DMD and IL1RAPL1: two large adjacent genes localized within a common fragile site (FRAXC) have reduced expression in cultured brain tumors

Common fragile sites (CFSs) are large regions of profound genomic instability found in all individuals. Spanning the center of the two most frequently expressed CFS regions, FRA3B (3p14.3) and FRA16D (16q23.2), are the 1.5 Mb FHIT gene and the 1.0 Mb WWOX gene. These genes are frequently deleted and/or altered in many different cancers. Both FHIT and WWOX have been demonstrated to function as tumor suppressors, both in vitro and in vivo. A number of other large CFS genes have been identified and are also frequently inactivated in multiple cancers. Based on these data, several additional very large genes were tested to determine if they were derived from within CFS regions, but DCC and RAD51L1 were not. However, the 2.0 Mb DMD gene and its immediately distal neighbor, the 1.8 Mb IL1RAPL1 gene are CFS genes contained within the FRAXC CFS region (Xp21.2-->p21.1). They are abundantly expressed in normal brain but were dramatically underexpressed in every brain tumor cell line and xenograft (derived from an intracranial model of glioblastoma multiforme) examined. We studied the expression of eleven other large CFS genes in the same panel of brain tumor cell lines and xenografts and found reduced expression of multiple large CFS genes in these samples. In this report we show that there is selective loss of specific large CFS genes in different cancers that does not appear to be mediated by the relative instability within different CFS regions. Further, the inactivation of multiple large CFS genes in xenografts and brain tumor cell lines may help to explain why this type of cancer is highly aggressive and associated with a poor clinical outcome.     

WWOX,large common fragile site genes,and cancer

WWOX is a gene that spans an extremely large chromosomal region. It is derived from within chromosomal band 16q23.2 which is a region with frequent deletions and other alterations in a variety of different cancers. This chromosomal band also contains the FRA16D common fragile site (CFS). CFSs are chromosomal regions found in all individuals which are highly unstable. WWOX has also been demonstrated to function as a tumor suppressor that is involved in the development of many cancers. Two other highly unstable CFSs, FRA3B (3p14.2) and FRA6E (6q26), also span extremely large genes, FHIT and PARK2, respectively, and these two genes are also found to be important tumor suppressors. There are a number of interesting similarities between these three large CFS genes. In spite of the fact that they are derived from some of the most unstable chromosomal regions in the genome, they are found to be highly evolutionarily conserved and the chromosomal region spanning the mouse homologs of both WWOX and FHIT are also CFSs in mice. Many of the other CFSs also span extremely large genes and many of these are very attractive tumor suppressor candidates. WWOX is therefore a member of a very interesting family of very large CFS genes.     

Differential loss of expression of common fragile site genes between oral tongue and oropharyngeal squamous cell carcinomas

The common fragile sites (CFSs) are large regions of profound genomic instability found in all individuals. A number of the CFSs have been found to span genes that extend over large genomic regions (>700 kb). The expression of these genes is frequently abrogated in a number of different cancers and several of them have already been shown to function as tumor suppressor genes, both in vitro and in vivo. We analyzed the expression of 14 large CFS genes in two distinct groups of head and neck cancers using real-time RT-PCR. The first were oral tongue squamous cell carcinomas (SCCs) and the second were base of tongue/tonsillar (oropharyngeal) SCCs. These two groups were previously examined for the presence of human papillomavirus (HPV) and while 46% of the oropharyngeal cancers were positive for HPV16 only one of 52 oral cancers contained HPV16 sequences. We observed a distinct pattern of loss of expression of the large CFS genes in the two groups of head and neck cancers. In addition, there was no correlation between the relative instability in different CFS regions and which genes were inactivated. Thus, this report demonstrates another distinction between these two groups of head and neck cancer. In addition, it suggests that there is selection for loss of expression of specific CFS genes in these cancers.     

