[Cancer Research 61, 6331-6334, September 1, 2001]
© 2001 American Association for Cancer Research
Loss of Annexin II Heavy and Light Chains in Prostate Cancer and Its Precursors1
Albert Chetcuti,
Sienna H. Margan,
Peter Russell,
Stephen Mann,
Douglas S. Millar,
Susan J. Clark,
John Rogers,
David J. Handelsman and
Qihan Dong2
Department of Medicine, the University of Sydney, NSW 2006 [A. C., S. H. M., Q. D.]; Departments of Anatomical Pathology [P. R.] and Urology [J. R.] and Kanematsu Laboratories [D. S. M., S. J. C.], Royal Prince Alfred Hospital, NSW 2050; and ANZAC Research Institute and Departments of Andrology [D. J. H.] and Anatomical Pathology [S. M.], Concord Repatriation General Hospital, NSW 2139, Australia
 |
ABSTRACT
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Annexin II mRNA coding for a calcium binding protein was found to be
absent in prostate cancer by subtractive hybridization and Northern analysis.
In contrast to high expression in normal and benign hyperplastic
glandular and basal epithelium, Annexin II heavy (p36) and light (p11)
chains in 31/31 prostate cancer specimens were lost
immunohistochemically. In glands involved by prostate intraepithelial
neoplasia, 65% lost both chains in glandular epithelial cells, whereas
basal cells were all positively stained. Southern analysis of cancer
DNA showed no noticeable deletion in p36 gene.
LNCaP cells treated with 5-azacytidine re-expressed p36, suggesting
methylation could be responsible for the silencing.
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Introduction
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Identification of genetic alterations is a necessity to gain
insight into neoplasia and can provide new tools for diagnosis,
treatment, and prevention. Prostate cancer is one of the most
frequently occurring carcinomas in men and has become the second most
common cause of cancer-related death. However, our understanding of its
etiology and the multistep progression is very limited. Oncogenes and
tumor suppressor genes known to be associated with other malignancies
have a remarkably low frequency of mutation or deletion in prostate
cancer. The most common change is the loss of Glutathione S-Transferase
pi-1 expression attributable to hypermethylation in the gene promoter
(1, 2, 3)
. We have now identified that Annexin II heavy (p36)
and light (p11) chains are also commonly lost in prostate cancer and in
some of its precursor,
PIN.3
The underlying cause is likely attributable to hypermethylation of the
p36 gene.
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Materials and Methods
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Prostate Specimens.
The entire prostate gland from radical prostatectomy patients was taken
to pathologists immediately after surgical removal. Biopsies were made
from the posterior prostate surface (4)
. Fresh normal
prostate tissue was obtained from two sources. One was a biopsy
specimen where no cancer cells were found. The other was a brain-dead
organ donor. The peripheral zone of the prostate from the organ donors
was dissected from the transitional/central zone using urethra and
ejaculatory ducts as the landmarks. Specimens used for
immunohistochemistry were fixed in 10% formaldehyde solution and
paraffin embedded. Benign hyperplastic tissue was obtained from
transurethral resection. Informed consent was obtained from the
patients or families, and the study protocol was approved by the
Central Sydney Area Health Service Ethics Review Committee.
RNA/DNA Extraction.
Before nucleic acid extraction, fresh cancer specimens and normal
prostate were microselected (5)
. The tissue was
homogenized in TRI-reagent (Sigma Chemical Co., Sydney, NSW,
Australia). Total RNA and DNA were isolated following the
manufacturer’s protocol with the following modification regarding DNA.
After precipitation of DNA, the pellet was washed 3 times with 75%
ethanol to remove residual phenol. The pellet was then digested
overnight at 55°C in a lysis buffer [50 mM Tris,
100 mM EDTA, 0.5% SDS, and 0.3 mg/ml proteinase K (pH
8)]. DNA was isolated by standard phenol:chloroform:isoamyl alcohol
technique.
Suppressive Subtractive Hybridization.
