Highlights
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Enhanced PYCR1 expression had certain clinical value in lung cancer.
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PYCR1 might serve as a marker for the diagnosis and prognosis of lung cancer.
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PYCR1 promotes the development of lung cancer through JAK-STAT3 pathway.
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PYCR1 promotes the expression of PD-L1 in lung cancer.
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PYCR1 significantly participated in process of lung cancer progression via the metabolism link between proline and glutamine.
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PYCR1 might be a novel therapeutic target for lung cancer.
Keywords: PYCR1, JAK-STAT3, PRODH, Glutamine, PD-L1
Abstract
Background
Lung cancer is a serious threat to human life. It is of great significance to elucidate the pathogenesis of lung cancer and search for new markers. This study evaluate the clinical value of pyrroline-5-carboxylate reductase 1 (PYCR1) and explore its role and mechanisms in the malignant progression of lung cancer.
Methods
PYCR1 expression and its relationship with prognosis were analyzed using a bioinformatics database. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were utilized to examine the expression of PYCR1 in lung cancer tissues and peripheral blood. PYCR1-overexpressing lung cancer cells were constructed, then the cell proliferative, migration, and invasion ability was examined by the MTT and Transwell assays. siRNA against PRODH and STAT3 inhibitor sttatic was used to further elucidate the underlying mechanisms. Luciferase and CHIP assays were carried out for validate the how PYCR1 regulated PD-L1 expression via STAT3. Xenograft experiment was performed to determine the role of PYCR1 in vivo.
Results
Database analysis showed that PYCR1 expression was significantly increased in lung cancer tissues, and its high expression predicted poor prognosis. Lung cancer tissue and peripheral blood of patients showed obviously increased PYCR1 expression, and the sensitivity and specificity of serum PYCR1 in the diagnosis of lung cancer were 75.7% and 60%, respectively. PYCR1 overexpression enhanced the proliferative, migration, and invasion abilities of lung cancer cells. Both PRODH silence and stattic effectively attenuated the function of PYCR1. Animal experiment and IHC data indicated that PYCR1 could activated STAT3 phosphorylation and PD-L1, as well as suppressed T cell infiltration in lung cancer. Finally, we also validated that PYCR1 promoted PD-L1 transcription by elevating STAT3 binding to the gene promoter.
Conclusion
PYCR1 has certain value in the diagnosis and prognosis of lung cancer. Moreover, through regulating JAK-STAT3 signaling pathway, PYCR1 significantly participated in process of lung cancer progression via the metabolism link between proline and glutamine, indicating that PYCR1 might be also a novel therapeutic target.
Introduction
Based on the latest cancer report [1], lung cancer remains the leading cause of cancer death, with a mortality rate as high as 18%, which seriously threatens human health. However, due to no clear symptoms at the early stage, most lung cancer patients are diagnosed in advanced stages, thereby losing access to the best treatment opportunities [2,3]. Thus, an early and accurate diagnosis, and timely and effective treatments are crucial to reducing the mortality of lung cancers. Currently, there is a lack of specific early diagnostic indicators for lung cancer in clinical practice, and the precise mechanisms used to explain its pathogenesis have not been fully elucidated. Thus, clarifying the underlying mechanism of lung cancer occurrence and develop, and identifying novel markers and therapeutic targets for its diagnosis, treatment, and therapy have crucial significance.
The abnormality of metabolism in glucose, amino acid, and fatty acid is a hallmark of cancer cells, which is also a potential target for cancer diagnosis and treatment [4]. Pyrroline-5-carboxylate reductase 1 (PYCR1) is one of the three isoenzymes of PYCR, a housekeeping gene widely present in humans and animals, which catalyzes the NAD(P)H-dependent conversion of pyrroline-5-carboxylate to proline [5]. PYCR1 is tightly associated with the development of bone, fat, and connective tissues. Its gene mutations can lead to abnormalities in the skin and skeletal system and cause skin laxity in humans [6]. Recent studies have demonstrated that PYCR1 significantly participates in the initiation and progression of multiple malignant tumors [7], [8], [9]. By biological information analysis, we found that PYCR1 expression is obviously increased in lung cancer tissues and is significantly associated with the prognosis of lung cancer patients. Increasing studies have reported that PYCR1 functioned as an important oncogene in lung cancer by promoting tumor cells migration, invasion, proliferation and inhibiting apoptosis via several mechanisms including by activating JAK/STAT signaling pathway [10,11]. However, the underlying mechanism by which PYCR1 promotes lung cancer progression is still poorly understood.
It is well known that glutamine plays a critical role in tumor development and progression, and most importantly this function is due in large part to its role in STAT3 activation [12,13]. On the other hand, researches have confirmed that proline and glutamine are interconvertible and linked in their metabolism [14]. However, whether the roles of PYCR1 in lung cancer progression were dependent on proline and glutamine interconversion, and the subsequent activation of JAK/STAT signaling pathway is still unknown. At the present study, we evaluated PYCR1 expression in lung and peripheral blood of lung cancer patients to assess its value for the diagnosis and prognosis. Furthermore, we tried to explore the role and mechanisms of PYCR1 in the lung cancer especially by focusing on proline and glutamine metabolism.
Materials and methods
Materials
Clinical specimens
70 lung cancer tissues and 34 normal lung tissues were collected. Meanwhile, serum samples from 111 lung cancer patients identified at our hospital from June 2019 to May 2021 and who did not undergo treatment were collected. Meanwhile, another 35 serum samples from benign lung patients were also collected (20 patients with inflammatory lesions, 6 with granulomatous lesions, 3 with hamartoma, 3 with atypical hyperplasia, 2 with intrapulmonary lymphadenitis, and 1 with pulmonary interstitial fibrosis) and 30 healthy individuals (control group). For tissue samples, after 10% neutral buffered formalin fixation and paraffin embedding, samples were cut into 4-μm sections and stored at 4 °C for further staining. For serum samples, after collection using vacuum blood collection tubes and centrifugation at 3500 rpm for 5 min, samples were recovered and stored at −20 °C for further analyses.
