Introduction
Many biological processes show a circadian rhythm, which is controlled by an endogenous clock that synchronizes with day and night phases of the solar day. The periodical rhythm is generated by a molecular oscillator located in the suprachiasmatic nuclei of the hypothalamus, which controls peripheral oscillators in virtually any other cell (1). The circadian clock is organized through a complex network of transcription-translational feedback loops that drive rhythmic expression patterns of core clock components in mammals (2).
The Timeless (TIM) protein interacts with clock proteins and is essential for generation of a circadian rhythm in flies (3). In addition, phylogenetic sequence analysis revealed the presence of a paralogue in D. melanogaster (4), which is not involved in the core clock machinery, but is important for the maintenance of chromosome integrity, growth control, and development. In contrast, a single TIM gene has been identified in mammals, which acquired all of these functions as indicated by its role in DNA damage response, replication, and circadian rhythm (5–9). Consistently, TIM knockout resulted in embryonic lethality in mice (10). In addition, TIM and other clock genes have been linked to human carcinogenesis (11–14). Using integrative molecular profiling we have recently identified that inactivation of Period homolog 3, a component of the clock machinery, occurs in human HCC indicating that dysregulation of this regulatory network may contribute to hepatocarcinogenesis (15).
The eukaryotic elongation factor 1-α (EEF1A), a member of the G protein family, represents one of the four subunits that constitute the eukaryotic elongation factor 1 (16). In humans, two EEF1A isoforms are known. While EEF1A1 is expressed in almost all tissues, EEF1A2 expression was only reported in heart, brain and skeletal muscle (17,18). However, EEF1A2 exerts an anti-apoptotic function, which may explain its role in tumorigenesis (19). We have previously identified EEF1A2 as a candidate oncogene in human hepatocarcinogenesis and demonstrated that EEF1A2 overexpression was strongly associated with the survival probability of HCC patients (20,21).
Here we analyzed the potential protumorigenic function of TIM in human hepatocarcinogenesis. Our data show that TIM overexpression promotes tumor cell proliferation via inhibition of CHEK2 phosphorylation. In addition, TIM directly interacts with the eukaryotic elongation factor 1A2 (EEF1A2), which promotes tumor cell migration and affects ribosomal protein biosynthesis.
Materials and methods
Tumor material and patient characteristics
Expression profiles were generated from 40 human HCCs as described previously (15). The specimen included 22 liver resections and 15 explant livers; median age at surgery was 56 years (range 16–78) and the male/female ratio was 3:1. Human tissue samples were provided by the tissue bank of the National Center for Tumor Diseases Heidelberg. All diagnoses were confirmed by histological re-evaluation and use of the samples was approved by the local ethics committee. According to the vote an informed consent was not required because only long-term archived (>5 years), pseudonymized samples were used for this study. From three patients two HCC nodules were included that previously showed different aCGH data indicating independent tumor development. Etiology was determined as previously described (20). The underlying etiologies were HBV (n=8), HCV (n=8), alcohol (n=7), cryptogenic (n=10), genetic hemochromatosis (n=3), and α1-antitrypsin deficiency (n=1). The patient characteristics are shown in Table I.
