PM2.5 promotes lung cancer progression through activation of the AhR‐TMPRSS2‐IL18 pathway

Abstract Particulate matter 2.5 (PM2.5) is a risk factor for lung cancer. In this study, we investigated the molecular mechanisms of PM2.5 exposure on lung cancer progression. We found that short‐term exposure to PM2.5 for 24 h activated the EGFR pathway in lung cancer cells (EGFR wild‐type and mutant), while long‐term exposure of lung cancer cells to PM2.5 for 90 days persistently promoted EGFR activation, cell proliferation, anchorage‐independent growth, and tumor growth in a xenograft mouse model in EGFR‐driven H1975 cancer cells. We showed that PM2.5 activated AhR to translocate into the nucleus and promoted EGFR activation. AhR further interacted with the promoter of TMPRSS2, thereby upregulating TMPRSS2 and IL18 expression to promote cancer progression. Depletion of TMPRSS2 in lung cancer cells suppressed anchorage‐independent growth and xenograft tumor growth in mice. The expression levels of TMPRSS2 were found to correlate with nuclear AhR expression and with cancer stage in lung cancer patient tissue. Long‐term exposure to PM2.5 could promote tumor progression in lung cancer through activation of EGFR and AhR to enhance the TMPRSS2‐IL18 pathway.


Introduction
Air pollution is contamination of the indoor or outdoor atmosphere by chemical, physical, or biological agents, and is one of the greatest environmental risks to human health. The combined effects of ambient (outdoor) and household air pollution were estimated by World Health Organization (WHO) to cause about 7 million premature deaths every year (P erez Velasco & Jarosi nska, 2022). The major components of air pollution are particulate matter (PM). Short-term or long-term exposure to PM is known to cause adverse health effects, including cardiovascular and respiratory disease, and cancers (Bazyar et al, 2019).
Particulate matter can be divided into PM10 or PM2.5 depending on whether the diameter of the PM particles is less than 10 or 2.5 lm. PM10 is mostly deposited in the nasal cavity and upper respiratory tract, while PM2.5 can enter the lower respiratory tract into the alveoli and enter the bloodstream (Sicard et al, 2019). PM2.5 enters the human body mainly through inhalation, thus causing various pathological effects on the respiratory system, such as chronic obstructive pulmonary disease, asthma, and multidrug resistance after tuberculosis treatment (Xing et al, 2016;Jeong et al, 2019;Yao et al, 2019). PM2.5 is composed of inorganic, organic, and biological compounds such as polycyclic aromatic hydrocarbons (PAHs), endotoxins, and fungi (Harrison & Yin, 2000). Among them, PAHs are the main components of PM2.5, accounting for approximately 80% (Javed et al, 2019). PAHs are mainly derived from fossil fuels such as petroleum and coal or manmade organic chemicals. PAHs are highly lipophilic and can enter cells directly through the cell membrane. PAHs can bind to cytoplasmic aryl hydrocarbon receptor (AhR) and then translocate to the nucleus to promote the expression of the cytochrome P450 family, which assists in the metabolism of carcinogens and environmental pollutants (Romagnolo et al, 2010). In addition, PAHs are known to photochemically react with sulfur oxides, nitrogen oxides, and ozone in the air to form secondary pollutants that are carcinogenic and genotoxic to the human body (Henkler et al, 2012).
Long-term exposure to air pollution is one of the causes of the high incidence of lung cancer (Pun et al, 2017). The inhalation of substances containing PAHs, such as PM2.5, is known to increase the risk of lung cancer (Singh et al, 2018). Exposure to PM2.5 can cause DNA damage and activation of AhR, epidermal growth factor receptor (EGFR), and the immune system, leading to tumorigenesis . Recent studies have shown that PM2.5 exposure promotes the expression of transmembrane serine protease 2 (TMPRSS2) , a type 2 transmembrane serine protease family (TTSP) that is known to promote SARS-CoV-2 infection and is also involved in the regulation of carcinogenesis (Martin & List, 2019;Hoffmann et al, 2020). Although many epidemiological studies have confirmed that PM2.5 is a risk factor for the occurrence of lung cancer (Al-Hamdan et al, 2017;Tseng et al, 2019), the definitive role of PM2.5 in the development of lung cancer remains largely unknown. In this study, we show that long-term exposure of lung cancer cells to PM2.5 can activate AhR to promote the expression of TMPRSS2 and IL18 and promote lung cancer progression.

