Dysregulation of ferroptosis may involve in the development of non‐small‐cell lung cancer in Xuanwei area

Abstract The Xuanwei area of Yunnan Province, China, is one of the regions suffering from the highest occurrence and mortality rate of lung cancer in the world. Local residents tend to use bituminous coal as domestic fuel, which causes serious indoor air pollution and is established as the main carcinogen. After the local government carried out furnace and stove reform work, lung cancer rate including incidence and mortality among residents remains high. We herein wonder if there are specific mechanisms at protein level for the development of non‐small‐cell lung cancer (NSCLC) in this area. We investigated the changes of protein profiling in tumour of the patients from Xuanwei area. Tandem mass tag (TMT) was employed to screen the differential proteins between carcinoma and para‐carcinoma tissues. We identified a total of 422 differentially expressed proteins, among which 162 proteins were significantly up‐regulated and 260 were downregulated compared to para‐carcinoma tissues. Many of the differentially expressed proteins were related to extracellular matrix (ECM)‐receptor interaction, focal adhesion, PI3K/AKT pathway and ferroptosis. Further experiments on the two differential proteins, thioredoxin 2 (TXN2) and haptoglobin (HP), showed that the change of their expressions could make the lung cancer cell lines more resistant to erastin or RSL‐induced ferroptosis in vitro, and promote the growth of tumour in nude mice. In conclusion, this study revealed that aberrant regulation of ferroptosis may involve in the development of lung cancer in Xuanwei area.

the highest in the world. Especially, the incidence of lung cancer among non-smokers is 400/100 000, which is 20 times higher than the national average. 2,3 Previous studies have associated this excessive incidence of lung cancer with the domestic combustion of 'smoky coal', especially burning the coals in unvented households. 4,5 The smoky coal typically means the locally obtainable Late Permian bituminous coal, 6 which releases high level of visible smoke upon combustion. This kind of coal is available from many local coal mines within Xuanwei region and constitutes the primary fuel source for native residents. Several researches have proposed that the smoky coal combustion products like polycyclic aromatic hydrocarbons (PAHs) and air pollution fine-grained matter such as crystalline quartz particulate are classified as mutagens to human cancer. 7,8 The Yunnan Provincial Government has carried out furnace and stove reform work in Xuanwei area, hoping to reduce the incidence of lung cancer by reducing indoor coal-burning air pollution. However, the results of the Third National Sampling Survey of Cause of Death showed that the lung cancer mortality rate in the whole Xuanwei area increased most significantly nationwide. 9 Therefore, there could be other more important carcinogens.
Research to date on alternative potential driver risks for prevalent lung cancer in Xuanwei has been revealed. The primary risk arises from environmental pollution. The roles of some metal elements, especially heavy metal elements, are partially known in carcinogenesis in the context of lung cancer. As studies of Yunnan soil are observed, heavy metal pollution with metals of V, Cd, Cr, Cu, Mn, Co, Ni, Pb, As and Zn exceeds the vicinities concentrations, which denotes these metals have heavily polluted the street dusts of Xuanwei. Among which, Cd, Cr, Ni, Cr and As, are considered to be carcinogenic for human with long-term dusts exposure, and Cr is especially relevant for local children's risk. 10 The toxicity of Pb on lungs has been found to related to the increases in Nuclear factor kappa B (NF-κB) and aryl hydrocarbon receptor (AHR) levels, which further lead to the increases of nitric oxide synthase (iNOS) and cytochromes of the 1 subfamily (CYP1A1). 11 Another study shows that Cr(VI) causes alterations in DNA modification and microRNAs. 12 Moreover, LUSC patients show lower Ti expression but higher miR-24-3p and miR-28-5p. 13 Some other studies have found the association between the high lung cancer incidence and the contamination of metal elements in Xuanwei area. 10,14 A recent study shows that the mean levels of heavy metals especially in the magnetic fractions within the road dust are rather higher than their background values. 15 Thus, we suggested that the geological composition of the Xuanwei area may have special molecular mechanisms in the development of lung cancer in these area . Furthermore, we would analyse the protein profiling changes of the lung cancer tissues form the patients of Xuanwei area, providing more insights to the large excess incidence of lung cancer in this area and finding specific bio-molecular mechanisms involving the regional lung cancer.