Epigenetic and Genetic Alterations Affect the WWOX Gene in Head and Neck Squamous Cell Carcinoma

Different types of genetic and epigenetic changes are associated with HNSCC. The molecular mechanisms of HNSCC carcinogenesis are still undergoing intensive investigation. WWOX gene expression is altered in many cancers and in a recent work reduced WWOX expression has been associated with miR-134 expression in HNSCC. In this study we investigated the WWOX messenger RNA expression levels in association with the promoter methylation of the WWOX gene and miR-134 expression levels in 80 HNSCC tumor and non-cancerous tissue samples. Our results show that WWOX expression is down-regulated especially in advanced-stage tumor samples or in tumors with SCC. This down-regulation was associated with methylation of the WWOX promoter region but not with miR-134 expression. There was an inverse correlation between the expression level and promoter methylation. We also analyzed whole exons and exon/intron boundries of the WWOX gene by direct sequencing. In our study group we observed 10 different alterations in the coding sequences and 18 different alterations in the non-coding sequences of the WWOX gene in HNSCC tumor samples. These results indicate that the WWOX gene can be functionally inactivated by promoter methylation, epigenetically or by mutations affecting the sequences coding for the enzymatic domain of the gene, functionally. We conclude that inactivation of WWOX gene contributes to the progression of HNSCC.     

WWOX, a new potential tumor suppressor gene

BACKGROUND: WWOX (WW domain-containing oxidoreductase) gene, located on chromosome 16q 23.3-24.1 in the region recognized as the common fragile site FRA16D is considered to be a tumor suppressor gene involved in various cancers: breast, ovarian, prostate, esophageal, lung, pancreatic, gastric and hepatic. The aim of this study was to describe (i) putative protein interactions of WWOX (ii) the molecular mechanisms of tumor suppressor activity (iii) present an overview of WWOX in relation to nervous system and breast, prostate and ovarian cancers. METHODS AND RESULTS: WWOX expression is up-regulated in endocrine organs indicating its importance in these tissues. In many cancers WWOX expression is down-regulated and low WWOX expression is related to poor prognosis. CONCLUSION: All the evidence suggest that WWOX can be considered as a new tumor suppressor gene and target for gene therapy due to the association of high WWOX expression with improved disease free survival.     

普通型脆性位点的研究进展

脆性位点是DNA合成被部分抑制之后,在中期染色体上发生的位点特异的缺口或断裂区域,它也是基因组不稳定的区域。通常脆性位点在体细胞中是稳定的,但它们在许多癌细胞中经常发生缺失或重排。在已发现的80多种脆性位点基因中,研究最多的是FHIT和WWOX,它们与肿瘤的发生发展密切相关。简要综述了普通型脆性位点的研究进展及其与肿瘤的关系。     

抑癌基因WWOX和FHIT在膀胱尿路上皮癌中的表达及其临床意义

目的探讨抑癌基因包含WW域的氧化还原酶(WWOX)和脆性组氨酸三联体(FHIT)在膀胱尿路上皮癌中的表达及临床意义。方法收集武汉大学人民医院病理科2006-2009年有完整临床和病理资料的膀胱尿路上皮癌存档蜡块50例和5例癌旁组织,采用免疫组织化学S-P法检测50例膀胱尿路上皮癌和5例癌旁组织中WWOX和FHIT的表达水平。采用HPIAS-1000高清晰度彩色病理图文报告管理系统,对WWOX和FHIT的表达进行定量分析,并用SPSS13.0软件对各组免疫组织化学反应阳性颗粒的平均光密度、阳性面积率做单因素方差分析和SNK(q)检验。结果 WWOX和FHIT的表达在膀胱尿路上皮癌中呈低表达,癌旁组织中呈高表达。膀胱尿路上皮癌与癌旁组织相比,差异有显著性(P<0.05)。结论 WWOX和FHIT在膀胱尿路上皮癌中的低表达是膀胱尿路上皮癌发生过程中的早期表现,通过联合检测可预测膀胱尿路上皮癌的预后。     

WWOX at the crossroads of cancer,metabolic syndrome related traits and CNS pathologies

WWOX was cloned as a putative tumor suppressor gene mapping to chromosomal fragile site FRA16D. Deletions affecting WWOX accompanied by loss of expression are frequent in various epithelial cancers. Translocations and deletions affecting WWOX are also common in multiple myeloma and are associated with worse prognosis. Metanalysis of gene expression datasets demonstrates that low WWOX expression is significantly associated with shorter relapse-free survival in ovarian and breast cancer patients. Although somatic mutations affecting WWOX are not frequent, analysis of TCGA tumor datasets led to identifying 44 novel mutations in various tumor types. The highest frequencies of mutations were found in head and neck cancers and uterine and gastric adenocarcinomas.     

