Modulation of oncogenic potential by alternative gene use in human prostate
cancer
Shrihari S. Kadkol1, Jonathan R. Brody1, Jonathan Pevsner2, 3, Jining Bai1
& Gary R. Pasternack1
1 The Division of Molecular Pathology, Department of
Pathology, The Johns Hopkins University School of Medicine,
720 Rutland Avenue, Baltimore, Maryland 21205,
USA
2 Department of Neuroscience, The Johns Hopkins University
School of Medicine, 720 Rutland Avenue, Baltimore,
Maryland 21205, USA
3 Department of Neurology, The Kennedy Krieger Institute,
Baltimore, Maryland 21205, USA
Correspondence should be addressed to Gary R. Pasternack gpastern@jhmi.edu
Only a small percentage of primary prostate cancers have genetic changes.
In contrast, nearly 90% of clinically significant human prostate cancers seems
to express high levels of the nuclear phosphoprotein pp32 by in situ
hybridization. Because pp32 inhibits oncogene-mediated transformation, we
investigated its paradoxical expression in cancer by comparing the sequence
and function of pp32 species from paired benign prostate tissue and adjacent
prostatic carcinoma from three patients. Here we demonstrate that pp32 is
expressed in benign prostatic tissue, but pp32r1 and pp32r2, closely-related
genes located on different chromosomes, are expressed in prostate cancer.
Although pp32 is a tumor suppressor, pp32r1 and pp32r2 are tumorigenic. Alternative
use of the pp32, pp32r1 and pp32r2 genes may modulate the oncogenic potential
of human prostate cancer.
In human prostate cancer, high-level expression of pp32 RNA occurs in nearly
90% of clinically significant prostate cancers, in contrast to the substantially
lower frequencies of alterations of other oncogenes and tumor suppressors1,
2,
3. This highly conserved nuclear phosphoprotein may act as a
tumor suppressor. Functionally, pp32 inhibits transformation in vitro
by a wide variety of oncogene pairs, including ras and myc, ras and mutant
p53, ras and E1a, ras and jun, and human papilloma virus E6 and E7 (refs. 4−7). It also inhibits
the growth of transformed cells in soft agar4. Ras-transfected
NIH3T3 cells previously stably transfected to overexpress normal human pp32
do not form foci in vitro and do not form tumors in nude mice, unlike
control cells. In contrast, reduction of endogenous pp32 in the same system
by an antisense pp32 expression construct augments tumorigenesis considerably
(J.B. et al., unpublished observations). Here we have addressed the
paradox of the apparent expression of high levels of an anti-oncogenic protein
by prostate cancers. We compared the sequence and function of pp32 species
from paired benign prostate tissue and adjacent prostatic carcinoma from three
patients, and found that prostate cancers express little or no pp32, but do
express other members of the pp32 family encoded by genes on separate chromosomes.
The alternative pp32 genes expressed in prostate cancer are tumorigenic, unlike
pp32.
pp32 mRNA in benign prostate tissue and prostate cancer In benign prostate tissue, basal cells express pp32, whereas pp32
mRNA is not detectable by in situ hybridization in differentiated glandular
cells (Fig. 1a). In contrast, there is strong
in situ hybridization to pp32 probes in nearly all clinically significant
human prostatic adenocarcinomas. Strongly hybridizing tumors show intense
immunopositivity with antibodies to pp32, indicating that they express pp32
or immunologically related proteins (Fig. 1a
and b). The restricted expression of pp32
mRNA in benign prostate is consistent with observations that pp32 has
a distinct pattern of expression in vivo4,
5,
6. The
expression of pp32 mRNA in normal peripheral tissues is restricted
to 'stem-like' cell populations, such as crypt epithelial cells in the gut
and basal epithelium in the skin; cerebral cortical neurons and Purkinje cells
also express pp32.
Figure 1.a, Detection of pp32-related mRNA in benign prostate tissue
and prostate cancer.
The pp32 mRNA was detected by stringent in situ hybridization with
a pp32 probe; the signal is cytoplasmic, as mRNA and not protein is detected
in this assay. Normal prostatic basal cells are positive, whereas the clear,
differentiated glandular cells are negative. In contrast, prostatic adenocarcinoma
(left arrow) is very positive. b, Prostate cancers stain intensely
with antibody against pp32. High-grade human prostate cancer stained with
affinity-purified rabbit polyclonal antibody against pp32 (17). Left, a representative field (original magnification,
250); rectangle, the area shown in computer-generated detail (right).
