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Meeting Report |
From the Department of Pathology and the University of Iowa Cancer
Center,*
The University of Iowa, and Veterans Affairs
Medical Center, Iowa City, Iowa; the Center for Scientific
Review,
National Institutes of Health,
Bethesda, Maryland; and the Department of Anatomy and Cell Biology, and
the University of Iowa Cancer Center,
The
University of Iowa, Iowa City, Iowa
The Pathology B study section sponsored a one-day
workshop focusing on new experimental models of prostate cancer
research and preceded the International Conference on Prostate Cancer
Research in Iowa City, Iowa. The workshop began with a discussion of
the integrin-mediated modulation of prostate cancer proliferation and
motility by Lucia R. Languino (Yale University, New Haven, CT).
Interactions between (cancer) cells and the extracellular matrix are
largely mediated by integrins, which have also emerged as key
regulators of cell proliferation, migration, and intracellular
signaling. ß1C and ß3 integrins may act as
"growth or motility modulators" in prostate cells and play a role
in modulating downstream intracellular signaling events. The
ß1C integrin is expressed in nonproliferative,
differentiated benign prostatic epithelium and is down-regulated in
prostatic adenocarcinoma, as well as in hyperplastic prostate glands.
The ß1C integrin is an alternatively spliced variant of
the ß1A subunit that, in contrast to ß1A,
inhibits fibroblast and epithelial cell proliferation. To investigate
whether ß1C plays a role in prostate epithelial cell
proliferation, the prostatic carcinoma cell line PC3 was transfected
with an inducible system with either ß1C or
ß1A cytoplasmic tails and normal epithelial cell
transfectants expressing ß1C. In contrast to
ß1A, expression of ß1C or its cytoplasmic
domain completely inhibited thymidine incorporation by serum
stimulation. Further results point to ß1C as an upstream
regulator of the cell cycle inhibitor p27kip1 expression.
Immunohistochemical and immunoblotting analysis of human prostate
epithelial cells reveal that ß1C is co-expressed with
p27kip1, the loss of which correlates with poor prognosis
in prostate cancer. In vitro, increased levels of
p27kip1 and inhibition of cyclin A-dependent kinase
activity were observed in normal prostate epithelial cells upon
expression of ß1C. These data show that
p27kip1 is a key downstream effector of ß1C,
in that ß1C inhibitory activity on cell proliferation is
completely prevented by p27kip1 antisense (but not
mismatch) oligonucleotides. In parallel studies, a role for the
vß3 integrin in the regulation of prostate cancer cell functions
has been identified. Specifically, vß3 is expressed in primary
cultures of prostate cancer epithelial cells, whereas it is
undetectable in normal prostate epithelial cells; and vß3 mediates
prostate cancer epithelial cell migration on ß3 integrin
substrates, such as vitronectin, an vß3 ligand expressed in mature
bone where prostate cancer cells preferentially metastasize. Exogenous
expression of vß3 induces LNCaP cells to adhere to and migrate on
vitronectin. In response to vß3 engagement, increased tyrosine
phosphorylation of focal adhesion kinase (FAK), a signaling molecule
activated by integrins and able to modulate cell migration, is
detected. Transfection of FAK-related non-kinase (FRNK), known to
compete with FAK for its correct localization and phosphorylation,
causes inhibition of ß3-LNCaP cell migration,
specifically on vitronectin. The study of the pathophysiological
relevance of ß1C and ß3 integrin downstream
effectors is likely to yield new insights into the mechanisms that
contribute to prostate cancer progression and metastatic spread.