Poly(A)+ RNA was isolated from total RNA of a
cancer specimen and a normal prostate of an organ donor using Dynabeads
oligo(dT)25 (DYNAL, Oslo, Norway). Using
PCR-Select cDNA subtraction kit (Clontech, Palo Alto, CA), subtracted
libraries were subcloned into pGEM T-easy vector (Promega, Madison,
WI). Individual recombinant colonies were randomly picked and
inoculated into 96-well plates containing L-broth and
ampicillin. Cloudy medium was used as template for PCR amplification of
the insert sequence. PCR products were dot blotted onto membranes.
Duplicate membranes were hybridized to subtracted radiolabeled cancer
and normal cDNA library, respectively. The sequence of differentially
expressed clones was determined by single direction sequencing.
RT-PCR.
Total RNA (2 µg each) was reverse transcribed into cDNA. For p36, PCR
was performed using primers derived from the nucleotides 971–991 and
1213–1229 (GenBank accession no. D00017). For p11, PCR was performed
using primers derived from the nucleotides 138–158 and 376–394
(GenBank accession no. M38591). HPRT was amplified as a loading control
(5'-TTACTTTTCTAACACACGGTGGTA-3' and 5'-TTGCTGACCTGCTGGATTACATCA-3').
Thermocycling included an initial denaturation at 94°C for 2 min; 24
cycles (p36 and p11) and 30 cycles (HPRT) of denaturation (94°C for
30 s); annealing (30 s) at 55°C (p36), 51°C (p11), and 52°C
(HPRT); and elongation (72°C for 45 s). A water control
consisted of all reagents except template was included in each
experiment.
Northern and Southern Analyses.
Total RNA (15 µg) was denatured for 15 min at 65°C and
electrophoresed using a 1% formaldehyde agarose gel. The RNA was
transferred onto Hybond N nylon membrane. A PCR product (nucleotide
971-1229) of p36 cDNA was used as a probe. The membrane was hybridized
in ExpressHyb solution (Clontech) containing 1 x 106/ml cpm of denatured probe overnight at
68°C. Genomic DNA (10 µg each) was digested using
HindIII, PstI, and BglII. After
overnight digestion at 37°C, DNA was electrophoresed in a 0.8%
agarose gel and transferred onto nylon membrane after depurination,
denaturation, and neutralization. Radiolabeled probes were prepared
from two overlapping PCR products covering the entire open reading
frame of p36 gene. Hybridization condition was same as
Northern.
Immunohistochemistry.
Tissue sections (5 µm) were incubated for 1 h at 37°C after
microwave antigen retrieval with a mouse monoclonal anti-p36 antibody
(Zymed Laboratories) diluted 1/400 in 1% preimmune goat serum.
Biotinylated goat antimouse IgG, diluted 1/200 in 1% preimmune goat
serum, was used as the secondary antibody. The signal was amplified
using the avidin-biotin-peroxidase complex system (Vector Laboratories,
Burlingame, CA) and visualized using the liquid 3,3'-diaminobenzidine
substrate-chromogen system (Dako, Carpinteria, CA). Isotype and method
controls were performed for each sample by substituting the primary
antibody with preimmune mouse IgG (Dako) and 1% preimmune goat serum,
respectively. For p11, a mouse monoclonal anti-p11 antibody
(Transduction Laboratories) diluted 1/1000 was used. For 34ßE12 and
PSA, the antibodies were diluted 1/100 (Dako) and 1/600 (Dako),
respectively. Dako Envision + peroxidase (mouse K4001 for
34ßE12 and rabbit K4003 for PSA) was used as the signal detection
system.
5-Azacytidine Treatment.
LNCaP cells (0.5 x 106) were
cultured in T-medium (6)
with 10% heat-inactivated
fetal calf serum (FCS) for 2 days. A final concentration of 5
µM of 5-azacytadine was added to the culture medium from
a freshly prepared 5 M stock solution in DMSO. After 5 days
the cells were harvested and RNA isolated using TRI-reagent. The RNA
was DNase treated, and RT-PCR was performed as described above using
p36 primers.
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Results
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Identification of Loss of Annexin II (p36) mRNA in Prostate Cancer.