| Ethical statement
The experimental protocols were approved by the Ethics Committee of The Third Affiliated Hospital of Kunming Medical University and followed the guidelines of the 1975 Declaration of Helsinki. All patients in this study provided written informed consent.

| Specimen collection
Total 20 lung cancer tissues and 20 adjacent normal lung tissues were collected from The Third Affiliated Hospital of Kunming Medical University. All subjects were born and lived in Xuanwei area for more than 3 generations and with local bituminous coal contact history over 10 years. The pathologic diagnosis was performed by a pathologist for all subjects of lung carcinoma. All the patients had not received any anti-tumour treatment before specimen collection. The clinic pathological manifestations were acquired from the medical records of the patients. All the collected specimens were stored at −80°C until use.

| Cell culture
All of the lung cancer cell lines, including A549 and NCI-H1299, were purchased from Shanghai Cellular Research Institute and maintained in RPMI-1640 medium (ScienCell) supplemented with 10% foetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin in a humidified incubator at 37°C with 5% CO 2 .

| Western blotting
The cells were treated under the indicated conditions and then efficient lysed with RIPA buffer at pH 8.0 (150 mM NaCl, 50 mM Tris, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS). All the samples were quantified using Pierce TM bicinchoninic acid (BCA) assay (Thermo Fisher Scientific Inc.) to ensure equal loading of proteins.
The cellular protein samples were separated by SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore). After blocked by 5% BSA, the membrane was probed with the primary antibody and then incubated with HRP-conjugated secondary antibody in TBST. The primary antibodies include anti-TXN2 (1:1000 dilution, Abcam, ab185544) and anti-HP (1:1000 dilution, Abcam, ab256454).

| Quantitative RT-PCR
RNA extraction with a kit (Qiagen) and the PCR was analysed using an ABI 7300 Real-Time PCR System (Applied Biosystems) with the DreamTaq Green PCR Master Mix (2×).

| Cell viability assay
Cell viability was measured by a CCK-8 kit (Beyotime) following the manufacturer's instruction.

| Iron quantification
Cells were seeded with 5 × 10 6 cells per plate and treated with erastin or RSL3 for 24 hours. The iron assay kit (ab83366) was performed according to the manufacturers' protocols. The concentration of intracellular ion was determined with a microplate reader at the absorption of 593 nm.

| Malondialdehyde (MDA) assay
As a product of lipid peroxidation, MDA was measured based on the reaction between MDA and thiobarbituric acid using a commercial kit (Beyotime Biotechnology). 18 The activity of enzyme was recorded as U/mg protein. The mean values from assays were from 3 independent experiments.

| High-performance liquid chromatography (HPLC) and mass spectrometry analysis
Each sample was prepared by subjecting to trypsin digestion and TMT labelling. After that, the resulted peptides were fractionated with a high-performance liquid chromatography (HPLC) system, and then, analysis of the peptides was performed with the Q Exactive TM mass spectrometer (Thermo Finnigan).
HPLC consisted Easy nLC system with C18 column (10 cm × 75 μm i.d., 3 μm particle size) (Thermo Scientific); mobile phase A (0.1% formic acid in H 2 O) and mobile phase B (85% acetonitrile with 0.1% formic acid in H 2 O) consisted the binary solvent gradient; and the elution procedure was performed as follows: initial A/B was 100:0 in volume, whereas B was linear from 0% to 35% at the first 50 minutes, and then reached to 100% at 55 minutes and held for another 5 minutes. The flow rate of mobile phase during gradient elution was 250 nL/min. Injection with an amount of 10 μL of samples and all samples was kept in an automatic injector at 4°C during the sequential analysis. Temperature of the column was kept at constant 25°C. Quality control samples were deployed in the sample queue to surveil the reliability of the raw data.
Mass spectrometry analyses were conducted with a Q-Exactive mass spectrometer with an orbitrap analyzer, using positive ESI. The ions with a range of 300 to 1800 m/z can be acquired. The duration of dynamic exclusion was set 40.0 seconds. The resolution for the HCD spectra of MS/MS was set to 17 500 at m/z of 200. The normalized collision energy was set at 30 eV, and the under-fill ratio at 0.1%. Quality control samples were ran at an interval of 7 samples.