Molecular analysis of pp32-related transcripts Given the localization data (Fig. 1a and b), it would seem that pp32 is expressed in both normal
and neoplastic prostatic epithelial cells, despite its ability to inhibit
neoplastic functions such as transformation. The explanation for this apparent
'discordant' expression is that prostate tumors do not usually express pp32;
rather, they express variant pp32 species that promote transformation, instead
of inhibiting it. RT−PCR of paired benign prostate tissue and prostatic
adenocarcinoma from three patients yielded amplification products ranging
from 889 to 907 bp (Fig. 2a). For this RT−PCR,
consensus primers capable of amplifying the full-length coding sequence from
pp32 and the two closely-related intronless genomic sequences pp32r1
(GenBank AF008216) and pp32r2 (GenBank U71084) were used. The
only difference between the samples was a lower amplicon yield from benign
tissue than from neoplastic tissue. Four human prostatic adenocarcinoma cell
lines, DU-145, LNCaP, PC-3 and TSUPR-1, also yielded products of a similar
size (data not shown). Qualitative differences between normal and neoplastic
tissue were demonstrated when the RT−PCR products were subcloned and
analyzed by cleavase fragment length polymorphism analysis (CFLP) and sequence
analysis. All clones of the RT−PCR products from benign prostate tissue
produced a normal CFLP pattern that corresponded precisely to that obtained
from previously cloned pp32 cDNA template (Fig. 2
b). Prostatic adenocarcinomas yielded four distinct CFLP patterns,
of which three were unique and one was like the normal pp32 pattern.
Figure 2.a, Amplification of pp32 and pp32 variants from human prostate
cancer.
Lanes: 1, 1-kb DNA 'ladder' (left margin, size in kb); 2, pCMV32; 3, FT-1,
without reverse transcription; 4, FN-1; 5, FT-1; 6, FN-2; 7, FT-2; 8, FN-3;
9, FT-3; 10, negative control with template omitted. FN, frozen benign prostate;
FT, frozen prostatic adenocarcinoma; numbers indicates patient designations.
b, Cleavase fragment length polymorphism (CFLP) analysis of pp32 detects
variant pp32 transcripts in human prostate cancer. CFLP analysis of cloned
cDNA amplified by RT−PCR from human prostatic adenocarcinoma and adjacent
benign prostate tissue, using primers derived from the normal pp32 cDNA sequence.
Lanes: 1, undigested normal pp32 cDNA; 2 and 3, normal pp32 cDNA; 4, FT1.11;
5, FT1.7; 6, FT2.2; 7, FT2.4; 8, FT3.18; 9, FT3.3;10, FN3.17; 11, FN2.1. FT,
frozen prostate cancer; FN, frozen benign prostate. The band shifts correspond
to sequence differences.
In contrast to benign prostate samples, which yielded only pp32
transcripts by sequence analysis, adjacent prostate cancers expressed little
or no pp32. Instead, four pp32-related transcripts with distinct
sequences encoding open reading frames were obtained from the adjacent prostate
cancers, varying from 92.4% to 95.9% nucleotide identity to normal pp32
cDNA. Of these, two transcripts corresponding to the products of the
pp32-related genes pp32r1 and pp32r2 were obtained repeatedly
from patient samples; thus, we studied them further. We compared the properties
of the pp32r1 and pp32r2 transcripts obtained from prostatic
adenocarcinomas (Table 1). The identical
pp32r1 sequences obtained from two patients differed by four nucleotides
from the pp32r1 genomic sequence. The pp32r2 sequences obtained
from two patients were also identical and differed by three nucleotides from
the pp32r2 genomic sequence. Both sets of differences are considered
consistent with polymorphic variation.
Multiple pairwise alignment of the predicted protein sequences8
demonstrated that pp32, pp32r1 and pp32r2 have sequence changes along their
entire lengths (Fig. 3). The 'pile-up' and pairwise
alignments show that there is a high degree of sequence conservation at the
predicted amino acid levels, corresponding to an underlying conservation of
nucleotide sequence; and that the sequence differences are distributed throughout
the length of the sequence without obvious clustering, 'hotspots' or segmentation
of sequence differences. No straightforward process of somatic mutation or
alternate splicing could explain these results. Instead, given the correspondence
of the variant sequences with previously identified genes on chromosomes 4
and 12, the data are consistent with alternative gene expression.
Figure 3. Alignment of pp32 with human prostate tumor-derived pp32r1 and pp32r2
sequences.