Results
Effects of short-and long-term exposure to PM2.5 on cell proliferation, EGFR signaling, and anchorage-independent growth of lung cancer cells The cytotoxic effects of PM2.5 on normal lung and lung cancer cells were evaluated by short-term 24-h exposure using the normal lung fibroblast cell lines MRC5 and IMR90 and lung cancer cell lines A549 (EGFR wild-type) and H1975 (EGFR L858R+T790M). As shown in Fig 1A, short-term exposure to PM2.5 appears to produce a greater cytotoxic effect on H1975 cells than on IMR90 (P < 0.001), MRC5 (P < 0.001), and A549 (P < 0.001) cells. The IC50s for IMR90, MRC5, and A549 were over 100 lg/ml, while the IC50 of H1975 was 68.6 AE 3.46 lg/ml ( Fig 1A). As EGFR activation is the main driver of lung cancer (Tumbrink et al, 2021), the effects of exposure to PM2.5 on EGFR signaling were examined next. As shown in Fig 1B, PM2.5 induced the activation of phosphor (p)-EGFR, and pSTAT3 in A549 cells but induced the activation of only pAKT in H1975 cells.
To examine the effects of long-term exposure on cancer cells, we cultured A549 and H1975 cells in the presence of PM2.5 at 50 lg/ml for 90 days before analysis of proliferation and EGFR activation. As shown in Fig 2A and B, long-term exposure to PM2.5 appeared to increase the proliferation of both EGFR wild-type and EGFR mutant cancer cells (P < 0.05). The effects of long-term exposure on EGFR activation were also examined at different times of continuous exposure to PM2.5 at 50 lg/ml in lung cancer cells. As shown in Fig 2C, the expression of pEGFR in H1975 cells was continuously upregulated at different times of exposure to PM2.5 and reached the highest level after 90 days. Exposure to PM2.5 also upregulated the expression of activated pEGFR in A549 cells (Fig 2D) but resulted in the highest activation on Day 1, followed by a show decrease to lower levels after longer exposure times. Next, we examined the effects of long-term exposure to PM2.5 on anchorage-independent growth in H1975 and A549 cells. As shown in Fig 2E and F, longterm exposure to PM2.5 substantially increased the ability of H1975 and A549 cells to undergo anchorage-independent growth, although the increased pattern did not reach statistical significance in A549 cells. Increased ability to proliferate and to undergo anchorageindependent growth was also observed in PC9 lung cancer cells exposed to PM2.5 for 60 days (Fig EV1). Thus, long-term exposure to PM2.5 increased the ability of cancer cells to proliferate and undergo anchorage-independent growth.
Long-term exposure of lung cancer cells to PM2.5 enhances tumorigenicity in vivo A previous study showed that A549 cells treated with PM2.5 for 10 days have an increased ability to migrate and invade and induce cancer stem cell properties (Wang et al, 2019). Here, we examined the effects of long-term exposure to PM 2.5 on H1975 cells. To determine whether the increased ability to proliferate and to undergo anchorage-independent growth by long-term exposure to PM2.5 may indeed impact tumor growth in vivo, we subcutaneously injected unexposed and PM2.5-exposed H1975 cells into the flanks of nude mice, and the tumor volumes were determined twice a week. At 16 days after injection, the mice were euthanized. As shown in Fig 3A and B, the tumor growth in the mice injected with PM2.5-exposed H1975 cells was considerably faster than that in the mice injected with untreated H1975 cells. Consistent with this finding, the levels of Ki-67, TMPRSS2, and IL18 were greatly increased in the tumors derived from the PM2.5-exposed cells compared to the tumors derived from the untreated cells ( Fig 3C). These results indicate that long-term exposure to PM2.5 enhanced the tumorigenic abilities of lung cancer cells in vivo.