| Data processing
Mascot (version 2.5, Matrix Science) was used to analyse the MS/ MS spectra. Then, the data set was searched with the SWISS-PROT

| Transmission electron microscopy (TEM)
The cells were washed with pre-cooled PBS (pH 7.4) and then postfixed in 2.5% glutaraldehyde and 1% osmium tetroxide in phosphate- were stained with UA and lead citrate. Ultrastructural images were acquired with a TEM (Hitachi HT7700).

| Statistical analysis
Statistic Package for Social Science (SPSS) 20.0 statistical software was employed to perform data analysis. The differences were denoted with one-way ANOVA and considered as significant at P < .05.

| Identification of differentially expressed gene in lung cancer
The scheme of the proteomic analysis is presented in Figure 1A. A total of 11 134 peptides in the clinic tissue samples were detected, which covers 3473 proteins; of these, 422 differentially expressed proteins (fold-change >1.2 or <0.83 in comparison with the paracarcinoma tissues, P < .05) were found in the analysis with quantitative information and were included in the next bioinformatics analysis. A volcano chart was drawn according to 2 factors, the foldchange and the p value obtained by t test, to show the significant difference in data between the two groups of samples ( Figure 1B). We used the hierarchical cluster to compare the differentially expressed proteins of the representative cases, showing with the heat map ( Figure 1C). The chart indicated that the threshold of fold-change set in the current study can effectively separate the cancer and the para-carcinoma groups, and the data of each case from the 2 groups were reproducible. According to the description about in GO terms   and several recently published studies, various proteins, including   TXN2, HP, PCNA, MYH7, POLG, TMEM62, TMEM16F, TMEM131L,   SLC34A2, SLC35A3, SLC9A3R2, SLC44A2, HSPH1, HSPA5, LPCAT1, PPP1R14A and PPP2R5C, were screened out ( Figure 1D) and subsequently subjected to qPCR validation ( Figure 1E).

| OncoPPi network and critical pathway hubs
The 422 proteins (162 up-regulated and 260 down-regulated versus para-carcinoma tissues) were annotated according to their biological process, cellular component and molecular function by BLAST2TO ( Figure 2A). Biological processes analysis showed that these proteins were mainly involved in single-organism process, localization, cellular component organization or biogenesis, biological regulation and multicellular organismal process. Cellular component analysis showed that most of the differential proteins were located in the extracellular region, membrane-enclosed lumen and organelle. Molecular function analysis revealed that a large proportion of these proteins played a role in molecular function regulator, structural molecule activity, protein binding, transporter activity and molecular transducer activity. Pathway annotation by KEGG analysis demonstrated that these differential proteins are mainly involved in ECM-receptor interaction, focal adhesion, small cell lung cancer, PI3K/AKT pathway and amoebiasis ( Figure 2B). The above pathways might be involved in the lung cancer in this area.
TXN2 and HP were relatively in the centre of the PPI network of all differentially expressed gene (DEGs), implying some important roles that they might play in the progression of NSCLC (red arrow, Figure 2C). Other various ferroptosis-related proteins that were indicated with blue arrows ( Figure 2C) were scattered on the network, further suggesting that ferroptosis probably involved in the progression of NSCLC. In addition, we picked out TXN2 and HP to analyse more potential association with other possible molecules in ferroptosis ( Figure 2D,E), and we noticed GPX4 (blue arrow), which is a documented key negative regulator of ferroptosis, and apolipoprotein E (APOE) (blue arrow), a well-known lipid peroxidation inhibitor, are closely implicated. In addition, we noticed that there are few studies about these two proteins TXN2 and HP in ferroptosis. Based on these findings, we decided to pay more attention on these two molecules in the following research.

| Altered expression of Txn2 and HP in lung cancer was associated with ferroptosis
We validated the changes of TXN2 and HP in proteomics using several lung cancer cell lines and clinic samples ( Figure 3A,B). We observed that TXN2 was significantly decreased but HP increased in the cells treated with the ferroptosis inducers erastin, RSL or sorafenib at both mRNA and protein levels ( Figure 3C,D), which indicated that the trend in proteomic analysis was reversed by the inducer. We hypothesized -that regulating the levels of TXN2 and HP in lung cancer cells would influence the cell proneness to ferroptosis.