Differences from the pp32 sequence are indicated with lower-case letters;
agreement with the pp32 sequence is indicated as a blank. The variant pp32r2
encodes a truncated protein (wavy lines indicate the truncated region). The
GCG Pileup and Pretty programs were used9.
Oncogenic potential of pp32, pp32r1 and pp32r2 A switch in the oncogenic potential of the expressed pp32 family members
accompanies the expression of alternative pp32 genes in prostate cancer. Expressed
pp32r1 and pp32r2 often fail to inhibit or, indeed, sometimes stimulate transformed
focus formation when co-transfected with ras and myc, compared with the number
of foci obtained when ras and myc are transfected with control vector (Fig. 4). In contrast, normal pp32 consistently suppresses
transformation. Similarly, both pp32r1 and pp32r2 are tumorigenic when stably
transfected into NIH3T3 cells, in contrast to pp32, which is non-tumorigenic
(Tables 1 and 2).
Figure 4. Effect of variant pp32 species on transformation: altered oncogenic
potential of pp32r1 and pp32r2.
Each data point represents the results from an individual flask expressed
as the percent foci obtained in the contemporaneous control of ras+myc+vector.
Data represent four separate experiments.
Discussion Species of pp32 have been found in a variety of biologic contexts, although
the relationship to neoplasia has not always been appreciated. An essentially
equivalent molecule, PHAPI, was cloned from an EBV-transformed human B-lymphoblastoid
cell line9; PHAPII, cloned by the same strategy, is unrelated
to pp32. PHAPI was identified through its association in solution with human
HLA class II protein, and was localized to the plasma membrane, cytoplasm
and nucleus. The PHAPI gene putatively localizes to chromosome 15q22.3-q23,
as shown by fluorescent in situ hybridization10. (
PHAPI and pp32 are equivalent). The protein pp32 has been variously identified
as I1PP2a, an inhibitor of protein phosphatase 2a (11; I2PP2a is unrelated to pp32); a cytoskeletally-associated cytosolic
protein in CHO cells12 (perhaps due to a difference in system,
or perhaps pp32 can localize to the cytoplasm under certain circumstances);
and LANP, a leucine-rich nuclear protein in the central nervous system13. There are reports of gene products with less homology to pp32
as well. PHAPI2a (EMBL HSPHAP12A) and PHAPI2b (EMBL HSPHAP12B) were also cloned
from an EBV-transformed human B-lymphoblastoid cell line. These variant pp32
sequences, different from the sequences reported here, represent APRIL (acidic
protein rich in leucines; 14; EMBL HSAPRIL),
a protein cloned from human pancreas that is shorter than PHAPI2a by two N-terminal
amino acids; PHAPI2b is identical to a subset of APRIL. Silver-stainable protein
SSP294 (GenBank HSU70439) was cloned from HeLa cells and is identical to PHAPI2a.
There are probably pp32 genes other than the three with known chromosomal
localization studied here; however, their characterization remains incomplete
and their precise number is not known. We obtained single isolates of tumor
cDNA encoding two additional tumorigenic pp32 variants that are undergoing
further characterization. The previously mentioned cell lines, DU-145, PC-3,
and TSUPr-1, yielded pp32r1 and pp32r2, but also yielded single isolates of
additional tumorigenic variants of unknown chromosomal origin that are undergoing
further analysis.
There seem to be at least four genes in the pp32 family in rodents,
which is consistent with the existence of a gene family of comparable size
in humans. This is a minimum estimate, as the expressed sequence tags detected
so far may not represent the complete extent of the pp32 gene family.
For example, a murine pp32 (4) (GenBank U73478)
has 89% amino acid identity with pp32, but less identity with pp32r1 and APRIL.
We have also identified expressed sequence tags predicted to encode pp32-related
proteins in Caenorhabditis elegans, schistosomes, zebrafish and Drosophila
(data not shown), indicating that pp32 family members effect fundamental functions
subject to phylogenetic conservation.
The human pp32 gene has been mapped to chromosome 15q22.3-q23 by
fluorescence in situ hybridization10. A Unigene entry
for pp32 (Hs. 76689; HLA-DR associated protein I) lists 93 expressed
sequence tags corresponding to this gene, 12 of which contain a mapped sequence-tagged
site. These sequence-tagged sites all map to chromosome 15, as do many of
the pp32 expressed sequence tags (http://www.ncbi.nlm.nih.gov).
