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.

Keywords: AhR; EGFR; PM2.5; TMPRSS2; lung cancer.

Figure 1. Effects of short‐term exposure to PM2.5 on cell viability and EGFR signaling in lung cancer cells

1. Normal lung cells (IMR90 and MRC5) and lung cancer cells (A549 and H1975) were treated with various concentrations of PM2.5 for 24 h, and cell viability was assessed by a Trypan blue assay. The values are the mean ± SD of three independent experiments.

2. H1975 and A549 cells were treated with various concentrations of PM2.5 for 24 h, and their cell lysates were analyzed for phosphor‐ERK (pERK), phosphor‐AKT (pAKT), phosphor‐STAT3 (pSTAT3), phosphor‐EGFR (pEGFR), ERK, AKT, STAT3, and EGFR by Western blotting. β‐actin served as the loading control. The results shown are from one of three similar experiments (left panel). The relative expression levels of pEGFR, pAKT, pSTAT3, and pERK were quantified by normalizing with β‐actin and are shown in the right panel. The values are the mean ± SD of three independent experiments. *P < 0.05, as analyzed with one‐sample t‐test and compared with untreated cells.

Source data are available online for this figure.

Figure 2. Effects of long‐term exposure to PM2.5 on cell proliferation, EGFR activation, and anchorage‐independent growth of lung cancer cells

1. A, B

   H1975 and A549 cells were treated with PM2.5 at 50 μg/ml for 90 days, and the proliferation of the treated cells was assessed by Trypan blue assays.

2. C, D

   H1975 and A549 cells were exposed to PM2.5 at 50 μg/ml for different lengths of time, and the cell lysates of treated cells were assessed for phosphorylated EGFR (pEGFR) and EGFR by Western blotting. β‐actin served as the loading control.

3. E, F

   H1975 and A549 cells were treated with PM2.5 at 50 μg/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.

Data information: The data shown are the means ± SDs from three independent experiments. *P < 0.05 and **P < 0.01, compared with untreated control cells. The results shown in (C and D) are from one of three similar experiments. (A and B) P‐values were determined by two‐sample t‐test. (E and F) P‐values were determined by one‐sample t‐test. Source data are available online for this figure.

Figure EV1. Effects of long‐term exposure to PM2.5 on cell proliferation and anchorage‐independent growth of PC9 lung cancer cells

1. A, B

   PC9 cells were treated with PM2.5 at 50 μg/ml for 60 days. The proliferation of treated cells was assessed by Trypan blue assay (A). The anchorage‐independent growth was assessed by a soft agar colony formation assay (B). The data shown are the means ± SDs from three independent experiments. *P < 0.05 and **P < 0.01, compared with untreated cells. (A) P‐values were determined by two‐sample t‐test. (B) P‐values were determined by one‐sample t‐test.

Source data are available online for this figure.

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 μg/ml PM2.5 for 90 days. Both unexposed and exposed cells were injected subcutaneously into the flank of each mouse (n = 6 per group)

1. 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).

2. C

   IHC staining of excised tumors for Ki‐67, IL18, and TMPRSS2 is shown in (C). Scale bars, 50 μm.

Data information: The results shown in (A) and (B) are presented as the means ± SDs of six mice. *P < 0.05 and **P < 0.01, compared with untreated group. (A) P‐values were determined by two‐way repeated measures ANOVA with pairwise comparison of post hoc analysis with Benjamini–Hochberg (BH) correction. (B) P‐values were determined by two‐sample t‐test. Source data are available online for this figure.

Figure 4. Effects of long‐term exposure to PM2.5 on the AhR‐TMPRSS2 axis in lung cancer cells

1. A, B

   H1975 cells were treated with 50 μg/ml PM2.5 for 90 days, and the expression levels of TMPRSS2 mRNA and protein were determined by real‐time RT–PCR (A) and Western blotting (B), respectively.

2. C

   The proteins from whole cell lysates (WCL) and nuclear fractions (CN) were analyzed for AhR, lamin B (nuclei marker), and vimentin (cytoplasm marker) by Western blotting.

3. D

   ChIP–qPCR analysis of AhR binding to the promoter of the TMPRSS2 locus. The chromatin of untreated or treated H1975 cells was immunoprecipitated using AhR antibody. Precipitated genomic DNA was amplified for the five sites (RG1a, 1d, 1f, 1 h, and 2a) in the proximal promoter of the TMPRSS2 locus by real‐time PCR. Data were normalized to the input and expressed as “Fold change” relative to the IgG control of H1975 cells.

4. E

   Western blot analysis of TMPRSS2 expression in cells treated with CH223191 at 10 μM for 48 h. The relative expression level of TMPRSS2 was quantified by normalizing with β‐actin and is shown in the right panel.

