The FHIT gene, recently cloned and mapped on chromosome 3p14.2, has frequently been found to be abnormal in several established cancer cell lines and primary tumours. As alterations of chromosome 3p are common events in ovarian cancers with breakpoint sites at 3
p14.2, we decided to investigate the role of FHIT in human ovarian tumorigenesis. Fifty-four primary ovarian carcinomas were studied by reverse transcription of FHIT mRNA followed by polymerase chain reaction (PCR) amplification and sequencing of products. The same tumours and matched normal tissues were also investigated for loss of heterozygosity using three microsatellite markers located inside the gene. We found an abnormal transcript of the FHIT gene in two cases (4%) and allelic losses in eight cases (15%). Twelve (22%) of the 54 tumours investigated belonged to young patients with a family history of breast/ovarian cancer. In none of these cases was the FHITgene found to be altered. Our results indicate that FHITplays a role in a small proportion of ovarian carcinomas.
Dannewitz Prosseda S, etal., Am J Respir Crit Care Med. 2019 Jan 1;199(1):83-98. doi: 10.1164/rccm.201712-2553OC.
RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by progressive narrowing of pulmonary arteries, resulting in right heart failure and death. BMPR2 (bone morphogenetic protein receptor type 2) mutations account for most familial PAH forms whereas reduced BMPR2 is present i
n many idiopathic PAH forms, suggesting dysfunctional BMPR2 signaling to be a key feature of PAH. Modulating BMPR2 signaling is therapeutically promising, yet how BMPR2 is downregulated in PAH is unclear. OBJECTIVES: We intended to identify and pharmaceutically target BMPR2 modifier genes to improve PAH. METHODS: We combined siRNA high-throughput screening of >20,000 genes with a multicohort analysis of publicly available PAH RNA expression data to identify clinically relevant BMPR2 modifiers. After confirming gene dysregulation in tissue from patients with PAH, we determined the functional roles of BMPR2 modifiers in vitro and tested the repurposed drug enzastaurin for its propensity to improve experimental pulmonary hypertension (PH). MEASUREMENTS AND MAIN RESULTS: We discovered FHIT (fragile histidine triad) as a novel BMPR2 modifier. BMPR2 and FHIT expression were reduced in patients with PAH. FHIT reductions were associated with endothelial and smooth muscle cell dysfunction, rescued by enzastaurin through a dual mechanism: upregulation of FHIT as well as miR17-5 repression. Fhit-/- mice had exaggerated hypoxic PH and failed to recover in normoxia. Enzastaurin reversed PH in the Sugen5416/hypoxia/normoxia rat model, by improving right ventricular systolic pressure, right ventricular hypertrophy, cardiac fibrosis, and vascular remodeling. CONCLUSIONS: This study highlights the importance of the novel BMPR2 modifier FHIT in PH and the clinical value of the repurposed drug enzastaurin as a potential novel therapeutic strategy to improve PAH.
The fragile histidine triad (FHIT) gene is a candidate tumour suppressor gene in breast and other cancers. We investigated deletions within the FHIT gene in lobular breast cancer and found that 16% of cases showed loss of he
terozygosity (LOH) within the gene. We compared LOH within FHIT in lobular and ductal breast tumours and found a significant association between LOH at FHIT and the ductal histological type (P<0.001). To determine whether genomic alteration of the FHIT gene in lobular breast cancer leads to Fhit inactivation we have assessed the level of Fhit expression by immunohistochemical detection and determined that 27% (15 of 55) consecutive sporadic lobular tumours showed negative or reduced Fhit expression. A significant association was found between LOH at the FHIT gene and reduced Fhit expression in lobular and ductal tumours (P=0.025 and P=0.001, respectively). Thus, genetic alterations within the FHIT gene, leading to loss of Fhit protein, may play an important role in the carcinogenesis of a significant number of sporadic lobular breast cancers, even though the apparent frequency of genomic alterations within the gene is lower than in ductal breast cancer.
Terry G, etal., Br J Cancer. 2007 Jan 15;96(1):110-7. Epub 2006 Dec 12.
Immunohistochemical staining for FHIT and PCNA proteins was carried out in 451 breast lesions showing nonproliferative benign breast disease (BBD) (n=263), proliferative BBD without atypia (n=128), proliferative BBD with atypia (n=11), carcinoma in situ (n=15) o
r invasive carcinoma (n=34) and for EGFR protein in a subset of 71 of these cases. FHIT underexpression was not detected in nonproliferative lesions, but occurred in 2% of proliferative BBD without atypia, 10% proliferative BBD with atypia, 27% of carcinoma in situ and 41% of invasive carcinoma, which suggests that it could be useful in assessing those carcinoma in situ lesions (ductal, DCIS and lobular, LCIS) that are more likely to progress to malignancy. Preliminary microarray comparisons on DCIS and invasive carcinoma samples dissected from formalin-fixed paraffin sections showed a consistent downregulation of two previously identified FHIT-related genes, caspase 1 and BRCA1 in lesions underexpressing FHIT.
The FHIT (fragile histidine triad) gene located at chromosome 3p14.2 has been proposed as a candidate tumor suppressor gene in human cancers. Fhit protein with the diadenosine 5',5'''-P1,P3-triphosphate (Ap3A) hydrolase acti
vity is the protein product of FHIT gene. The way in which Fhit exerts its tumor suppressor activity and the relationship of the Ap3A hydrolase activity to tumor suppression are not known. As a step toward understanding of the Fhit function in the cell we have explored its intracellular localization and distribution in the rat tissues. Data obtained from immunoblot analysis showed that Fhit protein was most abundant in spleen and brain. Moderate amount of Fhit was detected in kidney and liver, whereas the level of Fhit protein in heart, skeletal muscle and kidney glomeruli was undetectable. RT-PCR performed on RNA isolated from these tissues showed no product, whereas the level of Fhit mRNA in spleen, brain, kidney, liver and lung correlated with the Fhit protein level. The immunoblot analysis performed on subcellular fractions of various rat tissues obtained by differential and density-gradient centrifugation showed that Fhit protein was localized exclusively in nucleus and at the plasma membrane. Presented data showing nuclear and plasma membrane localization of Fhit may support the hypothesis concerning Fhit as a signaling molecule.