Before conducting the experiment in accordance with the declaration of Helsinki, we obtained the written informed consent of each patient and their families. It was also approved by the research ethics committee of Shaoxing people's hospital (The ethical permission number: 2019-K-Y-046–01).
Cells and reagents
Human lung cancer cell lines A549 and H1299 were employed in this study. The human PYCR1 monoclonal antibody was obtained from Abcam (UK, ab279385). PD-L1, and PRODH antibodies were purchased from Proteintech (Chicago, Illinois, USA; 66,248–1-lg and 22,980–1-AP respectively). The STAT3, p-STAT3, and β-actin antibodies were purchased from Cell Signaling Technology (Boston, Massachusetts, USA; 4904T, 9145S, and 4970S respectively). The CD3, CD4 and CD8 antibodies were obtained from Proteintech (Chicago, Illinois, USA; 60,181–1-Ig, 67,786–1-Ig, and 66,868–1-Ig respectively) . The ELISA kit for PYCR1 detection was sourced from Jiangsu Meimian Industrial Co., Ltd. (MM-60192H1, China), and Opti-MEM was purchased from Gibco (31,985–070, USA). Lipofectamine 2000 was purchased from Invitrogen (11,668–019, USA). Pierce® BCA Protein Assay Reagent A was purchased from Thermo Fisher (23,228, USA). The MTT reagent was purchased from Sigma (M2128, USA). The reverse transcription kit for qPCR was purchased from Applied Biosystems (4,309,155, USA). pcDNA3.1-PYCR1 overexpression plasmid, PYCR1 overexpression lentivirus, and luciferase reporter plasmids were obtained from Molecular Detection bio-tech (Hangzhou, China). siRNA against PRODH1 was obtained Hema bio-tech (Huzhou, China).
Methods
Bioinformatics database analysis
First, we used the GEPIA online database to analyze PYCR1 expression in normal and cancer lung tissues. Using normal tissues as controls, differential expression greater than 2-fold and with p < 0.001 were established as thresholds. Additionally, the associations between the expression of PYCR1 and the clinic pathological indicators of lung cancer were evaluated using 293 lung cancer patients with follow-up data in theGene Expression Omnibus (GEO) database and the corresponding relative PYCR1 expression data. The Kaplan-Meier plotter database was employed to identify PYCR1 expression and its relationship with the overall survival (OS) of patients. To analyze the relationships between the expression of PYCR1 and the OS of different lung cancer patients, the survival analysis was carried out using the medianPYCR1 expression as the cut-off.
Immunohistochemical staining
Briefly, after neutralization of the endogenous peroxidase using 3% H2O2 and antigen retrieval by microwave heating, the tissue was blocked with 10% normal goat serum and incubated with primary antibody (dilution rate: 1:400) at 4 °C overnight in a refrigerator. After washing with PBS, incubation with secondary antibody, 3,3′-diaminobenzidine (DAB) development, and counterstaining using hematoxylin, the slides were mounted. Five representative fields of view were selected for each section under high magnification and 100tumor cells and 500 total cells were counted in each field. Cells appeared as yellow or brownish-yellow particles in the nucleus, cell membrane, and cytoplasm was considered positive cells. A percentage of positive cells ≥ 5% was defined as positive, and a percentage of positive cells < 5% was defined as negative.
ELISA
After the addition of 50 μL standards and patient samples (1:5 dilution) to 96-well plates, 30 min of incubation at 37 °C, and washing with the washing buffer, 50 μL of the enzyme labeling reagent was added to the samples. Then, samples were incubated at 37 °C for 30 min and the plates were washed again. Chromogenic reagents A and B (50 μL each)were sequentially added to the plates, and color development was carried out for 10 min at 37 °C in the dark. Finally, after the addition of 50 μL stop solution, the values of absorbance at 450 nm were measured and recorded. For each plate, a standard curve was plotted based on the concentrations of the standard provided by the kit and the actual optical density (OD) values measured.
Cell culture
A549 and H1299 cells were cultured in FBS (10%), penicillin (100 U/mL)and streptomycin (100 μg/mL), and 1 × glutamine contained DMEM. Once grown to 80–90% confluence, the cells were passaged at a ratio of 1:2 or 1:4, then trypsinized and passaged every 2–3 d
Cell transfection
The transfection was carried out when cells reached 70–80% confluence in a 6-cm culture dish. Briefly, 2 μL of Lipofectamine 2000 was mixed with 50 μL of Opti-MEM by gently pipetting, and the mixture was incubated for 5 min. Then, 6 μg of plasmid or siRNA was mixed with 50 μL ofOpti-MEM by gently pipetting. These two solutions were mixed and incubated for 20 min. After removing the medium, two washes with PBS, and replacement with fresh medium (3 mL), this mixture was added dropwise to the cells and gently mixed. Finally, cells were placed in an incubator and subsequent experiments were performed at 24 or 48 h after transfection.
Western blot assay
First, the total cellular protein was extracted, and the concentration was measured using the bicinchoninic acid (BCA) assay. After separation with 12% or 15% SDS-PAGE, the protein bands were transferred to PVDF membranes. After 2 h blocking using 5% BSA solution and overnight incubation with indicated primary antibodies (dilution rates for all antibodies are 1:1000) at 4 °C, the membranes were washed using PBST. Then, after incubation with indicated secondary antibodies (dilution rate:1:5000), and three washes with PBST, the membranes were incubated with the enhanced chemiluminescence(ECL)substrate, exposed to X-ray film, visualized, and imaged. The Western blot results were quantitatively analyzed using ImageJ. Each experiment was repeated three times, and the relative protein levels were calculated as the average of the three experimental results.
