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Departments of Adult Oncology [P. S., H. G., J. E. G., K. P.], and Biostatistics [K. L. L.], Dana-Farber Cancer Institute, Boston, Massachusetts 02115; Brigham and Women’s Hospital [E. R., C. M. K., D. J. I.], Boston, Massachusetts 02115; Center for Cancer Risk Analysis, Massachusetts General Hospital, Charlestown, Massachusetts 02129 [D. W. B., D. A. H.]; Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts 02114 [S. M. N.]; Harvard Medical School, Boston, Massachusetts 02115 [P. S., D. W. B., H. G., S. M. N., E. R., C. M. K., D. J. I., J. E. G., D. A. H., K. P.]; Harvard School of Public Health, Boston, Massachusetts 02115 [K. L. L.]; and Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710 [J. R. M.]
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Sulfotransferases are involved in the metabolism of xenobiotics and
endogenous chemicals (steroids, catecholamines, and iodothyronines;
Refs. 6
, 7
). Sulfonation is generally associated with
inactivation of xenobiotics, although a large number of carcinogens and
mutagens are activated by this process. In humans, there are three
members of the phenol sulfotransferase family (SULT1A1, SULT1A2,
and SULT1A3). DNA sequences and the structure of these three
enzymes are highly homologous, and all three genes are localized
tightly linked on chromosome 16p12.1-p11.2 (8)
. Similar to
other drug-metabolizing enzymes (glutathione transferases,
N-acetyltransferases, and others), inherited differences in
the enzymatic activity of sulfotransferases are likely to influence the
individual’s risk for cancer (9)
, but this connection has
not yet been investigated. Interestingly, exon 7 of the
SULT1A1 gene contains a recently described, functionally
relevant polymorphism that significantly influences its enzymatic
activity (10)
. Cell extracts from individuals homozygous
for the low activity allele (SULT1A*2) have 
10-fold lower
phenol sulfotransferase activity compared with that of individuals with
the high activity (SULT1A*1) allele (11)
.
Similarly, there are several functionally relevant polymorphisms
described in the highly related (96% identity at the amino acid level)
but much lower activity SULT1A2 gene; however, thus far no
high throughput assay has been developed for their analysis
(11, 12, 13)
.
To determine whether SULT1A1 polymorphism and the resulting variability in enzymatic activity would influence breast cancer risk, we analyzed SULT1A1 genotypes in relation to breast cancer risk. To our knowledge, this is the first report investigating the association between polymorphism in sulfotransferases and cancer risk in humans.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Northern and Immunoblot Analysis.
For Northern blot analysis, RNA from ZR75-1 cells was probed with a 78
bp PCR-derived probe corresponding to the 1B alternatively spliced exon
that contained the 5'UTR of the SULT1A cDNA. poly(A) RNA was prepared
using µMACs (Miltenyi Biotech) kit following the manufacturer’s
instructions. RNA electrophoresis and hybridization were performed as
described (14)
. Immunoblot analysis of ZR75-1 cell
extracts was performed with antihuman SULT1A3 polyclonal antibody
(Oxford Biomed.)
Gene-specific RT-PCR Reactions.
To determine which SULT1A genes are expressed in
ZR75-1 cells, the following oligonucleotides were used for RT-PCR
analysis. The forward primers were SULT-F2 (5'UTR),
5'-gaggccaggttcccaagagc-3'; SULT-F3 (5'UTR),
5'-gcattccccacacaacaccc-3'; and SULT-FC (coding region),
5'-acatggagctgatccaggac-3'. The reverse primers were SULT1A1R
(specific for SULT1A1), 5'-ccctcaattcatattttattcttgagccg-3'; SULT1A2R1
(specific for SULT1A2), 5'-aacacaaatcatactttattctggagcct-3'; SULT1A2R2
(specific for SULT1A2), 5'-ggagcctcttggtcaggc-3'; and SULT1A2R3
(specific for SULT1A2), 5'-acccataggacacttctccag-3'. RT-PCR was
performed essentially as described (14)
. The identity of
the PCR fragments was confirmed by cyclosequencing (Thermosequenase,
USB).