Larson GP, etal., Cancer Res. 2005 Feb 1;65(3):805-14.
We conducted linkage analysis of 80 candidate genes in 201 brother pairs affected with prostatic adenocarcinoma. Markers representing two adjacent candidate genes on chromosome 3p, CDC25A and FHIT, showed suggestive evidence for linkage with single-point identit
y-by-descent allele-sharing statistics. Fine-structure multipoint linkage analysis yielded a maximum LOD score of 3.17 (P = 0.00007) at D3S1234 within FHIT intron 5. For a subgroup of 38 families in which three or more affected brothers were reported, the LOD score was 3.83 (P = 0.00001). Further analysis reported herein suggested a recessive mode of inheritance. Association testing of 16 single nucleotide polymorphisms (SNP) spanning a 381-kb interval surrounding D3S1234 in 202 cases of European descent with 143 matched, unrelated controls revealed significant evidence for association between case status and the A allele of single nucleotide polymorphism rs760317, located within intron 5 of FHIT (Pearson's chi(2) = 8.54, df = 1, P = 0.0035). Our results strongly suggest involvement of germline variations of FHIT in prostate cancer risk.
Mao X, etal., Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2016 Apr;33(2):186-90. doi: 10.3760/cma.j.issn.1003-9406.2016.02.013.
OBJECTIVE: To correlate the clinical characteristics with mutations of the STK11 and FHIT genes in 16 patients with Peutz-Jeghers syndrome (PJS). METHODS: Potential mutations in the coding regions and flanking sequences of the STK11 and FHIT
:700;'>FHIT genes were detected with PCR and Sanger sequencing. RESULTS: Of the 16 patients with PJS, 8 had novel mutations in the coding region of the STK11 gene, 1 had a previously reported mutation. 1 carried a mutation in the exon 10 of the FHIT gene, which is a non-coding region. None of the mutations was detected in the immediate family members. None of the patients with STK11 gene mutations had mutation in the FHIT gene. The mutation rate of the STK11 gene among patients with PJS was 56.25%. CONCLUSION: Mutations of the STK11 gene are the major cause of PJS. Few such patients had mutations of the FHIT gene. Mutations of the FHIT gene may play a part in the pathogenesis of PJS.
Malak CA, etal., Asian Pac J Cancer Prev. 2015;16(18):8197-201.
BACKGROUND: To investigate the expression of the fragile histidine triad (FHIT) gene in acute lymphoblastic leukemia and its clinical significance. MATERIALS AND METHODS: The level of expressed FHIT mRNA in peripheral blood
from 50 patients with acute lymphoblastic leukemia (ALL) and in 50 peripheral blood samples from healthy volunteers was measured via RT-PCR. Correlation analyses between FHIT gene expression and clinical characteristics (gender, age, white blood count, immunophenotype of acute lymphoblastic leukemia and percentage of blast cells) of the patients were performed. RESULTS: The FHIT gene was expressed at 2.49+/-7.37 of ALL patients against 14.4+/-17.9 in the healthy volunteers. The difference in the expression levels between ALL patients and healthy volunteers was statistically significant. The rate of gene expression did not significantly vary with immunophenotype subtypes. Gene expression was also found to be correlated with increase of total leukocyte and decrease in platelets, but not with age, gender, immunophenotyping or percentage of blast cells. CONCLUSIONS: FHIT gene expression is low in acute lymphoblastic leukemia and could be a useful marker to monitor minimal residual disease. This gene is also a candidate target for the immunotherapy of acute lymphoblastic leukemia.
Eyzaguirre EJ, etal., Mod Pathol. 1999 Oct;12(10):979-83.
BACKGROUND: The FHIT gene on human chromosome 3p14.2 is deleted in a variety of malignant tumors, including clear cell renal carcinomas (RCCs) resulting in a loss of expression of Fhit protein. The Fhit
00;'>Fhit expression in specific subtypes of renal carcinomas has not been characterized. We have investigated the association of Fhit expression with particular subtypes of renal tumors to determine the role and specificity of this putative tumor suppressor gene in renal neoplasia. MATERIAL AND METHODS: The immunohistochemical expression of Fhit was tested in normal kidneys and in 109 renal neoplasms consisting of 51 clear cell RCCs, 26 papillary RCCs, two chromophobe carcinomas, six oncocytomas, four pelvic transitional cell carcinomas and 20 Wilms' tumors from formalin fixed and routinely processed tissue. RESULTS: Normal renal tubules expressed Fhit strongly and consistently. The majority (78%) of clear cell RCCs showed reduced or absent expression of Fhit, whereas the majority (74%) of papillary carcinomas, all chromophobe renal cell carcinomas, and oncocytomas were strongly positive. Sixty-eight percent of low-grade (G1 plus G2) but only 9% of high-grade (G3 plus G4) clear cell carcinomas were Fhit negative. Wilms' tumors demonstrated focal staining in the epithelial component in 8 of 20 cases (40%). CONCLUSIONS: The loss of Fhit expression in a high percentage of clear cell RCCs with conservation of Fhit in other types of tumors supports the proposed role of FHIT alterations in the genesis of clear cell carcinomas in contrast to other types of renal epithelial tumors. FHIT expression may play a role in epithelial differentiation of nephroblastomas (Wilms' tumors).