MTT assay
After cell preparation and counting, 100 µL cell suspension was used for the MTT assay. The cell density was adjusted to guarantee that the cells covered approximately 50% of the surface of each well (generally 1000–10,000 cells/well). The wells at the edge of the plate were filled with sterile PBS. Then, 100 µL of a suspension of transfected cells was added to each well. Three replicate wells were prepared. After adhering to the wells, cells were cultured for 0, 24, 48, and 72 h. At the corresponding time points, the culture medium was aspirated, and 50 μL of the MTT solution (1 mg/mL in PBS) was added. After 3 h of incubation at 37 °C and the addition of 150 μL dimethyl sulfoxide (DMSO), the plate was placed on a shaker. The OD values were represented by the absorbance at 570 nm. GraphPad 6.02 was used for graph construction and statistical analysis.
Transwell assay
The membrane at the bottom of the upper chamber of a Transwell plate was coated with or without Matrigel for detection of cell invasion and migration respectively. Fifty microlitres of 50 mg/L Matrigel was diluted at a ratio of 1:6 and added to each well and incubated at 37 °C for 3 h. A 100-µl single cell suspension containing 1.5 × 105 cells in the serum-free culture medium was added to the upper chamber of the Transwell, and the culture medium containing 10% FBS was added to the lower chamber. The cells were routinely cultured for 48 h, fixed in 4% paraformaldehyde, and stained in Giemsa staining solution. Five fields were randomly selected and photographed under an inverted microscope. The cells were counted using Image J software, and each experiment was repeated 3 times.
qRT-PCR
Briefly, total RNA was extracted from cell samples by using Trizol reagent. The amplification primers were designed and the reverse transcription kit was used for real-time fluorescence quantitative PCR reaction Each gene was detected three times. Finally, the 2−ΔΔCT method was applied to normalize relative expression of target gene to β-actin. The primer sequences were as follows:
PD-L1: 5′-CAAGCAGTGACCATCAAGTCCTGAGT-3′ (Forward), R5’-CCATCATTCTCCCTTTTCTTAAACGG-3′ (Reverse);
β-actin: 5′-AGCGGGAAATCGTGCGTG-3′ (Forward), R5’-CAGGGTACATGGTGGTGCC-3′ (Reverse).
Xenograft experiment
For animal experiment, PYCR1 stably expressed H1299 cells were established using lentivirus and puromycin screening. Female BALB/c nude mice (4 weeks old, ∼15 g) were obtained from SLAC Animal(Shanghai, China) to generate the xenograft models. After one week of acclimatization, twelve BALB/c nude mice were injected subcutaneously with 5 × 106 PYCR1 stably expressed H1299 cells and their control cells, and each group contained six mice. Tumor width and length were measured by a caliper, and tumor volume was calculated by using the formula (Volume=tumor length × width2/2). At the end, mice were killed after anesthesia with pentobarbital (50 mg/kg weight), tumors were removed, and photographed and weighed. The animal experiment was approved by Ethics Committee of Shaoxing People's Hospital (2019-K-Y-046–01) .
Immunofluorescence staining
The sections were repaired with antigen repair solution and sealed with 3% BSA. Then the CD3 antibody was mixed with CD4 and CD8antibodies respectively, and then added into the section, and incubated at 4 °C overnight. Drop the corresponding second antibody and incubate it at room temperature in dark. After re-staining the nucleus, add anti-fluorescence quenching sealing agent. The photos were obtained using aImmunofluorescence microscope (Olympus, Japan).
Luciferase reporter gene assay
This experiment was performed using a Dual reporter luciferase assay kit (Promega) according to the protocol of manufactory. In brief, after cells were incubated overnight to allow cell attached, the luciferase reporter plasmids and PYCR1 overexpression plasmid were co-transfected for 24 h. The cmv-rennila plasmid was used as the internal control plasmid. Subsequently, 100 μL of 1X lysis buffer was added to cover the cells in each well of 24-well plates. Plates were punched to ensure that the lysis buffer completely lysed the cells. Then, cells were thoroughly scraped with pipet tips, collected in a 1.5 mL centrifuge tube, and centrifuged at 14,000 R/M for 20 min. Next, 20 μL of cell lysis products were added to a 96-well luminescence plate, followed by 50 μL LARII, and placed in the microplate detector (Promega). After measurement, 50 μL Stop &Glo®Reagent was added and placed in the detector again. The relative fluorescence intensity was calculated and compared with the no-load control to determine whether the influencing factor acted effectively on the target gene.
Chromatin immunoprecipitation assay
After treatment indicated, cells were cross linked and fixed with 1% formaldehyde and resuspended in SDS lysis buffer (1% SDS, 10 mM EDTA and 50 mM Tris [pH 8.1]). Dilute with immunoprecipitation (IP) buffer and use ultrasonic generator (SCIENTZ, Ningbo, China, JY92-IIN) to conduct ultrasonic treatment on ice to produce 100–500 bp DNA fragments. Add protein A/G agarose beads to H1299 cell lysate for pretreatment for 2 h, then add STAT3 antibody and IgG antibody and incubate overnight. Then wash with low-salt buffer, high-salt buffer, LiCl buffer, Tris-EDTA and elution buffer respectively. Incubate with NaCl for 4 h at 65 °C to reverse crosslinking. Then digest the protein with Core Mix(10µL 0.5 M EDTA, 20µL 1 M Tris·HCl(pH 6.5), 2µL 10 mg/ml Protein K). Finally, DNA was purified by phenol-chloroform extraction and ethanol precipitation. ChIP-enriched DNA was quantitated by real-time PCR with primers amplifying the PD-L1 promoter. The following primer pairs were used for PD-L1 ChIP: forward, 5′- ATTTCCTATTATACACCCA-3′; reverse, 5′- AAAGTCAGCAGCAGACCCA-3′.
