AhR Promotes the Development of Non-small cell lung cancer by Inducing SLC7A11-dependent Antioxidant Function

Objective: Aryl hydrocarbon receptor (AhR) is a transcription factor. It is reported that AhR is associated with non-small cell lung cancer (NSCLC), but the mechanisms underlying this relationship remain unclear. Therefore, we investigated the role of AhR in NSCLC to elucidate the underlying mechanisms. Methods: We collected clinical lung cancer samples and constructed AhR overexpression and knockdown cell lines to investigate the tumorigenicity of AhR in vivo and in vitro. Furthermore, we performed a ferroptosis induction experiment and chromatin immunoprecipitation experiment. Results: AhR was highly expressed in NSCLC tissue. AhR knockdown cells showed ferroptosis related phenomenon. Furthermore, Chromatin immunoprecipitation confirmed the correlation between AhR and solute carrier family 7 member 11 (SLC7A11) and ferroptosis induction experiment confirmed that AhR affects ferroptosis via SLC7A11. Specifically, AhR regulates ferroptosis-related SLC7A11, which affects ferroptosis and promotes NSCLC progression. Conclusions: AhR promoted NSCLC development and positively correlated with SLC7A11, affecting its actions. AhR bound to the promoter region of SLC7A11 promotes NSCLC by activating SLC7A11 expression, improving the oxidative sensitivity of cells, and inhibiting ferroptosis. Thus, AhR affects ferroptosis in NSCLC by regulating SLC7A11, providing foundational evidence for novel ferroptosis-related treatments.


Introduction
Globally and in China, lung cancer is one of the most common malignancies with the greatest fatalities. More than 80% of lung cancers are non-small cell lung cancer (NSCLC). Furthermore, air pollution is becoming increasingly worse as China enters the late stage of industrialization. Thus, the incidence of NSCLC in China will likely continue to increase over the next 30 years. Therefore, under-Ivyspring International Publisher standing the pathogenesis of and how to prevent NSCLC is crucial [1,2].
Aryl hydrocarbon receptor (AhR) is a member of the transcription factor family that requires ligand activation, and its classic high-affinity ligand, tetrachlorodibenzo-p-dioxin, is a common industrial pollutant [3]. AhR is a multi-functional protein involved in growth, development, inflammation, tumors, the cell cycle, and cell proliferation, differentiation, and migration [4][5][6]. Currently, controversy exists about AhR's role in cancer and cancer cells. AhR directly and indirectly regulates the expression of various genes, such as those for vital functional proteins (e.g., interleukin-6 and -21 [cytokines], vascular endothelial growth factor, epiregulin, and amphiregulin [growth factors], cyclin D, and matrix metalloproteinase [a matrix protein]) and transcription factors (e.g., Slug) [7]. Therefore, the functions and mechanisms of AhR often vary among tissues, cells, and their molecular microenvironments [8][9][10].
The latest research shows that benzopyrene, found in cigarette smoke and air pollutants, can induce programmed death-ligand 1 expression in lung epithelial cells through AhR; thus, it escapes the body's adaptive immunity, promoting tumor occurrence [11]. Our previous study found that benzopyrene activates AhR nuclear transport, leading to the malignant transformation of NSCLC [9]. Others have also reported that AhR has an important role in tumor development [7,12] and that inhibiting its signaling pathway with AhR antagonists significantly inhibited the malignant evolution and invasion of tumors [13][14][15]. Our previous research also found that AhR independently activates downstream target genes [16], AhR pathway activation has been associated with stem-like peculiarities and radiation resistance in tumor cells [17] and can be stabilized by de-ubiquitination [18]. Therefore, downstream molecules are recruited for various roles after exogenous factors activate AhR, which may affect the expression of ferroptosis-related genes, such as SLC7A11, and thus the ferroptosis process.
Cell death occurs at the end of a cell's life, with essential roles in the body's survival and development. Ferroptosis is a new form of cell death, differing from apoptosis, autophagy, and necrosis. This type of cell death primarily depends on iron metabolism and is characterized by the accumulation of reactive oxygen species (ROS) [19,20]. Intracellular signaling pathways closely regulate ferroptosis, including the iron homeostasis regulatory, RAS, and cystine transport pathways [21]. In addition, cystine transport plays an essential role in regulating ferroptosis [22]; cystine replaces xcintracellular glutamate via the cystine-glutamate antiporter system, and excessive lipid ROS causes ferroptosis [23]. Solute carrier family 7 member 11 (SLC7A11) is a key component of system xc -, and reports indicate that oxidative and metabolic stress can induce SLC7A11 expression [24,25]. Furthermore, the activity and stability of SLC7A11 regulate the cells' antioxidant capacity [26] and then affect ferroptosis [27]. Thus, SLC7A11 is a key regulatory molecule for ferroptosis.
To date, the mechanisms underlying the associations among AhR, NSCLC, and ferroptosis remain unclear. Therefore, this study investigated the potential effects of AhR on ferroptosis and NSCLC to elucidate the underlying mechanisms.