Eyzaguirre E and Gatalica Z, Mod Pathol. 2002 Oct;15(10):1068-72.
The FHIT gene, located at human chromosome 3p14.2, is frequently deleted in a number of human cancers, and interstitial deletions at this site were recently described in a significant proportion (41%) of testicular germ cell tumors. We studied the expression of
Fhit protein in the progression and differentiation of testicular germ cell tumors to further elucidate its role in this type of malignancy. Forty-five patients with testicular germ cell tumors and intratubular germ cell neoplasia (identified in 42/45 cases) were included in the study. Immunohistochemical staining with polyclonal rabbit IgG antibody to Fhit (ZR44, Zymed Laboratories) on formalin-fixed, paraffin-embedded tissues was used. Fhit was constitutively expressed in germ cells, Sertoli cells, and Leydig cells. All 42 cases of intratubular germ cell neoplasia revealed no expression of this protein. No expression of Fhit was observed in any case of pure seminoma or in the seminomatous component of mixed germ cell tumors. Unexpectedly, Fhit expression was frequently (16/18) observed in the glandular tissue of mature teratomatous component of mixed germ cell tumors, despite the absence of Fhit in the intratubular germ cell neoplasia, the presumed precursor lesion. The loss of Fhit expression is a consistent characteristic of intratubular germ cell neoplasia, which suggests a potential role in a maturation/differentiation defect early in the development of testicular germ cell tumors. Likewise, the lack of expression in seminomas is supportive of this view. However, re-expression of Fhit in well-differentiated glandular epithelium of teratomatous component of mixed germ cell tumors suggests that there is no loss of FHIT gene in this subset of neoplasia but rather that Fhit protein expression is differently regulated through the phases of germ cell tumor progression.
Yang Q, etal., Clin Cancer Res. 2002 Sep;8(9):2890-3.
PURPOSE: The FHIT gene, which spans the FRA3B fragile site at chromosome 3p14.2, is a candidate tumor suppressor gene in breast carcinomas. In this study, we would like to delineate more precisely its role in breast tumorigenesis. EXPERIMENTAL DESIGN: To confirm
the tumorigenic role of FHIT, 46 sporadic invasive ductal carcinomas of the breast were tested for the "two hits" required to inactivate this gene. Microsatellite loss of heterozygosity (LOH) was considered as the first hit. To examine the possibility that hypermethylation serves as the second hit for FHIT inactivation, methylation of 5'-CpG islands of FHIT was analyzed by methylation-specific PCR. RESULTS: LOH was detected in 8 of 40 informative tumors, and hypermethylation was observed in 22 of 46 (48%) cases. Aberrant FHIT protein expression was found in 31 of 46 (67%) cases examined. All seven tumors showing both LOH and hypermethylation showed complete loss of Fhit protein expression. In addition, a significant positive association was found between the existence of LOH and 5'-CpG island hypermethylation (P = 0.04), which was consistent with the two-hit model. CONCLUSIONS: To our knowledge, this study provides the first evidence that biallelic inactivation of FHIT by LOH and hypermethylation leads to the complete inactivation of FHIT gene in patients with breast cancer. Silencing of the FHIT gene by promoter hypermethylation occurs in primary breast carcinomas, especially those with LOH. These findings support a role for this tumor suppressor gene in sporadic breast tumorigenesis.
Burke L, etal., Cancer Res. 1998 Jun 15;58(12):2533-6.
The fragile histidine triad (FHIT) gene at chromosome 3p14.2 is a candidate tumor suppressor gene linked to cancers of the lung, breast, colon, pancreas, and head and neck. Reports of frequent allelic deletion and abnormal transcripts in primary lung tumors plus
recent evidence that it is targeted by tobacco smoke carcinogens suggest that it plays an important role in lung carcinogenesis. Non-small cell lung carcinoma still maintains a poor 5-year survival rate with the stage of disease at presentation as a major determinant of prognosis. We examined for allelic deletion at the FHIT locus in a series of 106 non-small cell lung carcinomas for which a full clinical, epidemiological, and 5-year survival profile was available. We found an allelic deletion frequency of 38% at one or two intragenic microsatellites. Allelic deletion of FHIT was related to tumor histology with 4 of 20 adenocarcinomas (20%) displaying loss of heterozygosity (LOH) compared with 12 of 22 (55%) nonadenocarcinomas (P = 0.03). We found that 63% of tumors with LOH of FHIT also had p53 missense mutations whereas only 26% with LOH had wild type p53 negative sequence (P = 0.02). We also found a significant trend toward poorer survival in patients with LOH of at least one locus of the FHIT gene (log rank, P = 0.01). This survival correlation is independent of tumor stage, size, histological subtype, degree of differentiation, and p53 mutation status. Our data support the hypothesis that the loss of the FHIT contributes to the molecular pathogenesis of human lung cancer and is an indicator of poor prognosis.
Gemmill RM, etal., Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9572-7.
The 3;8 chromosomal translocation, t(3;8)(p14.2;q24.1), was described in a family with classical features of hereditary renal cell carcinoma. Previous studies demonstrated that the 3p14.2 breakpoint interrupts the fragile histidine triad gene (FHIT) in its 5' no
ncoding region. However, evidence that FHIT is causally related to renal or other malignancies is controversial. We now show that the 8q24.1 breakpoint region encodes a 664-aa multiple membrane spanning protein, TRC8, with similarity to the hereditary basal cell carcinoma/segment polarity gene, patched. This similarity involves two regions of patched, the putative sterol-sensing domain and the second extracellular loop that participates in the binding of sonic hedgehog. In the 3;8 translocation, TRC8 is fused to FHIT and is disrupted within the sterol-sensing domain. In contrast, the FHIT coding region is maintained and expressed. In a series of sporadic renal carcinomas, an acquired TRC8 mutation was identified. By analogy to patched, TRC8 might function as a signaling receptor and other pathway members, to be defined, are mutation candidates in malignant diseases involving the kidney and thyroid.
