Keith W. Brown

Somatic Allelic Loss at the DCC, APC, nm23-H1 and P53 Tumor Suppressor Gene Loci in Human Prostatic Carcinoma

We present a restriction fragment length polymorphism (RFLP) analysis of 29 benign and 30 malignant prostatic tumors, using polymorphic DNA probes to the putative tumor suppressor genes DCC (Deleted in Colorectal Carcinoma; chromosome 18q21.3), nm23-H1 (17q21.3), APC (Adenomatous Polyposis Coli; 5q21) and p53 (17p13). Six of 23 evaluable cancers (26%) showed loss of heterozygosity (LOH) at DCC; 5 were advanced stage and one was clinically localized (p < 0.05). Mapping 18q deletions, another (advanced) cancer showed LOH at a locus distal to DCC (18q22), but no LOH at DCC. Three of 15 evaluable cancers (20%), all advanced, showed LOH at APC. Three of eight (38%) cancers, of which 2 were advanced, showed LOH at p53. One high grade/stage cancer of 21 (5%) showed LOH at nm23-H1 (and also at DCC). Combining data, allelic losses at either DCC, APC, or p53 genes were seen in 13% of localized cancers, but in 71% of advanced cancers (p < 0.002). Allelic loss involving nm23-H1 is rare in prostatic carcinoma. We suggest that loss of tumor suppressor genes DCC and/or an unidentified gene located distally on chromosome 18q, APC, or p53 may influence progression in prostatic carcinoma.

Loss of heterozygosity on chromosome 18q is associated with muscle-invasive transitional cell carcinoma of the bladder.

Somatic allelic loss is regarded as a hallmark of tumour-suppressor gene (TSG) inactivation. Thirty-one human bladder transitional cell carcinomas (TCCs) were examined for allelic loss at five chromosome 18q loci, including the DCC gene (deleted in colorectal carcinoma) and at chromosome 11p15 in a restriction fragment length polymorphism analysis. Allelic loss was observed at one or more 18q loci in 9/26 (35%) samples, associated with muscle-invasive disease (P < 0.02). Allelic loss was observed at DCC in 8/24 (33%) samples, associated with muscle-invasive disease (P = 0.05). Three out of the five evaluable recurrent TCCs exhibited allelic loss at DCC, two of which were superficial. No allelic losses were detected at other 18q loci in tumours which retained both DCC alleles. Allelic loss was observed at 11p15 in 5/20 (25%) tumours. These data suggest the presence of a late-acting TSG located on 18q in TCC bladder cancer. DCC is a candidate gene since it lies within the region of most common deletion (18q21.3-qter).

Autoregulation of the human WT1 gene promoter

The human Wilms tumour suppressor gene, WT1, encodes a zinc‐finger protein which can function as a transcriptional activator or suppressor. This study reports the analysis of the human WT1 gene promoter, and demonstrates that high levels of WT1 expression lead to autosuppression of the WT1 promoter. Deletion analyses of the promoter region implicate sequences 5′ and 3′ of the transcriptional start site as being crucial in WT1 autosuppression. Loss or alteration of this function of WT1 may be important in tumourigenesis.

Loss of heterozygosity at 7p in Wilms’ tumour development

Chromosome 7p alterations have been implicated in the development of Wilms’ tumour (WT) by previous studies of tumour cytogenetics, and by our analysis of a constitutional translocation (t(1;7)(q42;p15)) in a child with WT and radial aplasia. We therefore used polymorphic microsatellite markers on 7p for a loss of heterozygosity (LOH) study, and found LOH in seven out of 77 informative WTs (9%). The common region of LOH was 7p15–7p22, which contains the region disrupted by the t(1;7) breakpoint. Four WTs with 7p LOH had other genetic changes; a germline WT1 mutation with 11p LOH, LOH at 11p, LOH at 16q, and loss of imprinting of IGF2. Analysis of three tumour-associated lesions from 7p LOH cases revealed a cystic nephroma-like area also having 7p LOH. However, a nephrogenic rest and a contralateral WT from the two other cases showed no 7p LOH. No particular clinical phenotype was associated with the WTs which showed 7p LOH. The frequency and pattern of 7p LOH demonstrated in our studies indicate the presence of a tumour suppressor gene at 7p involved in the development of Wilms’ tumour.

Epigenetic gene deregulation in cancer

A mini-review of the literature concerning epigenetic gene regulation in cancer.

