2022 Volume 45 Issue 6 Pages 793-797
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of dioxins and polycyclic aromatic hydrocarbons. Recent studies have suggested that AhR is involved in cancer immunity. In the present study, we examined whether AhR regulates the expression of immune checkpoint genes in breast cancer cells. We discovered that the mRNA expression of V-set domain containing T cell activation inhibitor 1 (VTCN1) that negatively regulates T cell immunity was upregulated by AhR agonists in breast cancer cell lines, MCF-7 and T47D. Furthermore, AhR knockout or knockdown experiments clearly demonstrated that upregulation of VTCN1 gene expression by 3-methylcholanthrene was AhR dependent. Luciferase reporter and chromatin immunoprecipitation assays revealed that this upregulation of VTCN1 gene expression was induced by the recruitment of AhR to the AhR responsive element in the VTCN1 gene promoter in MCF-7 cells. Taken together, AhR directly regulates VTCN1 gene expression in MCF-7 cells.
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor.1) Upon binding to ligands, AhR translocates from the cytoplasm to the nucleus and forms a complex with the aryl hydrocarbon receptor nuclear translocator (Arnt). This complex binds to the xenobiotic response element (XRE) and upregulates the expression of its target genes such as CYP1A1 and CYP1B1.
We have investigated the roles of AhR in breast cancer cell lines. We reported that ligand-activated AhR suppressed tumorsphere (mammosphere) formation in MCF-7 cells.2–5) On the other hand, we also found that AhR promoted cell migration and mammosphere formation in human epidermal growth factor receptor 2 overexpressing breast cancer cell lines.6,7) In addition, AhR is involved in doxorubicin resistance in MDA-MB-231 cells.8) Thus, AhR shows complicated actions in breast cancer cells, and therefore, further investigations are necessary to understand the role of AhR in breast cancer cells.
In recent years, it has been increased interest in understanding the role of AhR in cancer immunity. AhR mediates tobacco-induced programmed cell death 1 ligand 1 (PD-L1) expression and is associated with response to immunotherapy.9) Moreover, AhR suppresses antitumor immune responses in oral squamous cell carcinoma.10) These studies suggest that AhR modulates cancer immunity.
The B7 gene family regulates both stimulatory and inhibitory effects on T cell responses. Therefore, the B7 gene family is important in cancer immunity. The B7 family comprises ten members, including CD80 (B7-1), CD86 (B7-2), CD274 (PD-L1/B7-H1), programmed cell death 1 ligand 2 (PDCD1LG2/B7-DC/PD-L2), inducible T cell costimulator ligand (ICOSLG/B7-H2), CD276 (B7-H3), V-set domain containing T cell activation inhibitor 1 (VTCN1/B7-H4/B7S1/B7x), V-set immunoregulatory receptor (VSIR/VISTA/B7-H5), natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1/B7-H6), and HERV-H LTR-associating 2 (HHLA2/B7-H7).11) It is reported that cigarette smoke, benzo[a]pyrene (B[a]P), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induce PD-L1 expression via the AhR.9,10) However, whether the other immune checkpoint genes are regulated by AhR is unknown.
In the present study, our attention was focused on the effect of AhR on the expression of immune checkpoint genes in order to obtain new insight into the role of AhR in breast cancer cells.
3-Methylcholanthrene (3MC) and B[a]P were obtained from FUJIFILM Wako (Osaka, Japan). β-Naphthoflavone (β-NF) was obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Indirubin and 6-formylindolo[3,2-b]carbazole (FICZ) were obtained from Cayman Chemical (Ann Arbor, MI, U.S.A.).
Cell CultureThe human breast cancer cell lines MCF-7 and T47D were purchased from the American Type Culture Collection (Manassas, VA, U.S.A.). AhR knockout MCF-7 (AhR-KO) cells were generated with the clustered regularly interspaced short palindromic repeats CRISPR-associated proteins 9 (CRISPR/Cas9) system.2) Cells were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Kyoto, Japan), supplemented with 10% fetal bovine serum (Sigma-Aldrich), penicillin (100 units/mL), and streptomycin (100 µg/mL) at 37 °C with 5% CO2.
