Alteration of the fragile histidine triad gene early in carcinogenesis: an update

An association between common chromosome fragile sites and frequent chromosomal deletions in cancer has been observed and led to the hypothesis that genes at fragile sites may play a role in tumor development. In 1996, the human fragile histidine triad gene, FHIT, was identified by positional cloning at 3p14.2, a chromosomal region spanning the carcinogen-sensitive, common fragile site FRA3B. FHIT gene is lost and inactivated in a large fraction of tumors and early in carcinogenesis. A group of ancestral cancerous cells that carry FHIT alterations, expanding in succeeding cell generations, exhibits a hallmark in carcinogenesis scenario.     

Common chromosomal fragile sites and cancer: focus on FRA16D

A growing body of experimental evidence supports the view that certain human chromosomal fragile sites have roles to play in cancer. The principle lines of evidence are at the level of mutation mechanism and gene function. Most research in this area has previously focussed on the FRA3B common fragile site and the FHIT gene that spans this site. Here we review recent progress in characterising the second most readily observed common fragile site, FRA16D, and the WWOX gene that spans it. Comparative analyses of FRA3B/FHIT and FRA16D/WWOX reveal some striking similarities suggesting that these sites and their associated genes may play a part in a normal protective response of cells to environmental stress.     

FHIT基因与肿瘤

脆性组氨酸三联体(FHIT)基因是一种定位于人类染色体3p14.2上的抑癌基因,在多种器官中都有表达,其包含的脆性位点FRA3B,是基因组中最脆弱的部分.FHIT可促进细胞凋亡,抑制细胞增殖和细胞癌变.FHIT基因高度甲基化、基因缺失、FRA3B脆性位点的改变以及蛋白合成过程中的异常,可导致FHIT基因功能缺失,促进多种肿瘤的发生与发展.将野生型FHIT导入FHIT表达降低或表达缺失的肿瘤细胞中可促进肿瘤细胞凋亡.FHIT基因与其他基因联合治疗可能为肿瘤的治疗提供新的方向.     

Identification of unstable sequences within the common fragile site at 3p14.2: implications for the mechanism of deletions within fragile histidine triad gene/common fragile site at 3p14.2 in tumors

The FRA3B, at 3p14.2, lies within the fragile histidine triad (FHIT) gene and is the most highly expressed of the common fragile sites observed when DNA replication is perturbed by aphidicolin. Common fragile sites are highly unstable regions of the genome. Large intragenic deletions within FHIT, localized within the FRA3B sequences, have been identified in a variety of tumor cells. To characterize the FRA3B deletions in tumor cells and identify FRA3B sequences that are required for fragile site induction, we used microcell-mediated chromosome transfer to isolate hybrid cell clones that retain chromosome 3 homologues with various deletions within FRA3B. Detailed molecular mapping of the FHIT/FRA3B locus in the resultant hybrid cells revealed a complex pattern of instability within FRA3B. Each tumor cell line contained multiple chromosome 3 homologues with variable deletion patterns, often with discontinuous deletions, suggesting that the process of breakage and repair within FRA3B is an ongoing one. By comparing the approximate location of the breakpoints in the hybrid clones, we identified 11 recurring breakpoint/repair regions within the FRA3B. A comparison of the frequency of breaks/gaps within FRA3B in the hybrid clones with various deletions of FRA3B sequences revealed that the loss of FRA3B sequences does not reduce the overall rate of breakage and instability within the remaining FRA3B sequences. The majority of breaks occurred in the proximal portion of the FRA3B, in a 300-kb interval between exon 4 and the proximal 50 kb of intron 5. Our observations suggest that there is no single sequence within the FRA3B that influences breakage or recombination within this region; however, we cannot rule out the presence of multiple "hot spots" within the FHIT/FRA3B locus. Together, the results suggest that factors other than the DNA sequence per se are responsible for the formation of DNA breaks/gaps.     

