G. Steven Bova

Major susceptibility locus for prostate cancer on chromosome 1 suggested by a genome-wide search.

Despite its high prevalence, very little is known regarding genetic predisposition to prostate cancer. A genome-wide scan performed in 66 high-risk prostate cancer families has provided evidence of linkage to the long arm of chromosome 1 (1q24-25). Analysis of an additional set of 25 North American and Swedish families with markers in this region resulted in significant evidence of linkage in the combined set of 91 families. The data provide strong evidence of a major prostate cancer susceptibility locus on chromosome 1.

Evidence for a prostate cancer susceptibility locus on the X chromosome.

Over 200,000 new prostate cancer cases are diagnosed in the United States each year, accounting for more than 35% of all cancer cases affecting men, and resulting in 40,000 deaths annually1. Attempts to characterize genes predisposing to prostate cancer have been hampered by a high phenocopy rate, the late age of onset of the disease and, in the absence of distinguishing clinical features, the inability to stratify patients into subgroups relative to suspected genetic locus heterogeneity. We previously performed a genome-wide search for hereditary prostate cancer (HPC) genes, finding evidence of a prostate cancer susceptibility locus on chromosome 1 (termed HPC1; ref. 2). Here we present evidence for the location of a second prostate cancer susceptibility gene, which by heterogeneity estimates accounts for approximately 16% of HPC cases. This HPC locus resides on the X chromosome (Xq27-28), a finding consistent with results of previous population-based studies suggesting an X-linked mode of HPC inheritance. Linkage to Xq27-28 was observed in a combined study population of 360 prostate cancer families collected at four independent sites in North America, Finland and Sweden. A maximum two-point lod score of 4.60 was observed at DXS1113, θ=0.26, in the combined data set. Parametric multipoint and non-parametric analyses provided results consistent with the two-point analysis. evidence for genetic locus heterogeneity was observed, with similar estimates of the proportion of linked families in each separate family collection. Genetic mapping of the locus represents an important initial step in the identification of an X-linked gene implicated in the aetiology of HPC.

Copy Number Analysis Indicates Monoclonal Origin of Lethal Metastatic Prostate Cancer

Many studies have shown that primary prostate cancers are multifocal and are composed of multiple genetically distinct cancer cell clones. Whether or not multiclonal primary prostate cancers typically give rise to multiclonal or monoclonal prostate cancer metastases is largely unknown, although studies at single chromosomal loci are consistent with the latter case. Here we show through a high-resolution genome-wide single nucleotide polymorphism and copy number survey that most, if not all, metastatic prostate cancers have monoclonal origins and maintain a unique signature copy number pattern of the parent cancer cell while also accumulating a variable number of separate subclonally sustained changes. We find no relationship between anatomic site of metastasis and genomic copy number change pattern. Taken together with past animal and cytogenetic studies of metastasis and recent single-locus genetic data in prostate and other metastatic cancers, these data indicate that despite common genomic heterogeneity in primary cancers, most metastatic cancers arise from a single precursor cancer cell. This study establishes that genomic archeology of multiple anatomically separate metastatic cancers in individuals can be used to define the salient genomic features of a parent cancer clone of proven lethal metastatic phenotype.