Using suppressive subtractive hybridization between two
microselection-derived prostate mRNAs, one from a previously untreated
64-year-old prostate cancer patient and the other from a 25-year-old
brain-dead, cancer-free organ donor, we identified a cDNA sequence that
was absent in prostate cancer. Sequencing revealed a full match with
Annexin II heavy chain (p36) from nucleotide position 657 to 1357
(GenBank accession no. D00017). RT-PCR and Northern analysis using
total RNA from additional six normal prostate and seven cancer
specimens confirmed that expression of p36 was significantly reduced in
prostate cancer (Fig. 1, A and B)
.
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Fig. 1. RT-PCR and Northern analyses of p36 and p11.
A, RT-PCR of p36 in individual normal prostate
(1–4 and 6 from cancer-free biopsy
specimens; and 5 from organ donor) and prostate
cancer-derived total RNA. HPRT was used to indicate comparable RNA
loading. B, Northern analysis of p36 in individual
normal prostate (1–3 from cancer-free biopsy specimens;
4 and 5 from organ donors) and prostate
cancer-derived total RNA. Ethidium bromide-stained 18S rRNA indicates
comparable RNA loading. C, RT-PCR of p11 in individual
normal prostate (same as p36) and prostate cancer-derived total RNA.
Three PCR cycles (21, 24, and 27) were used, and there was no
statistically significant difference in the intensity of PCR products
between normal prostate and prostate cancer. Depicted are products with
24 PCR cycles.
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Annexin II (p36) Protein in Normal Prostate, BPH, Prostate Cancer,
and PIN.
We analyzed p36 immunohistochemically in normal and diseased prostate
(Fig. 2)
. In peripheral (n = 10) and
transitional/central zones (n = 6) of normal
prostates from organ donors, the apical and lateral (i.e.,
circumferential) plasma membrane of glandular and ductal epithelial
cells showed high expression of p36. Similarly, the basal cells also
expressed p36 protein. In BPH tissue derived from transurethral
resection of prostate (n = 12), p36 was
expressed in the same cell type as normal tissue with comparable
intensity. In contrast, a loss of p36 expression in cancer cells was
observed in 31/31 randomly selected individual cancer blocks with a
Gleason score range of 3–9 (5.7 ± 1.4, mean ± SD), whereas adjacent normal and hyperplastic glands in the
same tissue block had positive expression. The proportion of cancer
cells in each block that had no p36 expression was nearly 100% in 28
cases and close to 50–75% in the other 3 cases. The Gleason score of
the 3 cases was 5, 6, and 5, respectively. One of them was the cancer
case 5 in the Northern analysis (Fig. 1B)
. Cytokeratin
34ßE12, a marker of prostate basal cells (7)
, was used
to verify that the p36-positive cells were indeed cancer cells, because
they lacked a participating basal cell component.
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Fig. 2. p36 and p11 protein expression in prostate tissues.
Depicted in each row are consecutive sections stained with H&E
(first column), p36 antibody (second
column), p11 antibody (third column), and
34ßE12 Ab (fourth column). A–D, glands
from peripheral zone of an organ donor (x20); E–H, BPH
from transurethral resection (x20), I–L, cancer from
radical prostatectomy (x20); M–P, p36/p11 negative PIN
from radical prostatectomy (x40); Q–T, p36/p11 positive
PIN (x40).
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We additionally examined the p36 expression in high-grade PIN.
Twenty-one of the 31 cancer blocks had foci of high-grade PIN at the
interface between cancer and non-neoplastic glands. Each block had 6 or
7 glands involved by high-grade PIN, making the total 130 glands
examined. There was a clear difference in p36 expression in this
morphologically indistinguishable, high-grade PIN. Of them, 65% showed
negative and 35% showed positive, apical staining whereas all of the
basal cells were positively stained. Persistent expression of 34ßE12
in p36-negative PIN ruled out the possibility of cancer (Fig. 2)
.
PSA-positive staining confirmed that p36-negative PIN was prostate
gland origin (data not shown).
Annexin II (p11) Protein in Normal Prostate, BPH, Prostate Cancer,
and PIN.
Because Annexin II light chain (p11) is always in complex with p36, the
absence of p36 expression in prostate cancer and in 65% of PIN
prompted us to examine p11 expression immunohistochemically (Fig. 2)
.
In normal and BPH, p11 was expressed in the same cell type as p36.
Prostate cancer cells had also lost p11 expression. The small fraction
of p36-positive cancer cells in three cases also expressed p11.
