DNA methylation, molecular genetic, and linkage studies in prostate cancer

The Prostate Supplement 6:36-44 (1996)

DNA Methylation, Molecular Genetic, and Linkage Studies in Prostate Cancer

David F. Jarrard, G. Steven Bova, and William B. Isaacs

Srady Urological Institute, johns Hopkins Hospital, Baltimore, Maryland

ABSTRACT: Molecular biologic studies have now identified a number of important genetic and epigenetic mechanisms that cause alterations in growth and differentiation genes in prostate cancer. In addition to DNA deletion and point mutation, DNA methylation represents a new paradigm for the inactivation of tumor suppressor or growth suppressor genes. The identification of new genes, including a prostate cancer susceptibility locus, may furnish further insight into the molecular characteristics of prostate cancer and permit the early identification of affected individuals. 0 1996 Wiley-Liss, Inc.

KEY WORDS: DNA methylation, linkage analysis, prostate cancer, tumor suppressor

INTRODUCTION

The field of molecular biology owes much of its foundation and progress to the study of cancer. Aris- ing from this study is one of the basic tenets of carcinogenesis: the development of a tumor requires a sequential series of genetic alterations. Among human cancers, this multistep nature has been most clearly demonstrated in the development of colon cancer [l]. In the human prostate, where the initiation of cancer is remarkably frequent, a distinct series of genetic alterations has not been similarly defined [2]. Experimentally, reproducible methods for the transformation of prostate epithelial cells have only recently been developed (3-61. Each of these experi- mental systems has demonstrated the requirement for an accumulation of genetic abnormalities in order to develop fully malignant prostate cancer. Several broad categories of molecular events that may lead to cancer include the activation of dominant oncogenes, inactivation of tumor suppressor gene activity, and repression of DNA repair genes. Recently, DNA methylation has created interest as a nonmutational mechanism of gene inactivation that complements deletion and mutation [7].

The importance of discovering the molecular mechanisms underlying the development of prostate cancer resides in the vast scope of this health prob- lem. Adenocarcinoma of the prostate will strike 244,000 men in the United States in 1995 and will directly cause the death of 40,400 [8]. Prostate cancer

has a number of unusual biologic features including a slow growth rate, a high prevalence in older men, and a marked propensity for multifocal, regional development in the peripheral prostate gland [9]. Ade- nocarcinoma of the prostate has an unpredictable course, remaining indolent and localized in some patients; however, other cases are characterized by rapid progression and lethality. In contrast to breast and colon cancer, a specific gene has not been identified for the estimated 9% of all cases of prostate cancer thought to be hereditary [lo]. This review dis- cusses novel genes and genomic regions potentially involved in prostate cancer progression. In addition, we focus on recent advances in the understanding of the genetic and epigenetic mechanisms for altering the expression of genes implicated in the initiation and progression of prostate cancer.

LINKAGE STUDIES AND HEREDITARY PROSTATE CANCER

One of our primary goals in prostate cancer re- search has been to identify the genetic locus or loci involved in the inherited predisposition to cancer. Hereditary cancers result from a single gene or genes,

Received for publication August 8, 1995; accepted October 5, 1995. Address reprint requests to Dr. W.B. Isaacs, Brady Urological In- stitute, Johns Hopkins Hospital, 600 N. Wolfe Street, Baltimore, MD 21287

0 1996 Wiley-Liss, Inc.

Molecular Genetics of Prostate Cancer 37

usually mutated tumor-suppressor genes, passed within a family that confer an increased susceptibility for the disease to develop. In most cases in which a cancer susceptibility gene has been identified, the critical studies that led to its discovery have been linkage studies. In colon cancer, linkage studies led to the identification of both the APC gene and the family of mut mismatch repair genes as the molecular events responsible for the development of familial adenomatous polyposis coli (FAP) [ll] and hereditary nonpolyposis colon cancer (HNPCC) [ 121, respectively. Analysis of early-onset breast cancer demonstrated a tight linkage to chromosome 17q21 and led to the recent identification of BRCAl [13]. Linkage analyses provide statistical evidence for the existence of such genes and information regarding their genomic loca- tion. Often, familial cancer genes appear to be inactivated in sporadic cancers (e.g., retinoblastoma, NF-1 and 2, Wilms tumor, FAP, and von Hippel-Lin- dau) [14] and have provided important insights into initiating lesions in the multistep pathogenesis of these cancers. However, BRCAl appears not to be mutated in sporadic breast cancers [15], suggesting that more complex scenarios must exist in some tumor systems. The identification of an inherited gene for prostate cancer through linkage analysis may furnish insight into the molecular characteristics of this cancer, as well as provide more effective methods for the identification of individuals at risk for the development of prostate cancer.