Wojdyla-Mamon AM and Guranowski A, Biosci Rep. 2015 Jun 25;35(4). pii: e00235. doi: 10.1042/BSR20150135.
Fhits (fragile histidine triad proteins) occur in eukaryotes but their function is largely unknown, although human Fhit is believed to act as a tumour suppressor. Fhits also exhibit dinu
cleoside triphosphatase, adenylylsulfatase and nucleoside phosphoramidase activities that in each case yield nucleoside 5'-monophosphate as a product. Due to the dinucleoside triphosphatase activity, Fhits may also be involved in mRNA decapping. In the present study, we demonstrate Fhit-catalysed ammonolysis of adenosine 5'-phosphosulfate, which results in the formation of adenosine 5'-phosphoramidate. This reaction has previously been associated with adenylylsulfate-ammonia adenylyltransferase (EC 2.7.7.51). Our finding shows that the capacity to catalyse ammonolysis is another inherent property of Fhits. Basic kinetic parameters and substrate specificity of this reaction catalysed by human Fhit are presented.
The present study was conducted to assess whether Fhit gene alterations are a feature of hepatocellular carcinomas (HCCs) induced by N-nitrosodiethylamine (DEN) in male Fischer 344 rats. Animals, 6 wk old, received a single intraperitoneal injection of DEN at a
dose of 10 mg/kg body weight, followed by combined treatment with partial hepatectomy and colchicine to induce cell-cycle disturbance and a selection procedure, consisting of 2-acetylaminofluorene and carbon tetrachloride. Fourteen HCCs were obtained 42 wk after the beginning of the experiment; total RNA was extracted for the assessment of aberrant transcription of the Fhit gene by reverse transcriptase-polymerase chain reaction analysis. Aberrant transcripts were detected in nine of the 14 HCCs (64.3%). Sequence analysis showed that these resulted from the absence of nt -9 to 279, nt -9 to 348, nt -98 to 279, nt -26 to 365, or nt -98 to 348. Western blot analysis demonstrated reduced expression of Fhit protein in six of 10 HCCs (60.0%), with a perfect correlation with Fhit gene alterations. These results indicated that changes in the Fhit gene occur frequently and may thus play some role in the development of HCCs induced by DEN in rats.
Lubet RA, etal., Carcinogenesis. 2005 Mar;26(3):571-8. Epub 2004 Dec 9.
The administration of 4-hydroxybutyl(butyl)nitrosamine (OH-BBN) to male B6D2F1 mice yielded a high incidence of large palpable urinary bladder cancers. Since prior studies demonstrated chemopreventive effects of non-steroidal anti-inflammatory drugs (NSAIDs), we further explored the efficacy of the
NSAID indomethacin using different treatment regimens. OH-BBN was administered twice per week for 12 weeks (the first week of treatment was designated week 1). In Experiment I continual indomethacin treatment (20 mg/kg diet) was initiated either prior to (week -1) or following (week 13) OH-BBN dosing. Palpable bladder masses (subsequently diagnosed as cancers) developed in 32% of carcinogen-treated only mice by 32 weeks, while mice administered indomethacin either prior to or after OH-BBN developed palpable masses in 3 and 6% of the animals, respectively. In Experiment II mice were treated with indomethacin beginning 1 week after OH-BBN for either 12 weeks (limited treatment, weeks 13-24) or for 30 weeks (weeks 13-42). Continual treatment resulted in a 77% decrease in palpable bladder masses and an 82% decrease in all cancers (palpable and microscopic), while limited treatment decreased palpable masses by 48% but failed to decrease the number of bladder cancers (palpable plus microscopic). In Experiment III OH-BBN-treated mice were followed for 61 weeks. Palpable masses developed in 66% of control mice, while 26% of mice treated with indomethacin continually from 1 week after OH-BBN (weeks 13-61) developed palpable masses. A separate group in this study treated with indomethacin beginning when 5% of the mice had palpable bladder masses continued to develop new masses for an additional 4 weeks. By 6 weeks after beginning indomethacin treatment, however, these animals showed a profound decrease in the development of additional cancers. The expressions of FHIT and survivin in normal urinary bladder epithelium and in bladder cancers were determined by immunohistochemical analysis. FHIT was expressed at high levels in normal epithelium, but was minimally expressed in cancers, and even showed decreased expression in papillomas. The anti-apoptotic protein survivin was not expressed in normal bladder epithelium, but was variably expressed in cancers. FHIT and survivin expressions were similar in cancers from indomethacin-treated and non-treated mice.
He D, etal., Med Oncol. 2015 Apr;32(4):92. doi: 10.1007/s12032-015-0525-y. Epub 2015 Feb 27.
