Figure 38-1 Examples of Gleason grade 3, 4, and 5 prostate cancer. Gleason grade 3 shows well-formed, separate glands (A). Gleason grade 4 shows merging or cribriform glands (B). Gleason grade 5 is the most poorly differentiated, and cancer cells no longer form glands but are visible as sheets of cells (C).
Molecular Pathology
Hereditary Prostate Cancer
Family history is one of the strongest risk factors for the development of prostate cancer, with a two- to eightfold higher risk of prostate cancer in men with an affected first-degree relative. 6 Prostate cancer associated with familial clustering and high incidence of cancer among multiple first-degree relatives with a diagnosis before age 60 is considered hereditary and/or familial prostate cancer (HPC/FPC). Approximately 9% of all cases are attributable to hereditary prostate cancer following an autosomal dominant susceptibility pattern. Prostate cancer susceptibility genes have been identified using lineage analysis of affected families. Significant linkage between chromosome 1q24-25, the HPC1 locus, and hereditary prostate cancer has been established. RNASEL, which lies within the HPC1 locus, encodes an endoribonuclease that mediates the activities of an interferon-inducible RNA degradation pathway. Polymorphisms of the RNASEL gene have been associated with increased prostate cancer risk. However, not all studies have confirmed these findings. Mutations in the RNASEL gene do not occur at a greater frequency in patients with familial prostate cancer compared with patients with sporadic prostate cancer. Recent genome-wide association studies (GWAS) consistently identified that several single-nucleotide polymorphisms (SNPs) in the 8q24 locus are associated with risk of HPC/FPC22. Genetic variants caused by polymorphisms or mutations in other genes, such as PALB2, BRCA2, the androgen receptor (AR), 5-α-reductase type II (SRD5A2), and CYP17, have also been implicated in the development of HPC/FPC.
Table 38-1
Oncogenes and Tumor Suppressors Implicated in Prostate Cancer
Gene Mutation
Early candidate gene approaches have implicated many different genes in prostate cancer. Germline mutations involving ELAC2 (HPC2), MSR1, and RNASEL genes have been reported in familial prostate cancer. The most common somatic mutations found in sporadic prostate cancer include TP53, PTEN, and AR. Recent whole-genome exon sequencing analyses identified significant mutated genes, including TP53, AR, ZFHX3, RB1, PTEN, APC, MLL2, OR5L1, and CDK12. Of these genes, MLL2, OR5L1, and CDK12 have unknown tumor suppressor functions in prostate cancer. 7 The most commonly affected signaling pathways by genetic alterations are the WNT signaling (TP53, APC, CTNNB1, MYC, and SMAD4) and the PTEN interaction network (PTEN, MAGI3, and HDAC11). 7
DNA Copy Number Variation
DNA copy number variation (CNV) is DNA structure alteration involving relatively large (at least 1 kb) regions. CNV can manifest as loss or gain of chromosomal regions. Earlier studies using fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) identified common losses at 1p, 6q, 8p, 10q, 13q, 16q, and 18q and gains at 1q, 2p, 7, 8q, 18q, and Xq. For example, MSR1, NKX3.1, and N33 are candidate tumor suppressor genes in prostate cancer lying within the most commonly deleted regions on chromosome 8p. MSR1 encodes a receptor on the macrophage cell surface that induces binding of oxidized low-density lipoprotein and other polyanionic ligands. Mutations, polymorphisms, or loss of the MSR1 gene may compromise global macrophage function, thereby exposing organs, including the prostate, to oxidative stress and damage. Although this gene does not code for prostatic proteins directly, oxidative stress has been implicated in the initiation of prostate carcinogenesis.
Loss of 8p23, a region that harbors the CUB and Sushi multiple domains 1 gene (CSMD1), has been associated with advanced prostate cancer. The retinoblastoma (RB1) gene is also a tumor suppressor gene and lies within the 13q locus. It is deleted in early prostate cancer development in animal models, prostate cancer cell lines, and some human prostate cancer specimens. RB1 inactivation in prostate cancer is the result of loss of heterozygosity and mutation. The 10q locus is lost in up to 45% of prostate cancers examined, and MXI1 and PTEN are two putative tumor suppressors in this region.
