{"paper_id":"c69e4da6-f9d4-4fd8-a41f-b9a78279acf0","body_text":"Polyfluoroalkyl substances (PFAS) have\nbeen manufactured for over\n80 years, and several thousand different individual PFAS have been\nsynthesized for industrial, consumer, food packaging, and cosmetic\napplications. 1 , 2  The widespread use of PFAS is\ndue to several factors which include their thermal and chemical stability,\nwater-repellent and flame-retardant activities, and amphipathic structures.\nThe extensive production, use, and disposal practices of PFAS have\nresulted in their contamination of the global ecosystem including\nthe marine and aquatic environments, fish, wildlife, food products,\nand humans. 3 , 4  PFAS exposures are complex, and in humans,\nthere is PFAS uptake from consumer products, food, environmental,\ncosmetic, and occupational exposures and contaminated water. Perfluorooctanoic\nacid (PFOA) and perfluorooctanesulfonate (PFOS) were among the most\nwidely produced PFAS, and although uses of these compounds are restricted\nor have been eliminated, these legacy PFAS are still detected with\nhigh frequency in environmental and human samples. 3 − 8  Several studies have investigated the association of PFAS levels\nin human serum samples with human diseases, and analysis of these\nstudies showed that higher PFAS levels were associated with increased\nincidence of metabolic disease, endocrine disorders, cardiovascular\ndisease, male and female reproductive tract problems, cancer, immune\neffects, urinary tract problems, and developmental toxicities. 8  For example, individuals with high serum PFAS\nexhibit a higher incidence of multiple cancers including pancreatic,\nrenal, thyroid, breast, and liver cancer. 9 − 23  The mechanisms of PFAS-mediated responses are complex and dependent\non the structure of the individual PFAS congener and the response.\nFor example, in laboratory animal studies, administration of PFOS\n(5 or 10 mg/kg/day) to wild-type and humanized (PPARα) mice\nfor 28 days resulted in changes in liver pathology and induction of\nACOX1 and CYP4A11 enzymes that are consistent with a PPARα-dependent\nresponse, and induction of these genes was not observed in PPARα\nknockout mice. However, liver toxicities such as hepatomegaly that\nare induced by PFOS were PPARα -independent. 24  There is also evidence that PFAS interacts with many other\nnuclear and cell surface receptors; however, direct linkages between\nthese interactions and PFAS-associated adverse health effects are\nlimited. 24 − 37\nStudies in this laboratory have identified a series of 1,1-bis(3′-indolyl)-1-(substitutedphenyl)methane\nanalogues (CDIMs) that bind the pro-oncogenic orphan nuclear receptor\n4A1 (NR4A1) and act as inverse NR4A1 agonists that inhibit NR4A1-dependent\ncancer cell growth and survival and enhance immune surveillance. 38 , 39  For several NR4A1-dependent responses such as decreased immunity,\nenhanced neurotoxicity, metabolic disease, endometriosis, stress/inflammation,\nand cancer, the effects of CDIMs and other NR4A1 ligands 38 − 46  on these responses are  inversely related  to\nthose observed for epidemiologic studies which show the association\nbetween increased incidence of these disease with individuals exposed\nto higher levels of PFAS. 9 − 23  For example, CDIMs act as inverse NR4A1 agonists and inhibit NR4A1-dependent\ncancer cell and tumor growth/viability, 38 , 47  whereas some\nPFAS such as PFOS induce cancer cell and tumor growth/viability. 48 , 49  Moreover, CDIMs downregulated the expression of the histone methyltransferase\ngene product G9a 45  in cancer cell lines,\nwhereas increased exposure to PFAS is associated with enhanced DNA\nmethylation. 50 − 55  Therefore, we hypothesize that NR4A1 plays a role in the toxicities\nassociated with PFAS.\nPrevious laboratory studies show that\nsome PFAS compounds enhance\nnontransformed breast and prostate cell growth and viability 48 , 49 , 53 , 56  but do not induce cell transformation. In contrast, the effects\nof PFOS in vivo and in cell culture are dependent on the animal model,\nconcentration of PFOS, and cancer cell context since inhibition or\ninduction and no effects on cancer cell growth have been observed\nin studies using PFOS. 49 , 57 − 62  This work investigates the effects of PFOS and structurally related\ncompounds as NR4A1 ligands and NR4A1 agonists in cancer cells.