Long-term exposure to PM2.5 elevates the expression of TMPRSS2 by activating AhR in lung cancer cells We used whole-transcriptome sequencing to further explore the mechanism of action affected by long-term exposure to PM2.5 in H1975 cells. A total of 292 genes exhibited differential expression over twofold greater or lesser in the PM2.5-treated H1975 cells. These differentially expressed genes were subjected to ingenuity pathway analysis, which revealed that PM2.5 affected transcriptional dysregulation in cancer, the hedgehog signaling pathway, viral protein interactions with cytokine and cytokine receptors, retinol metabolism, alanine, aspartate, and glutamate metabolism, cytokine-cytokine receptor interactions, hypertrophic cardiomyopathy, malaria, cell adhesion molecules, and the AGE-RAGE signaling pathway in diabetic complications ( Fig EV2). The 10 differentially expressed genes with the highest scores are listed in Table 1. Among them, TMPRSS2 is a known oncogene that promotes tumor progression and metastasis in prostate cancer (Stone, 2017;Ko et al, 2020). Previous studies have shown that PM2.5 exposure upregulates the expression of TMPRSS2 in pulmonary fibroblasts . We thus selected TMPRSS2 for further study. To address the potential importance of TMPRSS2 upregulation in PM2.5-treated H1975 cells, we first examined whether PM2.5  A, B H1975 and A549 cells were treated with PM2.5 at 50 lg/ml for 90 days, and the proliferation of the treated cells was assessed by Trypan blue assays. C, D H1975 and A549 cells were exposed to PM2.5 at 50 lg/ml for different lengths of time, and the cell lysates of treated cells were assessed for phosphorylated EGFR (pEGFR) and EGFR by Western blotting. b-actin served as the loading control. E, F H1975 and A549 cells were treated with PM2.5 at 50 lg/ml for 90 days. The anchorage-independent growth was assessed by a soft agar colony formation assay.
The number of colonies was scored, and the data are presented as the relative colony formation ability. induced TMPRSS2 upregulation by quantitative real-time RT-PCR and Western blotting. As shown in Fig 4A and B, TMPRSS2 was increased to a higher level in the PM2.5-treated 1975 cells than in the untreated cells. Polycyclic aromatic hydrocarbons can activate AhR and then regulate the transcriptional expression of related genes (Tsay et al, 2013). Studies have shown that there are putative binding sites for AhR in the promoter of TMPRSS2 (Watzky et al, 2021). Therefore, we hypothesized that long-term exposure to PM2.5 activates AhR translocation to the nucleus and then binds to the promoter of TMPRSS2. To test this hypothesis, we used nuclear fractionation and Western blot analysis to examine the effects of PM2.5 treatment on the cellular distribution of AhR in nuclear and whole-cell lysates.
As shown in Fig 4C, the levels of nuclear AhR were greatly increased in the PM 2.5-exposed H1975 cells. To verify the nuclear localization of AhR, we performed immunofluorescence staining of AhR in A549 and H1975 cell lines. As shown in Fig EV3, elevated nuclear localization of AhR was evident in the PM2.5-treated cells.
To determine whether the increased nuclear AhR may be associated with enhanced binding at the promoter of TMPRSS2, we performed ChIP-qPCR analysis in the PM2.5-treated and untreated H1975 cells. As shown in Fig 4D, AhR binding was enhanced at four distinct sites of the TMPRSS2 promoter in the PM2.5-exposed H1975 cells. These results suggest that long-term exposure to PM2.5 can activate AhR to enhance the expression of TMPRSS2 in lung cancer cells. Consistent with a role of AhR in the expression of TMPPSS2, we observed Figure 3. Effects of long-term exposure to PM2.5 on tumor growth of lung cancer cells in vivo. H1975 cells were exposed to 50 lg/ml PM2.5 for 90 days. Both unexposed and exposed cells were injected subcutaneously into the flank of each mouse (n = 6 per group).
