PTS is activated by ATF4 and promotes lung adenocarcinoma development via the Wnt pathway.

Background: The effects and mechanism of 6-pyruvoyl-tetrahydropterin synthase (PTS) on lung adenocarcinoma (LUAD) were studied in LUAD cells and mice with subcutaneously transplanted tumors.
Methods: PTS level in tissues and cells was tested by immunohistochemistry, western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). The impacts of PTS on cell viability, proliferation, apoptosis, invasion, and migration were determined by Cell Counting Kit-8 (CCK-8), colony formation assay, flow cytometry, transwell assay, and wound healing assay, respectively. The Cancer Genome Atlas (TCGA) analysis and dual luciferase assay were conducted to predict and verify the relationship between PTS and activating transcription factor 4 (ATF4). A mouse model was established by subcutaneous injection with cancer cells. Tumor volume was calculated as V = ab2/2. Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to measure cell proliferation and apoptosis in tumors.
Results: PTS was highly expressed in LUAD. Higher PTS level was correlated with late clinical stages and poor survival of patients. Down-regulation of PTS inhibited the viability and proliferation and induced apoptosis of LUAD cells. PTS was activated by ATF4, and up-regulation of ATF4 reversed the inhibitory effect of PTS silencing on LUAD cells. Silencing of PTS inhibited the Wnt pathway. Down-regulation of PTS inhibited tumor growth in mice. Conclusions: PTS was highly expressed in LUAD. PTS was activated by ATF4 and promoted LUAD development via the Wnt pathway. 2022 Translational Lung Cancer Research. All rights reserved.

Introduction

Lung cancer includes mainly two subtypes: non-small cell lung cancer (NSCLC), which accounts for 85% of lung cancers, and small cell lung cancer. Lung adenocarcinoma (LUAD) is the main subtype of NSCLC, accounting for about 60% of all NSCLC cases (1-3). Many potential therapeutic targets and corresponding targeted drugs have been discovered and applied in clinics for the treatment of LUAD, including epidermal growth factor receptor (EGFR), fibroblast growth factor receptor, and insulin-like growth factor (4-6). Nevertheless, there is an urgent need to discover more potential and effective target genes in LUAD to further improve the treatment strategy.

The 6-pyruvoyl-tetrahydropterin synthase (PTS) gene was cloned by Thöny et al. in 1992 and is located at 11q22.3-23.3 (7). At present, research on PTS is mainly focused on its effect on tetrahydrobiopterin deficiency (BH4D), which is caused by a lack of PTS (8). PTS gene levels are relevant to newborn screening, as a lack of PTS can lead (8): (I) a BH4 synthesis disorder, resulting in accumulation of phenylalanine; (II) a lack of L-dopa, epinephrine, 5-hydroxytryptophan, and other physiologically active substances; (III) a decreased nerve cell myelin protein synthesis and immunity. The PTS gene has been rarely studied in cancer. Zhao et al. (9) reported that PTS is up-regulated in early colorectal cancer; however, there has been no prior research on PTS expression in LUAD.

In order to study the upstream mechanism of PTS in lung cancer, we predicted the transcription factors upstream of PTS by bioinformatics analysis, and finally screened 7 transcription factors. Activating transcription factor 4 (ATF4) was selected according to the high prediction score and their correlation with PTS expression in The Cancer Genome Atlas (TCGA)-LUAD. ATF4, which belongs to the cAMP-response element binding (CREB) protein family, is activated by many stimuli, including oxygen deficiency, amino acid deficiency, endoplasmic reticulum stress, oxidative stress, and growth factor regulatory proteins (10,11). The role of ATF4 in lung cancer has been investigated, but its effect remains controversial. Du et al. (12) found that ATF4 may promote lung cancer cell proliferation and invasion, while Zhang et al. (13) reported that ATF4 promotes Noxa-dependent apoptosis in LUAD cells treated with andrographolide. The Wnt gene was first discovered as a proto-oncogene (int) in mice in 1982 and renamed Wnt due to its homology with the Drosophila wingless gene reported by Sharma (14). The Wnt pathway participates in embryonic development and some normal physiological processes, as well as in the induction and regulation of a variety of tumors, including lung cancer (15,16). Here, we focused on the function of PTS in LUAD, hypothesizing that PTS is activated by ATF4 and promotes LUAD progression via the Wnt pathway. We present the following article in accordance with the ARRIVE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-593/rc).

