TIMELESS (TIM) is a mammalian homolog of a Drosophila circadian rhythm gene, but its circadian properties in mammals have yet to be determined. TIM appears to be essential for replication protection and genomic stability. Recently, the involvement of TIM in human malignancies has been reported; therefore, we investigated the role of TIM in lung cancer. Microarray expression analysis of lung cancer cell lines showed that TIM expression was elevated 3.7-fold (P <0.001) in non-small cell lung cancer cell lines (n =116) compared to normal lung controls (n =59). In addition, small cell lung cancer cell lines (n =29) expressed TIM at levels 2.2-fold (P <0.001) higher than non-small cell lung cancer. Western blot analysis of 22 lung cancer cell lines revealed that all of them expressed TIM protein and that 20 cell lines (91%) expressed TIM protein at higher levels than a normal control line. Remarkably, immunohistochemistry of 30 surgically resected lung cancer specimens showed that all lung cancer specimens but no matched normal lung tissues were positive for TIM expression. Moreover, immunohistochemistry of surgically resected specimens from 88 consecutive patients showed that high TIM protein levels correlated with poor overall survival (P = 0.013). Mutation analysis for TIM in 23 lung cancer cell lines revealed no mutation. TIM knockdown suppressed proliferation and clonogenic growth, and induced apoptosis in H157 and H460 cells. Taken together, our findings suggest that TIM could be useful as a diagnostic and prognostic marker for lung cancer and targeting it would be of high therapeutic value for this disease.
Circadian rhythms of approximately 24-h periodicity are commonly observed in a variety of physiological functions of organisms from bacteria to humans. In Drosophila, period (per) and timeless (tim) are two main clock genes that participate in an intracellular transcriptional/translational feedback loop and create 24-h rhythmicity.[2, 3] In mammals, homologs of several of the fly clock genes have been identified and characterized. Mammalian Timeless (mTim) was cloned as a mammalian homolog of Drosophila tim, but its role in mammalian circadian clock systems has not been fully clarified. TIM knockdown in the rat suprachiasmatic nucleus (SCN) disrupted SCN neuronal activity rhythms and caused a reduction of PER1, PER2 and PER3 and an increase in CRYPTOCHROME (CRY)1and CRY2. Furthermore, full-length TIM protein exhibited a 24-h oscillation, whereas a truncated isoform was constitutively expressed, indicating the active role of mTIM in the circadian clock system. By contrast, several reports suggest that mTIM does not function as a clock protein.[6, 7] Database mining and phylogenetic sequence analysis showed that mTIM may not be a true ortholog of a Drosophila circadian rhythm gene, because it shares even greater sequence similarities with other TIM-related genes that do not function as clock genes, including Drosophila TIMEOUT, Bombyx mori TIMEOUT, and Caenorhabditis elegans TIM-1. In addition, a heterozygous Tim mutant mouse did not show a change in circadian phenotype despite reduced TIM protein levels. Thus, it is still unclear whether TIM functions as a clock protein.
Previous reports show the involvement of TIM in human cancers. First, screens of human breast cancers have identified TIM mutations. Second, a study has reported that TIM depletion sensitizes HCT116 colon cancer cells to doxorubicin-induced cytotoxicity. The authors conclude that this effect might be due to the requirement for TIM in the ataxia telangiectasia mutated-dependent Chk2 DNA damage-response pathway that causes cells to arrest at the G2/M phase. Third, Fu et al. provide data suggesting that TIM may contribute to the carcinogenesis of breast cancer. They report an association between breast cancer risk and single-nucleotide polymorphism (SNP) in TIM and increased levels of TIM in tumor tissues compared to controls. These findings suggest that TIM plays an important role in the development of human cancer. However, the role of TIM in lung cancer, one of the deadliest cancers, has not been investigated. Therefore, in the current study we investigate the role of TIM in lung cancer. We evaluate the expression level and mutational state of TIM in lung cancers and examine the effects of RNA interference (RNAi)-mediated TIM knockdown on growth, apoptosis, and sensitivity to doxorubicin and cisplatin using the lung cancer cell lines H157and H460. No mutation was found in the cell lines studied, but TIM was overexpressed in both lung cancer cell lines and clinical lung cancer specimens. A high TIM protein level correlated with poor overall survival in lung cancer patients. In addition, TIM knockdown suppressed growth and induced apoptosis in lung cancer cells. These findings suggest the potential of TIM as a prognostic marker and a therapeutic target for lung cancer.