Alterations in global DNA methylation and specific regulatory gene methylation are frequently found in cancer, but the significance of these epigenetic changes in EBV-associated gastric carcinoma (EBVaGC) remains unclear. We evaluated global DNA methylation status in 49 EBVaGC and 45 EBV-negative g
astric carcinoma (EBVnGC) tissue samples and cell lines by 5-methylcytosine immunohistochemical staining and methylation quantification. We determined promoter methylation status and protein expression for the p16, FHIT, CRBP1, WWOX, and DLC-1 genes in tissues and studied the correlation between CpG island methylator phenotype (CIMP) class and clinicopathological characteristics. Changes in gene methylation and mRNA expression in EBVaGC cell line SNU-719 and in EBVnGC cell lines SGC-7901, BGC-823, and AGS were assessed after treatment with 5-aza-2'-deoxycytidine (5-aza-dC), trichostatin A (TSA), or a combination of both, by methylation-specific PCR and quantitative real-time RT-PCR. Global genomic DNA hypomethylation was more pronounced in EBVnGC than in EBVaGC. Promoter methylation of all five genes was more frequent in EBVaGC than in EBVnGC (p < 0.05). p16 and FHIT methylation was reversely correlated with protein expression in EBVaGC. Most (41/49) EBVaGC exhibited CIMP-high (CIMP-H), and the prognosis of CIMP-H patients was significantly worse than that of CIMP-low (p = 0.027) and CIMP-none (p = 0.003) patients. Treatment with 5-aza-dC and/or TSA induced upregulation of RNA expression of all five genes in SNU-719; meanwhile, individual gene expression increased in EBVnGC cell lines. In summary, EBV-induced hypermethylation of p16, FHIT, CRBP1, WWOX, and DLC-1 may contribute to EBVaGC development. Demethylation therapy may represent a novel therapeutic strategy for EBVaGC.
Levin AM, etal., Cancer Epidemiol Biomarkers Prev. 2007 Jun;16(6):1294-7.
Many studies have established that loss of heterozygosity and/or altered expression of the fragile histidine triad (FHIT) gene is a common event in a number of tumor types including prostate carcinoma. Encompassing the most active fragile site in the human genom
e, FRA3B, FHIT has become the model fragile site-associated tumor suppressor gene. In a recent study, linkage and association between germline genetic variation in FHIT (specifically single nucleotide polymorphism rs760317) and prostate cancer were reported. We sought to confirm this finding in two independent samples: (a) a family-based sample of 817 men with (n = 434) and without (n = 383) prostate cancer from 323 Caucasian families, and (b) a community-based case-control sample of African American men with (n = 133) and without (n = 342) prostate cancer. Using a family-based association test, rs760317 was associated with prostate cancer in Caucasians (P = 0.031), with a reduction in the risk of prostate cancer among carriers of the minor allele (odds ratio, 0.66; 95% confidence interval, 0.42-1.04; P = 0.074). African American carriers experienced a similar risk reduction (odds ratio, 0.63; 95% confidence interval, 0.42-0.96; P = 0.032). These results are remarkably consistent across ethnic samples but are in opposition to results from the original study, which showed an association between the minor allele of rs760317 and an increased risk of prostate cancer. Taken together, the consistently significant but flipped association between single nucleotide polymorphism rs760317 and prostate cancer in three independent samples suggests that rs760317 may be in linkage disequilibrium with one or more prostate cancer susceptibility variants in or near FHIT.
Asensio AC, etal., Biochimie. 2006 May;88(5):461-71. Epub 2005 Nov 15.
We describe here the purification and characterisation of the human enzyme diadenosine triphosphatase isolated from human platelets and leukocytes, offering biochemical and immunochemical evidence to identify this enzyme with the novel tumour suppressor Fhit pro
tein, a homodimer composed of approximately 17 kDa monomers. It catalyses the Mg(2+)-dependent hydrolysis of diadenosine triphosphate, Ap(3)A, to AMP+ADP. The fluorogenic substrate di-ethenoadenosine triphosphate, epsilon-(Ap(3)A), and Fhit antibodies were used for enzymatic and immunochemical characterisations, respectively. Human Ap(3)Aase presents a native molecular mass of approximately 32 kDa and no significant differences were found in K(m) values (2 microM), activating effects by Mg(2+), Ca(2+), and Mn(2+), optimum pH (7.0-7.2) or inhibition by Zn(2+) and diethyl pyrocarbonate between the human enzyme and the recombinant Fhit protein. Suramin is a very potent competitive inhibitor of both human Ap(3)Aase and Fhit protein with K(i) values in the range 20-30 nM. Both human and rat Ap(3)Aase activity co-purifies with Fhit immunoreactivity under gel filtration, ion-exchange and affinity chromatography. Homogeneous human Ap(3)Aase preparations analysed by SDS-PAGE and Western blot analysis with Fhit antibodies elicit immunochemical responses corresponding to a approximately 17 kDa polypeptide, indicating a dimeric structure for the enzyme Ap(3)Aase. The strong inhibition of Fhit enzyme by the drug suramin, supports the need to investigate the therapeutic potential of Fhit-Ap(3)Aase mediated by its interaction with suramin or related drugs.
Tomizawa Y, etal., Cancer Res. 1998 Dec 1;58(23):5478-83.
Abnormalities in structure and expression of the FHIT gene have been detected in a considerable fraction of primary lung tumors. Previous reports indicated that FHIT gene alterations can be simply detected by immunohistochem
ical methods. Therefore, we investigated the association of Fhit expression with clinicopathological features and allelic imbalance (AI) at the FHIT locus in 105 stage I non-small cell lung cancers (NSCLC) by the immunohistological method and PCR analysis. Thirty-six of 105 (34%) tumors showed marked reduction of Fhit immunoreactivity. Fhit expression was markedly reduced in most squamous cell carcinomas (24 of 28, 86%), whereas such a reduction was detected only in a small subset of adenocarcinomas (7 of 67, 10%; P < 0.001). A marked reduction of Fhit protein expression was observed more frequently in patients with a smoking history (32 of 80, 40%) than in patients without a smoking history (4 of 25, 16%; P = 0.013). These results indicate that FHIT gene alterations preferentially occur in squamous cell carcinomas and in smokers. Furthermore, a reduction of Fhit protein expression in tumor cells was associated with a poorer survival of patients with stage I NSCLC, irrespective of histological subtypes of tumors (P = 0.005; log-rank test). Fhit expression was reduced preferentially in tumors with AI at the FHIT locus; however, AI at the FHIT locus did not correlate with patients' survival (P = 0.262; log-rank test). These results suggested that Fhit protein expression could be a useful molecular marker for the prognosis of patients with surgically resected stage I NSCLC.