Methods

Cell culture and transfection

Human bronchial cells (16HBE) and lung cancer cells (NCI-H1975, H23, H1299, and A549) were purchased from the American Type Culture Collection (ATCC) and were cultured with RPMI 1640 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Gibco) at 37 ℃ with 5% CO2.

The pc-DNA3.1-PTS vector was constructed by Tsingke Biotechnology Co., Ltd. (Beijing, China) small interfering RNAs (siRNAs) for PTS (si-PTS-1 and si-PTS-2), siRNA-negative control (si-NC) were synthesized by RiboBio (Guangzhou, China). short hairpin RNAs (shRNAs) for PTS (sh-PTS) and shRNA-negative control (sh-NC) were synthesized and purified by. Shanghai Genechem Co., Ltd. (Shanghai, China) H1299 and A549 cells were transfected with si-NC, si-PTS-1, and si-PTS-2. H23 cells were transfected with pc-DNA3.1 vector of PTS. A549 cells were transfected with cDNA clones of ATF4. sh-PTS and sh-NC were transfected into A549 cells. The above transfections were carried out using LipofectamineTM 2000 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) (17).

Clinical samples

LUAD tissue and adjacent tissue were collected from LUAD patients (n=6) admitted to Shandong Provincial Hospital Affiliated to Shandong First Medical University. All patients underwent tumor resection and were not treated with neoadjuvant therapies, including chemotherapy and radiotherapy. The patients were diagnosed with LUAD through pathological examination. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University (No. 2018-029). Informed written consent was obtained from patients and their families.

Animals and grouping

BALB/c nude mice (n=10, male, 4–5 weeks) were provided by the Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Jinan, China). Mice were divided into two groups: a sh-NC group (n=5) that was injected subcutaneously with cells transfected with sh-NC (5×107/mL, 0.1 mL) and a sh-PTS group (n=5) that was injected subcutaneously with cells transfected with sh-PTS (5×107/mL, 0.1 mL). Ten days after inoculation, subcutaneous tumor formation was visually observed, and the length and width of the tumors were observed and recorded at 12, 15, 18, 21, 24, 27, and 30 days. The tumor volume (V = ab2/2) was calculated, and the transplanted tumor growth curve was drawn. The mice were sacrificed, and the transplanted tumor was completely removed, photographed, weighed, and stored at −80 ℃ for further study. The protocol was prepared before the study without registration. Animal experiments were performed under a project license (No. 2018-030) granted by ethics board of Shandong Provincial Hospital Affiliated to Shandong First Medical University, in compliance with Shandong Provincial Hospital Affiliated to Shandong First Medical University guidelines for the care and use of animals.

Western blot

Proteins from clinical samples and cells were extracted with lysate, and protein concentration was tested using a BCA Kit (ab102536, Abcam, USA). Proteins were then taken and subjected to polyacrylamide gel electrophoresis. After electrophoresis, the proteins were electro transferred to a polyvinylidene fluoride membrane (PVDF) and sealed with 5% nonfat milk powder for 1 hour. After that, the membranes were incubated with the following primary antibodies for 24 hours at 4 ℃: PTS (ab96104, Abcam, USA), B-cell lymphoma 2 (Bcl-2; 26593-1-AP, Proteintech, Wuhan), poly-ADP-ribose polymerase (PARP1; ab191217, Abcam, USA), Bcl-2-associated X protein (Bax; #41162, CST, USA), Wnt family member 5A (WNT5A; ab229200, Abcam, USA), glycogen synthase kinase-3 beta (GSK-3β; #12456, CST, USA), β-catenin (ab32572, Abcam, USA), cyclin D1 (ab16663, Abcam, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 60004-1-Ig, Proteintech, Wuhan). Next, the membranes were washed with tris-buffered saline with Tween 20 (TBST) and incubated with horseradish peroxidase (HRP)-labeled secondary antibodies for 2 hours and then developed with enhanced chemiluminescence (ECL).

