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-Subunit Gene in Prostate Carcinoma
Monash Institute of Reproduction and Development (J.F.S., M.F., G.P.R.), Monash University, Clayton, Victoria 3168, Australia; Kanematsu Laboratories (D.S.M., J.S.P., S.L.C., G.P.R.), Royal Prince Alfred Hospital, Camperdown, New South Wales 2050; Melbourne Pathology (J.S.P.), Collingwood, Victoria 3066; Peter MacCallum Cancer Institute (D.J.V.), Melbourne 3002; and CSIRO Molecular Science (P.L.M.), North Ryde, New South Wales 1670, Australia
Address all correspondence and requests for reprints to: Dr. Gail P. Risbridger, Monash Institute of Reproduction and Development, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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- and a ß-subunit. Transgenic
studies assigned a tumor-suppressive role to the inhibin -subunit,
and in human prostate cancer inhibin -subunit gene expression was
down-regulated. This study examined the inhibin -subunit gene
promoter and gene locus to determine whether promoter hypermethylation
or LOH occurred in DNA from prostate cancer. The 5'-untranslated region
of the human inhibin -subunit gene was sequenced and shown to be
highly homologous to the bovine, rat, and mouse inhibin -subunit
promoter sequences. A 135-bp region of the human promoter sequence that
continued a cluster of CpG sites was analyzed for hypermethylation.
Significant (P < 0.001) hypermethylation of the
inhibin -subunit gene promoter occurred in DNA from Gleason pattern
3, 4, and 5 carcinomas compared with nonmalignant tissue samples. A
subset of the carcinomas with a cribriform pattern were unmethylated.
LOH at 2q32–36, the chromosomal region harboring the inhibin
-subunit gene, was observed in 42% of prostate carcinomas. These
data provide the first demonstration that promoter hypermethylation and
LOH are associated with the inhibin -subunit gene and gene locus in
prostate cancer. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit, resulting in
dimeric inhibin proteins (5, 6, 7). Inhibins and activins are
both implicated in endocrine-related cancers [see review by Risbridger
et al. (8)]. Functional studies using
transgenic mice identified the inhibin -subunit as a tumor
suppressor gene in the gonads and adrenals (9, 10, 11). Recent
studies identified subsets of patients with ovarian granulosa cell
tumors that showed down- regulation of inhibin -subunit
expression (12, 13), and in one of these studies there was
a correlation with disease-free survival (12). In the
prostate, inhibin -subunit protein and mRNA were detected in tissues
from men with benign prostatic hyperplasia (BPH) and regions of
nonmalignant tissue from men with prostate cancer (14, 15). In prostate cancer cells and tissues the expression of
inhibin -subunit protein and mRNA was down-regulated
(14).
The aim of the present study was to identify the molecular changes to
the inhibin -subunit gene in prostate carcinoma, i.e.
hypermethylation of the promoter and LOH, because these molecular
changes are often associated with silencing or loss of expression of
tumor suppressor genes. Aberrant methylation of the inhibin -subunit
gene promoter was reported in human cancers, but hypermethylation
of the promoters of other genes, e.g. the GSTP1 gene and the
ER and PR genes, occurred in prostate cancer (16, 17, 18). The
human inhibin gene was localized to the q33-q36 region of
chromosome 2 (19), and deletions of 2q were identified in
a number of human tumors, including prostate cancer
(20, 21, 22, 23). Deletions involving the 2q33–36 region have not
been reported for prostate carcinoma.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-Subunit Promoter
Regions-subunit gene
was determined by automated sequencing. Figure 1
shows the alignment of the human
sequence with the promoter sequences of the bovine (24),
rat (25), and mouse (26). Comparison of the
human sequence with the bovine, rat, and mouse sequences revealed
homologies of 73%, 65%, and 70%, respectively. High-sequence
homology (>78% between the human sequence and the other species) was
observed in a region of 297 bp immediately 5' of the ATG translation
start site in the human sequence. Consistent with the mouse, bovine,
and rat promoter regions, the human inhibin -subunit gene promoter
lacked an obvious TATA box, but conserved the sequence for the
specificity protein (Sp1) upstream promoter element. Inducible promoter
elements included a cAMP response element (CRE) and binding sites for
activator proteins 1, 2, and 3 (AP1, AP2, AP3), corresponding to those
in the rat, mouse, and bovine promoter sequences (24, 25, 26).
A potential Smad binding element (SBE) sequence was identified upstream
of the AP1 site (27).