BACKGROUND: Granulosa cell tumors (GCTs) are relatively rare and are subtypes of the sex-cord stromal neoplasms. Methylation induced silencing in the promoters of genes such as tumor suppressor genes, DNA repair genes and pro-apoptotic genes is recognised as a critical factor in cancer development.
METHODS: We examined the role of promoter hypermethylation, an epigenetic alteration that is associated with the silencing tumor suppressor genes in human cancer, by studying 5 gene promoters in 25 GCTs cases by methylation specific PCR and RT-PCR. In addition, the compatible tissues (normal tissues distant from lesion) from three non-astrocytoma patients were also included as the control. RESULTS: Frequencies of methylation in GCTs were 7/25 (28 % for FHIT), 6/25 (24% for FNACF), 3/25 (12% for Cyclin D2), 1/25 (4% for BRCA2) and 14/25 (56%) in RUNX3 genes. Correlation of promoter methylation with clinical characteristics and other genetic changes revealed that overall promoter methylation was higher in more advanced stage of the disease. Promoter methylation was associated with gene silencing in GCT cell lines. Treatment with methylation or histone deacetylation-inhibiting agents resulted in profound reactivation of gene expression. CONCLUSIONS: These results may have implications in better understanding the underlying epigenetic mechanisms in GCT development, provide prognostic indicators, and identify important gene targets for treatment.
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.
As current evidence suggests the involvement of epigenetic modification of tumour suppressor genes in human cancer, we investigated the aberrant promoter methylation of FHIT and RASSF1A genes in human papillomavirus (HPV)-mediated cervical cancer in Indian women
. We analysed 60 cervical cancer tissue biopsies of different clinical stage and histological grading and 23 healthy control samples with normal cervical cytology. Methylation-specific polymerase chain reaction (MSP) was performed to analyse the methylation status of FHIT and RASSF1A genes and confirmed by sequencing. Both patients and controls were screened for HPV infection and 98% of the HPV-infected cases showed positivity for HPV type 16. Aberrant promoter methylation of the FHIT gene was found in 28.3% (17/60) of cases and of the RASSF1A gene in 35.0% (21/60) of cases; promoter methylation of both the genes was found in 13.3% (8/60) of cervical cancer cases. Methylation was significantly (p<0.01) associated with the cervical cancer cases compared with controls. None of the 23 controls was found to be methylated in either of these genes. This is the first study indicating a correlation between the promoter methylation of FHIT and RASSF1A genes and the clinical stage and histological grading of cervical carcinoma in Indian women. Future studies are underway to examine the practical implications of these findings for use as a biomarker.
Paisie CA, etal., Cancer Sci. 2016 Apr;107(4):528-35. doi: 10.1111/cas.12887. Epub 2016 Feb 23.
Loss of expression of Fhit, a tumor suppressor and genome caretaker, occurs in preneoplastic lesions during development of many human cancers. Furthermore, Fhit-deficient mouse models are exquisitely susceptible to carcinoge
n induction of cancers of the lung and forestomach. Due to absence of Fhit genome caretaker function, cultured cells and tissues of the constitutive Fhit knockout strain develop chromosome aneuploidy and allele copy number gains and losses and we hypothesized that Fhit-deficient cells would also develop point mutations. On analysis of whole exome sequences of Fhit-deficient tissues and cultured cells, we found 300 to >1000 single-base substitutions associated with Fhit loss in the 2% of the genome included in exomes, relative to the C57Bl6 reference genome. The mutation signature is characterized by increased C>T and T>C mutations, similar to the "age at diagnosis" signature identified in human cancers. The Fhit-deficiency mutation signature also resembles a C>T and T>C mutation signature reported for human papillary kidney cancers and a similar signature recently reported for esophageal and bladder cancers, cancers that are frequently Fhit deficient. The increase in T>C mutations in -/- exomes may be due to dNTP imbalance, particularly in thymidine triphosphate, resulting from decreased expression of thymidine kinase 1 in Fhit-deficient cells. Fhit-deficient kidney cells that survived in vitro dimethylbenz(a)anthracene treatment additionally showed increased T>A mutations, a signature generated by treatment with this carcinogen, suggesting that these T>A transversions may be evidence of carcinogen-induced preneoplastic changes.
Waters CE, etal., Oncotarget. 2015 Feb 20;6(5):3409-19.
APOBEC cytidine deaminase activity is a major source of hypermutation in cancer. But previous studies have shown that the TC context signature of these enzymes is not observed in sizable fractions of cancers with overexpression of APOBEC, suggesting that cooperating factors that contribute to this
mutagenesis should be identified. The fragile histidine triad protein (Fhit) is a tumor suppressor and DNA caretaker that is deleted or silenced in >50% of cancers. Loss of Fhit protein activity causes replication stress through reduced Thymidine Kinase 1 expression, increased DNA breaks, and global genome instability in normal and cancer cells. Using data from The Cancer Genome Atlas (TCGA), we show that FHIT-low/APOBEC3B-high expressing lung adenocarcinomas display significantly increased numbers of APOBEC signature mutations. Tumor samples in this cohort with normal FHIT expression do not exhibit APOBEC hypermutation, despite having high APOBEC3B expression. In vitro, silencing Fhit expression elevates APOBEC3B-directed C > T mutations in the TP53 gene. Furthermore, inhibition of Fhit loss-induced DNA damage via thymidine supplementation decreases the TP53 mutation burden in FHIT-low/APOBEC3B-high cells. We conclude that APOBEC3B overexpression and Fhit-loss induced DNA damage are independent events that, when occurring together, result in a significantly increased frequency of APOBEC-induced mutations that drive cancer progression.