APRIL was also mapped to chromosome 15q25 (14; GenBank Y07969). The pp32r1 gene maps to chromosome
4, as determined by PCR of the NIGMS monochromosomal panel 2 (National Institute
of General Medical Sciences human genetic mutant cell repository; 15) followed by sequencing of the PCR product. The
full sequence of pp32r1, including 4,364 nucleotides of sequence upstream
of the putative translational start site , had more than 400 matches in a
'Blastn' search of the non-redundant GenBank database. These matches were
to two short regions of about 278 and 252 base pairs (nucleotides 674−952
and 2542−2794) that represent repeats in opposite orientations. The
repeats are related to elements on many chromosomes.
The data reported here indicate that the alternative use of pp32 genes
is a common feature of human prostate cancer, and that this alternative gene
use is accompanied by a change in oncogenic potential. Of prostate cancers
of Gleason Score 5 and above, 87% express pp32 or closely related transcripts3, which is in contrast to the much lower frequency of molecular alterations
in other widely studied oncogenes and tumor suppressor genes. For example,
myc overexpression2 occurs in about 60% of cases, and p53 is
abnormal in only about 25% of primary tumors1. In contrast to
these oncogenes and tumor suppressors, the high frequency of pp32 variant
expression indicates that alternative expression of variant pp32 species may
be involved in the etiology of human prostate cancer. These findings may have
important diagnostic and prognostic implications.
Modulation of oncogenic potential by alternative gene use has interesting
implications. Preliminary comparison of structures predicted by energy minimization
programs for pp32 and variant pp32 species indicates considerable structural
differences that might form the basis for interaction with different mechanistic
pathways by pp32 and the variant pp32 species. It is not yet apparent how
early in the neoplastic process the use of alternative genes of the pp32
family occurs. Because alternative pp32 gene use in prostate cancer
is common, it could be an early, important event in tumorigenesis.
Alternative gene use is potentially reversible, which has additional clinical
and mechanistic implications. The malignant potential of tumor cells might
eventually be modified by manipulating the pattern of expression of pp32 family
members in a form of endogenous gene therapy. Gene therapy by pharmacological
manipulation of the differential expression of pp32 family members must await
characterization of the switching mechanism, which is now unknown. Mutation
and loss of heterozygosity of the pp32 locus at 15q22.3-q23 could theoretically
lead to increased expression of pp32r1 and pp32r2; however,
loss of heterozygosity is unlikely, as chromosomal loss of 15q22.3-q23 is
not a common feature of prostate cancer. More likely possibilities involve
regulatory aberrations. Epigenetic regulation should be explored, as it could
lead to inactivation of the pp32 gene and concomitant activation of
the pp32r1 and pp32r2 genes by means such as methylation and
demethylation. Regulation by one or more transcription factors that act differentially
to repress pp32 while inducing pp32r1 and pp32r2 is possible.
Finally, post-transcriptional mechanisms could also be invoked, whereby differential
expression would be regulated by changes in mRNA or protein stability. For
carcinogenesis, all of the mechanisms involving regulatory aberrations would
contribute to tumorigenesis through a plastic, potentially reversible regulatory
change rather than an irreversible structural change in the genome. Thus,
members of the pp32 family may eventually be used as targets for pharmacologic
chemopreventive and therapeutic strategies in prostate cancer.
Methods In situ hybridization. Bases 1−298 of
the pp32 cDNA sequence (GenBank HSU73477) were subcloned into the Bluescript
vector by standard techniques. Digoxigenin-labeled antisense and sense RNA
probes were generated using a commercially available kit (Boehringer). Vector
DNA linearized with BamHI and XhoI served as template for the
generation of antisense and sense probes, respectively. DNA was transcribed
in vitro for 2 h at 37 °C in a final volume of 20 l containing
1 g of template DNA; 2 U/l of either T3 or T7 RNA polymerase; 1
U/l ribonuclease inhibitor; 1 mM each of ATP, CTP and GTP; 0.65 mM UTP;
0.35 mM digoxigenin-11-UTP; 40 mM Tris-HCl, pH 8.0; 10 mM NaCl; 10 mM DTT;
6 mM MgCl2; and 2 mM spermidine. The addition of 2 l of 0.2M EDTA, pH
8.0 stopped the reaction, and subsequent incubation for 30 min at −70
°C with 2.2 l of 4 M LiCl and 75 l of pre-chilled ethanol precipitated
the synthesized transcripts. RNA was pelleted by centrifugation, washed with
80% ethanol, partially dried, and dissolved in 100 l of DEPC-treated water.
Yields of labeled probe were determined by an enzyme linked immunoassay using
a commercially available kit (Boehringer). Nonradioactive in situ hybridization
used anti-sense and sense pp32 RNA probes generated by in vitro
transcription3. The signal is cytoplasmic, as mRNA and
not protein is detected in this assay.
