TIGAR promotes malignant proliferation of NSCLC by modulating deoxynucleotide anabolism via a YBX1-RRM2B axis

doi:10.21203/rs.3.rs-2845674/v1

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TIGAR promotes malignant proliferation of NSCLC by modulating deoxynucleotide anabolism via a YBX1-RRM2B axis

https://doi.org/10.21203/rs.3.rs-2845674/v1

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Background:Lung cancer is one of the most common causes of cancer-related death worldwide due to its rapid growth and metastasis. TIGAR plays a role in promoting survival in various cancer cells, but the molecular mechanisms on metabolic reprogram in tumor cells are not fully understood.

Methods: TIGAR expression was detected in lung cancer by western blot and immunohistochemistry. A shRNA interference system was used to knockdown the TIGAR in NSCLC cell lines to delineate its role in NSCLC tumor proliferation using in vitro functional assays and in vivo mouse models. Finally, transcriptomics and metabolomics were used to identify the mechanism of TIGAR.

Results: The expression of TIGAR in tumor tissues of NSCLC patients was significantly higher than that of adjacent tissues, which was associated with poor prognosis of NSCLC. The proliferation of NSCLC cells in vitro and the growth of xenografted tumors in vivo were significantly inhibited by TIGAR knockdown. With a combination of transcriptomics and metabolomics, we found that TIGAR maintained intracellular deoxyribonucleotide levels by regulating the expression of the ribonucleotide reductase (RNR) subunit RRM2B, a protein involved in deoxynucleotide synthesis. Further studies with LC-MS and co-immunoprecipitation revealed that TIGAR interacted with the transcription factor YBX1, participated in its phosphorylation and nuclear translocation to induce the expression of its downstream gene RRM2B. In addition, overexpression of RRM2B or exogenous supplementation of dNTPs effectively rescued the restriction of cell proliferation and DNA repair caused by TIGAR knockdown.

Conclusion: The present studies revealed a novel mechanism of TIGAR in promoting the proliferation and DNA repair of NSCLC cells through maintaining the intracellular deoxynucleotide level via the YBX1-RRM2B axis.

TIGAR

NSCLC

YBX1

RRM2B

dNTP

With the increasing incidence and mortality of lung cancer, it has become a major public health problem and a huge social burden worldwide [1]. Due to its major characteristics of rapid proliferation without restraint, tumor cells have a much higher demand for raw materials for DNA synthesis than normal cells. Meanwhile, frequent DNA damage occurs in tumor cells, thus a large amount of deoxyribonucleotide is needed in a short period of time to meet the demand for DNA repair [2]. Therefore, strategies that affect intracellular deoxyribonucleotide levels may be an effective strategy to inhibit tumorigenesis and progression.

As a Tp53-Induced Glycolysis and Apoptosis Regulator [3], TIGAR has been found to not only maintain the balance of energy metabolism [4], regulate autophagy [5] and stem cell differentiation [6], but also promote cell survival [7]. In multiple cancers, TIGAR has been implicated in cell proliferation [8], invasion [8], tumor resistance [9], and DNA damage and repair [10]. Mechanistically, the role of ROS regulated by TIGAR through the promotion of NADPH production has been thoroughly investigated. For example, in a pancreatic ductal adenocarcinoma model, TIGAR modulated the transition between tumor proliferation and metastasis through dynamic ROS control [8]. Nuclear TIGAR has also been found to regulate tumor resistance through redox rebalance by interacting directly with the antioxidant regulator NRF2 [9]. Our previous studies have shown that TIGAR regulated DNA damage and repair in cancer cells through the pentose phosphate pathway (PPP) and CDK5-ATM pathway [10]. However, as a metabolic enzyme of glycolytic pathway, the mechanisms of TIGAR's effects on the metabolic reprogramming of tumor cells have not been fully elucidated.

In this study, we focused on the effects of TIGAR on tumor cell proliferation and DNA repair in NSCLC, and found that TIGAR could interact with the transcription factor YBX1, promote its nuclear translocation, and activate the transcriptional expression of its downstream target gene RRM2B to maintain intracellular deoxyribonucleotide levels.

Patients and clinical samples

The cancer tissues and adjacent tissues of NSCLC patients used in this study were all from the First Affiliated Hospital of Soochow University (Suzhou, China). Fresh samples were obtained from patients undergoing pneumonectomy in the First Affiliated Hospital of Soochow University for Western blot assay, and paraformaldehyde fixed samples were used for IHC and immunohistochemistry. All experimental procedures were approved by the Medical Ethics Committee of the First Affiliated Hospital of Soochow University.

Subcutaneous tumor model

Nude mice were purchased from the Experimental Animal Center of Soochow University, and all experimental animals used in this study were authorized by the Animal Ethics Committee of Soochow University. A subcutaneous tumor model was established by injecting 1×10⁷ A549 cells with or without TIGAR knockdown (n = 6 per group) into the axilla of BALB/C male nude mice (4–5 weeks). The mice are maintained under SPF (Specific Pathogen Free) conditions with free access food and water. The tumor size was measured every 4 days and calculated with the formula: volume = (length × width²)/2.