Jack Schalken (University of Nijmegen, Nijmegen, The Netherlands) then spoke about E-cadherin as a prognostic parameter. Prostate cancer, like many solid tumors, is characterized by an unpredictable biological behavior. Some tumors remain indolent for many years, whereas others progress rapidly to a life-threatening disease. Clearly, the acquisition of a metastatic phenotype is a hallmark of clinical aggressiveness that often leads to an incurable disease. It is now well recognized that in the maintenance of epithelial integrity the calcium-dependent adhesion molecule E-cadherin plays a crucial role and that it can function as a suppressor of invasive ability. An ongoing prospective clinical trial (Biomed II MPC project) is underway to establish the clinical usefulness of E-cadherin immunohistochemistry as molecular marker for prostate cancer prognosis. The mechanism by which E-cadherin function is impaired is not yet fully resolved, although transcriptional down-regulation and concomitant up-regulation of other cadherins, particularly N-cadherin and caherin-11, appear to be common steps in the malignant progression of prostate cancer. It is important to note that this implies that the mechanisms might be reversible and that cadherins can be considered not only as molecular markers for prostate cancer prognosis but also as targets for therapy. Considering these observations, up-regulation of E-cadherin would result in prevention of progression of metastatic disease. This in fact provides a rational basis for differentiation therapy and the use of E-cadherin to evaluate and/or target new therapeutic modalities.
Gary J. Miller (University of Colorado, Denver, CO) presented some of his work on Vitamin D in prostate cancer. Although it is usually thought of as an androgen-dependent disease, it has recently become clear that numerous other hormones including 1,25-dihydroxyvitamin D3 (1,25D3) can regulate its growth and differentiation. In addition to its role in calcium homeostasis, 1,25D3 is also known to play pleiotropic roles in modulating the differentiation of various benign and malignant cells. Epidemiological data exist indicating that mortality from prostate cancer is inversely related to latitude and ultraviolet exposure. Prostate cancer incidence has also been tied to prediagnostic serum levels and vitamin D receptor polymorphisms. Specific receptors for 1,25D3 exist in all of the prostatic carcinoma cell lines examined to date. Furthermore, these receptors regulate both the antiproliferative and differentiating effects of 1,25D3 in various prostatic cancer cell lines. For example, 1,25D3 increases the production of prostate-specific antigen (PSA) and prostate-specific acid phosphatase in LNCaP while inhibiting their growth. Numerous nonhypercalcemic analogues of 1,25D3 have been synthesized that retain the differentiating effects (increasing the expression of prostate-specific antigen and prostate-specific acid phosphatase) of the parent compound. Those with hexafluorinated side chains have been found to enhance activity in prostatic carcinoma cells. Recent data also indicate that aging individuals, especially those with advanced stage prostate cancer, may have profound deficiencies of 1,25D3 even in the presence of dietary supplementation. A hypothesis was presented which suggested that the lack of response to chemotherapeutic agents may be related to vitamin D deficiency. Isobologram analyses of in vitro studies have revealed that 1,25D3 has a synergistic effect on the antiproliferative effects of cis- and carboplatin. A clinical trial is underway to determine whether such combined effects will also occur in patients. Finally, questions remain regarding the molecular signal transduction pathways that operate in prostatic cancer cells exposed to vitamin D. Of the cell cycle-related regulators, p21 is known to have a vitamin D response element. Ongoing studies indicate that the expression of p21 is up-regulated in prostatic cancer cells exposed to 1,25D3 and that the degree of this change is correlated with the observed antiproliferative effects. Regarding differentiation, Miller has also examined the HOX transcription factors and found that selected members are inappropriately expressed in prostatic carcinoma cells. In addition, preliminary results indicate that the expression of individual HOX genes, such HOXA10, is regulated by 1,25D3 in prostatic carcinoma cells. In summary, a substantial body of evidence exists indicating that 1,25D3 is important in modulating the malignant phenotype of prostatic carcinoma cells. In view of this, we should no longer think of prostatic carcinoma solely in terms of its response to androgens.
