 for polyp load in mid SI versus proximal or distal
SI, in VO-treated APCMin/þ mice. P , 0.001 for effects of 1a,25-(OH)2 D3,
(Vit D3), QW or BTW versus VO control on polyp number and polyp load
in APCMin/þ mice.
Fig. 4. VO-treated ApcMin/þ mice (solid line) developed lower mean serum
calcium levels than that of VO-treated Apcþ/þ littermates (short fragment
broken line) by 12 weeks although differences were not statistically
significant. All secosteroid treatments increased serum calcium levels in
ApcMin/þ mice (P , 0.001). 1a,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 Ca2þ values mg/dl).
significance (P 5 0.058; Figure 4; all serum Ca2þ values expressed
in milligrams per deciliters).
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 ApcMin/þ mice (1.7 ± 1.4 g; P , 0.001, ANOVA;
Figure 5). ApcMin/þ mice receiving QW or BTW but not 1a,25-(OH)2
D3 had significantly greater weight gain than VO-treated ApcMin/þ
mice (QW, 4.02 ± 1.03 gversus VO, 1.7 ± 1.36 g, P 5 0.004;
BTW, 3.65 ± 1.18 g; P 5 0.019 versus VO, 1a,25-(OH)2 D3,
2.46 ± 1.32 P 5 not significant versus VO ANOVA; Figure 5).
Discussion
Fig. 3. Colonic ACF formation. (a and b) Treatment of ApcMin/þ mice by
1a,25-(OH)2 D3, QW or BTW significantly inhibited ACF number
[(a) P , 0.001] and size [(b) P , 0.001].
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 1a,25-(OH)2 D3 (serum Caþþ at 8 and 12 weeks,
QW, 10.2 ± 0.9; 10.45 ± 0.84; BTW, 11.07± 0.77; 11.57 ± 0.9, 1a,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
1438
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 b-catenin stabilization, nuclear
b-catenin accumulation, enhanced b-catenin/Tcf binding and activity
(44). b-Catenin/Tcf target genes include Twist, (45) a coactivator of
CD44 (27). The present study shows that VO-treated ApcMin/þ mice
maintained on a vitamin D-deficient diet, expressed greater b-catenin
(total and nuclear), Tcf1, c-Myc, CD44 and OPN but lower E-cadherin
and VDR in tumour-free SI and colon than Apcþ/þ wt controls.
Although the molecular basis of these expression differences have
not been fully elucidated, the ApcMin/þ genotype also upregulates
specific histone deacetylases in flat mucosa (46) that may enhance
expression of OPN (47), synergize with b-catenin/Tcf signalling (48)
and corepress both E-cadherin (49) and VDR (50).
The heighted intestinal or colonic b-catenin/Tcf activity reported
here contrasts with a previous study showing unclear effects of
Apc1638Nþ/ 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
ApcMin modulation of vitamin D growth signalling
Fig. 5. Body weight increased over 12 weeks in all mice. Weight gain was
greatest in VO-treated Apcþ/þ mice (broken line short fragments) from
15.25 ± 1.04 to 24.2 ± 1.39 g. VO-treated ApcMin/þ mice (solid line) had least
weight gain, increasing from 15.13 ± 1.0 to 18.13 ± 1.3 g (P , 0.001 versus
Apcþ/þ mice). ApcMin/þ mice receiving QW (broken line, single long
fragments) or BTW (broken line, closely spaced grouped fragments) but not
1a,25-(OH)2 D3 (broken line, widely spaced grouped fragments) had
significantly greater weight gain than VO-treated ApcMin/þ mice
(QW P 5 0.004 versus VO; BTW P 5 0.019 versus VO).
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 b-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 ApcMin truncating mutation at Apc codon
850 retains these characteristics, acts as a dominant-negative APC,
promotes b-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 Apc1638N mutation
raised by insertion of a neomycin cassette at Apc codon 1638 produces very low amounts (1–2%) of trApcs (52). The Apc1638N protein
product neither homodimerize with the wt allele nor act as a dominantnegative APC (52). The Apc1638Nþ/ genotype is not associated with
intestinal intracellular b-catenin accumulation and produces only an
attenuated intestinal tumourigenesis phenotype (55). However,
Apc1638Nþ/ 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 b-catenin and its growth promoting target genes in vitro (10)
and inhibit tumour load in ApcMin/þ mice (58). Derivatized vitamin D
analogues can have important functional differences from the natural
1a,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 nongenomic 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 1a,25-(OH)2 D3. QW
retains retains transactivating capacity (31), whereas BTW has lower
VDR binding affinity than 1a,25-(OH)2 D3 (33). All treatments increased E-cadherin but inhibited nuclear b-catenin localization as
well as Tcf1, c-Myc and CD44 expression in ApcMin/þ SI and colon.
Our findings that QW and BTW were weaker in these effects than
1a,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 ApcMin/þ mice given a vitamin D-deficient
diet (37). We found that the number and load of polyps in VO-treated
ApcMin/þ mice were greater in the mid SI than other regions. Treatment by 1a,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 1a,25(OH)2 D3 or analogue treatment against tumour formation. We also
found differences in treatment effectiveness and site specificity. 1a,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. 1a,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 1a,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. ApcMin/þ tumourigenesis could impede intestinal transit and/or calcium absorption.
Although we found lower serum calcium levels in ApcMin/þ versus
Apcþ/þ 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 1a,25-(OH)2 D3 treatment involve calcium homeostasis.
1a,25-(OH)2 D3 treatment induced higher serum calcium levels than
QW or BTW in ApcMin/þ mice, after 4 weeks. Despite the greater
effects of 1a,25-(OH)2 D3 in activation of E-cadherin and inhibition
of SI tumour formation, weight gain was significantly less in ApcMin/þ
animals treated by 1a,25-(OH)2 D3 than QW or BTW. Hence, the
greater tumour suppressor effects of 1a,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 b-catenin accumulation invoked by
the ApcMin/þ 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 ApcMin/þ 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 b-catenin/
Tcf signalling (69).
Taken together, the present study shows that the ApcMin/þ 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 1a,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/
1439
H.Xu et al.
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).
Acknowledgements
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.
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Received March 24, 2010; revised May 7, 2010; accepted May 12, 2010
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