TARGETS IN THE DIAGNOSIS AND TREATMENT OF PROSTATE CANCER
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KAVITHA RAMACHANDRAN, MARK S. SOLOWAY, RAKESH SINGAL
of Urology (MM, MSS) and Sylvester Comprehensive and Cancer Center (MM,
KR, RS, MSS) Miller School of Medicine, University of Miami, Miami, Florida,
USA, and Miami VA Medical Center (RS), Miami, Florida, USA
cancer (PC) is one of leading cause of cancer related deaths in men. Various
aspects of cancer epigenetics are rapidly evolving and the role of 2 major
epigenetic changes including DNA methylation and histone modifications
in prostate cancer is being studied widely. The epigenetic changes are
early event in the cancer development and are reversible. Novel epigenetic
markers are being studied, which have the potential as sensitive diagnostic
and prognostic marker. Variety of drugs targeting epigenetic changes are
being studied, which can be effective individually or in combination with
other conventional drugs in PC treatment. In this review, we discuss epigenetic
changes associated with PC and their potential diagnostic and therapeutic
applications including future areas of research.
words: prostate cancer; DNA methylation; epigenesis, genetic
Int Braz J Urol. 2007; 33: 11-8
cancer (PC) is one of leading cause of cancer related deaths in men. In
the United States, an estimated 230,000 men were diagnosed with PC in
the year 2005 and approximately 30,000 are expected to die from this cancer
annually (1). Epigenetics refers to stable non inherited changes in the
gene expression without alterations in DNA structure (2).
With the advent of prostate specific antigen
(PSA) in 1998, there has been dramatic increase in the diagnosis of PC
(3). The American Cancer Society recommends annual screening of men above
the age of 50 for PC with PSA and rectal examination (4). However, it
is not clear whether the PSA is effective in the diagnosis of PC as it
lacks both specificity and sensitivity (5,6). About 25% men with normal
PSA may harbor PC (5) and PSA less than 20 ng/mL may not differentiate
between PC and benign conditions (6). This leads to unnecessary prostate
biopsies and on the other hand, we might miss PC in patients with low
PSA. Similarly, there is lack of effective prognostic markers to predict
the behavior of PC and outcome following definitive treatment. Novel biomarkers
based on epigenetic profiling are being explored to aid in the diagnosis
and management of PC (7-9).
Epigenetics is one of the rapidly expanding
fields in cancer related research. Recent studies have shown that epigenetics
plays an important role in cancer biology, somatic gene therapy, viral
infections, genomic imprinting. Epigenetic changes, particularly the DNA
methylation is found to be involved in a variety of cancers including
colon, lung, breast and ovarian cancers apart from prostate cancer (8).
Unlike passively transferred genetic mutations, the epigenetic changes
must be actively maintained and its “reversibility” makes
them a potential therapeutic target (10).
In this review, we discuss epigenetic changes
associated with prostate cancer and their potential diagnostic and therapeutic
applications including future areas of research.
Approximately 23,000 genes are contained
in the human genome. For proper functioning of the cells, these genes
should be expressed in specific cells at specific times (11). The chromatin
is a nucleoprotein complex made of nucleosomes. The nucleosomes are made
of DNA, which are wrapped around octamers of globular histone proteins
(12). The changes in the chromatin structure influence the gene expression.
When the chromatin is condensed, the gene expression is “switched
off” and when it is open, the gene expression is “switched
on” (13). The status of chromatin is dynamic and can be controlled
by reversible epigenetic mechanisms.
The two important, well studied epigenetic
mechanisms are DNA methylation and histone modifications such as acetylation.
These two processes can act independently and/ or together affecting the
gene expression and in turn the tumorigenesis.
Methylation in Prostate Cancer
DNA methylation refers to a covalent chemical
modification, resulting in the addition of a methyl (CH3) group at the
C-5 position of the Cytosine ring in the DNA. The human genome is not
uniformly methylated. “CpG islands” are small regions within
the genome that are rich in Cytosine and Guanine bases and are mostly
unmethylated (8). Epigenetic alterations target this region thereby affecting
gene expression. Both the hyper and hypomethylation can affect the gene
expression and the role of DNA methylation in oncogenesis has been studied
for several years.
DNA hypermethylation is a well established
epigenetic abnormality seen in several malignancies, more importantly
in prostate cancer (9). Carcinogenesis is a multi step process and hypermethylation
is hypothesized as an early event in the development and progression of
prostate cancer (14). Hypermethylation of the gene is facilitated by a
group of enzymes known as DNA methyltransferases (DNMT), which includes
DNMT1, DNMT1b, DNMT1o, DNMT1p, DNMT2, DNMT3a, DNMT3b and DNMT3L (15).
The hypermethylation involves the CpG islands in the promoter regions
that results in the silencing of the genes that are involved in tumor
suppressor activity, DNA repair and other critical cellular mechanisms.