Cell proliferation, migration and invasion, and plate colony-formation assays
We followed the previously described procedures by SY et al. and JY et al. [30,31].

Total and lipid ROS and intracellular iron measurements
We followed previously described procedures for these measurements [23].

Flow cytometry
Cells were stained with antibodies or fluorescent dyes following standard protocols (D3861-BODIPY™ 581/591 C11; Thermo Fisher Scientific, Waltham, MA, USA) before being loaded for flow cytometry. Fluorescein-green and Texas-red were used to detect the labeled cells. The analysis was performed using FlowJo software (FloJo LLC., Ashland, OR, USA).

Reverse transcription-quantitative real-time PCR
We followed previously described procedures [23].

Histology and immunohistochemistry (IHC)
The Pathology Department of Xiangya Hospital in China provided the lung cancer samples. Details of the immunohistochemical analysis of paraffin sections of the lung cancer tissue have already been published [23]. Two pathologists at Xiangya Hospital in Changsha, China, performed the differential quantifications.

Nude mice experiment
Four-week-old female severe combined immune-deficiency mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Hunan, China). The Animal Protection and Use Agency Committee of Xiangya Medical College of Central South University approved all animal experiment protocols, which complied with the legal authorization and federal animal protection and conservation guidelines.
Each mouse was subcutaneously injected with AhR overexpression or knockdown cells (1 × 10 6 cells/animal). The control mice were injected with the same concentration of cells transfected with a control plasmid. The tumor volume and body weight were measured every three days until day 31.

Statistical analyses
The experiments were repeated at least three times. The results are presented as means ± standard deviations, as indicated. All statistical analyses were performed using Prism GraphPad Software (version 8.0, GraphPad Software, San Diego, CA, USA). Student's t-tests were used to compare two groups, and an analysis of variance was used to compare three or more groups. P-values of <0.05 were considered statistically significant.

AhR is upregulated in NSCLC at the protein level
To clarify the role of AhR in NSCLC, we extracted mRNA from clinical non-small cell lung cancer samples for analysis, finding that the AhR mRNA level did not differ between lung adenocarcinoma (ADC; Fig. 1A) and squamous cell carcinoma (SCC; Fig. 1B) tissues and adjacent tissues. Subsequently, we evaluated AhR protein expression in ten NSCLC and adjacent normal tissue pairs by western blot (Fig. 1C), which showed upregulated AhR protein expression in both ADC and SCC tissues. This result suggested that AhR regulation occurred at the post-transcriptional level.
Simultaneously, we performed IHC analyses on NSCLC and adjacent normal tissue samples to confirm the AhR expression level. AhR expression was significantly higher in SCC and ADC tissues than in the normal adjacent tissue (Fig. 1D). Additionally, the AhR IHC scores in the SCC and ADC tissues were higher than those in adjacent normal tissues (Fig. 1E). These results suggested that AhR expression may increase in human NSCLC, and AhR may play a vital biological role.

AhR knockdown inhibits NSCLC progression in vitro and in vivo
We measured AhR protein expression levels in different lung cancer cell lines ( Fig. 2A). We found low AhR levels in the PC9 and SPC-A-1 cells and an increased level in the A549 cells.
Next, we stably knocked down AhR in the A549 cell line using four different AhR-targeted shRNAs; shAhR #1 and shAhR #2 had relatively high knockdown efficiencies (Fig. 2B). Therefore, all experiments were performed with these two shRNAs, unless otherwise noted. The AhR-knockdown cells grow more slowly than the control cells (Fig. 2C) and had significantly inhibited colony-forming abilities (Fig. 2D). Furthermore, the AhR-knockdown cells had lower invasion and migration capacities than the carrier cells (Fig. 2E, F).  Finally, we injected A549 cells into nude mice to investigate tumor formation in vivo. AhR knockdown significantly reduced the tumor size, volume, and weight compared to the control mice ( Fig. 2G-I), but body weight did not differ between the two groups (Fig. 2J). These results indicated that AhR promotes non-small cell lung cancer development and thus has carcinogenic properties.

AhR overexpression promotes NSCLC progression
To clarify the physiological effects of AhR in NSCLC, we overexpressed AhR in PC9 and SPC-A-1 cells. Then, we assessed the overexpression efficiencies by western blot (Fig. 3A). AhR overexpression promoted PC9 and SPC-A-1 cell growth in vitro (Fig.  3B, C) and significantly improved their colonyforming abilities (Fig. 3D, E). In addition, the cell lines overexpressing AhR had stronger invasion and migration capabilities than the carrier cells ( Fig. 3F-I).
These results confirmed that AhR overexpression is related to cell growth, colony formation and cell migration and has carcinogenic influences.