However, we noticed in a few cancer cells that weak p11 expression was
present, whereas p36 was lost. p36-negative or -positive PIN remained
negative or positive for p11. Interestingly, despite the absence of p11
protein in prostate cancer, p11 mRNA levels remained unchanged when
compared with normal prostate (Fig. 1C)
.
Association of Hypermethylation with Silencing of Annexin II (p36)
Gene Expression.
To address the question of whether the decrease in p36 mRNA level is a
result of a homozygous deletion in p36 coding regions in cancer DNA,
Southern analysis with three different endonucleases was conducted
using p36 open reading frame sequence as the probe on genomic DNA from
paired cancer tissue and blood leukocyte of the same patients
(n = 8). We found no difference between
cancer and blood DNA (data not shown). To establish if the
p36 gene has a CpG island spanning the promoter region and
could therefore be susceptible to hypermethylation, we used the
full-length p36 cDNA sequence to search for p36 genomic DNA sequences
from a high throughput genomic DNA database and obtained a sequence
from clone AC019146. Analysis of the upstream region of the gene has
shown a CpG island spanning the promoter, first exon, and intron of the
gene (Fig. 3)
. To determine whether methylation is associated with Annexin II
silencing in prostate cancer, we treated prostate cancer LNCaP cells,
which do not express p36, with 5-azacytidine (Fig. 4)
. p36 is expressed in normal prostate, blood, and DU145 prostate cancer
cells but is silent in prostate cancer and LNCaP cells. Annexin II
expression was reactivated in LNCaP cells after 5-azacytidine treatment
suggesting that methylation is associated with Annexin II silencing in
prostate cancer.
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Fig. 3. CpG map of p36 gene. Vertical lines
above bar indicate the relative position of the CpG sites.
Nucleotide position below the bar is based on the
sequence from GenBank (Accession no. AC019146). The orientation of
genomic DNA is reverse complimentary to p36 cDNA.
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Fig. 4. 5-Azacytine reactivation of p36 gene
expression in LNCaP cells. RT-PCR was used to determine p36 expression
using RNA isolated from (1) prostate cancer,
(2) normal prostate, (3) blood,
(4) DU145 cells, and (5) LNCaP cells
treated with and (6) without 5 azacytidine.
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Discussion
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Annexin II can be found in vivo as p36, p36p11, and
p362p112, and is involved
in endocytosis, exocytosis, and membrane trafficking (8)
.
p36 belongs to a family of calcium- and lipid-binding proteins and is a
substrate for receptor and nonreceptor protein kinases (9
, 10)
. Recently, membrane p36 has been shown to mediate steroid
rapid action (11)
. p11 is a small calcium binding protein
and shares sequence homologies with the S-100 family (12)
.
Overexpression of p36 increases p11 protein level with no change in its
mRNA levels (13)
, suggesting that p36 has a
post-translational stabilizing effect on p11 protein. Thus, it is
possible that the lack of p36 in prostate cancer gives rise to
substantial degradation of p11 so that the rate of translation from p11
mRNA fails to compensate. The observation that negative p36 expression
coexists with a weak expression of p11 in a small number of cancer
cells may represent the early stage of p11 degradation. As the loss of
p36 and subsequently p11 in prostate cancer obligates all forms and
functions of Annexin II, it is difficult to know which function of
Annexin II is related to prostate carcinogenesis. However, the
importance of calcium, the key element for Annexin II to function, is
well appreciated in programmed cell death in the prostate (14
, 15)
. The distinctive expression pattern of Annexin II among BPH,
PIN, and cancer indicates that the loss of Annexin II is specific for
prostate cancer. The loss of expression in 31/31 individual prostate
cancer blocks regardless of the Gleason score and 65% of high-grade
PIN suggests that Annexin II is involved in an early stage or common
pathway of prostate carcinogenesis. Recently, Annexin I has been found
to be absent in prostate cancer and PIN by Western blot and
immunohistochemistry (16)
, and Annexin VII can suppress
proliferation of prostate cancer cells and is significantly reduced in
metastatic and recurrent prostate cancer (17)
. Thus,
Annexin could represent a previously unrecognized mechanism involved in
prostate cancer. The clinical implication of residual Annexin
II-positive cancer cells in three cases will be closely followed up.