\n\nRh30 rhabdomyosarcoma, SW480, HCT116, RKO, and MC38 (mouse) colon\ncancer cells, MIA PaCa-2 pancreatic cancer cells, and CT26, U87MG,\nA172, and T98G glioblastoma cells were obtained from ATCC (Manassas,\nVA, USA). Cells were maintained in RPMI (St. Louis, MO, USA) medium\nsupplemented with 10% FBS (Gibco/Invitrogen) at 37 °C in the\npresence of 5% CO 2 . Cells were treated with PFAS generously\nprovided by Wellington Laboratories (Guelph, Ontario, Canada), and\nthese include PFOS (technical grade), sodium perfluorooctanesulfonate\n(PFOS-XST), sodium perfluorohexanesulfonate (PFHxS-XST), sodium perfluoroheptanesulfonate\n(PFHpS-XST), sodium perfluorononanesulfonate (PFNS-XST), and sodium\nperfluorodecanesulfonate (PFDS-XST). The XST designation indicates\nthat these compounds are linear and have been purified. Commercial\nPFOS contains some nonlinear impurities. The PPARγ inhibitor\nGW9662 was purchased from Tocris Biosciences (Minneapolis), and N-(4′-aminopyridyl-2-chloro-5-nitrobenzamide)\n(T007) was synthesized in the laboratory. The GAL4-NR4A1 chimera (LBD)\nand a UAS 5 -luc reporter construct were transfected into\ncancer cells, and induction of luciferase activity was determined\nas described. 43 , 46\nAt 25 °C, the Varian Cary\nEclipse Fluorescence Spectrophotometer was used to examine the quenching\nof fluorescence of a Trp residue in the NR4A1 ligand binding domain\nto determine direct ligand binding. 65  Different\nconcentrations of PFAS ligands were incubated with the ligand-binding\ndomain of NR4A1 (1.0 μM) in phosphate-buffered saline (PBS;\npH 7.4). Wavelengths of excitation (at 285 nm with a slit width of\n5 nm) and emission (between 300 and 420 nm with a slit width of 5\nnm) were used to obtain fluorescence. Sigma Plot was used to perform\ndata analyses. At a 330 nm emission wavelength, the concentration-dependent\nNR4A1 tryptophan fluorescence intensity was measured to quantify  R 2  and  K D  values.\nThe experimental\nprotocol provided by the manufacturer was carried out using the ChIP-IT\nExpress Kit (Active Motif, 53008). Rh30 cells were seeded on a plate\nfor 24 h, then treated with DMSO, 10 μM PFOS, and 12.5 μM\nDIM-3,5-Cl 2 . After 24 h, treated cells were fixed and lysed,\nand nuclei were collected for shearing by sonication. Sheared chromatin\nsamples were then immunoprecipitated overnight with antibodies using\nprotein G-conjugated magnetic beads. NR4A1 antibodies and mouse IgG\nwere used for the ChIP assay. Eluted chromatin was then purified using\nthe Chromatin IP DNA Purification Kit (58002). Purified DNA was analyzed\nusing amfiSure qGreen Q-PCR master mix (genDEPOT) for real-time PCR.\nThe primers used for detection of the Human PAX3-FOXO1 promoter region\nwere FOXO1 F 5′-TGCCTGTGCTTCACATTAGC-3′, FOXO1 R 5′-CAGATGGGGACAGAGACGC-3′,\nand G9a R 5′-CCCGGAGCATTGCACG-3′.\nRh30 cells\n(2 × 10 5 ) were seeded in RPMI medium supplemented\nwith 2.5% charcoal-stripped fetal bovine serum prior to the 24 h treatment\nperiod. Subsequent treatment of cells was performed using different\nconcentrations of PFOS for 24 h. Trypsinized counted cells (1 ×\n10 5 ) were loaded in a BioCoat 8.0 μm 24-well plate\nwith a growth factor reduced Matrigel invasion chamber from Corning\n(Bedford, MA). Cells were allowed to migrate for 48 h, followed by\nformaldehyde fixation and Crystal Violet staining. Migration of cells\nthrough the pores was quantified using ImageJ.\nRh30 cells (4 ×\n10 5 ) were seeded and allowed to attach on 6-well plates\nfor 24 h. RPMI medium was removed from the plates, and scratches were\nmade using a sterile 200 μL pipet tip. PBS was used to wash\nand remove the dead cells. Attached cells were treated with either\nDMSO or different concentrations of PFOS (2.5 and 10 μM) in\nRPMI medium supplemented with 2.5% charcoal-stripped fetal bovine\nserum. The medium was removed and replaced with PBS after 24–48\nh. Migrated cells were observed through the Evos digital inverted\nmicroscope, and images were taken to analyze the percent migration\nof Rh30 cells by using the ImageJ/Fiji wound healing size tool.\nHuman Rh30 rhabdomyosarcoma\ncells were grown in RPMI medium. Cells were seeded in 96-well plates\nwith a seeding density of 1.2 × 10 4  cells per well.