| Survival analysis of TXN2
Using the online tool DriverDBv3 (http://drive rdb.tms.cmu.edu.tw/), whose data source is TCGA, TXN2 expression was found enhanced in LUSC (lung squamous cell carcinoma) but not in LUAD (lung adenocarcinoma), compared to lung normal tissue ( Figure 4A Figure 4D, high-TXN2-expression patients also exhibited lower RFS (relapse free survival) in Figure 4G (PROBE ID: 209077_at). But for F I G U R E 1 The proteomic analysis for the clinic carcinoma and para-carcinoma tissues of the patients diagnosed as lung cancer in Xuanwei. A, The scheme of the proteomic analysis in this study. B, The volcano plot showed the differentially expressed genes in the clinic samples. C, Hierarchical clustering of the differentially expressed genes. For hierarchical clustering, blue and red indicate decreased and increased expression, respectively. The proteins were clustered by hierarchical clustering using the complete linkage algorithm and Pearson correlation metric in R. D, Heatmap of the proteins potentially related to ferroptosis. E, Relative protein level of the molecules potentially related to ferroptosis; data were collected from the proteomic analysis results LUSC, no significantly differences were found between low-TXN2 and high-TXN2 groups (data not shown).

| Survival analysis of HP
HP expression data in LUAD and LUSC from TCGA were analysed with online tool DriverDBv3 (http://drive rdb.tms.cmu.edu.tw/), and it was found that HP was notably downregulated in both LUAD and LUSC tissues, compared to lung normal tissue ( Figure 5A,B). The possible role of HP in tumour progression of NSCLC was revealed through analysis of its expression in NSCLC using GENT2 (http://gent2.appex.kr/ gent2/) based on GEO public repository. However, lower HP at later clinical stages was not frequently observed ( Figure 5C) Figure 5F,G). In addition, according the survival analysis results from GEO data sets, HP expression level showed no effect on LUSC patients' survival (data not shown).

| Altered TXN2 and HP linked to ferroptosis in lung cancer cell lines in vitro
We overexpressed TXN2 or interfered with HP in lung cancer cell lines (A549 and NCI-H11299). The efficiency of TXN2 overexpression and HP depletion was confirmed by Western blotting ( Figure 6A); the mRNA level of TXN2 overexpression and HP depletion induced by erastin or RSL was also detected, as shown in Figure 6B; and erastin or RSL were unable to eliminate TXN2 expression with TXN2 overexpression, whereas erastin or RSL can increase the ferroptosis rate of lung cancer cells.

| TXN2 and HP were associated with lung cancer development in vivo
In vitro, both knocking down of TXN2 and overexpression of HP were observed to modulate sensitivity of lung cancer cell lines to erastin and RSL. Afterwards, we constructed the siTXN2 but HPoverexpressing A549 cells and performed the tumorigenicity tests using the wild-type A549 and the co-transfected A549 cells on nude  Figure 8A. When the tumours reached 50 mm 3 at day 2, mice were treated with erastin for 26 days ( Figure 8B). We also found that the co-transfected cells were more sensitive to erastin or RSL treatment in vivo, which showed that the tumour volume was much smaller than the group of wild-type A549 at 28 days after implantation ( Figure 8C,D).
Therefore, promoting the development of lung cancer by inhibiting ferroptosis may be a potential and special mechanism for the high incidence of lung cancer in Xuanwei.

