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• W4239799318 abstract "Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract More than 90% of lung cancers are caused by cigarette smoke and air pollution, with polycyclic aromatic hydrocarbons (PAHs) as key carcinogens. In Xuanwei City of Yunnan Province, the lung cancer incidence is among the highest in China, attributed to smoky coal combustion-generated PAH pollution. Here, we screened for abnormal inflammatory factors in non-small cell lung cancers (NSCLCs) from Xuanwei and control regions (CR) where smoky coal was not used, and found that a chemokine CXCL13 was overexpressed in 63/70 (90%) of Xuanwei NSCLCs and 44/71 (62%) of smoker and 27/60 (45%) of non-smoker CR patients. CXCL13 overexpression was associated with the region Xuanwei and cigarette smoke. The key carcinogen benzo(a)pyrene (BaP) induced CXCL13 production in lung epithelial cells and in mice prior to development of detectable lung cancer. Deficiency in Cxcl13 or its receptor, Cxcr5, significantly attenuated BaP-induced lung cancer in mice, demonstrating CXCL13’s critical role in PAH-induced lung carcinogenesis. https://doi.org/10.7554/eLife.09419.001 eLife digest Lung cancer causes the most cancer deaths worldwide. For decades, people have known that lung cancer is associated with environmental factors, and both cigarette smoke and air pollution are known to cause cancers in humans. Smoke and air pollution both contain chemicals called polycyclic aromatic hydrocarbons (or PAHs). These chemicals cause chronic inflammation of the lung, which in turn is a major risk factor for developing lung cancer. However, it is unclear exactly how PAHs trigger inflammation and cancer. Xuanwei City in China is suited to the study of this question because until the 1970s its inhabitants used 'smoky coal' for cooking in unventilated indoor spaces; this produced high levels of small particles that contain high concentrations of PAHs. Women from this region, who traditionally do most of the cooking, have rates of lung cancer comparable to those of men. In other parts of China a woman’s chance of getting lung cancer is approximately half that of a man’s. Therefore, Xuanwei City provides a setting in which air pollution is a main contributor to lung cancer risk. Wang, Cheng et al. have now compared the levels of certain proteins (which are linked to inflammation) in lung cancer patients from Xuanwei City with those in control regions of China. The level of one such protein marker, called CXCL13, was particularly high in almost all patients from Xuanwei City, but only highly expressed in half of the patients from the control regions. Moreover, there was also a clear link between cigarette smoke and CXCL13 expression because, in control regions, smokers were much more likely to have high levels of CXCL13 than non-smokers. To test whether PAHs cause CXCL13 expression, Wang, Cheng et al. first exposed normal lung epithelial cells, cancer cells and then mice to a PAH. These experiments showed that CXCL13 levels did indeed increase and the mice developed lung tumours. However, when the genes for CXCL13 or its binding partner were deleted, the mice no longer got cancer when exposed to the PAH. This shows that CXCL13 signalling is an important mechanism by which PAHs cause lung cancer. Lastly, further experiments showed that CXCL13’s binding partner is highly expressed on some immune cells that can promote lung cancer. Importantly, the over-expression of CXCL13 occurred before the lung tumours developed. This might provide a new treatment strategy in which CXCL13 signalling could be inhibited after the exposure to PAHs. Future studies may now focus on discovering new drugs, or modifying existing drugs, to achieve this goal. https://doi.org/10.7554/eLife.09419.002 Introduction Air pollution is a diverse mixture of pollutants that originated from anthropogenic and natural sources, is comprised of particulate matter (PM), gases (e.g., sulfur oxides, carbon monoxide, ozone), organic compounds (e.g., polycyclic aromatic hydrocarbons, PAHs), metals (e.g., lead, vanadium, and nickel), and others, such as microbes (Akimoto, 2003; Huang et al., 2014). Air pollution is