Materials and Methods
Cell lines used in this study were purchased from American Type Culture Colllection (Manassas, VA, USA) or obtained from the Hamon Center collection (University of Texas Southwestern Medical Center, Dallas, TX, USA). These cell lines included H820, H1975, HCC44, HCC2279, H838, PC9, H3255, HCC4011, HCC2935, A549, H1650, HCC4006, HCC827, H1666,H358, H1299, H1155, H460, H157, H146, H526, H82, H740 and the cdk4/hTERT-immortalized normal human bronchial epithelial cell line HBEC4. Lung cancer cell lines were cultured in RPMI-1640 (Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% FBS, and HBEC4 was cultured in keratinocyte serum-free medium (Life Technologies, Gaithersburg, MD, USA) supplemented with 50 ng/mL bovine pituitary extract and 5 ng/mL epidermal growth factor.
Total RNA was isolated using Trizol (Life Technologies). For mRNA analysis, 5 μg of total RNA was reverse transcribed using a Super Script III First-Strand Synthesis System using a Random primer system (Life Technologies).
RNA quality and concentration were checked using the Experion Bioanalyzer (Bio-Rad, Hercules, CA, USA) according to the manufacturer's protocol. Total RNA (500 ng) from each sample was used to label the cRNA probes with the Illumina TotalPrep RNA Amplification kit (Cat# IL1791; Ambion, Austin, TX, USA). Amplified and labeled cRNA probes (1.5 μg) were hybridized to Illumina Human WG-6 v3.0 Expression BeadChip (Cat# BD-101-0203; Ambion) overnight at 58°C, then washed, blocked, and detected by streptavidin-Cy3 following the manufacturer's protocol. After drying, the chips were scanned using an Illumina iScan system (Ambion). Bead-level data were obtained and pre-processed using the R package Model-Based Background Correction (MBCB) for background correction and probe summarization. Pre-processed data were then quantile-normalized and log-transformed.
The sequences of primers used for reverse transcriptase PCR and for direct sequencing were: TIM-S101, GGA GCG GGC CTC ATC ATT TC; TIM-AS941, TGG CGC AAC ACC TCC AGT TC; TIM-S790, TCG TCT GCT GAG GAG CAA TG; TIM-AS 1652, GGC TGG CAT CTC TCA TCA AAC; TIM-S1509, GGC AAC AGT GAA TGA GAT GGA C; TIM-AS2442, AGG GTC ACT AAG CAG ACG ATT G; TIM-S 2293, CTG CTA CTA AGG AGC TAC CAG C; TIM-AS3175, TTG AAA GGA CTA AGC TAC CCT G; TIM-S3022, GCA TCC TCC ATC TTG CCA AAT G; and TIM-AS3929, TCC GTG AAA GAG CCT GGG ATT C. The reaction was performed in a 20 μL mixture containing primers (final concentrations 0.25 μM), HotStar Taq Master Mix (Qiagen, Tokyo, Japan; 10 μL) and cDNA (0.5 μL). Amplification was carried out in a TaKaRa PCR Thermal Cycler Dice (Takara Bio, Otsu, Japan). Cycling conditions were one cycle at 95°C for 5 min, followed by 35 cycles at 94°C for 30 s, 60°C (except for TIM-S2293 and TIM-AS3175) or 56°C (TIM-S2293 and TIM-AS3175) for 30 s and 72°C for 1 min. The final extension was at 72°C for 10 min. PCR products were purified using ExoSAP IT (GE Healthcare, Buckinghamshire, UK). PCR products were sequenced with the same primers as the PCR reaction or with internal primers using a BigDye Terminator v1.1 Cycle Sequencing Kit (Life Technologies). The sequences of the internal primers were: TIM-S470, CTA AGG AGC CCA GCT TTC GG; TIM-AS658, TGA CCA GCA GTA GGA TCC GTT C; TIM S1902, GGA CTC CGT GGT TCC CTT TG; TIM-AS2060, ACA TCT CCT TCA GGC CAC ACC; TIM-S 2638, CCC GAA GAA GAG GCT CAT C; TIM-AS2800, CCT TGA CAC TGT CAG CCA GTC C; TIM-S3295, CCA TTG GTG CCA CTC ACA GAG; and TIM-AS3558, TCG GTG CTC TTT ACA GTG CTC C. Samples were electrophoresed on an ABI Prism 310 Genetic Analyzer (Life Technologies) and analyzed using Sequencing Analysis Software Version 5.1 (Life Technologies).