Regions of chromosome amplification in advanced prostate cancer include 8q containing the MYC gene and Xq11-13 encoding the AR gene. Gene amplification at 11q13.1 has been associated with disease recurrence. There are several candidate genes in this location (Table 38-1 ), but only MEN1 and MAP4K2 correlate with disease progression.
Recent prostate cancer genome sequencing studies consistently show that the genes most commonly affected by loss of copy number are CHD1, NTSE, PTEN, RB1, and TP53, and the most gained genes are AR, MYC, PIK3CA, and the HOXA3 cluster. These aberrations are more prominent in castration-resistant prostate cancer compared to localized disease. 7,8
Gene Fusion
The prostate cancer genome can harbor an average of 90 chromosomal rearrangements involving many genes. One of the most frequent gene fusion events in prostate cancer is the fusion of the TMPRSS2 and ERG genes, which are located 3 Mb apart on chromosome 21q22.2. 9 TMPRSS2 is an androgen-responsive gene, and ERG encodes an erythroblast transformation-specific (ETS) transcription factor. Their fusion is believed to be stimulated by androgens that recruit AR and TOP2B topoisomerase to chromosomal sites where TOP2B introduces double-strand breaks in DNA. TMPRSS2-ERG fusion results in overexpression of ETS genes in prostate cancer. TMPRSS2-ERG has been identified in 40% to 70% of prostate cancer and correlated with metastasis and disease-specific mortality. Gene fusion events such as TMPRSS2-ERG may be used for prostate cancer diagnosis through simple PCR detection of gene fusions in urine sediment.
WNT Signaling and β-Catenin
The Wnt signaling pathway plays a key role in embryonic development and is essential for the maintenance of stem cells. Wnt is an extracellular protein that interacts with the membrane-bound frizzled receptor to initiate its biologic activity. Wnt signaling leads to stabilization of CTNNB1 and its nuclear accumulation. Nuclear CTNNB1 converts the TCF/LEF DNA-binding protein complex from a transcriptional repressor into a transcriptional activator. Inappropriate activation of the Wnt pathway is observed in many cancers and is putatively associated with tumor development.
In mice, CTNNB1 stabilization through targeted excision of CTNNB1 exon 3 induces prostate intraepithelial neoplasia (PIN)-like lesions that are similar to the early stages of human prostate cancer. In human prostate cancers, high levels of nuclear CTNNB1 are detectable by immunohistochemistry, whereas their levels are undetectable in normal prostate tissue. High levels of CTNNB1 expression are associated with the more aggressive prostate tumors. Together, these findings imply that inappropriate activation of the Wnt signaling pathway can contribute to prostate cancer and progression.
There are several mechanisms by which the Wnt pathway may be inappropriately activated in prostate cancer; DNA methylation plays a key role in several of these processes. The adenomatous polyposis coli (APC) gene is hypermethylated in prostate tumors relative to samples of benign prostatic hyperplasia (BPH; 64.1% vs. 8.7%). APC is a key component of the CTNNB1 degradation complex. Thus, methylation-dependent silencing of APC can lead to CTNNB1 accumulation and Wnt pathway activation.
E-cadherin (CDH1), a cell membrane protein, interacts with CTNNB1 and sequesters it at the inside surface of the cellular membrane. However, CDH1 expression is often lost in prostate cancers because of chromosomal loss or promoter hypermethylation. Thus, because CDH1 is no longer present, CTNNB1 is released into the cytoplasmic and nuclear compartments, leading to Wnt pathway activation. Finally, the secreted-frizzled related proteins (SFRPs) and Wnt inhibitory factor-1 (Wif-1) sequester Wnt and antagonize Wnt signaling. In this manner, loss of SFRP/Wif-1 expression can lead to Wnt pathway activation. The genes encoding several of the SFRPs and Wif-1 are epigenetically silenced by DNA methylation in colorectal, lung, bladder, and kidney cancers and lymphocytic leukemia. In prostate cancer, Wif-1 expression is strongly suppressed. The SFRP1 gene is also aberrantly hypermethylated in prostate tumors relative to BPH tissue and is partially to completely methylated in several human prostate cancer cell lines. These findings suggest that silencing of genes antagonist to Wnt may play a role in prostate cancer development.