Prostate cancer appears to demonstrate a strong familial component on the basis of several epidemiologic studies to assess the extent of familial aggrega- tion in prostate cancer [16-201. Prostate cancer was found to have a higher familiality than breast and colon carcinoma, both tumors recognized as having a strong genetic component. Men with an affected brother or father had a twofold increased risk of de- veloping prostate cancer, and a trend of increasing risk with increasing number of affected family members was noted (see review [21]). A segregation analysis performed by Carter et al. [lo] revealed that familial clustering was best explained by the dominant inheritance of a rare (q=0.003) high-risk allele. This model suggests that the inherited form of prostate cancer may account for a sigruficant portion (43%) of early-onset disease (<55 yr); however, it represents only a small proportion (-9%) of all prostate cancers. An analysis of the clinical features of hereditary prostate cancer revealed no differences in the clinical stage at presentation, preoperative prostate-specific antigen (PSA) level, final pathological stage and prostatic weight between individuals classified as having inherited disease, and a group with no other family members affected [21]. In addition, pathological fea-

tures, including multifocality of disease, do not appear to be different [22]. Therefore, with the excep- tion of an early age of disease onset, there appear to be no unique clinical or pathological characteristics in the inherited form of the disease. The empiric risk associated with familial prostate cancer at an early age suggests that individuals with a family history of affected probands should undergo screening and careful surveillance.

DNA METHYLATION

DNA methylation has recently been demonstrated to be important in the regulation of genes implicated in tumor and growth suppression [23-291. An “im- balance” of DNA methylation appears to develop in cancer cells, and this phenomenon may participate in the genetic instability that underlies tumor progression [q. In vertebrate genomes, the cellular enzyme 5‘-methyltransferase catalyzes the transfer of a methyl group from S-adenosyl methionine to the 5‘ position of a cytosine nucleotide adjacent to a guanine (see reviews [7,30,31]). The extent of DNA methylation increases with an increased complexity of the genome, and in humans roughly 70430% of cytosines 5’ to guanines are methylated. In developmental systems, DNA methylation functions to organize the chromatin and aids in the compartmentalization of DNA. It is responsible for the maintenance of X-chromosome inactivation in the female and has been implicated in the phenomenon of genomic imprinting (i.e., differential allele specific expression). During development, methylation appears to play a critical role in the dy- namic patterns of gene expression, perhaps by altering chromatin structure and the subsequent accessi- bility of DNA-binding regulatory sequences. A targeted ”knockout” mutation of the methyltransferase gene in mice resulted in an abnormal development of the embryo and death at midgestation [32]. Therefore, methyltransferase appears to play a critical role in the regulation of gene expression.

One aspect of the alteration in methylation patterns seen in cancer is a global loss of 5’-methylcytosines noted throughout the cell genome. This wide- spread hypomethylation, demonstrated in prostate cancer [33], appears to be a consistent molecular feature of cancer cell DNA; however, its significance is not entirely clear. A second feature of these methylation changes, and of potentially more importance, are regions of hypermethylation (Fig. 1). The most important site with respect to methylation and altered gene transcription occurs in regions of high- density C-G dinucleotide sequences, called CpG islands. These CpG islands are generally found in or near the 5’ region of genes and often contain the pro-

38 Jarrard et al.

7 - Methylated CpG

7 - Unmethylated CpG

Normal

5' T t t 3'

EXONl EXON2

+ Cancer

5' T P t

EXONl EXON2

Fig. 1. Model demonstrating the effects of DNA methylation on transcription in cancer and normal cells. The promoter region (exon I and 5' region) of this hypothetical gene contains many C-G dinucleotides consistent with the presence of a CpG island. In the normal cell (top), the lack of CpG methylation in the promoter region is consistent with an open chromatin conformation and has a permissive effect on transcriptional activity. In the tumor cell

moter and one or more exons of its associated gene [31]. Methylation of CpG islands may be associated with transcriptional inactivation of the affiliated gene. The recent observation that certain growth suppressor or tumor suppressor genes can be inactivated by CpG island methylation led Baylin and coworkers to propose that this is an important mechanism in tumor formation [7]. CpG island hypermethylation is associated with the inactivation of the growth regulatory genes Rb in retinoblastoma [MI and the VHL tumor-suppressor gene in renal cancer [29]. It is be- lieved that the functional loss of gene expression via regional hypermethylation may be an early event in the promotion of tumor progression and appears to complement other mechanisms of gene inactivation, including deletion and point mutation, in these tumor systems. Furthermore, methylation patterns are heritable, and de novo alterations may result in an inherited repression of gene transcription analogous to that seen in mutation or deletion.