Western blot analysis
Western blot analysis was performed as described previously using whole cell lysates. Primary antibodies used were rabbit polyclonal anti-actin (Sigma–Aldrich), rabbit polyclonal anti-cleaved caspase3 (Cell Signaling Technology, Boston, MA, USA), and rabbit monoclonal anti-timeless (Abcam, Cambridge, MA, USA). Actin protein levels were measured as a control for equality of protein loading. Anti-rabbit antibody (GE Healthcare) was used at 1:2000 dilution as a secondary antibody.
Surgically resected lung cancer samples were obtained from patients at Nagoya University Hospital. Before tissue samples were collected, ethical approval and fully informed written consent were obtained from all patients (ethical approval number: 285-4).
Tissue sections (4-μm thick) were cut, deparaffinized in xylene, rehydrated in graded alcohols, and blocked with 5% skim milk for 10 min. For the first set of 30 paired normal and tumor tissues, BenchMark XT (Ventana Medical Systems, Tucson, AZ, USA) was used. The antigens were retrieved by heating 60 min at 100°C in EDTA (pH 8.5). Sections were incubated with anti-timeless antibody (Abcam) at 37°C for 32 min or anti-Ki-67 antibody (Roche, Tokyo, Japan) at 37°C for 16 min, and then biotinylated using a secondary antibody at 37°C for 8 min and streptavidin-peroxidase conjugate at 37°C for 8 min. For the second set of tumor tissues that were consecutively obtained from 88 patients, immunohistochemistry (IHC) was performed manually. The antigens were retrieved by heating for 60 min at 98°C in citrate buffer (pH 6). Sections were incubated with anti-timeless antibody at room temperature for 60 min, and then biotinylated using a secondary antibody at room temperature for 30 min and streptavidin-peroxidase conjugate at room temperature for 30 min. Diaminobenzidine (DAB) substrate was used for color development. Cells with strongly stained nuclei were defined as positive. For the first set of 30 paired tissues, positive cells were divided into three categories: 1+, 2+ and 3+, indicating that <33%, 33–66% and >66%, respectively, of tumor cells were positive. For the second set of 88 tumor tissues, High and Low indicate that ≥50% and <50%, respectively, of tumor cells were positive.
Transfection of short interfering RNA
H157 and H460 cells (4 × 105) were plated in 6-well plates. The following day, cells were transiently transfected with either 10 nM predesigned short interfering RNA (siRNA) (Stealth Select RNAi, Life Technologies) targeting TIM or control siRNA (Life Technologies) using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer's protocol. After 48 h, the transfected cells were harvested for further analyses or plated for cell growth assays.
Cell growth assays
A colorimetric proliferation assay was performed using a WST-1 assay kit (Roche) according to the manufacturer's instructions. Liquid and soft agar colony formation assays were done as described previously.
Drug sensitivity assay
H157 and H460 cells were transfected with TIM RNAi or control oligos. Forty-eight hours after transfection, cells were seeded in 96-well plates at a density of 2 × 104 cells/mL (50 μL/well) and incubated for 24 h. Then the cells were treated with various doses of doxorubicin (Wako, Osaka, Japan) or cisplatin (Sigma–Aldrich) for 5 days, and cell viability was measured using a WST-1 assay.
Stat View version 5.0 (SAS Institute, Cary, NC, USA) was used for all statistical analyses in this study. The Mann–Whitney U-test was used for analyzing differences between two groups. Overall survival (OS; OS event, death resulting from any cause) were measured from the date of surgery and estimated using the Kaplan–Meier method. Differences were assessed using the log-rank test. Pearson's χ2-test and Fisher's exact test were used to evaluate the independency between TIM expression and disease stage or tumor histology, respectively.
TIMELESS is overexpressed in human lung cancers
We examined TIM mRNA levels using microarray expression analysis in a large panel of non-small cell lung cancer (NSCLC; n = 116), small cell lung cancer (SCLC; n =29) and normal lung controls (normal human lung cultures and immortalized normal human bronchial epithelial cell lines; n = 59; Fig. 1A). TIM expression was elevated 3.7-fold (Mann–Whitney U-test, P <0.001) in NSCLC cell lines compared to normal lung controls. SCLC expressed TIM more abundantly than NSCLC 2.2-fold (Mann–Whitney U-test, P <0.001).
To evaluate TIM expression at the protein level, we performed western blot analysis for TIM in 18 NSCLC cell lines and 4 SCLC cell lines. All 22 cell lines expressed TIM, and 20 cell lines (91%) expressed TIM at higher levels than the normal control line HBEC4 (Fig. 1B). TIM expression levels of SCLC were higher than those of NSCLC. We obtained tumor specimens and matched normal lung tissues from 30 patients with lung cancer who underwent surgery and performed IHC of TIM on them (Table 1, Fig. 2). All the lung cancer specimens studied were positively stained, but all the matched normal lung tissues were negative for TIM staining. These results demonstrated that TIM was overexpressed in lung cancer.