Subjects and Sample Preparation.
Patients were selected based on early onset of breast cancer [under
the age of 40 in the MGH cohort and under the age of 65 (majority of
the patients under the age 60) in the DFCI cohort]. Both these
women and the healthy blood donors provided written informed consent
for research under protocols approved by the institutional review
boards at each institution. The majority (>99%) of the subjects were
Caucasians, and most affected patients did not have a family history of
breast cancer. Genomic DNA was extracted using standard protocols.
PCR-RFLP Assay for SULT1A1 Genotype Analysis.
PCR amplifications and RFLP analysis were performed essentially as
described with minor modifications (10)
. A detailed
protocol is available from the authors upon request.
Statistical Analysis.
Pearson 
2
test was used to test for
independence of alleles (Hardy-Weinberg Equilibrium, HWE) within
each sample. Fisher’s exact test was used to test for differences in
genotype and allele frequencies among the three samples and between
patients and controls. Within each sample, Fisher’s exact test was
used to test for relationships between genotype and various available
predictors, such as race, ER status, and other tumors present. We used
ANOVA to determine whether the genotypes that coded four different ways
(three genotypes, dominant 1A1*1, dominant 1A1*2,
and the additive 1A1*1 allele) were significantly related to
age of onset in each sample. Because the data may not conform
well to the assumptions of ANOVA, we also performed the nonparametric
K-W test in each case.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. This SAGE tag can
correspond to SULT1A1, SULT1A2, or SULT1A3 (phenol-preferring
sulfotransferase 1, 2, and 3) because of their high degree of
similarity; therefore, we arbitrarily refer to this transcript as
SULT1A. To confirm the induction of SULT1A by tamoxifen, we performed
Northern blot analysis using poly(A) RNA and a 78 bp probe that
corresponded to an alternatively spliced exon 1B that contained the
5'UTR (Fig. 1B)
. Because of
the high similarity of the SULT1A cDNAs, we were unable to design a
gene-specific probe for Northern blot analysis. Sequence analysis of
PCR fragments determined that both SULT1A1 and SULT1A2 mRNAs contain
this alternatively spliced exon. As Fig. 1
shows, the mRNA levels of
SULT1A were induced by 24 h with an even more significant increase
detected at 72 h after 4-OH-tamoxifen treatment. However, even at
this time point the increase in the mRNA levels is modest (2–3-fold).
Surprisingly, estrogen treatment also increased SULT1A mRNA levels at
later time points (Fig. 1B)
, which were not detected by SAGE
because this analysis was performed at earlier (16 h) time points. To
determine which one of the three SULT1A genes is affected by
tamoxifen treatment, we performed RT-PCR and immunoblot analysis using
gene-specific primers and antibodies (when available). In these
experiments, we found that, although SULT1A3 was not affected, we were
not able to determine conclusively if SULT1A1, SULT1A2, or both mRNA
levels are affected by hormonal treatment in ZR75-1 cells (data not
shown). Because of the lack of commercially available antibodies, we
were unable to analyze the protein levels of SULT1A1 and SULT1A2.
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. We tested a total of 444
breast cancer patients from three different cohorts and 227 controls
(healthy blood donors, male and female) free of malignancy. Two of the
cohorts consisted of 378 early-onset breast cancer patients whose data
were collected at MGH (280 cases; <40 years of age at diagnosis) and
DFCI (98 cases; <57 years of age at diagnosis), whereas the third
cohort included 66 sporadic breast cancer patients whose data
were collected from consecutive surgeries at Brigham and
Women’s Hospital, MGH, and Duke University Medical Center. Age and
ethnicity information was not available for 98 controls, whereas 129
controls were ethnically and age matched to the MGH patient set.