Chromosome arm 8p and cancer: a fragile hypothesis

Chromosome arm 8p is one of the most frequently altered regions in human cancers. Several potential oncogenes and tumour suppressor genes have been identified but further investigations are needed to confirm which are bona fide oncogenic targets. In cancer cells, chromosome breaks may occur at fragile sites throughout the genome. Some fragile sites lie within genes that may have a role in cancer; the best example is FHIT at 3p14, which contains the fragile site FRA3B. We have found that chromosome breaks disrupt the NRG1 gene at 8p12 in breast and pancreatic cancers. We hypothesise that alteration of the NRG1 gene could occur through breakage at a non-common fragile site.     

Evidence that instability within the FRA3B region extends four megabases

FRA3B is the most frequently expressed common fragile site localized within human chromosomal band 3p14.2, which is frequently deleted in many different cancers, including cervical cancer. Previous reports indicate aphidicolin-induced FRA3B instability occurs over approximately 500 kb which is spanned by the 1.5 Mb fragile histidine triad (FHIT) gene. Recently an HPV16 cervical tumor integration, 2 Mb centromeric to the published FRA3B region, has been identified. FISH-based analysis with a BAC spanning the integration has demonstrated this integration occurs within the FRA3B region of instability. These data suggest that the unstable FRA3B region is much larger than previously reported. FISH-based analysis of aphidicolin-induced metaphase chromosomes allowed for a complete characterization of instability associated with FRA3B. This analysis indicates that fragility extends for 4 Mb. Within this region are a total of five genes, including FHIT. FRA3B gene expression analysis on a panel of cervical tumor-derived cell lines revealed that three of the five genes within FRA3B were aberrantly regulated. A similar analysis of genes outside of FRA3B indicated that the surrounding genes were not aberrantly expressed. These data provide additional support that regions of instability associated with CFSs and the genes contained within them, may play an important role in cancer development.     

FHIT as tumor suppressor: mechanisms and therapeutic opportunities

Chromosomal abnormalities including homozygous deletions and loss of heterozygosity at 3p l4.2 are commonly observed in most human tumors, including lung, breast and kidney cancers. This region also contains the most common human fragile site FRA3B, a familial kidney cancer-associated translocation breakpoint and papilloma virus integration sites. The FHIT gene is a tumor suppressor, which is frequently inactivated by mentioned genomic alterations at 3pl4.2. In the last few years considerable amount of data describing inactivation of FHIT in a variety of human malignancies and demonstrating the tumor suppressor potential of Fhit has accumulated. However, these have not yet led to major advances in uncovering the precise molecular mechanism of Fhit action. This review focuses on the most recent progress in understanding of Fhit function as a tumor suppressor and opportunities for gene cancer therapy with Fhit.     

The fragile histidine triad/common chromosome fragile site 3B locus and repair-deficient cancers

In various studies of sporadic breast cancers, 40-70% were strongly positive for fragile histidine triad (Fhit) protein expression, whereas only 18% of BRCA2 mutant breast cancers demonstrated strong Fhit expression, suggesting that the BRCA2 repair function may be necessary to retain intact fragile common chromosome fragile site 3B(FRA3B)/FHITloci. In the current study, 22 breast tumors with deleterious BRCA1 mutations were analyzed for Fhit expression by immunohistochemistry in a case-control matched pair analysis. Loss of Fhit expression was significantly more frequent in the BRCA1 cancers compared with sporadic breast tumors (9% Fhit positive versus 68% Fhit positive), suggesting that the BRCA1 pathway is also important in protecting the FRA3B/FHIT locus from damage. To investigate the relationship between repair gene deficiencies and induction of chromosome fragile sites in vitro, we have analyzed the frequency of aphidicolin induction of chromosome gaps and breaks in PMS2-, BRCA1-, MSH2-, MLH1-, FHIT-, and TP53-deficient cell lines. Each of the repair-deficient cell lines showed elevated expression of chromosome gaps and breaks, consistent with the proposal that proteins involved in mismatch and double-strand break repair are important in maintaining the integrity of common fragile regions. Correspondingly, genes at common fragile sites may sustain elevated levels of DNA damage in cells with deficient DNA repair proteins such as those mutated in several familial cancer syndromes.     

