Fragile Histidine Triad Loss Research Articles

Germinal epimutation of Fragile Histidine Triad (FHIT) gene is associated with progression to acute and chronic adult T-cell leukemia diseases

BackgroundHuman T cell Leukemia virus type 1 (HTLV-I) is etiologically linked to adult T cell leukemia/lymphoma (ATL) and an inflammatory neurodegenerative disease called HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP). The exact genetic or epigenetic events and/or environmental factors that influence the development of ATL, or HAM/TSP diseases are largely unknown. The tumor suppressor gene, Fragile Histidine Triad Diadenosine Triphosphatase (FHIT), is frequently lost in cancer through epigenetic modifications and/or deletion. FHIT is a tumor suppressor acting as genome caretaker by regulating cellular DNA repair. Indeed, FHIT loss leads to replicative stress and accumulation of double DNA strand breaks. Therefore, loss of FHIT expression plays a key role in cellular transformation.MethodsHere, we studied over 400 samples from HTLV-I-infected individuals with ATL, TSP/HAM, or asymptomatic carriers (AC) for FHIT loss and expression. We examined the epigenetic status of FHIT through methylation specific PCR and bisulfite sequencing; and correlated these results to FHIT expression in patient samples.ResultsWe found that epigenetic alteration of FHIT is specifically found in chronic and acute ATL but is absent in asymptomatic HTLV-I carriers and TSP/HAM patients’ samples. Furthermore, the extent of FHIT methylation in ATL patients was quantitatively comparable in virus-infected and virus non-infected cells. We also found that longitudinal HTLV-I carriers that progressed to smoldering ATL and descendants of ATL patients harbor FHIT methylation.ConclusionsThese results suggest that germinal epigenetic mutation of FHIT represents a preexisting mark predisposing to the development of ATL diseases. These findings have important clinical implications as patients with acute ATL are rarely cured. Our study suggests an alternative strategy to the current “wait and see approach” in that early screening of HTLV-I-infected individuals for germinal epimutation of FHIT and early treatment may offer significant clinical benefits.

Mechanisms shaping the mutational landscape of the FRA3B/FHIT‐deficient cancer genome

Genome instability is an enabling characteristic of cancer that facilitates the acquisition of oncogenic mutations that drive tumorigenesis. Underlying much of the instability in cancer is DNA replication stress, which causes both chromosome structural changes and single base-pair mutations. Common fragile sites are some of the earliest and most frequently altered loci in tumors. Notably, the fragile locus, FRA3B, lies within the fragile histidine triad (FHIT) gene, and consequently deletions within FHIT are common in cancer. We review the evidence in support of FHIT as a DNA caretaker and discuss the mechanism by which FHIT promotes genome stability. FHIT increases thymidine kinase 1 (TK1) translation to balance the deoxyribonucleotide triphosphates (dNTPs) for efficient DNA replication. Consequently, FHIT-loss causes replication stress, DNA breaks, aneuploidy, copy-number changes (CNCs), small insertions and deletions, and point mutations. Moreover, FHIT-loss-induced replication stress and DNA breaks cooperate with APOBEC3B overexpression to catalyze DNA hypermutation in cancer, as APOBEC family enzymes prefer single-stranded DNA (ssDNA) as substrates and ssDNA is enriched at sites of both replication stress and DNA breaks. Consistent with the frequent loss of FHIT across a broad spectrum of cancer types, FHIT-deficiency is highly associated with the ubiquitous, clock-like mutation signature 5 occurring in all cancer types thus far examined. The ongoing destabilization of the genome caused by FHIT loss underlies recurrent inactivation of tumor suppressors and activation of oncogenes. Considering that more than 50% of cancers are FHIT-deficient, we propose that FRA3B/FHIT fragility shapes the mutational landscape of cancer genomes.

Effect of FHIT loss and p53 mutation on HPV-infected lung carcinoma development.