Semba S, etal., Oncogene. 2006 May 11;25(20):2860-72.
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.
Siraj AK, etal., Ann Hematol. 2007 Dec;86(12):887-95. Epub 2007 Aug 22.
Diffuse large B cell lymphoma (DLBCL) is one of the most common non-Hodgkin's lymphoma types. Methylenetetrahydrofolate reductase (MTHFR) balances the pool of folate coenzymes in one carbon metabolism of deoxyribonucleic acid (DNA) synthesis and methylation; both are implicated in carcinogenesis of
many types of cancer including lymphoma. Two common variants in the MTHFR gene (C677T and A1298C) have been associated with reduced enzyme activity, thereby making MTHFR polymorphisms a potential candidate as a cancer-predisposing factor. The O6 methylguanine DNA methyltransferase (MGMT) and fragile histidine triad (FHIT) genes are transcriptionally silenced by promoter hypermethylation in DLBCL. These genetic differences are highly race specific and have never been screened in the Saudi DLBCL patients. We conducted a hospital-based case-control study including 160 DLBCL cases and 511 Saudi control samples analyzing the MTHFR C677T and A1298C functional polymorphisms by the restriction fragment length polymorphism method and their association with MGMT and FHIT genes promoter hypermethylation. Our data demonstrated that Saudi individuals carrying MTHFR genotype 1298CC (p < 0.001) and the 1298C allele (p = 0.012) had 4.23 and 1.73-fold higher risk of developing DLBCL, respectively. Additionally, combined genotype CCCC (MTHFR 677CC + MTHFR 1298CC) was associated with 3.489-fold, and CTCC (MTHFR 677 CT + 1298CC) was related to 9.515-fold higher risk, compared with full MTHFR enzyme activity. No significant association between MTHFR variant genotypes and methylation of MGMT and FHIT genes were observed. Our findings suggested that polymorphisms of MTHFR enzyme genes might be associated with the individual susceptibility to develop DLBCL. Additionally, the results indicated that MTHFR variants were not related to MGMT or FHIT hypermethylation in DLBCL.
Brenner C Biochemistry 2002 Jul 23;41(29):9003-14.
HIT (histidine triad) proteins, named for a motif related to the sequence HphiHphiHphiphi (phi, a hydrophobic amino acid), are a superfamily of nucleotide hydrolases and transferases, which act on the alpha-phosphate of ribonucleotides, and contain a approximately 30 kDa domain that is typically eit
her a homodimer of approximately 15 kDa polypeptides with two active-sites or an internally, imperfectly repeated polypeptide that retains a single HIT active site. On the basis of sequence, substrate specificity, structure, evolution, and mechanism, HIT proteins can be classified into the Hint branch, which consists of adenosine 5'-monophosphoramide hydrolases, the Fhit branch, which consists of diadenosine polyphosphate hydrolases, and the GalT branch, which consists of specific nucleoside monophosphate transferases, including galactose-1-phosphate uridylyltransferase, diadenosine tetraphosphate phosphorylase, and adenylyl sulfate:phosphate adenylytransferase. At least one human representative of each branch is lost in human diseases. Aprataxin, a Hint branch hydrolase, is mutated in ataxia-oculomotor apraxia syndrome. Fhit is lost early in the development of many epithelially derived tumors. GalT is deficient in galactosemia. Additionally, ASW is an avian Hint family member that has evolved to have unusual gene expression properties and the complete loss of its nucleotide binding site. The potential roles of ASW and Hint in avian sexual development are discussed elsewhere. Here we review what is known about biological activities of HIT proteins, the structural and biochemical bases for their functions, and propose a new enzyme mechanism for Hint and Fhit that may account for the differences between HIT hydrolases and transferases.
Chu TY, etal., Int J Cancer. 1998 Jan 19;75(2):199-204.
The genetic aberration involved in the loss of heterozygosity (LOH) at 3p14 has recently been attributed to the disruption of the FHIT gene in many cancers. This study analyzed HPV DNA and allelic status of 5 microsatellite markers spaning 3p13-3p25 in 57 cases
of cervical cancer. With no homozygous deletion found in any case, a 39% overall frequency of LOH was noted. The presence of tumorigenic HPV DNA (91%) did not correlate with the allelic loss at any marker, including THRB (3p22-24) and D3S1228 (3p14) which were found with high LOH rates of 43% (12/28) and 37% (11/30), respectively. Further analysis of FHIT mRNA in 29 cancers by reverse transcription (RT)-PCR showed a full-length transcript in all cases. However, additional minor transcripts were occasionally observed in cancer tissues (9/29) as well as in normal tissues (12/31) by nested PCR of the RT products. Sequence analysis of these transcripts showed exclusive internal exon deletions, suggesting a source of minor splicing variants. No apparent mutation of the mRNA sequences was found in 8 transcripts examined, except for a silent polymorphism and a site of alternative splicing. The results suggest that, although frequently reported to be abrogated in several cancers, the mRNA of FHIT remains intact in cervical cancer. Other genes closely linked to FHIT may be responsible for frequent LOH at 3p14 observed in cervical cancer.
Pekarsky Y, etal., Proc Natl Acad Sci U S A 1998 Jul 21;95(15):8744-9.