The role of early growth response genes in prostate cancer was
discussed by Donald J. Tindall, (Mayo Foundation, Rochester, MN). Using
a differential display reverse transcriptase-polymerase chain reaction
technique, a number of androgen-regulated genes that are differentially
expressed in prostatic carcinoma cell lines were isolated. A novel
gene, EGR- (closely related to TIEG), is
expressed to a higher extent in the androgen-independent cell lines
DU-145 and PC3 but not in the androgen-sensitive cell line LNCaP. These
observations led to a more detailed study of EGR-1 expression in
prostate cancer. In vitro, EGR-1 expression increases
concomitantly with an increase in the index of malignancy. These
results were further corroborated with immunocytochemistry, which
indicated that EGR-1 is expressed primarily in the basal epithelial
cells and detected primarily in the cytoplasm of normal and benign
epithelial cells, whereas it is predominantly nuclear in malignant
cells. To further corroborate the subcellular localization of EGR-1 in
cancer versus noncancerous prostate cells,
immunocytochemistry on two human prostate cell lines from benign and
cancerous origin was performed. EGR-1 protein is primarily localized in
the cytoplasm of BPH-1 cells and in the nuclei (mostly in the
nucleolus) of PC-3 cells. Interestingly, in all dividing cells EGR-1 is
preferentially associated with the mitotic spindle. Further studies
have focused on the effect of calcium on EGR-1 expression. LNCaP was
maintained in DCC-FBS medium for 24 hours and then treated with
10-7 M thapsigargin and EGR-1 levels reached a maximum of
fourfold induction between 40 and 60 minutes of treatment. This
expression is blocked by staurosporine, suggesting a role of PKC in
EGR-1 regulation in prostate cancer cells. In addition, the calmodulin
inhibitor, trifluoroperazin, also suppressed the thapsigargin-inductive
effect. These data suggest that a calmodulin-mediated pathway may be
involved in the regulation of EGR-1 expression in prostate cancer
cells. Moreover, since thapsigargin induces apoptosis in LNCaP, EGR-1
may also be involved in this process in prostate cancer cells. These
findings have led to a working hypothesis that EGR-1, in an active
nuclear form, is involved in the initiation and progression of prostate
cancer.
In the afternoon there were several presentations about apoptosis in
prostate cancer. Michael B. Cohen (The University of Iowa, Iowa City,
IA) began with a review of his work on TNF receptor family-mediated
apoptosis in prostate cancer. Of 6 human prostatic carcinoma cell lines
examined by flow cytometric analysis, all were found to be positive for
Fas (CD95) antigen. Furthermore, all of the prostate tissue specimens
studied revealed Fas expression in benign and malignant epithelial
cells. Agonistic anti-Fas monoclonal antibody induced apoptosis in only
2 of 6 cell lines investigated. Subsequent effort has focused on
identifying the mechanism of resistance to Fas-mediated apoptosis in
prostate cancer, because treatment with the protein synthesis inhibitor
cycloheximide (CHX) converted the phenotype of resistant cell lines
from Fas resistant to Fas sensitive. Subsequently, Fas-mediated
apoptosis in cell hybrids between resistant and sensitive cell lines
was investigated. All three types of F1 hybrid cells investigated were
found to be resistant to Fas-mediated apoptosis at the same level as
the corresponding parental resistant cell lines. Furthermore, treatment
with CHX converted the phenotype of the hybrids from resistant to
sensitive. These results indicate that resistance to Fas-mediated
apoptosis dominates over sensitivity in cell hybrids and suggest that
an apoptosis suppressor factor or factors acting in resistant but not
in sensitive cells may regulate resistance. Finally, they have also
investigated the sequential activation of caspase family members, to
gain insight into the likely site of action of the suppressor
protein(s). In prostate cancer cell lines, caspase-8 activation is
followed by caspase-7; caspase-3 does not appear to be involved. These
results suggest that an inhibitory protein or proteins, which suppress
apoptosis in Fas-resistant cell lines, presumably act at the apex of
apoptotic cascade by preventing the activation of caspase-8. In
addition, Fas ligation results in the release of cytochrome C and
activation of caspase-9, and the timing suggests that this is an early
event. Current studies are targeted at identifying the inhibitory
protein(s) responsible for resistance to Fas-mediated apoptosis.