(bottom), methylation of the CpG dinucleotides in this region has taken place, leading to a transcriptional inactivation of this gene. In addition, scattered losses of CpG methylation characteristically occur throughout the cancer cell genome, represented by a loss of 5'-methylcytosines downstream to the regulatory region. Adapted from Baylin et al. [7].

One additional aspect of the involvement of DNA methylation in cancer is the demonstration that 5'- methylcytosine nucleotides are susceptible to undergo deamination to thymine. This may account for the frequent C-T mutations seen in a heavily methylated region of exon 7 in the p53 gene in colon cancer [35]. This observation may also explain the relative paucity of the CpG dinucleotide throughout other ar- eas in the genome.

Bird and coworkers have examined methylation in human and rodent cultured cell lines by creating a representative library of clones containing CpG islands and found evidence that tens of thousands of genes are stably inactivated by the de novo hypermethylation of CpG island promoters that are nor- mally free of methylation [36]. These workers hypoth- esize that this abnormal methylation may be one mechanism by which differentiation genes are inactivated, thus leading to the clonal selection of immor- talized cells. Using this technique to create a genomic


library, we have examined the methylation patterns of a series of CpG island clones associated with unknown genes and found several cancer-specific methylation patterns when prostate tumors were compared to normal peripheral prostate tissue [37l. Surprisingly, we also noted a high frequency (-70%) of CpG island methylation in histologically normal human adult prostate as compared to other nonprostate tissues. This high incidence of methylated genes in the prostate may reflect altered control mechanisms and may potentially contribute to the high incidence of growth abnormalities seen in the prostate. Further analysis and characterization of these CpG island associated genes are in progress.

Examination of the methylation status of genes sus- pected to participate in the development of cancer in the prostate may further provide insight into the marked tendency of the prostate for abnormal growth. Examination of a specific known polymorphic locus on chromosome 17, D17S5 within the Hic-1 gene (for hypermethylated in cancer), by Morton et al. [38] has demonstrated hypermethylation at this CpG-rich site in 14 of 15 prostate tumors and 4 of 5 prostate cancer cell lines. This locus is commonly hypermethylated in colon, lung, and kidney cancers and is frequently deleted in breast cancers, consistent with the presence of a tumor-suppressor gene in this region [28,39,40]. Cu- riously, this locus is also commonly methylated in normal prostate tissue, suggestive of a tissue-specific pattern of gene expression (or inactivation) that may be involved in the high incidence of prostatic hyper- plasias. We have also examined the p16 (~16'"~&/mts- lkdkn2) tumor-suppressor gene, a gene homozy- gously deleted in 20% of prostate cancers [41], for alterations in methylation patterns. p16, located at 9p21, is a member of a novel class of cyclin-dependent kinase inhibitory proteins that control cell passage through the G1 phase of the cell cycle [a]. Methyl- ation inactivation of an exon 1 CpG island correlates with a lack of expression in the prostate cancer cell lines TSU-PR1 and PC3, and methylation appears to be an alternative mechanism for the inactivation of the p16 gene in primary prostate cancers [43]. Further- more, treatment of the prostate cell lines TSU-PR1 and PC3 with the demethylating agent 5-deoxyazacytidine leads to reactivation of this gene in culture. These provocative studies suggest that methylation-associated alterations in gene expression may play an important role in the process of prostate tumorigenesis and establish 5' CpG island methylation as a new paradigm for the inactivation of tumor suppressor or growth suppressor genes.

Recently, Lee et al. [23] examined the methylation status of the promoter of the glutathione S-transferase (GSTIT) gene, located on chromosome llq13, in

normal and cancerous prostate. Originally examined as a mechanism to explain resistence to chemothera- peutic drugs, GSTIT was unexpectedly found to be downregulated in virtually every specimen of prostate cancer examined. Analysis of a CpG island within the promoter region revealed hypermethylation in all 20 prostate cancer DNAs examined. This methylation pattern was not detected in any DNA samples from either normal or hyperplastic prostate tissue. Therefore, methylation of this region appears to be the most common genetic alteration yet seen in prostate cancer.