Cell culture

A549, PC9, NCI-H1299 and HEK293T cells were purchased from ATCC. A549 and HEK293T cells were cultured in DMEM (Gibco BRL, Gaithersburg, MD, United States) supplemented with 10%FBS and 100 µg/ml antibiotics (penicillin and streptomycin). PC9 and NCI-H1299 cells were cultured in RPMI 1640 (Gibco BRL) supplemented with 10%FBS and 100 µg/ml antibiotics (penicillin and streptomycin). The cell incubator controls the cell culture environment at 37 ℃ temperature, 70–80% humidity, and 5% CO2.

Construction of cell lines

In this study, TIGAR was knocked down by infection with shRNA virus particles. The TIGAR-shRNA virus was purchased from Genepharma (Shanghai, China) and the sequence (5 'to 3') was GATTAGCAGCCAGTGTCTTAG. According to the manufactuerer’s instructions, viruses (LV-NC and sh-TIGAR) and polybrane were mixed in proportion to infect cells. The green fluorescence was observed for 48 h to reflect the infection of viruses. After 72 h, puromycin (2 µg/ml) was used for screening. The medium used for subsequent cells should be supplemented with puromycin to ensure stable TIGAR knockdown.

Cell viability

A549 cells from TIGAR knockdown and the controls were irradiated (X-ray irradiator X-RAD320IX, irradiation dose rate of 1 Gy/min), and the irradiated cells were returned to the incubator for further culture. Cell viability was detected with Cell Counting Kit-8 (CCK-8) (Dojindo, Japan) after 48 h and 72 h, respectively. CCK-8 and medium were prepared into a working solution at a ratio of 1:10 and incubated with the cells for 1–2 h. Then the absorbance was detected at 450 nm using a multiscan microplate reader (Tecan, Switzerland) for statistical analysis.

Cell proliferation

A549 and PC9 cells with stably TIGAR knockdown and corresponding controls were counted and planted in 24-well plates at the density of 1×10⁴ cells per well. Cells in 24-well plates were counted daily for one week, and then cell proliferation curves were fitted.

Colony formation assay

A549 cells and PC9 cells with stably TIGAR knockdown and corresponding controls were counted and planted in 6-well plates with 500 cells per well. Cells were incubated in an incubator for two weeks to determine the size of the clones and then stained. The cells were washed twice with 1×PBS, and then fixed with an appropriate amount of 4% paraformaldehyde for 20 min. The cells were stained with 0.1% crystal violet (Beyotime Biotechnology, China) for 2 h at room temperature. After washing the residual staining solution with 1×PBS, the clones were photographed and counted.

EdU incorporation assay

A549 cells and PC9 cells with stably TIGAR knockdown and corresponding controls were counted and planted in 24-well plates. EdU incorporation efficiency of cells was measured using BeyoClick™ EdU-647 Cell Proliferation Assay Kit (Beyotime Biotechnology, China) as per the manufacturer’s instructions.

Cell cycle analysis

A549 cells and PC9 cells were counted and then the plates were seeded. When the cell density reached about 90% confluence, the cell culture medium was replaced with DMEM to synchronize the cell cycle. After 24 h, the cells were obtained and fixed with precooled 70% ethanol, and the cells were fixed in a refrigerator at 4 ℃ for 24 h. After fixation, the cells were stained with diluted propidium iodide (Beyotime Biotechnology, China), incubated at room temperature for 30 min in the dark, and the cell cycle distribution was analyzed with flow cytometry.

Protein sample preparation and western blot

After the cells were digested with trypsin, cells were collected in a cell lysis solution (1% sodium deoxycholate, 1% SDS, 1% Triton X-100, 150mM NaCl and 10mM Tris-HCl, pH 7.4) with a protease inhibitor cocktail (Roche, Switzerland) on ice. The cells were then broken by ultrasound in the ice and added to loading buffer, and the samples were boiled in boiling water for 15 minutes to denature the protein for western blot samples. When using tissue samples from lung cancer patients, the cancer tissues and adjacent tissues should be cut into small pieces and placed in tubes and on ice. After weighing, the cell lysate was added according to the ratio, and the cells were broken by ultrasound in the ice until the lysate was cleared. Then the lysate was centrifuged at 13500 rpm at 4 ℃ for 20 min. After protein concentration determination of the supernatant by BCA Protein Assay Kit (Thermo Fisher Scientific, United States), the protein concentration was adjusted to be consistent, and the loading buffer was added respectively. Western blot samples were obtained.

Proteins in the samples were separated by 10% SDS-PAGE gel and transferred to nitrocellulose membrane. The membranes were blocked in 1×TBST containing 10% skimmed milk for 1 h, followed by incubation with primary antibody at 4℃ overnight. After incubating the primary antibody, the membrane was washed three times for 10 min with 1×TBST at room temperature on a shaker, and then incubated with the secondary antibody for 2 h at room temperature. After incubation of the secondary antibodies, the membranes were washed three times for 10 min with 1×TBST at room temperature on a shaker, and then immunoreaction signal was determined using Odyssey Two-Color Infrared Fluorescence Imaging System (LICOR Biosciences, United States). The list of antibodies and article numbers used is shown in Supplementary Table S1.

