Hyper-methylation of RIZ1 tumor suppressor gene is involved in the early tumorigenesis of hepatocellular carcinoma

The retinoblastoma protein-interacting zinc finger gene RIZ1 is a putative tumor suppressor gene, and the inactivation of the RIZ1 is frequently found in tumors through a loss of mRNA expression. In order to understand the role of RIZ1 inactivation in the tumorigenesis of hepatocellular carcinoma (HCC), we detected the RIZ1 promoter methylation status in 39 HCCs using a methylation specific PCR (MSP) method, and carried out LOH study with marker P704. We also assessed the associations between the methylation status and clinicopathological parameters, tumor size, tumor differentiation, and fractional allelic loss (FAL). The results showed that the RIZ1 promoter methylated both in advanced tumors (>3 cm), (18/31, 58.0%) and in early tumors (<3 cm), (4/8, 50.0%). There were 54.6% (12/22) tumors with hyper-methylation in the low FAL group and 45.5% (10/22) in the high FAL group. Moreover, the DNA methylation of the RIZ1 promoter was found not only in the poorly differentiated tumors (12/22, 54.6%), but also in the well differentiated tumors (10/22, 45.5%). Among the 22 HCCs (22/39, 56.4%) that showed hyper-methylation at the RIZ1 promoter region, 3 cases showed biallelic methylation. Interestingly, one case showed hyper-methylation on one allele and a loss of heterozygosity (LOH) on the other allele. In other words, 4 HCCs showed the biallelic inactivation of the RIZ1. These results suggest that the inactivation of the RIZ1 by DNA methylation at its promoter region is involved in the tumorigenesis of HCC, particularly in the early stage of disease.     

Allelic expression of the putative tumor suppressor gene p73 in human fetal tissues and tumor specimens

p73, a proposed tumor suppressor, shares significant amino acid sequence homology with p53. However, p73 is rarely mutated in tumors but it has been suggested that p73 is monoallelically expressed in some tissues. This latter feature would predispose p73 to gene inactivation because a single genetic 'hit' or the loss of the expressed parental allele would leave the cell without p73 activity. We examined the allelic expression of p73 in normal fetal tissues and in ovarian cancer and Wilms' tumor. We found that p73 was biallelically expressed in all fetal tissues, except in brain, where differential expression of the two parental alleles was observed. Biallelic expression of p73 was also observed in paired samples of ovary cancer and Wilms' tumor. Loss of heterozygosity of p73 occurred at relatively low rates in tumors: one of 11 informative samples (9.1%) of ovarian cancer and two of 19 (10.1%) Wilms' tumors. These data demonstrate that p73 is biallelically expressed in most tissues, thus excluding genomic imprinting as a molecular mechanism to predispose to allelic inactivation of p73 in human tumors.     

Allelotype of human thyroid tumors: loss of chromosome 11q13 sequences in follicular neoplasms.

Two classes of genes are the targets of mutations involved in human tumorigenesis: oncogenes, the activation of which leads to growth stimulation, and tumor suppressor genes, which become tumorigenic through loss of function, often through allelic deletion. To obtain evidence for a role for tumor suppressor genes in thyroid tumorigenesis, we examined DNA from 80 thyroid neoplasms for loss of heterozygosity in multiple chromosomal loci using 19 polymorphic genomic probes. None of the informative thyroid tumors studied had allelic loss detected with probes for chromosome 2q (D2S44), 3p (D3F15S2, D3S32), 3q (D3S46), 4p (D4S125), 6p (D6S40), 8q (D8S39), 9q (D9S7), 12p (D12S14), 13q (D13S52), 17p (D17S30), or 18q (D18S10). One of eight of the follicular adenomas had a 10q deletion detected with marker D10S15, and one of 26 had a 10q deletion detected with D10S25. One of two of the follicular carcinomas had an 11p deletion in the H-ras locus. The most significant findings were on chromosome 11q13, the site containing the putative gene predisposing to multiple endocrine neoplasia type I. Four of 27 follicular adenomas had loss of heterozygosity for probes in this region. Allelic deletions were detected with the following probes: D11S149, PYGM, D11S146, and INT2. None of 13 informative papillary carcinomas and none of two follicular carcinomas had loss of heterozygosity detectable with these 11q13 markers. Allelic loss is a relatively infrequent event in human thyroid tumors. Deletions of chromosome 11q13 are present in about 14% of follicular, but not papillary, neoplasms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental defects in Gorlin syndrome related to a putative tumor suppressor gene on chromosome 9.