A, B The tumor volume and excised tumor weight were measured. The sizes of tumors excised from each group are shown at the top of (B). C IHC staining of excised tumors for Ki-67, IL18, and TMPRSS2 is shown in (C). Scale bars, 50 lm. that the AhR antagonist CH223191 inhibited the expression of TMPRSS2 in PM2.5-treated cells ( Fig 4E).
Effects of TMPRSS2 depletion on the anchorage-independent growth and in vivo tumor growth of lung cancer cells TMPRSS2 is associated with the development of prostate cancer and the promotion of SARS-CoV-2 infection of lung cells (Hoffmann et al, 2020). However, the biological function of TMPRSS2 in lung cancer is unclear, and the current research results are inconsistent Schneider et al, 2022). To determine whether the expression level of TMPRSS2 is increased in lung cancer cells, we examined the endogenous protein level of TMPRSS2 in four established lung cancer cell lines and two normal lung fibroblasts by Western blotting. As shown in Fig 5A, a high level of TMPRSS2 protein was detected in three lung cancer cell lines (H460, A549, and H1975). A low level of TMPRSS2 was also detected in H1299 lung cancer cells and normal IMR90 fibroblasts but was undetectable in normal MRC5 fibroblasts. To determine whether the level of TMPRSS2 may impact tumorigenicity in lung cancer cells, we examined the effect of stable depletion of TMPRSS2 on anchorageindependent growth of H1975 cells. As shown in Fig 5B and C, the levels of TMPRSS2 and anchorage-independent growth were greatly reduced in the cells stably expressing shTMPRSS2-1. The cells stably expressing shTMPRSS2-2 had only a slight reduction in the expression of TMPRSS2 and a slight inhibition of anchorageindependent growth. To determine whether TMPRSS2 affects lung cancer cell tumorigenesis in vivo, we performed a xenotransplantation assay in nude mice. As shown in Fig 5D, the growth of the shTMPRSS2-1 H1975 cell-derived xenograft tumors was slower than that of the vector control cell-derived xenograft tumors. Similarly, the average weight of the excised shTMPRSS2-1 tumors was lower than that of the vector control tumors (Fig 5E). These results show that the downregulation of TMPRSS2 inhibited lung cancer cell tumor growth in vitro and in vivo.
Transcriptomic profiling of TMPRSS2-regulated genes in H1299 cells To explore the molecular effects of TMPRSS2 on lung cancer cells, we examined the effects of overexpressing TMPRSS2 using H1299 cells, which express low levels of endogenous TMPRSS2 ( Fig 5A). First, we employed whole-transcriptome sequencing to compare the gene expression profiles of H1299 cells transfected with or without TMPRSS2 ( Fig 6A). A total of 169 putative genes were identified in the H1299 cells that overexpressed TMPRSS2 and showed a significant twofold difference in the expression level. With ingenuity pathway analysis, the pathways that were enriched in the TMPRSS2overexpressing H1299 cells were determined and included coronavirus disease-COVID-19, legionellosis, ribosome, influenza A, lipid and atherosclerosis, Yersinia infection, malaria, hedgehog signaling pathway, NOD-like receptor signaling pathway, and pathogenic Escherichia coli infection ( Fig EV4). Table 2 shows the top 10 upregulated genes in the TMPRSS2-overexpressing H1299 cells. Among them, IL18 has been shown to play a role in carcinogenesis and tumor progression (Zitvogel et al, 2012). IL18 expression showed positive correlations with TMPRSS2 in various human tissues (Cao et al, 2021). To further confirm the correlation of IL18 upregulation by TMPRSS2 overexpression, we performed RT-qPCR and Western blot analysis to detect IL18 mRNA and protein levels in the TMPRSS2-overexpressing H1299 cells. As shown in Fig 6B and C, the IL18 mRNA and protein levels were increased in the TMPRSS2overexpressing H1299 cells. Interestingly, we found that the mRNA and protein levels of IL18 and TMPRSS2 were also increased in the H1975 cells and A549 cells exposed to PM2.5 for 90 days, although the protein-increased level of IL18 did not reach the statistical significance in A549 cells (Fig 6D-H). These results suggest that enhanced expression of IL18 is correlated with increased expression of TMPRSS2.