Real-Time PCRTotal RNA was extracted from the cells using ISOGEN II (Nippon Gene, Tokyo, Japan). Reverse transcription was performed using the ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). PCR was performed using Quick Taq HS Dye Mix (Toyobo) and TaKaRa PCR Thermal Cycler Dice Gradient (94 °C for 2 min, 40 cycles of 94 °C for 30 s, 55 °C for 30 s, and 68 °C for 30 s). Genes whose amplification was confirmed by PCR were evaluated by real-time PCR, and genes whose amplification could not be confirmed were judged as not detected. Real-time PCR was performed on a Thermal Cycler Dice Real Time System Lite (TaKaRa, Shiga, Japan) using TB Green Premix Ex Taq II (TaKaRa). Real-time PCR conditions were as follows: 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60–64 °C for 60 s. CYP1A1 promoter region was as follows; 95 °C for 30 s, 40 cycles of 95 °C for 5 s, 50 °C for 30 s, and 72 °C for 30 s. The expression of each target mRNA relative to ribosomal protein lateral stalk subunit P0 (RPLP0) was calculated based on the standard curve method. Primer sequences are listed in Supplementary Table 1.
AhR Knockdown ExperimentT47D cells were transfected with negative control dicer-substrate short interfering RNA (siNC) (DsiRNA, Integrated DNA Technologies (Coralville, IA, U.S.A.)) or AhR-specific DsiRNA (siAhR) (hs.Ri.AHR.13.2, Integrated DNA Technologies) using Lipofectamine RNAiMAX Reagent (Invitrogen, Carlsbad, CA, U.S.A.). The final concentration of DsiRNA used was 5 nM. After 2 d, the cells were treated with 2 µM 3MC for 24 h. Then, the cells were harvested, and total RNA was extracted.
Plasmid Construction and Reporter Gene AssayThe XRE-driven luciferase reporter plasmid (pGL3-XRE) was created as previously described.12) A fragment of the 5′-regulatory region (−465 to +72) of human VTCN1 gene was amplified by PCR using PrimeSTAR Max DNA Polymerase (TaKaRa). The amplified fragment was ligated into the Kpn I and Xho I sites of the firefly luciferase reporter vector, pGL4.10 (Promega, Madison, WI, U.S.A.). The plasmid thus obtained was designated as VTCN1-wt. VTCN1 promoter without the XRE sequence (−56 to −52 bp) plasmid (VTCN1-mut) was created by inverse PCR using VTCN1-wt as a template. Primer sequences are listed in Supplementary Table 1. The reporter plasmids and Renilla luciferase vector, pGL4.74 (Promega), were transfected into MCF-7 and AhR-KO cells using PEI Max reagent (Polysciences, Warrington, PA, U.S.A.). The luciferase activity was determined with the Dual Luciferase Reporter Assay System (Promega).
Chromatin Immunoprecipitation (ChIP) AssayCells were treated with 2 µM 3MC for 24 h. Then, the cells were harvested and subjected to ChIP assays using a Simple ChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology (Beverly, MA, U.S.A.)). Chromatins were immunoprecipitated with anti-AhR (#83200, Cell Signaling Technology) or normal immunoglobulin G (IgG) (PM035, MBL Life Sciences, Nagoya, Japan) antibodies overnight at 4 °C. After immunoprecipitation, the DNA fragments were amplified by real-time PCR. Primer sequences are listed in Supplementary Table 1. The CYP1A1 promoter region (−528 to −416) and VTCN1 promoter region (−171 to +55) were amplified by real-time PCR.
Statistical AnalysisStatistical analyses were performed with Student’s t-test or Dunnett’s multiple comparison test using KaleidaGraph software, version 4.1.1 (Synergy Software, Eden Prairie, MN, U.S.A.). A value of p < 0.05 was regarded as statistically significant.