a global environmental health risk that affects the populations in developed and developing countries alike, and satellite observations suggest that 80% of the global population resides in locations where the ambient pollutant concentrations exceed the World Health Organization (WHO) Air Quality Guideline (van Donkelaar et al., 2010). Outdoor air pollution in cities and rural areas was estimated to cause 3.7 million premature deaths annually worldwide in 2012, including 220,000 deaths due to lung cancer (WHO, 2014). Recently, outdoor (Loomis et al., 2013) and indoor (WHO, 2010) air pollution has been classified as a Group 1 carcinogen in humans by the International Agency for Research on Cancer (IARC) of WHO. Indeed, the risk of lung cancer rises by 18% for every increase of 5 μg/m3 of PM smaller than 2.5 μm in diameter (PM2.5) in the environment; the risk increases by 22% for every increase of 10 μg/m3 in PM smaller than 10 μm (PM10) (Raaschou-Nielsen et al., 2013). There have been efforts to investigate how air pollution causes human cancers by exposing cells and animals to PM or related chemicals (Straif et al., 2012), but the carcinogenic mechanism remains elusive, at least in part, due to the difficulty of identifying human lung cancers that are causally associated with air pollution. Xuanwei City of the Yunnan Province in China provides a significant association between air pollution and lung cancer (Lan et al., 2002; Sinton et al., 1995; Mumford et al., 1987). Until the 1970s, residents of this region used smoky coal in unvented indoor fire pits for domestic cooking and heating, which are processes that release high concentrations of PM2.5/PM10 that contain high concentration PAHs (Mumford et al., 1987). Indoor air pollution in Xuanwei is associated with a significant increase in the absolute lifetime risk of developing lung cancer (Xiao et al., 2012), and the lung cancer incidence in this region is among the highest in China (Mumford, et al., 1987; Xiao et al., 2012; Li et al., 2011). In particular, nearly all women in this region are non-smokers and cook food on the household stove, and the lung cancer incidence in women is high. In Xuanwei, the male-to-female ratio of lung cancer incidences is 1.09:1, while that of China’s national average reaches 2.08:1 (Xiao et al., 2012). Tobacco smoke was weakly and non-significantly associated with lung cancer risk in this region (Kim et al., 2014). In the 1990s, a reduction in the lung cancer incidence was noted after stove improvement, supporting the association between indoor air pollution and lung cancer (Lan et al., 2002). These findings had been cited by the IARC monograph to classify indoor emissions from household coal combustion as ‘carcinogenic to humans (Group 1) (WHO, 2010). However, lung cancer incidence in Xuanwei is still increasing (Li et al., 2011), possibly due to pollutants generated by coal-burning industrial plants that have moved into the area (Cao and Gao, 2012). Therefore, the population in this highly polluted region (HPR) provides a unique opportunity to dissect air pollution-related lung carcinogenesis. Furthermore, the key carcinogens of smoky coal combustion are PAHs (Lv et al., 2009), which are also the key carcinogens of tobacco smoke that causes more than 85% of global lung cancer deaths (Hecht, 2012). Xuanwei lung cancer is, therefore, applicable to elucidate tobacco smoke-induced lung carcinogenesis. Chronic inflammation can promote cancer formation, progression and metastasis by inducing oncogenic mutations, genomic instability, and enhanced angiogenesis (Grivennikov et al., 2010). Studies show that PAHs can cause immune suppression (Zaccaria and McClure, 2013) and induce the secretion of cytokines/chemokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, CC chemokine ligand 1 (CCL1), chemokine (C-X-C motif) ligand 1 (CXCL1), and CXCL5 (N'Diaye et al., 2006; Umannová et al., 2011; Chen et al., 2012; Dreij et al., 2010). These factors may facilitate cancer initiation and progression. However, most of these factors were identified in cellular and animal PAH exposure models. Clinically relevant inflammation factors should be identified to shed insights into PAH-related