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-subunit gene promoter identified a
cluster of seven CpG sites within a 135-bp region from -149 to -284
of the ATG start (Fig. 1
); four of these CpG sites (CpG1, CpG2, CpG5,
CpG6) were unique to the human sequence. CpG5 differed from the bovine,
rat, and mouse sequences by a T-C change and was within a region found
to bind AP2 in the bovine sequence (28). CpG3, CpG4, and
CpG7 were conserved between species and lay within the Sp1, CRE, and
AP3 binding sites, respectively. CpG4 was within an AP1 site and
adjacent to the SBE site.
Methylation Analysis of the Inhibin -Subunit Gene in
Microdissected Human Prostate Tissues
Methylation was determined for the seven CpG sites in the 135-bp
region from -149 to -284 of the ATG site in the human inhibin
-subunit gene promoter. An overall comparison of DNA from
nonmalignant and malignant prostate samples showed significant
(P < 0.0001) hypermethylation of the inhibin
-subunit gene promoter in prostate cancer (Fig. 2A). The mean percent methylation in
malignant tissues was 34.26 ± 2.76%, whereas in nonmalignant
tissues it was 14.78 ± 2.17%.
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). Figure 2C shows a comparison between methylation in prostate
carcinomas of cribriform-pattern (Gleason pattern 3 and 4) and
small-gland pattern (Gleason pattern 3–5) prostate carcinomas and
nonmalignant prostate tissues. Small-gland prostate carcinomas
(47.43 ± 3.74%) were significantly hypermethylated
(P < 0.0001) relative to nonmalignant samples
(14.78 ± 2.17%) and prostate carcinomas of cribriform cell
arrangement (10.89 ± 1.36%). There was no difference between
cribriform carcinomas and nonmalignant prostate tissues.
Figure 3A shows a typical pattern of
methylation at the seven individual inhibin -subunit gene
promoter CpG sites in nonmalignant and malignant tissue samples. The
percent methylation at each CpG site showed variation (Fig. 3B). Of the
seven CpG sites, CpG5 was rarely methylated in either nonmalignant or
malignant samples. The percent methylation at the remaining sites
varied, but overall analysis showed significant (P <
0.05) hypermethylation at sites CpG1–4 and CpG7 in malignant compared
with nonmalignant samples. CpG6 had lower methylation levels, and there
was no significant difference between nonmalignant and malignant tissue
samples.
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).
The quality of the DNA and the suitability of the methodology
for the detection of LOH were validated using PCR primers for the 8p21
microsatellite marker (D8S136). LOH at 8p21 was observed in 60% of
prostate carcinoma samples (Table 1) and is consistent with that
previously reported (29). Using the same DNA samples,
analysis of the 2q chromosome arm revealed that LOH occurred with at
least one microsatellite marker at 2q32–36 in 42% of prostate
carcinomas (Table 1). Patients A, C, F, G, and L were also analyzed for
methylation. Patient A showed both hypermethylation and LOH, patients
C, F, and G showed only hypermethylation, whereas patient L showed only
LOH.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit
gene in prostate carcinoma. Sequence analysis of the inhibin
-subunit promoter region identified a number of potential sites for
methylation. Significant hypermethylation of five of seven of these
sites occurred in DNA from samples of Gleason pattern 3, 4, or 5
prostate carcinoma. In addition, 42% of prostate carcinomas showed LOH
at chromosome 2q32–36. These results support the hypothesis that the
inhibin -subunit is tumor suppressive in the prostate and are
consistent with previous studies using transgenic mice that identified
this subunit as a gonadal and adrenal tumor suppressor (9, 10, 11, 30, 31, 32).
Sequence determination for the human inhibin -subunit gene promoter
was required to identify putative targets for methylation,
i.e. CpG sites. Comparison of the human sequence with the
bovine, mouse, and rat inhibin -subunit gene promoter sequences
revealed a high degree of sequence homology, particularly over a region
of 297 bp immediately upstream of the ATG translation start site
(26, 28, 33, 34). Studies with the bovine
(24), mouse (26), and rat (28)
showed that this region had promoter activity in vitro.
Several regulatory elements were conserved between the species and
included a CRE site with an overlapping AP1 site, an Sp1 site, an AP2
site, and an AP3 site. A putative SBE site adjacent to the AP1, similar
to that previously reported to occur within the JunB gene
promoter (27), was also identified. Many of these putative
transcription factor-binding sites had a CpG site within their
sequence.