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shows the SULT1A1 genotype and allele frequency
distribution among the three patient samples and in controls. No
evidence for differences in allele or genotype frequencies was found
between each of the three patient samples and controls (Fisher’s exact
test; P = 0.75, 0.55, 0.47 for the MGH, DFCI,
and Other sample set, respectively). Similarly, no evidence for the
association of genotype or allele frequency with various other
parameters (ER status and tumor stage at diagnosis) was found in any of
the three samples. Previous data indicated that SULT1A1
allele frequency may be influenced by ethnicity (10)
. In
accordance with this, in the MGH sample set we found some evidence of
allele frequency differences between Caucasian and non-Caucasian
(11)
allele frequency (0.70 and 0.93, respectively;
P = 0.08), but this did not reach statistical
significance and is based on only seven non-Caucasians in the sample
versus 273 Caucasians. Interestingly, in the MGH patient
set, there were 27 patients who had a history of other tumors in
addition to breast cancer at the time of diagnosis. These tumors
included cervical (n = 1), vulval
(n = 1), ovarian (n = 6), colon (n = 1), thyroid
(n = 1), and basal cell
(n = 8) carcinomas; osteosarcomas
(n = 2); melanomas (n = 4); and Hodgkin’s lymphomas (n = 3).
None of the breast/osteosarcoma patients had Li-Fraumeni syndrome, and
none of the breast/thyroid cancer patients had Cowden syndrome. One of
the breast/ovarian cancer patients was found to have a BRCA1 mutation,
and another patient had a strong family history of breast cancer that
indicated the potential involvement of a high-penetrance breast
cancer susceptibility gene. Although high-penetrance genes might
confound the weaker effect of the SULT1A1 polymorphism, removal of
these two (or even all six) cases from the analysis would not influence
the overall result. Statistical analysis revealed that patients are
more likely to have SULT1A1*1/1A*1 (20 cases) or
SULT1A1*1/1A*2 (7 cases) genotype if they have other tumors
in addition to breast cancer than if they do not
(P = 0.022). Using allele data, the
1A*1 allele is associated with having other tumors present
(P = 0.004; odds ratio = 3.02;
95% confidence interval, 1.32, 8.09). This may indicate that in
certain genetic background or environmental conditions, high
sulfotransferase enzymatic activity may increase cancer risk in
general, or it may modify the penetrance of mutations in certain tumor
suppressor gene(s). This observation is not unexpected because several
animal and in vitro studies have found an association
between sulfonation activity and incidence of chemically induced
cancers (15)
.
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and 3)
. By
this approach in the MGH sample set, there is some borderline
significant evidence that genotype affects age of onset, using a
dominant SULT1*1 allele model (ANOVA, P = 0.05; K-W, P = 0.06). In the DFCI
sample set, there is some evidence that genotype affects age of onset,
using a recessive SULT1A1*1 allele model (ANOVA and K-W,
P = 0.03). We found no evidence of a genotype
effect on age of onset using any genotype coding in the sporadic
patients’ sample set. Because prior analysis indicated that the
SULT1A1 genotype and allele frequency is not statistically
different in the different patient sample sets, we combined the early
onset MGH and DFCI sample sets and performed a two-way ANOVA that
accounted for genotype and sample. The three-genotype ANOVA model has
P = 0.04 for genotype. Hence, although the
evidence for dominance/recessiveness of the SULT1A1*1 allele
in the two sample sets is different, the two do not cancel each other
out. Thus, the combined sample does provide evidence that genotype has
an effect.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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45 kb), alleles for SULT1A1 are in linkage disequilibrium
with alleles for SULT1A2 (8
, 13)
. Moreover, high activity
alleles for SULT1A1 are linked to high activity alleles for SULT1A2;
the same is true for the low activity alleles (13)
. In
addition, the enzymatic activity of SULT1A2 is much lower than that of
SULT1A1 (13)
. On the basis of all of this data, if SULT1A
enzymatic activity influences breast cancer risk, analysis of SULT1A1
polymorphism in relation to breast cancer is likely to be informative
and is not likely to be confounded by the potential effect of the
SULT1A2 gene, although this possibility cannot be completely
excluded. Our analysis of 444 breast cancer patients and 227 controls revealed no evidence of increased risk of breast cancer associated with any SULT1A1 genotype. The control group consisted of 129 healthy blood donors, ethnically and age matched to the MGH patient set. An additional 98 controls were healthy blood donors with no data available on age and ethnicity. However, on the basis of the average patient population of the institutions where the samples were collected, we have no reason to believe that the patient and control groups would be significantly different.