carcinogenesis and provide novel therapeutic targets for lung cancer. To systematically investigate air pollution-induced lung carcinogenesis, we used Xuanwei lung cancers to analyze the abnormalities in the cancer genomes (Yu et al., 2015), genome-wide DNA methylation, non-coding RNAs (miRNAs [Pan et al., 2015] and lncRNAs), and inflammation factors. The abnormalities found in the HPR lung cancers were tested in patients from control regions (CRs) where smoky coal was not used, to compare the difference between HPR and control region (CR) lung cancers. In this study, we explored the abnormal inflammatory factors in HPR non-small cell lung cancers (NSCLCs). Results Abnormal inflammatory factors in HPR NSCLCs Using a microarray analysis of 84 cytokines/chemokines in tumor samples and their adjacent normal lung tissues of eight HPR NSCLCs, we found that the expression of four cytokines (IL-1F5, IL-1F9, MIF, and SPP1) and seven chemokines (CXCL13, CCL7, CCL20, CCL26, CXCL6, CXCL9, and CXCL14) was increased in tumors compared with their counterpart normal lung tissues (Figure 1A). Among them, CXCL13 was the most significantly up-regulated gene, with an average of 63-fold (10.48–173.65) higher expression in the tumor samples than in the normal controls. The expression of three cytokines (IL-1α, IL-5, and TNF-α) and nine chemokines (CCL4, CCL17, CCL18, CCL21, CCL23, CXCL1, CXCL2, CXCL3, and CXCL5) was lower in the tumor samples than in the normal controls (Figure 1A). Figure 1 with 1 supplement see all Download asset Open asset CXCL13 expression in lung cancer. (A) A PCR array was used to detect the expression of 84 cytokines/chemokines in eight highly polluted region (HPR) lung cancers. (B) The ratios of CXCL13 in tumor samples to their counterpart normal lung tissues from both the HPR and control region (CR) non-small cell lung cancers (NSCLCs). (C) Comparison of the CXCL13tumor/CXCL13normal values of the HPR patients with the CR cases. (D, E) CXCL13 expression was detected by immunohistochemistry (IHC) in HPR and CR patients (D), and the immunoreactivity score was calculated (E). (F) Western blot analyses of lysates from the tumors and adjacent normal lung tissues harvested from CR NSCLCs. (G) The concentrations of CXCL13 in the blood samples from healthy donors (HDs) and HPR and CR patients were detected by ELISA. (H, I) CXCL13 expression in Oncomine reports. (H) CXCL13 expression was detected by microarrays in tumor samples and normal lung tissues. AD, adenocarcinoma; A+S, adenocarcinoma and squamous cell carcinoma; Ca, Canada; It, Italy; Jp, Japan; SC, squamous cell carcinoma; Tw, Taiwan, China. (I) The expression of CXCL13 was detected in tumor tissues of smokers and non-smokers. (J) In mouse Gene Expression Omnibus (GEO) data sets, the expression of cxcl13 in indicated mice was detected by microarray. (K) The relationship between the CXCL13 expression and the tumor stages of lung cancer patients. (L) Overall survival of 54 CR patients (see Table 3 for their baseline demographic characteristics). The median follow-up was 1087 days (range, 187–1845 days). https://doi.org/10.7554/eLife.09419.003 Figure 1—source data 1 Sequences of primers for real-time PCR and ChIP, and siRNA. https://doi.org/10.7554/eLife.09419.004 Download elife-09419-fig1-data1-v2.docx Overexpression of CXCL13 in NSCLCs We expanded these observations, and found that the expression of CXCL13 was elevated in the tumor tissues from 63/70 (90%) HPR patients (Figure 1B) and 71/131 (54.2%) CR patients (Table 1) compared with their normal controls. CXCL13 expression was much higher in the HPR patients than the CR cases (p<0.002; Figure 1C). Using immunohistochemistry (IHC) and immunoreactivity scoring, we showed that the expression of the CXCL13 protein was significantly higher in the tumor samples than their adjacent normal controls (Figure 1D, E). CXCL13 expression was higher in the HPR NSCLCs than the CR patients, and the CR smoker NSCLCs had higher CXCL13 levels than the CR non-smoker patients (Figure 1B, D, E, and Table 1). Using Western blot assays, we found that CXCL13 was much higher in the tumor samples than their adjacent normal lung tissues (Figure 1F). ELISA showed that the CXCL13 serum