Reverse transcription and polymerase chain reaction
RNA was isolated from 100 mg of snap-frozen tissue after histological validation using the RNeasy Midi-Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. One microgram of total RNA was reversely transcribed with the RevertAid™ H minus Reverse Transcriptase (Fermentas, St. Leon-Rot, Germany) and analyzed using the ABI PRISM 7300 Real-Time PCR System (Sequence Detection software v1.2.2, Applied Biosystems, Foster City, CA, USA) with Absolute SYBR Green ROX Mix (ABgene, Epsom, UK). Calculations of efficacy, normalization, and relative quantification versus 18s rRNA were done according to published algorithms (22). The following primer sequences were used (Eurofins MWG Operon, Ebersberg, Germany): TIM-fw 5′-GCC CTC AAT GTG AGG CTC TT-3′, TIM-rev 5′-CCC GAA GCA GGT GAT CCT TT-3′, MDM4-fw 5′-CAG CAG GTG CGC AAG GTG AA-3′, MDM4-rev 5′-CTG TGC GAG AGC GAG AGT CTG-3′, MDM2-fw 5′-TCT GTG AGT GAG AAC AGG TGT CAC-3′, MDM2-rev 5′-ACA CAC AGA GCC AG GCT TTC-3′, p21-fw 5′-CAC CGA GAC ACC ACT GGA GG-3′, p21-rev 5′-GAG AAG ATC AGC CGG CGT TT-3′, puma-fw 5′-AAA CGG CTA CCA CAT CCA AG-3′, puma-rev 5′-CCT CCA ATG GAT CCT CGT TA-3′, bax-fw 5′-TGG AGC TGC AGA GGA TGA TTG-3′, bax-rev 5′-AAA CAT GTC AGC TGC CAC TCG-3′, EEF1A2-fw 5′-AGA TGT CGA TGG TGA TGC-3′, EEF1A2-rev 5′-AGA TGT CGA TGG TGA TGC-3′, 18s-fw 5′-AAA CGG CTA CCA CAT CCA AG-3′, 18s-rev 5′-CCT CCA ATG GAT CCT CGT TA-3′. Reaction temperatures and periods were used according to the manufacturer’s instructions (ABgene).
DNA microarray hybridization and analysis
Quality and integrity of the total RNA was controlled using an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). Total RNA (200 ng) was applied for Cy3-labelling reaction using the one color Quick Amp Labeling protocol (Agilent Technologies). Labeled cRNA was hybridized to Agilent human 8×60k microarrays at 68°C for 16 h and scanned using the Agilent DNA Microarray Scanner (Agilent Technologies). Expression values were calculated by the software package Feature Extraction 10.5.1.1. Complete data are available online (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE50579).
Western blot analysis and immunoprecipitation
Cell samples were homogenized in lysis buffer (no. 9803, Cell Signaling Technology, Danvers, MA, USA) containing the complete protease inhibitor cocktail (Roche, Mannheim, Germany) and sonicated. Protein concentrations were determined using Nanodrop ND1000 (Thermo Scientific, Waltham, MA, USA). For Western blotting, aliquots of 100 μg were denatured by boiling in Tris-glycine SDS sample buffer (Invitrogen, Karlsruhe, Germany), separated by SDS-PAGE, and blotted onto nitrocellulose membranes (Invitrogen). Membranes were blocked in 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h and probed with the following specific antibodies: mouse monoclonal anti-β-actin (1:10,000; MP Biomedicals, Santa Ana, CA, USA), rabbit polyclonal anti-AKT (1:1,000; Cell Signaling Technology), mouse monoclonal anti-CHK1 (1:1,000; Cell Signaling Technology), rabbit monoclonal anti-CHK2 (1:1,000; Cell Signaling Technology), rabbit monoclonal anti-EEF1A2 (1:1,500, Abcam, Cambridge, UK), mouse monoclonal anti-MDM2 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit polyclonal anti-MDM4 (1:3,000; Abiocode, Agoura Hills, CA, USA), rabbit monoclonal anti-pAKT (Serine 473, 1:500; Cell Signaling Technology), rabbit polyclonal anti-PARP (1:500; Cell Signaling Technology), rabbit monoclonal anti-pCHK1 (Serine 345, 1:1,000; Cell Signaling Technology), rabbit polyclonal anti-pCHK2 (Threonine 68, 1:1,000; Cell Signaling Technology), rabbit polyclonal anti-TIM (1:2,000; Bethyl Laboratories, Montgomery, TX, USA), mouse monoclonal anti-α-tubulin (1:2,000; Sigma-Aldrich, St. Louis, MO, USA). Each incubation with a primary antibody was followed by incubation with a horseradish peroxidase-conjugated secondary antibody for 1 h (1:2,000; Cell Signaling Technology) and visualized using the Super Signal West Pico (Pierce Chemical, New York, NY, USA). For quantification the protein densities were calculated by ImageQuaNT 5.1 software (GE Healthcare, Piscataway, NJ, USA) and normalized to α-tubulin to determine the relative expression levels. For immunoprecipitation (IP) experiments NP40 buffer (Tris-HCl 50 mM, NACl 150 mM, NP40 1%) was used for protein isolation. Three milligrams of HCC cell protein lysate were immunoprecipitated with 6 μg of protein A agarose-bound EEF1A2 rabbit polyclonal antibody (Santa Cruz Biotechnology) at 4°C overnight. After immunoblotting, the membranes were incubated with a rabbit monoclonal anti-EEF1A2 antibody (1:1,500; Abcam), rabbit polyclonal anti-TIM (Bethyl Laboratories) or mouse monoclonal anti-actin antibody (MP Biomedicals). To exclude unspecific binding IP was carried out without protein input as negative control. Respectively, mouse monoclonal anti-rabbit and goat anti-mouse light chain IgG (1:7,000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were used as secondary antibodies to detect EEF1A2 bound proteins.
