Inactivation of the FHIT gene favors bladder cancer development.

The fragile histidine triad (FHIT) gene located on chromosome 3p14.2 is frequently deleted in human tumors. We have previously reported deletions at the FHIT locus in 50% of bladder carcinoma derived cell lines and reduced expression in 61% of primary transitional carcinomas of the urinary bladder. To additionally investigate the role of FHIT alterations in the development of bladder cancer, we used heterozygous and nullizygous Fhit-deficient mice in a chemically induced carcinogenesis model. Results showed that 8 of 28 (28%) and 6 of 13 (46%) of the Fhit -/- and +/-, respectively, versus 2 of 25 (8%) Fhit +/+ mice developed invasive carcinoma after treatment with N-butyl-N-(4-hydroxybutyl) nitrosamine. To explore the possibility of a FHIT-based gene therapy for bladder cancer, we studied the effects of restored Fhit protein expression on cell proliferation, cell kinetics, and tumorigenicity in BALB/c nude mice, with human SW780 Fhit-null transitional carcinoma derived cells. In vitro transduction of SW780 Fhit-negative cells with adenoviral-FHIT inhibited cell growth, increased apoptotic cell population, and suppressed s.c. tumor growth in nude mice. These findings suggest the important role of Fhit in bladder cancer development and support the effort to additionally investigate a FHIT-based gene therapy.     

Differential susceptibility of renal carcinoma cell lines to tumor suppression by exogenous Fhit expression

Hemizygous deletions of the fragile histidine triad (FHIT) gene at human chromosome band 3p14.2 and down-regulation of its gene product are found in the majority of renal cell carcinomas (RCCs). Functional tumor suppressive activity of Fhit in renal cancer cells previously was observed in RCC cell line RC48, which lacks endogenous Fhit expression. To further investigate the potential role of FHIT as a tumor suppressor gene in RCC, we transfected FHIT cDNA expression constructs into RCC cell lines RCC-1 and SN12C, which show low-level expression of endogenous Fhit and reveal an intact von Hippel-Lindau (VHL) gene. Stable transfectants of both cell lines showed no alterations of cell morphology, proliferation kinetics, or cell cycle parameters in vitro. The FHIT gene transfer rate, however, was significantly lower in RCC-1 cells compared with SN12C cells, suggesting a selection against exogenous Fhit expression. In addition, in nude mouse assays, a significant delay of tumor formation was observed for FHIT-transfected RCC-1 cell lines, with outgrowing tumors demonstrating loss of Fhit expression in the majority of cells. In contrast, tumorigenicity of FHIT-transfected SN12C cell clones was not suppressed, despite stable transgene expression. In conclusion, our results demonstrate a selective tumor suppressive activity of Fhit in RCC cells in vivo and suggest that the susceptibility to suppression is not restricted to cancer cells with complete loss of Fhit expression.     

Restoration of fragile histidine triad (FHIT) expression induces apoptosis and suppresses tumorigenicity in breast cancer cell lines

The fragile histidine triad (FHIT) gene at chromosome 3p14.2 is a tumor suppressor gene that is altered mainly by deletion in a large fraction of human tumors, including breast cancers. To evaluate the potential of FHIT gene therapy in this type of cancer, we have studied the biological effects of adenoviral FHIT transduction (Ad-FHIT) in breast cancer cell lines. The results showed that, after FHIT restoration in BT-549, MDA-MB-436, and HCC1806 cells, they underwent apoptosis by activation of the intrinsic pathway. In all three cell lines infected with Ad-FHIT, we have found activation of caspase-2, which is required for permeabilization of mitochondria, release of cytochrome c, and apoptosis. Furthermore, Fhit overexpression produces alteration in cell cycling properties, as well as reduction of the tumorigenic potential in nude mice.     

Altered expression of Fhit in carcinoma and precarcinomatous lesions of the esophagus

