Long-term exposure to BAY2416964 reduces proliferation, migration and recapitulates transcriptional changes induced by AHR loss in PyMT-induced mammary tumor cells

Abstract

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor which in certain cancer types drives pro-survival processes that facilitate tumorigenesis, malignant cell migration, invasion, and metastasis. Much of AHR's pro-tumorigenic action is due to its activation by the oncometabolite, kynurenine. Because of this AHR antagonists are being actively investigated as new anti-tumor therapy. In this study we compared the effects of treatment with the AHR antagonists, BAY2416964 and GNF351, to that of AHR knockout in PyMT murine mammary cancer cells. BAY2416964 and GNF351 effectively inhibited kynurenine-dependent increases in Cyp1a1 and Cyp1b1 mRNA levels. CRISPR/Cas9-generated PyMT Ahr^KO cells exhibited reduced cell proliferation compared with controls, but treatment with 1 μM BAY2416964 for 96 h had no effect on the proliferation of wildtype cells. To further examine the differences between AHR knockout and short term BAY2416964, we generated long-term BAY2416964 (LT-BAY) cells by exposing wildtype cells to 1 μM BAY2416964 for at least 6 weeks. Similar to Ahr^KO cells, LT-BAY cells exhibited reduced cell proliferation and migration compared with wildtype cells. No differentially expressed genes (DEGs) were identified in wildtype cells exposed to 1 μM BAY2416964 for 24 h; however, 46.4% of DEGs overlapped between Ahr^KO and LT-BAY cells including gene regulated cell proliferation. Our data reveal long-term pharmacological inhibition of AHR by BAY2416964 closely resembles AHR loss in a mouse model of breast cancer.

Keywords: BAY2416964; GNF351; PyMT; aryl hydrocarbon receptor; breast cancer; kynurenine; proliferation.

Figure 1

BAY2416964 inhibits kynurenine-induced AHR activity in PyMT mouse mammary cancer cells. Kynurenine increased Cyp1a1 (A), Cyp1b1 (B) and Parp7 (C) mRNA levels in a dose-response manner in PyMT cells as measured by RT-qPCR. Relative mRNA levels of Cyp1a1 (D) and Cyp1b1 (E) after dose-response treatment with BAY2416964. Cyp1a1 (F) and Cyp1b1 (G) mRNA levels after treatment with increasing doses of GNF351. Relative Cyp1a1 and Cyp1b1 mRNA levels after treatment with increasing amounts of BAY2416964 (H, I) or GNF351 (J, K) in the presence of 100 µM kynurenine. RT-qPCR results are generated by samples treated 6 h with test compounds and presented as mean ± S.E.M n=3. (L) Western blot of PyMT cells treated for 6 h with kynurenine, BAY2416964 or GNF351 at the concentrations indicated. Representative image n=3. (M) Quantification of western blot. (N) AHR protein in cytoplasmic and nuclear fractions after 6 h treatment with BAY2416964 or GNF351. Representative image n=3. (O) Protein quantification of cytoplasmic AHR relative to loading control (Tubulin). (P) Protein quantification of nuclear AHR relative to loading control (Lamin A/C). *p<0.05 compared with control (DMSO).

Figure 2

The murine pancreatic cancer cell line CR705 responds to BAY2416964 similarly as PyMT cells. Basel levels of Cyp1a1 (A) and Cyp1b1 (B) mRNA level decreased with 0.1 µM and 1 µM BAY2416964 and increased with 10 µM BAY2416964 in CR705 cells treated for 6 h. RT-qPCR of Cyp1a1 (C) and Cyp1b1 (D) mRNA levels in CR705 cells treated 6 h with kynurenine alone, or in combination with increasing doses of BAY2416964. RT-qPCR data presented as mean ± S.E.M. n=3 p<0.05. (E) Western blot of CR705 cells treated with 50 µM kynurenine or 10 µM BAY2416964. Representative image n=2. (F) Protein quantification of western blot presented in (E). *p<0.05 compared with control (DMSO).