Loss of FHIT protein expression is a marker of adverse evolution in good prognosis localized breast cancer

The FHIT tumor suppressor gene, which encompasses the fragile site FRA3B at 3p14.2, is altered frequently in many types of human cancers. To determine its importance as a prognostic marker in breast cancer, the expression of the FHIT protein was studied in a series of 452 breast carcinomas by using immunohistochemistry on sections of tissue microarrays. Three distinct levels of FHIT expression were observed: in 154 cases (34.1%) expression was unchanged as compared to normal level; in 78 (17.2%) no expression was found; in the remaining 220 cases (48.7%), expression was intermediate. Overall, two-thirds of the cases had abnormal levels of the protein. Absence of FHIT was significantly associated with a higher grade (p < 0.01) and absence of hormone receptors (p < 0.001). The patients were separated into Group I (153 node-negative good prognosis patients who did not receive adjuvant chemotherapy) and Group II (226 high-risk patients treated by adjuvant chemotherapy) according to the St.-Gallen conference consensus. The median follow-up was 48 months. Among Group I but not Group II patients, a multivariate analysis showed that FHIT expression was significantly associated with disease-free survival. The relative risk of recurrence for FHIT-negative Group I patients was 2.37 (1.21-4.64; p = 0.03). Thus, among the patients who present with tumors of apparent good prognosis, FHIT is an independent prognostic factor that distinguishes a subgroup of patients who could benefit from adjuvant treatment.     

Down-regulation of fragile histidine triad expression in prostate carcinoma

BACKGROUND: The fragile histidine triad (FHIT) gene is a tumor suppressor gene that belongs to the histidine triad family of nucleoside binding proteins. The gene encompasses the common human chromosomal fragile site, the FRA3B locus at chromosome 3p14.2, and is expressed in most normal adult tissues and tumor cell lines. Numerous studies have indicated that the FHIT gene on chromosome 3p may play an important role in human neoplasia, although very few studies have investigated the FHIT gene in prostate carcinoma. METHODS: Using immunohistochemical analyses, the authors studied the expression of FHIT in prostate tumors from 84 radical prostatectomy specimens to determine whether there were any correlations between FHIT expression and various clinicopathologic characteristics. RESULTS: The percentages of cells stained with antibody to FHIT were significantly lower overall for tumor cells compared with normal cells (P = 0.0001). FHIT immunostaining intensity also was significantly lower for tumor cells compared with normal cells (P = 0.0001). A weak but statistically significant correlation (P = 0.045) was demonstrated with the presence of extraprostatic extension in the patient samples. No other significant correlation was seen between the percentage of cells stained for FHIT or FHIT immunostaining intensity and Gleason grade, tumor stage, tumor size, lymph node metastasis, surgical margins, vascular invasion, perineural invasion, or the presence of high-grade prostatic intraepithelial neoplasia. CONCLUSIONS: The data presented indicate a down-regulation of the FHIT tumor suppressor gene in prostate carcinoma and, thus, propose a potential target for therapeutic intervention.     

FHIT基因与皮肤癌

FHIT基因是组氨酸三联体基因家族的成员,在嘌呤代谢中参与编码一个AP3A水解酶。该基因在3号染色体上包含常见的脆性部位FRA3B,并且致癌物质引起的损伤可以导致染色体易位从而致使基因的转录本异常。事实上,这种基因的异常转录本在所有的食管、胃和结肠癌中发现大约有一半出现异常。研究显示FHIT基因可能通过调控细胞周期,诱导细胞凋亡发挥抑制肿瘤的作用。其失活机制主要表现为启动子区域CPG岛甲基化、缺失及异常转录。同时有研究表明FHIT基因与皮肤癌的发生、发展、转移和预后有着密切的关系。本文就FHIT基因与皮肤癌关系研究最新进展做出综述。     

FHIT基因与皮肤癌

FHIT基因是组氨酸三联体基因家族的成员,在嘌呤代谢中参与编码一个AP3A水解酶。该基因在3号染色体上包含常见的脆性部位FRA3B,并且致癌物质引起的损伤可以导致染色体易位从而致使基因的转录本异常。事实上,这种基因的异常转录本在所有的食管、胃和结肠癌中发现大约有一半出现异常。研究显示FHIT基因可能通过调控细胞周期,诱导细胞凋亡发挥抑制肿瘤的作用。其失活机制主要表现为启动子区域CPG岛甲基化、缺失及异常转录。同时有研究表明FHIT基因与皮肤癌的发生、发展、转移和预后有着密切的关系。本文就FHIT基因与皮肤癌关系研究最新进展做出综述。     