concentration was higher in the HPR patients compared with the CR patients, while the latter was higher than the healthy donors (Figure 1G). Table 1 Baseline demographic characteristics of the 201 patients who underwent CXCL13 analyses. https://doi.org/10.7554/eLife.09419.006 CharacteristicsTotalHighly polluted region (HPR)Control region (CR)p values (HPR vs CR)Case, nCXCL13 high, n (%)p valuesCase, nCXCL13 high, n (%)p valuesCase, nCXCL13 high, n (%)p valuesTotal201134 (66.7)7063 (90)13171 (54.2)0.0000003G: Male13488 (65.7)0.674741 (87.2)0.278747 (54)0.950.0001 Female6746 (68.7)2322 (95.7)4424 (54.5)0.0006A: <65 y14098 (70)0.215651 (91.1)0.558447 (56)0.70.000009 ≥65 y5634 (60.7)1412 (85.7)4222 (52.4)0.03Unknown52 (40)52 (40)S: Smoker10775 (70.1)0.183631 (86.1)0.177144 (62)0.040.01 Non-smoker8753 (60.9)2726 (96.3)6027 (45)0.000006 Unknown76 (85.7)76 (85.7)H: Adenocarcinoma (AD)13191 (69.5)0.454844 (91.7)0.378347 (56.6)0.840.00003Squamous cell carcinoma (SCC)6139 (63.9)1916 (84.2)4223 (54.8)0.03Others94 (44.4)33 (100)61 (16.7)Tumor node metastasis (TNM): I8851 (58)0.0072923 (79.3)0.025928 (47.5)0.050.004 II2717 (63)98 (88.9)189 (50)0.005 III5843 (74.1)1717 (100)4126 (63.4)0.004 IV2219 (86.4)1111 (100)118 (72.7)0.06 Unknown64 (66.7)44 (100)20 (0) G, gender; A, age; S, smoke; H, histology. Analysis of association between CXCL13 expression and clinical characteristics CXCL13 expression was not significantly different in smoker and non-smoker HPR patients (p=0.17; Table 1), suggesting that severe air pollution had a carcinogenic effect on humans. In NSCLCs from CRs, however, the expression of CXCL13 was significantly higher in smokers (44/71, 62%) than in non-smokers (27/60, 45%, p=0.04; Table 1), suggesting a potential association between tobacco smoke and CXCL13 expression. The multivariate logistic analyses showed that among the 201 NSCLCs, CXCL13-high was associated with HPR (p=4.6×10–6) and tobacco smoke (p=0.032; Table 2). Table 2 Multivariate logistic analyses of the association between CXCL13 high expression and clinical characteristics. https://doi.org/10.7554/eLife.09419.007 Highly polluted region (HPR) patients, n=70 Variable Odds ratio 95.0% confidence interval P value Age1.4830.177–12.4520.716Gender4.7110.488–45.510.18Smoking0.6520.115–3.6820.628Histology0.370.069–1.9920.247TNM stage3.0920.765–12.5020.113Control region (CR) patients, n=131 Variable Odds ratio 95.0% confidence interval P value Age0.9140.455–1.8320.799Gender2.150.772–5.9930.143Smoking0.5130.251–1.0520.06Histology1.1480.569–2.3130.7TNM stage2.3551.157–4.7930.018Total (HPR and CR patients), n=201 Variable Odds ratio 95.0% confidence interval P value Region7.9083.272–19.1144.6×10-6Age0.9640.5–1.8610.914Gender2.2540.897–5.6620.084Smoking0.3940.168–0.9250.032Histology0.9960.52–1.9070.99TNM stage2.7071.401–5.2320.003 To investigate CXCL13 expression in NSCLCs of other cohorts, a cancer microarray database Oncomine (Rhodes et al., 2004) (www.oncomine.org) was applied. We found that in several works of this database (Okayama et al., 2012; Bhattacharjee et al., 2001; Hou et al., 2010; Landi et al., 2008; Selamat et al., 2012; Talbot et al., 2005; Su et al., 2007; Stearman et al., 2005), CXCL13 in tumor samples was elevated compared with their paired normal lung tissues or other normal controls (Figure 1H). CXCL13 was also higher in smoker NSCLCs than non-smoker patients in some studies (Okayama et al., 2012; Landi et al., 2008; Selamat et al., 2012) (Figure 1I). In microarray data sets GSE6135 (Ji et al., 2007), GSE21581 (Carretero et al., 2010), and GSE54353 (Xu et al., 2014) deposited in the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) from genetically engineered mouse models of lung cancer, Cxcl13 was increased in KrasG12DStk11-/- mice (especially in metastatic tumors), as compared with KrasG12DStk11wt mice (Figure 1J). These results suggest that CXCL13 overexpression was not specific to the Chinese cohorts, and Cxcl13 may play a role in Stk11-related lung tumorigenesis. We showed patients of both HPR and CR regions with relatively earlier disease (stages I and II) had lower CXCL13, while those with advanced disease (stages III and IV) had higher CXCL13 (Figure 1K), and multivariate