RNA extraction and quantitative PCR

Cells were lysed and digested with RNAiso Plus according to the instructions, followed by centrifugation and extraction to obtain total RNA. The total RNA was then reverse transcribed into cDNA by Strand cDNA Synthesis Kit (vazyme, China). RT-qPCR was carried out using Taq Pro universal SYBR qPCR Master Mix (vazyme, China) in ABI-7500 quantitative PCR system (Applied Biosystems, United Kingdom) with holding stage of 95 ℃ for 10 min followed by 40 cycles of 95 ℃ for 15 s and 60 ℃ for 1 min. The data were calculated and analyzed by ΔΔCt value. The sequences of PCR primers are shown in Supplementary Table S2.

RNA-sequencing analysis

Total RNA was extracted from A549 cells infected with virus stably TIGAR knockdown. The concentration and quality of total RNA were detected, and the next generation sequencing library was constructed. After passing the quality inspection, the sample double-ended sequencing data were sequenced using I llumina HiSeqTM 2500 sequencer. The differential genes were obtained by Trimmomatic (v 0.36) analysis on the original data, and the two parameters of p < 0.05 and | log2 Fold Change | > 1 were screened to obtain the statistically significant genes for subsequent bioinformatics analysis and GSEA analysis, and the heat map of cluster analysis was drawn for each group.

Untargeted metabolome profiling and analysis

Six samples of A549 cells from TIGAR knockdown and the corresponding controls were taken from each group, and the cell number of each sample was ensured to be above 1×10⁶. Subsequently, the mass spectrum samples were extracted following the manufacturer’s manual, and the metabolites were analyzed with LC-MS/MS (SHIMADZU LC-30AD/Thermo Fisher Scientific QE). The enrichment map of metabolic pathways can be obtained through the analysis function of MBRole pathway, and p < 0.05 was considered to be statistically significant.

Alkaline comet assay

A549 cells with stable TIGAR knockdown and their corresponding controls were pretreated with different doses of dNTP for 24 hours in advance. When the cells were overgrown, ionizing irradiation was performed. Then the comet assay was carried out using Single Cell Gel Electrophoresis Assay Kit (R&D Systems, United States) as per the manufacturer’s instructions. The tail distance of comet was viewed and photographed under red light using a fluorescence microscope (Nikon, Germany). CASP Comet Assay (V1.2.3 beta 2) software was used for analysis and quantitative statistics, in which 50 cells were randomly selected for each sample.

Nucleus-cytoplasm fractionation assay

A549 cells with TIGAR knockdown and its controls were harvested and added with 800 µl NP40 nuclear extraction buffer [11], mixed and placed on ice. The tube was shaken every 5 minutes, and centrifuged at 600 rpm for 15 min at 4 ℃ after 30 minutes. The supernatant was the cytosolic fraction. The supernatant was transferred to a new tube, the loading buffer was added proportionally after ultrasound in ice, and the samples were boiled in boiling water for 10 min to obtain cytoplasmic samples. The precipitate was the nuclear component, and washed with 800 µl of nuclear extraction buffer. The cells were centrifuged at 600 rpm at 4 ℃ for 10 min. After the supernatant was discarded, the precipitate was mixed with 300 µl cell lysate. After ultrasonication on ice, loading buffer was added and the samples were boiled in boiling water for 10 min. The protein expression in the cytoplasm and the nucleus was detected with western blot analysis.

Dual-luciferase reporter assay

A549, PC9 and 293T cells were transfected with RRM2B promoter reporter plasmid for 36 h, and then screened with G418 (2.5 µg/ml) to obtain cell lines stably expressing RRM2B promoter reporter elements. Subsequently, several cell lines were used to knock down TIGAR with sh-TIAGR and LV-NC, and the activities of promoters were detected with Dual-Luciferase Reporter Gene Assay Kit (Beyotime Biotechnology, China) by using Luminoskan™Ascent (Thermo Fisher Scientific, United States) as per the manufacturer’s instructions.

Immunofluorescence

A549 cells seeded in 24-well plates were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 for 10 min. After blocking with 2.5% BSA for 1 h, the cells were incubated with the corresponding primary antibody at 4 ℃ overnight. The primary antibodies were washed with 1×PBS for 3 times, and the secondary antibodies were incubated for 1 h at room temperature in the dark. After washing with 1×PBS for 3 times, the cells were stained with DAPI for 5 min. Finally, the distribution of corresponding proteins within the cell was visualized by using LSM 710 Laser Scanning Confocal Microscope (Carl Zeiss, Germany).

Immunohistochemistry

Cancer tissues and adjacent tissues from NSCLC patients were first fixed with 4% paraformaldehyde for 3 days at 4 ℃ and then dehydrated in 20% and 30% sucrose for 3 days, respectively. After freezing overnight at -80 ℃, 20 µm-thick slices were obtained at -20°C using a CM1950 Freezing Microtome (Leica, Germany) and directly attached to adhesive slides. The surface was washed with 1×PBS, and then the slices were permeabilized with 0.5% Triton X-100 for 3 times. After blocking with 2.5% FBS for 2 h, the slices were incubated with the primary antibody at 4 ℃ overnight. After incubation with the corresponding secondary antibody for 2–3 h, the nuclei were stained with DAPI. Finally, the distribution of corresponding proteins within the tissues was visualized by using LSM 710 Laser Scanning Confocal Microscope (Carl Zeiss, Germany).