Statistical methods
All the data collected in this study were analyzed using SPSS 19.0 software. Count data are expressed as the number of cases and percentages, and measurement data are as P50 (P25-P75). To compare two different groups,the nonparametric Mann-Whitney U test was applied. To compare more than two groups, the Kruskal-Wallis test was performed. The Dunn's test was used after Kruskal-Wallis to evaluate the differences between individual groups among the three groups. Percentages were compared using the χ2 test. The p value less than 0.05 was considered as significant difference. To analyze the efficiency of serum PYCR1 in the diagnosis of lung cancer, the receiver operating characteristic (ROC) curve was conducted.
Results
Bioinformatics database analysis
The bioinformatics analysis showed that, compared to normal lung tissues, lung cancer tissues presented significantly increased PYCR1 expression (p < 0.001). Meanwhile, a more significant difference in the PYCR1 expression was detected between lung adenocarcinoma and normal lung tissues compared to the difference between lung squamous cell carcinoma and normal lung tissues (Fig. 1A). Additionally, significant associations were detected between PYCR1 expression and sex (p = 0.012), as well as the histological classification (p < 0.001). Regarding the Tumor, Node, and Metastasis (TNM) staging, a significant correlation between PYCR1 expression and N staging (p = 0.015) was found, but not for T or M staging (Table 1). Compared to low PYCR1-expressed patients, high PYCR1-expressed lung cancer and lung adenocarcinoma patients presented significantly poorer prognoses (p = 0.0018 and p < 0.0001, respectively) (Fig. 1B, 1C). However, no association was found between PYCR1 expression and the prognosis of lung squamous cell carcinoma patients (Fig. 1D).
Fig. 1.
The results of the bioinformatics database analysis. A.
PYCR1 expression was significantly higher in lung cancer tissue than in the normal lung tissue; B. Lung cancer patients with high PYCR1 expression had a poorer prognosis; C. Lung adenocarcinoma patients with high PYCR1 expression had a poorer prognosis; D. In patients with lung squamous cell carcinoma, PYCR1 expression was not correlated with prognosis. The y-axis indicates the survival probability (overall survival rate).
Table 1.
The relationship between PYCR1 mRNA expression in lung cancer tissue and clinical case data in the GSE30219 dataset.
| Pathological index | Number of patients | PYCR1 |
Chi-square value | P | |
|---|---|---|---|---|---|
| Low expression | High expression | ||||
| Age (years) | 0.112 | 0.738 | |||
| ≥60 | 174 | 85 | 89 | ||
| <60 | 118 | 60 | 58 | ||
| Sex | 6.253 | 0.012 | |||
| Male | 250 | 117 | 133 | ||
| Female | 43 | 29 | 14 | ||
| Histological type | 48.743 | <0.001 | |||
| Lung adenocarcinoma | 85 | 26 | 59 | ||
| Lung squamous cell carcinoma | 61 | 28 | 33 | ||
| Basal cell carcinoma | 39 | 24 | 15 | ||
| Large cell endocrine carcinoma | 56 | 22 | 34 | ||
| Small cell carcinoma | 21 | 18 | 3 | ||
| Other types | 31 | 28 | 3 | ||
| T staging | 3.266 | 0.325 | |||
| 1 | 166 | 75 | 91 | ||
| 2 | 69 | 39 | 30 | ||
| 3 | 31 | 17 | 14 | ||
| 4 | 21 | 9 | 12 | ||
| N staging | 10.467 | 0.015 | |||
| 0 | 198 | 86 | 112 | ||
| 1 | 53 | 32 | 21 | ||
| 2 | 30 | 18 | 12 | ||
| 3 | 10 | 8 | 2 | ||
| M staging | 0.102 | 0.75 | |||
| 0 | 282 | 140 | 142 | ||
| 1 + 2 | 11 | 6 | 5 | ||
Expression of PYCR1 in clinical specimens
Expression of PYCR1 in the tissue of lung cancer patients
In lung cancer cells, most of the PYCR1 was expressed in the cytoplasm and was occasionally observed in the membrane and nucleus. Yellow or brownish-yellow particles were considered positive signals (Fig. 2A, 2B, 2D, 2E), and no PYCR1 expression was found in the most of the normal lung tissues (Fig. 2C, 2F). As showed in Table 2, patients were grouped based on smoking history, sex, age, clinical stage, and pathological classification and the χ2 test was used to compare expression differences among groups (Table 2). The expression of PYCR1 was dramatically enhanced in lung cancer tissues (58/70, 82.9%) than in normal lungtissues (6/34, 17.6%) (χ2 = 41.115, p = 0.000) (Table 2). Additionally, tumor tissues from lung cancer patients with stages III and IV presented significantly enhanced expression of PYCR1 compared to patients with stages I and II (χ2 = 4.729, p = 0.027). However, the expression of PYCR1 was not related to smoking history, age, sex, and pathological type (p > 0.05).
Fig. 2.
Expression of PYCR1 in clinical specimens of lung cancer. A, D.
PYCR1 was expressed in lung adenocarcinoma tissue (× 100, × 400); B, E. PYCR1 was expressed in lung squamous cell carcinoma tissue (× 100, × 400); C, F. PYCR1 was not expressed in normal lung tissue (× 100, × 400); G. The serum PYCR1 concentration was higher in patients with lung cancer than in patients with benign lung diseases and healthy controls (P< 0.001); H. ROC curve for PYCR1 in the diagnosis of lung cancer.