Some of the important genes that are frequently
hypermethylated in prostate cancer are listed in Table-1. Glutathione
S-transferase P1 (GSTP1) is a protector gene and silencing this gene by
hypermethylation leads to DNA damage and cancer initiation (7,16). Methyl
Guanine DNA methyl transferase (MGMT) is another DNA repair gene which
are silenced by hypermethylation (17). Inactivation of putative tumor
suppressor genes by hypermethylation, such as Ras association domain family
1 gene (RASSF1A) (18,19), KAI 1 (20), Inhibin-alpha (21) and DAB21P (22).
Hypermethylation promotes carcinogenesis in prostate cancer by affecting
cell cycle control, hormonal response, cell adhesions and architecture
DNA hypomethylation is a second type of
methylation related epigenetic aberration seen in variety of malignancies
including prostate cancer (23). Hypomethylation is facilitated by enzyme
group demethylases which includes 5-methylcytosine glycosylase and MBD2b
(24). Methylation of normal genomes act as defensive mechanisms against
cancer, for example, the oncogenes can be transcriptionally silenced and
prevented from propogating by being methylated. The hypomethylation causes
breakdown of this defense mechanism and is implicated in the tumor genesis.
The hypomethylation can be “global”
or “localized”. Global hypomethylation refers to overall decrease
in methylation content in the genome. Bedford et al. reported that global
hypomethylation is significantly lower in patients with metastatic prostate
cancer compared to non metastatic prostate cancer (23). Localized or gene
specific hypomethylation refers to a decrease in cytosine methylation
relative to normal levels. This affects the specific regions within genome
such as promoter regions of oncogenes which are highly methylated (9).
Histones have emerged as important regulators
of chromatin, thereby controlling gene expression. In each nucleosome,
two super helical turns of DNA containing around 146 base pairs wrap an
octomer of histone core made of four histone partners (an H3-H4 tetramer
and two H2A-H2B diamers (25). Histones consist of a globular domain and
a more flexible and charged NH2 terminal called as histone “tail”.
These tails which are placed peripherally are susceptible for a variety
of covalent modifications, such as acetylation, methylation, phosphorylation
and ubiquitination. These modifications are referred as “the histone
code” and is effective epigenetic mechanism regulating gene expression
Histone acetylation and deacetylations are
mediated by histone acetyl transferases (HAT) and histone deacetylases
(HDAC) respectively. Huang et al. and Tsubaki et al. reported that treatment
of prostate cancer cells with HDAC inhibitors results in increased expression
of specific genes such as CPA3 (27) and Insulin like growth factor binding
protein 3 (28). Coxsackie and adenovirus receptor (CAR) gene and Vitamin
D receptor gene have been shown to be affected by histone acetylation
in prostate cancer. Decreased CAR expression is associated with an increased
Gleason score (29).
Histone methylation affects the chromatin
function depending on the specific amino acid being modified and the extent
of methylation (30). Methylation of H3 at lysine 4 is associated with
inactive transcription of the PSA gene in prostate cancer cell line LNCaP
and decreased di and trimethylated H3 at lysine 4 is associated with AR
mediated transcription of the PSA gene (9). No histone demethylases have
been described so far and it is postulated that histone methylation may
be relatively stable and even irreversible (30).
Methylation - Histone Code Interplay
DNA promoter methylation and histone deacetylation
can act synergistically resulting in inactive chromatin state resulting
in suppression of gene expression (Figure-1). Methylated DNA binding proteins
such as MeCP2 may play an important role. Retinoic acid receptor beta
gene (RARB) which is silenced in prostate cancer tissues and cell lines
is regulated by both methylation and histone acetylation. This indicates
that combined treatment targeting methylation and histone acetylation
may result in reversal of epigenetic silencing of tumor suppressor genes
(9,31). Similarly, DNA methylation and histone methylation may interact
to facilitate chromatin silencing. However, it is unclear which event
takes place first (9).
Epigenetic Diagnostic Markers
Prostate specific antigen (PSA) is a less
than optimal tumor marker and cannot effectively differentiate between
prostate cancer and other conditions such as prostatitis, benign prostatic
hyperplasia. The false positive results lead to expensive and invasive
investigations such as transrectal prostate biopsy. This provides the
opportunity to the researchers to identify potential epigenetic markers
in the diagnosis of prostate cancer.
Epigenetic markers, particularly aberrant
DNA methylation, have the potential as an useful diagnostic tumor marker.
These markers can be detected in cancer tissues, serum and body fluids.
The methylation markers have several advantages over the mutation based
genetic markers. The detection of these markers is technically simple
and can be sensitively detected both quantitatively and qualitatively
by polymerase chain reaction (PCR). Furthermore, the incidences of aberrant
DNA methylation are higher than those of mutations are and can be discovered
by genome wide screening procedures (32).