Additional work has focused on the role of p53 in TNF--mediated
apoptosis. LNCaP is a human prostatic carcinoma cell line that
expresses wild-type p53 and is sensitive to TNF- treatment. To
analyze the role of p53 in TNF--mediated apoptosis, a LNCaP subline
has been created, termed LN-56, that expresses GSE-56, a
dominant-negative element of p53. p53 inactivation in LN-56 was
associated with an increased resistance to apoptosis induced by TNF-
treatment. Caspase-7 activation and PARP proteolysis were delayed in
LN-56, TNF- treatment increased p53 in LNCaP, but not in LN-56, and
resulted in up-regulation of p21/WAF1, which was accompanied by
p21/WAF1 proteolysis in LNCaP, but not in LN-56; this proteolysis was
inhibited by a pan-caspase inhibitor (Z-VAD-FMK). Interestingly,
accumulation of p53 was decreased in the presence of Z-VAD-FMK,
indicating a new role of activated caspases in acceleration of p53
response. Mdm2 as not found to be a target for caspase-mediated
degradation. In summary, these results suggest that p53 plays an
important role in TNF--mediated apoptosis.
Next, Martin Tenniswood (University of Notre Dame, South Bend, IN)
spoke on the implications of the changes in biogenesis of clusterin
during anti-androgen induced apoptosis, and put forth a very intriguing
hypothesis. Glandular tissues, such as the prostate, regress when
deprived of their trophic factors. Previous studies have shown that a
number of genes involved in the destruction of the extracellular matrix
and basement membrane are required for the apoptotic death of the
epithelial cells that occurs during tissue regression. Metastatic cells
express many of the same proteins. In cells that initiate the apoptotic
pathway, extracellular matrix proteases are expressed and the DNA is
fragmented. Metastatic cells, on the other hand, express elevated
levels of the proteases but do not fragment their DNA. This has led to
the hypothesis that the invasive phenotype might arise after the
initiation of apoptosis by anticancer agents if the DNA in the affected
cells was not fragmented appropriately. To test this hypothesis he has
established the androgen-dependent LNCaP prostate cancer cell line in
defined serum-free medium. Treatment of these cells with Casodex
(bicalutimide), a pure nonsteroidal anti-androgen, or TNF-, induced
apoptosis. In addition, cognate mRNA levels and activity of several
extracellular matrix proteases, including MMP-2 and MMP-9, are
increased during Casodex induced apoptosis, and there is a
corresponding decrease in the level of TIMP-I. Furthermore, Casodex
induces a dose-dependent, statistically significant increase in the
invasive potential of the surviving cells. He estimates that between
0.1 and 0.2% of the surviving cells acquire the ability to invade
following anti-androgen treatment; this is not seen when the cells are
treated with TNF-. Although this represents a very small percentage
of the surviving cells, these cells represent a clinically significant
population, because it is these cells that have the potential to give
rise to metastatic disease. Clonally selected invasive sublines,
referred to as LNCaPcasinv sublines, have been developed
and retain their invasive ability, suggesting that these cells may
serve as a model of late stage, androgen-resistant metastatic disease.