DNA methylation has also been implicated in the control of imprinting, or allele-specific expression, seen in selected genes, including the insulin-like growth factor I1 (IGF-11) gene on chromosome llp13 [44]. Recently, a loss of IGF-I1 imprinting (LOI) was found in sporadic Wilms tumors [45] and lung carcinomas [46] and this molecular event may contribute to the pathogenesis of these tumors. We have recently reported that in prostates removed at radical surgery for localized adenocarcinoma, both the cancer and the associated normal peripheral zone tissue have a pronounced loss of imprinting of the IGF-I1 gene [47]. Biallelic expression of IGF-I1 RNA was demonstrated in 10 of 12 (83%) tumor samples, and in 7 of 11 matched peripheral zone prostate samples. However, the IGF-I1 imprint was maintained in benign prostatic hyperplasia (BPH) samples from the central prostate. This finding of a biallelic expression of IGF-I1 in both cancerous and adjacent peripheral prostate specimens suggests a tissue-specific molecular event that may predispose peripheral prostatic tissue to the development of carcinogenesis and furthermore implicates alterations in methylation patterns in prostate diseases.

The altered patterns of methylation demonstrated in malignant cells suggest that 5'-methyltransferase, the enzyme that establishes and maintains methylation, may be important in tumorigenesis. 5'-Meth- yltransferase overexpression has been noted in many neoplastic tissues and cell lines [48]. In a recent study, a reduction in DNA methyltransferase activity reduced the formation of Apc?"-induced neoplasia in the Min mouse, suggesting that regional DNA hypomethylation may permit the re-expression of silenced tumor-suppressor genes [49]. Similarly, trans- fection of a murine tumor cell line with an antisense to methyltransferase resulted in a reversal of the tu- morigenic phenotype [MI. Recently, several demethylating agents have been demonstrated in vitro and in vivo to re-express silenced genes [51,52]. These agents have potential as new therapeutic strategies for the re-expression of inactivated tumor suppressor genes.

40 larrard et al.

TUMOR SUPPRESSOR GENES

While DNA methylation appears to be an important nonmutational mechanism of gene alteration, previ- ous molecular genetic studies in cancer have generally focused on alterations in tumor suppressor genes and oncogenes. The initiation and progression of prostate cancer clearly require not only gains in critical oncogene function (for review, see [53-55]), but a loss of growth suppressive or tumor suppressive functions. Deletional analysis has supported the involvement of several known and unknown tumor suppressor gene candidates in prostate cancer [56,57]. Located on chromosome 17, p53 encodes a phosphoprotein that pre- vents cell progression through the G1 phase of the cell cycle and is therefore thought to permit cells with damaged genomes to either growth arrest or initiate programmed cell death [58]. Mutations of p53 have been demonstrated in three of five prostate cancer cell lines, and the growth of these lines is suppressed by the introduction of a wild-type copy of this gene [59]. Mutations in primary prostate cancer appear to occur at a low frequency (-10-20%) as compared to other cancers [60-621, although some controversy exists with respect to this value [63]. Immunostaining to detect mutant p53 protein accumulation has led to similar conclusions [64-661. In studies of advanced disease, mutant p53-immunostained deposits were associated with a worse clinical outcome, higher disease stage, and the transition to hormone-refractory disease [65-671. These results suggest that p53 mutations occur as a late event in the progression of prostate cancer.

Rb tumor suppressor gene, first demonstrated to be inactivated in retinoblastoma, encodes a protein involved in the regulation of cell cycle progression, primarily by regulating the action of the E2f family of transcription factors [68]. Initial studies with prostate cancer cell lines demonstrated a deletion in exon 21 in the DU145 prostate cancer cell line, and reintroduc- tion of the wild-type Rb gene suppressed tumorigenicity of this cell line [69]. Analysis of the Rb promoter in 24 prostate cancers and exon 21 in 7 cancers failed to reveal any alterations [70]. Recently, a VNTR in intron 20 of the gene was examined, and 11 of 47 (27%) of primary tumors contained allelic loss of one copy [71]. This suggests that Rb inactivation may be important in the development of a subset of prostate cancers. Deleted in colorectal carcinoma (DCC) demonstrated loss of heterozygosity (LOH) in 6 of 23 (26%) of prostate tumors, with 5 of these deletions in advanced-stage cancers [72]. LOH at 5q21 was recently noted at the adenomatous polyposis coli (APC) locus in 20-63% of informative prostate cancers [ 72,731. Involvement of these tumor suppressor

genes in prostate cancer, first identified in other tumors, suggests that common pathways exist between certain cancer phenotypes.

A new group of cancer-associated genes, those involved in DNA repair (e.g., hMSH2), have been demonstrated to be mutated in the germlines of patients with HNPCC [ 121. Tumors from these individuals are characterized by thousands of spontaneous errors in replication manifested by alterations in the repeat length of simple sequence repeats (i.e., microsatellite instability) [74]. We have examined sporadic prostate cancer DNA replication errors, similar to those in HNPCC, and have found that a minor subset of prostate cancers ( ~ 5 % ) do manifest the replication error phenotype (RER+) [75]. Others have reported a higher frequency of microsatellite instability in prostate cancer [76]. The incidence of the RER+ phenotype is markedly increased in HNPCC and in inherited nonmelanoma skin cancers [77,78], and it will be of interest to analyze men with hereditary forms of prostate cancer for this type of genetic instability.