Common fragile genes

Common chromosome fragile sites show susceptibility to DNA damage, leading to alterations that contribute to cancer development. The cloning and characterization of fragile sites have demonstrated that fragile sites are associated with genes that relate to tumorigenesis. Identification of the basis of instability at fragile sites and the related genes provides an entree to understanding of important aspects of chromosomal instability, a prominent feature of neoplastic genomes. FHIT/FRA3B and WWOX/FRA16D, the most sensitive common fragile genes in the human genome, function as tumor suppressor genes. The common features of these two common fragile genes are summarized, and suggest clues to understanding the relation between genomic instability and tumor biology.     

Conditional inactivation of the mouse Wwox tumor suppressor gene recapitulates the null phenotype

WW domain‐containing oxidoreductase (WWOX) is highly conserved in both human and murine. WWOX spans the second most common human chromosomal fragile site, FRA16D, and is commonly inactivated in multiple human cancers. Modeling WWOX inactivation in mice revealed a complex phenotype including postnatal lethality, defects in bone metabolism and steroidogenesis and tumor suppressor function resulting in osteosarcomas. For better understanding of WWOX roles in different tissues at distinct stages of development and in pathological conditions, Wwox conditional knockout mice were generated in which loxp sites flank exon 1 in the Wwox allele. We demonstrated that Cre‐mediated recombination using EIIA‐Cre, a Cre line expressed in germline, results in postnatal lethality by age of 3 weeks and decreased bone mineralization resembling total ablation of WWOX as in conventional null mice. This animal model will be useful to study distinct roles of WWOX in multiple tissues at different ages. J. Cell. Physiol. 228: 1377–1382, 2013. © 2012 Wiley Periodicals, Inc.     

Components of DNA damage checkpoint pathway regulate UV exposure-dependent alterations of gene expression of FHIT and WWOX at chromosome fragile sites

Common chromosome fragile sites are highly recombinogenic and susceptible to deletions during the development of environmental carcinogen-induced epithelial tumors. Previous studies showed that not only genetic but also epigenetic alterations in cancerous cells are involved in inactivation of the genes FHIT and WWOX at chromosome fragile sites, reported to be potential tumor suppressor genes. Here we investigated the effect of UV light on the gene expression. After exposure to UV, the mRNA and protein of the two genes in murine embryonic fibroblasts (MEF) were unstable, apparently at the G1-S phase of the cell cycle, which was consistent with nuclear run-on assay. A study of MEFs synchronized via a double thymidine block indicated that, after the exposure, the expression of Fhit and Wwox was reduced in E2f-1-deficient cells and markedly in wild-type cells, whereas the reduction was partially inhibited in Trp53-deficient cells; cells at the S phase seemed to be sensitive to exogenous FHIT, suggesting a role of the checkpoint at the G1-S phase in the stability of gene expression and a possible involvement of FHIT function at the S phase. The transfection experiment showed that the UV-induced decrease in expression was partially inhibited by transfection of kinase-dead Atr (ataxia telangiectasia mutated and Rad3 related), which is a sensor of UV-induced damage. Taken together, the present study showed that UV-induced alterations of the fragile site gene expression are involved at least partially in the checkpoint function, suggesting the role in the process of carcinogenesis after exposure to UV.     