Gorlin syndrome is an autosomal dominant disorder that predisposes to basal cell carcinomas of the skin, ovarian fibromas, and medulloblastomas. Unlike other hereditary disorders associated with cancer, it features widespread developmental defects. To investigate the possibility that the syndrome is caused by mutation in a tumor suppressor gene, we searched for loss of heterozygosity in 16 sporadic basal cell carcinomas, 2 hereditary basal cell carcinomas, and 1 hereditary ovarian fibroma and performed genetic linkage studies in five Gorlin syndrome kindreds. Eleven sporadic basal cell carcinomas and all 3 hereditary tumors had allelic loss of chromosome 9q31, and all informative kindreds showed tight linkage between the Gorlin syndrome gene and a genetic marker in this region. Loss of heterozygosity at this chromosomal location, particularly in hereditary tumors, implies that the gene is homozygously inactivated and normally functions as a tumor suppressor. In contrast, hemizygous germline mutations lead to multiple congenital anomalies.     

Allelic loss mapping and physical delineation of a region harboring a thymic lymphoma suppressor gene on mouse chromosome 16

Our previous mapping of allelic loss in gamma-ray induced thymic lymphomas in F(1) hybrid and backcross mice between BALB/c and MSM strains identified three regions with high frequencies of allelic loss which probably harbor a tumor suppressor gene. One region, Tlsr7, exists near the D16 Mit122 locus on chromosome 16. This study has further localized Tlsr7 by constructing a physical map and scanning a total of 587 thymic lymphomas. The map consists of 13 overlapping BAC clones and isolation of BAC-derived polymorphic probes leads to fine mapping of allelic losses. Eleven lymphomas show informative breakpoints of allelic loss regions relative to the flanking markers on the map. Pulsed-field gel electrophoresis of NotI digests of the clones shows that the commonly lost region is localized within an approximately 300 kb interval near D16Mit192. This map is invaluable to facilitate the identification of genes in the Tlsr7 region.     

Pooled analysis of loss of heterozygosity in breast cancer: a genome scan provides comparative evidence for multiple tumor suppressors and identifies novel candidate regions

Somatic loss of heterozygosity (LOH) has been widely reported in breast cancer as a means of identifying putative tumor-suppressor genes. However, individual studies have rarely spanned more than a single chromosome, and the varying criteria used to declare LOH complicate efforts to formally differentiate regions of consistent versus sporadic (random) loss. We report here the compilation of an extensive database from 151 published LOH studies of breast cancer, with summary data from >15,000 tumors and primary allelotypes from >4,300 tumors. Allelic loss was evaluated at 1,168 marker loci, with large variation in the density of informative observations across the genome. Using studies in which primary allelotype information was available, we employed a likelihood-based approach with a formal chromosomal instability and selection model. The approach seeks direct evidence for preferential loss at each locus compared with nearby loci, accounts for heterogeneity across studies, and enables the direct comparison of candidate regions across the genome. Striking preferential loss was observed (in descending order of significance) in specific regions of chromosomes 7q, 16q, 13q, 17p, 8p, 21q, 3p, 18q, 2q, and 19p, as well as other regions, in many cases coinciding with previously identified candidate genes or known fragile sites. Many of these observations were not possible from any single LOH study, and our results suggest that many previously reported LOH results are not systematic or reproducible. Our approach provides a comparative framework for further investigation of regions exhibiting LOH and identifies broad genomic regions for which there exist few data.     

The type of somatic mutation at APC in familial adenomatous polyposis is determined by the site of the germline mutation: a new facet to Knudson's 'two-hit' hypothesis.