The Wnt signaling pathway may interact with other signaling pathways such as the AR-CTNNB1, which in turn can upregulate AR transcriptional activity in an androgen-dependent manner. Subsequently, AR enhances nuclear translocation of CTNNB1. In addition, PI3K/Akt can modulate the activity of CTNNB1 by phosphorylation of CTNNB1 by GSK3B, a substrate of Akt.
MicroRNA and Other Noncoding RNA in Prostate Cancer
Only a small fraction of the transcription output in human genome encodes for proteins. Noncoding RNAs (ncRNAs) are arbitrarily classified into two major classes based on their size: small (microRNA) and long ncRNA (lncRNA).
MicroRNA and Prostate Cancer
Small ncRNAs, exemplified by microRNA (miRNA), are known to regulate diverse biological processes in stem cells, development, differentiation, metabolism, and disease such as cancer. Several hundred miRNAs have been identified in human cells, and these are transcribed from the genome as long, primary miRNAs ranging in size from hundreds to thousands of nucleotides. miRNAs depend on multiple proteins for their biogenesis and function. Aberrations in any of these proteins will affect miRNA-mediated gene regulation. Expression of Dicer, a key gene in the biosynthesis of miRNA, is upregulated in a significant fraction of prostate cancer and is associated with aggressive cancer features. Knockout of Dicer in the mouse prostate impairs prostate stem cell activity and causes prostate atrophy. Some other components of the miRNA machinery (XPO5, Ago1, Ago2, HSPCA, MOV10, and TNRC6B) are also upregulated in prostate cancer.
Processed miRNAs have been found to regulate gene expression by inhibiting protein translation and/or enhancing the degradation of target gene mRNA with which the miRNAs have imperfect sequence complementarity in the 3′UTR region. It has been predicted that each miRNA can target hundreds of genes, and a third of human protein-coding genes are regulated by miRNA. An increasing body of evidence suggests that miRNAs are involved in the initiation and development of different types of cancers, including prostate cancer.
Expression Signature of miRNA in Prostate Cancer
High-throughput miRNA expression profiling studies have found altered miRNA expression in prostate cancer, providing evidence for the involvement of miRNAs in this disease. miRNAs that have increased expression or amplification in cancer can act as oncogenes to enhance cell proliferation and survival by the inhibition of protein-coding tumor suppressor genes. They are thus called oncomiRs. Several such oncomiRs have been identified in prostate cancer cell lines including miR-21, miR-291, miR-221, miR-222, and the well-known miR-17-92 cluster, which contains six members: miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92a-1. Some common target genes of these oncomiRs include PTEN, BIM, RB1, p21, and p27. On the other hand, miRNAs that normally target and suppress are regarded as tumor suppressor miRNAs (ts-miRNAs). In prostate cancer, the miR-15a/16-1 cluster that targets BCL2 and CCND1 is downregulated in prostate cancer. Forced expression of these miRNAs can lead to suppression of cell proliferation and induction of apoptosis. 10 Other commonly identified tumor suppressor miRNAs include miR-34a/b/c, miR-145, miR-205, and let-7.
miRNA and the AR Signaling Pathway
The AR signaling pathway and miRNAs are engaged in reciprocal regulation via multiple interaction points. miRNA can suppress AR expression through the canonical miRNA pathway or affect AR transcriptional activity by modulating other AR cofactors such as MYC. A systemic analysis of miRNAs identified a number of miRNAs that could suppress AR expression post-transcriptionally by targeting the AR 3′UTR element. Similar to protein coding genes, miRNA genes are also under the regulation of many transcription factors, including AR, which binds to ARE in the promoter of many miRNAs. As the first example, AR can bind to the ARE of miR-21, a prostate oncomiR, and directly regulate its expression as a downstream signaling effector. 11 Several other miRNAs were later found to be also under the regulation of AR, including miR-101, miR-141, miR-27a, miR-32, and miR-148a.
miRNA as Biomarkers for Prostate Cancer Diagnosis
miRNAs derived from cancer cells can exist as circulating extracellular or cell-free RNA and are remarkably stable in body fluids such as plasma/serum. These extracellular miRNAs can be detected and quantified using either PCR-based methods or other high-throughput approaches, including miRNA microarray and next-generation sequencing. A number of studies have performed miRNA profiling in body fluids including serum, plasma, and urine and have identified several miRNAs whose levels can serve as diagnostic and/or prognostic markers for prostate cancer patients. 12,13 Some of the miRNAs that consistently exist in prostate cancer patients across different studies include miR-141 and miR-375.