Table 1. Clinical features of 30 patients with lung cancer
1+, 2+ and 3+ indicate that <33%, 33–66% and >66%, respectively, of tumor cells were positively stained.
Cells positively stained for TIM were distributed mainly in the invasive front of the tumor in several specimens (Fig. 3, Pt6, Pt13, Pt16, upper panels). We performed IHC on serial sections for TIM and Ki-67, an established marker for cellular proliferation. A correspondence was observed between the distributions of cells stained for TIM and Ki-67 (Fig. 3). This suggests the possible role of TIM as a proliferation marker.
TIMELESS expression correlates with poor overall survival in non-small cell lung cancer
Immunohistochemistry of TIM was performed on surgically resected NSCLC tissues from 88 consecutive patients. The operations were performed from January 2004 to April 2005, and the median follow-up period was 84 months. Patients whose tumor expressed higher levels of TIM protein had significantly shorter overall survival (P = 0.013; Fig. 4). Five-year survival rates for high TIM and low TIM expression groups were 79.2% and 50.2%, respectively. TIM expression did not correlate with disease stage or histological subtype (P = 0.87, P = 0.75, respectively).
No TIMELESS mutation was found in lung cancer cell lines
We searched for a mutation in TIM by direct sequencing. We analyzed the whole coding sequence of TIM. A total of 19 NSCLC cell lines (H820, H1975, HCC44, HCC2279, H838, PC9, H3255, HCC4011, HCC2935, A549, H1650, HCC4006, HCC827, H1666, H358, H1299, H1155, H460 and H157) and 4 SCLC cell lines (H146, H526, H82 and H740) were analyzed, but no mutation of TIM was found in cDNA from these cell lines (data not shown).
TIMELESS knockdown suppresses growth of lung cancer cells and induces apoptosis
To evaluate the role of TIM expression in the pathogenesis of lung cancer cells, a transient knockdown of TIM by RNAi was done in the H157 and H460 cell lines (Fig. 5A). To minimize the possibility of off-target effects, we used three non-overlapping synthesized oligos targeting TIM. To examine the effect of TIM knockdown on cellular proliferation, we performed WST-1 colorimetric assays and found that the TIM knockdown suppressed proliferation to 7–35% in H157 cells and 23–62% in H460 cells of that of the controls (Fig. 5B). The effect of TIM knockdown on clonal growth was measured by liquid colony formation assay. TIM knockdown suppressed colony formation to 3–5% in H157 cells and 6–16% in H460 cells (Fig. 5C). Next, to evaluate the effects of the TIM knockdown on anchorage-independent growth, we carried out a soft agar colony formation assay. TIM knockdown suppressed growth in soft agar to 7–15% in H157 cells and 6–21% in H460 cells (Fig. 5D). Western blot analysis revealed increased levels of cleaved caspase3 after TIM knockdown, suggesting that apoptosis was involved in growth inhibition (Fig. 5E).
Sensitivity to doxorubicin and cisplatin is increased by TIM knockdown in H157 cells but not in H460 cells
Yang et al. () show that TIM knockdown leads to chemosensitization of HCT116 colon cancer cells to doxorubicin. Therefore, we decided to explore the sensitivity of H157 and H460 cells to doxorubicin as well as cisplatin, which is one of the most widely used chemotherapeutic drugs for the treatment of NSCLC, following TIM knockdown. As shown in Figure 6A and Table 2, TIM RNAi exhibited increased sensitivity to doxorubicin and cisplatin in H157, which is consistent with Yang et al. By contrast, TIM RNAi did not increase sensitivity to these drugs in H460 (Fig. 6B and Table 2). These results suggest that TIM knockdown may exert different effects on the sensitivity to cytotoxic drugs in lung cancer, possibly depending on the cellular context.
Table 2. IC50 for doxorubicin and cisplatin after TIM knockdown
Doxorubicin (IC50 nM)
Cisplatin (IC50 μM)
In the present study, we found that TIM was overexpressed in a panel of human lung cancer cell lines as well as clinical lung cancer specimens compared to normal lung controls. Fu et al., using public databases, reported the overexpression of TIM in breast tumor tissue compared to adjacent normal tissue. The authors also found TIM promoter hypomethylation in peripheral blood samples from stage II, III and IV breast cancer patients. Although they did not analyze the methylation status of the corresponding tumor tissue, further investigation of the association between TIM promoter methylation status and the expression level of TIM in lung cancer may be warranted because TIM hypomethylation is one possible molecular mechanism accounting for TIM overexpression in lung cancer cells.