Tissue microarrays and immunohistochemistry
A tissue microarray (TMA) containing tissue from normal livers (n=27), non-tumor liver tissue of HCC patients (n=86), and HCCs (n=102; Table II) was constructed as previously described (23), and immunohistochemistry was performed on 5-μm sections. From eleven patients two independent HCC nodules were included. The following primary antibodies were used for incubation: rabbit monoclonal EEF1A2 (1:1,500; Abcam), rabbit polyclonal Timeless (1:2,000; Bethyl Laboratories). Antigens were retrieved using citrate buffer (pH 6.1; Dako, Glostrup, Denmark). For detection the EnVision method (Dako) was used. Counterstaining was performed using hemalum. Staining was assessed using the immunoreactive score as described previously (20): 0, absent; 1–4, weak; 5–8, moderate; 9–12, strong expression.
Cell lines, transfection and functional analyses
HepG2, Hep3B, HLE, HLF, HuH6, HuH7, PLC/PRF/5, SNU182 and SNU387 cells were cultured either in DMEM, MEM or RPMI medium, supplemented with 10% fetal bovine serum (PAA, Pasching, Austria) and 1% penicillin-streptomycin (10 mg/ml, PAA) at 37°C (5% CO2) and passaged every 3–4 days. All siRNA transfections were performed using oligofectamine (Invitrogen) according to the manufacturer’s protocol. The following small interfering RNAs were used (siRNA, Eurofins MWG Operon): siTIM1 5′-AGA AGA GAA GGA AGA AGA A-dTdT3′, siTIM3: 5′-GCC UAC AUG UGC UAG AGA U-dTdT3′, neutral control siRNA (siNC) 5′-UUC UCC GAA CGU GUC ACG U-dTdT3′. Transient transfection experiments of Hep3B cells with EEF1A2 cDNA in pCMV6-XL5 vector (OriGene Technologies, Rockville, MD, USA) was performed following the manufacturer’s protocol using FuGene HD transfection reagent (Promega, Mannheim, Germany). For functional assays HCC cell lines were seeded at a density of 6×103 cells in 96-well plates and were transfected with siRNA after 24 h. Cell viability (MTT-assay; M2128, Sigma, Deisenhofen, Germany), apoptosis (FACS-assay; FACS Calibur™, BD Bioscience, Heidelberg, Germany), and migration (2D-scratch-assay) were determined as described previously (15,20). For the treatment with chemical inhibitors, HCC cells were plated at a density of 1.5×105 cells/well in 6-cm plates and were incubated with the following drugs after 24 h, as indicated: cycloheximide (protein synthesis inhibitor; Merck, Darmstadt, Germany), MG132 (proteosomal inhibitor, 450 μmol/l; Enzo Life Sciences, Lörrach, Germany). For all cell based assays, results were confirmed in three independent experiments.