Clinical correlation of TMPRSS2 expression with nuclear AhR, IL18, and overall cancer staging in human lung cancer specimens To evaluate the role of TMPRSS2 expression in lung cancer progression in vivo, we examined the expression levels of TMPRSS2, IL18, and nuclear AhR by IHC in tumor specimens from 25 lung cancer patients. Examples of scoring the intensity of immunoactivity in IHC are shown in Fig EV5. Representative immunostaining of normal tissue revealed almost no to very weak expression of TMPRSS2, IL18, and AhR ( Fig 7A). IHC staining of two representative tumor specimens revealed that a high expression level of TMPRSS2 was accompanied by a high expression level of nuclear AhR and IL18, while a low expression level of TMPRSS2 was associated with a low   expression level of nuclear AhR and IL18 (Fig 7B). Statistical analysis of all specimens showed a significant correlation between the expression level of TMPRSS2 and nuclear AhR (P < 0.01) and IL18 (P < 0.05) ( Table 3). Elevated expression of TMPRSS2 was also positively correlated with the advanced overall stages of lung cancer patients (P < 0.05; Table 3). Notably, the expression level of TMPRSS2 also correlated with sex (P < 0.05; Table 3).

Discussion
Most studies of air pollution on the progression of lung cancer are based on "population-based" epidemiology. In this study, lung cancer cells exposed to PM2.5 for 90 days were employed as a cellular model to evaluate the effects of long-term exposure to PM2.5 on lung cancer progression. As exposure to PM2.5 can cause EGFR  , we first examined the effects of exposure to PM2.5 on EGFR signaling. Our results revealed that short-term exposure to PM2.5 for 24 h indeed activated the EGFR pathway in lung cancer cells (Fig 1). Long-term exposure to PM 2.5 also upregulated the expression of activated EGFR (pEGFR) in lung cancer cells harboring EGFR mutations (H1975) (Fig 2C). Long-term exposure to PM2.5 enhanced the ability of H1975 cells to undergo anchorage-independent growth and to promote tumor cell growth both in vitro and in vivo (Figs 2 and 3). Therefore, activation of EGFR signaling can be one of the factors that contribute to the enhanced ability to promote tumor growth in PM2.5-exposed lung cancer cells.
To further explore the mechanism involved in the enhanced ability to promote tumor growth, we conducted wholetranscriptome sequencing analysis in the H1975 cells exposed to PM2.5 for 90 days. A total of 292 genes exhibited differential expression with twofold increases or decreases in the PM2.5treated H1975 cells. Among the 10 differentially expressed genes with the highest scores, TMPRSS2 is a known oncogene that promotes tumor progression and metastasis in prostate cancer (Stone, 2017;Ko et al, 2020) and was recently shown to be activated by PM2.5 exposure . Therefore, we examined whether TMPRSS2 may be involved in the enhanced ability to promote tumor growth in lung cancer cells. We confirmed that the expression of TMPRSS2 mRNA and protein was increased in the PM2.5-treated H1975 cells (Fig 4A and B). As PM2.5 is known to activate the translocation of AhR into the nucleus, the increased expression of TMPRSS2 may be attributed to the binding of activated AhR to the TMPRSS2 promoter. Indeed, we confirmed that the level of AhR was increased in the nucleus of the PM2.5-treated cells (Fig 4C). Furthermore, we showed that the binding of AhR to the TMPRSS2 promoter was enhanced in the PM2.5-treated cells (Fig 4D), suggesting that the upregulation of TMPRSS2 expression is correlated with the enhanced binding of AhR to the promoter of TMPRSS2.