Figure 3

Loss of AHR prevents increases in kynurenine-induced AHR target genes and reduces proliferation of PyMT cells. (A) Ahr mRNA levels in PyMT Ahr^KO cells compared with WT cells as determined by RT-qPCR. Significance was determined with Student’s t-test, n=3 (B) Western blot of WT and Ahr^KO cells detected no AHR protein in the PyMT Ahr^KO cell line. Representative image of n=3. AHR activity was determined in the PyMT WT and Ahr^KO cell lines by treatment with 100 µM kynurenine, 10 µM BAY2416964 or combination for 6 hours. Kynurenine and high concentration of BAY2416964 failed to increase Cyp1a1 (C), Cyp1b1 (D) and Parp7 (E) expression levels in Ahr^KO cells. RT-qPCR results are presented as mean ± S.E.M. of n=3. Significance was determined using Two-Way ANOVA, p<0.05. * Significant from PyMT WT control (DMSO). (F) Ahr^KO cells proliferate more slowly than WT cells. (G) BAY2416964 did not affect proliferation of PyMT WT cells. (H) Proliferation of PyMT WT cells treated with increasing doses of GNF351. Treatment with 10 µM GNF351 decreased proliferation of PyMT WT cells. Proliferation of Ahr^KO cells treated with increasing concentrations of BAY2416964 (I) or GNF351 (J). Only 10 µM GNF351 affected proliferation of Ahr^KO cells compared to DMSO control. WT was added for comparison. Data are presented as mean ± S.E.M of n=12, measured by IncuCyte. Significance was determined using Area under curve with p<0.05.

Figure 4

Characterization of Ahr^KO and long-term BAY2416964 treated PyMT cells. (A) Western blot of PyMT WT, PyMT Ahr^KO and PyMT LT-BAY cells show AHR protein levels are not changed in PyMT LT-BAY cells compared to WT cells. Representative image of n=3. (B) Quantification of western blot from figure (A), presented as mean ± S.E.M of n=3. Relative Cyp1a1 (C) and Cyp1b1 (D) mRNA levels in PyMT WT, Ahr^KO and LT-BAY cells after exposure to 100 µM kynurenine for 6 hours, measured by RT-qPCR. Data are presented as mean ± S.E.M of n=3. *p<0.05 relative to WT DMSO, and ^#p<0.05 between Ahr^KO and LT-BAY determined by one-way ANOVA for DMSO samples only. ^$p<0.05 compared with WT DMSO and ^ap<0.05 significance between indicated comparisons determined by two-way ANOVA. (E) Proliferation of PyMT WT, Ahr^KO and LT-BAY cells measured by IncuCyte. Data are presented as mean ± S.E.M. of n=16. (F) Migration of WT, Ahr^KO and LT-BAY cells in a transwell assay measured by xCELLigence. Data are presented as mean ± S.E.M. of n=8. Significance was determined by area under curve, p<0.05.

Figure 5

RNA sequencing revealing expressional effects of AHR inhibition. mRNA from PyMT WT, Ahr^KO and LT-BAY cells was sequenced and analyzed of n=4, p<0.01. (A) Principal component analysis (PCA) plot of the 4 replicates. (B) Heatmap illustrating the relative gene expression profiles of overlapping differentially expressed genes in Ahr^KO and LT-BAY cells relative to WT for each replicate. There is close homogeneity among the replicates, in addition to a greater effect of the genetic loss of Ahr, compared to pharmacological inhibition with BAY2416964. (C) Venn diagram of significantly changed genes in Ahr^KO or LT-BAY cells compared with WT. Top up- and downregulated genes in Ahr^KO (D) and in LT-BAY (E) compared with WT. Upregulated genes are marked in red, and downregulated genes in blue, with the size of the dot indicating the level of significance. (F) Presentation of AHR target genes that are significantly different in Ahr^KO compared to WT, and in LT-BAY compared to WT. (G) Comparison of the top 12 commonly regulated pathways in Ahr^KO or LT-BAY vs WT identified by ingenuity pathway analysis. Significantly changed pathways are defined by a Z-score >2 or <-2.

Figure 6

Ectopic AHR expression partially rescued the top up- and downregulated genes in the PyMT Ahr^KO cell line. The top two up- and downregulated genes in Ahr^KO cells were tested by RT-qPCR in WT, Ahr^KO and LT-BAY cells. (A) Cdh2 was strongly upregulated in PyMT Ahr^KO cells, and slightly upregulated in LT-BAY cells compared with WT. (B) Grb10 was upregulated in Ahr^KO cells, but not in LT-BAY cells. Both Mmp2 (C) and Gas1 (D) were downregulated in Ahr^KO and LT-BAY cells compared to WT cells. Data are presented as mean ± S.E.M. of n=3. (E) Western blot of Ahr^KO cells transfected with empty vector (-) or pcDNA3.1-Ahr. Representative image of n=3. Cdh2 (F) and Grb10 (G) mRNA levels were decreased after overexpression of AHR compared with empty plasmid at both time points. Mmp2 (H) and Gas1 (I) mRNA levels were increased 72 h after overexpression of AHR. Data are presented as mean ± S.E.M. of n=3, p<0.05.