Immunofluorescence microscopy
After 24 h of culturing on glass coverslips HCC cells were transfected using siRNAs as described above. Seventy-two hours after transfection cells were washed repeatedly in PBS containing 2 mM MgCl2 at 37°C, and fixed with methanol/acetone. Fixed cells were again washed with PBS for 30 min, permeabilized in PBS with 0.05% Tween-20 for 10 min, and processed as described previously (24) using a mouse monoclonal β-actin antibody (1:5,000; MP Biomedicals). Fluorescence was detected using an Olympus BX40 microscope (Olympus, Hicksville, NY, USA) equipped with a Leica DFC365FX camera (Leica, Wetzlar, Germany).
Statistical analyses
The correlation between gene expression and clinicopathological parameters was tested by Wilcoxon-signed rank tests and measured by Spearman’s rank correlations. P<0.05 was considered statistically significant. Statistical analyses were conducted using SPSS 20.0 (SPSS, Chicago, IL, USA).
Results
TIM is upregulated in a subset of human HCCs
According to our previous expression profiling (15) upregulation of TIM mRNA (>2-fold) was observed in 55% of HCCs (n=22), while only 1 tumor (2.5%) showed lower expression (<50%) compared to normal liver (Fig. 1A). These findings were validated using quantitative real-time RT-PCR (Fig. 1B). Thus, there was a significant upregulation of TIM mRNA in human HCCs compared to normal liver (P=0.004). Tumors measuring >5 cm in diameter showed significantly higher TIM mRNA levels compared to smaller lesions (P=0.01). Although there was a trend for increased TIM expression with tumor dedifferentiation, the results did not reach statistical significance (rho=0.3, P=0.07). No significant association was found with gender, etiology, vascular invasion and UICC stage (P>0.05).
Next we determined the TIM protein expression in human HCCs using TMA (Fig. 1C and E). Weak TIM expression was seen in 52% (n=14) of normal liver tissues and the remaining were negative. In non-tumorous liver tissues of HCC patients (n=86), 47% displayed no detectable TIM signal at all, whereas half of the samples showed weak (n=43) and 3% showed moderate expression of TIM. Regarding HCCs (n=103), 40% did not show any, 50% showed weak, 9% moderate, and 2% showed strong TIM staining. There was a trend towards higher TIM protein expression in primary HCCs that had developed extrahepatic metastasis compared to non-metastasized tumors (P=0.05). No significant association was found with gender, etiology, vascular invasion, and tumor size (P>0.05).
TIM promotes cell viability and tumor cell migration
TIM protein was expressed in all HCC cell lines analyzed. High TIM expression levels were observed in Hep3B, HepG2, HuH6, HuH7, and PLC/PRF/5 cells (Fig. 1D). To test whether TIM exerts protumorigenic functions in vitro, we used gene-specific siRNAs to knock down TIM expression in HepG2 and Hep3B cells, which resulted in significant reduction of cell viability compared to mock transfected cells [0.61±0.03 (siTIM1) and 0.37±0.11 (siTIM3) in HepG2; 0.50±0.10 (siTIM1) and 0.59±0.17 (siTIM3) in Hep3B cells, P<0.01 respectively; Fig. 2A], and was associated with a mild induction of apoptosis as shown by FACS analysis [1.87±0.09-fold (siTiM1) and 2.07±0.10-fold (siTIM3) in HepG2 and 3.56±0.10-fold (siTIM1) and 1.62±0.10-fold (siTIM3) in Hep3B compared to Mock transfected cells, each P<0.01; Fig. 2B] as well as cleavage of PARP in Western immunoblotting (Fig. 3A). In addition, knockdown of TIM resulted in a significant G2 arrest in both cells lines analyzed [20.3±0.2% (siTIM1)/25.8±0.5% (siTIM3) vs. 12.8±1.2% (siNC) in HepG2 and 50.5±2.1% (siTIM1)/37.4±0.4% (siTIM3) vs. 20.2±0.3% in Hep3B cells, P<0.01 respectively; Fig. 2C]. Finally, migration was significantly reduced in TIM-depleted compared to siNC-transfected Hep3B cells [0.41±0.08 (siTIM1) and 0.72±0.05 (siTIM3), P<0.01 respectively; Fig. 2D]. Due to their multilayer and colony forming growth characteristics the migration of HepG2 cells could not be explored in a standardized manner.