High-risk human papillomavirus (HPV)16/18 infection in the development of lung cancer has previously been identified, and fragile histidine triad (FHIT) loss and p53 mutation are frequently observed in the disease. However, the association between these factors has not been well studied. The present study aimed to further investigate the significance of HPV infection, FHIT loss and p53 mutations in the development of lung cancer and their possible associations. DNA was extracted from paraffin-embedded specimens from 88 cases of squamous cell carcinoma (SCC), 56 of adenocarcinoma (AC), 36 of small cell lung carcinoma (SCLC) and 110 non-cancer control cases of lung neoplasms. The prevalence of HPV infection was determined by polymerase chain reaction analysis, and FHIT loss and p53 mutations were detected by immunohistochemistry. The χ2, Fisher's exact and Pearson correlation tests were applied for statistical analysis. The results of the present study demonstrated that HPVL1 (the major capsid protein of HPV), HPV16 and HPV18 infection were more prevalent in the lung cancer samples compared with the non-cancer controls (all P<0.001). FHIT loss occurred more frequently in the lung cancer samples (44.44%) compared with the non-cancer controls (7.25%) (P<0.001). FHIT loss in the HPVL1-positive group was significantly increased compared with the HPVL1-negative group in the lung cancer cases and the non-cancer controls (P<0.05). In the lung cancer cases, the p53 mutation rates in the HPVL1- and HPV16/18-positive groups were significantly increased compared with the HPVL1- and HPV16/18-negative groups (P<0.05). In the 180 lung cancer cases, the coexistence rate of FHIT loss and a history of smoking was 38.33% (69/180; Pearson contingency coefficient of r=0.318; P<0.001). FHIT loss and p53 mutation exhibited a synergistic effect on HPV-associated lung cancer (Pearson contingency coefficient r=0.357, P<0.001). The present study demonstrated that FHIT loss may be important in the occurrence of lung cancer, particularly in lung SCCs. FHIT loss may therefore be used as an early indicator for lung cancer, particularly for patients with a history of smoking. HPV infection in lung tumorigenesis may, at least in part, be mediated through FHIT loss. FHIT loss and p53 mutation may coordinate together in the development of HPV-associated lung cancer, and accelerate the occurrence and development of lung cancer.

Molecular damage and lung tumors in cigarette smoke-exposed mice.

Cigarette smoke (CS) induces lung cancer through a multistep process that is now being depicted by molecular analyses. During the early phase (weeks), DNA damage occurs in nuclear and mitochondrial DNA, triggering adaptive responses activated by transient microRNA downregulation in the expression of defensive genes and proteins. During the intermediate phase (months), damaged cells are removed by apoptosis and the resulting cell loss is counteracted by a recruitment of stem cells that are highly sensitive to genotoxic damage. In parallel, microRNA downregulation becomes irreversible because of an accumulation of molecular damage in DICER. During the late phase (years), apoptosis efficacy is decreased by fragile histidine triad loss, while irreversible microRNA downregulation triggers the expression of mutated oncogenes, resulting in adenoma appearance. Furthermore, deletions occur in microRNA-encoding genes, causing carcinoma formation and uncontrolled growth. All reported pathogenic steps are required to obtain a fully developed lung cancer. This complex pathogenesis develops over a long period of time; therefore, it is difficult to induce cancer in short-living animals exposed to CS, whereas in humans there is a long latency from the start of smoke exposure to the onset of cancer.

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.

A knockdown with smoke model reveals FHIT as a repressor of Heme oxygenase 1

Fragile histidine triad (FHIT) gene deletions are among the earliest and most frequent events in carcinogenesis, particularly in carcinogen-exposed tissues. Though FHIT has been established as an authentic tumor suppressor, the mechanism underlying tumor suppression remains opaque. Most experiments designed to clarify FHIT function have analyzed the consequence of re-expressing FHIT in FHIT-negative cells. However, carcinogenesis occurs in cells that transition from FHIT-positive to FHIT-negative. To better understand cancer development, we induced FHIT loss in human bronchial epithelial cells with RNA interference. Because FHIT is a demonstrated target of carcinogens in cigarette smoke, we combined FHIT silencing with cigarette smoke extract (CSE) exposure and measured gene expression consequences by RNA microarray. The data indicate that FHIT loss enhances the expression of a set of oxidative stress response genes after exposure to CSE, including the cytoprotective enzyme heme oxygenase 1 (HMOX1) at the RNA and protein levels. Data are consistent with a mechanism in which Fhit protein is required for accumulation of the transcriptional repressor of HMOX1, Bach1 protein. We posit that by allowing superinduction of oxidative stress response genes, loss of FHIT creates a survival advantage that promotes carcinogenesis.

