Loss of fragile histidine triad (Fhit) protein expression alters the translation of cancer-associated mRNAs.
ABSTRACT: In?>?50% of cancers tumor development involves the early loss of Fhit (fragile histidine triad) protein expression, yet the mechanistic pathway(s) by which Fhit mediates its tumor suppressor functions are not fully understood. Earlier attempts to identify a Fhit-deficient gene expression profile relied on total cellular RNA and microarray analysis. The data here used RNA sequencing (RNA-Seq) of Fhit-negative and Fhit-positive cells as proof of principle for the impact of Fhit on specific mRNAs, and to lay the foundation for a study using ribosome profiling to identify mRNAs whose translation is affected by FHIT loss.RNA-Seq was performed on RNA from lines of Fhit-expressing and Fhit-deficient lung cancer cells. This identified changes in the levels of mRNAs for a number of cell survival and cell cycle progression genes. Polysome profile analysis performed on cytoplasmic extracts from Fhit-negative and Fhit-positive cells showed changes in the sedimentation of select mRNAs consistent with changes in translation efficiency. The impact of differential Fhit expression on the turnover of selected cancer-linked mRNAs was determined by RT-qPCR of cytoplasmic RNA isolated at intervals after treating cells with a transcription inhibitor.
Project description:FHIT is a genome caretaker/tumor suppressor that is silenced in >50% of cancers. Although it was identified more than 20 years ago, questions remain as to how FHIT loss contributes to cancer, and conversely, how FHIT acts to maintain genome integrity and suppress malignancy. Fhit belongs to the histidine triad family of enzymes that catalyze the degradation of nucleoside 5',5'-triphosphates, including the m7GpppN 'caps' that are generated when mRNAs undergo 3'-5' decay. This raised the possibility that Fhit loss might affect changes in the translation of cancer-associated mRNAs, possibly as a consequence of increased intracellular concentrations of these molecules.Ribosome profiling identified several hundred mRNAs for which coding region ribosome occupancy changed as a function of Fhit expression. While many of these changes could be explained by changes in mRNA steady-state, a subset of these showed changes in translation efficiency as a function of Fhit expression. The onset of malignancy has been linked to changes in 5'-UTR ribosome occupancy and this analysis also identified ribosome binding to 5'-untranslated regions (UTRs) of a number of cancer-associated mRNAs. 5'-UTR ribosome occupancy of these mRNAs differed between Fhit-negative and Fhit-positive cells, and in some cases these differences correlated with differences in coding region ribosome occupancy.In summary, these findings show Fhit expression impacts the translation of a number of cancer associated genes, and they support the hypothesis that Fhit's genome protective/tumor suppressor function is associated with post-transcriptional changes in expression of genes whose dysregulation contributes to malignancy.
Project description:FHIT is a genome caretaker gene that is silenced in >50% of cancers. Loss of Fhit protein expression promotes accumulation of DNA damage, affects apoptosis and epithelial-mesenchymal transition, though molecular mechanisms underlying these alterations have not been fully elucidated. Initiation of genome instability directly follows Fhit loss and the associated reduced Thymidine Kinase 1 (TK1) protein expression. The effects on TK1 of Fhit knockdown and Fhit induction in the current study confirmed the role of Fhit in regulating TK1 expression. Changes in Fhit expression did not impact TK1 protein turnover or transcription from the TK1 promoter, nor steady-state levels of TK1 mRNA or turnover. Polysome profile analysis showed that up-regulated Fhit expression resulted in decreased TK1 RNA in non-translating messenger ribonucleoproteins and increased ribosome density on TK1 mRNA. Fhit does not bind RNA but its expression increased luciferase expression from a transgene bearing the TK1 5'-UTR. Fhit has been reported to act as a scavenger decapping enzyme, and a similar result with a mutant (H96) that binds but does not cleave nucleoside 5',5'-triphosphates suggests the impact on TK1 translation is due to its ability to modulate the intracellular level of cap-like molecules. Consistent with this, cells expressing Fhit mutants with reduced activity toward cap-like dinucleotides exhibit DNA damage resulting from TK1 deficiency, whereas cells expressing wild-type Fhit or the H96N mutant do not. The results have implications for the mechanism by which Fhit regulates TK1 mRNA, and more broadly, for its modulation of multiple functions as tumor suppressor/genome caretaker.