Immunohistochemistry and Ki67 detection

Specimens were fixed with formaldehyde (4%) for 30 min, routinely embedded in paraffin, and sections (5 µm) were prepared. The sections were dewaxed and incubated in 3% H2O2 for 5–10 minutes at 37 ℃. After antigen retrieval in a pressure cooker, a PTS primary antibody (12150-1-AP, Proteintech) was added, and the sections were incubated overnight at 4 ℃. Then, the sections were incubated with HRP-labeled secondary antibody (37 ℃, 30 minutes). After washing with phosphate-buffered saline (PBS; 2 minutes) for three times, the sections were color developed with diaminobenzidine (DAB) solution (Elabscience, Wuhan, China). PBS was used as a negative control (NC). Ki67 primary antibody (27309-1-AP, Proteintech) was used to detect cell proliferation of subcutaneous tumors.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was isolated using Trizol (Invitrogen). cDNA was synthesized using a Reverse Transcription Kit (Vazyme. Nanjing, China). Then, the genes were amplified using a SYBR Green System (Vazyme. Nanjing, China), with GAPDH used as the internal reference. Relative gene expression was calculated by the comparative Ct method (2ΔΔCT). The sequences of the primers were as follows: PTS, F: 5'-TATGAATCTGGCTGATCTCAA-3'; R: 5'-TGCAAAGTATGGCACATCC-3'; ATF4, F: 5'-ATGACCGAAATGAGCTTCCTG-3'; R: 5'-GCTGGAGAACCCATGAGGT-3'; GAPDH, F: 5'-CCATGGGGAAGGTGAAGGTC-3'; R: 5'-TCGCCCCACTTGATTTTGGA-3'.

Cell Counting Kit-8 (CCK-8)

After transfection, cells (H23, H1299, and A549) were seeded into 96-well plates at 2×103 cells/well at 37 ℃ with 5% CO2. After culturing for 24, 48, 72 and 96 h, 10 µL CCK-8 solutions (Beyotime, Shanghai, China) were added to the plates. After mixing, the cells were incubated 2 hours, and the optical density (OD) of each well was detected at 450 nm.

Flow cytometry

After transfection, H1299 and A549 cells were collected and prepared into a single cell suspension. After washing with precooled PBS, the cells were labeled with fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide (PI) double antibody (BD Biosciences). Flow cytometry (AccuriC6, BD Biosciences) was used to measure the total apoptosis rate of the cells.

Colony formation assay

After transfection, the cells (H23, H1299, and A549) were digested and inoculated in a 6-well plate. The 6-well plate was taken out after culturing at 37 ℃ with 5% CO2 for 2 weeks. Methanol (2 mL) was added to each well for 30 minutes. After the methanol was discarded, crystal violet (0.1%, 2 mL) was added to each well for 10 minutes. After the crystal violet was washed off, a picture was taken with a digital camera, and the colonies were counted under a light microscope.

Wound healing assay

After treatment, H23, H1299, and A549 cells were digested and inoculated evenly in 6-well plates overnight. The cell layer was scratched vertically with a sterile pipette tip, and cells scratched by the pipette tip were washed with sterile PBS. The cells were cultured in an incubator (37 ℃, 5% CO2) for 24 hours, and then the cells were taken out. The width of the scratches was observed and measured under a microscope and photographed. Image J software (National Institutes of Health, Bethesda, MD, USA) was used for data analysis.

Dual-luciferase reporter assay

The sequence of wild type or mutant PTS promoter including ATF4 binding sites was subcloned into a pGL3-luciferase reporter vector. The promoter sequence was synthesized by Genscript Biotech Corporation. The wildtype and mutated PTS promoter were synthesized separately. The luciferase reporter vector and ATF4 were cotransfected into HEK-293T cells using Lipofectamine 3000 reagent (Invitrogen). After culturing at 37 ℃ for 48 hours, the cells were evaluated by a Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s instructions.

