Background
‘Fragile’ sites are among the most frequently deleted loci in cancers [
1].
FHIT was identified more than 20 years ago [
2] at a locus that is deleted or otherwise silenced in >50% of most human cancer types [
3,
4]. Loss of
FHIT alleles occurs early in malignant transformation [
5,
6], and is associated with decreased apoptosis [
7], increased EMT [
8,
9], increased resistance to genotoxic agents [
10], and altered control of reactive oxygen species production [
11]. Nevertheless, the mechanisms through which Fhit protein affects these functions have remained elusive.
Fhit is a small cytoplasmic protein that does not interact with known tumor suppressors or oncogenes. Its name (Fragile Histidine Triad) derives from a His-X-His-X-His-XX motif characteristic of nucleoside hydrolases. Fhit cleaves diadenosine triphosphate (Ap3A) in vitro to yield ADP and AMP [
12], and Ap3A accumulates in Fhit-deficient cells [
13]. Recently Taverniti and Seraphin [
14] identified Fhit as a scavenger decapping enzyme. Scavenger decapping enzymes are responsible for degrading m
7GpppN cap dinucleotides that are generated by 3′-5’ mRNA decay. Because they can be bound by eIF4E such mRNA decay remnants can inhibit translation initiation if they accumulate to a level that can compete with mRNA 5′ ends. DcpS is the major scavenger decapping enzyme [
15], and the identification of Fhit as another of this type of enzyme is consistent with previous work describing Fhit’s ability to cleave GpppBODIPY, an mRNA cap-like molecule [
16].
Translation plays a critical role in cancer [
17], and a recent report showed that, for a number of cancer-associated mRNAs, changes in ribosome occupancy of upstream open reading frames precedes the appearance of detectable tumors [
18]. As noted above, the key biochemical property of Fhit is its ability to hydrolyze nucleoside 5′,5′-triphosphates, and while catalytically-inactive forms of Fhit are unable to function as tumor suppressors, a mutant (H96N) that binds to but does not hydrolyze nucleoside 5′,5′-triphosphates is nearly as effective as wild-type Fhit in suppressing tumor formation [
12].
FHIT allele loss can occur prior to appearance of detectable preneoplastic lesions or tumors [
5,
6,
19]. Since few direct targets of Fhit loss have been identified [
11,
20], we wondered if its tumor suppressor or genome caretaker effects might be related to its function in degrading nucleoside 5′,5′-triphosphates, and to the impact of these molecules on the translation of a limited number of mRNAs.
The current study used Fhit expression-negative H1299 lung cancer cells carrying an inducible
FHIT transgene to examine the impact of Fhit protein and its loss on the scope of translating mRNAs. Ribosome profiling and RNA-Seq showed that Fhit loss is associated with changes in steady-state level and ribosome occupancy of several hundred mRNAs, but little change in translation efficiency of most of the transcriptome. Rescuing Fhit expression resulted in changes in average ribosome density of a limited number of cancer-associated genes and this was reflected by changes in protein expression. We also identified additional cancer-associated genes for which 5’-UTR ribosome occupancy changed when Fhit expression was restored. This result is consistent with transformation-associated changes in 5’-UTR ribosome occupancy in [
18] and with loss of Fhit expression as an early step in this process.
Discussion
Despite more than 1100 papers on Fhit the molecular mechanism(s) by which it acts to affect these processes are poorly understood. We approached this from the perspective that Fhit might act indirectly to alter gene expression as a function of its ability to clear nucleoside 5′,5′-triphosphates and act as a scavenger decapping enzyme [
14]. We reasoned that Fhit loss might increase the intracellular levels of these dinucleotides, including the m
7G caps generated by mRNA 3′-5′ decay, and these in turn might affect the translation of some mRNAs. To address this we performed ribosome profiling of Fhit-deficient H1299 lung cancer cells carrying an inducible
FHIT transgene and a matching cell line carrying empty vector. Changes in Fhit expression impacted ribosome occupancy, with 67 mRNAs showing statistically-significantly increased ribosome binding and 103 mRNAs showing decreased binding (Fig.
1d and Additional file
7). For the most part changes in ribosome occupancy could be accounted for by corresponding changes in mRNA level. Once this was taken into account 6 mRNAs were identified for which Fhit increased coding region average ribosome density (ARD) and 4 mRNAs for which ARD decreased (Fig.
1e, Table
1).