Project description:FHIT (fragile histidine triad), a candidate tumour suppressor gene, has recently been identified at chromosomal region 3p14.2, and deletions of the gene have been reported in many types of human cancer. However, the biological function of the Fhit protein has not been fully characterized yet. Using the yeast two-hybrid screen to search for proteins that interact with Fhit in vivo, we identified a protein that is specifically associated with Fhit. This association was confirmed in both immunoprecipitation and glutathione S-transferase pull-down assays. The sequence of the protein is identical with that of human ubiquitin-conjugating enzyme 9 (hUBC9). The last 21 amino acids at the C-terminus of hUBC9 appear to be unimportant for its biological activity, since an hUBC9 mutant harbouring a deletion of these amino acids could still restore normal growth of yeast containing a temperature-sensitive mutation in the homologue UBC9 gene. Mutational analysis indicated that hUBC9 was associated with the C-terminal portion of Fhit. Neither a single amino acid substitution at codon 96 (His-->Asn) nor triple amino acid substitutions (His-->Asn) at a histidine triad (codons 94, 96 and 98) affected the association, whereas Fhit triphosphate (diadenosine 5',5"'-P(1),P(3)-triphosphate) hydrolase activity has been reported to be eliminated by either type of mutation, suggesting that the interaction between Fhit and hUBC9 is independent of Fhit enzymic activity. Given that yeast UBC9 is involved in the degradation of S- and M-phase cyclins, Fhit may be involved in cell cycle control through its interaction with hUBC9.
Project description:The tumor suppressor gene FHIT is inactivated by genetic and epigenetic changes in the majority of common human cancers. The human Fhit protein undergoes phosphorylation on tyrosine residue 114 by Src and related kinases both in vitro and in vivo. Src is a key cytoplasmic tyrosine kinase downstream to several growth factor receptors, including those of the EGF receptor family, which are overexpressed and activated in about one-third of human breast and ovarian carcinomas. However, the biological significance of Fhit phosphorylation by Src has remained elusive. In the present study, we demonstrate that FHIT acts as a checkpoint in cell proliferation mediated by activated tyrosine kinase receptors that recruit Src. Activation of EGF receptor family members induced Fhit phosphorylation by Src and the subsequent proteasome degradation of the phosphorylated Fhit protein. Indeed, the use of the Fhit mutant Y114F, which carries a phenylalanine instead of a tyrosine at position 114, unable to be phosphorylated on tyrosine 114 by Src, prevents Fhit degradation. Moreover, Fhit protein reduction is transient and occurs in a specific temporal window. During the signaling pathway of activated tyrosine kinase receptors, the phosphorylation of Fhit induces its degradation and the subsequent reduction in Fhit protein levels allows the transmission of the mitogenic signal; immediately thereafter, Fhit protein levels are restored. Such a scenario would suggest a key role for Fhit in the balance of proliferation/survival/apoptosis signals.
Project description: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 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.
Project description:The FHIT gene, encompassing an active common fragile site, FRA3B, is frequently silenced in preneoplasia and cancer, through gene rearrangement or methylation of regulatory sequences. Silencing of Fhit protein expression causes thymidine kinase 1 downregulation, resulting in dNTP imbalance, and spontaneous replication stress that leads to chromosomal aberrations, allele copy number variations, insertions/deletions, and single-base substitutions. Thus, Fhit, which is reduced in expression in the majority of human cancers, is a genome "caretaker" whose loss initiates genome instability in preneoplastic lesions. To follow the early genetic alterations and functional changes induced by Fhit loss that may recapitulate the neoplastic process in vitro, we established epithelial cell lines from kidney tissues of Fhit-/- and +/+ mouse pups early after weaning, and subjected cell cultures to nutritional and carcinogen stress, which +/+ cells did not survive. Through transcriptome profiling and protein expression analysis, we observed changes in the Trp53/p21 and survivin apoptotic pathways in -/- cells, and in expression of proteins involved in epithelial-mesenchymal transition. Some Fhit-deficient cell lines showed anchorage-independent colony formation and increased invasive capacity in vitro. Furthermore, cells of stressed Fhit-/- cell lines formed s.c. and metastatic tumors in nude mice. Collectively, we show that Fhit loss and subsequent thymidine kinase 1 inactivation, combined with selective pressures, leads to neoplasia-associated alterations in genes and gene expression patterns in vitro and in vivo.