Table 2.
PYCR1 protein expression in pathological tissue.
| Group | Number of cases (n) | With PYCR1 expression | Without PYCR1 expression | Positive rate (%) | χ2 | P value | |
|---|---|---|---|---|---|---|---|
| Normal lungtissue | 34 | 6 | 28 | 17.6 | 41.115 | 0.000 | |
| Lung cancer tissue | 70 | 58 | 12 | 82.9 | |||
| Age (year) | 0.192 | 0.754 | |||||
| >60 | 39 | 33 | 6 | 84.6 | |||
| ≤60 | 31 | 25 | 6 | 80.6 | |||
| Sex | 0.008 | 0.927 | |||||
| Male | 30 | 25 | 5 | 83.3 | |||
| Female | 40 | 33 | 7 | 82.5 | |||
| Smoking history | 0.004 | 0.950 | |||||
| Yes | 18 | 15 | 3 | 83.3 | |||
| No | 52 | 43 | 9 | 82.7 | |||
| Clinical stage | 4.729 | 0.027 | |||||
| I + II | 45 | 34 | 11 | 75.6 | |||
| III + IV | 25 | 24 | 1 | 96.0 | |||
| Pathological type | 0.298 | 0.813 | |||||
| Adenocarcinoma | 43 | 35 | 8 | 81.4 | |||
| Squamous carcinoma | 18 | 15 | 3 | 83.3 | |||
| Otherpathological types | 9 | 8 | 1 | 88.9 |
PYCR1 secretion in serum of lung cancer patients
The protein levels of PYCR1 presented a skewed distribution in all groups. Thus, the P50 (P25-P75) was used to represent the concentrations in each group. The differences in the indices were compared using a nonparametric test (the Mann-Whitney U test) (Fig. 2G, Table 3). In comparison to the healthy control group (p = 0.000) and patients with benign lung cancer (p = 0.006), lung cancer patients showed significantly increased PYCR1 production. No significant difference was detected in the protein levels of PYCR1 between the benign lung disease and the healthy control groups (p = 0.108). Additionally, the protein levels of PYCR1 were not correlated with smoking history, sex, age, clinical stage, and pathological type (p-values of 0.096, 0.816, 0.835, 0.519, and 0.653, respectively) (Table 3).
Table 3.
Comparison of the serum PYCR1 concentrations among the 3 groups, P50 (P25-P75).
| Group | Number of individuals (n) | PYCR1(pg/ml) |
|---|---|---|
| Lung cancer group | 111 | 50.25(43.58 ∼ 62.97) |
| Age (years) | ||
| >60 | 78 | 50.64(44.93 ∼ 63.27) |
| ≤60 | 33 | 47.56(36.69 ∼ 61.17) |
| Sex | ||
| Male | 63 | 50.38(42.17 ∼ 68.87) |
| Female | 48 | 49.51(44.32 ∼ 55.61) |
| Smoking history | ||
| Yes | 46 | 50.51(43.22 ∼ 69.30) |
| No | 65 | 49.61(43.42 ∼ 56.79) |
| Clinical stage | ||
| Stage I+II | 63 | 48.71(42.77 ∼ 69.41) |
| Stage III+IV | 48 | 50.51(44.80 ∼ 58.34) |
| Pathological classification | ||
| Adenocarcinoma | 80 | 49.32(42.87 ∼ 62.52) |
| Squamous carcinoma | 22 | 51.26(44.73 ∼ 67.29) |
| Other | 9 | 48.59(38.11 ∼ 59.37) |
| Benign lung disease group | 35 | 43.07(35.54 ∼ 53.27) |
| Healthy control group | 30 | 38.51(33.81 ∼ 45.37) |
Diagnostic value of PYCR1 protein in lung cancer
The diagnostic value of PYCR1 protein in lung cancer diagnosis was evaluated using aROC curve and the AUC was calculated. For positive screening, we selected the maximum Youden index as the optimal cut-off value. The PYCR1 cut-off value for the diagnosis of lung cancer was 43.42 pg/mL, the AUC was 0.709, the sensitivity was 75.7%, and the specificity was 60.0% (Fig. 2H).
PYCR1 promotes proliferation, migration, and invasion of lung cancer cells
Verification of PYCR1 overexpression production in lung cancer cells
A549 and H1299 cells were transfected with empty pcDNA3.1 vector or pcDNA3.1-PYCR1 overexpression plasmid for 24 h, and the expression of PYCR1 was verified by Western blot. Compared to the control cells, the PYCR1-overexpressed A549 and H1299 cells showed significantly increased production of PYCR1 (p < 0.01) (Fig. S1).
Effects of PYCR1 overexpression on the proliferative, migration and invasion ability of lung cancer cells
We first detected the activation of STAT3 in lung cancer cells with PYCR1 overexpression. The data indicated that the ectopic expression of PYCR1 not only promoted the phosphorylation of STAT3, but also elevated the expression of PD-L1 protein (Fig. 3A). As expected, the data of qPCR revealed that the expression of PD-L1 mRNA was also up-regulated by PYCR (Fig. 3B). Next, MTT and Transwell assays were performed to determine the role of PYCR1 on cell proliferation, migration, and invasion of lung cancer cells. As showed in Fig. 3C, PYCR1 overexpression enhanced the proliferative ability of human A549 and H1299 lung cancer cells compared to empty vector-treated cells at 48 and 72 h. In comparison to that of the control cells, the migration and invasion abilities of PYCR1-overexpressed A549 and H1299 cells were significantly increased transfection (Fig. 3D).