\nCells were grown to ∼70% confluency and then treated with various\nconcentrations of PFAS and other compounds as indicated. After 24\nh, 0.02 mg/mL resazurin was added to each well and incubated for 4\nh. End point fluorescent activity (excitation 540 nm and emission\n590 nm) was measured as the reduction of resazurin to resorufin, an\nindicator for metabolic activity. The final concentration of DMSO\nin each well was 0.0032% to minimize DMSO-induced toxicity. Controls\nincluded on this plate included a vehicle control (DMSO) and an untreated\ncontrol.\nRh30 cells (3 × 10 5 ) were seeded and allowed to attach for 24 h on 6-well plates, followed\nby a 24 h treatment with either DMSO or different concentrations of\nPFOS. RIPA buffer that contained protease and phosphatase inhibitors\nwas added to lyse cells, and 4–20% Mini-PROTEAN TGX Gels (BioRad,\n4561094) were prepared to resolve whole-cell lysates. Polyvinylidene\nfluoride membrane was used to transfer proteins through wet blotting,\nblocked in 5% milk, followed by their incubation with primary and\nsecondary antibodies. Protein bands, in the presence of Immobilon\nwestern Chemiluminescence HRP-substrates, were visualized using the\nBioRad ChemiDoc imaging system, and the antibodies were used as described:\nPAX3FOXO1 (C2944), G9a (C5688515), PARP (CS9532), N-Myc (SC2236) (Cell\nSignaling Technologies, Danvers, MA), c-Myc (SC-40) and β1-integrin\n(CS96995) (Santa Cruz, CA), TXNDC5 (GTX106914) (GeneTex, Irvine, CA)\nand NR4A1 (ab283264)(Abcam). 45 , 63 , 64\nIn six-well\nplates, Rh30 cells (1.5 × 10 5 ) were seeded and allowed\nto reach approximately 60% confluency in 24 h. Lipofectamine RNAiMAX\nwas used for cell transfection. A transfection mixture prepared with\nsiRNA along with Lipofectamine RNAiMAX Reagent (Invitrogen; 56531)\nand Opti-MEM (Gibco; 31985–062) following the Lipofectamine\nRNAiMAX reagent protocol was used after 24 h. Replacement of the Opti-MEM\nwith fresh medium was performed after 6 h of transfection, and cells\nwere incubated (at 37 °C, 5% CO 2 ) for an additional\n72 h. Harvested cells were used to determine the expression of proteins\nand RNA analysis. Western blots were performed to determine the efficiency\nof NR4A1 knockdown by siRNAs targeting NR4A1 that were purchased from\nSigma-Aldrich. siRNAs used were siNR4A1 (NR4A1_C and NR4A1_D and Scrambled\nsiRNA (CGU ACG CGG AAU ACU UCG A (Sigma-Aldrich).\nThe animal study protocols were approved\nby the Institutional Animal Care and Use Committee (IACUC) at Texas\nA&M University. Four-week-old male athymic nude mice were obtained\nfrom The Jackson Laboratory (Bar Harbor, ME) and housed at the Laboratory\nAnimal Resources and Research facility, Texas A&M University.\nMale mice were chosen for this study based on their enhanced responsiveness\nto PFOS in a preliminary study; future research will confirm male\nvs female responsiveness to PFAS using xenograft and syngeneic mouse\nmodels. Mice were allowed to acclimate for 1 week and were fed a standard\nchow diet. Each mouse received an injection of 2 × 10 6  Rh30 cells suspended in 100 μL of a 1:1 Matrigel and PBS solution\ninto each flank subcutaneously. Once tumors reached a palpable size\n(approximately 50 to 100 mm 3 ), the mice were randomly assigned\nto control and treatment groups. Mice in the control group were administered\n100 μL of a DMSO:corn oil (1:4) solution by oral gavage daily.\nMice in the treatment groups were administered 100 μL of a PFOS\nsolution prepared in DMSO:corn oil (1:4) by oral gavage daily at doses\nof 20, 10, 0.5, and 0.2 mg/kg/day. The mice were weighed regularly,\nand where possible, their tumor volumes were measured using a Vernier\nCaliper ( V  =  L  ×  W  ×  H  mm 3 ) every week. After 4 weeks\nof drug administration, the mice were euthanized, and their tumors\nwere excised and weighed. A portion of each tumor was homogenized\nin lysis buffer, and the resulting extract was used for Western blot\nanalysis.\nStatistical analysis was conducted\nusing the  t  test to assess differences between the\ngroups. To compare the median survival rates of tumor-bearing animal\ncohorts, the log-rank (Mantel-Cox) test was applied with the analysis\nperformed using Prism 9 software. All in vitro experiments were repeated\nthree times to ensure the reliability and consistency of results.\nIn vitro results are presented as the ± SD, and in vivo results\nare means ± SE. To determine statistical significance, a one-way\nanalysis of variance (ANOVA) with Dunnett’s posthoc test was\nused, and a  P  value <0.05 was considered statistically\nsignificant.