lncRNA and Prostate Cancer
The longer class of ncRNAs is known as long intergenic RNA (lncRNA), with sizes ranging from a few hundred to thousands of nucleotides. lncRNAs can regulate protein-encoding genes by affecting transcription and chromatin state by mechanisms distinct from those used by small ncRNAs. Thus genetic alterations and aberrant expression of lncRNAs can be a causal factor in disease. Several lncRNAs pertinent to prostate cancer have been identified. A 3.7-kb lncRNA known as PCA3 (DD3) has been mapped to chromosome 9q21-22 and shown to be highly overexpressed in prostate cancer samples. Genetic variation in lncRNAs has been found to affect prostate cancer risk. Microarray profiling of intronic transcripts identified many ncRNAs expressed in prostate cancer samples, and the expression levels of some correlate with the extent of prostate tumor cell differentiation. 14 By high-throughput RNA sequencing of 102 prostate tissues and cell lines, Prensner and colleagues identified 121 unannotated prostate cancer–associated ncRNA transcripts (PCATs) and have characterized one of them, PCAT-1, as a prostate cancer–specific ncRNA functionally implicated in disease progression.
Given the regulatory role of lncRNA in prostate cancer, their aberrant expression suggests that they may serve as tumor biomarkers. In this regard, Cui and co-workers identified an lncRNA termed PlncRNA that expresses higher in prostate cancer cells compared to normal prostate epithelial cells. 15 Knockdown of PlncRNA has an inhibitory effect on prostate cancer cells, potentially through its regulation of AR activity
Epigenetic Alterations in Prostate Cancer
Epigenetic alterations contribute to the malignant transformation and progression of prostate cancer. Figure 38-2 shows the potential contribution of epigenetic events to the development of prostate cancer and its progression to advanced and castration-resistant cancer. Initially DNA methylation was regarded as a new type of promising biomarker for prostate cancer diagnosis and prognosis, and a therapeutic target. Despite intensive research efforts in the past decade, results from clinical studies evaluating DNA methylation as a biomarker have been disappointing, and so far no biomarker has advanced into the clinical arena.
Hypermethylation
DNA hypermethylation is one of the most common and best-characterized epigenetic abnormalities in prostate cancer. Genes including classic and putative tumor suppressor genes as well as genes involved in a number of cellular pathways, such as hormonal responses, cancer cell invasion/tumor architecture, cell cycle control, and DNA damage repair, can be hypermethylated in prostate cancer. For many of these genes, promoter hypermethylation is often the mechanism responsible for their functional loss in prostate cancer. Inappropriate silencing of these genes can contribute to cancer initiation, progression, invasion, and metastasis. Some commonly hypermethylated genes in prostate cancer are discussed next (Table 38-2 ).
DNA Damage-Repair Genes
Antioxidants and DNA repair pathways protect the genome and maintain genome stability during replication or following DNA damage. Hypermethylation of genes important for such processes, such as glutathione S-transferase Pi (GSTP1) and O-6-methylguanine DNA methyltransferase (MGMT), has been frequently documented in prostate cancer. GSTP1, located at chromosome 11q13, belongs to a supergene family of glutathione S-transferases (GSTs) that play an important role in the detoxification of carcinogens and cytotoxic drugs by catalyzing their conjugation to glutathione. GSTP1 inactivation may lead to increased cell vulnerability to oxidative DNA damage and the accumulation of DNA base adducts, which can precede carcinogenesis.
Figure 38-2 Epigenetic alterations in the development of prostate cancer Multiple factors are associated with the development of prostate cancer, including genetic predisposition, environmental factors, diet, ethnicity, and aging. Many of these factors modify the genome through epigenetic effects, and DNA methylation may be an early event causing inactivation of DNA damage repair genes such as GSTP1 and MGMT. Subsequent inactivation of cell-cycle control genes provides a growth advantage leading to locally advanced prostate cancer. Functional loss of genes in the cell adhesion pathway, such as CD44, may allow for metastasis. Ultimately, inactivation of AR via DNA hypermethylation allows cancer cells to become androgen insensitive. From Li LC, Okino ST, Dahiya R. DNA methylation in prostate cancer. Biochim Biophys Acta. 2004;1704:87-102, with permission.