Interestingly, we did find evidence that SULT1A1 genotype influences the age of onset of the disease in patients preselected for early-onset breast cancer (MGH and DFCI) but not in unselected (Other) cases. Because these early-onset breast cancer patients may have a genetic predisposition to breast cancer attributable to a high-penetrance gene or to other low-penetrance genes, the influence of the SULT1A1 polymorphism on the age of onset may indicate a genetic interaction between SULT1A1 genotype and other breast cancer susceptibility genes. Similarly, the intriguing association between SULT1A1 genotype and the presence of multiple different tumors in the same patient may suggest such an interaction between SULT1A1 and a higher penetrance cancer susceptibility gene(s). Because these other cancer types were of diverse origin, SULT1A1 may modify the effect of several different cancer-predisposing genes, an observation that is likely to stimulate additional studies.
This type of genetic interaction could explain the differing effect the SULT1A1*1 allele has on the age of onset in the MGH and DFCI sample sets. In the case of the MGH patients (age of onset <40), the SULT1A1*1 allele behaves as a dominant allele with both SULT1A1*1 homozygotes and heterozygotes having an earlier onset. Conversely, in the DFCI patients (<65 years of age), it behaves as a recessive allele with only the SULT1A1*1 homozygotes demonstrating an earlier onset. Although this difference between the two samples sets can be attributable to various exogenous (exposure to carcinogens) or endogenous (estrogen levels and others) factors as well, at this point we have no data available to differentiate between the above-mentioned possibilities.
Currently, we do not know the mechanism through which SULT1A1 activity influences the development of breast cancer and other cancer types. One hypothesis is that certain environmental agents (alkylphenols, octylephenol, bisphenol A, and others), either by interfering with the metabolism of endogenous steroid hormones or by becoming mutagenic upon sulfonation, increase the probability of acquiring a mutation in oncogenes or tumor suppressor genes. It is notable in this respect that phenolic compounds have been shown to influence sexual development and reproductive function in lower vertebrates. In addition, several animal and in vitro studies have found an association between high sulfotransferase activity and the risk of developing chemically induced cancers (15) .
Because of the high frequency of the high activity SULT1A1 allele in Caucasian populations and the large number of xenobiotics sulfonated by this enzyme, SULT1A1 may turn out to be an important low-penetrance cancer-predisposing gene. The possible biological interaction of breast cancer preventive agents such as tamoxifen with SULT1A1 genotype merits additional attention because of their widespread use in high-risk patients.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by the Sydney Kimmel Cancer
Foundation, by the American Society of Clinical Oncology Career
Development Award, and by the Claudia Adams Barr-Weaver Award from the
Dana-Farber Cancer Institute (to K. P.) and by the Dana-Farber-Harvard
Gilette Women’s Cancer Program (to D. A. H. and J. E. G.). 
2 To whom requests for reprints should be
addressed, at Department of Adult Oncology, Dana-Farber Cancer
Institute, 44 Binney Street D740C, Boston, MA 02115. Phone:
(617) 632-2106; Fax: (617) 632-4005; E-mail: Kornelia_Polyak{at}dfci.harvard.edu
3 The abbreviations used are: ER, estrogen
receptor; SAGE, serial analysis of gene expression; SULT1A,
sulfotransferase 1A; DFCI, Dana-Farber Cancer Institute; MGH,
Massachusetts General Hospital; UTR, untranslated region;
RT-PCR, reverse transcription-PCR; K-W, Kruskal-Wallis.
4 Seth et al. manuscript in
preparation.
Received 7/25/00. Accepted 10/23/00.
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0.55 for patient control
genotype differences. The three different samples are listed as MGH
(early onset, <40 years of age), DFCI (early onset, <57 years of
age), and Other (unselected cases).