\n\nPFOS and some analogues are legacy PFAS compounds\ndetected in most\nhuman samples and were chosen as models for this study. Initial studies\nexamined the binding of PFOS and structurally related compounds to\nthe ligand binding domain (LBD) of NR4A1 using a fluorescent assay\nwhich measures quenching of the fluorescence of a Trp residue in the\nLBD of NR4A1 as previously described. 65 Figure  1  summarizes\nthe binding curves generated from PFAS ligands and their interactions\nwith the LBD of NR4A1. Commercial PFOS ( Figure  1 A), the linear PFOS-XST ( Figure  1 B), the linear hexafluoro (PFHxS-XST;  Figure  1 C), linear nonafluoro\n(PFNS-XST;  Figure  1 D), and decafluoro (PFDS-XST;  Figure  1 E) alkyl sulfonates differentially decreased fluorescence\nassociated with a Trp residue in the LBD. Only minimal displacement\nwas observed for PFHxS which was considered to be inactive in the\nquenching assay. The KD values observed for binding of PFOS, PFOS-XST,\nPFNS-XST, and PFDA-XST were 2.99, 0.92, 0.24, and 9.12 μmol/L,\nrespectively. The commercially available PFOS contains some branched\nPFOS isomers; however, the binding and  K D  values were similar to those of the purified linear PFOS-XST.\nPFAS compounds\nbind to NR4A1. PFOS (A), PFOS-XST (B), PFHxS-XST\n(C), PFNS (D), and PFDS (E) were incubated with the ligand binding\ndomain of NR4A1, and the binding curves were generated as outlined\nin the  Materials and Methods  section. Results\nobtained for the compounds alone (▼), ligand plus receptor\nuncorrected (●), and (ligand + receptor)–(ligand alone)\ncorrected (○). The  K D  values were\ndetermined for all compounds that bound NR4A1 and not PFHxS-XST which\nexhibited minimal quenching of fluorescence; the  K D  values were 2.99 (PFOS), 0.92 (PFOS-XST), 0.24 (PFNS-XST),\nand 9.12 (PFDS-XST) μmol/L.\nPrevious studies have associated human exposures\nto higher levels\nof PFOS with increased levels of cancer, whereas in cancer cell lines,\nthe growth-promoting activities of PFAS are highly variable. For example,\nPFOS alone at low doses (10 –10  – 10 –5  M) did not affect T47D breast cancer cell growth, but higher concentrations\n(>10  –5  M) inhibited growth. 58  In A549 lung cancer cells, there was a >10% increase\nin\ncell proliferation by 50 and 100 μM PFOS, and cytotoxicity was\nobserved at higher concentrations (200–1000 μM). 66  In this study, we compared the cytotoxicity\nof the inverse agonist 1,1-bis(3′-indolyl)-1-(3,5-dichlorophenyl)methane\n(DIM-3,5-Cl 2 ) and PFOS in A549 cells ( Figure  2 A). DIM-3,5-Cl 2  (25\nμM) inhibited A549 cell viability, and this has previously been\nobserved for CDIM compounds in lung and other cancer cell lines where\nthe CDIMs inhibit NR4A1-dependent growth 67  ( Figure  2 B). In contrast,\n100 nM–5 μM PFOS did not increase cell viability, whereas\nhigher concentrations (10 and 25 μM) inhibited the growth of\nA549 cells as previously reported. 57  This\nexperiment was repeated in Rh30 rhabdomyosarcoma cells and DIM-3,5-Cl 2  (10 and 25 μM) inhibited cell growth as previously\nreported for CDIMs, 47 , 63 , 64  whereas 10 and 25 μM PFOS induced >2.5-fold increase in\nRh30\ncell proliferation over a 24 h treatment period ( Figure  2 C). In addition, PFOS induced\nand DIM-3,5-Cl 2  decreased the luciferase activity in Rh30\ncells transfected with GAL4-NR4A1 and UAS-Luc constructs. These observations\ndemonstrate that PFOS induces Rh30 cell proliferation and NR4A1-dependent\ntransactivation. Induction of cell growth by PFOS was cell context-dependent\nin A549 and Rh30 cells. In contrast, DIM-3,5-Cl 2  decreased\nthe proliferation of both A549 and Rh30 cells, and the growth-promoting\neffects of PFOS were inversely related to the growth-inhibiting effects\nof DIM-3,5-Cl 2 .\nComparative induction and growth-promoting effects\nof DIM-3,5-Cl 2  and PFOS. (A) Structures of DIM-3,5-Cl 2  and PFOS.\nComparative effects of DIM-3,5-Cl 2  and PFOS on the growth\nof A549 lung cancer (B) and Rh30 rhabdomyosarcoma (C) cells after\ntreatment for 24 h. (D) Rh30 cells were transfected with GAL4-NR4A1\nand UAS-Luc plasmids, and after treatment with PFOS or DIM-3,5-Cl 2  luciferase activity was determined as outlined in the Methods.\nResults are expressed as means ± SD for at least 3 replicates\nfor each treatment groups, and significant ( p  <\n0.05) induction or inhibition of growth or luciferase activity is\nindicated (*).