Supplemental Figure S4. NR4A1 antagonist inhibit 4T1-luc cell growth and invasion, and induce apoptosis. 4T1-Luc cells were treated with different concentrations of CDIM8 or Cl-OCH3 and effects on cell proliferation (A), invasion (B) and induction of Annexin V staining (C) were determined as outlined in the Materials and Methods. Results are expressed as means {plus minus} SD for at least 3 replicate determinations per treatment group and significant (p<0.05) effects caused by the NR4A1 antagonists are indicated (*). The control (solvent) results are set at 1.0.
ARTICLE ABSTRACT
PD-L1 is expressed in tumor cells and its interaction with PD-1 plays an important role in evading immune surveillance; this can be overcome using PD-L1 or PD-1 immunotherapy antibodies. This study reports a novel approach for targeting PD-L1. In human breast cancer cell lines and 4T1 mouse mammary tumor cells, PD-L1 expression was regulated by the nuclear receptor NR4A1/Sp1 complex bound to the proximal germinal center (GC)-rich region of the PD-L1 gene promoter. Treatment of breast cancer cells with bis-indole–derived NR4A1 antagonists including 1,1-bis(3′-indolyl)-1-(3-chloro-4-hydroxy-5-methoxyphenyl)methane (Cl-OCH3) decreased expression of PD-L1 mRNA, promoter-dependent luciferase activity, and protein. In in vivo studies using a syngeneic mouse model bearing orthotopically injected 4T1 cells, Cl-OCH3 decreased tumor growth and weight and inhibited lung metastasis. Cl-OCH3 also decreased expression of CD3+/CD4+/CD25+/FoxP3+ regulatory T cells and increased the Teff/Treg ratio. Therefore, the potent anticancer activities of NR4A1 antagonists are also accompanied by enhanced antitumor immunity in PD-L1–expressing triple-negative breast cancer and thus represent a novel class of drugs that mimic immunotherapy.
These findings show that the orphan nuclear receptor NR4A1 controls PD-L1 expression and identify a chemical probe capable of disrupting this regulatory axis.
