Loss of aryl hydrocarbon receptor and long-term, but not short-term treatment with BAY2416964 alters gene expression profiles and reduces proliferation and migration of PyMT mammary cancer cells
The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that 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. BAY2146964 and GNF351 effectively inhibited kynurenine-dependent increases in Cyp1a1 and Cyp1b1 mRNA levels. However, single doses of 10 mM BAY2416964, but not 10 mM GNF351, exhibited agonist activity reflected by weak increases Cyp1a1 and Cyp1b1 mRNA levels. CRISPR/Cas9-generated PyMT AhrKO cells exhibited reduced cell proliferation compared with controls, but treatment with 1 mM BAY2416964 for 96 h had no effect on the proliferation of wildtype cells. To examine the differences between AHR knockout and short term BAY2416964 further, we generated long-term BAY2416964 (LT-BAY) by exposing wildtype cells to 1 mM BAY2416964 for at least 6 weeks. Similar to AhrKO cells, LT-BAY cells exhibited reduced cell proliferation and migration compared with wildtype cells; although not to the same extent as that observed with AhrKO cells. RNA sequencing studies identified 714 differentially expressed genes (DEGs) that overlapped between LT-BAY and AhrKO cells, but none after 24 h exposure with 1 mM BAY2416964. Our data reveal BAY2416964 exhibits weak AHR agonist activity in PyMT cells and that long-term pharmacological inhibition of AHR more closely mimicked AHR loss. Overall design: PyMT WT, AhrKO and LT-BAY cells were plated in a 12-well plate at a density of 1×10^5 cells/well in quadruplicates. Total RNA was isolated with the Aurum™ Total RNA isolation kit (BioRad), following manufacturer's protocol and were subjected to RNA sequencing