Perfluorooctanoic acid (PFOA) as a stimulator of estrogen receptor-negative breast cancer MDA-MB-231 cell aggressiveness: Evidence for involvement of fatty acid 2-hydroxylase (FA2H) in the stimulated cell migration

Abstract

Detailed in vitro studies on the effects of perfluorooctanoic acid (PFOA) have demonstrated that activation of peroxisome proliferator-activated receptor α (PPARα) is a key process by which PFOA affects the malignancy of estrogen receptor α (ERα)-positive breast cancer cells. However, there is very little information on the PPARα-regulated genes responsible for the effects of PFOA in ERα-negative breast cancer cell malignancy. We recently demonstrated that fatty acid 2-hydroxylase (FA2H) stimulates the migration of ERα-negative human MDA-MB-231 cells, and PPARα is a key factor for the induction of FA2H in these cells. However, evidence for the relationship between PFOA exposure and PPARα-FA2H axis-driven migration has not been obtained. Here we analyzed the effects of PFOA on PPARα transcription and FA2H expression in relation to MDA-MB-231 cell migration. We found that simultaneously with stimulated migration, PFOA upregulated FA2H and activated the transcription of PPARα. FA2H-selective siRNA, but not siRNA control, clearly dampened PFOA-mediated cell migration. There is an inhibitory interaction between PPARα and PPARβ/δ (i.e., PPARβ/δ can suppress PPARα-mediated transcription) in MDA-MB-231 cells, but even in the presence of PPARβ/δ expression, PFOA appeared to free PPARα to upregulate FA2H. Collectively, our findings show that i) PFOA activates PPARα-mediated transcription, ii) PFOA stimulates migration dependent on FA2H expression, and iii) mechanistically, PFOA relieves PPARβ/δ suppression of PPARα activity to upregulate FA2H in MDA-MB-231 cells.

INTRODUCTION

Perfluorooctanoic acid (PFOA), a man-made chemical, was frequently used in industrial and consumer products because of properties such as its stain resistance and water repellency, but it is now restricted by Persistent Organic Pollutants (POPs) regulation. Some perfluoroalkyl substances (PFASs), including PFOA, are recognized as endocrine-disrupting chemicals (EDCs) that can influence estrogen homeostasis (Jensen and Leffers, 2008; Bonefeld-Jorgensen et al., 2011; Sonthithai et al., 2016). PFOA has been categorized as a possible carcinogen for humans (Group 2B) by the International Agency for Research on Cancer (IARC) (IARC, 2016). When considering the effects of PFOA on cancer biology, some epidemiological studies have linked PFOA toxicity to tumorigenesis in various organs (Lau et al., 2007; Bonefeld-Jorgensen et al., 2011; Barry et al., 2013; Vieira et al., 2013; Wielsøe et al., 2017). Specifically, epidemiological analyses have demonstrated that among chemicals such as the investigated PFASs, PFOA exposure correlates most with breast cancer risk including estrogen receptor α (ERα)-negative breast cancer (Mancini et al., 2020). Experiments using human breast epithelial cells (MCF-10A; an ERα-positive line), which can be malignantly transformed, revealed that treatment of the cells with PFOA (0.1 μM – 1 mM) increased their migration and invasion, effects dependent on the activation of peroxisome proliferator-activated receptor α (PPARα) (Pierozan et al., 2018). However, there is very little information on the “execution factor(s)” after PPARα activation by PFOA (i.e., PPARα-regulated genes responsible for the malignant effects of PFOA) in ERα-negative breast cancer cells.

To date, our studies have demonstrated that i) fatty acid 2-hydroxylase (FA2H) is a PPARα-regulated gene in MDA-MB-231 cells that are ERα-negative, a model of triple-negative breast cancer (TNBC) (Takeda et al., 2013a, 2014; Hirao-Suzuki et al., 2019b) and ii) FA2H can positively stimulate the migration of MDA-MB-231 cells (Hirao-Suzuki et al., 2020a). PPARs are nuclear hormone receptors that include three subtypes, PPARα, PPARβ/δ, and PPARγ; PPARs play key roles in regulating the biological activities of cancer cells, such as apoptosis, proliferation, and survival (Peters et al., 2012). Thus, endogenous or exogenous (synthetic) ligands of PPARs can alter the behavior of cancer cells. Based on a report demonstrating that PPARβ/δ can suppress the transcriptional activity of PPARα when they are co-expressed in cells (Shi et al., 2002), we subsequently revealed that this interaction is functional in MDA-MB-231 cells, and chemicals that can induce FA2H may attenuate the PPARβ/δ-mediated inhibition of PPARα (Hirao-Suzuki et al., 2019b). PFOA has previously been shown to activate human PPARα transiently expressed in an African green monkey kidney fibroblast-like cell line (COS-1 cells) (Wolf et al., 2008, 2012). However, it is unclear whether PFOA activates PPARα in human breast cancer cells as observed in the case of COS-1 cells, especially in the MDA-MB-231 cells co-expressing PPARα and PPARβ/δ.

