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From the Center of Study on Mitochondria and Energy
Metabolism,*
Consiglio Nazionele delle Ricerche, Bari,
Italy; the Chair of Urology
and the
Institute of Pathological Anatomy,§
School of
Medicine, University of Bari, Bari, Italy; and the Department of
Pathology,
Yale University School of Medicine,
New Haven, Connecticut
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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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Integrins are a superfamily of cell surface adhesion receptors that play a critical role in tumor progression and metastases as well as in a number of physiological processes such as inflammation, cell adhesion, migration, proliferation, survival, and differentiation.2-5 Integrins are receptors for extracellular matrix proteins such as fibronectin, vitronectin, collagen, and laminin.6 In addition to mediate cell adhesion to the extracellular matrix, integrins also transduce biochemical signals into the cell thus regulating cell proliferation and differentiation.7,8
Integrins are transmembrane glycoproteins composed of 
and ß
subunits that associate to form a heterodimer; 16 subunits and
eight ß subunits, that associate to form at least 22 different
receptors, have been discovered to date.7,8
Each subunit
has a large extracellular domain, a single transmembrane domain and a
short cytoplasmic domain.9
The role of the integrin
cytoplasmic domain in modulating integrin functions and signaling
events is well established.10,11
Alternatively spliced variants of the integrin cytoplasmic domain have
been described for some of the and ß subunits.11
Alternative splicing events between exon 6 and exon 7 of the ß1
integrin subunit generate four different isoforms.11
A
ß1 isoform, ß1C, was found to be expressed in normal prostate
epithelial cells.11,12
Its cytoplasmic domain consists of
26 amino acids encoded by exon 6, and 48 amino acids derived from an
additional exon, exon C and part of exon 7, in the ß1 integrin gene.
It has been demonstrated that ß1C expression inhibits cell
proliferation and causes growth arrest in the late
G1 phase of the cell cycle.13-15
Recent studies have also demonstrated that ß1C causes up-regulation
of the cyclin kinase inhibitor p27Kip1 protein
levels in prostate cells shedding new lights into the molecular
mechanisms underlying prostate cancer progression.16
Although several groups have analyzed integrin expression in prostate cancer in vitro or in vivo17-20 at the protein levels, very few studies have described integrin mRNA expression in prostate malignant transformation either in vitro or in vivo.21,22 We have studied ß1C and ß1 expression at both mRNA and protein levels by Northern and immunoblotting analysis using specimens from 33 patients affected by prostatic carcinoma. Our results demonstrate for the first time that ß1C and ß1 mRNA expression is down-regulated in prostate carcinoma, whereas only ß1C protein levels are reduced.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study was performed using 38 prostate specimens (Table 1)
obtained from either patients who
underwent radical cystoprostatectomy for bladder carcinoma noninvolving
the prostate (five specimens) or patients with prostate cancer (33
specimens) hospitalized at the Department of Urology of the University
of Bari, School of Medicine, in the years 1998 to 1999. Informed
consent was obtained from all patients. Soon after surgical removal of
the prostate, a sample was taken from all specimens, snap-frozen, and
cryopreserved in liquid nitrogen for RNA extraction and immunoblotting
analysis. The remaining tissue samples were fixed in 10%
neutral-buffered formalin for 12 to 24 hours, embedded in paraffin, and
stained with hematoxylin and eosin (H&E). H&E-stained sections were
reviewed, and the tumor grade, according to Gleason’s
criteria23
and the stage of tumor, according to TNM
system,24
were estimated in each tumor sample.
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RNA Extraction and Northern Blot Analysis
Frozen tissue samples were pulverized to a fine powder and
cellular RNA was extracted using the guanidinium isothiocyanate-cesium
chloride procedure.25
Total RNA (25 µg) isolated from
the tissues was electrophoresed through 1% denaturing agarose gel
containing 660 mmol/L formaldehyde, and transferred26
to a
nylon membrane (Hybond N+; Amersham, Milan,
Italy). The filters were subsequently prehybridized overnight at 42°C
with a buffer consisting of 50% formamide, 5x Denhardt’s solution
(1% Ficoll 400, 1% polyvinylpyrrolidone, 1% bovine serum albumin),
5x sodium chloride/sodium phosphate/ethylenediaminetetraacetic acid
(SSPE) (3 mol/L NaCl, 200 mmol/L
Na2H2PO4,
pH 7.0, 19 mmol/L ethylenediaminetetraacetic acid), 0.5% sodium
dodecyl sulfate (SDS) and 100 µg/ml of sonicated salmon sperm DNA.