In prostate cancer, methylation of the GSTP1 gene promoter is the most frequently detected epigenetic alteration. Elevated CpG methylation has been detected in prostate cancer tissues as early as at the stage of atypia and PIN. GSTP1 promoter hypermethylation can also be readily detected in the serum/plasma, urine, and ejaculates of prostate cancer patients with high specificity but unsatisfying sensitivity. Current findings from a large number of studies do not support the feasibility of using GSTP1 as an independent tumor biomarker, but it may complement prostate-specific antigen (PSA) screening for prostate cancer diagnosis.
Table 38-2
Epigenetic Changes in Prostate Cancer
Hormone Receptors
AR mediates testosterone and dihydrotestosterone activity, which is essential for the development and maturation of the prostate gland and prostate cancer. Most prostate cancer is initially androgen dependent, but eventually becomes androgen independent after androgen-deprivation therapy. Androgen-independent prostate cancers are characterized by a heterogeneous loss of AR expression. Genetic alterations that alter the sensitivity of the receptor to androgen, such as AR gene mutation and, more commonly, amplification without loss of AR expression, are thought to play key roles in the development of androgen-independent prostate cancer. The prostate expresses two types of estrogen receptors (ERs): ERα (ESR1) and ERβ (ESR2), whose functional role in the prostate and prostate cancer remains controversial. Hypermethylation of ERs leading to decreased expression increases with aging and can contribute to tumor progression in prostate cancer patients.
Cell Cycle Control Genes
An important characteristic of tumor cells is unbalanced proliferation due to impaired regulation of the cell cycle. The multiple checkpoints that control the cell cycle include the retinoblastoma protein, cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CDKIs). CDKIs are potential tumor suppressor genes that act as molecular brakes on cell cycle progression. Failure of cell cycle arrest due to alterations in CDKI expression has been implicated in prostate cancer. CDKIs are grouped into two families: the INK4 family and the CIP/KIP (kinase inhibitor protein) family. The INK4 family includes CDKN2A (p16), CDKN2B (p15), CDKN2C (p18), and CDKN2D (p19) and inhibits cyclin D–associated kinases (CDK4 and CDK6). The CIP/KIP family, which includes CDKNIA (p21), CDKN1B (p27), and CDKN1C (p57), inhibits most CDKs.
CDKN2A can be inactivated in prostate cancer by a variety of mechanisms, including deletion, point mutation, and hypermethylation. Methylation-mediated inactivation of the CDKN2A gene has been reported in prostate cancer cell lines and tissues at a very low frequency (0% to 16%). Inactivation of other cell cycle genes such as CDKN2B, CDKN1A, and CDKN1B by hypermethylation is rare in prostate cancer.
The Ras-association domain family-1 gene (RASSF1) is located at 3p21.3 and encodes a protein similar to the RAS effector proteins. A tumor suppressor role has been proposed for RASSF1. RASSF1 promoter methylation is a common event in prostate cancer and high-grade PIN, occurring in 54% to 96% of tumors, and increases with higher Gleason scores.
Tumor Invasion and Tumor Architecture Genes
The cadherin-catenin adhesion system is critical for the preservation of normal tissue architecture and is regulated by a family of proteins collectively termed cell adhesion molecules (CAMs). Decreased expression of CDH1 and other CAMs has been reported to have prognostic significance in various human cancers, including prostate cancer. In prostate cancer, expression of CDH1 is markedly suppressed and its promoter is methylated to varying degrees. In addition, methylation of the CDH1 promoter is increased in advanced prostate cancer, making it a potential biomarker for cancer progression.
CD44 is an integral membrane protein involved in matrix adhesion and signal transduction. Loss of CD44 expression correlates with methylation of its gene promoter in prostate cancer and is associated with stage and prognosis. Other genes involved in the cadherin-catenin adhesion system have also shown methylation-mediated inactivation in prostate cancer, such as H-cadherin, adenomatous polyposis coli (APC), caveolin-1 (CAV1), laminin α-3 (LAMA3), laminin β-3 (LAMB3), and laminin γ-2 (LAMC2).