AhR regulates ferroptosis through SLC7A11
When we knock down AhR, we found that there was a phenomenon related to ferroptosis in cells, for example, the total ROS level of A549 cells knocked down AhR would increas (Fig. 4A), suggesting that AhR may be related to ferroptosis.
Therefore, we performed a preliminarily ferroptosis-related gene expression analysis in lung ADC cells, finding Significant differential SLC7A11 expression after treating the cells with an AhR agonist and antagonist (Fig. 4B), suggesting that AhR may be related to SLC7A11.Furthermore, in order to explore the relationship between AhR and SLC7A11,we used the University of California Santa Cruz Genome Browser database and bioinformatics analyses showed that there might be AhR binding sites in the promoter region of SLC7A11 (Fig. 4C). We also identified a positive correlation between AhR and SLC7A11 (Fig. 4D). The Cancer Genome Atlas database analysis also showed higher SLC7A11 expression in ADC and SCC tissue than in adjacent tissues (Fig. 4E, F). Furthermore, high SLC7A11 expression was unfavorable for survival prognoses (Fig. 4G, I). Together, these results indicated that AhR might inhibit tumor cell ferroptosis by regulating SLC7A11 expression.

SLC7A11 positively correlates with AhR in NSCLC
Owing to previous analysis and experiments, we hypothesized a positive correlation between AhR and SLC7A11. To clarify this relationship, we detected the SLC7A11 protein level (by immunoblot) in lung cancer cell lines after overexpressing and knocking down AhR. The SLC7A11 protein level increased in the AhR overexpression cells (Fig. 5A) and decreased in the AhR knockdown cells (Fig. 5B). For further clarification, we transfected the AhR expression plasmid into the AhR knockdown A549 cell line, and then detected the AhR and SLC7A11 protein levels by western blot, which confirmed their positive relationship (Fig. 5C).
We also performed a ChIP analysis to detect AhR in the promoter region of SLC7A11. In PC9 cells, AhR was recruited into the SLC7A11 promoter. Furthermore, Ahr recruitment into the SLC7A11 promoter region was greater in PC9 cells overexpressing AhR than in the control cells (Fig. 5D).

AhR overexpression inhibits ferroptosis in NSCLC
SLC7A11 is a key molecule regulating ferroptosis, and we found that AhR positively correlated with SLC7A11, prompting us to explore the role of AhR in ferroptosis. First, we treated PC9 and SPC-A-1 cell lines overexpressing AhR with Erastin, a SLC7A11 inhibitor, to evaluate cell growth inhibition, the drug concentration followed previously described instructions [28,29]. Ahr overexpression decreased the growth inhibition of Erastin-treated PC9 and SPC-A-1 cell lines (Fig. 6A, B). Morphological observations of control cells (PC9 cells treated with DMSO) versus Erastin-treated PC9 and SPC-A-1 cells overexpressing AhR supported this result (Fig. 6C, D). These results suggested that AhR inhibits Erastin-induced cancer cell death.
Iron, glutathione (GSH), and lipid ROS concentrations in cells are surrogate markers of ferroptosis [21,32]. We observed that PC9 and SPC-A-1 cells treated with Erastin had a higher concentration of iron than the control group, but AhR overexpression inhibited this increase (ferrous ions and total iron concentrations; Fig. 6E-H). Furthermore, Erastin treatment decreased the total GSH level, but AhR overexpression inhibited this reduction (Fig. 6I, J). Finally, Erastin treatment increased the lipid ROS level, but AhR overexpression inhibited this increase (Fig. 6K, L). These findings indicated that AhR overexpression inhibits Erastin-induced cancer cell ferroptosis.

AhR knockdown promotes ferroptosis in NSCLC
To further clarify the role of AhR in ferroptosis in NSCLC, we tested the effects of Erastin on growth inhibition of AhR-knockdown A549 cells treated with Erastin. Knocking down AhR enhanced the Erastininduced inhibitory effect on A549 cell growth (Fig.   7A); the morphological observation aligned with this finding (Fig. 7B).  . Aryl hydrocarbon receptor (AhR) regulates ferroptosis via solute carrier family 7 member 11 (SLC7A11). A) The total ROS level was higher in AhR-knockdown A549 cells than in A549 cells without knockdown. B) AhR binding site predictions in the SLC7A11 promoter region using a University of California Santa Cruz Genome Browser database analysis and Algorithmics and Genetics Group (i.e., ALGGEN) website. C) Bioinformatics analysis identified a positive correlation between AhR and SLC7A11. D) SLC7A11 expression in A549 cells was significantly upregulated after treatment with an AhR agonist; expression did not change after treatment with an AhR agonist. E, F) The Cancer Genome Atlas database analysis showed that SLC7A11 expression in LUAD and LUSC is higher than that in adjacent tissues. G, I) High SLC7A11 expression is unfavorable for survival and prognosis. We also measured the total iron and ferrous concentrations in A549 cells after knocking down AhR, which increased compared to cells without AhR knockdown (Fig. 7C, D). Furthermore, the total GSH level was obviously lower in AhR knockdown cells than in those without knockdown (Fig. 7E). Finally, the lipid ROS level of A549 cells after AhR knockdown significantly increased after Erastin treatment compared to the DMSO group (Fig. 7F). Therefore, AhR knockdown promotes ferroptosis of Erastininduced cancer cells.
Overall, AhR may regulate iron, GSH, and lipid ROS levels in lung cancer cells by promoting SLC7A11 transcription, thereby inhibiting lung cancer cell ferroptosis and promoting non-small cell lung cancer development (Fig. 8).