A mouse model of the fragile gene FHIT: From carcinogenesis to gene therapy and cancer prevention

Mouse models of tumor suppressors are increasingly useful to investigate biomedical aspects of cancer genetics. Some tumor suppressor genes are located at common fragile sites that are specific chromosomal regions highly susceptible to DNA lesions. The tumor suppressor gene FHIT, at the fragile site FRA3B, is the first fragile gene with a developed and characterized mouse knockout model. The human gene FHIT is frequently deleted in cancers and cancer cell lines of many epithelial tissues, and Fhit protein is absent or reduced in most cancers. The mouse Fhit ortholog is also located at a common fragile site, Fra14A2 on murine chromosome 14, and sustains homozygous deletions in murine cancer cell lines. The Fhit knockout mouse is, therefore, an adequate model to study human FHIT function. To establish an animal model and to explore the role of FHIT in tumorigenesis, we have developed a mouse strain carrying one or two inactivated Fhit alleles. Insights into Fhit mouse genetics that have emerged in the last 7 years, and are reviewed in the present article, allowed for development of new tools in carcinogenesis and gene delivery studies.     

Ever since Knudson

Many publications have documented loss of heterozygosity (LOH) on many different chromosomes in a wide variety of tumours, implicating the existence of multiple tumour suppressor genes (TSGs). Knudson's two-hit hypothesis predicts that these LOH events are the second step in the inactivation of both alleles of a TSG. However, to date the number of TSGs identified that are inactivated mainly at the somatic level in cancers and are not inherited has remained disappointingly small. Here we postulate that the accurate mapping of LOH events in a series of tumours to define a common LOH region is greatly confounded by deficient LOH detection, genetic instability and intertumour heterogeneity. Finding the TSGs in chromosomal regions of frequent LOH might require 'brute-force' genomic approaches.     

Molecular genetic alterations of FHIT and p53 genes in benign and malignant thyroid gland lesions

Several oncogenes and tumor-suppressor genes are involved either as early or late event in thyroid gland carcinogenesis. Human FHIT (fragile histidine triad) gene is highly conserved gene whose loss of function may be important in the development and/or progression of various types of cancer. We undertook this study to analyze FHIT and p53 gene status in different benignant and malignant thyroid tumors. Status of these genes as well as intensity of apoptosis was analyzed in tumor tissues by molecular genetic methods, immunohistochemistry, and FACS-scan analysis. The majority of the malignant thyroid cancers displayed aberrant expression of FHIT gene, concominant with p53 gene inactivation. This is followed by low rate of apoptosis, which may be important in the development and/or progression of thyroid cancer. We found higher incidence of p53 mutation and aberrant processing of FHIT mRNA in malignant tumors (papillary, follicular, medullary and anaplastic carcinomas) and in those tumors with distant metastasis. The growth of p53(-)/FHIT(-) follicular carcinoma of human origin was much faster in nude mice than p53(+)/FHIT(+) follicular carcinoma, and mice had shorter survival rate. Our results show a correlation between aberrant FHIT and p53 expression, low rate of apoptosis, and malignancy. Concomitant aberration of FHIT gene and p53 could be responsible for development of highly malignant types of thyroid cancer and may be considered as a prognostic marker for these tumors.     

FHIT Suppresses Epithelial-Mesenchymal Transition (EMT) and Metastasis in Lung Cancer through Modulation of MicroRNAs

Metastasis is the principal cause of cancer death and occurs through multiple, complex processes that involve the concerted action of many genes. A number of studies have indicated that the Fragile Histidine Triad (FHIT) gene product, FHIT, functions as a tumor suppressor in a variety of common human cancers. Although there are suggestions of a role for FHIT loss in progression of various cancers, a role for such loss in metastasis has not been defined. Here, via in vivo and in vitro assays, we reveal that the enforced expression of FHIT significantly suppresses metastasis, accompanied by inhibition of the epithelial-mesenchymal transition (EMT), a process involved in metastasis through coordinate modulation of EMT-related genes. Specifically, miR-30c, a FHIT-upregulated microRNA, contributes to FHIT function in suppression of EMT and metastasis by directly targeting metastasis genes Metadherin (MTDH), High-mobility group AT—hook 2 (HMGA2), and the mesenchymal markers, Vimentin (VIM) and Fibronectin (FN1), in human lung cancer. Finally, we demonstrate that the expression pattern of FHIT and miR-30c is inversely correlated with that of MTDH and HMGA2 in normal tissue, non-metastatic and metastatic tumors, serving as a potential biomarker for metastasis in lung cancer.