\nWe further examined the effects of several polyfluorinated\nalkyl\ncompounds on the growth of 8 different cancer cell lines using a range\nof concentrations from 0.1 to 25 μM ( Figure  3 ). The results show that for a number of\ncancer cell lines, PFOS induced a >2-fold increase in RKO, CT26,\nMC38,\nand U87 cell growth, whereas a <2-fold increase was observed in\nHCT116, A172, T98G, and MIA PaCa-2 cells. PFOS induced some proliferation\nof most cancer cell lines; however, the responsiveness of these cells\nwas variable. One possible explanation for the different responsiveness\nof cancer cell lines to PFOS-induced cell proliferation may be due\nto the expression of PPARγ which also binds PFOS. Since PPARγ\nligands primarily inhibit cancer cell growth, we cotreated Rh30 and\nA549 cells treated with PFOS alone and in combination with the PPARγ\ninhibitors T007 and GW9662 expecting that by blocking PPARγ,\nPFOS-induced growth would be enhanced. The results showed the PPARγ\ninhibitors had minimal effects on PFOS-induced growth of Rh30 and\nA549 cells, and therefore, PPARγ expression was not related\nto the cell context-dependent growth-promoting effects of PFOS. Interestingly,\nthe >3-fold induction of growth by PFOS in HCT116, RKO, MC38, and\nU87G cells is unusually high and exceeds the effects of most growth\nfactors in cancer cells.\nPFOS inducing cancer cell proliferation screening.\nHCT116 and RKO\n(A), CT26 and MC38 (B), U87MG, and A172 (C), and T98 and MIA PaCa-2\n(D) were treated with PFOS (0.1–25 μmol/L) for 24 h,\nand cell proliferation was determined using the resazurin assay as\noutlined in the Methods. Results are expressed as means ± SD\nfor at least 3 replicates for each treatment groups, and significantly\n( p  < 0.05) increased or decreased growth is indicated\n(*).\nThe effects of PFOS and structurally related sulfonates\non the\ngrowth of cancer cells were investigated over a broad range of concentrations\n( Figure  4 A). PFOS significantly\ninduced the proliferation of Rh30 cells at concentrations between\n2.5 and 10 μM, whereas in SW480 cells, PFOS concentrations as\nlow as 100 nM and as high as 25 μM significantly induced SW480\ncell proliferation, indicating that the SW480 cell line was also highly\nresponsive to the growth-promoting activity of PFOS. We also examined\nthe effects of a series of perfluoroalkyl sulfonates containing 9\nand 6 ( Figure  4 B) and\n7 and 10 ( Figure  4 C)\ncarbon atoms on the proliferation of Rh30 cells. This cell line was\nchosen as a model since previous studies show that it is NR4A1-responsive\nwith respect to cell growth and related pro-oncogenic pathways/genes. 45 − 47 , 63  Both the nona- and hepta-compounds\n(PFNS and PFHpS) enhanced cell proliferation, whereas this was not\nobserved for the deca- and hexa- (PFDS and PFHxS) sulfonates. The\nlack of activity for PFDS was surprising based on the binding data\nfor this compound which exhibited a low  K D  value and significant fluorescence quenching in the receptor binding\nassay, whereas PFHxS had minimal effects on cell growth and exhibited\nminimal binding in the fluorescence quenching assay ( Figure  1 ). The maximal magnitude of\ngrowth enhancement by the active polyfluoroalkyl sulfonates varied\nfrom 2- to 4-fold in Rh30 cells, and the magnitude of this response\nwas greater than the effects previously observed in Rh30 cells for\ntransforming growth factor β in previous studies. 47 , 63 , 64  Results in  Figure  4 D show that knockdown of NR4A1 (siNR4A1)\ndecreased the growth of Rh30 cells, and in the NR4A1-deficient cells,\ninduction of growth by 2.5 or 10 μM PFOS was inhibited. The\nefficiency of NR4A1 knockdown is shown in Western blot ( Figure  4 D). This confirms a role for\nNR4A1 in mediating the growth-promoting effects of PFOS, and results\nin  Figure  4 E show that\nknockdown of NR4A1 also blocks the growth-promoting effects of several\nstructurally related perfluoroalkyl sulfonates. These results suggest\nthat the perfluoroalkyl sulfonates act as NR4A1 agonists to enhance\nNR4A1-dependent proliferation responses.