In some cases, the patterns of IHC staining for TIM and Ki-67 showed similarities, implying a possible role for TIM as a marker of cellular proliferation. Conversely, Xiao et al. find no significant association between the patterns of proliferating cell nuclear antigen staining and TIM on IHC analysis of serial sections of mouse embryonic lungs. This might reflect a possible difference between tissues during morphogenesis and developed tissues. However, this point needs further study for clarification.
Recurrence of NSCLC is observed in 30–75% of patients who have undergone complete resection, depending on pathologic stage. TIM might help stratify patients for appropriate adjuvant therapy. Our results were based on retrospective data and we observed only 29 deaths in the follow-up period. Because of the relatively small number of observed deaths, we did not perform multivariate analysis. To verify the usefulness of TIM expression as a prognostic factor, the present results need to be confirmed by an adequately designed prospective study with an appropriate multivariate analysis taking into account the classical well-defined prognostic factors for survival in lung cancer patients. While we are writing this manuscript, Schepeler et al. reported that abundant TIM expression in non-muscle-invasive bladder cancer correlated with risk of progression to muscle-invasive disease. This result is in line with our findings and supports our notion that TIM could be used as a prognostic marker.
Sjöblom et al. found mutations of TIM in two breast cancer xenografts of 35 breast cancer cell lines or xenografts during a comprehensive mutation search. However, we did not detect TIM mutation in the 23 lung cancer cell lines examined. Our provisional results suggest that TIM mutations might not contribute to the development of lung cancer. Taking into consideration the relatively small number of cell lines used in our study, mutation analysis for TIM in a larger panel of lung cancer samples might be necessary. An SNP in the TIM promoter was associated with breast cancer risk among 441 breast cancer cases and 479 cancer-free controls. It would be interesting to examine whether such an association is also seen in lung cancer cases.
Silencing of TIM suppressed cell proliferation and clonogenic growth and induced apoptosis in lung cancer cell lines. Previous published studies demonstrate the critical role of TIM in DNA replication and intra-S checkpoint. From these findings, we speculated that the growth-suppressive effect of TIM knockdown may occur through inhibition of DNA replication, and, therefore, we evaluated DNA synthesis by BrdU incorporation analysis. However, we did not see significant differences in BrdU incorporation between cells treated with TIM knockdown oligos and control cells (data not shown), implying that the growth inhibitory effects of TIM knockdown may not be attributable primarily to inhibition of DNA synthesis. Instead, we assume that the apoptosis induced by TIM knockdown may mainly suppress growth. We speculate that TIM knockdown-induced apoptosis might result from an impaired intra-S checkpoint that leads to apoptosis. Replication protein A (RPA), a single-stranded DNA binding protein, might be involved in this process because RPA was shown to interact with DNA damaged by cisplatin, and Gotter et al. demonstrate that TIM-Tipin complex binds directly to RPA.
We observed different effects of TIM knockdown on drug sensitivity between H157 and H460 cells. TIM knockdown enhanced the sensitivity to doxorubicin and cisplatin in H157 cells but not in H460 cells. Cellular capacity for DNA damage response is an important determinant of drug sensitivity in cancer cells. H157 and H460 cells differ in regards to the status of p53 protein, which plays a pivotal role in DNA damage response; H157 has a p53 nonsense mutation, resulting in non-functional p53 protein, while H460 has the wild-type p53 gene. Therefore, it is possible that the observed difference in the effects of TIM knockdown between these two cell lines was due to their different p53 statuses.
In conclusion, TIM is overexpressed in lung cancer and its expression is associated with poor patient survival. Moreover, TIM knockdown inhibited tumor growth, and resulted in apoptosis in cultured lung cancer cells. These results suggest that TIM is a promising diagnostic and prognostic marker and an attractive therapeutic target for lung cancer.
This work was supported by a Grant-in-Aid for Scientific Research (C) 23591145 (to M. Sato), a Grant-in-Aid for Scientific Research (C) 23591144 (to M. Kondo), and a Grant-in-Aid for Scientific Research (B) 21390257 (to Y. Hasegawa) from the Japan Society for the Promotion of Science and Global COE Program at Nagoya University Graduate School of Medicine, which is funded by Japan's Ministry of Education, Culture, Sports, Science and Technology.
The authors have no conflict of interest to declare.