The FHIT gene, located at chromosome 3p14.2, is a tumor suppressor gene often involved in tumors resulting from exposure to environmental carcinogens. We studied 46 pairs of esophageal primary tumors and corresponding normal squamous mucosa specimens by molecular genetic and immunohistochemical methods to investigate the role of the FHIT gene in esophageal carcinoma. In addition, we studied several different types of lesions, such as carcinoma in situ or dysplasia by immunohistochemistry. Loss of heterozygosity at or around the FHIT gene was observed in 35 (76%) primary tumors. Immunohistochemical detection of Fhit protein in the primary tumors demonstrated that 14 (30%) were positive and 32 (70%) were negative. We observed concordance between loss of Fhit protein and loss of heterozygosity and between loss of Fhit protein and RNA abnormalities. Because the FHIT/FRA3B locus is susceptible to damage by environmental carcinogens, we investigated the correlation between Fhit expression and smoking or alcohol habits. In this relatively small study, the patients who were both heavy users of tobacco and alcohol showed a significantly higher frequency of loss of Fhit expression than those who were light users. Noncarcinomatous squamous epithelium showed positive Fhit reactivity in most cases; however, five showed negative Fhit reactivity. Interestingly, all of these five patients had habits of heavy use of tobacco and alcohol. Eight of 12 carcinomas in situ, 2 of 4 severe dysplasias, 4 of 8 moderate dysplasias, and 3 of 9 mild dysplastic lesions showed negative Fhit reactivity. These findings indicated that loss of Fhit expression may be an early event in the development of human esophageal carcinoma and may occur even in normal-appearing squamous epithelium in some patients heavily exposed to environmental carcinogens.     

Epigenetic modulation of endogenous tumor suppressor expression in lung cancer xenografts suppresses tumorigenicity

Epigenetic changes involved in cancer development, unlike genetic changes, are reversible. DNA methyltransferase and histone deacetylase inhibitors show antiproliferative effects in vitro, through tumor suppressor reactivation and induction of apoptosis. Such inhibitors have shown activity in the treatment of hematologic disorders but there is little data concerning their effectiveness in treatment of solid tumors. FHIT, WWOX and other tumor suppressor genes are frequently epigenetically inactivated in lung cancers. Lung cancer cell clones carrying conditional FHIT or WWOX transgenes showed significant suppression of xenograft tumor growth after induction of expression of the FHIT or WWOX transgene, suggesting that treatments to restore endogenous Fhit and Wwox expression in lung cancers would result in decreased tumorigenicity. H1299 lung cancer cells, lacking Fhit, Wwox, p16(INK4a) and Rassf1a expression due to epigenetic modifications, were used to assess efficacy of epigenetically targeted protocols in suppressing growth of lung tumors, by injection of 5-aza-2-deoxycytidine (AZA) and trichostatin A (TSA) in nude mice with established H1299 tumors. High doses of intraperitoneal AZA/TSA suppressed growth of small tumors but did not affect large tumors (200 mm(3)); lower AZA doses, administered intraperitoneally or intratumorally, suppressed growth of small tumors without apparent toxicity. Responding tumors showed restoration of Fhit, Wwox, p16(INKa), Rassf1a expression, low mitotic activity, high apoptotic fraction and activation of caspase 3. These preclinical studies show the therapeutic potential of restoration of tumor suppressor expression through epigenetic modulation and the promise of re-expressed tumor suppressors as markers and effectors of the responses.     

脆性组氨酸三联体基因及其表达蛋白在肺癌发病机制中的研究进展

脆性组氨酸三联体（FHIT）基因是一种重要的抑癌基因,在非小细胞肺癌（NSCLC）早期和癌前病变中常见到其蛋白缺失,反映了FHIT基因改变是肺癌发生的中早期分子事件。在FHIT蛋白的参与下,促进铁氧化还原蛋白的电子传递功能,促进了癌细胞在氧化应激环境中的凋亡,FHIT蛋白再表达与铂剂联合增强了肿瘤细胞对凋亡的应答。FHIT／FRA3B的异常变化出现早、频率高、检测方便,对阐明人种、遗传多态性、地理因素、生活环境、呼吸疾病等在肺癌发病机制中的影响具有重要的意义。     

Reduced Fhit expression occurs in the early stage of esophageal tumorigenesis: no correlation with p53 expression and apoptosis

The FHIT gene, encompassing the FRA3B fragile site at chromosome 3p14.2, is a candidate tumor suppressor gene involved in multiple tumors, including esophageal carcinoma. We analyzed Fhit expression using an immunohistochemical method in invasive carcinoma, carcinoma in situ (CIS) and dysplasia, in paraffin sections of 75 esophageal squamous cell carcinomas (ESCs) to further elucidate the role of Fhit protein in esophageal carcinogenesis. In addition, we also examined whether Fhit expression correlated with p53 expression and apoptosis. Compared to adjacent normal mucosa, significant loss or reduction of Fhit expression was noted in 67 of 75 (89.3%) invasive ESCs, in 13 of 19 (68.4%) CIS lesions, and in 10 of 23 (43.5%) dysplastic lesions. There was a progressive loss or reduction of Fhit expression with progressive increases in the severity of histopathological changes (p < 0.001). However, there was no association between Fhit expression and clinicopathological findings, including tumor stage, lymph node metastasis, or overall survival. Moreover, Fhit expression was not significantly associated with p53 expression and apoptosis. These results indicate that abnormal Fhit expression is a common event in the early stage of ESC development and may occur independently of p53 expression and apoptosis mechanisms.     