Perilobar Nephrogenic Rests Are Nonobligate Molecular Genetic Precursor Lesions of Insulin-Like Growth Factor-II-Associated Wilms Tumors

Purpose: Perilobar nephrogenic rests (PLNRs) are abnormally persistent foci of embryonal immature blastema that have been associated with dysregulation at the 11p15 locus by genetic/epigenetic means and are thought to be precursor lesions of Wilms tumor. The precise genomic events are, however, largely unknown. Experimental Design: We used array comparative genomic hybridization to analyze a series of 50 PLNRs and 25 corresponding Wilms tumors characterized for 11p15 genetic/epigenetic alterations and insulin-like growth factor-II expression. Results: The genomic profiles of PLNRs could be subdivided into three categories: those with no copy number changes (22 of 50, 44%); those with single, whole chromosome alterations (8 of 50, 16%); and those with multiple gains/losses (20 of 50, 40%). The most frequent aberrations included 1p- (7 of 50, 14%) +18 (6 of 50, 12%), +13 (5 of 50, 10%), and +12 (3 of 50, 6%). For the majority (19 of 25, 76%) of cases, the rest harbored a subset of the copy number changes in the associated Wilms tumor. We identified a temporal order of genomic changes, which occur during the insulin-like growth factor-II/PLNR pathway of Wilms tumorigenesis, with large-scale chromosomal alterations such as 1p-, +12, +13, and +18 regarded as “early” events. In some of the cases (24%), the PLNRs harbored large-scale copy number changes not observed in the concurrent Wilms tumor, including +10p, +14q, and +18. Conclusions: These data suggest that although the evidence for PLNRs as precursors is compelling, not all lesions must necessarily undergo malignant transformation.

Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells

Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over‐expression. Here we present data showing that short‐interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell‐lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN‐negative SH‐SY5Y NB cell‐line, or in two immortalized human fibroblast cell‐lines. Immunoblotting of NB cell‐lines shows that high PRMT5 expression is strongly associated with MYCN‐amplification (P < 0.004, Mann–Whitney U‐test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN‐amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN‐overexpressing cells, including the SHEP‐21N cell‐line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN‐associated cell‐death in SHEP‐21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post‐transcriptional level. Reciprocal co‐immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK‐N‐BE(2)C and NGP cell lines. By using liquid chromatography – tandem mass spectrometry (LC‐MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post‐translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small‐molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene.

Transactivation of the WT1 antisense promoter is unique to the WT1[+/-] isoform

The Wilms’ tumour suppressor gene, WT1, encodes a zinc finger transcription factor that has been shown to repress a variety of cellular promoters via binding to cognate DNA elements. Our earlier work identified an antisense WT1 promoter that contains WT1 consensus sites, but is transcriptionally activated by WT1. In this study, we demonstrate that, unlike previous reports of transcriptional regulation by WT1, transactivation of the antisense promoter is unique to a single isoform of WT1. Of the four alternatively spliced isoforms in which exon 5 (at splice I) or amino acid residues KTS (at splice II) are inserted or omitted, only the WT1 isoform containing splice I and omitting splice II (WT1[+/−]) displays transactivation. We demonstrate that transregulation variations observed with WT1 isoforms are not solely attributable to differential DNA binding by [+KTS] or [−KTS] isoforms. Thus, the transactivation of the antisense promoter displays an absolute requirement for exon 5, suggesting that interaction between WT1 and other cellular factors is necessary for this regulatory function.

Wilms' tumor: a paradigm for the new genetics.

Carcinogenesis can be triggered by a diverse range of molecular lesions, a variety of which can be illustrated by Wilms tumor (WT), a pediatric kidney cancer. Molecular defects observed in WTs include several independent targets and mechanisms best exemplified by changes on the short arm of chromosome 11. This article will review the molecular pathology of WT and emphasize the broader ramifications for cancer genetics. Consideration will be given to carcinogenic pathways, novel cellular molecules, and technologies that will assist in the rapid interpretation and assimilation of DNA sequence data arising from the sequencing of the human genome.

MYCN is recruited to the RASSF1A promoter but is not critical for DNA hypermethylation in neuroblastoma

Tumor suppressor genes such as RASSF1A are often epigenetically repressed by DNA hypermethylation in neuroblastoma, where the MYCN proto‐oncogene is frequently amplified. MYC has been shown to associate with DNA methyltransferases, thereby inducing transcriptional repression of target genes, which suggested that MYCN might play a similar mechanistic role in the hypermethylation of tumor suppressor genes in neuroblastoma. This study tested that hypothesis by using co‐immunoprecipitation and ChIP to investigate MYCN–DNA methyltransferase interactions, together with MYCN knock‐down and over‐expression systems to examine the effect of MYCN expression changes on gene methylation, employing both candidate gene and genome‐wide assays. We show that MYCN interacts with DNA methyltransferases and is recruited to the promoter region of RASSF1A. However, using four model systems, we showed that long‐term silencing of MYCN induces only a small loss of DNA methylation at the RASSF1A promoter in MYCN amplified neuroblastoma cell lines and over‐expression of MYCN does not induce any DNA methylation, suggesting that MYCN is not critical for DNA hypermethylation in neuroblastoma.