logistic analyses showed that CXCL13-high was associated with TNM stage (Table 2, p=0.003). In 54 CR patients whose survival information was available (Table 3), the median survival time of CXCL13-high patients (965 days) was much shorter than the CXCL13-low cases (1193 days, p=0.03; Figure 1L). Kaplan-Meier estimates of survival of patients with NSCLC according to age (Figure 1—figure supplement 1A), cancer stage (Figure 1—figure supplement 1B), and histology (Figure 1—figure supplement 1C) confirmed that patients with stages III–IV lung cancer had shorter survival time than those with earlier stages of NSCLCs. Table 3 Baseline demographic characteristics of 54 control region (CR) lung cancer patients whose survival information was available. https://doi.org/10.7554/eLife.09419.008 CharacteristicsCase, n CXCL13-high, n (%)P* ValueTotal 5425 (46.3)Gender Male3816 (42.1)0.34Female169 (56.3)Smoking Smoker3316 (48.5)0.69Non-smoker219 (42.9)Age, years 0.72<653817 (44.7)≥65168 (50)Histology Adenocarcinoma3215 (46.9)0.83squamous cell carcinoma189 (50)small cell lung cancer10 (0)Others31 (33.3)TNM stage 0.65I2612 (46.2)II62 (33.3)III189 (50)IV42 (50) BaP induces CXCL13 production in vitro and in vivo PAHs were reported to be the major carcinogens in the PM2.5/PM10 in HPR, as well as in the PM at urban locations in Beijing, Shanghai, Guangzhou and Xi’an in China during January 2013 (Huang et al., 2014). Clinically, a long latency is required for individuals to develop lung cancer since they were first exposed to smoking or air pollution. To test the effects of PAHs on cytokine/chemokine production, the normal human lung epithelial 16HBE cells (Cozens et al., 1994) were exposed to a representative PAH compound benzo(a)pyrene (BaP) at 1 μM for a long period of time (30 days). We found that CXCL13 was the most significantly up-regulated gene among the 84 cytokines/chemokines (Figure 2A). We confirmed that BaP up-regulated CXCL13 at both the mRNA and protein levels in a dose- and time-dependent manner in 16HBE and A549 lung cancer cells (Figure 2B, C). Figure 2 with 1 supplement see all Download asset Open asset Benzo(a)pyrene (BaP) induces CXCL13 in vitro and in vivo. (A) A PCR array analysis of the expression of 84 cytokines/chemokines in 16HBE normal lung epithelial cells treated with 1 μM BaP for 30 days. (B) The cells were treated with BaP at 10 μM for indicated time points or with the indicated concentrations for 72 hr, and CXCL13 expression was assessed by real-time RT-PCR. The experiments were conducted in triplicate and repeated three times. The error bars represent the SD. (C) The cells were treated with BaP as described in (B), and the concentration of CXCL13 in the supernatants was evaluated by ELISA. (D) The A/J mice were treated with BaP and/or dexamethasone (DEX) for 5 weeks (see also Figure 2—figure supplement 1A) and sacrificed 6 months later. The lung tissues were isolated and analyzed by Hematoxylin and eosin (HE) staining or immunohistochemistry (IHC) using an anti-Cxcl13 antibody (left panel). The immunoreactivity score was calculated (right panel). (E) Cxcl13 expression was detected in the lung tissues by real-time PCR. (F) The concentration of Cxcl13 in mouse serum was assayed by ELISA. (G) IHC assays of mice’ lung tumor tissues using anti-Cd68, anti-Ttf1, and anti-Cxcl13 antibodies. (H) Immunofluorescence assay of mice’ lung tumor tissues using antibodies against Cxcl13 (green), Cd68 (red), and Ttf1 (white). 4',6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus (blue). (I) The survival curves of the mice treated with BaP and/or DEX (n=8 for each group). https://doi.org/10.7554/eLife.09419.009 To test the in vitro observations in vivo, A/J mice were treated with vehicle control (corn oil) or 50–100 mg/kg BaP (Figure 2—figure supplement 1A, modified from previous studies (Wattenberg and Estensen, 1996). All BaP-treated animals developed lung cancer within 6 months, as detected by a microCT scan (Figure 2—figure supplement 1B,C) and histopathology (Figure 2D). We found that Cxcl13 was significantly up-regulated in the tumor samples compared with their normal counterparts at both the mRNA (Figure 2E) and protein (Figure 2D) levels, which