Other Putative Tumor Suppressor Genes
Other possible tumor suppressor genes that are subject to epigenetic inactivation in prostate cancer include KAI1 (a prostate-specific tumor metastasis suppressor gene), inhibin-α (a member of the TGF-β family of growth and differentiation factors), and DAB2IP, a novel GTPase-activating protein for modulating the Ras-mediated signal pathway. 16 Recent high-throughput DNA methylation profiling in prostate cancer cell lines and tissues unveiled methylated genes such as SLC15A3, KRT7, TACSTD2, GADD45b 17 OXD3 and BMP7 18 PR83, ADCY4, LOC63928, and D4S234E; 19 however, these genome-wide analyses seem not to have yielded consistent information identifying new prostate cancer biomarkers.
Hypomethylation
Both global and gene-specific hypomethylation have been implicated in human malignancy. The PLAU gene encodes urokinase plasminogen activator and is highly expressed in most prostate cancer tissues and invasive prostate cancer cell lines. DNA methylation and gene amplification may participate in the regulation of the PLAU gene in prostate cancer. Hypomethylation of the PLAU promoter is associated with increased expression in hormone-independent prostate cancer cells, higher invasive capacity in vitro, and increased tumorigenesis in vivo. Other hypomethylated genes in prostate cancer include CAGE, a novel cancer/testis antigen gene; heparanase (HPSE), CYP1B1, and Melanoma antigen gene protein-A11 (MAGE-11). HPSE, an endo-β-D-glucuronidase, and CYP1B1 are overexpressed and substantially hypomethylated in prostate cancer compared with benign prostatic hyperplasia samples. Aberrant hypomethylation of repetitive DNA elements such as LINE-1 and ncRNA such as XIST also occurs in prostate cancer.
Histone Modification
DNA is organized into a nucleoprotein complex termed chromatin. The basic chromatin unit is the nucleosome, which is composed of 146 bp of DNA wrapped around four pairs of histone proteins. The N-terminal tails of histones are positioned outside the nucleosome core and are thus susceptible to covalent modifications including acetylation and methylation. Acetylation and deacetylation of histone tails are catalyzed by histone acetyltransferase (HATs) and deacetylases (HDACs), respectively. Through histone acetylation, HATs have been shown to increase the activity of several transcription factors, including nuclear hormone receptors, which facilitate promoter access to the transcriptional machinery. Conversely, HDACs cause histone deacetylation, which is associated with transcriptional repression. Histone methylation is facilitated by histone methyltransferases (HMTs), which use S-adenosylmethionine as a methyl donor group to the lysine and arginine residues of histone protein pairs H3 and H4. Like histone acetylation, histone methylation is reversible and is facilitated by at least two enzymes: lysine-specific demethylase1 (LSD1) and JmjC domain-containing histone demethylase1 (JHDM1).
The expression of many genes is aberrantly regulated in prostate cancer through histone modification. Tumor-specific alterations in the enzymes that modify histone states can alter global histone modification profiles. For example, MLL2 encodes an H3K4-specific histone methyltransferase that is recurrently mutated in multiple cancers including prostate cancer. Also, the loss of acetylation of H3 and H4 resulting from increased HDAC activity may also be of importance in prostate cancer. Treatment of prostate cancer cells with HDAC inhibitors increased the expression of specific genes such as insulin-like growth factor binding protein-3 and carboxypeptidase A3 (CPA3), thereby suggesting a role for histone acetylation in aberrant gene regulation.
Polycomb Group Transcriptional Repression
The Polycomb group (PcG) proteins are developmental regulators that silence chromatin through H3K27 methylation. Enhancer of Zeste 2 (EZH2) and SUZ12 are members of the PcG proteins that are overexpressed in prostate cancer and are highly associated with tumor aggressiveness. Other studies reveal that EZH2 is overexpressed and associated with aggressiveness in cutaneous melanoma, endometrial cancer, bladder cancer, and breast cancer. In addition, other PcG proteins, BMI1 and RING1, are also overexpressed in aggressive prostate cancers. The EZH2 complex silences gene expression by catalyzing H3K27 methylation to generate an inaccessible, heterochromatic chromatin configuration. In addition, EZH2 was found to control DNA methylation through direct physical contact with DNA methyltransferase. It is unclear how EZH2 is overexpressed in prostate cancer. EZH2 overexpression in prostate cancer causes the silencing of developmental regulators and tumor suppressor genes such as ADRB2, CDH1, DAB2IP, SNCA, and SOCS via histone methylation, conferring on cancer cells a stem cell–like epigenetic state, because PcG is a stem cell–specific marker including cancer stem cells and plays an important role in maintaining the undifferentiated state of embryonic stem (ES) cells. Pharmacologic disruption of polycomb repressive complex 2 has been shown to inhibit prostate tumorigenicity and tumor progression in animal models of prostate cancer.