FHIT loss confers cisplatin resistance in lung cancer via the AKT/NF-κB/Slug-mediated PUMA reduction.

Fragile histidine triad (FHIT) loss by the two-hit mechanism of loss of heterozygosity and promoter hypermethylation commonly occurrs in non-small cell lung cancer (NSCLC) and may confer cisplatin resistance in NSCLC cells. However, the underlying mechanisms of FHIT loss in cisplatin resistance and the response to cisplatin-based chemotherapy in NSCLC patients have not yet been reported. In the present study, inhibition concentration of 50% cell viability induced by cisplatin (IC50) and soft agar growth and invasion capability were increased and decreased in FHIT-knockdown and -overexpressing cells, respectively. Mechanistically, Slug transcription is upregulated by AKT/NF-κB activation due to FHIT loss and, in turn, Slug suppresses PUMA expression; this decrease of PUMA by FHIT loss is responsible for cisplatin resistance. In addition, cisplatin resistance due to FHIT loss can be conquered by AKT inhibitor-perifosine in xenograft tumors. Among NSCLC patients, low FHIT, high p-AKT, high Slug and low PUMA were correlated with shorter overall survival, relapse-free survival and poorer response to cisplatin-based chemotherapy. Therefore, the AKT inhibitor perifosine might potentially overcome the resistance to cisplatin-based chemotherapy in NSCLC patients with low-FHIT tumors, and consequently improve the outcome.

Detection of FHIT and p16 mRNA Deletion in Biopsy Specimens Obtained by Bronchoscopy for the Diagnosis of Lung Cancer

The purpose of this study was to investigate the diagnostic value of the deletion of fragile histidine triad (FHIT) and p16INK4a (p16) mRNA in biopsies obtained by bronchoscopy. Biopsies were analyzed using RT-PCR in 52 patients with lung cancer and 19 patients with benign lung disease. The results showed that the detection rates of FHIT and p16 gene transcript deletion were significantly higher in lung cancer patients than in patients with benign lung disease (65.4% versus 10.5%, p=0.001 and 59.6% versus 5.3%, p<0.001, respectively). The sensitivities for detecting FHIT and p16 transcript deletion in biopsies were 65.4% and 59.6% (combined 80.8%), respectively, which were markedly better than those of histology and cytology (42.3% and 34.6%, respectively; combined 57.7%). In 22 lung cancer patients with negative histology and cytology at initial bronchoscopy, FHIT and p16 mRNA loss was detected in 40.9% (9/22) and 36.4% (8/22) cases, respectively. FHIT mRNA loss was associated with smoking status in lung cancer patients. In conclusion, deletion of FHIT and p16 mRNA can be identified in biopsies obtained during bronchoscopic procedures. FHIT and p16 mRNA deletion can be used as biomarkers in the clinical diagnosis of lung cancer and may serve as adjuncts to histology and cytology in lung cancer diagnosis.

Diagnostic value of the FHIT and p16 mRNA loss and the K-ras gene mutation in pleural fluids for malignant pleural effusion

Inactivation of the tumor suppressor genes and activation of oncogenes are involved in the development of cancer. The aim of this study was to evaluate the diagnostic value of the fragile histidine triad (FHIT) and p16 mRNA loss and the K-ras gene mutation in distinguishing malignant from benign pleural effusion. A total of 50 patients with malignant pleural effusion and 30 patients with benign pleural effusion were enrolled in this study. All pleural fluid specimens were evaluated in parallel by cytology, reverse transcriptase-PCR for the loss of FHIT and p16 mRNA, and PCR-SSCP (single-stranded conformation polymorphism) for the mutation of K-ras gene. The detection rates of FHIT and p16 mRNA loss were significantly higher in malignant than in benign pleural effusion (P < 0.001 and P = 0.001). The K-ras mutations were more frequent in malignant than benign pleural effusion (P = 0.006). The sensitivity and specificity were 58% and 93% for FHIT loss, 48% and 90% for p16 loss, and 44% and 87% for the K-ras mutation, respectively. In combined evaluation with both FHIT and p16 loss, the sensitivity was 68%, and specificity was 90%. The combination of the three molecular markers reached 74% sensitivity, whereas the combined use of the cytology and the three markers increased the diagnostic yield of the former by 38%. More than one third of cytology negative malignant pleural effusion could be identified by at least one of the three markers. These results suggest that the detection of FHIT and p16 mRNA loss and the k-ras gene mutation in pleural fluid could be helpful adjunct to cytology in the diagnosis of malignant pleural effusion.