Mechanisms involved in pro-proliferative and anti-apoptotic function of TIM
As TIM has been reported to affect cell proliferation either via CHEK1-or CHEK2-mediated mechanisms (8,25), we analyzed both expression and phosphorylation of these proteins. Indeed, both CHEK2 protein levels as well as CHEK2 phosphorylation were increased in HepG2 and Hep3B cells after knockdown of TIM expression (Fig. 3A). Interestingly, we observed that numerous other proteins including CHEK1 (Fig. 3A), EEF1A2, v-akt murine thymoma viral oncogene homolog (AKT), and β-actin (Fig. 3B) were downregulated following TIM knockdown suggesting that TIM may activate the protumorigenic EEF1A2/AKT/MDM4 axis (21). In line, transcriptional activation of some p53 target genes was seen following TIM knockdown in p53-wild-type cells indicating reactivation of p53 function in these cells (Fig. 4).
EEF1A2 is a crucial mediator of protumorigenic TIM functions
EEF1A2 has been shown to promote oncogenic functions in HCC (20) and was downregulated upon TIM knockdown. In addition, we observed a correlation (rho=0.37, P>0.001) between EEF1A2 and TIM protein as determined by immunohistochemistry (Fig. 1E). As EEF1A2 is involved in ribosomal function as well as actin dynamics, we hypothesized that EEF1A2 could be a crucial mediator of the protumorigenic properties of TIM. To explore whether EEF1A2 was able to revert the TIM-induced migratory phenotype, we transiently expressed an EEF1A2 cDNA after TIM knockdown. Both, EEF1A2 mRNA and protein levels were increased (Fig. 5A and B), which was paralleled by an increase in migratory capacity compared to Mock transfected cells. However, both the migration of siTIM1-treated Hep3B cells as well as their EEF1A2 protein level remained lower compared to siNC-treated Hep3B cells (Fig. 5B and C).
Immunoprecipitation experiments revealed a direct interaction of EEF1A2 with both TIM and β-actin (Fig. 6A). In addition, filopodia formation was impaired following TIM knockdown (Fig. 7) indicating that the TIM-EEF1A2 interaction may promote actin dynamics and thus tumor cell migration. As TIM was expressed predominantly in the nucleus, while EEF1A2 was present in the cytoplasm (Fig. 1E), we further explored in which cellular compartment TIM and EEF1A2 might interact. Cellular fractionation confirmed that EEF1A2 was mainly localized in the cytoplasm, while TIM expression was observed in both compartments suggesting that the TIM-EEF1A2 interaction occurs in the cytoplasm (Fig. 6B).
Although TIM seemed to stabilize EEF1A2 via direct interaction, this finding did not explain the reduction of other proteins like CHEK1, AKT, or β-actin following TIM knockdown. In humans, two EEF1A protein isoforms (EEF1A1 and EEF1A2), which have similar elongation activity, are known (17,18). Thus, we evaluated the effect of TIM knockdown on protein biosynthesis. EEF1A2 protein levels were significantly reduced after cycloheximide treatment in siTIM-compared to siNC-treated cells (Fig. 6C), while inhibition of proteasomal degradation using MG132 did not prevent reduction of EEF1A2 expression following TIM knockdown (Fig. 6D) indicating that impaired protein biosynthesis, and not increased proteasomal degradation, may be responsible for this phenotype.
Thus, our data show that TIM exerts protumorigenic functions in human HCC, which are crucially mediated by physical interaction with EEF1A2 and downstream activation of the AKT signaling cascade as well as promotion of tumor cell migration. A schematic model of the protumorigenic mechanisms activated by TIM overexpression is shown in Fig. 8.