\nInduction of Rh30 cell\ngrowth by polyfluoroalkyl sulfonates. Rh30\nand SW480 (A) cells were treated with PFOS-XST for 24 h, and cell\nviability was determined as outlined in the  Materials\nand Methods  section. Rh30 cells were treated with PFNS-XST\nand PFHxS-XST (B) and PFHpS-XST and PFDS-XST (C) for 24 h, and cell\nviability was determined as outlined in the  Materials\nand Methods  section. (D) Rh30 cells were transfected with siCt1\nor siNR4A1 and treated with 2.5 or 10 μM PFOS, and cell proliferation\nand Western blot analyses on whole cell lysates were determined as\noutlined in the Methods. (E) Cells were also transfected with siNR4A1\nalone and after treatment with 2.5 μM polyfluoroalkyl sulfonates,\nand cell viability was determined as outlined in the Methods. Results\nare expressed as means ± SD for at least 3 replicate determinations\nfor each treatment group, and significant ( p  <\n0.05) induction or inhibition is indicated (*). The XST designation\nfor these compounds indicates that they are purified linear polyfluoroalkyl\nsulfonates.\nThe effects of PFOS on several NR4A1-regulated\nresponses in Rh30\ncells were investigated, and this includes their effects on Rh30 cell\nmigration using a scratch assay in which cells were treated with DMSO\n(control) and 2.5 or 10 μM PFOS for 24 and 48 h. The results\nshowed that the relative migration of Rh30 cells was increased after\ntreatment for 24 or 48 h ( Figure  5 A); however, significant induction of cell migration\nwas only observed for the 2.5 μM dose. Results in  Figure  5 B show that 2.5 μM PFOS\nenhances the invasion of Rh30 cells in a Boyden chamber assay, and\nthis complements the enhanced migration observed in the scratch assay.\nIn previous studies, the CDIM/NR4A1 inverse agonists modulated the\nexpression of several NR4A1-regulated gene products in Rh30 cells,\nand these include the PAX3-FOXO1 fusion oncogene, c-Myc and N-Myc. 47 , 63 , 64  Results illustrated in  Figure  6 A show that 10 or\n25 μM PFOS induces levels of PAX3-FOXO1 and N-Myc proteins in\nRh30 cells and 25 μM PFOS also induces c-Myc levels in this\ncell line. In addition, treatment of Rh30 cells with 10 or 25 μM\nor both concentrations of PFOS for 24 h also increased levels of several\nother NR4A1-regulated gene products including G9a (25 μM), β1-integrin\n(25 μM), thioredoxin domain containing 5 (TXNDC5) (10 and 25\nμM), and PARP cleavage (10 and 25 μM). DIM-3,5-Cl 2  acts as an inverse NR4A1 agonist in cancer cells, and in\nRh30 cells, induction of proliferation by PFOS is inhibited by DIM-3,5-Cl 2  ( Figure  6 C).\nMoreover, DIM-3,5-Cl 2  also inhibits PFOS-induced NR4A1-regulated\nPAX3-FOXO1 (FOXO1) and G9a gene products in Rh30 cells ( Figure  6 D). Previous studies in this\nlaboratory showed that CDIM compounds decreased interactions of NR4A1\nwith the transcriptionally active region of the G9a gene promoter\nin a ChIP assay. 45  Results in  Figure  6 E also show that\nafter treatment with 10 μM PFOS, there is no change in NR4A1\ninteractions with the G9a promoter compared to that observed in the\nuntreated cells, whereas DIM-3,5-Cl 2  (12.5 μM) significantly\ndecreased NR4A1-G9a gene promoter interactions. This is another example\nof the inverse relationship between the effects of PFOS and CDIMs,\nwhich is also observed for Rh30 cell proliferation, migration/invasion,\nand gene product expression, indicating that PFOS is acting as an\nNR4A1 agonist.\nPFOS induces cell migration. (A) Rh30 cells were treated\nwith DMSO\n(control) 2.5 or 10 μM PFOS for 24 h, and cell migration was\ndetermined in scratch assay and quantitated as outlined in the  Materials and Methods  section. A Boyden chamber\nassay (B) was also carried out in Rh30 cells as outlined in the Methods.\nThe assays were carried out in triplicate; results are expressed as\nmeans ± SE, and significantly ( p  < 0.05)\nenhanced migration is indicated (*).\nPFOS induces NR4A1-dependent gene products in the Rh30\ncells. Rh30\ncells were treated with 10 or 2.5 μM PFOS for 24 h, and whole\ncell lysates were analyzed for PAX3-FOXO1, N-Myc, and c-Myc (A) and\nother NR4A1-regulated gene products (B) by Western blots. Rh30 cells\nwere treated with PFOS and DIM-3,5-Cl 2  alone or in combination\nand effects on cell proliferation (C), and gene products (D) were\ndetermined as outlined in the Methods. (E) Cells were treated with\nDMSO (control) PFOS (10 μM) and DIM-3,5-Cl 2  (12.5\nμM), and interactions of NR4A1 with the G9a gene promoter were\ndetermined in a ChIP assay as outlined in the Methods. The Western\nblots were carried out in triplicate, and band intensities (means\n± SD) were determined relative to GAPDH (control), and significant\n( p  < 0.05) induction or inhibition is indicated\n(*).