APC is often cited as a prime example of a tumor suppressor gene. Truncating germline and somatic mutations (or, infrequently, allelic loss) occur in tumors in FAP (familial adenomatous polyposis). Most sporadic colorectal cancers also have two APC mutations. Clues from attenuated polyposis, missense germline variants with mild disease and the somatic mutation cluster region (codons 1,250-1,450) indicate, however, that APC mutations might not result in simple loss of protein function. We have found that FAP patients with germline APC mutations within a small region (codons 1,194-1,392 at most) mainly show allelic loss in their colorectal adenomas, in contrast to other FAP patients, whose 'second hits' tend to occur by truncating mutations in the mutation cluster region. Our results indicate that different APC mutations provide cells with different selective advantages, with mutations close to codon 1,300 providing the greatest advantage. Allelic loss is selected strongly in cells with one mutation near codon 1,300. A different germline-somatic APC mutation association exists in FAP desmoids. APC is not, therefore, a classical tumor suppressor. Our findings also indicate a new mechanism for disease severity: if a broader spectrum of mutations is selected in tumors, the somatic mutation rate is effectively higher and more tumors grow.     

Search for putative suppressor genes in meningioma: significance of chromosome 22

Summary Cytogenetic and molecular studies of various solid tumors have indicated that a series of different chromosomal regions may be deleted in the tumor genome. Usually, losses of heterozygosity are observed and, from this finding, the presence of specific genes acting as tumor suppressors has been deduced. In particular tumors, however, only a single chromosome site appears to be affected. Therefore, we have carried out a study of human meningioma, investigating 7 such putative suppressor regions by applying twelve site-specific DNA markers. In 6 out of 19 tumors, we exclusively found loss of heterozygosity for markers of the long arm of chromosome 22; none of the tumors showed statistically significant additional allelic losses for the regions 1p, 3p, 5p, 5q, 11p, 13q, 17p. Our data support the long-standing observation that only losses of or within chromosome 22 are associated with the development of meningiomas. Other suppressor regions are apparently not involved.     

Analysis of the candidate tumor suppressor Ris-1 in primary human breast carcinomas

Frequent chromosome 3 losses have been described in several tumors types, which strongly suggest the presence of one or several tumor suppressor genes. Recently, a novel candidate tumor suppressor gene termed Ris-1 (for Ras-induced senescence 1) has been identified at chromosomal position 3p21.3. Ris-1 has been proposed to participate in anti-tumor responses that resemble cellular senescence and that are elicited by oncogenes such as Ras. To analyze the role of Ris-1 as a putative tumor suppressor gene in human breast cancer, we have performed a real-time quantitative analysis of its mRNA expression in 60 patients. Moreover, we carried out a first approach to evaluate the most common inactivation mechanism that can affect expression levels of tumor suppressor genes (mutation, promoter hypermethylation and allelic losses). Furthermore, a correlation study between expression as well as inactivating mechanisms of Ris-1 and several clinico-pathological parameters of the tumors was designed, with the objective of appraising the prognostic value of Ris-1 status. Decreased expression of Ris-1 was observed in 23% of the cases and overexpressed Ris-1 was detected in 15% of the primary breast tumors. Our data showed high frequency of LOH (30%) at one of the markers used. Nevertheless, a polymorphism related with the expression levels was described. Statistically significant correlations were found between decreased Ris-1 expression and negative progesterone receptors, as well as between overexpressing Ris-1 tumors and high histological grade. Despite all these data, we conclude that the suggested role of Ris-1 as tumor suppressor gene is not evident, at least in breast cancer. Future and larger series studies in different tumor types are necessary to clarify Ris-1 function in human cancer.     

Mapping of a cadherin gene cluster to a region of chromosome 5 subject to frequent allelic loss in carcinoma

Cadherin adhesion molecules define cellular interactions during embryogenesis and morphogenesis, while later in life they are responsible for maintaining tissue integrity. Mutation and loss of expression of cadherins have been implicated in the progression of some malignant tumors, suggesting that cadherins may also act as tumor/metastasis suppressor genes. To determine the extent to which cadherin loci could be affected by allelic losses, we used radiation hybrid mapping to define the chromosomal position of five cadherin genes. A cadherin gene cluster consisting of three genes was identified on the short arm of chromosome 5. This region of the genome is subjected to frequent allelic loss in malignant disease.     