Immunohistochemistry. Formalin-fixed, paraffin-embedded
tissue was cut into sections 4 M in thickness, deparaffinized, hydrated,
processed for heat-induced antigen retrieval at 95 °C in 0.01 M citrate
buffer, pH 6.0, for 20 min (16), then incubated
overnight at room temperature with a 1:20 dilution of antibody against pp32.
After being washed, slides were sequentially developed with biotinylated swine
antibody against rabbit IgG at a diltuion of 1:100 (Dako, Carpinteria, California),
streptardin peroxidase (Dako, Carpinteria, California) and diaminobenzidine.
RT−PCR and CFLP. Sequences were 'reverse-transcribed'
and amplified using bases 32−52 of HSU73477 as a forward primer and
bases 919−938 of the same sequence as a reverse primer in conjunction
with the Titan One-Tube RT−PCR kit (Boehringer). Reverse transcription
was done at 50 °C for 45 min followed by incubation at 94 °C for 2
min; the subsequent PCR comprised 45 cycles of 92 °C for 45 s, 55 °C
for 45 s and 68 °C for 1 min with a final extension at 68 °C for 10
min in a PTC 100 thermocycler (MJ Research, Watertown, Massachusetts). Template
RNA was isolated from cell lines or frozen tumor samples using RNAzol B (Tel-Test,
Friendswood, Texas) according to the manufacturer's instructions, then digested
with RNAse-free DNAse I (Boehringer). The plasmid pCMV32 was used as a positive
control without reverse transcription. After RT−PCR, amplicons were
cloned into pCR2.1 or pCR3.1 (Invitrogen, Carlsbad, California) for further
analysis. Cloned inserts were analyzed by cleavase fragment length polymorphism
(CFLP) assay according to the manufacturer's specifications (Life Technologies),
with digestion at 55 °C for 10 min in 0.2 mM MnCl2, and were
electrophoresed on a 6% denaturing polyacrylamide sequencing gel.
Figure 2b, lane 1 is an undigested control whose band migrated
substantially slower than the digestion products; samples in all other lanes
were digested with cleavase as described.
Sequence analysis. Some comparisons of sequences to
determine nucleotide identity were done using the Wisconsin Package, version
9.1 (1997; Genetics Computer Group, Madison, Wisconsin).
Transformation assay Rat embryo fibroblasts were transfected
with constructs as described4 and transformed foci were counted.
For each experiment, approximately 1 106 cells were
plated per 75-cm2 flask and incubated for 2−3 d before
transfection, to achieve approximately 40% confluency. For each flask of primary
rat embryo fibroblasts, the following amount of plasmid was added: pEJ-ras,
5 g; pMLV-c-myc, pCMV32, pCMVneo or variant pp32 constructs in pCR3.1
(Invitrogen, Carlsbad, California), 10 g. Plasmids were prepared in two
volumes of lipofectin (2 l lipofectin per g DNA; Life Technologies),
then gently mixed by inversion in 1.5 ml OPTIMEM (Life Technologies) in sterile
15-ml polystyrene tubes, and allowed to incubate at room temperature for >15
min. For experiments with more than one flask, mixtures of all reagents were
increased in proportion to the numbers of flasks required for each transfection.
Cells were washed once with OPTIMEM, and then incubated in 6 ml of OPTIMEM
and 1.5 ml of the DNA/lipofectin mix. After overnight incubation, the cells
were grown in standard media and supplied with fresh media twice weekly. Foci
were counted 14 days after transfection.
Received 21 December 1998; Accepted 15 January 1999
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Acknowledgments We thank D. Coffey, C.V. Dang, T.J. Kelly Jr., S. Kern, A. Owens Jr., D.
Pardoll, H. Shin and S. Sisodia for reading the manuscript, G.S. Bova for
providing the paired tissue samples from human prostate cancer patients, and
R. Ashworth of The Johns Hopkins DNA Analysis Facility for automated sequencing.
This work was supported by USPHS Grant RO1 CA 54404. In accordance with institutional
policies, we wish to disclose the following information. G.R.P. is an inventor
on US Patents 5756676, 5734022, and 5726018, which encompass the antecedent
and present technology described in this manuscript. G.R.P., S.S.K. and J.R.B.
are inventors on a pending US Patent application related to the present technology.
There is no current licensee or other financial interest for the aforementioned
technology.
250); rectangle, the area shown in computer-generated detail (right).
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1 