\nIn initial studies, it was observed that higher\nconcentrations\nof PFOS (10 and 20 mg/kg/day) significantly inhibited tumor growth\nin an athymic nude mouse model using Rh30 cells as xenografts ( Figure  7 A). The doses of\nPFOS were then lowered to 0.2 and 0.5 mg/kg/day and tumor volumes\nwere observed over a period of 4 weeks after injection of the cells.\nA summary of the results demonstrates that after 3 or 4 weeks of treatment\nwith 0.5 but not 0.2 mg/kg/day PFOS, there was a significant induction\nof tumor volumes compared to the control (corn oil-treated) mice ( Figure  7 A). Body weights\nwere not significantly different between the control and PFOS-treated\nmice ( Figure  7 B), and\nwhile relative tumor weights were increased in the 0.5 mg/kg/day treatment\ngroup, the effect was not significantly different than the controls\nor mice treated with 0.2 mg/kg/day PFOS ( Figure  7 C) due to interindividual animal variability.\nResults in  Figure  7 D show that in tumor lysates from the 0.5 mg/kg/day treatment group\nlevels of NR4A1-responsive genes were significantly induced compared\nto controls, and this further supports that PFOS is acting through\nNR4A1.\nIn vivo studies. Athymic nude mice bearing Rh30 cells were treated\nwith 20, 10, 0.5, and 0.2 mg/kg/day and tumor volumes (A), body weight\n(B), relative tumor weights (C), and Western blot analysis of tumor\nlysates (D) were determined as outlined in the  Materials\nand Methods  section. Results (A–D) are expressed as\nmeans ± SE, and significant ( p  < 0.05) induction\nis indicated (*).\n\nPFOS is an important legacy PFAS compound\nthat is routinely identified\nas a major PFAS component in environmental samples and human serum.\nAlthough PFOS induces multiple responses in cell culture and laboratory\nanimals, the collective effects of PFOS and related PFAS in humans\nindicate that higher levels of these compounds are associated with\nan increased incidence of several diseases. As indicated in the Introduction,\ndiseases associated with exposures to high levels of PFAS have a\nlinkage to NR4A1 and these adverse responses are ameliorated after\ntreatment with an NR4A1 ligand such as celastrol, cytosporone B and\nrelated compounds, and CDIMs. 38 − 46  For example, CDIM ligands act as inverse receptor agonists that\ninhibit NR4A1-dependent pro-endometriotic and pro-carcinogenic responses, 42 , 43  whereas higher PFAS levels in humans are associated with increased\nendometriosis 68 , 69  and cancer, 9 − 23  respectively.\nPrevious reports show that PFOS induces the\nproliferation of nontransformed\nbreast and prostate cells but does not induce their transformation\ninto cancer cells, whereas the growth-promoting effects of PFOS on\ncancer cells and tumors are highly variable. 56 − 62  In contrast, CDIMs act as inverse NR4A1 agonists in most solid tumor-derived\ncell lines and inhibit NR4A1-regulated pro-oncogenic genes/pathways,\nand in this study, Rh30 and other cancer cells have been used as a\n“mechanistic model” to investigate whether PFOS is acting\nas an NR4A1 agonist that enhances NR4A1-mediated pro-oncogenic activities.\nThis approach has its limitations in terms of explaining the complete\ncarcinogenic activity of PFAS since the results show effects on cancer\ncells and tumors but not on the role of PFOS in the transformation\nof normal cells.\nResults illustrated in  Figure  1  demonstrate that PFOS and other polyfluoroalkyl\nsulfonates\ndirectly bind the LBD of NR4A1 using a fluorescent quenching assay\nas described in previous studies. 65  Decreased\nfluorescence was observed for the polyfluoro deca-, nona-, octa-,\nand heptaalkyl sulfonates ( Figure  1 ), and with the exception of the PFDS congener, these\ncompounds also induced cell proliferation ( Figure  4 ). The reason for this “outlier”\neffect for PFDS is not known and is being investigated. PFHxS exhibits\nminimal receptor binding and effects on growth, suggesting that for\nthe linear polyfluoroalkyl sulfonates, the NR4A1-active compounds\nmust have greater than 6 carbons. Previous studies in cancer cell\nlines showed that low concentrations of PFOS (1–100 nM) induced\nthe proliferation of T47D breast cancer cells; however, the antiestrogen\nfasoldex inhibited this response, suggesting that the proliferative\nactivity was related to the estrogenic activity of PFOS and PFOS alone\ndid not affect cell growth. 