Approximately 70% of all breast cancer is estrogen receptor (ER)-positive.13) The ER-positive breast cancer cell lines, MCF-7 and T47D, are widely used as experimental models for breast cancer research in vitro.14) Therefore, we evaluated whether AhR agonists affect the expression of immune checkpoint genes in MCF-7 and T47D cells. For the treatment conditions of 3MC, we referred to previous studies.2–4) As shown in Figs. 1A and B, the mRNA expression levels of CYP1A1 and VTCN1 were upregulated by 3MC in MCF-7 and T47D cells. NCR3LG1 mRNA expression was decreased by 3MC in MCF-7 cells but upregulated in T47D cells. CD276 mRNA expression was decreased in T47D cells, but not in MCF-7 cells. The mRNA expression levels of VSIR and PD-L2 were upregulated by 3MC in MCF-7 cells, but not detected in T47D cells. CD80, CD86, and HHLA2 mRNAs were not detected in MCF-7 and T47D cells (data not shown). Previously, cigarette smoke, B[a]P, and TCDD were found to induce PD-L1 expression via the AhR.9,10) However, PD-L1 mRNA expression was not changed by 3MC in MCF-7 and T47D cells (Figs. 1A, B). It is conceivable that the mechanism of AhR-induced PD-L1 expression may be different in tissues or cell types. Consequently, further investigations are necessary to clarify the mechanism of regulation of PD-L1 gene expression by AhR.
(A, B) MCF-7 and T47D cells were treated with solvent (0.1% DMSO) or 3MC (2 µM) for 24 h. The mRNA levels were determined by real-time PCR. Data are presented as the mean ± standard deviation (S.D.) over the solvent control (n = 3 or 4). ∗∗ p < 0.01. Plots indicate individual sample data. The graph shows the representative result of two independent experiments. N.D. means not detected.
As shown in Figs. 1A and B, only VTCN1 mRNA expression was increased by 3MC in both MCF-7 and T47D cells. VTCN1 negatively regulates T cell immunity.15) In addition, VTCN1 promotes cancer progression.16) Therefore, we focused on the regulation of VTCN1 gene expression by AhR. As shown in Figs. 2A and B, 3MC upregulated CYP1A1 mRNA expression in a dose dependent manner. VTCN1 mRNA expression was upregulated by 3MC at a concentration of 0.5 µM, and further upregulation was slightly dose dependent manner. Next, we investigated the effect of AhR agonists, other than 3MC, on the expression of VTCN1. All the AhR agonists tested including B[a]P, β-NF, indirubin, and FICZ significantly upregulated VTCN1 mRNA expression in MCF-7 cells (Fig. 2C). VTCN1 mRNA expression was upregulated by 3MC, B[a]P, indirubin, and FICZ, but not by β-NF in T47D cells (Fig. 2C).
(A, B) MCF-7 and T47D cells were treated with 3MC (0.5, 1, and 2 µM) or solvent (0.1% DMSO) for 24 h. (C) MCF-7 and T47D cells were treated with solvent (0.1% DMSO), 3MC (2 µM), B[a]P (2 µM), β-NF (10 µM), indirubin (1 µM) or FICZ (1 µM) for 24 h. The mRNA levels were determined by real-time PCR. Data are presented as the average of MCF-7 and T47D cells over the solvent control (mean ± S.D., n = 3 or 4). ∗∗p < 0.01. ∗p < 0.05. Plots indicate individual sample data. The graph shows the representative result of three independent experiments.
Figure 1 shows that gene expression regulation by 3MC, such as the NCR3LG1 gene, is different between MCF-7 and T47D cells. In addition, there was a large difference in the induction ratio of CYP1A1 by 3MC (0.5 µM) between MCF-7 and T47D cells (Figs. 2A, B). These results suggest that the mechanism of AhR-mediated regulation of gene expression may differ among AhR ligands and cell lines. Further research is needed to understand the differences between MCF-7 and T47D cells in the gene regulation mechanisms by AhR.
Next, we examined whether AhR is involved in the induction of VTCN1 gene expression by AhR agonists. Previously, we generated AhR-knockout MCF-7 (AhR-KO) cells. We found that 3MC did not upregulate the mRNA expression of either CYP1A1 or VTCN1 in AhR-KO cells (Fig. 3A). In T47D cells, we performed an AhR knockdown experiment using a short interfering RNA technique. As shown in Fig. 3B, AhR mRNA expression was inhibited by AhR-targeting siRNA (siAhR). Upregulation of CYP1A1 and VTCN1 mRNA expression by 3MC was attenuated by AhR knockdown (Fig. 3B). These results indicate the upregulation of VTCN1 mRNA expression by 3MC is mediated by AhR.