In the current study, we sought to investigate whether i) PFOA can activate PPARα, ii) PFOA can induce FA2H, coupled with increased cell migration, and iii) PFOA can attenuate PPARβ/δ inhibition of PPARα transcription, using the MDA-MB-231 cell line, a TNBC model.

MATERIALS AND METHODS

Reagents

PFOA (purity: ≥ 95%) (CAS: 335-67-1) was purchased from Sigma-Aldrich (St. Louis, MO, USA). GW6471 (purity: ≥ 98%) (CAS: 880635-03-0) was purchased from Cayman Chemicals (Ann Arbor, MI, USA). Both PFOA and GW6471 were dissolved in dimethyl sulfoxide (DMSO).

Cell cultures and treatments

PFOA exposure has been shown to be correlated with breast cancer risk including that of ERα-negative breast cancer (Mancini et al., 2020), and MDA-MB-231 cells can be utilized as a model of ERα-negative breast cancer. The MDA-MB-231 cells were purchased from the American Type Culture Collection (Rockville, MD, USA). The cell culture methods were based on previously described procedures (Takeda et al., 2013b; Hirao-Suzuki et al., 2020a). These cells were routinely cultured in phenol red-containing minimum essential medium α (MEMα) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 5% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin, in a humidified incubator with 5% CO₂ at 37°C. Prior to chemical treatments, the culture medium was changed to phenol red-free MEMα supplemented with 10 mM HEPES, 5% dextran-coated charcoal-treated FBS (DCC-FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. MDA-MB-231 cells were seeded and incubated for 4 hr, followed by the treatment with PFOA. After exposure to PFOA for 48 hr, 60 hr, or 72 hr, the cells were used for expression analysis, cell migration assay, and MTS assay (Supplemental Fig. 1A). The cells were then treated with PFOA and GW6471 at the final concentrations indicated in each figure legend. The mean blood PFOA concentrations of occupationally exposed populations are approximately three orders of magnitude higher than those of the general population, and maximum concentrations of > 100,000 ng/mL (approximately 242 μM) have been detected (Fromme et al., 2009). In vitro studies on PFOA interactions with MCF-10A cells have revealed that PFOA concentrations higher than 250 μM lead to reduced cell viability (Pierozan et al., 2018). Based on these findings, PFOA was used at a maximum concentration of 100 μM in the present study.

Transfection and dual-luciferase assay

The experiments were performed as previously described, with minor modifications (Takeda et al., 2016; Hirao-Suzuki et al., 2019b; Hirao-Suzuki et al., 2020c). Each plasmid was transfected using Lipofectamine^® LTX with PLUS™ reagent (Thermo Fisher Scientific, Waltham, MA, USA). The DNA mixtures containing 300 ng of the rat acyl-CoA oxidase PPAR response element (PPRE)-Luc plasmid and 2 ng of the Renilla luciferase reporter plasmid (pRL-CMV) were co-transfected with human PPARα (100 ng), PPARβ/δ (25 or 50 ng), and RXRα (100 ng) expression plasmids. The PPRE reporter plasmid and the expression plasmids were a kind gift from Prof. Curtis J. Omiecinski (Pennsylvania State University, PA, USA). At 24 hr post-transfection, the cells were washed with phosphate-buffered saline (PBS) and cultured in phenol red-free MEMα supplemented with 5% DCC-FBS. The cells were then treated with PFOA and GW6471. After 24 hr, the cell extracts were prepared in 100 μL passive lysis buffer (Promega, Madison, WI, USA), and 20 μL of these extracts were used for the firefly luciferase and Renilla luciferase assays (Dual-Luciferase Reporter Assay System, Promega). The ratio of firefly luciferase activity to Renilla luciferase activity was calculated as the relative luciferase activity.

Analysis of real-time reverse transcription-polymerase chain reaction (real-time RT-PCR)

Total RNA preparation and real-time RT-PCR were performed as described previously (Hirao-Suzuki et al., 2019a). The following primers were used: FA2H (sense) 5′-AAC GAG CCT GTA GCC CTT GA-3′, FA2H (antisense) 5′-ACT GCC ACC GTG TAC TCT GTT-3′; PAPRα (sense) 5′-CTT CGC AAA CTT GGA CCT GA-3′, PAPRα (antisense) 5′-TGA GCA CAT GTA CAA TAC CCT C-3′; PAPRβ/δ (sense) 5′-GCC TCT ATC GTC AAC AAG GAC-3′, PAPRβ/δ (antisense) 5′-GCA ATG AAT AGG GCC AGG TC-3′; β-actin (sense) 5′-GGC CAC GGC TGC TTC-3′, β-actin (antisense) 5′-GTT GGC GTA CAG GTC TTT GC-3′. The primers for FA2H, PAPRα, and β-actin were utilized in our previous reports (Takeda et al., 2013a, 2014; Hirao-Suzuki et al., 2020b). The FA2H, PAPRα, and PAPRβ/δ mRNA expression levels were normalized to β-actin mRNA expression levels.

