Mutation analysis of the DBC2 gene in sporadic and familial breast cancer.
TL;DR: The sequence variations found within the promoter and 5′-UTR region of the gene warrant expression analysis and screening more tumor samples at this region, and no deleterious mutations were detected in the coding sequence.
Abstract: The expression of the recently identified tumor suppressor gene, DBC2 (Deleted in Breast Cancer 2), is frequently extinguished in breast cancer cells or tissues. Mutation analysis of the essential promoter region, all exons and exon/intron boundaries of the DBC2 gene was performed in 100 sporadic breast cancer cases by PCR-SSCP, and DHPLC, followed by direct sequencing. An additional 17 breast cancer families, who were negative for the BRCA1/2 mutations, were analyzed by direct sequencing. Three novel mutations were observed in the promoter and 5′-untranslated region (UTR) of the gene; a germ-line G>A transition in the promoter at nt −238 from the transcription start site, and two tumor-specific mutations at nt −121C>T and nt +48G>A. No deleterious mutations were detected in the coding sequence of the gene in familial and sporadic breast cancer cases. The sequence variations found within the promoter and 5′-UTR region of the gene warrant expression analysis and screening more tumor samples at this region.
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TL;DR: A model emerges in which RhoBTB proteins are required to maintain constant levels of putative substrates involved in cell cycle regulation or vesicle transport through targeting for degradation in the 26S proteasome.
Abstract: RhoBTB proteins constitute a subfamily of atypical members within the Rho family of small guanosine triphosphatases (GTPases). Their most salient feature is their domain architecture: a GTPase domain (in most cases, non-functional) is followed by a proline-rich region, a tandem of 2 broad-complex, tramtrack, bric a brac (BTB) domains, and a conserved C-terminal region. In humans, the RhoBTB subfamily consists of 3 isoforms: RhoBTB1, RhoBTB2, and RhoBTB3. Orthologs are present in several other eukaryotes, such as Drosophila and Dictyostelium, but have been lost in plants and fungi. Interest in RhoBTB arose when RHOBTB2 was identified as the gene homozygously deleted in breast cancer samples and was proposed as a candidate tumor suppressor gene, a property that has been extended to RHOBTB1. The functions of RhoBTB proteins have not been defined yet, but may be related to the roles of BTB domains in the recruitment of cullin3, a component of a family of ubiquitin ligases. A model emerges in which RhoBTB proteins are required to maintain constant levels of putative substrates involved in cell cycle regulation or vesicle transport through targeting for degradation in the 26S proteasome. RhoBTB proteins are engrossing the list of Rho GTPases involved in tumorigenesis. Unlike typical Rho GTPases (usually overexpressed or hyperactive), RhoBTB proteins appear to play a part in the carcinogenic process through a mechanism that involves the decreased or abolished expression of the corresponding genes, or more rarely, mutations that result in impaired functioning of the protein, presumably leading to the accumulation of RhoBTB substrates and alterations of the cellular homeostasis.
70 citations
TL;DR: The GTPase domain, which does not bind GTP, is able to interact with the BTB domain region, thus preventing proteasomal degradation of RhoBTB, and fits into a model in which an intramolecular interaction maintains Rho BTB in an inactive state, preventing the formation or the functionality of Cul3-dependent complexes.
69 citations
TL;DR: Restoration of normal RhoBTB1 expression rescues Golgi morphology and dramatically inhibits breast cancer cell invasion and hence loss of normal polarity.
Abstract: RhoBTB1 and 2 are atypical members of the Rho GTPase family of signaling proteins. Unlike other Rho GTPases, RhoBTB1 and 2 undergo silencing or mutation in a wide range of epithelial cancers; however, little is known about the consequences of this loss of function. We analyzed transcriptome data to identify transcriptional targets of RhoBTB2. We verified these using Q-PCR and then used gene silencing and cell imaging to determine the cellular function of these targets downstream of RhoBTB signaling. RhoBTB1 and 2 regulate the expression of the methyltransferases METTL7B and METTL7A, respectively. RhoBTB1 regulates the integrity of the Golgi complex through METTL7B. RhoBTB1 is required for expression of METTL7B and silencing of either protein leads to fragmentation of the Golgi. Loss of RhoBTB1 expression is linked to Golgi fragmentation in breast cancer cells. Restoration of normal RhoBTB1 expression rescues Golgi morphology and dramatically inhibits breast cancer cell invasion. Loss of RhoBTB1 expression in breast cancer cells leads to Golgi fragmentation and hence loss of normal polarity.
53 citations
Cites background from "Mutation analysis of the DBC2 gene ..."