This small number of mRNAs was unexpected, but their identity was significant. Of the mRNAs showing increased translation in association with Fhit expression, CDKN2C is a member of the INK4 cyclin-dependent kinase inhibitors with medium to low expression in lung cancers (see [
27,
28]. IGSF9 is involved in cellular adhesion, TP53I3 is induced by TP53 and thought to generate reactive oxygen molecules, MECP2 binds methyl-CpG sites in chromatin and CSRP2 is a LIM domain protein thought to be involved in regulating cell growth. Mecp2 is also the key protein that is lost or mutated in Rett syndrome [
29]. We also identified a number of mRNAs for which coding region ribosome occupancy was higher in Fhit-negative versus Fhit-positive cells. The greatest differential was seen for EEF2, a GTP-binding protein that catalyzes the movment of peptide-bound tRNAs from the ribosome A site to the P site during translation. This is notable because EEF2 is highly expressed in numerous cancers [
30], where it promotes cell proliferation and EMT [
26].
Because we were interested in the relationship between Fhit loss and protein expression our initial work focused on coding region ribosome occupancy. However, this changed with the report by Sendoel et al. [
18], showing the onset of malignancy is preceded by changes in 5’-UTR ribosome occupancy of a number of cancer-associated mRNAs. This was first evident in Fig.
2, where CDKN2C, ATG16L2 and MECP2 had higher 5’-UTR ribosome occupancy in Fhit-negative vs Fhit-positive cells. Restoring Fhit expression shifted this pattern, resulting in higher coding region and lower 5’-UTR ribosome occupancy of these mRNAs. A related picture was seen for TP53I3, but in Fhit-negative cells there appears to be a single stalled ribosome bound at the start codon that is released by Fhit expression. These findings are consistent with results in [
31] and [
32] in which a uORF regulates downstream translation, which in our case is affected by the presence or absence of Fhit.
Futher examination identified 5 mRNAs for which changes in 5’-UTR ribosome occupancy increased with Fhit rescue, 2 mRNAs for which 5’-UTR ribosome occupancy decreased (Fig.
4), and 13 mRNAs for which Fhit expression changed the ratio of 5’-UTR vs coding region ribosome occupancy. Each of these mRNAs has a potential or verified uORF, the sequences of which are listed in Additional file
9. As in [
18], these uORFs have mostly non-canonical (CUG, GUG, UUG) start codons, and some extend into the coding region, raising the possibility that loss of Fhit leads to changes in ribosome occupancy that can affect the protein products translated from these mRNAs.
In total, this study identified 30 genes for which
FHIT loss alters ribosome occupancy of the 5’-UTR or the coding region of their respective mRNAs (Table
3). These are notable for the breadth of pathways they impact. A number of genes have been directly linked to malignancy, including ADAM9, CDKN2C, DCAF5, KPNB1, MECP2, RACK1, SERTAD3, TBC1D7, TP53I3 and TIAM1. H2AF7 was recently identified as a major regulator of EMT [
33]. Others that have been indirectly linked to cancer include HIST1H2AD, two zinc finger proteins (ZCCHC3, ZNF552), and CSRP2, CREBL2, and SERTAD3. Intermediary metabolism and protein synthesis have essential roles in malignancy, and these processes are represented in our dataset by ACSL1 and FASN, which encode enzymes that function in fatty acid synthesis and lipid metabolism, and TROVE2, EEF2, RACK1 and RPL37A, each of which impact RNA metabolism or translation. This analysis also identified HINT2, which is a histidine triad containing protein that functions in mitochondrial cell death signaling. Based on results presented here we propose that past challenges in identifying how loss of
FHIT leads to cancer may be partially due to the fact that Fhit and its loss exert pleiotropic effects on the translation of genes that function in a multitude of cellular processes.
