Perfluorooctane Sulfonate (PFOS) and Related Compounds Induce Nuclear Receptor 4A1 (NR4A1)-Dependent Carcinogenesis

Abstract

Polyfluoroalkyl substances (PFAS) are widely used industrial compounds that have been identified as contaminants in almost every component of the global ecosystem, and in human studies, higher levels of PFAS have been correlated with increased incidence of multiple diseases. Based on the results of human and laboratory animal studies, we hypothesize that the orphan nuclear receptor 4A1 (NR4A1) may be a critical target for some PFAS such as the legacy linear polyfluorooctanesulfonate (PFOS) and other sulfonates. We show that PFOS and related compounds bound the ligand binding domain (LBD) of NR4A1 and induced the growth of several cancer cell lines and enhanced tumor growth in an athymic nude mouse model. Using NR4A1-responsive rhabdomyosarcoma Rh30 cells as a model, PFOS induced NR4A1-dependent cell proliferation and Rh30 cell migration and invasion. Moreover, in Rh30 cells, PFOS also induces several NR4A1-regulated genes including the PAX3-FOXO1 oncogene and downstream gene products, and in a chromatin immunoprecipitation assay, PFOS does not decrease NR4A1 binding to the promoter. These results demonstrate that PFOS is an NR4A1 ligand and enhances tumorigenesis through the activation of this receptor.

Figure 1

PFAS compounds bind to NR4A1. PFOS (A), PFOS-XST (B), PFHxS-XST (C), PFNS (D), and PFDS (E) were incubated with the ligand binding domain of NR4A1, and the binding curves were generated as outlined in the Materials and Methods section. Results obtained for the compounds alone (▼), ligand plus receptor uncorrected (●), and (ligand + receptor)–(ligand alone) corrected (○). The K_D values were determined for all compounds that bound NR4A1 and not PFHxS-XST which exhibited minimal quenching of fluorescence; the K_D values were 2.99 (PFOS), 0.92 (PFOS-XST), 0.24 (PFNS-XST), and 9.12 (PFDS-XST) μmol/L.

Figure 2

Comparative induction and growth-promoting effects of DIM-3,5-Cl₂ and PFOS. (A) Structures of DIM-3,5-Cl₂ and PFOS. Comparative effects of DIM-3,5-Cl₂ and PFOS on the growth of A549 lung cancer (B) and Rh30 rhabdomyosarcoma (C) cells after treatment for 24 h. (D) Rh30 cells were transfected with GAL4-NR4A1 and UAS-Luc plasmids, and after treatment with PFOS or DIM-3,5-Cl₂ luciferase activity was determined as outlined in the Methods. Results are expressed as means ± SD for at least 3 replicates for each treatment groups, and significant (p < 0.05) induction or inhibition of growth or luciferase activity is indicated (*).

Figure 3

PFOS inducing cancer cell proliferation screening. HCT116 and RKO (A), CT26 and MC38 (B), U87MG, and A172 (C), and T98 and MIA PaCa-2 (D) were treated with PFOS (0.1–25 μmol/L) for 24 h, and cell proliferation was determined using the resazurin assay as outlined in the Methods. Results are expressed as means ± SD for at least 3 replicates for each treatment groups, and significantly (p < 0.05) increased or decreased growth is indicated (*).

Figure 4

Induction of Rh30 cell growth by polyfluoroalkyl sulfonates. Rh30 and SW480 (A) cells were treated with PFOS-XST for 24 h, and cell viability was determined as outlined in the Materials and Methods section. Rh30 cells were treated with PFNS-XST and PFHxS-XST (B) and PFHpS-XST and PFDS-XST (C) for 24 h, and cell viability was determined as outlined in the Materials and Methods section. (D) Rh30 cells were transfected with siCt1 or siNR4A1 and treated with 2.5 or 10 μM PFOS, and cell proliferation and Western blot analyses on whole cell lysates were determined as outlined in the Methods. (E) Cells were also transfected with siNR4A1 alone and after treatment with 2.5 μM polyfluoroalkyl sulfonates, and cell viability was determined as outlined in the Methods. Results are expressed as means ± SD for at least 3 replicate determinations for each treatment group, and significant (p < 0.05) induction or inhibition is indicated (*). The XST designation for these compounds indicates that they are purified linear polyfluoroalkyl sulfonates.

Figure 5

PFOS induces cell migration. (A) Rh30 cells were treated with DMSO (control) 2.5 or 10 μM PFOS for 24 h, and cell migration was determined in scratch assay and quantitated as outlined in the Materials and Methods section. A Boyden chamber assay (B) was also carried out in Rh30 cells as outlined in the Methods. The assays were carried out in triplicate; results are expressed as means ± SE, and significantly (p < 0.05) enhanced migration is indicated (*).

Figure 6

PFOS induces NR4A1-dependent gene products in the Rh30 cells. Rh30 cells were treated with 10 or 2.5 μM PFOS for 24 h, and whole cell lysates were analyzed for PAX3-FOXO1, N-Myc, and c-Myc (A) and other NR4A1-regulated gene products (B) by Western blots. Rh30 cells were treated with PFOS and DIM-3,5-Cl₂ alone or in combination and effects on cell proliferation (C), and gene products (D) were determined as outlined in the Methods. (E) Cells were treated with DMSO (control) PFOS (10 μM) and DIM-3,5-Cl₂ (12.5 μM), and interactions of NR4A1 with the G9a gene promoter were determined in a ChIP assay as outlined in the Methods. The Western blots were carried out in triplicate, and band intensities (means ± SD) were determined relative to GAPDH (control), and significant (p < 0.05) induction or inhibition is indicated (*).

Figure 7

In vivo studies. Athymic nude mice bearing Rh30 cells were treated with 20, 10, 0.5, and 0.2 mg/kg/day and tumor volumes (A), body weight (B), relative tumor weights (C), and Western blot analysis of tumor lysates (D) were determined as outlined in the Materials and Methods section. Results (A–D) are expressed as means ± SE, and significant (p < 0.05) induction is indicated (*).