Vivek M. Rangnekar (University of Kentucky, Lexington, KY) discussed
the mechanism of prostate apoptosis response-4 (Par-4) gene-dependent
apoptosis. The Par-4 gene was identified in a differential screen for
genes induced during apoptosis of prostate cancer cells. Par-4 is
widely expressed in the nucleus and evolutionarily conserved in
vertebrates. Interestingly, Par-4 is exclusively induced in in
vitro and in vivo paradigms of apoptosis, but not
during necrosis, growth arrest, or growth stimulation. It is induced in
the secretory epithelium of the involuting prostate in castrated rats,
in myoblasts of the tadpole tail undergoing resorption during
metamorphosis, and in the web area between the digits during limb bud
development. It is also up-regulated in degenerating neurons of
Alzheimer’s disease patients. The deduced amino acid sequence of Par-4
predicts a protein with a leucine zipper sequence at its carboxy
terminus. Functionally, Par-4 is necessary but not sufficient for
apoptosis: inhibition of Par-4 with antisense oligomers or with a
dominant-negative mutant results in abrogation of insult-driven
apoptosis. Overexpression of Par-4 sensitizes cells to apoptosis.
Interestingly, Par-4 expression is decreased in some but not all
tumors. Moreover, oncogenes down-regulate Par-4, and replenishment of
Par-4 prevents oncogene-induced cellular transformation. The mechanism
of Par-4-mediated apoptosis is currently being studied with Bc1-2,
NF-
B, and ERK as potential downstream targets. For example, Par-4
and Bcl-2 expression are inversely correlated. Investigations are
underway on Par-4 function in neuronal degeneration,
ischemia/reperfusion-induced injury, and development, in addition to
cancer paradigms.
Finally, Timothy C. Thompson (Baylor College of Medicine, Houston, TX) presented some of his work on caveolin-1, a gene involved in prostate cancer metastasis. The high level of mortality from prostate cancer results in large part from the inexorable growth of overt or occult metastasis present at the time of diagnosis. To better understand the metastatic phenotype in prostate cancer, a strategy to identify mRNAs that are expressed differentially in cell lines derived from primary versus metastatic mouse prostate cancer (MPR mouse prostate reconstitution model) using differential display-polymerase chain reaction has been developed. In using this system a number of metastasis-related sequences were identified, including a cDNA that encodes caveolin-1. Caveolin-1 was found to be overexpressed not only in metastatic mouse prostate cancer, but also in human metastatic disease. Recent studies have indicated that suppression of caveolin-1 expression induces androgen sensitivity in high caveolin, androgen-sensitive mouse prostate cancer cells derived from metastases. Conversely, overexpression of caveolin-1 leads to androgen insensitivity in low caveolin-1, androgen-sensitive mouse prostate cancer cells. Caveolin-1, therefore, is both a metastasis-related gene as well as a candidate androgen resistance gene for prostate cancer in man. Interestingly, recent studies also point to a potential role for caveolin-1 in the resistance of various malignancies to multiple antineoplastic agents. The linkage of caveolin-1 expression with the androgen-resistant phenotype in prostate cancer and the multidrug resistance phenotype in various solid tumors establishes a novel paradigm for understanding these clinically important and now potentially related processes in malignant progression. Additional studies are ongoing to more broadly define the role of caveolin-1 as an apoptosis resistance gene in prostate cancer.
The studies presented at this workshop consisted of some of the
contemporary lines of investigation being pursued to address the
fundamental questions about prostate cancer biology. New information
was presented regarding the role of integrins in modulating cell
biology, studies focusing on Vitamin D and EGR as agents in
androgen-independent growth, several new lines of investigation in
apoptosis (including receptor ligand-mediated cell death, PAR-4), and
studies in the area of metastasis–a possible link between apoptosis
and metastasis, the role of caveolin-1, and the use of selected
prognostic markers (E-cadherin) in predicting metastases. The work
presented highlights exciting avenues for further investigation. In
addition, although many of the studies focused on basic molecular
cancer biology, it is clear that translational components are at
varying stages of development. This is indeed an exciting
prospect.
Footnotes
Address reprint requests to Michael B. Cohen, M.D., Department of Pathology, 1117ML, The University of Iowa, Iowa City, IA 52242-1087. E-mail: michael-cohen{at}uiowa.edu
The workshop was held June 24, 1999 in Iowa City, Iowa.
Accepted for publication October 20, 1999.
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