paralleled the increase in Cxcl13 serum concentrations (Figure 2F). Notably, BaP increased Cxcl13 concentrations in peripheral blood of the mice at 1 month after the beginning of the treatment (Figure 2F), at which point no tumors were observed (Figure 2—figure supplement 1D). To determine the source of Cxcl13, we performed IHC and immunofluorescence assays in lung cancer tissues of the mice. To do this, thyroid transcription factor-1 (Ttf-1) which is expressed in the lung epithelial cells (Lazzaro et al., 1991), was used to mark lung cancer cells, and Cd68 stain was employed to mark macrophages which can produce Cxcl13 in inflammatory lesions with lymphoid neogenesis (Carlsen et al., 2004). By IHC assay, we found that both the Ttf1-positive lung cancer cells and Cd68-positive macrophages were stained positive for Cxcl13, but Ttf1-positive cells constituted more than 95% of the cellular component of the lung tumor tissues of the mice (Figure 2G). This observation was confirmed by immunofluorescence assay (Figure 2H) using antibodies against Cxcl13 (green), Cd68 (red), and Ttf1 (white). These results indicate that Ttf1 positive lung cancer cells were the main source of Cxcl13 in mice exposed to BaP. We further showed that the anti-inflammatory drug dexamethasone (DEX) (Wattenberg and Estensen, 1996) down-regulated Cxcl13 mRNA and protein, and reduced the Cxcl13 serum concentration (Figure 2D–F). DEX reduced the tumor burden (Figure 2—figure supplement 1B,C, Figure 2D) and prolonged the survival (Figure 2I) of the mice. Cxcl12, which was shown to be associated with lung cancer (Teicher and Fricker, 2010), was not significantly changed in the BaP-treated mice (Figure 2—figure supplement 1E). BaP induces CXCL13 production by facilitating AhR translocation to the nucleus We screened for transcription factors that could regulate CXCL13, and found potential aryl hydrocarbon receptor (AhR) (Figure 3A) and V-Rel Avian Reticuloendotheliosis Viral Oncogene Homolog A (RelA) (data not shown) binding sites in its promoter. AhR is a ligand-activated transcription factor that binds the xenobiotic-responsive element (XRE) or aryl hydrocarbon response element (AHRE) to regulate genes in response to the planar aromatic hydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Fujisawa-Sehara et al., 1987; Lo and Matthews, 2012; Shimizu et al., 2000). The potential XRE-like sequence, 5’-GCCCAGGCTGGAGTGCAG-3’, is located at 1.7 kb downstream from the transcription start site (TSS; Figure 3A). The potential interaction between AhR and CXCL13 was analyzed by the chromatin immunoprecipitation (ChIP) assay, and the results showed that AhR could not bind CXCL13 in A549 cells without BaP treatment. Interestingly, AhR-CXCL13 interaction was detected and CXCL13 was significantly up-regulated in the presence of BaP, revealed by quantitative PCR (qPCR) (Figure 3B). However, significant interaction between AhR and CXCL12, CXCL14, CXCL11, or CXCL2 was not detected (Figure 3B). BaP significantly induced luciferase activity driven by the CXCL13 promoter containing XRE-like sequence; deletion of the XRE-like element, co-incubation with the AhR antagonist α-NF (Wilhelmsson et al., 1994) or transfection of an AhR-specific siRNA significantly reduced luciferase activity (Figure 3C–E). Figure 3 Download asset Open asset CXCL13 is a target gene of aryl hydrocarbon receptor (AhR). (A) The AhR binding site is located at 1.7 kb downstream of the CXCL13 transcription start site (TSS). (B) A chromatin immunoprecipitation (ChIP) assay was performed in BaP-treated or untreated 16HBE cells. The enriched CXCL13 was detected by qPCR. (C) The A549 cells were transfected with the wild-type (WT) or mutant (deletion mutation (mut) in the XRE-like sequence) CXCL13 promoter-luciferase reporter construct, treated with BaP and/or α-NF for 48 hr, and assessed by the luciferase assays. (D, E) A549 cells were transfected with AhR-specific siRNAs, and western blot was performed to detect the expression of AhR. Three siRNAs were used, and the result of one was shown (D). Luciferase assays were performed in A549 cells transfected with the WT CXCL13 promoter-luciferase reporter construct and siRNAs in the absence or presence of