Co-immunoprecipitation

After transfection of GFP-TIGAR and Flag-TIGAR in A549 cells for 48 hours, the cells were harvested and added to IP lysis buffer[11]. After ultrasound treatment on ice, a small amount of lysate was taken as Input sample. All remaining lysates were added to Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, United States) previously conjugated with antibodies, and mixed overnight at 4℃. After centrifugation at 3000 rpm at 4 ℃ for 5 min, the supernatant lysate was discarded. The remaining precipitation was washed with lysis buffer for 3 times for 5 min. After discarding the supernatant, the loading buffer was added, followed by boiling in boiling water for 10 min and centrifugation at 5000 rpm for 5 min. The supernatant was then collected as the IP sample. The interaction between TIGAR and YBX1 was detected with western blot analysis.

Chromatin immunoprecipitation (ChIP)

The ChIP experiment was performed using the BeyoChIP™ ChIP Assay Kit (Beyotime Biotechnology, China) according to the manufacturer's instructions. A549 cell lysates were used for the ChIP experiment, and the antibodies and primers are listed in Supplementary Table S1 and Supplementary Table S2, respectively.

Silver staining

Proteins interacting with TIGAR were purified by immunoprecipitation in A549 cells and subsequently separated by SDS-PAGE. Following the silver staining procedure [12], the protein in the gel can be visualized and photographed.

Mass spectrometry (MS) analysis

The silver-stained samples were divided into small pieces according to their molecular weight and destained. Subsequently, the protein is reduced, alkylated and digested according to the in-gel trypsin digestion protocol [13]. The final sample was dissolved in 0.1% formic acid to obtain the MS sample.

Use of standardized datasets

Kaplan-Meier Plotter was used to analyze disease-free survival of TIGAR in lung cancer, Kaplan-Meier plotter [Lung] (kmplot.com). Oncomine was used to analyze the expression of TIGAR copy number in different lung cancer patients, https://www.oncomine.org/. GEPIA2 was used for gene correlation analysis, GEPIA 2 (cancer-pku.cn).

Statistical Analysis

The results of western blotting and colony formation assay were quantified by gray scale and counting analysis using Image J (V 1.8.0, National Institutes of Health). CaspLab Comet Assay (V 1.2.3 beta 2, Krzysztof Końca) was used to analyze the tail distance of single cell gel electrophoresis. Data were analyzed and plotted using Prism (V 8.0, GraphPad Software), and all data were expressed as mean ± SEM. The paired t test was used to compare two sets of data from cancer tissues and adjacent tissues of the same patient. The other two sets of data were compared with the unpaired t test. Data from multiple groups were compared between groups by two-way ANOVA Sidak's Multiple bar test. Correlations were determined by calculating Pearson correlation coefficients. p < 0.05 was considered to be statistically significant.

High expression of TIGAR in NSCLC was associated with malignant proliferation and poor prognosis

To determine the clinical significance of TIGAR in patients with NSCLC, immunohistochemical staining was used to detect TIGAR expression in cancer tissues and adjacent tissues of 35 patients with NSCLC (Fig. 1a, b). The intensity of the immune response to TIGAR was generally higher in most cancer tissues than that in adjacent tissues. In addition, western blot analysis of TIGAR and Ki67 in cancer tissues and adjacent tissues of 10 lung cancer patients (Fig. 1c) showed that the expression of TIGAR in cancer tissues was significantly higher than that in adjacent tissues (Fig. 1d). As an antigen associated with proliferating cells, Ki67 is commonly used to characterize rapidly proliferating tumors. Correlation analysis showed that the expression of TIGAR and Ki67 was significantly correlated in cancer tissues and adjacent tissues, indicating that TIGAR might be related to the malignant proliferation of NSCLC (Fig. 1e). A search of the oncomine database also revealed that TIGAR gene copy numbers were significantly higher in both lung adenocarcinoma and lung squamous cell carcinoma than that in normal lung tissue (Fig. 1f). Moreover, high expression of TIGAR in lung cancer was significantly associated with shorter disease-free survival time (Fig. 1g). Taken together, these results indicated that TIGAR was significantly associated with malignant proliferation and poor prognosis of NSCLC.

TIGAR regulated proliferation and cell cycle of NSCLC cells in vitro

To further determine the role of TIGAR in the proliferation of NSCLC cells, the cell proliferation of A549 and PC9 cells was determined after knocking down TIGAR with shRNA viral particles for one week. As shown in Fig. 2a, the cell proliferation began to significantly decrease on the fifth day after TIGAR knockdown. Cell proliferation was also detected by colony formation and EdU incorporation, and the results showed that TIGAR knockdown in A549 and PC9 cells significantly decreased cell proliferation (Fig. 2c-g). Cell cycle analysis revealed that TIGAR knockdown resulted in cell cycle arrest at s-phase (Fig. 2b). Taken together, these results indicated that TIGAR played an important role in the regulation of tumor cell proliferation.