Table 3
Targets of Fhit-mediated changes in ribosome occupancy
ACSL1 | Acyl-CoA synthetase, functions in lipid metabolism by converting long chain fatty acids into their CoA esters | Increased 5’-UTR |
ATG16L2 | Autophagy Related 16 Like 2, paralog of ATG16L1, which functions in a complex needed for autophagy | Increased CDS |
ADAM9 | A Disintegrin And Metalloproteinase Domain 9, involved in cell-cell interactions, cell-matrix interactions | Increased 5’-UTR |
CALR | Calreticulin, a major Ca(2+)-binding (storage) protein | Decreased 5’-UTR, CDS unchanged |
CDKN2C | Cyclin Dependent Kinase Inhibitor 2C, a member of the INK4 family of cyclin-dependent kinase inhibitors that regulate cyclin-dependent kinases | Increased CDS |
CLPTM1 | Cleft Lip And Palate Associated Transmembrane Protein 1, a paralog of CLPTM1, has a role in susceptibility to cisplatin | Increased 5’-UTR, CDS unchanged |
CREBL2 | cAMP Responsive Element Binding Protein Like 2, transcription factor associated with lung cancers | Decreased 5’-UTR, increased CDS |
CSRP2 | Cysteine And Glycine Rich Protein 2, a member of the CSRP family encoding LIM domain proteins involved in cellular differentiation | Increased CDS |
DCAF5 | DDB1 And CUL4 Associated Factor 5, a ubiquitin ligase that regulates cell proliferation, survival, DNA repair, and genomic integrity | Increased 5’-UTR and CDS |
EEF2 | Eukaryotic Translation Elongation Factor 2, GTP-binding translation factor that facilitates movement of tRNA-bound peptides from the ribosome A site to the P site. | Decreased CDS |
FASN | Fatty Acid Synthase, catalyzes the synthesis of palmitate from acetyl-CoA and malonyl-CoA. Also an mRNA-binding protein. | Increased 5’-UTR, CDS unchanged |
FBLN1 | Fibulin 1, a secreted glycoprotein that is incorporated into the extracellular matrix | Decreased 5’-UTR, CDS unchanged |
GDA | Guanine Deaminase | Decreased CDS |
HINT2 | Histidine Triad Nucleotide Binding Protein 2, a member of the superfamily of histidine triad proteins, functions in mitochondrial cell death signaling | Increased 5’-UTR, CDS unchanged |
HIST1H2AD | Histone Cluster 1 H2A Family Member D | Increased 5’-UTR |
H2AFZ | H2A Histone Family Member Z, regulates epithelial-mesenchymal transition, evidence for a role in repressing lncRNA | Increased 5’-UTR |
IFIT1 | Interferon Induced Protein With Tetratricopeptide Repeats 1, antiviral protein targeting RNAs with 5′-triphosphate ends | Decreased CDS |
IGSF9 | Immunoglobulin Superfamily Member 9, molecule involved in cell-cell interaction | Increased CDS |
KPNB1 | Karyopherin Subunit Beta 1, subunit of the importin alpha complex that binds the nuclear localization signal, functions in importing proteins into the nucleus | Increased 5’-UTR, CDS unchanged |
LRRC73 | Leucine Rich Repeat Containing 73, no known function | Decreased CDS |
MECP2 | Methyl-CpG Binding Protein 2, mediates transcriptional silencing by binding methyl CpG in DNA, mutations result in Rett syndrome, neurological | Increased CDS |
RACK1 | Receptor For Activated C Kinase 1, component of the 40S ribosome subunit, involved in signaling between protein kinase pathways and translation | Decreased 5’-UTR |
RPL37A | Ribosomal Protein L37A, constituent of the large ribosome subunit | Decreased 5’-UTR |
SERTAD3 | SERTA Domain Containing 3, a transcriptional coactivator | Increased 5’-UTR and CDS |
TBC1D7 | Subunit of the tuberous sclerosis TSC1-TSC2 complex | Increased 5’-UTR and CDS |
TP53I3 | Tumor Protein P53 Inducible Protein 3, an oxidoreductase that is induced by TP53 | Increased CDS |
TIAM1 | T-cell Lymphoma Invasion And Metastasis 1, encodes a RAC1-specific guanine nucleotide exchange factor | Increased 5’-UTR and CDS |
TROVE2 | TROVE Domain Family Member 2, binds misfolded ncRNAs including Y RNAs | Increased 5’-UTR |
ZCCHC3 | Zinc finger protein of unknown function, interacts with a large number of proteins, including ELAV1, LIN28B, PAIP1, RACK1, NANOG | Increased 5’-UTR and CDS |
ZNF552 | Zinc Finger Protein 552, unknown function | Decreased 5’-UTR, increased CDS |
It remains to be determined how Fhit or its loss affects changes in translation. The 5’-UTR is a nexus for post-transcriptional gene regulation [
34], and with changes in 5’-UTR ribosome occupancy being the feature most characteristic of the targets identified here. We speculate that sequences or structural elements within this region play a major role in determining specificity for their regulation by Fhit. Fhit does not contain any recognizable RNA binding domains, nor has it been identified as a constituent of the mRNP proteome [
35‐
37]. As a member of the histidine triad family Fhit catalyzes the hydrolysis of nucleoside 5′,5′-triphosphates, including m
7G caps that are the remnants of 3′-5’ mRNA decay [
14]. The H1299 cells used here have low levels of the main scavenger decapping enzyme DcpS, and we hypothesize that nucleoside 5′,5′-triphosphates (including but not necessarily limited to m
7G caps) accumulate in Fhit-negative cells to a level sufficient to compete with eIF4E binding to the capped ends of a limited number of mRNAs. As noted above, sequences or structural elements within the 5’-UTR likely determine susceptibility to this disruption. Indirect targeting of gene expression by the intracellular level of nucleoside 5′,5′-triphosphates is consistent with the observation that Fhit/H96N, which binds nucleoside 5′,5′-triphosphates with high affinity, is nearly as effective as wild-type Fhit in suppressing tumor formation [
38,
39].