Hypermethylation of CpG islands within the regulatory regions of tumor
suppressor genes is a common aberration in human cancers
(35, 36, 37) and is often associated with gene silencing
(16, 38, 39, 40, 41). The current study focused on a 135-bp region
(within the 297-bp region discussed above) of the inhibin -subunit
gene promoter that was highly conserved between the species. This
region contained a cluster of seven CpG sites and housed numerous
potential transcription factor binding sites; by analogy to bovine,
rat, and mouse this region is likely to be essential for promoter
activity (24, 25, 26). Overall, this cluster of CpGs was
hypermethylated in prostate cancer samples relative to nonmalignant
epithelium and BPH samples. Hypermethylation of the inhibin
-subunit promoter was observed in lower grade prostate cancer
(Gleason pattern 3) as well as in high-grade (Gleason pattern 4 and 5)
prostate cancer. Whether or not methylation of this subunit is a cause
or a consequence of malignancy remains to be determined, but loss of
the inhibin -subunit was believed to initiate gonadal and adrenal
tumor formation in inhibin -subunit null mice (9, 11, 30).
In the pathological assessment of the samples used for microdissection,
a subset of Gleason pattern 3 and 4 tumors showed a cribriform
arrangement of cells. It was noted that hypermethylation of the inhibin
-subunit gene promoter did not occur in these samples. Thus,
molecular analysis of the methylation status of the inhibin -subunit
gene promoter provides further evidence for a distinction between small
gland carcinomas and cribriform carcinomas of the prostate
(42). The significance of this finding lies in the report
that cribriform carcinomas have poor prognosis and patient outcome
(43, 44, 45, 46).
The degree of methylation varied between the seven CpG sites examined
in the inhibin -subunit gene promoter. CpG sites 1–4 and 5 were
significantly hypermethylated in prostate carcinoma, and CpG6 showed
some hypermethylation, but the difference was not significant. CpG5 was
consistently unmethylated in both nonmalignant and malignant samples.
CpG5 lay within a site shown to bind AP2 in the bovine inhibin
-subunit gene promoter sequence (28). AP2 binding at an
AP2 site within the tau gene promoter prevented access of
DNA methyltransferase (47), and therefore binding of AP2
at CpG5 may account for the consistent observation that this site was
unmethylated.
CpG4 lay within a CRE and AP1 transcription factor-binding site. The
CRE is required for cAMP-induced up-regulation of inhibin -subunit
expression (24, 25, 26). Functional studies demonstrated that
CpG methylation blocked transcription factor binding at CRE sites
(48, 49, 50). The AP1 site was adjacent to an SBE recognition
sequence. Both AP1 and SBE are involved in signaling by members of the
TGFß superfamily (27, 51, 52) and located in close
proximity to each other within the promoters of a number of genes
regulated by TGFß (27, 51, 53, 54). The
identification of adjacent AP1 and SBE binding sites in the inhibin
-subunit gene promoter region suggests that the inhibin -subunit
gene may be another target for regulation by TGFß or other members of
the TGFß superfamily. Methylation of CpG4 in prostate carcinoma could
block both CRE and AP2 transcription factor binding and alter inhibin
-subunit gene expression. This would be consistent with our previous
report that inhibin -subunit immunoreactivity was down-regulated
in prostate carcinoma (14).
As well as methylation, this study reported LOH at 2q32-q36 in 42% (6 of 14) of prostate carcinomas. Changes at chromosome 2q occur in prostate carcinoma (55, 56), although allelic loss involving this specific region was not previously reported. In other human tumors, deletions at 2q correlated with disease progression and outcome. For example, in bladder carcinoma (57) and head and neck squamous cell carcinoma (58), 2q deletions correlated with advanced disease and poor prognosis.
The functional consequences of the loss of inhibin -subunit gene
expression are worth consideration. We previously showed the inhibin
ß-subunits were expressed and localized to tumor cells in specimens
from men with prostate cancer. Therefore, in the absence of inhibin
-subunit, the tumor cells retain the capacity to produce activins
but not inhibins. Activins are generally growth inhibitory and induced
apoptosis in the androgen-dependent cell line LNCaP. However, the
androgen-independent cell line PC3 is resistant to the
growth-inhibitory actions of activins. Resistance to activins, like
resistance to TGFß, commonly occurs in tumor cells, and many of the
components of the activin-signaling pathway are tumor suppressive. It
is tempting to speculate whether or not there is a sequence of changes
to the inhibins/activins that contributes to malignancy in the prostate
gland, starting with the loss of inhibin -subunit expression and
followed by the onset of resistance to activins.