Fragile histidine triad expression delays tumor development and induces apoptosis in human pancreatic cancer.

The fragile histidine triad (FHIT) gene is a tumor suppressor gene that is altered by deletion in a large fraction of human tumors, including pancreatic cancer. To evaluate the potential of FHIT gene therapy, we developed recombinant adenoviral and adenoassociated viral (AAV) FHIT vectors and tested these vectors in vitro and in vivo for activity against human pancreatic cancer cells. Our data show that viral FHIT gene delivery results in apoptosis by activation of the caspase pathway. Furthermore, Fhit overexpression enhances the susceptibility of pancreatic cancer cells to exogenous inducers of apoptosis. In vivo results show that FHIT gene transfer delays tumor growth and prolongs survival in a murine model mimicking human disease.     

5' CpG island methylation of the FHIT gene is correlated with loss of gene expression in lung and breast cancer

Allele loss and loss of expression of fragile histidine triad (FHIT), a putative tumor suppressor gene located in chromosome region 3p14.2, are frequent in several types of cancers. Tumor-acquired methylation of promoter region CpG islands is one method for silencing tumor suppressor genes. We investigated 5' CpG island methylation of the FHIT gene in 107 primary non-small cell lung cancer (NSCLC) samples and corresponding nonmalignant lung tissues, 39 primary breast carcinomas, as well as in 49 lung and 22 breast cancer cell lines by a methylation-specific PCR assay. In addition, we analyzed brushes from the bronchial epithelium of 35 heavy smokers without cancer. FHIT methylation was detected in 37% of primary NSCLCs, 31% of primary breast cancers, and 65% of lung and 86% of breast cancer cell lines. The frequency of methylation in small cell and NSCLC cell lines were identical. Methylation was found in 9% of the corresponding nonmalignant lung tissues and in 17% of bronchial brushes from heavy cigarette smokers. FHIT methylation was significantly correlated with loss of FHIT mRNA expression by Northern blot analysis in lung cancer cell lines and with loss of Fhit expression in NSCLC and breast tumors by immunostaining. We conclude that methylation of FHIT is a frequent event in NSCLC and breast cancers and is an important mechanism for loss of expression of this gene. Methylation of FHIT commences during lung cancer pathogenesis and may represent a marker for risk assessment.     

FHIT基因及其与食管癌关系的研究进展

脆性组氨酸三联体（FHIT）基因是一个候选抑癌基因,其抑癌作用可能通过诱导凋亡而实现。在多种肿瘤特别是环境致癌物引起的肿瘤中常有FHIT基因的异常改变。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。此文拟就FHIT基因与食管癌的发生、发展、治疗及预后等几方面进行综述。     

Fhit modulation of the Akt-survivin pathway in lung cancer cells: Fhit-tyrosine 114 (Y114) is essential

The Fhit tumor suppressor binds and hydrolyses diadenosine polyphosphates and the Fhit-substrate complex has been proposed as a proapoptotic effector, as determined by infection of susceptible cancer cells with adenoviruses carrying wild-type fragile histidine triad (FHIT) or catalytic site mutants. The highly conserved Fhit tyrosine 114 (Y114), within the unstructured loop C-terminal of the catalytic site, can be phosphorylated by Src family tyrosine kinases, although endogenous phospho-Fhit is rarely detected. To explore the importance of Y114 and identify Fhit-mediated signaling events, wild-type and Y114 mutant FHIT-expressing adenoviruses were introduced into two human lung cancer cell lines. Caspase-dependent apoptosis was effectively induced only by wild-type but not Y114 mutant Fhit proteins. By expression profiling of FHIT versus mutant FHIT-infected cells, we found that survivin, an Inhibitor of Apoptosis Protein (IAP) family member, was significantly decreased by wild-type Fhit. In addition, Fhit inhibited activity of Akt, a key effector in the phosphatidylinositol 3-OH kinase (PI3K) pathway; loss of endogenous Fhit expression caused increased Akt activity in vitro and in vivo, and overexpression of constitutively active Akt inhibited Fhit-induced apoptosis. The results indicate that the Fhit Y114 residue plays a critical role in Fhit-induced apoptosis, occurring through inactivation of the PI3K-Akt-survivin signal pathway.     