TIGAR regulated deoxyribonucleotide production in NSCLC cells

To investigate the underlying mechanism of TIGAR in regulating the proliferation of NSCLC cells, we established a stable TIGAR knockdown cell line by infecting A549 cells with virus particles LV-NC and sh-TIGAR, followed by 2 weeks of screening with puromycin (Fig. 3a). The knockdown of TIGAR in the cell line was confirmed by western blot (Fig. 3b). After harvesting cells, 266 differential metabolites were identified by LC-MS untargeted metabolomics analysis, among which 234 metabolites were up-regulated and 318 metabolites were down-regulated (Fig. 3c, Supplementary Table S3). As shown in Fig. 3d, the enrichment of metabolic pathways was plotted by GO and KEGG analysis, with the red line indicating a p-value of 0.01 and the blue line indicating a p-value of 0.05. All the signaling pathways listed are significantly different, with the most significant effects on purine and pyrimidine metabolism. Since the final metabolites of purine and pyrimidine metabolism are several deoxyribonucleotides, metabolomics data were further mined and discovered that the intracellular levels of several deoxyribonucleotides were significantly decreased after TIGAR knockdown compared with controls (Fig. 3e, f).

TIGAR regulated the gene expression in purine metabolism pathway

Furthermore, transcriptomic analysis was performed by reverse transcription of total RNA into cDNA extracted from the TIGAR knockdown stable A549 cell line. Principal component analysis diagram (PCA) showed the similarity degree among the samples of this sequencing, excluding the difference in expression abundance caused by the variation between the samples (Figure S1a). Cluster analysis of the genes screened by sequencing showed that 520 genes were down-regulated and 390 genes were up-regulated after TIGAR knockdown compared with controls (Fig. 4a, Supplementary Table S4). Since the results of metabolomics showed that TIGAR knockdown had the most significant effect on several metabolic pathways, transcriptome data were further used for Gene Set Enrichment Analysis (GSEA) on purine and pyrimidine metabolic pathways (Supplementary Table S5, Table S6). Interestingly, it was found that the genes related to purine metabolism pathway were significantly down-regulated after TIGAR knockdown (Fig. 4b), while the genes related to pyrimidine metabolism pathway were also down-regulated, but did not reach the statistical significant level (Figure S1b).

After being integrated, these results suggested that TIGAR may regulate the expression of genes involved in purine metabolism pathway to maintain intracellular deoxyribonucleotide levels. Therefore, we compared the most significant genes affected in purine metabolic pathway (Figure S1c), and further verified their expression in A549, PC9 and NCI-H1299 cells after TIGAR knockdown. The results showed that TIGAR knockdown decreased several genes in several cell lines, with the most significant effects on RNR subunit RRM2B and mitochondrial thymidine kinase TK2 (Fig. 4c-e).

Subsequently, protein levels were also determined and found that RRM2B was significantly down-regulated after TIGAR knockdown in several cell lines, while RRM1 and RRM2, two other subunits of RNR, were not significantly changed (Fig. 4f, g).

Studies have shown that RNR encoded by RRM2B is a key rate-limiting enzyme in de novo deoxyribonucleotide synthesis pathway in vivo [14]. RNR has a unique catalytic function and is the only catalytic enzyme that can convert ribonucleotides (NDPs) into deoxyribonucleotides (dNDPs) [14]. Moreover, deoxyribonucleotides are substrates for DNA replication, repair and synthesis in vivo. In summary, TIGAR regulated intracellular deoxynucleotide levels through RRM2B to maintain normal DNA replication and tumor proliferation.

TIGAR interacted with the transcription factor YBX1 and participated in its nuclear translocation to activate the transcriptional expression of RRM2B

To further determine the relationship between TIGAR and RRM2B, the correlation between TIGAR and RRM2B gene expression in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) databases was analyzed by the correlation analysis module of GEPIA2 (Gene Expression Profiling Interactive Analysis), and a strong positive correlation was found (Figure S2a, b). Moreover, the determination of TIGAR and RRM2B proteins in cancer tissues and adjacent tissues of lung cancer patients also found that they had a significant positive correlation (Figure S2c, d). The co-localization of TIGAR and RRM2B with high expression in lung cancer tissues was also confirmed with immunofluorescence (Figure S2e).

Since TIGAR knockdown reduced the mRNA level of RRM2B, we speculated that TIGAR could regulate the transcription of RRM2B. Therefore, luciferase reporter gene assay of RRM2B promoter was performed and found that RRM2B gene promoter activity was effectively reduced by TIGAR knockdown in A549, PC9 and HEK293T cells (Figure S3a-c).

However, existing studies have not shown that TIGAR acts as a transcription factor, so the hypothesis of its interaction with a transcription factor has been raised. To explore the potential, we isolated the TIGAR-interacting proteins in A549 cells with MS analysis (Figure S4a). The silver staining indicated that TIGAR was successfully isolated by immunoprecipitation under our experimental conditions and enriched to its interacting proteins (Figure S4b). After the purification of the TIGAR-interacting proteins, we performed MS analysis and identification proteins of two biological replicates by high-throughput LC-MS/MS. We used the label-free quantification (LFQ) incorporated in the MaxQuant software suite to quantify the relative protein abundance, and identified 307 interacting-proteins in total (Supplementary Table S7). The relative abundance (Log₂(LFQ_Ctrl/LFQ_Expt)) was used to construct a scatter plot, which was set to > 1 in both samples to indicate significant differences (Fig. 5a). Finally, 149 proteins were considered as potential TIGAR-interacting proteins, including the transcription factor YBX1.