Fig. 3.
Overexpression of PYCR1 promotes proliferation, migration, and invasion of lung cancer cells. A.
p-STAT3, STAT3 and PD-L1 protein levels were detected by Western blot; B. PD-L1 mRNA levels were determined by RT-PCR; C. MTT assay analysis of proliferation ability; D. Transwell assay analysis of migration and invasion ability. All data were used as mean ± SD. *P < 0.05, **P < 0.01.
PYCR1 promoted cell proliferation, migration, and invasion of lung cancer cells via elevating glutamine production in a PRODH-dependent manner
PRODH is a mitochondrial enzyme catalyzes the first step of proline catabolismin which proline is converted into pyrroline-5-carboxylate (P5C) and responsible for the conversion of proline into glutamate/glutamate [15]. A lot of evidence demonstrated that PRODH plays complex role in different cancer types, and it acts a pro-tumor gene in lung cancer by promoting EMT and inflammation related gene expression [16]. Therefore, we next tried to confirm whether PRODH and glutamine production play indispensable roles of PYCR1 in lung cancer cells. At first, siRNA against PRODH was used to transfect lung cancer cells to silence the gene expression and the silence efficiency was determined by WB experiment. As shown in Fig. 4A, siRNA-2 could effectively down-regulate the PRODH expression at 24 and 48 h in both two cell lines. Therefore, this siRNA was used to testified the role of PRODH in pro-tumor function of PRCY1 in lung cancer cells. Our data indicated that silence of PRODH effectively attenuated the phosphorylation of STAT3 and PD-L1 expression activated by PYCR1 overexpression in A549 and H1299 cells (Fig. 4B). On the other hand, the ectopic expression of PYCR1 significantly increased the level of glutamine in H1299 and A549 cells, and silence of PRODH obviously decreased the level of glutamine activated by PYCR1 overexpression in lung cancer cells (Fig. 4C, and not shown). As STAT3 is a key transcription factor for PD-L1 expression, qPCR was also used to determine the mRNA expression of PD-L1. As shown in Fig. 4D, the PD-L1 mRNA levels were up-regulated by PYCR1 overexpression but significantly recovered by the silence of PRODH. Similar results were observed in cell proliferation, migration, and invasion in A549 and H1299 cells (Fig. 4E, F). These findings suggested that PYCR1 might promoted lung cancer cells progression in PRODH dependent manner and involved the glutamine accumulation.
Fig. 4.
PYCR1 promotes the malignant phenotypes of lung cancer cells by up-regulating PRODH. A.
A549 and H1299 cells were transfected with NC siRNA or PRODH-siRNA for 24 h after. Then PRODH silence efficiency were detected by Western blot; B. A549 and H1299 cells were transfected with PYCR1 expression plasmid alone or co-transfected with PRODH-siRNA for 24 h. Then p-STAT3, STAT3 and PD-L1 protein levels were detected by Western blot; C. H1299 cell was transfected with PYCR1 expression plasmid alone or co-transfected with PRODH-siRNA for 24 h.Then glutamine levels were determined; D. A549 and H1299 cells were transfected with PYCR1 expression plasmid alone or co-transfected with PRODH-siRNA for 24 h. Then PD-L1 mRNA levels were determined by RT-PCR; E. MTT assay analysis of proliferation ability in A549 and H1299 transfected with PYCR1 expression plasmid alone or co-transfected with PRODH-siRNA for 24 h; F. Transwell assay analysis of migration and invasion ability in A549 and H1299 transfected with PYCR1 expression plasmid alone or co-transfected with PRODH-siRNA for 24 h. All data were used as mean ± SD. *P < 0.05, **P < 0.01.
PYCR1 promoted cell proliferation, migration, and invasion of lung cancer cells via STAT3 activation
To further confirm if the activation of STAT3 is the key step for PYCR1 to function as a pro-tumor gene in lung cancer, STAT3 inhibitor stattic was included in our study. As shown in Fig. 5A, stattic obviously restored the function of PYCR1 overexpression in STAT3 phosphorylation and PD-L1 protein expression in A549 and H1299 cells. The mRNA levels of PD-L1 up-regulated by PYCR1 overexpression were also significantly attenuated by stattic (Fig. 5B). MTT and Transwell assays were then performed to determine whether STAT3 activation was necessary for the functions of PYCR1 in lung cancer cell proliferation and movement. As indicated in Fig. 5C, D, stattic effectively impaired the role of PYCR1 overexpression on cell proliferation, migration, and invasion in both two lung cancer cell lines. Our data implied that functions of PRCY1 exerted in lung cancer is dependent on the phosphorylation of STAT3.
Fig. 5.
PYCR1 affects the malignant phenotypes of lung cancer cells through activated STAT3 signal pathway.
A549 and H1299 cells were transfected with PYCR1 expression plasmid alone or co-treated with stattic (2 μM) for 24 h. A. p-STAT3, STAT3 and PD-L1 protein levels were detected by Western blot; B. PD-L1 mRNA levels were determined by RT-PCR; C. MTT assay analysis of proliferation ability in A549 and H1299 cells; D. Transwell assay analysis of migration and invasion ability in A549 and H1299 cells. All data were used as mean ± SD. *P < 0.05, **P < 0.01.
Here, we also further verified whether PYCR1 promoted PD-L1 in a STAT3 dependent manner. The core fragment of PD-L1 promoter was subclone into the luciferase reporter vector PGL3, and the luciferase assay revealed that PYCR1 overexpression significantly elevated the transcription activity of PD-L1 promoter in both A549 and H1299 cells (Fig. S2A). To confirm if STAT3 was directly took part in the regulation of PD-L1 expression by PYCR1, STAT3 antibody was used to perform CHIP experiment in H1299 cell with PYCR1 overexpressed or control cells. As shown in Fig. S2B, PYCR1 overexpression promoted the STAT3 binding to the PD-L1 promoter. All these data supported that PYCR1 activated PD-L1 expression via STAT3 in lung cancer.