Fhit expression in gastric adenocarcinoma: correlation with disease stage and survival

BACKGROUND: The FHIT gene is inactivated by deletion in a large fraction of human tumors, including gastric carcinomas, and the Fhit protein has been proposed to act as a tumor suppressor in multiple tumor types. A large fraction of gastric adenocarcinomas have lost expression of the candidate tumor suppressor protein, Fhit, whereas normal gastric epithelial cells are strongly positive and Fhit loss has been found to correlate with alterations of the FHIT locus. Because the majority of gastric tumors in the current study were found to be entirely negative for Fhit protein, it is possible that alteration of the carcinogen-susceptible fragile region within the FHIT gene is an early event in gastric carcinoma, as it is in lung carcinoma. METHODS: To determine whether the absence of Fhit protein correlates with expression of tumor markers or with clinical parameters, such as grade, stage, and survival time, the authors assessed Fhit expression using immunohistochemistry in a well characterized set of 55 gastric adenocarcinomas resected over several years, with longitudinal follow-up of patients for outcome. RESULTS: In this set of 55 gastric cancers, the absence of Fhit protein correlated with higher tumor stage (P = 0.003) and higher histologic grade (P = 0.007). In addition, patients whose tumors had lost expression of Fhit died of disease significantly earlier than those with Fhit positive tumors (P = 0.017). The absence of Fhit expression did not correlate with the expression of any tumor markers. CONCLUSIONS: Larger studies will be required to elucidate further the relation between tumor stage, grade, and Fhit loss and to determine whether inclusion of Fhit antiserum in immunophenotyping of gastric adenocarcinomas will be a useful indicator of post-diagnosis prognosis.     

The expression of Fhit protein is related inversely to disease progression in patients with breast carcinoma

BACKGROUND: The FHIT gene, located at human chromosome 3p14.2, frequently is deleted in a number of human tumors, including breast carcinoma. Its protein product (Fhit) is presumed to have tumor suppressor function. Loss of expression of a tumor suppressor gene is an important step in tumor progression from premalignant, to in situ, to invasive carcinoma. METHODS: In the current study, Fhit expression was examined in invasive carcinomas and in epithelial lesions representing stages of carcinoma progression in 50 mastectomy specimens using immunohistochemical methods. RESULTS: Normal ductal and lobular epithelium consistently and strongly expressed Fhit. A complete loss of or a significant reduction in Fhit expression was observed in 72% of breast carcinomas. A statistically significant, negative correlation in Fhit expression among the stages of disease progression in Fhit negative breast carcinomas was observed (normal epithelium > hyperplasia > atypical hyperplasia and carcinoma in situ > invasive carcinoma), whereas no loss of Fhit expression in precursor lesions was observed in Fhit positive tumors. CONCLUSIONS: These observations are consistent with the observed role of FHIT as a tumor suppressor gene in the pathogenesis of specific subsets of carcinomas.     

Induction of apoptosis and inhibition of tumorigenicity and tumor growth by adenovirus vector-mediated fragile histidine triad (FHIT) gene overexpression.

We studied the effects of fragile histidine triad (FHIT) gene overexpression mediated by an adenoviral vector, Ad-FHIT, on cell proliferation, apoptosis, and cell cycle kinetics in human cancer cells and on tumorigenicity and tumor growth in nude mice. Overexpression of the FHIT gene significantly inhibited cell growth in various Ad-FHIT-transduced human lung cancer cells and head and neck carcinoma cells with FHIT gene abnormalities, but not in normal human bronchial epithelial cells. Fewer than 20% of cells in all Ad-FHIT-transduced cells survived at 7 days after transduction. Overexpression of the FHIT gene induced cell apoptosis and altered cell cycle processes. The apoptotic cell population markedly increased, and cells accumulated in S phase after Ad-FHIT transduction. The tumorigenicity of human H1299 lung cancer cells transduced by Ad-FHIT, in comparison with that of the control transductants and untreated cells, was eliminated in vivo. Subcutaneous tumor growth in nude mice who received intratumoral injections of Ad-FHIT, at a total dose of 3 x 10(10) plaque-forming units/tumor for H1299 tumors and 4 x 10(10)/tumor for A549 tumors, were suppressed by more than 85% and 90%, respectively, compared with that in nude mice who received injections of empty vector at the same dose or with PBS alone. Together, our results suggest that the FHIT gene, when delivered at high efficiency by a recombinant adenoviral vector, functions as a tumor suppressor gene both in vitro and in vivo.