Molecular analysis of the FHIT gene in human prostate cancer.

A variety of studies suggest that the FHIT gene, which encompasses the fragile site at 3p14.2, is a candidate tumor suppressor gene in several forms of human cancer. To determine whether the FHIT gene is altered in prostate carcinomas, we examined 15 prostate tumors, four normal prostate tissue specimens and RNA from a pool of 62 normal human prostate tissues (Clontech) for the integrity of FHIT transcripts, using a robust single-stage PCR and a nested PCR method. In each case a major FHIT-specific full-length product was observed. Additional aberrantly sized products, which were more numerous in the nested PCR strategy, were present at a far lower level than the full-length transcripts in 14 of 15 tumors, three of four normal human prostate tissues and in the pooled normal prostate RNA. Sequence analysis revealed that these aberrant products corresponded to alternatively spliced FHIT transcripts, which were neither more numerous nor more prominent in the tumors than in the normal prostate specimens. Deletion at the FHIT locus was also evaluated by using three intragenic polymorphic markers (D13S1481, D3S1300, and D3S1234). Allelic loss was observed in two tumors, but these genomic alterations did not correspond to the aberrant FHIT transcripts. DNA analysis, furthermore, suggested that the tumor heterogeneity was not a likely explanation for presence of normal and alternatively spliced FHIT transcripts in the prostate tumors. In conclusion, we detected neither frequent loss of heterozygosity nor tumor specific transcripts of the FHIT gene in human prostate cancer.

PTEN/MMAC1/TEP1 involvement in primary prostate cancers.

The PTEN/MMAC1/TEP1 gene, located at 10q23.3, is a tumor suppressor gene responsible for the familial cancer syndromes Cowden disease and Bannayan-Zonana syndrome, and is commonly somatically mutated in several types of cancers. Mutations of the PTEN gene have been found in prostate cancer cell lines and LOH at 10q22-24 in prostate tumors have also been described with a high frequency. To determine the role of this gene in prostate tumorigenesis, we therefore analysed 22 primary tumors for loss of heterozygosity (LOH) within the 10q22-23 region such that tumors hemizygous at those loci may be examined for somatic PTEN mutations. Losses of heterozygosity of at least one locus was found in 12 (55%) of the 22 tumors DNAs. Among these, six tumors exhibited allele loss in the interval between D10S1765 and D10S541 wherein lies the PTEN gene. We searched the entire coding region of PTEN for somatic mutations in these six tumors. One somatic mutation (17%), a 1 bp deletion, was detected in exon 7 of the gene, in one tumor, indicating that somatic mutations of the PTEN gene may occur in primary prostate tumors.

Loss of heterozygosity at chromosome arm 13q and RB1 status in human prostate cancer.

Aberrations of the long arm of chromosome 13 are common in prostate cancer and were initially attributed to alterations of the RB1 gene in band q14 of the chromosome. However, prostate tumors generally yield normal p110RB1 nuclear staining despite loss of heterozygosity (LOH) at the RB1 locus. Our previous analysis of chromosome arm 13q showed allelic loss in 41% of primary prostate tumors. To refine our knowledge of 13q, we extended our previous LOH study by using more polymorphic markers to analyze more prostate tumors. Sixty human prostate carcinomas were screened for allelic loss on 13q by using 13 13q-specific markers. LOH on the long arm of chromosome 13 was found in 39 (65%) of the 60 tumors. Furthermore, 33 of these 39 tumors had evidence of allelic loss involving a region of 13q14 containing RB1. Because immunohistochemical assessment of pRb expression is controversial in prostate tumors, we used a quantitative reverse transcription polymerase chain reaction (RT-PCR) method to determine whether RB1 is the target tumor suppressor gene in this region. RB1 mRNA steady-state levels were determined in 12 prostate tumors preselected on the basis of presumed deletion at the RB1 locus and four prostate tumors without LOH at the RB1 locus; five normal prostate specimens were used as controls. One of the 12 assessable prostate tumors with presumed LOH at RB1 showed a corresponding decreased in RB1 mRNA expression, whereas none of the four tumors without LOH at RB1 locus showed such a decrease. This study, based on another technical approach, confirms that RB1 is not the main target of the observed LOH at 13q14.3, and raises the possibility that another tumor suppressor gene in this region plays a key role in prostate cancer.

VEGF overexpression in clinically localized prostate tumors and neuropilin-1 overexpression in metastatic forms.

Studies comparing tumor neovascularity with pathological findings suggest that angiogenesis contributes to the pathogenesis of prostate cancer. We have examined 42 primary sporadic prostate tumors at different clinical stages, together with 3 prostate cancer cell lines (DU145, PC3 and LNCaP), for expression of VEGF and the gene encoding the recently identified VEGF165 isoform-specific receptor neuropilin-1, by using a quantitative reverse transcription (RT)-PCR method. We also evaluated the VEGF transcription pattern. Upregulation of VEGF and neuropilin-1 was observed in 12 and 14 tumors, respectively. The VEGF165 isoform was slighly overrepresented in tumors that overexpressed VEGF. VEGF overexpression correlated with stage II disease (p < 0.05); neuropilin-1 overexpression correlated with advanced disease (p < 0. 01) and a high Gleason grade (p < 0.02). Our observations suggest that VEGF expression could be used as a prognostic marker in early-stage prostate tumors, whereas neuropilin-1 overexpression might be a marker of aggressiveness.

Expression and mutational analysis of the MADR2/Smad2 gene in human prostate cancer.

BACKGROUND: Loss of heterozygosity (LOH) on chromosome arm 18q is common in sporadic prostate cancer and may be involved in cancer development through inactivation of tumor-suppressor genes (TSG). Recent identification, at 18q21.1, of MADR2/Smad2, a key component in transforming growth factor beta (TGFbeta)-family signaling pathways, led us to investigate the role of this gene in prostate tumorigenesis. METHODS: Sporadic primary prostate tumors from 25 patients with clinically localized tumors and 7 with metastatic forms were examined for MADR2/Smad2 mutations by using polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis of cDNA, and for gene expression by quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: We detected no mutation in MADR2/Smad2 and no abnormal mRNA expression. CONCLUSIONS: Despite recent evidence indicating that MADR2/Smad2 acts as a tumor-suppressor gene, our findings suggest a limited role of this gene in prostate tumorigenesis, at least in the early stages. Another key tumor-suppressor gene may therefore be the main target of the observed LOH at 18q21.1.