Discussions
This study provides new evidence that AhR plays an important role in non-small cell lung cancer development. For the first time, we demonstrate that AhR, a ferroptosis inhibitor, influences carcinogenesis by promoting SLC7A11, an antioxidant system-related gene. Ferroptosis is a type of programmed cell death and a promising new target for treating tumors. Studies have reported strong relationships between ferroptosis and tumor progression [32][33][34][35][36], for example, ferroptosis-mediated radiation therapy for tumors [32] and tumor killing by immune cells [37]. Additionally, reports indicate that ferroptosis-related genes have vital roles in pancreatic cancer [38][39][40], suggesting that ferroptosis may be a novel way to inhibit cancer development.   Transcription factor AhR is a protein with several functions [41][42][43], including cell growth, development, proliferation, and tumor differentiation [7,44]. Its functions and mechanisms often vary based on tissue and cell type and their molecular microenvironment [45][46][47][48][49]. AhR is highly expressed in NSCLC, but there is no change in RNA level, the previous research results of our group have confirmed that this is based on the effect of de-ubiquitination [18]. Therefore, AhR is important for tumorigenesis, but how it affects tumor progression remains unclear. Elucidating these mechanisms will allow the identification of new targets for treating AhR-positive cancer. Similarly, AhR expression varies among NSCLCs, and the mechanism underlying the abnormal AhR expression remains unclear. Additionally, the results are inconsistent, with several reports indicating AhR downregulation in lung cancer [50], but others reported AhR overexpressed [51,52], consistent with our findings. Furthermore, some reports indicating AhR overexpression promoted NSCLC development.
Ferroptosis is characterized by an overwhelming, iron-dependent accumulation of lethal lipid ROS [53][54][55]. We demonstrated that AhR overexpression decreased the lipid ROS and iron concentrations and increased the total GSH concen-tration in human lung ADC cell lines, supporting AhR inhibition in ferroptosis. Ferroptosis is also characterized by an increase in lipid peroxide, which may be caused by compound-mediated inhibition of GSH peroxidase 4 (GPX4) or indirectly targeting GPX4 through GSH consumption. Additionally, system xcis an important antioxidant system in cells, in which SLC7A11 is responsible for the main transport activity with high specificity for cystine and glutamate [22,55]. System xcintracellular glutamate is used to exchange extracellular cystine [24,25], and then cystine is synthesized into GSH, catalyzed by glutamate-cysteine ligase and GSH synthetase. GSH is a reducing cofactor of the membrane lipid repair enzyme GPX4. Thus, increasing the activity of SLC7A11 increases the absorption of cystine [56,57], which affects GSH synthesis, increasing GPX4 activity [58,59]. Consequently, the antioxidant capacity of the cells is enhanced, inhibiting ferroptosis. Erastin induces ferroptosis, inducing ferroptosis by inhibiting the cysteine/glutamate reverse transporter system [60]. Erastin also inhibits SLC7A11, providing a reasonable way to study the role of AhR in ferroptosis in NSCLC. In this study, Erastin was mainly used to inhibit SLC7A11 and induce ferroptosis.In addition, the limitation of this experiment is that a complete pathway has not been found.
In conclusion, we demonstrated that AhR inhibits iron-dependent cell death (i.e., ferroptosis) in human non-small cell lung cancer cells by binding to the promoter region of SLC7A11, a transcription factor. Thus, it promotes SLC7A11 transcription, influencing tumor cell ferroptosis. Specifically, AhR upregulated SLC7A11 expression. Moreover, high SLC7A11 levels increased cystine absorption, promoted GSH synthesis, increased GPX4 activity, enhanced the antioxidant capacity of lung cancer cells, inhibited lung cancer cell ferroptosis, and promoted cancer development.
These findings highlight the indispensable role of AhR in tumor development via ferroptosis regulation, which may be a new treatment strategy for NSCLC.