Data information: The data shown represent the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01, and ***P < 0.001, compared with untreated cells. ^# P < 0.05, compared with PM2.5‐treated cells. The results shown in (B, C, and E) are from one of three similar experiments. (A) P‐values were determined by one‐sample t‐test. (D) One‐way ANOVA and Tukey's multiple‐comparisons test of post hoc analysis were used when homogeneity of variance across groups (RG1a, RG1d, and RG1f) occurred; Welch's ANOVA and Games–Howell test of post hoc analysis were used when variance across groups was not equal (RG1h and RG2a). (E) P‐values were determined by one‐sample t‐test. Source data are available online for this figure.

Figure EV2. The top 10 enriched biological processes in long‐term exposure to PM2.5

H1975 cells were exposed to 50 μg/ml PM2.5 for 90 days, and total RNA was subjected to whole‐transcriptome analysis as described in the Materials and Methods. Functional classification of the differentially expressed genes in PM2.5‐treated H1975 cells, as assessed using ingenuity pathway analysis.Source data are available online for this figure.

Figure EV3. Immunofluorescence staining of AhR

The subcellular distribution of AhR was assessed by immunofluorescence staining in H1975 and A549 cells treated with 50 μg/ml PM2.5 for 90 days. DAPI was used as a nuclear stain. Scale bar: 10 μm.Source data are available online for this figure.

Figure 5. Effects of TMPRSS2 depletion on the anchorage‐independent growth and in vivo tumor growth of lung cancer cells

1. A

   Expression of TMPRSS2 was examined by Western blots of four lung cancer cell lines (H460, A546, H1299, and H1975) and two normal fibroblasts (MRC5 and IMR90).

2. B, C

   H1975 cells were infected with sh‐TMPRSS2 (sh‐TMPRSS2‐1 and sh‐TMPRSS2‐2) or empty vector (Vector). The stable clones of TMPRSS2 knockdown cells were analyzed for the expression of TMPRSS2 by Western blots (B) and their ability to perform anchorage‐independent growth in soft agar (C).

3. D, E

   H1975‐shTMPRSS2‐1 cells were injected subcutaneously into mice (n = 9 per group), and the tumor growth of the implanted cells was measured (D). The excised tumors and their weights are shown in (E).

Data information: The results shown in (A and B) are from one of three similar experiments. β‐actin was used as the loading control. The data shown in (C) represent the means ± SDs from three independent experiments. The results shown in (D) and (E) are presented as the means ± SDs of nine mice. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with vector control. (C) P‐values were determined by one‐sample t‐test. (D and E) P‐values were determined by two‐sample t‐test. Source data are available online for this figure.

Figure 6. Effects of TMPRSS2 overexpression on the induction of IL18

1. A–C

   H1299 cells were transfected with TMPRSS2‐HA or the empty vector (Vector). After 48 h, the transfected cells were assayed for the expression of TMPRSS2 (A) and the induction of IL18 by qRT–PCR (B) and Western blotting (C).

2. D–H

   The expression level of IL18 was examined in H1975 cells (D, E) and A549 cells (F–H) exposed to PM2.5 at 50 μg/ml for 90 days by qRT–PCR (D, F, and G) and Western blotting (E and H).

Data information: The results shown in (A, C, E, and H) are from one of three similar experiments. β‐actin was used as the loading control. The data shown in (E and H) were normalized to β‐actin from three independent experiments. The data shown in (B, D–H) represent the means ± SDs from three independent experiments; *P < 0.05 and **P < 0.01, as analyzed with one‐sample t‐test and compared with vector control or untreated cells. Source data are available online for this figure.

Figure EV4. The top 10 enriched biological processes in TMPRSS2 overexpression

H1299 cells were transfected with TMPRSS2‐HA or the empty vector (Vector). After 48 h, the transfected cells were subjected to whole‐transcriptome analysis. Functional classification of the differentially expressed genes in TMPRSS2‐overexpressing H1299 cells, as assessed using ingenuity pathway analysis.Source data are available online for this figure.

Figure 7. Expression levels of TMPRSS2 and nuclear AhR in normal and cancer lung tissues

1. IHC staining of TMPRSS2, IL18, and nuclear AhR in a representative normal lung tissue section. Scale bars, 2.5 mm (low magnification) and 100 μm (high magnification).

2. 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 μm (high magnification).

3. 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.

Figure EV5. Representative IHC staining images showing immunoreactivity

1. A–D

   (A) Score 0, (B) score 1, (C) score 2, and (D) score 3 of TMPRSS2, IL18, and AhR in lung cancer tissue. Scale bars, 2.5 mm (low magnification) and 100 μm (high magnification).

Source data are available online for this figure.