Cancer Letters

Cancer Letters

Volume 225, Issue 1, 8 July 2005, Pages 121-130
Cancer Letters

Mutation analysis of the 8p candidate tumour suppressor genes DBC2 (RHOBTB2) and LZTS1 in bladder cancer

https://doi.org/10.1016/j.canlet.2004.10.047Get rights and content

Abstract

Genomic deletions of the short arm of chromosome 8 are common in many human cancers and are frequently associated with a more aggressive tumour phenotype. One of the regions of loss of heterozygosity (LOH) on 8p22 identified in bladder cancer contains two genes, LZTS1 (FEZ1) and DBC2 (RHOBTB2) that have been shown to be mutated at low frequency in other cancers. We screened a panel of bladder tumours and bladder tumour-derived cell lines for mutations in these genes. Forty two percent of the tumours were found to have LOH in the 8p22 region and many of the cell lines have known loss of 8p. Several known polymorphisms and novel polymorphisms were detected. One possible mutation of LZTS1 (G374S) was found in a cell line. The functional significance of this is unknown but the novel serine residue created may represent a novel phosphorylation site. In DBC2, we found a single somatic mutation in a tumour (E349D) that lies in a highly conserved region of the protein. mRNA levels for both genes were reduced in the majority of bladder cancer cell lines. We conclude that neither LZTS1 nor DBC2 is commonly mutated in bladder cancer. However, neither can yet be excluded as the target of 8p22 LOH. The finding of a somatic mutation of DBC2 in a tumour sample and the down-regulation of both gene transcripts in bladder tumour cell lines may indicate that an alternative mechanism of inactivation of the second allele, for example promoter hypermethylation, is more common than mutation and this must now be examined.

Introduction

Genomic deletions involving the short arm of chromosome 8 are common in a wide range of epithelial malignancies including prostate [1], [2], [3], colorectal [4], [5], breast [6], [7], [8], head and neck [9], [10], [11] and bladder [12], [13], [14] carcinomas. In many of these cancers, loss of heterozygosity (LOH) is associated with a more aggressive tumour phenotype indicating the possible presence of a tumour suppressor gene or genes involved in epithelial tumour progression. In transitional cell carcinoma (TCC) of the bladder, deletions of 8p have been identified by comparative genomic hybridisation (CGH) analysis [15] and LOH is found in 23–52% of cases [13], [14], [16], [17], [18], [19] with a clear association with higher tumour grade and stage. Commonly, approximately half of tumours of stage T1 or T2 have been found to have 8p LOH [14], [17], [18].

Several common regions of deletion on 8p have been mapped by LOH analysis in TCC. These are a telomeric region mapped at 8p23 [16], a region at 8p22 [18], [19] and at least three small non-coincident regions on 8p11-p12 [13], [20]. The region of LOH on 8p22 overlaps a region mapped in oesophageal cancer within which a candidate gene LZTS1 (originally called FEZ1) was identified [21]. This gene, which has a leucine zipper and similarities to the cAMP-responsive binding protein, was found not to be expressed at the RNA level in >60% of epithelial cancers. Mutations were found in primary oesophageal cancers and a prostate cancer cell line [21]. Subsequently, assessment of protein levels showed reduced expression in 44% of gastric carcinomas by immunohistochemistry and a missense mutation in one of these tumours. Hypermethylation was also found in the promoter region in gastric carcinoma cell lines [22]. The same gene was identified following a functional screen of YAC clones spanning a mapped region of LOH in prostate cancer. YAC clones were identified that reduced colony-forming efficiency of a rat prostate carcinoma cell line and isolated LZTS1 cDNA was shown to reduce colony-forming efficiency of these cells. Similarly, introduction of LZTS1 into LZTS1-negative human cancer cells resulted in suppression of tumorigenicity and reduced cell growth with accumulation of cells at late S-G2/M stage of the cell cycle [23]. More recently, levels of LZTS1 protein were measured in bladder cancer cell lines and primary transitional cell carcinomas (TCC). In four of five cell lines and 37 of 60 TCC samples, protein expression was reduced or absent. Restoration of protein expression in a bladder tumour cell line inhibited growth, altered cell cycle progression and suppressed tumorigenicity in nude mice indicating a likely role in the development of TCC [24]. To date, bladder tumours have not been screened for LZTS1 mutation.

A second candidate gene mapped to the 8p22 region of deletion is DBC2 (deleted in breast cancer 2), also known as RHOBTB2. This gene was cloned from a region of 8p22 found to be homozygously deleted in breast cancer [25]. Mutation analysis identified two missense mutations in breast tumour samples and expression analysis revealed loss of expression in many breast cancer cells. This gene contains a ras-related domain at its 5′ end and 2 BTB/POZ domains which in other proteins function as protein–protein interaction motifs. Expression of wildtype and mutant DBC2 cDNA in T-47D breast carcinoma cells indicated a potent anti-proliferative effect of the wildtype protein, confirming its potential role as a tumour suppressor.

Both these candidate genes map within the 8p22 region of LOH mapped in bladder cancer and the protein expression and gene transfer study of Vecchione et al. [24] has already provided strong evidence that LZTS1 may have a tumour suppressor role in the bladder. In this study, we have assessed a series of 54 bladder tumour samples for 8p22 LOH and have screened these and 34 bladder cell lines for mutations in both candidate genes.

Section snippets

Tissue samples

Tissues were obtained with informed consent from patients undergoing cystoscopic resection for bladder cancer. Biopsies were taken with cold cup biopsy forceps at cystoscopy or selected from the resected tumour tissue immediately after resection. Samples were snap frozen in liquid nitrogen. Frozen sections were cut and stained with haematoxylin and eosin. Consecutive sections from regions with at least 80% tumour cell content were used directly for DNA extraction. DNA was extracted using the

Results

Forty two tumours were assessed for LOH on 8p22 using the microsatellite markers NEFL, D8S258 and LPL (Fig. 1). All tumours except one were informative for at least one marker and 18 (43%) showed LOH for at least one marker. Two tumours showed apparent breakpoints within this small region of 8p22. One of these showed retention of heterozygosity for LPL and D8S258 and LOH for NEFL. The other showed retention of heterozygosity for LPL, was non-informative for D8S258 and showed LOH for NEFL. In

Discussion

LZTS1 and DBC2 map to a region of frequent LOH on 8p22 in bladder and other cancers, both have potential functions that would be consistent with tumour suppressor activity and mutations have been identified at low frequency in several cancers analysed. Therefore, we assessed them as candidate tumour suppressor genes for bladder cancer.

Our tumour panel and cell lines contained many samples with 8p22 LOH but only a low frequency of somatic mutation was found. The frequency of LOH detected in

Acknowledgements

We thank Dr Catherine Reznikoff for providing the cell lines 92-1, 97-6, 97-29, 97-7, 97-18, 97-24, 96-1, 97-21 and 97-1, Joanne Robinson for excellent maintenance of our tumour tissue collection and Urologists at St James's Hospital Leeds for their continued interest and support of this work.

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