To delineate the role of TMPRSS2 in promoting tumor growth, we examined the effects of TMPRSS2 depletion on tumor growth. As shown in Fig 5, stable depletion of TMPRSS2 in H1975 cells reduced the ability to undergo anchorage-independent growth in vitro and slowed tumor growth in vivo. Therefore, TMPRSS2mediated signaling is clearly involved in promoting tumor growth in lung cancers. To systematically explore the effect of TMPRSS2 on lung cancer cells, we used whole-transcriptome analysis to analyze the effects of TMPRSS2 overexpression on gene expression. As shown in Fig EV4, we identified several pathways that were highly enriched in the TMPRSS2-overexpressing H1299 cells, including those connected with immune, cell growth, and proliferation signatures. The immune-associated pathways included coronavirus disease-COVID-19, legionellosis, influenza A, Yersinia infection, malaria, NOD-like receptor signaling, and pathogenic Escherichia coli infection. The pathways associated with cell growth and proliferation included ribosome and hedgehog signaling. Interestingly, transcriptome sequencing analysis of the PM2.5-treated cells also revealed that PM2.5 affected immune and cell growth and proliferation signatures (Fig EV2). The immuneassociated pathways included viral protein interactions with cytokines and cytokine receptors and cytokine-cytokine receptor interactions. The pathways associated with cell growth and proliferation included transcriptional dysregulation in cancer and the hedgehog signaling pathway (Fig EV2). This finding indicates that upregulated TMPRSS2 expression was positively correlated with cell proliferation and immune modulation in lung cancer progression.
COVID-19, which is a global pandemic caused by the infection of the new coronavirus SARS-CoV-2, has a high diagnosis rate in cities with severe air pollution, and the patient's condition is more complicated and serious. PM is associated with the incidence and severity of COVID-19 (Yao et al, 2020;Zhu et al, 2020). SARS-CoV-2 enters the host cell by binding the spike (S) protein on the surface of the virus to the receptor angiotensin-converting enzyme 2 (ACE2) on the surface of the host cell, and then cleaving the S protein by another receptor protein TMPRSS2 on the surface of the host cell to make SARS-CoV-2 fuse into the host cell membrane, allowing the virus to enter (Hoffmann et al, 2020). Exposure to PM 2.5 promoted the upregulation of TMPRSS2 and ACE2 in lung fibroblasts, which in turn increased the infectivity of SARS-CoV-2 and the severity of COVID-19 . Our study shows that PM2.5 induces TMPRESS2 expression, which may make people more susceptible to infection with SARS-CoV-2. Therefore, TMPRSS2 appears to play an important role in PM 2.5 causing the more severe effects of COVID-19 and lung cancer.
IL18 was identified as one of the top 10 upregulated genes in the TMPRSS2-overexpressing H1299 cells (Table 2). We confirmed that the expression of IL18 is highly correlated with the expression of TMPRSS2 (Fig 6 and Table 3). The secretion of IL18 is known to be controlled by inflammasomes, which are composed of sensor proteins. The first family of sensor proteins revealed to form inflammasomes was NOD-like receptors (Karki et al, 2017). We found that NOD-like receptor signaling is highly enriched in the TMPRSS2-overexpressing H1299 cells (Fig EV4). Therefore, these data may indicate that the increased expression of TMPRSS2 in lung A IHC staining of TMPRSS2, IL18, and nuclear AhR in a representative normal lung tissue section. Scale bars, 2.5 mm (low magnification) and 100 lm (high magnification). B IHC staining of TMPRSS2, IL18, and nuclear AhR in lung cancer tissues that displayed high or low expression of TMPRSS2. Scale bars, 2.5 mm (low magnification) and 100 lm (high magnification). C A schematic representation summarizing the mechanism by which particulate matter upregulates TMPRSS2 to promote lung cancer progression.