Transwell invasion assay

First, the upper chamber was coated with Matrigel (50 µL), and then the cell suspension (400 µL, 8×104 cells) was added to the upper chamber. Next, RPMI-1640 medium (700 µL, containing 20% FBS) was added to the lower chamber and cultured at 37 ℃ with 5% CO2 for 24 hours. After that, the chamber was taken out, and the cells in the upper layer of the pore membrane were wiped off with a cotton swab. The chamber was then fixed with 4% paraformaldehyde for 20 minutes, stained with crystal violet (0.1%, 2 mL, 30 minutes), and counted using a microscope after drying.

Hematoxylin and eosin (HE) staining

Tissue sections were baked at 70 ℃ for 30 minutes and deparaffinized with xylene. The slices were then hydrated with ethanol (95%, 85%, and 75%, 5 minutes per concentration). The sections were stained with hematoxylin, rinsed with water, and differentiated with 1% hydrochloric acid alcohol. Next, the sections were stained with eosin for 10 minutes. After that, the sections were dehydrated with 70%, 80%, 90%, and 100% alcohol for 5 minutes each, rinsed with xylene for 5 minutes, and observed under a microscope.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

Tumor tissues were embedded in paraffin and cut into sections (5 µm). The sections were then analyzed using a TUNEL Apoptosis Assay Kit (Beyotime Biotechnology, Shanghai, China). Pictures were taken under a fluorescence microscope.

Bioinformatics analysis

PTS messenger RNA (mRNA) expression data in the LUAD dataset were downloaded from The Cancer Genome Atlas (TCGA; https://tcga-data.nci.nih.gov/tcga/). PTS expression level in LUAD based on individual cancer stages was analyzed by the University of ALabama at Birmingham CANcer data analysis Portal (UALCAN) tool (http://ualcan.path.uab.edu/index.html). The overall survival curve of PTS in LUAD was analyzed using Gene Expression Profiling Interactive Analysis (GEPIA; http://gepia.cancer-pku.cn/). The HumanTFDB online prediction tool (http://bioinfo.life.hust.edu.cn/HumanTFDB/#!/tfbs_predict) was used to predict the intersection of transcription factors upstream of PTS and the genes positively correlated with PTS expression in TCGA-LUAD. Gene Set Enrichment Analysis (GSEA) was performed on the sequenced data using GSEA software (using the dataset h.all.v7.1.symbols.gmt).

Statistical analysis

Data were analyzed using SPSS 21.0 software (IBM Corp., Armonk, NY, USA). Survival analysis was performed using Kaplan-Meier and log-rank tests. T-test was used to compare two groups; one-way analysis of variance (ANOVA) was used to compare multiple groups. Correlations were explored using Pearson’s correlation analysis. P<0.01 and P<0.05 were considered statistically significant.

Results

PTS was highly expressed in LUAD

Data analysis of the TCGA dataset using GEPIA found that PTS level was significantly up-regulated in LUAD tissue compared to that in normal lung tissue (). Meanwhile, PTS mRNA expression in LUAD patients was correlated with tumor stage (). In addition, higher PTS was found to be related with lower overall survival (log-rank P=0.012; ). Furthermore, we found that PTS was more highly expressed in LUAD tissues than in normal paracancerous tissue (). In addition, PTS level in LUAD cells was higher than that in 16HBE cells (P<0.01; ). Taken together, these results suggested that PTS was highly expressed in LUAD.