PSA
The PSA (KLK3) gene contains an androgen receptor response element in its 5′ regulatory region. Methylation of H3K4 is associated with transcriptional inactivation of the PSA gene in the prostate cancer cell line LNCaP, and transcription of the PSA gene is accompanied by rapid decreases in di- and trimethylated H3 at lysine 4. In addition, a lysine-specific demethylase (LSD1) has been found to interact with the androgen receptor to stimulate the AR-dependent transcription of PSA in LNCaP cells by removing the methyl group at H3K9. An inhibitor of LSD1, pargyline, can block AR-dependent transcription by blocking histone demethylation. The net effect of methylation on the PSA gene needs to be carefully considered, however, because PSA is a marker of disease progression rather than a causal factor.
Aberrant Translational Control in Prostate Cancer Etiology and Progression
In addition to the genomic and transcriptional alterations that drive cancer initiation and progression, there is an emerging appreciation for how altered protein synthesis may have a direct causal role in cancer etiology. The understanding of translational control has undergone a paradigm shift towards a greater appreciation for specificity in this step of gene expression regulation. For example, genes that encode distinct factors involved in translation initiation, the first and most highly regulated step of protein synthesis, are often found aberrantly expressed in human cancers. 19a–19c Furthermore, translation of mRNA networks involved in tumor suppression and oncogenic transformation is controlled by the presence of regulatory elements in their 5’- and 3’UTRs such as internal ribosome entry sites (IRESes), structured RNA sequences, RNA binding protein domains, and miRNA binding sites. Most importantly, activity of an entire repertoire of translational components is controlled by oncogenic signal transduction pathways such as RAS, PI3K-AKT-mTOR, and MYC, which are master regulators of protein synthesis. These oncogenic signaling pathways are commonly deregulated in human prostate cancer and have been shown to promote cancer initiation and progression. 19d–19h A striking example is the convergence of RAS, PI3K-AKT-mTOR, and MYC pathways on translation initiation. In this context, these pathways share a common regulatory node: oncogenes 19i and translational initiation factor eIF4E, which controls global protein synthesis as well as the translation of specific mRNA targets. 19b,19g–19l eIF4E is the best characterized and rate-limiting factor of the eIF4F translation initiation complex. The activity of eIF4E is negatively regulated by the tumor suppressor eIF4E binding proteins (4EBPs), which are phosphorylated and inhibited by the mTOR kinase.
Deregulation of the 4EBP/eIF4E axis has been linked to prostate cancer initiation and progression. In particular, it has been shown that eIF4E phosphorylation, which directs eIF4E activity, is necessary for tumorigenesis in a mouse model of prostate cancer driven by PI3K-AKT-mTOR hyperactivation. 20 Restraining the oncogenic activity of eIF4E downstream of hyperactive mTOR to normal levels inhibits tumor progression and leads to overall increased survival rates. 21 One of the molecular mechanisms underlying eIF4E’s oncogenic activity is its ability to upregulate the translation of pro-survival factors. For example, distinct mRNAs with highly structured 5′UTRs (which is an obstacle for translation initiation), such as the anti-apoptotic protein Mcl-1, are more efficiently translated on eIF4E hyperactivation. 19b Mechanistically, eIF4E recruits the eIF4A helicase to unwind these 5′UTR structured elements, leading to the translational upregulation of specific mRNAs such as Mcl-1. 22,23 Thus, eIF4E hyperactivation provides a survival advantage for cancer cells.