Next, we used biochemical approaches to test whether the transcription factor YBX1 indeed interacts with TIGAR. To do so, we used co-immunoprecipitation of endogenous and exogenous proteins. Forward and reverse immunoprecipitation and subsequent immunoblotting of TIGAR and YBX1 in A549 cells were performed and found that they did interact with each other (Fig. 5b, Figure S4c). To further confirm their interaction, we performed immunoprecipitation for immunoblotting analysis by overexpressing exogenous tagged proteins. On the one hand, GFP-TIGAR was transfected into A549 cells and anti-YBX1 antibody magnetic beads were used for immunoprecipitation (Fig. 5c). On the other hand, after transfection of FLAG-TIGAR, anti-FLAG antibody magnetic beads were used for immunoprecipitation (Figure S4d). The forward and reverse immunoprecipitation and immunoblotting experiments clearly demonstrated that TIGAR interacted with the transcription factor YBX1. Meanwhile, immunofluorescent staining also showed the colocalization of endogenous TIGAR (green) and YBX1 (Red) in A549 cells (Fig. 5d).

Furthermore, considering that YBX1 activity was mainly determined by its phosphorylation status and the level of nuclear translocation [15, 16], participation of TIGAR in the phosphorylation and nuclear localization of YBX1 was examined. We examined the expression of YBX1 and its phosphorylated form at ser102 in several lung cancer cells after TIGAR knockdown. The results showed that TIGAR knockdown effectively reduced the expression of p-YBX1 without affecting its total protein in A549, PC9, and NCI-H1299 (Fig. 5e). Intriguingly, nucleus-cytoplasm fractionation assay showed that YBX1 was present in both nuclear and cytoplasmic compartments of A549 cells, and TIGAR knockdown significantly decreased YBX1 levels in the nucleus (Fig. 5f). Meanwhile, immunofluorescence staining confirmed that the nuclear staining of YBX1 decreased after TIGAR knockdown (Figure S4f).

Since the transcription factor YBX1 has been reported to be involved in the transcriptional expression of many genes in tumor cells, including TK1, RRM2, TYMS and so on [17]. However, whether the transcriptional expression of RRM2B is also regulated by YBX1 is still unknown. Therefore, we further confirmed that YBX1 can bind to the promoter region of RRM2B and promote its transcriptional activation by chromatin immunoprecipitation (Fig. 5g, h).

These results indicated that TIGAR interacted with YBX1 and participated in the regulation of its biological function and transcription of the downstream gene RRM2B.

The inhibitory effect of TIGAR on NSCLC cell proliferation was dependent on levels of RRM2B and intracellular deoxynucleotide

The previous results confirmed that TIGAR knockdown inhibited tumor cell proliferation and down-regulated the transcriptional expression of RRM2B. Therefore, we hypothesized that TIGAR regulated proliferation of NSCLC cells through limiting availability of deoxynucleotide. We then constructed a plasmid containing the RRM2B transcript sequence (EGFP-RRM2B) and transfected it into TIGAR knockdown A549 and PC9 cells to ensure that RRM2B had been overexpressed (Figure S5a). The proliferation ability of tumor cells was detected by fluorescence observation and flow cytometry analysis after EdU incorporation (Fig. 6a, Figure S5b), combined with colony formation assay (Fig. 6b, Figure S5c). These data showed that overexpression of RRM2B could effectively antagonize the effects of TIGAR knockdown on the proliferation of A549 and PC9 cells.

Since RRM2B functions as a small subunit of RNR through the production of deoxyribonucleotide and TIGAR knockdown reduced the intracellular level of deoxyribonucleotide, we assumed that exogenous supplementation of deoxyribonucleotide could rescued the proliferation of NSCLC cells. Subsequently, A549 and PC9 cells with TIGAR knockdown were supplemented with 50 and 100 µM dNTP for 24 h. The proliferation ability of tumor cells was detected by fluorescence observation and flow cytometry analysis after EdU incorporation (Fig. 6c, Figure S6a), combined with colony formation assay (Fig. 6d, Figure S6b). Similarly, these data showed that dNTP supplementation effectively rescued the proliferation inhibition caused by TIGAR knockdown in A549 and PC9 cells.

The aggravation of DNA damage caused by TIGAR knockdown was rescued by dNTP

Deoxyribonucleotides in cells provide the raw materials for DNA synthesis. Tumor cells with rapid proliferation demand a large supply for deoxyribonucleotide to meet the need for DNA replication and repair. Based on the above results, we considered whether DNA repair was also impaired after TIGAR knockdown. We selected a DNA damage model of lung cancer cells induced by moderate ionizing radiation (IR) to investigate the role of TIGAR in DNA repair after DNA damage. We first investigated the effects of different doses of IR (0, 2.5, 5, 10 Gy) on cell survival of A549 cells with TIGAR knockdown. The results showed that the survival of A549 cells was significantly inhibited after 48 h and 72 h of IR in a dose-dependent manner, and the damage was worsened after TIGAR knockdown (Figure S7a, b).

As a classic marker of DNA damage, single-cell gel electrophoresis is used to directly reflect the damage of single and double strand DNA breaks [18]. Studies have shown that the single and double strands of DNA were rapidly broken after irradiation with 10 Gy IR [19]. However, due to the cell repair mechanisms, most of the damage can be repaired within 2 h, and the rest of the damage can be completely repaired within 24 h. The present results showed that the length of comet tail gradually decreased with time after 10 Gy IR, while TIGAR knockdown increased the length of tail distance, prolong the cell repair time, and increase DNA damage (Fig. 7a, b). Meanwhile, the signature markers γH2AX and p-ATR in DNA damage signaling pathway were also significantly upregulated during DNA damage and gradually decreased with time, while TIGAR knockdown significantly increased the expression of γH2AX and p-ATR, indicating that the damage was aggravated (Fig. 7c, d).