Source data are available online for this figure.
Ó 2023 The Authors EMBO Molecular Medicine 15: e17014 | 2023 cancer cells may affect the formation of inflammasomes to enhance the secretion of IL18. The increased secretion of IL18 may in turn promote cell proliferation, proinflammation, and cancer progression. A recent study indicated that mice with EGFR mutations are more likely to develop cancer after exposure to pollutant particles mediated by an inflammatory protein called interleukin 1 beta, which is part of the body's immune response to PM2.5 exposure (Swanton et al, 2022). The role of PM2.5-induced inflammatory response in cancer progression should be further explored. Finally, the role of TMPRSS2 expression in lung cancer was examined in tumor specimens from patients. A positive correlation was observed between high levels of TMPRSS2 and nuclear AhR (Table 3), confirming that AhR is associated with the expression of TMPRSS2 in vivo. Increased TMPRSS2 expression was also positively correlated with the expression of IL18 (Table 3). Interestingly, increased TMPRSS2 expression was positively correlated with advanced stages of lung cancer, supporting our finding that TMPRSS2 plays an important role in tumor progression of lung cancer. Surprisingly, we observed that high expression of TMPRSS2 was found preferentially in female patients (Table 3). At present, we cannot offer any explanation for this finding.
In conclusion, we have shown that long-term exposure to PM2.5 promotes tumor progression in lung cancer. Based on the results presented in this study, we propose a schematic model (Fig 7C) in which the mechanism involved in promoting tumor progression by exposure to PM2.5 may include (i) activation of EGFR signaling and (ii) activation of AhR to upregulate TMPRSS2, which in turn upregulates its downstream targets, such as IL18.

Methods and Protocols
Cell lines and culture A549, H1975, H460, H1299, IMR90, and MRC5 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The lung cancer cell line PC9 was kindly provided by Dr. Chih-Hsin Yang (National Taiwan University). All of the cells were cultured in RPMI-1640 medium. The short tandem repeat assay was used to confirm the identity of all cell lines. The absence of mycoplasma contamination was tested by a polymerase chain reaction (PCR)-based enzyme-linked immunosorbent assay (ELISA; Sigma).
Source and preparation of PM2.5 The source of PM2.5 used in this work is the National Institute of Standards and Technology (NIST, USA) particulate matter SRM 1650b and was obtained from Sigma-Aldrich (St. Louis, MO, USA). The major components of this PM2.5 are PAHs and nitro-PAHs. PM2.5 was dissolved in DMSO at 25 mg/ml, sonicated for 30 min, and used within 1 h for the experiments performed (Piao et al, 2018). The PM2.5 was diluted 100-fold with double-distilled water, and the average diameter was measured by a laser-scattering method (Nano ZS90, Malvern). The diameter of the prepared PM 2.5 measured was 992 AE 157.2 nm.

Plasmids and establishment of stable knockdown subclones
Full-length TMPRSS2 with HA tagged at the C-terminus was cloned into the pcDNA3.1 vector using NotI and XbaI restriction enzymes (TMPRSS2-HA). Plasmid DNAs were transfected into cells using Lipofectamine 2000 (Invitrogen, Waltham, Massachusetts, USA) according to the manufacturer's protocol. Packed lentiviruses expressing short hairpin RNA (shRNAs) designed to knock down the expression of TMPRSS2 (TRCN0000000265 and TRCN0000000266) were obtained from the RNA interference consortium (National RNAi Core Facility, Academia Sinica, Taipei, Taiwan). The stable knockdown H1975 cells were designated shTMPRSS2-1 and shTMPRSS2-2, while the control was named the vector (control).
Whole-transcriptome sequencing and functional enrichment analysis Total RNA was extracted from H1975/H1975-PM 2.5 cells treated for 90 days and H1299/H1299 cells overexpressing TMPRSS2 using TRIzol â Reagent (Invitrogen, USA). Whole-transcriptome sequencing and functional enrichment analysis were performed as described in a previous study (Leu et al, 2020). The RNAsequencing data generated in this study have been deposited in the Gene Expression Omnibus under the accession GSE220252 and GSE220306.