| D ISCUSS I ON
The Xuanwei area in Yunnan Province is a high-risk area for lung cancer. As it is the main coal mining area in south-western China, several geochemical studies have shown that mineral elements are associated with a high incidence of lung cancer in this area. 10,16 To date, the cause of high occurrence of lung cancer in Xuanwei area remains far from clear.
To identify the vital candidates for lung cancer pathogenesis of patients from Xuanwei, bioinformatic methods were applied firstly.
Microarray study was used to analyse the altered protein profiling between carcinoma and para-carcinoma tissues from Xuanwei area, and then, pathway enrichment was used to explore the interactions of these differential proteins and the present study showed pathways enriched in 5 processes including small cell lung cancer, ECM-receptor interaction, focal adhesion, PI3K/AKT and amoebiasis. Apart from amoebiasis, the above pathways like ECM-receptor interaction and adhesive attraction 17 and PI3K/AKT 18 are greatly noted in lung cancer previously, which may be responsible for lung cancer cell invasion and metastasis. In addition, centrally involved TXN2 and HP as two key factors of the PPI network were identified.
Also, our network analysis implicated that TXN2 associated with the established negative ferroptosis regulator GPX4 and HP interconnected with the lipid peroxidation inhibitor APOE; moreover, both TXN2 and HP are ferroptosis-related molecules. Ferroptosis is a recently identified form of cell death, which differs from apoptosis, cell necrosis and autophagy. It is caused by the accumulation of the products of iron-dependent lipid peroxidation. 19,20 Considering the growth of cancer cell is iron dependency, which means cancer cell including lung cancer can evade apoptosis but to be more susceptible to an iron-induced ferroptosis. Morphologically, the cells showed a decrease in volume, an increase in mitochondrial membrane density and disappearance of mitochondrial cristae during ferroptosis. 21 At the molecular level, phospholipid-bound polyunsaturated fatty acids (PL-PUFAs) are peroxidized during ferroptosis to generation ROS and achieve cell kill, which can be eliminated by 3 parallel pathways: GPX4-glutathione, fibroblastspecific protein 1 (FSP1)-CoQ10 and guanosine triphosphate cyclohydrolase 1 (GCH)1-BH4. 22 Several other molecules have been found to regulate ferroptosis in lung cancer, such as the erythroid 2-related factor 2 (Nrf2) 23 and serine threonine tyrosine kinase 1 (STYK1). 24 Besides, growing evidences suggest an important lethal role for iron and ferroptosis in lung cancer. 25 Given that the main emitted chemical particle from coal combustion is iron in Xuanwei, which is reducible and could generate ROS, a study focused on the valence state of iron in size-resolved particle found that oxidizable of ·OH was higher in fine particles than coarse particles. 26 Thus, we guess ion may link to lung cancer, and ferroptosis may be responsible for Xuanwei lung cancer.
As an important antioxidant defence mechanism, TXN system has attracted researchers' attention to its role in ferroptosis. 27 The persistent activation of antioxidant systems and overexpression of thioredoxin via genetic alterations in Nrf2 and Keap1 also contributes to carcinogenesis. 28 Moreover, a recent study reported genetic knockdown of TXN leads to accumulation of lipid ROS levels and induces an antioxidant response to cause ferroptosis, a potential pro-ferroptotic agent due to its inhibition of TXN would be an anticancer therapeutic strategy. 29 HP is a plasma protein that has the ability to combine with free haemoglobin, which allows hepatic recycling of heme iron to function in preventing kidney from damage.
HP also has antibacterial activity and plays an essential role in regulating responses under the acute phase. 30 The expression of HP was found increased after intracerebral haemorrhage in vivo, which could prevent haemoglobin-induced neuronal ferroptosis is as an antioxidant. 31 HP polymorphism causes change in haemoglobin-bind capacity, antioxidant and iron-recycling activities, which may be important for effective oxidative stress response in lung cancer pathogenic process. 32 Specifically, in the present study, we supposed that HP and TXN2 may be involved in ferroptosis and contribute to lung cancer in Xuanwei area.
It was revealed that TXN2 up-regulated and HP downregulated in lung cancer cell tissues of Xuanwei patients and 3 lung cancer cell lines. Public data also supported that TXN2 enhanced in LUSC but not in LUAD, HP downregulated in both LUAD and LUSC.
Online data showed higher TXN2 levels at later clinical stages, whereas lower HP at later clinical stages was not frequently observed. Kaplan-Meier survival analysis suggested that higher TXN2 levels and lower HP levels might be unfavourable factors of in LUAD prognosis.
In addition, the results also indicated that ferroptosis inducer

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are included in the manuscript and supplementary materials.