The filters were then hybridized for 20 hours at 42°C by adding
3 x 106
cpm of
32P-labeled probe/ml to the prehybridization
solution. The filters were washed once with 2x SSPE, 0.1% SDS for 10
minutes at room temperature, then with 1x SSPE, 0.1% SDS at 42°C,
followed by several washes in 0.1x SSPE, 0.1% SDS, at 65°C and
finally exposed at -80°C overnight or longer to Kodak X-Omat AR 5
film (Kodak, Rochester, NY). Radiolabeled probes were generated using
the Megaprime DNA labeling kit (Amersham), 5 µl of
-32P-dCTP (3,000 Ci/mmol,
Amersham)27
and 25 ng of double-stranded either 116-bp
fragment specific for the ß1C integrin or a full-length human ß1
cDNA.12
The specific 116-bp ß1C fragment (nucleotides
2435 to 2550)12
was generated by polymerase chain reaction
using pBluescript ß1C plasmid as template and the resulting fragment
was subcloned in the pBluescript vector. mRNA levels were normalized
using ribosomal 28S RNA, a constitutively expressed
gene.28
For this purpose, blots were stripped in 0.1%
boiling SDS and reprobed with the radiolabeled
32P-28S cDNA probe. Quantitative analysis was
performed by densitometric scanning of the autoradiographs using a
Bio-Rad GS-700 densitometer (Bio-Rad, Richmond, CA); multiple exposures
of the same Northern blots in a linear range were performed. The ratio
between the 4.2-kb long ß1C mRNA levels and the 28S rRNA levels was
calculated for each sample to take into account differences in RNA
loading. The average of either ß1C or ß1 mRNA expression levels in
control normal prostate derived from five patients was set at 100
(arbitrary units). ß1C or ß1 mRNA levels in neoplastic prostate
were calculated as percentage of normal prostate mRNA levels hybridized
on the same filter. For each specimen, the mean value (±SEM) of
results obtained in at least three experiments was calculated.
Immunoblotting
Either normal or tumor frozen tissue specimens obtained from radical prostatectomy were homogenized in lysis buffer containing 0.1% SDS, 1% Nonidet P-40, 50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 200 mmol/L LiCl, 5 mmol/L ethylenediaminetetraacetic, 10% glycerol, 10 µg/ml aprotinin, 120 µg/ml leupeptin, 170 µg/ml phenylmethylsulfonyl fluoride. The homogenate was sonicated for 20 seconds, then centrifuged for 30 minutes at 14,000 x g at 4°C. Two-mercaptoethanol (1%) was added to each lysate for 30 minutes at 4°C to further solubilize potentially cross-linked molecules and 150 µg of tissue extracts were electrophoresed on 7.5% SDS-polyacrylamide gel electrophoresis under reducing conditions. Immunoblotting was performed as previously described20 using either 5 µg/ml rabbit polyclonal affinity-purified antibody to ß1C integrin or 1 µg/ml mouse monoclonal antibody to ß1 integrin (Transduction Laboratories, Milan, Italy) or 10 µg/ml of antibody to ß-tubulin (Sigma, St. Louis, MO) for 16 hours at 4°C in Tris-buffered saline/Tween 20 (TBS-T) (20 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 0.2% Tween-20). The membrane was then washed three times in TBS-T and incubated with horseradish-peroxidase-conjugated goat affinity-purified antibody to either rabbit or mouse IgG (Amersham), in TBS-T for 1 hour at room temperature. After three washes in TBS-T, the proteins were visualized using the Amersham enhanced chemiluminescent system according to the manufacturer’s instructions. Densitometric values for immunoreactive bands were quantified using a GS-700 Imaging Densitometer (Bio-Rad). ß1C and ß1 protein levels were calculated as percentage of control (normal prostate tissue) upon normalization using ß-tubulin as control for protein loading.