PYCR1 promoted the growth of A549 cells-derived xenografts and the activation of STAT3 and PD-L1 expression in vivo
Subsequent, animal experiment was performed using PYCR1 stably expressed A549 cells in nude mice. As shown in Fig. 6A, the tumor size of PYCR1 stably expressed A549 cell-derived xenografts is bigger than that of control group. The tumor volumes and weights were also detected at the end of the experiment, and the data presented that both of them in PYCR1 overexpression group were significantly higher than those in control group (Fig. 6B). Moreover, WB experiments were performed in tumor tissues to confirm the role of PYCR1 on STAT3 activation and PD-L1 expression. As indicated in Fig. 6C, the ectopic expression of PYCR1 indeed elevated the phosphorylation of STAT3 and PD-L1 expression in vivo.
Fig. 6.
PYCR1 promoted the tumor growth, and activated STAT3 phosphorylation and PD-L1 expression in vivo. A.
Overall view of xenografts derived from PYCR1 stably expressed A549 cells and control cells by injecting subcutaneously in BALB/c nude mice; B. Histogram quantitative analysis of tumor volume and weight; C. The protein expression of PYCR1, p-STAT3, STAT3, and PD-L1 was detected in tumor tissues by Western blot. All data were used as mean ± SD. *P <0.05, **P < 0.01.
We also examined the association between the expression of PYCR1 and PD-L1 in cancer patient tissues using IHC assay. The results displayed that PYCR1 higher expressed in cancer tissue compared to paracancerous one (Fig. 7A), and more importantly, there was an obvious positive correlation between PYCR1and PD-L1 expression (Fig. 7B). Due to the role of PD-L1 in tumor-immune suppression, immunofluorescence experiment was performed to confirm if PYCR1 could play a role in T cell infiltration in lung cancer. As shown in Fig. 7C, more CD3+CD4+ and CD3+CD8+T cells were observed in lung cancer tissue with low level of PYCR1 compared with the high one. Taken together, our data suggested that PYCR1 not only promoted the lung cancer growth, but also induced immune suppression by activating PD-L1 expression and inhibiting T cell infiltration.
Fig. 7.
Clinical samples validate PYCR1 promote PD-L1 expression and suppresses T cell infiltration in lung cancer. A.
Graph showing PYCR1 expression in Lung cancer and paracancer tissue detected by Immunohistochemistry. Brown color displays PYCR1 protein levels, with counterstaining by hematoxylin in blue; B. Graph showing the expression level of PD-L1 in lung cancer tissues with high or low expression of PYCR1 detected by Immunohistochemistry. Brown color displays PYCR1 and PD-L1 protein levels, with counterstaining by hematoxylin in blue; C. Graph showing the expression level of CD3, CD4, CD8 in lung cancer tissues with high and low expression of PYCR1 detected by Immunofluorescence. Red color displays CD3 protein levels, green color displays CD4 and CD8 protein levels.
Discussion
PYCR1 is one of the key enzymes in proline metabolism by catalyzing the conversion of pyrroline-5-carboxylate (P5C) to proline using NAD(P)H. Additionally, PYCR1 also converts piperideine-6-carboxylic acid to pipecolic acid [17]. Proline significantly participates in the protection of cells against stress and the maintenance of redox balance [18,19]. Besides, proline generated from the degradation of collagen in the extracellular matrix provides adenosine triphosphate (ATP) to meet the energy needs of cells. In the presence of hypoxia or hypoglycemia, the utilization of proline contributes to the production of ATP and triggers autophagy by promoting ROS production. Therefore, the synthesis and metabolism of proline are tightly participated in tumor initiation and progression [14,20,21]. Thus, PYCR1 affects the growth and development of tumor cells mainly through the biosynthesis of proline.
The bioinformatics database analysis observed increased PYCR1 expression and significant association of high PYCR1 expression with poor prognosis. Then, we detected significantly increased PYCR1 expression in 70 lung cancer tissues. Compared to the stages I and II lung cancer patients, the tumor tissues from the stages III and IV lung cancer patients expressed more PYCR1. Additionally, no correlations were detected between the expression of PYCR1 and age, sex, smoking history, or pathological type. However, our analysis indicated that PYCR1 expression was dramatically correlated with sex and pathological stage in lung cancer, inconsistent with our present results. The underlying reason for this difference might be involved in the fact that the lung cancer cases included here were mainly lung adenocarcinoma (61.7%), and there were fewer other pathological types, which might have resulted in certain deviations in the statistical results. Therefore, it is necessary to increase the sample size to further verify our findings. We also found significantly high serum PYCR1 in lung cancer patients compared to normal controls, but no correlations with age, sex, smoking history, clinical stage, and pathological type were observed. The serum PYCR1 cut-off value for the diagnosis of lung cancer was 43.42 pg/mL, the AUC was 0.709, the sensitivity was 75.7%, and the specificity was 60.0%. These results indicated that PYCR1 has clinical value for the diagnosis and prognostic prediction of lung cancers.
Furthermore, we constructed PYCR1-overexpressing A549 and H1299 lung cancer cells and performed MTT and Transwell assays to examine their proliferative, migration and invasion abilities, respectively. These results indicated that all malignant phenotypes were enhanced in lung cancer cells that overexpressed PYCR1. JAK-STAT signaling pathway is a common pathway of intracellular signal transmission of various cytokines and growth factors and participates in mediating different biological processes, such as apoptosis, migration, differentiation, proliferation, and immune regulation. Additionally, the activation of this pathway can lead to the occurrence of malignant tumors [22,23]. Our current results indicated that PYCR1 overexpression significantly enhanced STAT3 phosphorylation in lung cancer cells, indicating the activation of JAK/STAT3 pathway, which is consistent with the previous study [10].