Figure 1

PTS was highly expressed in LUAD. (A) PTS expression level in LUAD in TCGA data. (B) PTS expression level in LUAD based on individual cancer stages. (C) Correlation analysis of PTS level and prognosis of LUAD patients. Expression of PTS level in LUAD tissues was measured by Western blot (D) and immunohistochemistry (stained with PTS antibody; magnification 40×) (E). PTS mRNA and protein level in LUAD cells was tested by qRT-PCR (F) and Western blot (G). **, P<0.01, compared with the indicated group. TCGA, The Cancer Genome Atlas; LUAD, lung adenocarcinoma; mRNA, messenger RNA; PTS, 6-pyruvoyl-tetrahydropterin synthase; HR, hazard ratio; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; qRT-PCR, quantitative real-time polymerase chain reaction.

Silencing of PTS inhibited LUAD cell viability and proliferation and accelerated apoptosis

We regulated the expression of intracellular PTS by exogenous transfection, and the results indicated that PTS level in the si-PTS-1 and si-PTS-2 groups was markedly lower than that in the si-NC group, while PTS level in the PTS group was higher than that in the vector group (). Furthermore, our results found that down-regulation of PTS suppressed cell viability and proliferation and accelerated apoptosis. Moreover, overexpression of PTS improved cell viability and proliferation and inhibited apoptosis (P<0.01; ). In addition, the Western blot results found that silencing of PTS decreased Bcl-2, increased Bax level and promoted the cleavage of PARP (). Collectively, these results suggested that silencing of PTS inhibited LUAD cell proliferation and accelerated apoptosis.

Figure 2

Silencing of PTS inhibited LUAD cell viability and proliferation and accelerated apoptosis. (A) PTS protein level was tested by Western blot. Cell viability and apoptosis were determined by CCK-8 (B), colony formation assay (stained with crystal violet; magnification 100×) (C), and flow cytometry (D), respectively. (E) Levels of Bcl-2, PARP1, and Bax were tested by Western blot. *, P<0.05; **, P<0.01, compared with the si-NC group or vector group. PTS, 6-pyruvoyl-tetrahydropterin synthase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; si-NC, siRNA-negative control; siRNA, small interfering RNA; si-PTS, siRNA for PTS; OD, optical density; PARP1, poly-ADP-ribose polymerase; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; LUAD, lung adenocarcinoma; CCK-8, Cell Counting Kit-8.

Silencing of PTS inhibited LUAD cell migration and invasion

The results showed that down-regulation of PTS suppressed cell migration and invasion. Moreover, overexpression of PTS improved cell migration and invasion (P<0.01, ). In addition, the Western blot results revealed that silencing of PTS decreased N-cadherin and Vimentin levels but increased E-cadherin level (). Collectively, these results suggested that silencing of PTS inhibited LUAD cell migration and invasion.

Figure 3

Silencing of PTS inhibited LUAD cell migration and invasion. (A) Cell migration was tested by wound healing assay (magnification 4×). Transwell assay was used to test cell migration (B) (stained with crystal violet; magnification 10×) and invasion (C) (stained with crystal violet; magnification 10×). (D) Levels of E-cadherin, vimentin and N-cadherin were tested by Western blot. **, P<0.01, compared with the indicated group. si-NC, siRNA-negative control; siRNA, small interfering RNA; si-PTS, siRNA for PTS; PTS, 6-pyruvoyl-tetrahydropterin synthase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LUAD, lung adenocarcinoma.

ATF4 is an upstream transcription factor of PTS

Analysis using the HumanTFDB online prediction tool showed that 7 upstream genes were positively correlated with PTS level, including ATF4, chromobox 3 (CBX3), histone deacetylase 2 (HDAC2), nuclear transcription factor Y subunit beta (NFYB), enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), ring finger protein 2 (RNF2), and zinc finger protein 143 (ZNF143; ). Correlation analysis found that ATF4 and PTS had the highest score and the largest correlation coefficient, so ATF4 was selected to be included in the study (R2=0.18; ). Furthermore, we verified the binding of ATF4 and PTS promoter in HEK-293T cells by dual luciferase experiments and found that ATF4 induced a bigger change in the luciferase activity of the PTS promoter-WT than that in the PTS promoter-Mut (P<0.05; ). We altered the expression of ATF4 in A549 cells by transfection and showed that overexpression of ATF4 increased the expression of PTS (P<0.01; ). We further verified the effect of ATF4 overexpression on LUAD cells and found that up-regulation of ATF4 reversed the inhibitory effect of PTS silencing on cells, leading to the promotion of cell proliferation and invasion and the inhibition of cell apoptosis (P<0.01; ). Taken together, these results suggested that ATF4 targeted PTS promoter and promoted the expression of PTS in LUAD.