Roles of FHIT and WWOX fragile genes in cancer

It was hypothesized as early as 1986, that the recently discovered common fragile sites could facilitate recombination events, such as deletions and translocations, that result in clonally expanded cancer cell populations with specific chromosome alterations in specific cancer types. A natural extension of this hypothesis is that the clonal expansion must be driven by alteration of genes at recombination breakpoints whose altered functions actually drive clonal expansion. Nevertheless, when the FHIT gene was discovered at FRA3B, the most active common chromosome fragile region, and proposed as an example of a tumor suppressor gene altered by chromosome translocations and deletions, a wave of reports suggested that the FHIT gene was altered in cancer simply because it was in a fragile region and not because it had contributed to the clonal expansion, thus turning the original hypothesis upside down. Now, after nearly ten years and more than 500 FHIT reports, it is apparent that FHIT is an important tumor suppressor gene and that there are genes at other fragile regions that contribute significantly to development of cancer. A second fragile gene with a demonstrated role in cancer development is the WWOX gene on chromosome 16q; alterations to the WWOX gene contribute to development of hormone responsive and other cancers. Results of our recent studies of these two fragile tumor suppressor genes were summarized at the first Fragilome meeting in Heidelberg, Feb. 2005.     

Correlated break at PARK2/FRA6E and loss of AF-6/Afadin protein expression are associated with poor outcome in breast cancer

Common fragile sites (CFSs) are regions of chromosomal break that may play a role in oncogenesis. The most frequent alteration occurs at FRA3B, within the FHIT gene, at chromosomal region 3p14. We studied a series of breast carcinomas for break of a CFS at 6q26, FRA6E, and its associated gene PARK2, using fluorescence in situ hybridization on tissue microarrays (TMA). We found break of PARK2 in 6% of cases. We studied the PARK2-encoded protein Parkin by using immunohistochemistry on the same TMA. Loss of Parkin was found in 13% of samples but was not correlated with PARK2 break. PARK2 break but not Parkin expression was correlated with prognosis. Alteration of PARK2/FRA6E may cause haplo-insufficiency of one or several telomeric potential tumor suppressor genes (TSG). The AF-6/MLLT4 gene, telomeric of PARK2, encodes the Afadin scaffold protein, which is essential for epithelial integrity. Loss of Afadin was found in 14.5% of cases, and 36% of these cases showed PARK2 break. Loss of Afadin had prognostic impact, suggesting that AF-6 may be a TSG. Loss of Afadin was correlated with loss of FHIT expression, suggesting fragility of FRA6E and FRA3B in a certain proportion of breast tumors.     

Expression of common chromosomal fragile site genes, WWOX/FRA16D and FHIT/FRA3B is downregulated by exposure to environmental carcinogens, UV, and BPDE but not by IR

Common chromosomal fragile sites are unstable genomic loci susceptible to breakage, rearrangement, and are highly recombinogenic. Frequent alterations at these loci in tumor cells led to the hypothesis that they may contribute to cancer development. The two most common chromosomal fragile sites FRA16D and FRA3B which harbor WWOX and FHIT genes, respectively, are frequently altered in human cancers. Here we report that environmental carcinogens, ultraviolet (UV) light, and Benzo[a]pyrene diol epoxide (BPDE), significantly downregulate expression of both genes. On the other hand, we observe that ionizing radiation (IR) does not affect expression of these genes, suggesting that the effect of repression exerted by UV and BPDE is not just a consequence of DNA damage but may be a result of different signaling pathways triggered by specific DNA lesions. Such downregulation correlates with an induction of an S-phase delay in the cell cycle. Treatment of UV-irradiated cells with caffeine abrogates the S-phase delay while concomitantly overcoming the repression phenomenon. This suggests the involvement of unique cell cycle checkpoint mechanisms in the observed repression. Therefore, it is hypothesized that protracted downregulation of the putative tumor suppressor genes WWOX and FHIT by environmental carcinogens may constitute an additional mechanism of relevance in the initiation of tumorigenesis.