Expression of Vitamin D Receptor and 25-Hydroxyvitamin
D3-1A-Hydroxylase in Normal and Malignant
Damien Matusiak,1,2Genoveva Murillo,3Robert E. Carroll,1,2Rajendra G. Mehta,3
and Richard V. Benya1,2
1Department of Medicine, University of Illinois at Chicago;
3Carcinogenesis and Chemoprevention Division, Illinois Institute of Technology Research Institute, Chicago, Illinois
2Jessie Brown Veterans Administration Medical Center; and
Considerable evidence exists to support the use of vitamin D
to prevent and/or treat colorectal cancer. However, the routine
use of bioactive vitamin D, 1,25-dihydroxyvitamin D3, is
limited by the side effect of toxic hypercalcemia. Recent
studies, however, suggest that colonic epithelial cells express
25-hydroxyvitamin D3-1A-hydroxylase, an enzyme that con-
verts nontoxic pro-vitamin D, 25-hydroxycholecalciferol
[25(OH)D3], to its bioactive form. Yet, nothing is known as to
the cellular expression of 1A-hydroxylase and the vitamin D
receptor (VDR) in the earliest histopathologic structures
associated with malignant transformation such as aberrant
crypt foci (ACF) and polyps [addressing the possibility of
using nontoxic 25(OH)D3 for chemoprevention], nor is any-
thing known as to the expression of these proteins in colo-
rectal cancer as a function of tumor cell differentiation or
metastasis [relevant to using 25(OH)D3 for chemotherapy]. In
this study, we show that 1A-hydroxylase is present at equal
high levels in normal colonic epithelium as in ACFs, polyps,
and colorectal cancer irrespective of tumor cell differentiation.
In contrast, VDR levels were low in normal colonic epithelial
cells; were increased in ACFs, polyps, and well-differentiated
tumor cells; and then declined as a function of tumor cell
de-differentiation. Both 1A-hydroxylase and VDR levels were
negligible in tumor cells metastasizing to regional lymph
nodes. Overall, these data support using 25(OH)D3 for
colorectal cancer chemoprevention but suggest that pro-
vitamin D is less likely to be useful for colorectal cancer chemo-
Vitamin D’s ability to prevent colorectal cancer has been
suspected for over a quarter of a century. One of the earliest
studies supporting this link came from the observation that
there was an inverse relationship between mean solar
radiation and age-adjusted colorectal cancer death rates (1).
Since that time, a number of epidemiologic studies have
suggested a link between vitamin D and/or calcium levels and
the incidence of human colorectal cancer formation (reviewed
in ref. 2). The validity of these epidemiologic observations was
significantly enhanced by the results of the Calcium Polyp
Prevention Study, a double-blind placebo-controlled study
that showed a decreased recurrence rate of colorectal adeno-
mas in patients receiving calcium carbonate (3, 4). Patients
receiving the greatest benefit from calcium supplementation
were those with decreased serum levels of 25-hydroxychole-
calciferol [25(OH)D3; ref. 3], highlighting the importance of
vitamin D in inhibiting colorectal cancer formation.
measuring serum 25(OH)D3 levels, this nontoxic pro-vitamin
requires hydroxylation at carbon 1 by 25-hydroxyvitamin D3-
1a-hydroxylase to generate bioactive 1,25-dihydroxyvitamin
D3 [1,25(OH)2D3]. Whereas the primary function of
1,25(OH)2D3 is to regulate calcium absorption and maintain
mineral homeostasis, upon binding to its cognate vitamin D
receptor (VDR), 1,25(OH)2D3 also decreases the proliferation
and enhances the differentiation of colorectal cancer cells (5) as
well as alters the transcription of a large number of genes
involved in inhibiting carcinogenesis (6, 7).