Figure 4

ATF4 is an upstream transcription factor of PTS. (A) PTS upstream transcription factors were predicted by HumanTFDB. (B) Linear analysis of PTS and ATF4. (C) Interaction between ATF4 and PTS was verified by dual luciferase assay. ATF4 mRNA (D) and PTS mRNA (E) were tested by qRT-PCR. Cell proliferation, apoptosis, and invasion were tested by colony formation assay (stained with crystal violet; magnification 100×) (F), flow cytometry (G), and transwell assay (stained with crystal violet; magnification 200×) (H). #, P<0.05, ##, P<0.05, compared with the si-PTS-1 group. *, P<0.05; **, P<0.01, compared with the si-NC group. PTS, 6-pyruvoyl-tetrahydropterin synthase; ATF4, activating transcription factor 4; CBX3, chromobox 3; HDAC2, histone deacetylase 2; NFYB, nuclear transcription factor Y subunit beta; EZH2, enhancer of zeste 2 polycomb repressive complex 2 subunit; RNF2, ring finger protein 2; ZNF143, zinc finger protein 143; NC, negative control; WT, wild type; Mut, mutant; mRNA, messenger RNA; si-NC, siRNA-negative control; siRNA, small interfering RNA; si-PTS, siRNA for PTS; qRT-PCR, quantitative real-time polymerase chain reaction.

Knockdown of PTS inhibited the Wnt pathway

We knocked down PTS using siRNA in A549 cells and performed transcriptome sequencing (https://www.novogene.com/). The results showed that knockdown of PTS affected the expression of lots of genes (). Further GSEA analysis found that knockdown of PTS inhibited the Wnt pathway (). Western blot analysis suggested that down-regulation of PTS inhibited the protein levels of WNT5A, GSK-3β, β-catenin, and cyclin D1, while up-regulation of PTS improved the protein levels of WNT5A, β-catenin, and cyclin D1 but decreased GSK-3β level (). To further verify the effect of PTS on the Wnt pathway, cells were treated with Wnt pathway activator BML-284 (1 µmol/L, MedChemExpress, Monmouth Junction, NJ, USA). The results showed that down-regulation of PTS inhibited the protein levels of WNT5A, β-catenin, and cyclin D1 but increased GSK-3β level (). Furthermore, BML-284 reversed the functional effects on LUAD cells of silencing PTS, leading to enhanced cell viability and the promotion of cell migration and invasion (P<0.01; ). In summary, down-regulation of PTS inhibited cell viability, migration, and proliferation by inhibiting the Wnt pathway.

Figure 5

Knockdown of PTS inhibited the Wnt pathway. (A,B) Transcriptome sequencing analysis after knockdown of PTS. (C,D) GSEA of PTS. (E,F) Protein levels of WNT5A, GSK-3β, β-catenin, and cyclin D1 were tested by Western blot. Cell viability, migration, and proliferation were detected using CCK-8 (G), wound healing assay (measured under a microscope, magnification 200×) (H), and transwell assay (stained with crystal violet; magnification 200×) (I). ##, P<0.05, compared with the si-PTS-1 group. *, P<0.05; **, P<0.01, compared with the si-NC group. sh-NC, shRNA-negative control; shRNA, short hairpin RNA; sh-PTS, shRNA for PTS; PTS, 6-pyruvoyl-tetrahydropterin synthase; GSEA, Gene Set Enrichment Analysis; GSK-3β, glycogen synthase kinase-3 beta; WNT5A, Wnt family member 5A; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; si-NC, siRNA-negative control; siRNA, small interfering RNA; si-PTS, siRNA for PTS; CCK-8, Cell Counting Kit-8.