Discussion
By demonstrating the epigenetic inactivation of Period homolog 3 we have recently shown that dysregulation of clock machinery components occurs in human HCC (15). We hypothesized that altered clock function may be a hallmark of hepatocarcinogenesis and found six clock genes (CLOCK, CSNK1E, PER1, PER2, RORA and TIM; Gene Expression Omnibus Series GSE50579) to be differentially expressed on the mRNA level between HCC and normal liver. We focused on TIM as it has been shown to be important for the maintenance of chromosome integrity and growth control (5,6), and a recent meta-analysis revealed a transcriptional timeless-dependent network and suggested a role of TIM in development and progression of various epithelial cancer types (e.g., bladder, breast, cervical, colorectal, gastric, head and neck, kidney, lung and prostate) (26). Here, we observed that TIM is overexpressed in a subset of human HCCs, which is contradictory to a study reporting downregulation of TIM in HCC (27). Our functional analysis supports an oncogenic function as TIM knockdown resulted in reduced viability of HCC cells due to the induction of apoptosis and cell cycle arrest in vitro. The effect on proliferation may be related to the fact that TIM-TIPIN complexes have been shown to stabilize the replication fork during early S phase. Depletion of TIM resulted in defects of mitotic progression, impaired sister chromatid cohesion and chromosome fragmentation (28). In line with a previous report showing that TIM is required for ATM-dependent CHEK2 activation and G2/M checkpoint control (8), we observed that siRNA-mediated inhibition of TIM in HCC cells increased CHEK2 phosphorylation resulting in cell cycle arrest. Together, these data provide evidence that upregulation of TIM in HCC cells promotes tumor cell proliferation. Although systemic targeting of TIM might exert antitumoral activity in overexpressing tumors, such an approach is probably limited by its physiological function in the replication fork (28), suggesting that the spectrum of unwanted side-effects following targeting of TIM might be similar to conventional chemotherapy due to broad unspecific targeting of any proliferating cell. Accordingly, specific targeting of the relevant ‘non-canonical’ mediators of the oncogenic TIM function might be a more promising therapeutic strategy.
Recent data indicated that EEF1A2 overexpression is a hallmark of prostate and ovarian cancer and plays an important role in mammary carcinogenesis (29–31). We have previously identified EEF1A2 as a candidate oncogene in human hepatocarcinogenesis (20). EEF1A2 acts as an upstream inducer of the PI3K/AKT/mTOR axis leading to functional inactivation of p53 by posttranslational stabilization of the mouse double minute homolog 4 (MDM4) in human HCCs (21).
Interaction of EEF1A with the cytoskeleton is important for efficient translation and EEF1A is involved in the organization of the actin cytoskeleton in vivo (32,33).
Herein, we demonstrate that EEF1A2 directly interacts with TIM and β-actin. Since our data indicate that the TIM-EEF1A2 interaction occurs likely in the cytoplasm, we speculate that TIM exerts its oncogenic function at least partially in this compartment. In line, knockdown of TIM reduced EEF1A2 expression and impaired tumor cell migration. EEF1A2 has been shown to induce filopodia formation in cell lines and enhance migration in an AKT- and PI3K-dependent manner (34,35) indicating that the reduced activation of the EEF1A2/PI3K/AKT/mTOR axis may contribute to the migratory phenotype observed in this study.
In addition to its canonical function in translational elongation EEF1A exerts a plethora of non-canonical activities including export of proteins from the nucleus (36), a role in viral replication (37), and interaction with the proteasome which is increased when translation is inhibited (38). As EEF1A2 protein levels could not be rescued by pharmacological inhibition of the proteasome after TIM knockdown, the latter function is unlikely to contribute to the decreased expression of various proteins observed here. The most likely explanation is that the EEF1A2 knockdown impaired ribosomal function resulting in reduced protein biosynthesis in HCC cells as indicated by significantly decreased EEF1A2 protein levels following cycloheximide treatment.
In conclusion, TIM exerts protumorigenic functions in human HCC, which are crucially mediated by physical interaction with EEF1A2 and the activation of its downstream signaling cascade as well as promotion of tumor cell migration via EEF1A2-mediated activation of actin dynamics. Although targeting of TIM and EEF1A2 was effective in vitro, EEF1A2 seems to be the more promising target as its physiological expression level is restricted to non-proliferating cells and de novo expression is frequently found in human cancers. Future studies are needed to evaluate both the efficacy as well as potential unwanted effects of these treatment approaches in vivo.