Hypermethylation of CpG Islands in Primary and Metastatic Human Prostate Cancer

Aberrant DNA methylation patterns may be the earliest somatic genome changes in prostate cancer. Using real-time methylation-specific PCR, we assessed the extent of hypermethylation at 16 CpG islands in DNA from seven prostate cancer cell lines (LNCaP, PC-3, DU-145, LAPC-4, CWR22Rv1, VCaP, and C42B), normal prostate epithelial cells, normal prostate stromal cells, 73 primary prostate cancers, 91 metastatic prostate cancers, and 25 noncancerous prostate tissues. We found that CpG islands at GSTP1, APC, RASSF1a, PTGS2, and MDR1 were hypermethylated in >85% of prostate cancers and cancer cell lines but not in normal prostate cells and tissues; CpG islands at EDNRB, ESR1, CDKN2a, and hMLH1 exhibited low to moderate rates of hypermethylation in prostate cancer tissues and cancer cell lines but were entirely unmethylated in normal tissues; and CpG islands at DAPK1, TIMP3, MGMT, CDKN2b, p14/ARF, and CDH1 were not abnormally hypermethylated in prostate cancers. Receiver operator characteristic curve analyses suggested that CpG island hypermethylation changes at GSTP1, APC, RASSF1a, PTGS2, and MDR1 in various combinations can distinguish primary prostate cancer from benign prostate tissues with sensitivities of 97.3–100% and specificities of 92–100%. Hypermethylation of the CpG island at EDNRB was correlated with the grade and stage of the primary prostate cancers. PTGS2 CpG island hypermethylation portended an increased risk of recurrence. Furthermore, CpG island hypermethylation patterns in prostate cancer metastases were very similar to the primary prostate cancers and tended to show greater differences between cases than between anatomical sites of metastasis.

The evolutionary history of lethal metastatic prostate cancer

Cancers emerge from an ongoing Darwinian evolutionary process, often leading to multiple competing subclones within a single primary tumour. This evolutionary process culminates in the formation of metastases, which is the cause of 90% of cancer-related deaths. However, despite its clinical importance, little is known about the principles governing the dissemination of cancer cells to distant organs. Although the hypothesis that each metastasis originates from a single tumour cell is generally supported, recent studies using mouse models of cancer demonstrated the existence of polyclonal seeding from and interclonal cooperation between multiple subclones. Here we sought definitive evidence for the existence of polyclonal seeding in human malignancy and to establish the clonal relationship among different metastases in the context of androgen-deprived metastatic prostate cancer. Using whole-genome sequencing, we characterized multiple metastases arising from prostate tumours in ten patients. Integrated analyses of subclonal architecture revealed the patterns of metastatic spread in unprecedented detail. Metastasis-to-metastasis spread was found to be common, either through de novo monoclonal seeding of daughter metastases or, in five cases, through the transfer of multiple tumour clones between metastatic sites. Lesions affecting tumour suppressor genes usually occur as single events, whereas mutations in genes involved in androgen receptor signalling commonly involve multiple, convergent events in different metastases. Our results elucidate in detail the complex patterns of metastatic spread and further our understanding of the development of resistance to androgen-deprivation therapy in prostate cancer.

Germline mutations and sequence variants of the macrophage scavenger receptor 1 gene are associated with prostate cancer risk

Deletions on human chromosome 8p22–23 in prostate cancer cells and linkage studies in families affected with hereditary prostate cancer (HPC) have implicated this region in the development of prostate cancer. The macrophage scavenger receptor 1 gene (MSR1, also known as SR-A) is located at 8p22 and functions in several processes proposed to be relevant to prostate carcinogenesis. Here we report the results of genetic analyses that indicate that mutations in MSR1 may be associated with risk of prostate cancer. Among families affected with HPC, we identified six rare missense mutations and one nonsense mutation in MSR1. A family-based linkage and association test indicated that these mutations co-segregate with prostate cancer (P = 0.0007). In addition, among men of European descent, MSR1 mutations were detected in 4.4% of individuals affected with non-HPC as compared with 0.8% of unaffected men (P = 0.009). Among African American men, these values were 12.5% and 1.8%, respectively (P = 0.01). These results show that MSR1 may be important in susceptibility to prostate cancer in men of both African American and European descent.