It is not known whether the inhibin -subunit null mice develop
prostate cancer or other premalignant changes. Prostate cancer
development requires androgens and is normally slow to develop, with
aggressive androgen-independent tumors generally emerging late in life.
The inhibin null transgenic mouse models develop gonadal tumors,
and the adrenal tumors emerge only upon castration. In the absence of
androgens, these mice will not develop prostate cancer. Furthermore,
the inhibin null mice died by 14 wk of age, and prostate cancer is a
disease with a long latency period. Hence, it is unlikely that prostate
cancer would emerge in the inhibin null mice in early adulthood,
but these mice should be examined for premalignant changes such as
prostatic intraepithelial neoplasia.
In summary, the data presented in this study demonstrate that
molecular change to the inhibin -subunit gene occurs in prostate
carcinoma and provides further evidence to support the hypothesis that
the inhibin -subunit gene is a tumor suppressor. Further molecular
studies that evaluate a larger patient group with known clinical
outcome would identify whether hypermethylation of the inhibin
-subunit gene promoter and/or LOH at 2q32-q36 provide markers of
survival and disease outcome for prostate carcinoma.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Sequence Determination of the Human Inhibin -Subunit Gene
5'-UTR
The sequence of the inhibin -subunit 5'-UTR was determined
from a genomic clone and partial sequence supplied by Dr. David Irving
(Biotech Australia Pty. Ltd., Roseville, NSW, Australia). The sequence
was determined using the BigDye Terminator Cycle Sequencing Kit
(PE Applied Biosystems, Foster City, CA) and the automated
ABI PRISM 377 DNA Sequencer (PE Applied Biosystems).
Detection of Methylation
Methylation was assessed by PCR and sequence analysis of
bisulfite-treated DNA using methodology similar to that previously
described (16, 59). The bisulfite reaction converted
unmethylated cytosines to uracil, whereas methylated cytosines were
unchanged. DNA was isolated from microdissected tissue lysates by
phenol chloroform extraction and ethanol precipitation in the presence
of 10 µg of tRNA. For bisulfite conversion, precipitated DNA was
resuspended in 20 µl PCRTE (10 mM Tris, 0.1
mM EDTA, pH 8.8), 2.2 µl of 3 M NaOH,
and 208 µl of 2 M metabisulfite, and 12 µl 10
mM quinone were added and the reaction incubated at 55 C
for 16 h (59). tRNA (1 µg) was added to each sample
and the DNA was purified using Wizard DNA Clean-Up System desalting
columns (Promega Corp., Madison, WI), eluted in 50 µl of
H2O and incubated with 5.5 µl 3 M
NaOH at 37 C for 15 min. The solutions were neutralized by the addition
of 33.5 µl NH4OAC, pH 7.0, ethanol
precipitated, and resuspended in 10 µl PCRTE. The inhibin -subunit
5'-UTR region was amplified by nested PCR using primers designed to the
bisulfite converted sequence. Primer sequences 1 (5'-GATAAGAGT-
TTAGATTGGTTTTATTGGTT-3') and 4 (5'-ACACCATAACTCACCTAACCCTACTAATAA-3')
were used for the first round of PCR and primer sequences 3
(5'-ACCCCTTCTACCAA- AATCTACCCAAAA-3') and 7
(5'-GAAGGTGTTGTATGTTTGTATGTGTGAGTT-3') were used for the second round
of PCR. The first round of PCR was performed in 25 µl reactions with
2 µl of bisulfite-converted DNA, PCR buffer (67 mM
Tris/HCl, 16.6 mM ammonium sulfate, 1.7 mg/ml BSA, and 10
mM ß-mercaptoethanol in PCRTE buffer), 1.5 mM
MgCl2, 0.2 mM each of dATP, dCTP,
dGTP, and dTTP, 6 ng/µl of each of the PCR primers 1 and 4, and 1 U
AmpliTaq DNA polymerase (PE Applied Biosystems). PCR
cycles consisted of 95 C for 5 min followed by 5 cycles of 95 C for 1
min, 50 C for 2 min, and 72 C for 3 min and followed by 30 cycles of 95
C for 1 min, 55 C for 2 min, and 72 C for 2 min with a final incubation
step of 72 C for 10 min. A sample of 2 µl from the first PCR was
amplified in a 25 µl reaction as above except that primers 3 and 7
were used. PCR cycling conditions were as for the first reaction, with
the exception that the annealing temperature was increased to 60 C. PCR
products were gel purified, ligated into the pCR 2.1 cloning vector,
and cloned using the TA Cloning Kit according to the manufacturer’s
instructions (Invitrogen, Carlsbad, CA). For each PCR,
10–13 clones were sequenced and the percentage methylation at each of
the seven CpGs was determined. Overall percent methylation for each
sample was determined as the mean of the percent methylation at the
seven individual CpG sites.