The significant and tight association of the JAK-STAT3 pathway with the regulation of PD-L1 expression was also observed [24,25]. PD-L1 is expressed in tumor cells and disrupts T-cell-mediated immune surveillance, thereby promoting the occurrence and metastasis of lung cancers [26,27].We also observed increased PD-L1 expression, enhanced activation of the PD-L1 promoter, and the PD-L1 transcription in the PYCR1-overexpressed lung cancer cells, xenografts tissues, as well as in clinical tissues. Based on these results and literature review, we hypothesized that PYCR1 might affect the PD-L1 expression and promote the malignant phenotypes of lung cancer cells by enhancing JAK-STAT3 pathway activation, thereby affecting tumor progression. This was further supported by that STAT3 inhibitor stattic could effectively impaired the role of the ectopic expression of PYCR1 in lung cancer cells. However, the underlying mechanism for PYCR1 affecting the PD-L1 expression remains to be further confirmed.
Researches have indicated that glutamine took part in regulating JAK/STAT signaling pathway activation by virous mechanisms, including promoting the activation of EGFR singling pathway [28], and neutralizing ROS [12]. It is also well known that there is an interconversion between proline and glutamine with multiple enzymes involved [14,15,29]. However, whether activated STAT3 by PYCR1 in lung cancer cells was related to the metabolism link of proline and glutamine, and the potential mechanism are still unknown. Here, we tried to illustrated if the enzyme PRODH was involved in this process as it is the first enzyme which initiated the synthesize of glutamine using proline as source [15]. By using siRNA against PRODH, we silenced its expression in lung cancer cells with PYCR1 overexpressed to determine the STAT3 activation, PD-L1 expression, and the malignant phenotypes of A549 and H1299 cells. Our data indicated that knockdown of PRODH effectively attenuated the role of PYCR1 in almost all aspects. The most important was the elevated glutamine level induced by PYCR1 overexpression was effectively attenuated by PRODH silence. These findings suggested PRODH might promote the conversion from proline synthesized by PYCR1 to glutamine, and activated STAT3, PD-L1, as well as the progression of lung cancer at least partially (Fig. 8).
Fig. 8.
Schematic diagram of PYCR1 promoting malignant progression of lung cancer.
In summary, the levels of PYCR1 in tumor tissues and peripheral blood can be a potential marker for lung cancer diagnosis and prognosis. Moreover, PYCR1 promotes the malignant progression of lung cancers through the JAK-STAT3 signaling pathway via PRODH-dependent glutamine synthesize from proline, indicating that PYCR1 can serve as a novel potential target for the treatment and therapy of lung cancer and is worthy of further in-depth investigations.
Funding
This work was supported by the Project of Basic Public Welfare Research of Zhejiang Province (LGF19H200004), Medical Science and Technology Project of Zhejiang Provincial Health Commission (2020KY326), Medical Science and Technology Project of Zhejiang Provincial Health Commission (2021KY359).
Consent for publication
Consent for publication was obtained from the participants.
Data availability
The data used to support the findings of this study are available from the corresponding author upon request.
Fig. S1. PYCR1 overexpressed in lung cancer cells. A549 and H1299 cells transfected with PYCR1 expressed plasmid or pcDNA3.1 vector for 24 h. A. PD-L1 protein levels were detected by Western blot; B. Histogram quantitative analysis of PYCR1 protein expression. All data were used as mean ± SD. **P < 0.01.
Fig. S2. PYCR1 promoted PD-L1 transcription via STAT3
A. Relative luciferase activities was examined by luciferase assay in A549 and H1299 cells transfected with the PGL3 vector or PD-L1 promoter luciferase reporter plasmid for 24 h with or without overexpression of PYCR1; B. CHIP analysis was used to determine the binding of STAT3 to PD-L1 promoter region in H1299 cells transfected with PYCR1 expressed plasmid or pcDNA3.1 vector for 24 h. All data were used as mean ± SD. *P < 0.05, **P < 0.01.
CRediT authorship contribution statement
Lihong Zhang: Conceptualization, Writing – review & editing, Methodology. Xinyu Zhao: Methodology, Formal analysis, Data curation. Enqin Wang: Methodology. Ye Yang: Methodology. Liangfeng Hu: Methodology. Hongkun Xu: Formal analysis, Data curation. Baojun Zhang: Conceptualization, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Footnotes
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tranon.2023.101667.
Appendix. Supplementary materials
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The data used to support the findings of this study are available from the corresponding author upon request.
Fig. S1. PYCR1 overexpressed in lung cancer cells. A549 and H1299 cells transfected with PYCR1 expressed plasmid or pcDNA3.1 vector for 24 h. A. PD-L1 protein levels were detected by Western blot; B. Histogram quantitative analysis of PYCR1 protein expression. All data were used as mean ± SD. **P < 0.01.
Fig. S2. PYCR1 promoted PD-L1 transcription via STAT3
A. Relative luciferase activities was examined by luciferase assay in A549 and H1299 cells transfected with the PGL3 vector or PD-L1 promoter luciferase reporter plasmid for 24 h with or without overexpression of PYCR1; B. CHIP analysis was used to determine the binding of STAT3 to PD-L1 promoter region in H1299 cells transfected with PYCR1 expressed plasmid or pcDNA3.1 vector for 24 h. All data were used as mean ± SD. *P < 0.05, **P < 0.01.