BaP (E). (F, G) Western blot analyses of AhR in the cytoplasm and nucleus of 16HBE cells co-incubated with BaP, with or without α-NF treatment (F) or siRNA transfections (G). (H, I) Immunofluorescence assays of AhR expression in 16HBE cells co-incubated with BaP, with or without α-NF treatment (H) or siRNA transfections (I). (J, K) CXCL13 mRNA (detected by qPCR; J) and protein (in supernatants of the cells detected by ELISA; K) levels in the AhR-silenced 16HBE cells treated with BaP and/or α-NF. https://doi.org/10.7554/eLife.09419.011 TCDD can activate AhR by inducing its translocation from the cytoplasm to nucleus (Pollenz et al., 1994). We reported that α-NF and siAhR drastically inhibited BaP caused translocation of AhR to nucleus in 16HBE cells (Figure 3F–I). These results might explain why α-NF and siAhR significantly decreased CXCL13 expression (Figure 3J) and concentration in the supernatant (Figure 3K) in BaP-treated 16HBE cells. Knockdown of Cxcl13 attenuates BaP-induced lung cancer To uncover the role of CXCL13 in BaP-induced lung cancer, Cxcl13 knockout mice (Cyster et al., 2000) were treated with 100 mg/kg BaP twice a week for 8 weeks (Figure 4—figure supplement 1A,B), and the tumor burden of the mice was evaluated. First, tumor nodules in histologic sections of mice upon BaP treatment were analyzed as described (Tan et al., 2013), and the results showed that at treatment time points of 120 days, 180 days, and 240 days, Cxcl13-/- mice had much fewer lesions than Cxcl13+/- and Cxcl13+/+ mice (Figure 4A). Then, microCT was used to detect visible tumors in mice 240 days after BaP treatment. We found that Cxcl13-/- mice harbored much fewer lung tumors than Cxcl13+/- and Cxcl13+/+ mice (Figure 4B). Consistent with this observation, the tumor volume of Cxcl13-/- mice was significantly smaller than Cxcl13+/- and Cxcl13+/+ mice (Figure 4C). In Cxcl13+/+ and Cxcl13-/+ mice, the Cxcl13 serum concentrations were elevated 3 months after BaP treatment, at which time point no tumors were detected (Figure 4D). Moreover, the life span of BaP-treated Cxcl13-/- mice was prolonged compared with the Cxcl13+/+ and Cxcl13-/+ mice (Figure 4E). These results indicate that CXCL13 is required for BaP-induced lung cancer. Figure 4 with 1 supplement see all Download asset Open asset Cxcl13 and Cxcr5 are critical to benzo(a)pyrene (BaP)-induced lung cancer. (A) Cxcl13 deficiency mice were treated with BaP, sacrificed 120 days, 180 days or 240 days later, and the tumor nodules in histologic sections were analyzed. See also (Figure 4—figure supplement 1). (B) MicroCT scanning images and HE staining of lung sections from the BaP-treated Cxcl13 wild-type (WT) or knockout mice. (C) Tumor volume of the microCT scanning of the mice. (D) Serum concentrations of Cxcl13 in the BaP-treated Cxcl13 WT or knockout mice. (E) Life span of the BaP-treated Cxcl13+/+, Cxcl13+/-, and Cxcl13-/- mice. (F, G) Cxcr5 expression in non-small cell lung cancers (NSCLCs, n=24; F) and in A/J mice treated with BaP (n=6 for each group; G). (H) Cxcr5 deficiency mice were treated with BaP, sacrificed 120 days, 180 days or 240 days later, and the tumor nodules in histologic sections were analyzed. See also Figure 4—figure supplement 1. (I) MicroCT scanning images and HE staining of lung sections from BaP-treated Cxcr5 WT or knockout mice. (J) Tumor volume of the microCT scanning of the mice. (K) Life span of the BaP-treated Cxcr5 WT or knockout mice. *p<0.05; **p<0.01. https://doi.org/10.7554/eLife.09419.012 CXCL13-CXCR5 signaling is critical for BaP-induced lung cancer CXCL13 primarily functions by binding to the G protein coupled receptor CXCR5, and CXCL13 is currently the only known ligand of CXCR5 (Förster et al., 1996; Lazennec and Richmond, 2010). We tested the expression of CXCR5 in 24 NSCLCs, and found that it was" @default.
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• W4239799318 date "2015-09-02" @default.
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• W4239799318 title "Decision letter: The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution" @default.
• W4239799318 doi "https://doi.org/10.7554/elife.09419.019" @default.
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