In the recent years, the role of GSTP1 is
being studied as a tumor marker widely. Gossel et al. reported that GSTP1
hypermethylation is seen in the serum of 72% patients with of Prostate
cancer patients (33). They also examined the urine after prostatic massage
and methylation was detected in 68% patients with early prostate cancer
and 78% of patients with locally advanced cancer. Table-2 shows methylation
of GSTP1 in different tissue and body fluids. Harden et al. reported 73%
GSTP1 methylation in prostate cancer tissue samples. They also reported
that methylation assay with histological analysis improves the diagnostic
specificity (34). Methylation of several other genes have been studied
in the diagnosis of prostate cancer including, RARB, CD44, E- cadherin
(ECAD), RASSF1A, APC and tazarotene induced gene 1 (T1G1) (7,35). Recent
studies reported by Yegnasubramanian et al. (36) and others have reported
that use of a panel of methylation markers including GSTP1 improves the
diagnosis of prostate cancer both in body fluids and tissues. Further
studies are needed before these markers can be used as diagnostic markers
in the routine clinical practice.
Kollerman et al. demonstrated that GSTP1
hypermethylation is seen in 40% of pre operative bone marrow aspirate
in patients with advanced PC (37). They also found evidence of GSTP1 hypermethylation
in 90% of PC patients with lymph node involvement where as in only 11%
of lymph nodes in non cancer group. Genes such as CAV1, CDH1, CD 44 and
T1G1 may exhibit specific methylation in high risk and Metastatic tumors
that can be used in the molecular staging and predictors of disease progression
(14). Prostate cancers with high Gleason score are correlated with a higher
degree of methylation of many genes, such as RARβ, RASSF1A, GSTP1
and CDH13 (8). Further studies also indicate that use of panel of multiple
methylation makers can be better predictors than individual genes (38).
changes are heritable and potentially reversible. Hence, it is reasonable
to expect that these can be used as potential therapeutic targets. Currently
there are several drugs which are at different stages of development.
They can be broadly classified in two groups: (i) DNMT inhibitors and
(ii) Histone Deacetylase (HDAC) inhibitor. Some of the drugs in both groups,
which are being tested and used currently, are shown in Table-3.
5-aza-2’ - Deoxycytidine (5-aza-dC)
is one of the early drugs identified as DNMT inhibitor after being as
cytotoxic drug around 1990. This drug forms irreversible covalent bonds
with DNMT1 after its incorporation in to DNA, thereby inducing degradation
of DNMT1 (39) Issa et al. (40) demonstrated that low dose continuous administration
is more effective than higher doses. Myelosuppression is a known side
effect of this drug which is otherwise well tolerated. 5-aza-dC has been
recently approved by FDA for clinical use in certain hematological conditions.
Another drug in the same group, Zebularine can be administered orally
or intraperitoneally. It has to be given in high doses, however, it is
chemically stable and has low toxicity (41). Other drugs in this which
are being studied include Epigallocatechin-3 - Gallate (EGCG), Procainamide,
Procaine and MG 98 (32).
Deacetylases (HDAC) Inhibitors
A variety of natural products exhibit HDAC
inhibitory activity. Commonly used HDAC inhibitors which are being tested
include trichostatin A (TSA), Suberoylanilide hydroxamic acid (SAHA) and
valproic acid (9). Many of these drugs have exhibited antitumor activity.
SAHA and sodium butyrate have shown prostate cancer inhibition in animal
models (42,43). Overall, low toxicity rates of these drugs are encouraging
in conducting further studies.
The combination of HDAC and DNMT inhibitors
has synergistic effect in the reactivation of silenced gene (9). Another
interesting possibility is the combination of epigenetic drugs and conventional
anti androgens and chemotherapeutic agents. It should be cautioned that
the epigenetic drugs currently lack gene specificity and some of them
are associated with significant toxicity. Hence, efforts are being made
to develop gene specific epigenetic drugs (32).
changes in prostate cancer are being studied extensively at present and
genome wide screening will lead to development of novel epigenetic markers.
Epigenetic changes are early event in cancer development and hence can
be used to assess the risk of developing cancer. Li et al. suggest that
genes such as CAV1, CDH1, CD44 and T1G1 should be explored further as
“risk markers”, particularly to differentiate the indolent
tumors from others with bad prognostic potential (14). Epigenetic molecular
classification will help to identify patients at high risk of recurrence
following definitive treatments such as radical prostatectomy. Therapeutic
drugs which reverse these epigenetic changes have the potential to be
an effective adjunct treatment for prostate cancer. However, they need
to be studied both for its efficacy and safety profile. Gene specific
epigenetic drugs need to be developed for better targeting of the disease.
As the epigenetic changes are early event in the PC development, these
drugs have a potential to play a role in disease prevention. Two main
features of epigenetic changes, “reversibility” and being
an “early event” in tumorigenesis, makes epigenetic targeting
as an important future research area for cancer diagnosis, risk stratification,
treatment and prevention resulting in effective cancer control.
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September 9, 2006
Dr. Murugesan Manoharan
Associate Professor of Urology
University of Miami School of Medicine
P.O. Box 016960 (M814)
Miami, Florida, 33101, USA
Fax: + 1 305 243-4653