Silencing of PTS inhibited tumor formation in mice

As demonstrated in , silencing of PTS markedly suppressed the weight and volume of tumors in the sh-PTS group in contrast with those in sh-NC group (P<0.01). The results of Ki67 assay found that the Ki67-positive rate in the sh-PTS group was markedly decreased than that in the sh-NC group (P<0.01; ). However, the apoptosis rate of the sh-PTS group was markedly increased than that of the sh-NC group (P<0.01; ). In summary, silencing of PTS inhibited tumor development in mice.

Figure 6

Silencing of PTS inhibited tumor formation in mice. Subcutaneous xenograft (A), weight (B), and volume (C) of tumor were monitored. (D) Ki67 assay for proliferation (magnification 200×). (E) Apoptosis was measured by TUNEL (magnification 200×). *, P<0.05; **, P<0.01, compared with the sh-NC group. sh-NC, shRNA-negative control; shRNA, short hairpin RNA; sh-PTS, shRNA for PTS; PTS, 6-pyruvoyl-tetrahydropterin synthase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Discussion

According to the 2018 global tumor statistics, approximately 18.19 million novel malignant tumors cases and 9.6 million deaths occurred worldwide, with lung cancer accounting for 11.6% of the incidence rate and 18.4% of the fatality rate (18). LUAD cells are mainly characterized by immortality, migration, and loss of contact inhibition, which are the main reasons for tumor refractory treatment (19). Studies have found that chromosomal changes, gene mutations, epigenetic changes, and other gene-level changes play a vital role in the occurrence and development of LUAD. At the same time, these gene changes also provide potential targets for the treatment of the disease (20-22). In this study, TCGA data analysis indicated that PTS is highly expressed in LUAD, and that high PTS is related to advanced stage and lower overall survival. Meanwhile, PTS was found to be up-regulated in LUAD tissue and cells. Hence, we speculated that PTS might be a novel potential target of LUAD, and we carried out experiments to study its effect and mechanism on LUAD. PTS has been extensively studied as an important cofactor for enzymes such as hepatic phenylalanine hydroxylase (23,24). Similarly, knockdown of PTS has been reported to suppress colorectal tumor cells growth (9).

Our study found that in LUAD, ATF4 level was positively correlated with PTS, and up-regulation of ATF4 reversed the inhibitory effect of silencing of PTS on LUAD cells. ATF4 has been reported to be up-regulated in lung cancer cells and to promote cancer progression (12), which is consistent with our study. Taken together, our results suggest that PTS is activated by ATF4 and promotes LUAD progression.

Further GSEA analysis showed that the Wnt pathway was inhibited by down-regulation of PTS in LUAD. To verify this finding, we found that down-regulation of PTS inhibited the levels of Wnt pathway downstream target proteins (WNT5A, β-catenin, and cyclin D1). The Wnt pathway is often abnormally activated and regulates tumor proliferation, migration, and invasion in the occurrence and development of LUAD (25). Interestingly, we found that silencing PTS inhibited LUAD development via inhibiting the Wnt pathway. Similarly, chromobox 4 has been found to promote tumorigenesis of LUAD through activation of the Wnt/β-catenin pathway (26). Tumor endothelial marker 8 has also been found to facilitate LUAD proliferation and metastasis via the Wnt/β-catenin pathway (27). We also explored the impact of PTS on LUAD in vivo. Down-regulation of PTS inhibited tumor growth in the mouse model, which was in agreement with our findings in cells. One limitation of this study is that although it appears that PTS is activated by ATF4 and this promotes LUAD’s progression by regulating the Wnt pathway, further investigation on the mechanism in vivo is missing, something in our follow-up research agenda.

Conclusions

Our study is the first work to investigate the expression and impacts of PTS in LUAD. Higher PTS is associated with poor prognosis. PTS is activated by ATF4 and facilitates LUAD progression by regulating the Wnt pathway, indicating that PTS may be a novel target for the treatment of LUAD.

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