Unfortunately, 1,25(OH)2D3 has a narrow therapeutic index,
with its propensity for causing toxic hypercalcemia precluding
its routine use in otherwise healthy patients. Recently,
however, a number of investigators have suggested that 1a-
hydroxylase is present in normal (8, 9) and malignant (10, 11)
epithelial cells lining the adult human colon, suggesting that
nontoxic 25(OH)D3 might represent an efficacious treatment
modality for treating patients with known colorectal polyps or
cancer (i.e., chemotherapy), or preventing these lesions from
even forming in the first place (i.e., chemoprevention). Yet, the
studies done to date have not systematically evaluated VDR
and 1a-hydroxylase expression in colon cancer as a function of
tumor cell differentiation or metastasis, critical to assessing
25(OH)D3’s potential as a chemotherapeutic agent, nor have
they evaluated the expression profile of these proteins in early
neoplastic lesions such as aberrant crypt foci (ACF), critical to
assessing 25(OH)D3’s potential as a chemopreventive agent.
To assess 25(OH)D3’s potential for colorectal cancer chemo-
prevention and/or chemotherapy, we herein report on cellu-
lar VDR and 1a-hydroxylase protein expression in human
ACFs, polyps, and colorectal cancer’s of defined differentia-
tion along with associated lymph node metastases. To do
this, we used quantitative immunohistochemistry to precisely
determine VDR and 1a-hydroxylase expression in formalin-
fixed surgically resected tissues at the cellular level. We herein
show that 1a-hydroxylase levels are consistently high in all
nonmetastatic tissues, irrespective of histology. In contrast,
we show that VDR levels are increased in ACFs and well-
differentiated tumors but progressively diminish with colo-
rectal cancer de-differentiation. Lastly, we show that both
1a-hydroxylase and VDR expression is low to negligible in
metastatic tissues, regardless of the histopathologic stage
assigned to either the primary tumor or tumor contained
Cancer Epidemiol Biomarkers Prev 2005;14(10). October 2005
Received 4/13/05; revised 7/13/05; accepted 8/11/05.
Grant support: Veterans Administration Merit Review (R.E. Carroll and R.V. Benya) and NIH
grants CA103861 (G. Murillo), CA82316 (R.G. Mehta), and CA-094346 (R.V. Benya).
The costs of publication of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
Requests for reprints: Richard V. Benya, Department of Medicine, University of Illinois at
Chicago, 840 South Wood Street (M/C 716), Chicago, IL 60612. Phone: 312-569-7439;
Fax: 312-569-8114. E-mail: email@example.com
Copyright D 2005 American Association for Cancer Research.
chemotherapy. Although the significance of this finding is not
clear, it is possible, given the observation that VDR polymor-
phisms can result in nonfunctional protein (40, 41) and that
tumor de-differentiation might be associated with increasing
mutation of the VDR gene. If so, this would be similar to what
has been observed for the gene for the colon cancer morphogen,
gastrin-releasing peptide receptor (13).
This study is also the first to evaluate 1a-hydroxylase and
VDR expression in the earliest lesion associated with malig-
nant transformation in the colon, ACF. ACFs were first defined
in the colons of rodents exposed to carcinogens and identified
soon thereafter in human colons (20). ACFs are associated with
adenomatous polyp (42) and colorectal cancer formation in
humans (20) and have become an accepted biomarker for
assessing the efficacy of chemopreventive drugs in various
nonhuman colorectal cancer models (43). Whereas polyps are
relatively fixed as lesions, ACF seem fluid and can readily
change in number over time (44). Thus, our finding that ACFs,
as well as premalignant polyps, express high levels of 1a-
hydroxylase and VDR, suggest that pro-vitamin D may be of
value as a chemopreventive agent.
In summary, we herein show that whereas (a) 1a-hydrox-
ylase is present in uniformly high levels in normal and
neoplastic colonic epithelia, except for tumor cells metastatic to
regional lymph node; (b) VDR expression is low in normal
colonic epithelial cells, increases with malignant transforma-
tion and then declines with progressive tumor de-differenti-
ation. Lastly, we show that (c) declining ratios of VDR
expression in the nucleus compared with the cytoplasm in
neoplastic lesions suggests that less of this protein translocates
to the nucleus as tumors progress. Overall, then, these data
support using pro-vitamin D analogues in colorectal cancer
chemoprevention, whereas they are less likely to be useful for
colorectal cancer chemotherapy.
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