Assays for cell viability and anchorage-independent growth
Viable cell counts by trypan blue exclusion assays and anchorageindependent growth by soft agar colony formation assays were performed as described previously (Chen et al, 2009(Chen et al, , 2016.

Chromatin immunoprecipitation-qPCR
Chromatin immunoprecipitation assays were performed using an Immunoprecipitation Assay Kit (Merck Millipore, Temecula, CA) according to the manufacturer's instructions. Each of the purified DNAs (10 pg) was used as a template for qPCR via five primers across potential TMPRSS2 regulatory regions (

RT-PCR, Western blotting, and immunofluorescence staining
The cell lysate for analysis by RT-PCR and Western blotting was performed as described previously (Chen et al, 2013;Wang et al, 2017). The immunofluorescence staining of cells and tumor sections was performed as described previously (Wang et al, 2017).

In vivo tumor xenograft study
The detailed procedure for tumor xenografts study was performed as previously described (Chen et al, 2018 Immunohistochemistry (IHC) Immunohistochemistry was performed as described previously (Jan et al, 2011). The scoring of IHC staining results was performed by two expert pathologists blinded to the clinical details. Immunoreactivity was classified into four classes: scored 0 for undetectable staining, scored 1 for weak staining, scored 2 for low intensity, and scored 3 for high intensity.

Statistical analysis
The presented results were representative of three independent experiments with similar results. Data in this study all followed normal distribution tested by Shapiro-Wilk test (P > 0.05). Onesample t-test was used to test whether relative phosphor protein expression, relative RNA expression, relative protein expression, relative proliferation of experiment group, and relative colony formation ability under null hypotheses mean equal to 1. To compare differences in proliferation, tumor weight, and tumor volume on the last observation day between treated group and control group, the variance equality between the two groups was examined by F-test, and a further two-sample t-test with or without equal variance was applied. For the comparison of fold change of AhR binding to different TMPRSS2 promoter regions, the Levene's test was used first for evaluating homogeneity of variance across promoter regions. If P-value of Levene's test > 0.05, the one-way analysis of variance (ANOVA) with post hoc analysis (Tukey's multiple-comparison test) was applied to compare differences between three groups (RG1a, RG1d, and RG1f). Otherwise, Welch's ANOVA and Games-Howell test of post hoc analysis was used (RG1h, RG2a). To assess relative cell ability among different cell lines (A549, IMR90, MRC5, and H1975) and PM2.5 concentration, the two-way ANOVA and Tukey's multiple-comparisons test of post hoc analysis were used. The two-way repeatedmeasurement ANOVA was used to evaluate treated effects over time on tumor volume, and pairwise comparison of post hoc analysis with Benjamini-Hochberg (BH) correction was conducted to the evaluated treated effect of PM2.5 on tumor volume in different time points. If multiple statistical tests were conducted in one experiment, BH method for adjusting multiple hypothesis testing was done. All tests were two tails and P-value < 0.05 was considered statistically significant. The scoring of IHC staining results was done in a blinded fashion by two pathologists.
Expanded View for this article is available online.

Results
In this study, lung cancer cells exposed to PM2.5 for 90 days were used as a cellular model of the effects of long-term exposure to PM2.5 on lung cancer. In mice, tumor progression was enhanced by longterm exposure to PM2.5. The molecular mechanism identified may include (i) activation of EGFR signaling and (ii) activation of AhR to upregulate TMPRSS2, which in turn upregulates its downstream targets, such as IL18.

Impact
Long-term exposure to PM2.5 could promote tumor progression in lung cancer through activation of EGFR and AhR to enhance the TMPRSS2-IL18 pathway.

Disclosure and competing interests statement
The authors declare that they have no conflict of interest.