Germline and Somatic Mutations of the STK11/LKB1 Peutz-Jeghers Gene in Pancreatic and Biliary Cancers

Peutz-Jeghers syndrome (PJS) is an autosomal-dominant disorder characterized by hamartomatous polyps in the gastrointestinal tract and by pigmented macules of the lips, buccal mucosa, and digits. Less appreciated is the fact that PJS also predisposes patients to an increased risk of gastrointestinal cancer, and pancreatic cancer has been reported in many PJS patients. It was recently shown that germline mutations of the STK11/LKB1 gene are responsible for PJS. We investigated the role of STK11/LKB1 in the development of pancreatic and biliary cancer in patients with and without the PJS. In a PJS patient having a germline splice site mutation in the STK11/LKB1 gene, sequencing analysis of an intestinal polyp and pancreatic cancer from this patient revealed loss of the wild-type allele of the STK11/LKB1 gene in the cancer. Inactivation of STK11/LKB1, by homozygous deletions or somatic sequence mutations coupled with loss of heterozygosity, was also demonstrated in 4-6% of 127 sporadic pancreatic and biliary adenocarcinomas. Our results demonstrate that germline and somatic genetic alterations of the STK11/LKB1 gene may play a causal role in carcinogenesis and that the same gene contributes to the development of both sporadic and familial forms of cancer.

Androgen-induced TOP2B-mediated double-strand breaks and prostate cancer gene rearrangements

DNA double-strand breaks (DSBs) can lead to the development of genomic rearrangements, which are hallmarks of cancer. Fusions between TMPRSS2, encoding the transmembrane serine protease isoform 2, and ERG, encoding the v-ets erythroblastosis virus E26 oncogene homolog, are among the most common oncogenic rearrangements observed in human cancer. We show that androgen signaling promotes co-recruitment of androgen receptor and topoisomerase II beta (TOP2B) to sites of TMPRSS2-ERG genomic breakpoints, triggering recombinogenic TOP2B-mediated DSBs. Furthermore, androgen stimulation resulted in de novo production of TMPRSS2-ERG fusion transcripts in a process that required TOP2B and components of the DSB repair machinery. Finally, unlike normal prostate epithelium, prostatic intraepithelial neoplasia cells showed strong coexpression of androgen receptor and TOP2B. These findings implicate androgen-induced TOP2B-mediated DSBs in generating TMPRSS2-ERG rearrangements.

Evaluation of Non-Formalin Tissue Fixation for Molecular Profiling Studies

Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analyzed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. Complete protocols and additional discussion of relevant issues are available on an accompanying website (http://cgap-mf.nih.gov/).

GSTP1 CpG Island Hypermethylation Is Responsible for the Absence of GSTP1 Expression in Human Prostate Cancer Cells

GSTP1 CpG island hypermethylation is the most common somatic genome alteration described for human prostate cancer (PCA); lack of GSTP1 expression is characteristic of human PCA cells in vivo. We report here that loss of GSTP1 function may have been selected during the pathogenesis of human PCA. Using a variety of techniques to detect GSTP1 CpG island DNA hypermethylation in PCA DNA, we found only hypermethylated GSTP1 alleles in each PCA cell in all but two PCA cases studied. In these two cases, CpG island hypermethylation was present at only one of two GSTP1 alleles in PCA DNA. In one of the cases, DNA hypermethylation at one GSTP1 allele and deletion of the other GSTP1 allele were evident. In the other case, an unmethylated GSTP1 allele was detected, accompanied by abundant GSTP1 expression. GSTP1 CpG island DNA hypermethylation was responsible for lack of GSTP1 expression by LNCaP PCA cells: treatment of the cells with 5-azacytidine (5-aza-C), an inhibitor of DNA methyltransferases, reversed the GSTP1 promoter DNA hypermethylation, activated GSTP1 transcription, and restored GSTP1 expression. GSTP1 promoter activity, assessed via transfection of GSTP1 promoter-CAT reporter constructs in LNCaP cells, was inhibited by SssI-catalyzed CpG dinucleotide methylation. Remarkably, although selection for loss of GSTP1 function may be inferred for human PCA, GSTP1 did not act like a tumor suppressor gene, as LNCaP cells expressing GSTP1, either after 5-aza-C treatment or as a consequence of transfection with GSTP1 cDNA, grew well in vitro and in vivo. Perhaps, GSTP1 inactivation may render prostatic cells susceptible to additional genome alterations, caused by electrophilic or oxidant carcinogens, that provide a selective growth advantage.