Since dNTP can maintain normal DNA replication to maintain tumor cell proliferation, we predicted that dNTP might also protecte DNA repair. The single cell gel electrophoresis and DNA damage markers were used to detect DNA damage. The results showed that exogenous dNTP reduced the DNA damage caused by IR in a dose-dependent manner, reduced the comet tail distance (Fig. 8a, b), inhibited the up-regulation of γH2AX and p-ATR (Fig. 8c, d), and reduced the fluorescence intensity of γH2AX (Fig. 8e). In particular, it had a significant protective effect on TIGAR knockdown cells.

Taken together, these results suggested that TIGAR regulated the repair process of DNA damage by maintaining the production of deoxyribonucleotide in cells.

TIGAR deficiency inhibited the growth of lung cancer xenografts by reducing the expression of RRM2B in vivo

To investigate the effect of TIGAR on tumor growth in vivo, A549 cells were infected with LV-NC and sh-TIGAR to construct a stable TIGAR knockdown cells, and then subcutaneously xenografted tumor model was established in nude mice. Knockdown of TIGAR significantly inhibited the growth of xenograft tumors as compared with the controls (Figure S8a), with a significant difference in the growth rate of tumors beginning on day 36 (Figure S8c) and a significant reduction in the size and weight of tumors (Figure S8b, d). Immunofluorescence staining further showed that knockdown of TIGAR reduced the number of proliferating cells in the xenografted tumors as compared with the controls (Figure S8e).

Subsequently, we further examined whether RRM2B mediates the effect of TIGAR on xenograft tumor growth in vivo with overexpressing RRM2B in transplanted A549 cells. Overexpression of RRM2B in xenograft tumor cells significantly increased tumor growth rate, size and weight in TIGAR knockdown cells (Fig. 9a-c), which was consistent with the role of RRM2B in mediating TIGAR’s role NSCLC cell proliferation in vitro. These results suggest that TIGAR regulated tumor proliferation and growth through RRM2B in vivo.

TIGAR plays an important role in regulating glycolytic flux and pentose phosphate pathway flux to generation of NADPH and ribose [20]. Many previous studies have reported a pro-survival role of TIGAR in tumor cells [7, 21]. The novel findings of the present study are: TIGAR interacted with the transcription factor YBX1, increased its phosphorylation and the nuclear localization to promote the transcriptional expression of its downstream gene RRM2B, a subunit of RNR involved in production of deoxynucleotides; knockdown of TIGAR resulted in reduction in deoxyribonucleotide, impaired DNA synthesis and repair and proliferation of NSCLC cells, the effects were rescued by reconstituting RRM2B and supplementing dNTP. This study thus revealed a novel mechanism of TIGAR in regulating tumor proliferation and survival (Fig. 9d).

TIGAR was originally discovered as a Tp53-induced glycolysis and apoptosis regulator to reverse glycolysis by catalyzing the hydrolysis of fructose 2, 6-diphosphate to fructose 6-phosphate, which in turn promotes the participation of glucose 6-phosphate in the pentose phosphate pathway and increases the production of NADPH and ribose [22]. Recent studies have found that TIGAR regulated various metabolic reprogramming processes in tumor cells [23, 24]. It had been found that the high expression of TIGAR in breast cancer promoted the catabolism of lactic acid and glutamine in mitochondria by upregulating lactate dehydrogenase B (LDH B) and glutaminase 1 (GLS1) [23]. Similarly, it had been found in esophageal squamous cell carcinoma that high expression of TIGAR induced phosphorylation and activation of AMPK, which in turn promoted the expression of GLS and induced catabolic metabolism of glutamine to enter the citric acid cycle and other metabolic pathways [24]. Our study demonstrated for the first time that TIGAR was involved in the intracellular deoxyribonucleotide synthesis in NSCLC cells, and identified with MS that TIGAR interacted with the transcription factor YBX1 to promote its function.

The transcription factor YBX1 has been shown to play an important role in tumorigenesis and progression [25]. In NSCLC, YBX1 has been found to regulate tumor growth [26], proliferation [27], metastasis [26, 28], invasion [29] and cell stemness [28]. YBX1 is a multifunctional protein involved in gene transcription [30, 31], mRNA splicing [32] and translation [33] by shuttling between the nucleus and the cytoplasm, and its active form is phosphorylated at ser102 [15]. In our results, although TIGAR had no effect on the overall expression level of YBX1, it was striking that the phosphorylation and nuclear entry of YBX1 were significantly reduced after TIGAR knockdown. Mechanistically, it is still unclear how TIGAR induces the phosphorylation and the nuclear localization of YBX1. Interestingly, TIGAR has been shown to bind AKT and promote its phosphorylation activation [21]. More importantly, it was previously proposed that activated Akt could bind to and phosphorylate domain at ser102 of YBX1, thereby facilitating its nuclear translocation [34]. Therefore, TIGAR may interact with AKT and YBX1 by forming a ternary complex to induce YBX1 phosphorylation and function. In addition, our previous study showed that TIGAR increased its nuclear localization under stress conditions [10]. However, how TIGAR promotes nuclear localization of YBX1 remains unclear.