To address the question of whether the >50% decrease in p36 mRNA
level is a result of a homozygous deletion in the p36 coding region in
cancer DNA, we conducted Southern analysis on genomic DNA from paired
cancer tissue and blood leukocyte. Because our prostate cancer cases
are sporadic, a somatic homozygous deletion in the p36 coding region in
the cancer DNA should be revealed by comparing it with the paired blood
DNA. Any point mutations or small deletions within the coding region of
the p36 gene, which Southern analysis may not be able to
detect, should not cause such a substantial decrease in p36 mRNA
levels. We found no difference thus far between cancer and blood DNA,
suggesting that the decrease in p36 mRNA level is likely attributable
to alterations affecting the expression or processing of mRNA. Because
DNA hypermethylation is commonly associated with silencing of
tumor-related genes (1, 2, 3)
, a preliminary study was
conducted to examine the possible association of methylation with
Annexin II silencing in prostate cancer. The finding that the
p36 gene does have a CpG island in the promoter region and
in the first exon and, more importantly, that p36 expression could be
reactivated by treatment of prostate cancer LNCaP cells with a
demethylation agent indicates that hypermethylation could be
responsible for silencing of the p36 gene in
vivo. However, because 5-azacytidine can also affect Sp1
transcriptional activity (18)
, additional study by
bisulphite sequencing of normal and cancerous prostate-derived DNA is
needed to verify and identify methylation sites that distinguish cancer
cells from normal cells. Although p36-positive cancer cells of case 5
was demonstrated by both Northern analysis and immunohistochemistry, it
remains to be seen whether additional mechanisms besides mRNA
transcription are involved in the down-regulation of the p36 protein,
considering the fact that 28 of 31 cases had no p36 protein, whereas
one of five cases showed unchanged p36 mRNA.
This study also provides evidence that high-grade PIN is not
biochemically homogenous. It is known that not all PIN will progress to
cancer (19)
; however, the lack of a progression-associated
"marker" has prevented pathologists from stratifying the
morphologically undistinguishable PIN. Although additional studies are
required to correlate the p36-negative and -positive PIN with the
incidence of cancer and prognosis, the fact that the vast majority of
cancer cells show negative expression for p36 suggests that the
p36-negative PIN is the true precursor of cancer. We speculate that
p36-positive PIN cannot progress to cancer without loss of p36
expression first. However, because we have encountered three cases
containing a small fraction of p36-positive cancer cells, it remains a
possibility that p36-positive PIN can develop directly to p36-positive
cancer.
A differential diagnosis among well-differentiated cancer, PIN, and
non-neoplastic lesion is often required for needle-core biopsy, in
particular when the number of glands is insufficient and/or the quality
of a specimen is unsatisfactory (20)
. Currently, 34ßE12
is the "gold standard" by highlighting the absence of basal cells
in infiltrating cancer. However, it is problematic to additionally
differentiate PIN from non-neoplastic glands because basal cells are
present in both. The unique expression pattern of Annexin II-negative
PIN, absent in glandular epithelial but present in basal cell, is in
contrast to benign hyperplasia in which Annexin II is expressed in both
cells and to the vast majority of cancer cells in which Annexin II
expression is abolished and basal cells are no longer attached. Thus,
identification of Annexin II provides a potential diagnostic marker for
needle-core biopsy specimens.
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FOOTNOTES
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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.
1 This study was supported by the Medical
Foundation, and Endocrinology & Diabetes Research Foundation,
University of Sydney and National Health Medical Research Council. 
2 To whom requests for reprints should be
addressed, at Department of Medicine, D06, University of Sydney, NSW
2006, Australia. Phone: 612-95155186; Fax: 612-95161273; E-mail: qhd{at}med.usyd.edu.au
3 The abbreviations used are: PIN, prostate
intraepithelial neoplasia; RT-PCR, reverse transcription-PCR; HPRT,
hypoxanthine phosphoribosyltransferase; DAB,
3,3'-diaminobenzidine; PSA, prostate-specific antigen; BPH, benign
prostatic hyperplasia.
Received 4/27/01.
Accepted 7/11/01.
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ABSTRACT
Introduction