High-resolution Whole-Genome Analysis of Skull Base Chordomas Implicates FHIT Loss in Chordoma Pathogenesis

Chordoma is a rare tumor arising in the sacrum, clivus, or vertebrae. It is often not completely resectable and shows a high incidence of recurrence and progression with shortened patient survival and impaired quality of life. Chemotherapeutic options are limited to investigational therapies at present. Therefore, adjuvant therapy for control of tumor recurrence and progression is of great interest, especially in skull base lesions where complete tumor resection is often not possible because of the proximity of cranial nerves. To understand the extent of genetic instability and associated chromosomal and gene losses or gains in skull base chordoma, we undertook whole-genome single-nucleotide polymorphism microarray analysis of flash frozen surgical chordoma specimens, 21 from the clivus and 1 from C1 to C2 vertebrae. We confirm the presence of a deletion at 9p involving CDKN2A, CDKN2B, and MTAP but at a much lower rate (22%) than previously reported for sacral chordoma. At a similar frequency (21%), we found aneuploidy of chromosome 3. Tissue microarray immunohistochemistry demonstrated absent or reduced fragile histidine triad (FHIT) protein expression in 98% of sacral chordomas and 67%of skull base chordomas. Our data suggest that chromosome 3 aneuploidy and epigenetic regulation of FHIT contribute to loss of the FHIT tumor suppressor in chordoma. The finding that FHIT is lost in a majority of chordomas provides new insight into chordoma pathogenesis and points to a potential new therapeutic target for this challenging neoplasm.

Role of fragile histidine triad protein expression in pathogenesis of malignant pleural mesothelioma

To investigate the relationship of fragile histidine triad (FHIT) and Ki-67 expression with clinicopathological variables of patients with malignant pleural mesothelioma (MPM). Formalin-fixed, paraffin-embedded tissue sections of 30 asbestos induced MPM (epithelial and biphasic) patients were examined for FHIT and Ki-67 expression using immunohistochemical techniques and results were compared with clinicopathological variables. Immunohistochemical study results were as follows: 12 (40%) cases showed low FHIT expression and 18 (60%) showed high expression. There was no significant relationship between FHIT and age, gender or histological subtypes (p > 0.05). Ki-67 expression was 'low' in 13 (43.3%) cases and 'high' in 17 (56.7%) cases. No correlation could be demonstrated between Ki-67 expression and age, gender or histological subtypes (p > 0.05). No significant association was observed between FHIT and Ki-67 expression in MPM. The results support the role of FHIT as a tumour suppressor gene in asbestos induced MPM. There is no significant correlation between FHIT and cell proliferation marker expressions in malignant pleural mesothelioma. Therefore, it can be concluded that loss of FHIT does not interfere with tumour proliferation. This can be accepted as evidence for an early role of FHIT loss in carcinogenesis; however, it needs to be strengthened by further studies.

Fragile Histidine Triad Expression in Oral Squamous Cell Carcinoma and Precursor Lesions