Frequent loss of the BLID gene in early-onset breast cancer

The BH3-like motif-containing inducer of cell death (BLID) is an intronless gene localized on 11q24.1. Loss of that region has frequently been reported in early-onset breast cancer and is significantly associated with poor prognosis and reduced survival. Downregulation of BLID is associated with younger age, triple-negative phenotype, and reduced disease-free and overall survival of breast cancer patients. In this study, we investigated allelic loss of BLID in breast tumor specimens from 78 women with invasive breast cancer using 2 dinucleotide polymorphic markers closely linked to the BLID gene (no intragenic marker for BLID is available). Seventy-three cases were informative. Overall, loss of heterozygosity (LOH) at the BLID locus was detected in 32% of the informative cases (23/73). However, in patients 40 years old and younger, LOH was detected in 50% of the cases (9/18). Patients aged 40 years and younger were significantly more likely to experience LOH than those aged 41-55 years (p = 0.04). Specifically, the odds of BLID loss for patients aged 40 years and younger were 3.7 times the odds of loss for patients aged 41-55 years (95% CI, 1.1-13). Our findings suggest a tumor suppressor role of the BLID gene in early-onset breast cancer.     

The application of single nucleotide polymorphism microarrays in cancer research

The development of microarray technology has had a significant impact on the genetic analysis of human disease. The recently developed single nucleotide polymorphism (SNP) array can be used to measure both DNA polymorphism and dosage changes. Our laboratory has applied SNP microarray analysis to uncover frequent uniparental disomies and sub-microscopic genomic copy number gains and losses in different cancers. This review will focus on the wide range of applications of SNP microarray analysis to cancer research. SNP array genotyping can determine loss of heterozygosity, genomic copy number changes and DNA methylation alterations of cancer cells. The same technology can also be used to investigate allelic association in cancers. Therefore, it can be applied to the identification of cancer predisposition genes, oncogenes and tumor suppressor genes in specific types of tumors. As a consequence, they have potential in cancer risk assessment, diagnosis, prognosis and treatment selection.     

Meningioma: a cytogenetic model of a complex benign human tumor, including data on 394 karyotyped cases

Meningioma is the most frequent tumor of neuroectodermal origin in humans. It is usually benign. Only a minority of cases shows progression to an anaplastic tumor (WHO grade II and III). Meningioma is generally a sporadic tumor. Multiple and familial cases are rare and mostly associated with (hereditary) neurofibromatosis 2 (NF2). Meningiomas show an unexpectedly high recurrence rate. Also, completely removed low-grade tumors can recur. Recurrence and multiplicity are correlated with the formation of a peritumoral edema. On the cytogenetic level, meningioma is the best-studied tumor in humans. Grade I tumors show either uniform monosomy 22 or a diploid karyotype. The majority of high-grade, but only a minority of low-grade, meningiomas show loss of merlin, a cytoskeleton-cytoplasm-linker protein. Merlin is the product of the NF2 gene located on chromosome 22. A second tumor suppressor gene on chromosome 22 has not yet been detected. In contrast to other solid tumors, progression of meningiomas is correlated with increasing hypodiploidy, showing characteristic clonal evolutions that mostly include chromosomes 14, 18, and 19 and, more rarely, 6 and 10. Structural aberrations are infrequent, except for the loss of the short arm of chromosome 1, which appears to be the decisive step for anaplastic growth. Comparative histochemical and molecular cytogenetic studies point to the alkaline phosphatase gene (ALPL, liver-bone-kidney type) located on 1p36.1-->p34 as a candidate tumor suppressor gene. A model is proposed that tries to explain - with a minimum number of essential steps - the origin, progression, infiltration, and recurrence of meningiomas.     