...Many studies have reported downregulation of RhoBTB2 expression in breast cancer, most commonly due to methylation of the RhoBTB2 promoter [7, 10, 11, 23, 24]....
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TL;DR: De novo missense variants clustering in the BTB-domain-encoding region of RHOBTB2 are established as causative for a developmental and epileptic encephalopathy and the role of atypical Rho GTPase RhoBTB in Drosophila neurological function and possibly dendrite development is elucidated.
Abstract: Although the role of typical Rho GTPases and other Rho-linked proteins in synaptic plasticity and cognitive function and dysfunction is widely acknowledged, the role of atypical Rho GTPases (such as RHOBTB2) in neurodevelopment has barely been characterized. We have now identified de novo missense variants clustering in the BTB-domain-encoding region of RHOBTB2 in ten individuals with a similar phenotype, including early-onset epilepsy, severe intellectual disability, postnatal microcephaly, and movement disorders. Three of the variants were recurrent. Upon transfection of HEK293 cells, we found that mutant RHOBTB2 was more abundant than the wild-type, most likely because of impaired degradation in the proteasome. Similarly, elevated amounts of the Drosophila ortholog RhoBTB in vivo were associated with seizure susceptibility and severe locomotor defects. Knockdown of RhoBTB in the Drosophila dendritic arborization neurons resulted in a decreased number of dendrites, thus suggesting a role of RhoBTB in dendritic development. We have established missense variants in the BTB-domain-encoding region of RHOBTB2 as causative for a developmental and epileptic encephalopathy and have elucidated the role of atypical Rho GTPase RhoBTB in Drosophila neurological function and possibly dendrite development.
42 citations
TL;DR: Microarray analysis of global gene expression shows that loss of RhoBTB2 results in downregulation of CXCL14—a chemokine that controls leukocyte migration and angiogenesis, and whose expression is lost through unknown mechanisms in a wide range of epithelial cancers.
Abstract: The Rho family of small GTPases control cell migration, cell invasion and cell cycle. Many of these processes are perturbed in cancer and several family members show altered expression in a number of tumor types. RhoBTB2/DBC2 is an atypical member of this family of signaling proteins, containing two BTB domains in addition to its conserved Rho GTPase domain. RhoBTB2 is mutated, deleted or silenced in a large percentage of breast and lung cancers; however, the functional consequences of this loss are unclear. Here we use RNA interference in primary human epithelial cells to mimic the loss of RhoBTB2 seen in cancer cells. Through microarray analysis of global gene expression, we show that loss of RhoBTB2 results in downregulation of CXCL14—a chemokine that controls leukocyte migration and angiogenesis, and whose expression is lost through unknown mechanisms in a wide range of epithelial cancers. Loss of RhoBTB2 expression correlates with loss of CXCL14 secretion by head and neck squamous cell carcinoma cell lines, whereas reintroduction of RhoBTB2 restores CXCL14 secretion. Our studies identify CXCL14 as a gene target of RhoBTB2 and support downregulation of CXCL14 as a functional outcome of RhoBTB2 loss in cancer.
38 citations
Cites background from "Mutation analysis of the DBC2 gene ..."
...…in RhoBTB2 were also detected in breast tumor samples and breast cancer cell lines (Hamaguchi et al., 2002), and subsequent studies have identified further sporadic mutations of the RhoBTB2 coding region and promoter in bladder (Knowles et al., 2005) and breast (Ohadi et al., 2007) tumor samples....
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TL;DR: Functional analysis revealed that DBC2 expression in breast cancer cells lacking D BC2 transcripts causes growth inhibition, and expression of a somatic mutant discovered in a breast cancer specimen does not suppress the growth of breast cancers cells.
Abstract: A previously uncharacterized gene, DBC2 (deleted in breast cancer), was cloned from a homozygously deleted region at human chromosome 8p21. DBC2 contains a highly conserved RAS domain and two putative protein interacting domains. Our analyses indicate that DBC2 is the best candidate tumor suppressor gene from this region. It lies within the epicenter of the deletions and is homozygously deleted in 3.5% (7/200) of breast tumors. Mutation analysis of DBC2 led to discovery of two instances of somatic missense mutations in breast tumor specimens, whereas no missense mutations were found in other candidates from the region. Unlike other genes in the region, expression of DBC2 is often extinguished in breast cancer cells or tissues. Moreover, our functional analysis revealed that DBC2 expression in breast cancer cells lacking DBC2 transcripts causes growth inhibition. By contrast, expression of a somatic mutant discovered in a breast cancer specimen does not suppress the growth of breast cancer cells.
199 citations
Journal Article•
TL;DR: This study suggests that breast cancer progression is clonal with regard to CIN, but different breast cancers would present distinct molecular profiles resulting from genetic heterogeneity caused by CIN.