58  In contrast,\n25–200 μM PFOS decreased the growth of A549 lung cancer\ncells, 57  and this response would be comparable\nto that observed for NR4A1 inverse agonists 47 , 63 , 64  ( Figure  2 ). In this study, PFOS induced proliferation of several\ncancer cell lines by greater than 2-fold, and the fold induction of\nothers varied from <2-fold to minimal induction of cell proliferation\neffects ( Figure  3 ).\nThe variable responsiveness of these cell lines to the growth-promoting\neffects of PFOS is not uncommon for other growth-promoting substances;\nwe hypothesized that the variability may be due to PFOS-mediated growth\ninhibition by activating PPARγ since PFOS binds PPARγ.\nHowever, this is unlikely since PPARγ inhibitors do not enhance\nPFOS-induced cancer cell growth ( Figure S1 ). We are now further investigating the underlying mechanisms causing\nthese cell context-dependent differences observed for inducing cancer\ncell growth by PFOS. The structure-dependent binding of PFOS and related\ncompounds to NR4A1 and the structure-dependent induction of Rh30 cell\nproliferation by the hepta-, octa-, and nonafluoroalkyl sulfonates\nis consistent with a role for NR4A1 in the induction of cell proliferation\nby these PFAS compounds.\nWe used the NR4A1-responsive Rh30 cell\nline as a model to further\ninvestigate the role of this receptor in mediating PFOS-induced cell\nproliferation. Knockdown of NR4A1 by RNA interference resulted in\nthe loss of growth-promoting activity for not only PFOS ( Figure  4 D) but also the related\nperfluoroalkyl sulfonates in Rh30 cells after knockdown by NR4A1 ( Figure  4 E). Thus, the effects\nof PFOS and related compounds on Rh30 cell proliferation were inversely\nrelated to those observed for CDIMs which are inverse NR4A1 agonists\nthat inhibit NR4A1-dependent cell growth. 47 , 63 , 64  The effects of PFOS on other pro-oncogenic\nand genomic responses that are inhibited by CDIMs were also investigated,\nand there was an inverse functional relationship between PFOS and\nCDIMs as NR4A1 ligands. PFOS induced cell migration in a scratch and\nBoyden chamber assay and induced the NR4A1-responsive PAX3-FOXO1 oncogene\nand related gene products in Rh30 cells, whereas the opposite effects\nwere previously observed for CDIMs in this cell line. 47 , 63 , 64  Moreover, DIM-3,5-Cl 2 , an NR4A1 inverse agonist, inhibits PFOS-induced growth of Rh30\ncells ( Figure  6 C) and\nPFOS-induced (NR4A1-dependent) gene products ( Figure  6 D) in the same cell line. The inverse effects\nof PFOS vs DIM-3,5-Cl 2  is also confirmed in a ChIP assay\nwhere PFOS had no effect on NR4A1 interactions with the G9a promoter,\nwhereas DIM-3,5-Cl 2  decreased the interaction of NR4A1\nwith the transcriptionally active GC-rich region of the G9a promoter.\nRh30 cells were also used as xenografts in athymic nude mice to\ninvestigate the effects of PFOS on tumor growth. Higher doses of 20\nand 10 mg/kg/day of PFOS inhibited tumor growth, and this was consistent\nwith the results of previous studies on PFOS. However, in mice treated\nwith 0.5 mg/kg/day, there was a significant increase in tumor volume,\nwhereas in mice receiving 0.2 mg/kg/day, tumor growth was not significantly\ndifferent than the control group, indicating a narrow range of PFOS-enhanced\ncarcinogeneses. Previous reports on the effect of PFOS on tumor growth\nin rodent models are variable. For example, in genetic mouse models\nfor colon cancer, 10 and 250 mg/kg/day or 200 mg/kg total dose of\nPFOS decreased intestinal tumor growth, 59 , 60  whereas PFOS\n(10 mg/kg/day) induced tumor growth in athymic nude mice bearing tumorigenic\nRWPE-2 prostate cancer cells as xenografts. 48  Differences between studies on the carcinogenicity of PFOS are unknown;\nhowever, higher concentrations of PFOS are known to be cytotoxic and\nthis response may be due, in part, to induction of reactive oxygen\nspecies (ROS). 57  In this study using Rh30\ncells as a model, there is now evidence that low concentrations and/or\ndoses of PFOS enhance tumorigenesis, and this response is, in part,\nNR4A1-dependent. The contributions of NR4A1 to other toxicities associated\nwith PFAS are not yet known and are currently being investigated.","source_license":"CC-BY-4.0","license_restricted":false}