LOH Analysis
LOH was determined using microsatellite markers on 2q32-q33
(D2S389), 2q33-q36 (D2S128), and 8p21 (D8S136) and the sequences from
the genome database (http://gdbwww. gdb.org/gdb). Oligonucelotide
primer sequences for each microsatellite marker were: D2S389
5'-TAAAGCCTAGTGG- AAGATCATC-3', 5'-GCTGAGTTAACAGTTATCAACAATT-3'; D2S128
5'-AAACTGAGATTTGTCTAAGGGG-3', 5'-AGCCAGGAATTTTTGCTATT-3' and D8S136
5'-CCTGAGCCC AAAGAGGAGAATAA-3', 5'-TGCTCTGTTTCCACACCGAA- GC-3'. PCR was
performed in 15-µl reactions consisting of 1 µl of tissue lysate
prepared as above, PCR buffer (10 mM Tris-HCl, pH 8.3 and
50 mM KCl), 2.5 mM MgCl2,
0.2 mM each of dATP, dCTP, dGTP, and dTTP, 0.45 µg
forward primer, 0.5 µg reverse primer, 0.05 µg
32P-labeled forward primer, and 0.3 U AmpliTaq
Gold (PE Applied Biosystems). PCR using the 8p12 primers
also included 5% dimethylsulfoxide. PCR cycles consisted of 95 C for 5
min followed by 10 cycles of 95 C for 60 sec, 60 C for 90 sec, and 72 C
for 90 sec followed by 25 cycles in which the annealing temperature was
reduced to 55 C for 90 sec. PCR products were detected by 6% PAGE and
autoradiography. For each patient, several regions of microdissected
tissue were examined individually for LOH. The regions were selected to
include at least two regions of nonmalignant epithelium or stroma and
at least three regions of prostate carcinoma. LOH for a patient was
deemed to be present if at least two regions of carcinoma showed
allelic loss.
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ACKNOWLEDGMENTS |
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-subunit genomic clone, Mr. Simon Bardill and the Wellcome Trust
Sequencing Centre for DNA sequence analyses, and Dr. Melissa Southey
and Mr. Leigh Batten (Peter MacCallum Cancer Institute) for helpful
technical advice. |
FOOTNOTES |
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Abbreviations: AP1, -2, -3, Activator proteins 1, 2, 3; BPH, benign prostatic hyperplasia; CRE, cAMP response element; PCRTE, 10 nM Tris, 0.1 mM EDTA, pH 8.8; SBE, Smad-binding element; Sp1, specificity protein 1; UTR, untranslated region.
Received for publication August 13, 2001. Accepted for publication October 10, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-Inhibin is a tumour-suppressor gene with gonadal specificity in
mice. Nature 360:313–319[CrossRef][Medline]
-subunit
in high grade prostate cancer. J Clin Endocrinol Metab 83:969–975 and ER-ß in normal and malignant prostatic
epithelial cells: regulation by methylation and involvement in growth
regulation. Cancer Res 60:3175–3182, ßA, and ßB on human and
mouse chromosomes and studies of jsd mice. Genomics 5:91–99[CrossRef][Medline]
-inhibin locus on chromosome 2q is not
a feature of human granulosa cell tumors. Gynecol Oncol 65:387–390[CrossRef][Medline]
-inhibin gene and its regulation in the ruminant gonad: inverse
relationship to oxytocin gene expression. Biol Reprod 50:401–412[Abstract]
inhibin gene by cyclic adenosine
3',5'-monophosphate after transfection into rat granulosa cells. Mol
Endocrinol 5:521–534[Abstract]
gene and its promoter. Biochem Biophys Res Commun 186:293–300[CrossRef][Medline]
-, ßA- and ßB-inhibin/activin
genes. Identification of transcription factor AP-2-binding sites in the
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- and ß-B-subunit genes suggests two
different regulatory mechanisms. Mol Endocrinol 3:1914–1925[Abstract]
-inhibin gene. Mol Cell Endocrinol 68:121–128[CrossRef][Medline]
gene promoter
region alters with age to downregulate transcriptional activity in
human cerebral cortex. Neurosci Lett 275:89–92[CrossRef][Medline]
2(1) collagen to transforming
growth factor-ß1. J Biol Chem 272:19738–19745This article has been cited by other articles:
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