Abstract: Breast cancer is considered to display a high degree of intratumor heterogeneity, without any obvious morphological and pathological steps to define sequential evolution, and its progression may vary among individual tumors. In an attempt to elucidate these etiological and phenotypic complexities, the present study, based on the fundamental concept that genomic instability is the engine of both tumor progression and tumor heterogeneity, was conducted to test the hypothesis that breast cancer pathogenesis is driven by double-strand break (DSB)-initiated chromosome instability (CIN). The rationale underlying this hypothesis is derived from the clues provided by family breast cancer syndromes, in which susceptibility genes, including p53, ATM, BRCA1 and BRCA2, are involved within the common functional pathway of DSB-related checkpoint/ repair. Because genomic deletion caused by DSB is reflected in the genetic mechanism of loss of heterozygosity (LOH), this genome-wide LOH study was conducted, using 100 tumors and 400 microsatellite markers. To minimize the effect of heterogeneity within tumors, the experimental technique of laser capture microdissection was used to ensure that genetic and phenotypic examinations were based on the same tumor cells. Support for our hypothesis comes from the observations that: (a) the extent of DSB-initiated CIN in tumors significantly increased as tumors progressed to poorer grades or later stages; (b) in the sequential steps toward CIN, the loci of p53 and ATM, the key checkpoint genes against DSB, were lost at the earliest stage; and (c) many loci identified to be important in breast tumorigenesis were the genomic sites possibly harboring the genes involved in DSB-related checkpoint/repair (including RAD51, RAD52, and BRCA1) or CIN (including FA-A, FA-D, and WRN), and a higher number of these loci showing LOH was significantly associated with increased level of DSB-initiated CIN (P < 0.0001). Breast cancers are thus considered to be sequentially progressive with CIN. However, CIN might also cause genetic heterogeneity, which was revealed by the findings that LOH at some markers was observed only in the component of ductal carcinoma in situ but not in the invasive component of the same tumors. In addition, some markers were found to preferentially lose at specific tumor grades, implying their contribution to genetic heterogeneity during tumor development. Therefore, this study suggests that breast cancer progression is clonal with regard to CIN, but different breast cancers would present distinct molecular profiles resulting from genetic heterogeneity caused by CIN.
182 citations
"Mutation analysis of the DBC2 gene ..." refers background in this paper
...Genome-wide loss of heterozygosity (LOH) analysis of sporadic breast cancers indicates highest frequency of LOH for chromosome arms 1q, 4p, 8p, 8q, 11q, 13q, 16q, 17p, 17q and 22q [1]....
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TL;DR: 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, provides a comparative framework for further investigation of regions exhibiting LOH and identifies broad genomic regions for which there exist few data.
Abstract: 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.
108 citations
"Mutation analysis of the DBC2 gene ..." refers background in this paper
...Pooled analysis of LOH in breast cancer provides comprehensive evidence for tumor suppressor genes at this region (pB/10 (9)) [3]....
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TL;DR: It is concluded that neither LZTS1 nor DBC2 is commonly mutated in bladder cancer, however, neither can yet be excluded as the target of 8p22 LOH.
78 citations
"Mutation analysis of the DBC2 gene ..." refers background in this paper
...A single study of the gene in bladder cancer [6] has also reported a possible somatic mutation in an isolated tumor at exon 5:E349D, which was absent, in our sample series....
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...Three previously reported SNPs, IVS3 /16G /A, IVS3 /29T /G and c864 C /T [6], were also detected in our case and control samples....
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Journal Article•
TL;DR: In an attempt to identify chromosomal regions harboring putative breast cancer genes, allelotyping in 82 familial breast carcinomas was performed and the most frequently involved chromosomal arms were 8p, 16q, 17p, 17q, and 19p.
Abstract: Three loci have been implicated in the etiology of familial breast cancer; the BRCA1 locus on 17q, the p53 gene on 17p, and the androgen receptor gene on the X chromosome. However, it has been estimated that in approximately 50% of all breast cancer families the predisposing genetic defect is not linked to any of these three loci. In an attempt to identify chromosomal regions harboring putative breast cancer genes we performed allelotyping in 82 familial breast carcinomas. Polymorphic markers representing 45 different loci were analyzed and the most frequently involved chromosomal arms were 8p, 16q, 17p, 17q, and 19p.
52 citations
"Mutation analysis of the DBC2 gene ..." refers background in this paper
...Familial breast cancer LOH studies exhibit a different pattern of loss with the following chromosome arms being most frequently lost: 8p, 16q, 17p, 17q and 19q [2]....
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