HL60 Cells
Human leukemia HL60 cells were grown in RPMI 1640 (Gibco, Life Technologies, Milan, Italy), with 50 µg/ml gentamicin, 2 mmol/L glutamine, and 15% inactivated fetal calf serum, at 37°C in presence of 5% CO2. Total RNA from differentiated cells was prepared 24 hours after incubation with 160 nmol/L TPA (or PMA phorbol-12- myristate-13-acetate; Sigma) as previously described.29
Statistical Analysis
Data are reported as the mean ± SEM for the indicated experiments. Statistical analysis was performed using the Student’s t-test. All experiments were repeated at least three times.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study was performed using 38 prostate specimens obtained from
patients with either prostate or bladder cancer. The patients were
divided into two groups (Table 1)
. The first group included five
patients (N1 to N5; age range, 60 to 72 years) with normal prostate who
underwent radical cystoprostatectomy for bladder carcinoma noninvolving
the prostate. The second group included 33 patients (K1 to K33; age
range, 57 to 77 years) with histologically proven prostatic
adenocarcinoma, who underwent radical prostatectomy. In the first
group, normal prostatic tissue was histologically confirmed. In the
second group, the grade of tumor differentiation and the stage of tumor
were estimated in each tumor sample according to Gleason’s criteria
and to TNM system, respectively. Based on Gleason’s
criteria23
the prostate specimens were divided in five
moderately differentiated (Gleason’s score <7) and 28 poorly
differentiated (Gleason’s score 
7) prostate tumors; based on TNM
system,24
the specimens were divided in 11 T2N0M0 (stage
II), 14 T3N0 M0 (stage III), and six T3N1M0, one T4N0M0, and one T4N1M0
(stage IV) prostate tumors. A neo-adjuvant hormonal therapy consisting
of the association of luteinizing hormone releasing hormone analogue
(goserelin depot, 3.6 mg/q 28 days) and a nonsteroidal anti-androgen
(bicalutamide, 50 mg/day) was administered to nine patients affected by
prostate carcinoma (Table 1)
before surgery to reduce prostate and
tumor volume and to obtain downstaging of the tumor, as reported in
preliminary clinical trials.30,31
Expression of ß1C and ß1 was examined at the RNA level in 38
prostate tissues using either a ß1C-specific probe (Figure 2)
or a
ß1 full-length probe that hybridizes with all of the ß1
variants.11
Steady-state levels of ß1C (Figure 1
and not shown) and ß1 (Figure 3
and
not shown) mRNA were evaluated by Northern blotting analysis of total
RNA isolated from 33 neoplastic and five normal tissues (Table 1)
.
Because of the low amount of RNA obtained from the tissue samples,
total RNA rather than poly(A+) RNA was analyzed. A 4.2-kb transcript
was detected in all samples (Figure 1)
. This band corresponds to the
ß1C mRNA because the probe used is specific for exon C, which is
found only in ß1C (Figure 2)
. Total RNA
extracted from TPA-differentiated HL60 cells and from human liver, was
used as positive and negative controls for ß1C mRNA expression,
respectively (Figure 1
, lanes 1 and 2), because ß1C is expressed in
TPA-differentiated HL60 cells and is barely detectable in normal human
liver.12,15
As expected, low levels of ß1C mRNA were
found in normal liver compared with HL60 cells (Figure 1
, lanes 1 and
2). Northern blotting analysis showed a significant decrease (49
± 4% decrease) of ß1C mRNA levels in neoplastic tissues (Figure 1
,
lanes 8 to 20) compared with normal prostate tissues (Figure 1
, lanes 3
to 7). Decreased ß1C steady-state mRNA levels were detected in 94%
of the prostatic carcinoma specimens compared with normal prostate
samples and the differences were statistically significant
(P < 0.005;
Figure 4
). In one instance (K16), ß1C
mRNA expression was found increased (121 ± 8%) compared with the
levels in normal prostate tissues, whereas in a different specimen
(K10), ß1C mRNA levels were comparable (100 ± 4%) to normal
prostate levels (Figure 4)
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and not shown). The results show that ß1C as well as ß1 mRNA levels are reduced in prostatic adenocarcinoma compared with normal prostate tissues.
ß1C mRNA Expression and Clinical Progression
To investigate whether ß1C expression is associated with tumor
stage, correlation of ß1C mRNA levels with clinicopathological
parameters (Gleason’s grade and tumor stage) was evaluated. As shown
in Figure 5A
, ß1C mRNA expression was
comparable in patients with Gleason’s grade <7 (57 ± 7%,
n = 5) and Gleason’s grade 7 (50 ± 5%,
n = 28). In parallel, the specimens were analyzed using
the TNM system for tumor stage classification (Figure 5B)
. The
differences in mRNA levels in specimens at different stages were not
statistically significant (P > 0.05); in fact,
as shown in Figure 5B
, ß1C mRNA expression was comparable, although
lower than normal prostate controls, in stage II, III, and IV tumors
(48 ± 6%, 52 ± 8%, and 56 ± 7%, respectively).
Moreover, we investigated whether a correlation between ß1C mRNA
expression and hormonal therapy occurred. As shown in Figure 6
, the differences between patients with
3- or 6-month hormonal treatment and patients who did not undergo
hormonal therapy were not significant (P > 0.05
in both cases) although they were reduced versus normal
prostate controls (64 ± 10%, 38 ± 7%, 52 ± 5%,
respectively). In the only available case where 1-month hormonal
therapy had been administered, there was a statistically significant
increase (156 ± 4%) with respect to the patients that had not
received any therapy; however, the results related to short-term (1
month) therapy need to be further investigated using a larger number of
cases, when available.