Project description:Loss of Fhit expression, encoded at chromosome fragile site FRA3B, leads to increased replication stress, genome instability and accumulation of genetic alterations. We have proposed that Fhit is a genome 'caretaker' whose loss initiates genome instability in preneoplastic lesions. We have characterized allele copy number alterations and expression changes observed in Fhit-deficient cells in conjunction with alterations in cellular proliferation and exome mutations, using cells from mouse embryo fibroblasts (MEFs), mouse kidney, early and late after establishment in culture, and in response to carcinogen treatment. Fhit (-/-) MEFs escape senescence to become immortal more rapidly than Fhit (+/+) MEFs; -/- MEFs and kidney cultures show allele losses and gains, while +/+ derived cells show few genomic alterations. Striking alterations in expression of p53, p21, Mcl1 and active caspase 3 occurred in mouse kidney -/- cells during progressive tissue culture passage. To define genomic changes associated with preneoplastic changes in vivo, exome DNAs were sequenced for +/+ and -/- liver tissue after treatment of mice with the carcinogen, 7,12-dimethylbenz[a]anthracene, and for +/+ and -/- kidney cells treated in vitro with this carcinogen. The -/- exome DNAs, in comparison with +/+ DNA, showed small insertions, deletions and point mutations in more genes, some likely related to preneoplastic changes. Thus, Fhit loss provides a 'mutator' phenotype, a cellular environment in which mild genome instability permits clonal expansion, through proliferative advantage and escape from apoptosis, in response to pressures to survive.
Project description:Inactivation of the fragile histidine triad (Fhit) gene has been reported in the majority of human cancers, particularly in lung cancer. The role of Fhit as a tumor suppressor gene has been well documented, and restoration of Fhit expression suppresses tumorigenicity in tumor cell lines and in mouse models by inducing apoptosis and inhibiting proliferation of tumor cells. Autophagy is a catabolic pathway, whereby cytoplasmic proteins and organelles are sequestered in vacuoles and delivered to lysosomes for degradation and recycling. Although autophagy is necessary for cell survival under stress conditions, recent studies have shown that autophagy can also promote cell death. Due to the fact that both autophagy induction and Fhit expression are commonly associated with nutrient starvation, we hypothesized that Fhit expression may be related to autophagy induction. In the present study, we assessed whether Fhit overexpression by gene transfer induces autophagy in Fhit-deficient non-small cell lung cancer (NSCLC) cells. The results of our study indicate that Fhit protein induces autophagy in NSCLC cells, and that this autophagy prevents apoptotic cell death in vivo and in vitro in a 14-3-3? protein-dependent manner. To the best of our knowledge, this is the first report to describe Fhit-induced autophagy. Suppressing autophagy might be a promising therapeutic option to enhance the efficacy of Fhit gene therapy in NSCLC.
Project description:Characterizing signal pathway alterations that contribute to cellular transformation induced by loss of Fhit protein Abstract: The FHIT gene, encompassing an active common fragile site, FRA3B, is frequently silenced in preneoplasia and cancer, through gene rearrangement or methylation of regulatory sequences. Silencing of Fhit protein expression causes thymidine kinase 1 downregulation, resulting in dNTP imbalance, and spontaneous replication stress that leads to chromosomal aberrations, allele copy number variations, insertions/deletions, and single‐base substitutions. Thus, Fhit, which is reduced in expression in the majority of human cancers, is a genome “caretaker” whose loss initiates genome instability in preneoplastic lesions. To follow the early genetic alterations and functional changes induced by Fhit loss that may recapitulate the neoplastic process in vitro, we established epithelial cell lines from kidney tissues of Fhit−/− and +/+ mouse pups early after weaning, and subjected cell cultures to nutritional and carcinogen stress, which +/+ cells did not survive. Through transcriptome profiling and protein expression analysis, we observed changes in the Trp53/p21 and survivin apoptotic pathways in −/− cells, and in expression of proteins involved in epithelial–mesenchymal transition. Some Fhit‐deficient cell lines showed anchorage‐independent colony formation and increased invasive capacity in vitro. Furthermore, cells of stressed Fhit−/− cell lines formed s.c. and metastatic tumors in nude mice. Collectively, we show that Fhit loss and subsequent thymidine kinase 1 inactivation, combined with selective pressures, leads to neoplasia‐associated alterations in genes and gene expression patterns in vitro and in vivo. Overall design: mouse kidney epithelial cell lines were derived from fhit knockout mice and subcultured with and without carcinogenic and nutritional stress
Project description:Environmental agents induce intragenic alterations in the FRA3B/FHIT chromosome fragile site, resulting in fragile FHIT allele loss early in cancer development. Fhit knockout mice are predisposed to tumor development and Fhit gene therapy reduces tumor burden. Repair-deficient cancers are likely to be Fhit-deficient and Fhit-deficient cells show enhanced resistance to ultraviolet C, mitomycin C, camptothecin and oxidative stress-induced cell killing. Loss of Fhit leads to alterations in the DNA damage response checkpoint and contributes to DNA instability. Hsp60/Hsp10 are Fhit interactors, suggesting a direct role for Fhit in stress responses. Fhit also interacts with and stabilizes ferrodoxin reductase (Fdxr), a mitochondrial flavoprotein that transfers electrons from NADPH to cytochrome P450, suggesting a role for Fhit in the modulation of reactive oxygen species production and of genomic damage.