The tumor suppressor gene FHIT encompasses the common human chromosomal fragile site at 3p14.2 and numerous cancer cell biallelic deletions. To study Fhit function we cloned and characterized FHIT
HIT genes from Drosophila melanogaster and Caenorhabditis elegans. Both genes code for fusion proteins in which the Fhit domain is fused with a novel domain showing homology to bacterial and plant nitrilases; the D. melanogaster fusion protein exhibited diadenosine triphosphate (ApppA) hydrolase activity expected of an authentic Fhit homolog. In human and mouse, the nitrilase homologs and Fhit are encoded by two different genes: FHIT and NIT1, localized on chromosomes 3 and 1 in human, and 14 and 1 in mouse, respectively. We cloned and characterized human and murine NIT1 genes and determined their exon-intron structure, patterns of expression, and alternative processing of their mRNAs. The tissue specificity of expression of murine Fhit and Nit1 genes was nearly identical. Because fusion proteins with dual or triple enzymatic activities have been found to carry out specific steps in a given biochemical or biosynthetic pathway, we postulate that Fhit and Nit1 likewise collaborate in a biochemical or cellular pathway in mammalian cells.
Liu L, etal., Biomed Mater Eng. 2015;26 Suppl 1:S2217-22. doi: 10.3233/BME-151527.
The development of carcinoma has been found to be associated with epigenetic modifications. The aim of this study was to estimate the methylation levels of FHIT and BRCA1 promoters using the bisulphite sequencing method (BSP) and high-resolution melting curve an
alysis (HRM) in the serum of patients with ductal breast carcinoma as a biomarker for the possible application of early diagnosis of breast cancer. The results showed that the methylation levels of both BRCA1 and FHIT promoters were higher in the serum of the breast ductal carcinoma group (BDC group) than those of the breast fibroadenoma group (BFA group), and the healthy individuals group (HI group). However, the methylation levels of the BRCA1 promoters were very low in all three groups compared to the levels of FHIT. The advanced quantitative detection of the samples with HRM showed that the FHIT promoter methylation level of the cfDNA in each serum was also very high in the BDC group compared to the HI group. The methylation level of FHIT was found to be significantly associated with breast cancer (p < 0.05). In conclusion, the methylation quantitative detection of FHIT promoter in serum may be useful for the early diagnosis of ductal breast carcinoma.
Su Y, etal., Drug Des Devel Ther. 2015 Oct 1;9:5439-45. doi: 10.2147/DDDT.S89861. eCollection 2015.
FHIT is a bona fide tumor-suppressor gene and its loss contributes to tumorigenesis of epithelial cancers including breast cancer (BC). However, the association and clinicopathological significance between FHIT promoter hype
rmethylation and BC remains unclear. The purpose of this study is to conduct a meta-analysis and literature review to investigate the clinicopathological significance of FHIT methylation in BC. A detailed literature search was performed in PubMed, EMBASE, Web of Science, and Google Scholar databases. The data were extracted and assessed by two reviewers independently. Odds ratios with 95% corresponding confidence intervals were calculated. A total of seven relevant articles were available for meta-analysis, which included 985 patients. The frequency of FHIT hypermethylation was significantly increased in invasive ductal carcinoma compared to benign breast disease, the pooled odds ratio was 8.43, P<0.00001. The rate of FHIT hypermethylation was not significantly different between stage I/II and stage III/IV, odds ratio was 2.98, P=0.06. In addition, FHIT hypermethylation was not significantly associated with ER and PR status. FHIT hypermethylation was not significantly correlated with premenopausal and postmenopausal patients with invasive ductal carcinoma. In summary, our meta-analysis indicated that the frequency of FHIT hypermethylation was significantly increased in BC compared to benign breast disease. The rate of FHIT hypermethylation in advanced stages of BC was higher than in earlier stages; however, the difference was not statistically significant. Our data suggested that FHIT methylation could be a diagnostic biomarker of BC carcinogenesis. FHIT is a potential drug target for development of demethylation treatment for patients with BC.
Wu X, etal., Drug Des Devel Ther. 2016 Feb 15;10:699-709. doi: 10.2147/DDDT.S85253. eCollection 2016.
Emerging evidence indicates that FHIT is a candidate tumor suppressor in many types of tumors including non-small-cell lung carcinoma (NSCLC). However, the prognostic value and correlation between FHIT hypermethylation and c
linicopathological characteristics of NSCLC remains unclear. In this report, we performed a meta-analysis to evaluate the effects of FHIT hypermethylation on the incidence of NSCLC and clinicopathological characteristics of human NSCLC patients. Final analysis of 1,801 NSCLC patients from 18 eligible studies was performed. FHIT hypermethylation was found to be significantly higher in NSCLC than in normal lung tissue. The pooled odds ratio (OR) from ten studies included 819 NSCLC and 792 normal lung tissues (OR =7.51, 95% confidence interval [CI] =2.98-18.91, P<0.0001). Subgroup analysis based on ethnicity implied that FHIT hypermethylation level was higher in NSCLC tissues than in normal tissues in both Caucasians (P=0.02) and Asians (P<0.0001), indicating that the difference in Asians was much more significant. FHIT hypermethylation was also correlated with sex status, smoking status, as well as pathological types. In addition, patients with FHIT hypermethylation had a lower survival rate than those without (hazard ratio =1.73, 95% CI =1.10-2.71, P=0.02). The results of this meta-analysis suggest that FHIT hypermethylation is associated with an increased risk and poor survival in NSCLC patients. FHIT hypermethylation, which induces the inactivation of FHIT gene, plays an important role in the carcinogenesis and clinical outcome and may serve as a potential diagnostic marker and drug target of NSCLC.