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ß1C Protein Expression in Neoplastic and Normal Prostate Tissues
Among the specimens showing down-regulation of ß1C mRNA, 13 were
selected for immunoblotting analysis of ß1C and ß1 integrins.
Figure 7, A and B
, shows the results of
our immunoblotting analysis. ß1C and ß1 were both expressed in
normal (Figure 7A
, lanes 1 and 2) and in tumor prostate tissue (Figure 7A
, lanes 3 to 15) as described previously.16,20
The
results in this set of specimens show a dramatic down-regulation of
ß1C in lysates from neoplastic tissues compared with normal tissues:
ß1C protein levels ranged from 8 to 28% (with an average value of
19 ± 4%) of the levels found in normal tissues (Figure 7B)
. In
contrast, ß1 protein levels were comparable in normal and prostatic
adenocarcinoma tissues (Figure 7, A and B)
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Alterations of integrin expression in prostate cancer have been previously described by several groups.17-22 In normal prostate, it has been shown that ß1 and ß4 are found in epithelial cells with redistribution to the whole surface (ß1) or loss of expression (ß4) associated with the malignant phenotype.11 In this study, a selective down-regulation of ß1C at the protein level is shown. ß1C is one of the four known ß1 variants; among these, the ß1B and ß1D variants are unlikely to be found in prostate cancer tissue, because ß1B is restricted to skin and liver tissues, whereas ß1D is found only in striated muscle cells.11 The two remaining ß1 integrin subunits, ß1C and ß1A, are pathophysiologically important for cancer growth because they differentially affect cell proliferation; ß1C inhibits proliferation, whereas ß1A promotes it. Thus, it is conceivable that a strong pressure in prostate cancer to maintain selectively reduced mRNA and protein levels of ß1C would occur. In contrast, although reduced at the mRNA level, ß1 protein levels, that are likely to reflect ß1A levels, are maintained constant in normal and neoplastic prostate tissues by compensatory mechanisms that remain to be identified.
The results reflect a specific down-regulation of ß1C in prostate
epithelial cells because this molecule is expressed in a
cell-type-specific manner and is not found in the
stroma.20
This sophisticated regulation of the ß1C
variant in prostate cancer suggests a tight control at the
transcriptional and/or translational level of its expression to prevent
inhibition of prostate epithelial cell proliferation. It is conceivable
that either a transcriptional or a posttranscriptional regulation of
ß1C expression might be responsible for the decreased mRNA levels,
whereas at the protein level, translation activities or protein
degradation that are expected to be increased in cancer cells, might be
accounted responsible for the decreased ß1C protein levels. In
vitro, regulation of integrin mRNA and protein expression has been
shown to be modulated by the protein kinase C activator PMA. PMA
was shown to stimulate adhesion of tumor cells to fibronectin and
fibrinogen by modulating IIbß3 expression in human prostatic
adenocarcinoma cells.21
Moreover, PMA determined changes
of V mRNA expression in leukemia cells34
and caused
increase of 2 integrin cell surface expression in tumor progression
by enhancing 2 integrin transcription.35
It is
conceivable that protein kinase C might act also as a modulator of
ß1C integrin expression in vitro and in vivo,
and this remains to be investigated. Androgen-deprivation therapy did
not seem to interfere with ß1C integrin expression, thus indicating
that androgen-mediated mechanisms act through pathways that do not
involve ß1C. Since, in addition to androgens, mitogens regulate cell
proliferation and integrin expression, they may be important
autocrine-paracrine modulators of the neoplastic phenotype and of ß1C
expression in vivo.14,36-40
Because ß1C is a spliced variant of the ß1 integrin subfamily, down-regulation of its mRNA could have been a reflection of an altered splicing mechanism occurring in prostate cancer. Our data show that altered splicing mechanisms are unlikely to explain the reduced ß1C mRNA levels in prostate cancer because all ß1 integrin mRNAs were found down-regulated. In this regard, Tamura et al41 and Belkin et al42 have demonstrated that splicing mechanisms control specific integrin expression at different stages of differentiation; our results show that ß1C mRNA and protein levels are down-regulated in differentiated (low to intermediate Gleason’s grade) as well as poorly differentiated (high Gleason’s grade) prostate cancer and are not regulated in a differentiation-dependent manner.