Genetic and pharmacological inhibition of the 4EBP1/eIF4E axis downstream of oncogenic mTOR also significantly inhibits the invasive and metastatic potential of prostate cancer. 19l The molecular mechanism by which hyperactive eIF4E exploits specific cellular processes to drive cancer progression is also mediated through the selective translation of distinct nodes of mRNAs. In this context, the translation of a novel signature of metastasis-associated mRNAs is found to be upregulated during prostate cancer invasion. This cancer cell invasion network includes the intermediate filament protein vimentin, 24 whose expression is increased during cancer progression; CD44, an antigen involved in cell migration that is also associated with cancer metastasis; 25 metastasis associated protein 1 (MTA1), which promotes neoangiogenesis in metastatic forms of cancer; 26,27 and Y-box binding protein 1 (YB-1). 28 The eIF4E-mediated translation of this invasion signature is dependent on a newly identified regulatory element termed a pyrimidine-rich translational element (PRTE) in the 5’UTR of these mRNAs. Importantly, a new and potent class of compounds that blocks mTOR oncogenic activity through inhibition of eIF4E (known as mTOR ATP site inhibitors) decreases the expression of the invasion signature and demonstrates therapeutic efficacy during all stages of prostate cancer progression and metastasis. Additional mRNA targets that direct cell invasion are also controlled by eIF4E including matrix metalloproteinase 9 (MMP9), an enzyme that aids breakdown of extracellular matrix (ECM), and heparanase, which degrades the interior lining of blood vessels. Thus, deregulation of translational control represents a highly specific mechanism for targeting critical nodes of gene expression that can steer prostate epithelial cells towards transformation and cancer progression. 19l,29,30 Interestingly, recent studies have uncovered that YB-1 in turn promotes cap-independent modes of translation for other mRNAs involved in metastasis.
Components of the translational machinery are increasingly being recognized as potential biomarkers of disease progression and attractive therapeutic targets in prostate cancer. eIF4E overexpression is common in multiple cancer types, including malignancies of the prostate, breast, stomach, colon, lung, skin, and the hematopoietic system. 30 In prostate cancer, eIF4E protein and phosphorylation levels as well as 4EBP1 phosphorylation status correlate positively with Gleason grade. 31,32 Furthermore, in postprostatectomy prostate cancer patients, elevated eIF4E expression and increased 4EBP1 phosphorylation are predictive of worse overall survival. 32 Beyond the translational machinery itself, specific downstream mRNA targets of translation initiation factors, including the eIF4E-directed invasion signature described earlier, are also candidate biomarkers for prostate cancer progression. For example, prostate cancer patients exhibit stepwise increases in YB-1 expression at every stage of the disease from normal prostate to castration-resistant prostate cancer. 19l Furthermore, the matrix metalloproteinase, MMP3, which is a translationally regulated mRNA responsive to eIF4E phosphorylation, is highly expressed in prostate cancer. 31 Other initiation factors are also emerging as biomarkers, including eIF3H, a component of the translation pre-initiation complex that is highly overexpressed at the protein level in human prostate cancer. 33 In addition to the translation initiation machinery, there is evidence suggesting that the abundance of rRNA (ribosomal RNA), a critical component of the ribosome, increases with advanced prostate cancer. 34,35 Moreover, the gene that encodes a key enzyme that modifies specific rRNA nucleotides, known as DKC1, is significantly overexpressed in prostate cancers, particularly in patients with aggressive disease. Therefore, multiple components of the translation machinery as well as downstream mRNA targets may have significant predictive power that could help delineate between prostate cancer patients with indolent and those with aggressive forms of the disease. Most importantly, next-generation therapeutics have shown great promise in preclinical and Phase I clinical trials at targeting the oncogenic translation machinery in prostate cancer. 37,38
Future Directions
The search for new biomarkers is under way to aid in the diagnosis, prognosis, and decision-making process of men with prostate cancer. Because of the multitude of mechanisms of carcinogenesis, many approaches can be taken to accomplish these aims. Because epigenetic events are theoretically reversible, novel therapies that target hyper- or hypomethylated genes or histone acetylation implicated in prostate cancer aggressiveness and progression could bring new hope to patients with metastatic disease. Although the past two decades have been largely dedicated to the use and importance of PSA, the future of prostate cancer detection and treatment will be refined by discoveries in molecular biology.
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