(A) AhR-KO cells were treated with solvent (0.1% DMSO) or 3MC (2 µM). (B) T47D cells were transfected with negative control siRNA (siNC) or AhR-targeting (siAhR). The next day, cells were treated with solvent (0.1% DMSO) or 3MC (2 µM) for 24 h. The mRNA levels were determined by real-time PCR. Data are presented as the average of AhR-KO and T47D cells over that in solvent control (mean ± S.D., n = 3 or 4). ∗∗ p < 0.01. Plots indicate individual sample. The graph shows the representative result of three independent experiments.
In silico analysis identified a putative XRE (CACGC) was present 52 to 56 bp upstream of the transcription start site of the VTCN1 gene. To identify whether the XRE of VTCN1 promoter was involved in the induction of VTCN1 gene expression by AhR, we constructed two luciferase reporter plasmids with the VTCN1 gene promoter region (−465/ + 72 bp); one with XRE (VTCN1-wt) and the other without XRE (VTCN1-mut) (Fig. 4A). pGL3-XRE plasmid (XRE-luc) was used as a positive control. The luciferase activity of XRE-luc or VTCN1-wt was increased by 3MC in MCF-7 cells. The induction of VTCN1-mut luciferase activity was attenuated compared to that of VTCN1-wt luciferase activity (Fig. 4B). 3MC did not increase luciferase activity in AhR-KO cells transfected with XRE-luc, VTCN1-wt, or VTCN1-mut (Fig. 4C). To determine whether AhR was recruited to the VTCN1 promoter region containing XRE, we performed ChIP assay. As shown in Fig. 4D, 3MC-activated AhR was recruited to both the CYP1A1 and VTCN1 promoter regions in MCF-7 cells, but not in AhR-KO cells. These results demonstrate that activated AhR directly regulates VTCN1 gene expression at the transcriptional level.
(A) Schematic representation of the reporter gene plasmids. (B, C) MCF-7 and AhR-KO cells were co-transfected with XRE-luc, VTCN1-wt or VTCN1-mut luciferase reporter plasmids, and pGL4.74 plasmid. The next day, cells were treated with solvent (0.1% DMSO) or 3MC (2 µM) for 24 h. Luciferase activity was determined using the Dual-Luciferase Reporter Assay System. The experiments were performed in three or four independent wells and repeated three times. ∗∗p < 0.01. Plots indicate individual sample data. (D) MCF-7 and AhR-KO cells were treated with solvent (0.1% DMSO) or 3MC (2 µM) for 24 h. ChIP assay was carried out and the precipitates were analyzed by real-time PCR. Data are presented as the mean ± S.D. (n = 3). * p < 0.05. Plots indicate individual sample data.
Previously, VTCN1 expression has been shown to be increased by hypoxia via the hypoxia-inducible factor-1α (HIF-1α).17) ChIP assay demonstrated that HIF-1α binds to the hypoxia response element (HRE, TGCACGCAC) within the VTCN1 gene promoter.17) Because XRE overlaps with HRE, the deletion of XRE in the VTCN1 promoter plasmid in our study was the same region. It is thought that VTCN1 gene expression is regulated by AhR and HIF-1α in the same region. Arnt forms a heterodimer complex with AhR and HIF-1α, and may be required for VTCN1 gene expression. A previous study reported that the AhR agonist, 4-n-nonylphenol upregulates the VTCN1 via the release of interleukin-2 (IL-2) that is functionally relevant as inhibition of VTCN1 prevented the induction of regulatory T cells.18) ChIP-sequencing analysis suggested that TCDD-activated AhR may regulate VTCN1 expression,19) but the details were unknown. Ours is the first report of the transcriptional regulatory mechanism of VTCN1 gene expression by AhR in MCF-7 cells.
Collectively, we have shown that AhR directly regulates VTCN1 gene expression at the transcriptional level. Overexpression of VTCN1 gene is linked to suppression of tumor immunity. AhR-dependent upregulation of VTCN1 gene should be further studies in vitro with wide variety of cell lines but also in vivo.
This work was supported by JSPS KAKENHI Grant Nos. 19K23811 and 21K15307 (to N.Y.).
The authors declare no conflict of interest.
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