Consistent with our recent experimental evidence ruling out a role for
ß1C cytoplasmic domain in ß heterodimer assembly or in
determining ligand specificity but affecting selectively intracellular
signaling,43
our findings suggest that the regulated
expression of different integrin-variant cytoplasmic domains might
provide a highly specialized mechanism to control cell proliferation
and intracellular signaling pathways in normal and pathological
conditions. Recent studies have demonstrated that the ß1C variant,
using a unique signaling mechanism, selectively inhibits the
mitogen-activated protein kinase pathway by preventing Ras activation
without affecting either survival signals stimulated by integrins or
cellular interactions with the extracellular matrix.43
Specifically, in human prostate epithelial cells, ß1C is co-expressed
with the cell cycle inhibitor p27kip1, the loss
of which correlates with poor prognosis in prostate
cancer;16
furthermore, exogenous expression of ß1C
in vitro inhibits prostate cell proliferation, and is
accompanied by an increase in
p27kip1.16
These findings suggest a
role for ß1 specific cytodomain sequences in maintaining an
intracellular balance of proliferation and survival signals and point
to ß1C as an upstream regulator of p27kip1
expression and, therefore, as a potential target for tumor suppression
in prostate cancer.
Efforts to confirm the prognostic value of ß1C are in progress. Future studies will indicate whether loss of ß1C expression in association with other traditional or novel markers has greater prognostic potential than each factor alone.
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Acknowledgements |
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Footnotes |
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Supported by Consiglio Nazionele delle Ricerche-PB Italy/USA (to E. P.), by Institutional Funds from the University of Bari, (to P. D.), by National Institutes of Health Grants CA-71870 and DK-52670 (to L.R. L.), and by Army Prostate Cancer Research Program grant DAMD 17-98-1-8506 (to L. R. L. and M. F.) and by a fellowship from CNR (#203.04.171) (Tol.M.).
Accepted for publication July 27, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3ß1 and 6ß4 integrins in highly invasive and metastatic variants of human prostate carcinoma cells selected by in vitro invasion through reconstituted basement membrane. Clin Exp Metastasis 1993, 11:391-400[Medline]
6 expression in human prostate carcinoma cells is associated with a migratory and invasive phenotype in vitro and in vivo. Clin Exp Metastasis 1995, 13:481-491[Medline]
IIß3 integrin. Cancer Res 1996, 56:5071-5078 subunit and comparative expression of adhesion receptor mRNAs. J Biol Chem 1987, 262:14080-14085 and interferon 
modulate the expression of the vitronectin receptor (integrin ß3) in human endothelial cells. J Biol Chem 1991, 266:7638-7645This article has been cited by other articles:
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  H. L. Goel, J. Li, S. Kogan, and L. R Languino Integrins in prostate cancer progression Endocr. Relat. Cancer, September 1, 2008; 15(3): 657 - 664. [Abstract] [Full Text] [PDF]
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 A. Gattelli, M. N. Zimberlin, R. P. Meiss, L. H. Castilla, and E. C. Kordon Selection of Early-Occurring Mutations Dictates Hormone-Independent Progression in Mouse Mammary Tumor Lines J. Virol., November 15, 2006; 80(22): 11409 - 11415. [Abstract] [Full Text] [PDF]
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 I. Lee, M. A. Skinner, H.-b. Guo, A. Sujan, and M. Pierce Expression of the Vacuolar H+-ATPase 16-kDa Subunit Results in the Triton X-100-insoluble Aggregation of {beta}1 Integrin and Reduction of Its Cell Surface Expression J. Biol. Chem., December 17, 2004; 279(51): 53007 - 53014. [Abstract] [Full Text] [PDF]
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L. Moro, E. Perlino, E. Marra, L. R. Languino, and M. Greco Regulation of {beta}1C and {beta}1A Integrin Expression in Prostate Carcinoma Cells J. Biol. Chem., January 16, 2004; 279(3): 1692 - 1702. [Abstract] [Full Text] [PDF]
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 M. Lovecchio, E. Maiorano, R. A. Vacca, G. Loverro, M. Fanelli, L. Resta, S. Stefanelli, L. Selvaggi, E. Marra, and E. Perlino {beta}1C Integrin Expression in Human Endometrial Proliferative Diseases Am. J. Pathol., December 1, 2003; 163(6): 2543 - 2553. [Abstract] [Full Text]
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 R. A. Vacca, E. Marra, G. Loverro, E. Maiorano, A. Napoli, M. Lovecchio, L. Selvaggi, and E. Perlino Differential Expression of {beta}1c Integrin Messenger Ribonucleic Acid and Protein Levels in Human Endometrium and Decidua during the Menstrual Cycle and Pregnancy J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 720 - 729. [Abstract] [Full Text] [PDF]
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