Recently, using the microcell transfer technique, a gene from chromosome llp11.2 was isolated (desig- nated KAI-1) and shown to suppress metastases when introduced into the rat AT6.1 prostate cancer model [79]. KAI-1 has a reduced expression in metastatic cell lines and appears to be responsible for the metastatic suppressive activity of this chromosome. A particularly well-characterized gene, E-cadherin, mapped to 16q22.1, is a cell adhesion molecule found to be important in neoplastic progression, particularly as a suppressor of invasion (for review, see [SO]). This gene also appears to be inactivated by 5’ CpG island methylation in the prostate “1.

LOSS OF HETEROZYGOSITY

Putative tumor suppressor genes may also be identified through studies involving loss of heterozygosity. We studied allelic loss in prostate cancer using polymorphic DNA probes and found the majority (61%) to exhibit allelic loss on at least one of the chromosomes examined [56]. This study and others [82] have identified chromosomes 8, 10, and 16 as the most common sites of allelic loss in prostate cancer. Concentrating on 8p, a high rate of loss of alleles (60%) was demonstrated in prostate cancers examined [57]. A deletional “hot spot” was identified at locus 8p22 in 70% of tumors and this, and another more proximal region [83], are hypothesized to contain one or more tumor suppressor genes important in prostate cancer and possibly other common human tumors, including colorectal and lung cancers [84,85].

Cytogenetic analyses of prostate cancer specimens have demonstrated few consistent regions of chromo-


soma1 deletion [86]. The analysis is difficult in part due to the low mitotic index and the preferential growth of nonmalignant cells. Recently, Visakorpi et al. [87] used comparative genomic hybridization (CGH), a new method that surveys both deletions and gains of chromosomal material throughout the entire genome. In primary, untreated prostate cancers, they found chromosome 8p and 13q to be the most frequently deleted (32%), followed by 6q, 16q, 18q, and 9p. Chromosomal gains were less frequent; however, in a subset of 9 advanced hormone unre- sponsive prostate tumors, there was a significant increase in chromosomes, including 8q (79%), 7p, and X, as well as a notable increase in deletions on 8p and 5q. Using CGH with Southern and microsatellite analysis, Cher et al. [88] analyzed 20 advanced prostate cancers (primarily lymph node metastases) and found frequent deletions on 8p (68%) and 13q (70%), followed by chromosomes 5q, 6q, 2q, lOq, and 16q. Amplifications in copy number were noted on 8q in 80% of cases, with gains noted in other regions as well. Although CGH analysis was unable to detect a known homozygous deletion at 8p22 in one sample, the concordance between CGH and Southern or microsatellite analysis was excellent, with agreement at 92% of informative loci [a]. This powerful new tool therefore provides important confirmation of previ- ous studies, as well as identifymg other chromosomal regions potentially containing tumor suppressor genes or oncogenes.

CONCLUSION

It is apparent from the increasing number of important findings arising from careful molecular bio- logical studies in the prostate that the initiation and progression of prostate cancer are complex and require a series of genetic events. There are now several identified genetic and epigenetic mechanisms for causing alterations in specific gene expression central to cellular growth and differentiation. The demonstration of DNA methylation represents a new paradigm for the inactivation of tumor suppressor or growth suppressor genes. It joins the "rogues gal- lery" of DNA deletion and point mutation as an important mechanism in altering growth regulatory genes involved in cancer progression. The familial clustering of prostate cancer suggests an inherited basis for a subset of the disease; identification of this inherited cancer susceptibility allele may further en- hance our ability to understand cancer predisposition at the molecular level. Understanding genetic alterations involved in the pathogenesis of prostate cancer will lead to the identification of markers for progression and the development of directed therapeutic

strategies for the treatment of this disease. The rap- idly increasing number of patients who will be diag- nosed with, and those who will die of, prostate cancer lend urgency to understanding the sequential molecular alterations involved in the development of prostate cancer.

ACKNOWLEDGMENTS

Support is provided by the American Foundation for Urologic Disease/George Sheehan Foundation (D.J.), NIH training grant DK07552, NCI CA59457 (G.B.), and SPORE grant CA58236 (W.I.).

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DNA methylation, molecular genetic, and linkage studies in prostate cancer

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