Fragile histidine triad (FHIT) expression in precursor oral lesions (POL) and oral squamous cell carcinomas (OSCC) was studied with regard to (a) the frequency of loss of FHIT expression, (b) whether loss of FHIT expression correlates with degree of dysplasia in POLs, (c) whether FHIT loss predicts high-risk POLs that are more likely to transform, and (d) whether FHIT loss in OSCCs correlates with survival. Ninety-four POLs and 86 OSCCs were immunostained for FHIT. Survival analysis was done for cases with validated clinical outcomes. By optimizing the immunostaining protocol, we found that FHIT is expressed in a distinctive strong nuclear and weak cytoplasmic pattern in oral tissues. Loss of FHIT expression was found in 42 of 94 (45%) POLs and in 66 of 86 (77%) OSCCs. We observed a statistically significant positive correlation between frequency of FHIT loss and increasing grade of dysplasia (chi2=13.8; degrees of freedom=4; P=0.008). Loss of FHIT expression in POLs that progressed to malignancy was more frequent than in those that did not [17 of 25 (68%) versus 12 of 29 (41.4%), respectively]. This difference was statistically significant (chi2=3.8; degrees of freedom=1; P=0.046). In OSCCs, loss of FHIT staining indicated a worse prognosis (survival rate, 36.2%) than when positive FHIT staining was observed (survival rate, 50%), but the difference was not statistically significant (P=0.546, Kaplan-Meier, log-rank). FHIT seems to localize to both nuclear and cytoplasmic domains. FHIT inactivation occurs early in oral carcinogenesis and may be useful molecular marker for progressive dysplastic oral lesions.

Expression of fragile histidine triad and p53 in non-small cell lung cancer

ObjectiveTo investigate the expression of fragile histidine triad (FHIT) and p53 protein in non-small cell lung cancer (NSCLC) and explore the relationship between their expressions and the clinicopathological features.MethodsFHIT protein and p53 protein were detected by immunohistochemistry in 76 cases of NSCLCs and matched normal lung tissues.ResultsFifty-one cases (67.1%) showed negative expression of FHIT (apparent reduction or loss) and thirty-seven cases (48.7%) showed p53 positive expression (overexpression). The difference was significant (P=0.04). However, there was no significant difference in FHIT expression between the p53-positive group and the p53-negative group (64.9% versus 69.2%, P=0.686). The negative rate of FHIT protein expression was higher in squamous cell carcinoma than in adenocarcinoma, in moderately and poorly differentiated carcinoma than in well-differentiated carcinoma, and in cases with smoking history than in cases without smoking history (P<0.05). There was no relationship between FHIT expression and clinical stage or lymph node metastasis. The negative FHIT expression was not an independent predictor of overall survival (P=0.0338).ConclusionThe frequency of negative expression of FHIT protein is higher than that of positive expression of p53 in NSCLCs. The negative expression of FHIT is independent of the expression of p53. The change of expression of FHIT may play a role in the smoking related lung tumorigenesis while it may have no relationship with the progress of NSCLC or prognosis of the patients.

Decreased Fragile Histidine Triad Gene Protein Expression Is Associated With Worse Prognosis in Oral Squamous Carcinoma

Fragile histidine triad (FHIT) gene is thought to be a tumor suppressor; abnormalities in expression have been reported in a variety of neoplasms. To determine whether abnormalities of FHIT protein expression or loss of heterozygosity in the FHIT gene were correlated with survival or other clinical parameters in patients with oral cavity squamous cell carcinoma. Fifty-three patients with initial surgical treatment of oral cavity squamous cell carcinoma were followed a minimum of 5 years or until death. The FHIT protein expression was studied by immunohistochemistry in all patients, and a subset of 20 patients was studied for allelic loss of heterozygosity and microsatellite instability using formalin-fixed, paraffin-embedded tissue. Sixty-one percent of patients whose tumors had reduced FHIT expression were dead of disease, and 37% of patients whose tumors exhibited preserved FHIT expression were dead of disease at 5-year follow-up. Log-rank analysis showed that patients retaining FHIT expression had a longer overall survival (P = .03) and disease-free survival (P = .01). The FHIT expression was not correlated with node status or clinical stage. Loss of heterozygosity was seen in 10 (50%) of 20 tumors, low levels of microsatellite instability in 4 (20%) of 20 tumors, and high levels of microsatellite instability in 1 (5%) of 20 tumors tested. The FHIT gene was associated with a worse survival outcome when its expression was reduced in patients with oral cavity squamous cell carcinoma. Loss of heterozygosity in the gene was common, but no correlation with protein expression was found. Neither loss of heterozygosity nor microsatellite instability was found to correlate with survival. Because genomic alterations involving loss of heterozygosity of the FHIT gene were not associated with protein expression in these tumors, the presence or absence of FHIT expression may be controlled by other factors.