Accurate DNA replication and repair are essential for normal growth and tumor survival in all organisms, and an adequate and balanced pool of dNTPs is a fundamental requirement for genome stability. Notably, RNR is a key rate-limiting enzyme in the de novo deoxyribonucleoside pathway in vivo and is of great significance because of its unique catalytic function to reduce ribonucleotides to their corresponding deoxyribonucleotides, which are substrates for DNA synthesis and replication [14]. Mammalian RNR consists of two homodimeric subunits, α and β subunits, which combine to form holoenzymes [14]. The α subunit is encoded by the RRM1 gene, and the β subunit comes in two different forms, one encoded by RRM2 and the other by RRM2B. The subunit encoded by RRM2B is regulated by Tp53 and is therefore sometimes referred to as the Tp53-inducible ribonucleotide reductase subunit, p53R2 [35]. In our study, TIGAR could regulate the transcriptional expression of RRM2B. Furthermore, YBX1 has also been found to be involved in the transcriptional expression of multiple nucleotide metabolism genes in tumor cells, including TK1, RRM2 and TYMS [17]. Our ChIP results also found that the transcription factor YBX1 bound to the promoter region of RRM2B and activated its transcription. The important mechanism of RRM2B regulated by TIGAR was further determined.

The level of deoxyribonucleotide in cells is essential for cell growth, proliferation and DNA repair. NSCLC cells after TIGAR knockdown in vitro showed that cell proliferation and cell repair following IR damage were significantly reduced. However, we found that overexpression of RRM2B or exogenous supplementation of dNTPs effectively rescued the restriction of cell proliferation and DNA repair induced after TIGAR knockdown. The biological importance of regulatory effect of TIGAR on intracellular dNTP levels via RRM2B was confirmed.

To further verify the importance of TIGAR in tumor growth, the role of RRM2B in mediating TIGAR on tumor growth was further identified in vivo. The present study demonstrated that the growth rate of tumors was significantly reduced, as was the size and weight of tumors, after TIGAR knockdown. This is consistent with the high expression of TIGAR in tumor tissues of patients with NSCLC. In addition, overexpression of RRM2B in xenografted tumor cells reversed the effects of TIGAR knockdown in NSCLC.

Overall, this study found that TIGAR knockdown in vivo and in vitro significantly inhibited the proliferation and DNA repair of NSCLC cells. TIGAR interacted with the transcription factor YBX1, promoted its nuclear translocation and function, induced the transcriptional expression of RRM2B, and maintained the intracellular deoxyribonucleotide levels. As RNR is currently a recognized target for cancer treatment, and RNR inhibitors (gemcitabine [36], clofarabine [37], hydroxyurea [38], 3-AP [39], etc.) have been used in the clinical treatment of multiple cancers. Therefore, we propose that TIGAR-targeted therapy for NSCLC may be an effective therapeutic strategy.

We have identified a novel mechanism by which TIGAR promotes the malignant proliferation of NSCLC cells, revealing its role along the TIGAR-YBX1-RRM2B axis in regulating intracellular deoxynucleotide anabolism and governing the cancer cell growth.

Availability of data and materials

The datasets used and/or analyzed during the current study are available within the manuscript and its supplementary information files.

Ethics statement

Patients and clinical samples were approved by the Medical Ethics Committee of the First Affiliated Hospital of Soochow University. The animal study was reviewed and approved by the Animal Ethics Committee of Soochow University.

Acknowledgements

This work is supported by the Natural Science Foundation of China (No. 81730092 and 81730098)

Author contributions

ZHQ and CM conceptualized, supervised, and acquired funding for the study. ZHQ oversaw the project administration. JF, BLi, YW, LG and QL were engaged in sample collection and compiled research resources. JF performed most RNA-seq, metabolome profiling, functional experiments and experiment data analysis and interpretation. JF, BLiu, SH and JW performed the formal analysis and data visualization. JF and GX performed the mass spectrometry. JF drafted and ZHQ revised and prepared the final manuscript. All authors approved the manuscript.

Conflict of interest

The authors declare no competing interests.

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TIGAR promotes malignant proliferation of NSCLC by modulating deoxynucleotide anabolism via a YBX1-RRM2B axis

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Abstract

Figures

Background

Methods

Patients and clinical samples

Subcutaneous tumor model

Cell culture

Construction of cell lines

Cell viability

Cell proliferation

Colony formation assay

EdU incorporation assay

Cell cycle analysis

Protein sample preparation and western blot

RNA extraction and quantitative PCR

RNA-sequencing analysis

Untargeted metabolome profiling and analysis

Alkaline comet assay

Nucleus-cytoplasm fractionation assay

Dual-luciferase reporter assay

Immunofluorescence

Immunohistochemistry

Co-immunoprecipitation

Chromatin immunoprecipitation (ChIP)

Silver staining

Mass spectrometry (MS) analysis

Use of standardized datasets

Statistical Analysis

Results

High expression of TIGAR in NSCLC was associated with malignant proliferation and poor prognosis

The aggravation of DNA damage caused by TIGAR knockdown was rescued by dNTP

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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