Cloning and Gene Mapping of the Chromosome 13q14 Region Deleted in Chronic Lymphocytic Leukemia

Frequent deletions and loss of heterozygosity in a segment of chromosome 13 (13q14) in cases of B-cell chronic lymphocytic leukemia (CLL) have suggested that this malignancy is caused by inactivation of an unknown tumor suppressor gene located in this region. Toward the identification of the putative CLL tumor suppressor, we have constructed a high-resolution physical map of YAC, PAC, and cosmid contigs covering 600 kb of the 13q14 genomic region. In addition to densely positioned genetic markers and STSs, this map was further annotated by localization of 32 transcribed sequences (ESTs) using a combination of exon trapping, direct cDNA selection, sample sequencing of cosmids and PACs, and homology searches. On the basis of these mapping data, allelic loss analyses at 13q14 using CLL tumor samples allowed narrowing of the genomic segment encompassing the putative CLL gene to <300 kb. Twenty-three ESTs located within this minimally deleted region are candidate exons for the CLL-associated tumor suppressor gene.     

KIF1Bbeta functions as a haploinsufficient tumor suppressor gene mapped to chromosome 1p36.2 by inducing apoptotic cell death

Deletion of the distal region of chromosome 1 frequently occurs in a variety of human cancers, including aggressive neuroblastoma. Previously, we have identified a 500-kb homozygously deleted region at chromosome 1p36.2 harboring at least six genes in a neuroblastoma-derived cell line NB1/C201. Among them, only KIF1Bbeta, a member of the kinesin superfamily proteins, induced apoptotic cell death. These results prompted us to address whether KIF1Bbeta could be a tumor suppressor gene mapped to chromosome 1p36 in neuroblastoma. Hemizygous deletion of KIF1Bbeta in primary neuroblastomas was significantly correlated with advanced stages (p = 0.0013) and MYCN amplification (p < 0.001), whereas the mutation rate of the KIF1Bbeta gene was infrequent. Although KIF1Bbeta allelic loss was significantly associated with a decrease in KIF1Bbeta mRNA levels, its promoter region was not hypermethylated. Additionally, expression of KIF1Bbeta was markedly down-regulated in advanced stages of tumors (p < 0.001). Enforced expression of KIF1Bbeta resulted in an induction of apoptotic cell death in association with an increase in the number of cells entered into the G(2)/M phase of the cell cycle, whereas its knockdown by either short interfering RNA or by a genetic suppressor element led to an accelerated cell proliferation or enhanced tumor formation in nude mice, respectively. Furthermore, we demonstrated that the rod region unique to KIF1Bbeta is critical for the induction of apoptotic cell death in a p53-independent manner. Thus, KIF1Bbeta may act as a haploinsufficient tumor suppressor, and its allelic loss may be involved in the pathogenesis of neuroblastoma and other cancers.     

Loss of heterozygosity and K-ras gene mutations in gastric cancer

In order to identify relevant genetic lesions in gastric carcinoma, we searched for tumor suppressor gene inactivation and K-ras gene mutations by analyzing tumor and control DNAs from 34 patients. These were from an epidemiologically defined area of Italy characterized by one of the world's highest incidences of stomach cancer. Allele losses were investigated by the Southern blotting procedure at 16 polymorphic loci on 11 different chromosomes. Our data demonstrate that chromosomal regions 5q, 11p, 17p and 18q are frequently deleted, and that 7q and 13q chromosome arms are also involved, although at a lower frequency. Loss of heterozygosity (LOH) at region 11p was not found during other surveys carried out on patients of different geographic origins. No specific combination of allelic losses could be recognized in the samples analyzed, the only exception being that tumors with 17p allelic loss also showed LOH on the 18q region. When matching frequent LOH events and the stage of progression of the tumors, we observed a trend of association between advanced stages and allelic losses on 17p and 18q chromosome arms. The analysis of K-ras, carried out by the polymerase chain reaction and denaturing gradient gel electrophoresis, demonstrated transforming mutations in only 3 out of 32 cases. Colorectal tumorigenesis proceeds by the accumulation of genetic alterations, including K-ras mutations and inactivation of tumor suppressor genes on the 5q, 17p and 18q regions. Our data indicate that, although gastric and colorectal neoplasias share common genetic alterations, they probably progress through different pathways.