Antibodies and western blot analysis

Antibodies specific for FA2H (15452-1-AP; Proteintech Group, Inc., Rosemont, IL, USA) and β-tubulin (018-25044; FUJIFILM Wako Pure Chemical Corporation) were used. The western blotting and quantification of band intensities were performed as described previously (Hirao-Suzuki et al., 2019a; Hirao-Suzuki et al., 2020a). The obtained values were normalized to those of β-tubulin, a loading control.

Cell migration assay

The cancer cell wound-healing assay was performed as described previously, with minor modifications (Takeda et al., 2012; Hirao-Suzuki et al., 2020a). Briefly, MDA-MB-231 cells were seeded into 6-well plates at a density of approximately 3 × 10⁵ cells/well and incubated for 4 hr; the cells were then treated with PFOA. Twenty-four hours after PFOA treatment, FA2H siRNA (sc-93418; Santa Cruz Biotechnology, Dallas, TX, USA) and Control siRNA (sc-37007; Santa Cruz Biotechnology, Inc.) were transfected using Lipofectamine™ RNAiMAX reagent (Thermo Fisher Scientific). Forty-eight hours after PFOA treatment (i.e., 24 hr after siRNA transfection), the monolayers were scratched with plastic tip. To visualize cell migration into the scratched region, the cells were imaged using an Olympus CKX41 inverted microscope (Tokyo, Japan) and an Olympus DP22 digital camera connected to a DP2-SAL camera controller after incubation for 0 hr, 12 hr, and 24 hr (i.e., treatment with PFOA for 48 hr, 60 hr and 72 hr, respectively) (Supplemental Fig. 1B).

Cell proliferation analysis (MTS assay)

The MTS assay was performed as described previously (Hirao-Suzuki et al., 2020b). The cells were treated with PFOA at the final concentrations indicated in the figure legend.

Data analysis

Differences were considered significant at P values of less than 0.05. The statistical significance of the difference between two groups was determined using the Student’s t-test. The statistical significance of the difference between multiple groups was determined using one-way or two-way ANOVA followed by the Dunnett’s or Tukey-Kramer post-hoc test. These calculations were performed using StatView 5.0J software (SAS Institute Inc., Cary, NC, USA). The details are indicated in individual figure legends.

RESULTS AND DISCUSSION

PFOA stimulates PPARα-mediated transcription in MDA-MB-231 cells

It has been demonstrated that, among the three human PPAR subtypes (α, β/δ, and γ), PFOA can preferentially activate PPARα-driven transcription in both mouse 3T3-L1 fibroblasts and African green monkey kidney COS-1 cells (Vanden Heuvel et al., 2006; Takacs and Abbott, 2007). Due to the lack of experimental evidence demonstrating whether PFOA (Fig. 1A) can activate human PPARα-dependent transcriptional activity in MDA-MB-231 cells, we performed luciferase reporter assays of PPARα-mediated PPRE activation in the presence or absence of up to 100 μM PFOA. In this study, to preferentially detect PPARα-driven transcriptional activity, we used a PPRE-Luc plasmid containing the rat acyl-CoA oxidase PPRE (Kane et al., 2006; Takeda et al., 2014; Hirao-Suzuki et al., 2019b). As shown in Fig. 1B, when compared with vehicle-treated control (Ctl.), PFOA stimulated the transcription mediated by PPARα/PPRE in a concentration-dependent manner, especially at 100 μM (1.57-fold). To further obtain evidence whether PFOA directly activates PPARα transcription in MDA-MB-231 cells, we utilized GW6471, a PPARα competitive antagonist that can also recruit corepressors (Xu et al., 2002). It was found that PFOA (100 μM)-driven transcriptional activity was abrogated by GW6471 in a concentration-dependent manner from 100 nM to 10 μM. Specifically at GW647 concentrations > 1 μM, the activity of PPARα was decreased below basal levels (Fig. 1C), implicating the GW6471-mediated recruitment of corepressors to PPARα. Thus, PFOA can directly stimulate the transcriptional activity of PPARα in MDA-MB-231 cells.

Fig. 1

PFOA directly stimulates PPARα-mediated transcriptional activity in MDA-MB-231 cells. (A) The structure of PFOA. (B and C) Effects of PFOA on PPARα-mediated transcriptional activity (B) and effects of GW6471 on PFOA-stimulated PPARα activity (C) in MDA-MB-231 cells. The cells were transiently transfected with a PPRE-luciferase reporter plasmid in combination with the PPARα expression plasmid. After transfection, the cells were treated with vehicle (indicated as Ctl.) or PFOA (1 to 100 μM) for (B), and vehicle (indicated as −/−) or 100 μM PFOA in combination with GW6471 (0.1 nM to 10 μM) for (C). After 24 hr, the cells were harvested and assayed for luciferase activity; all transfections were normalized for efficiency using the internal Renilla control plasmid. Data are presented as the mean ± S.E. (n = 3) of the fold induction relative to the vehicle-treated control. Significant differences (two-way ANOVA, followed by Dunnett’s post-hoc test) compared with the vehicle-treated control for (B) or PFOA treatment alone (+/−) for (C) are marked with asterisks (*P < 0.05).

PFOA upregulates expression of FA2H in MDA-MB-231 cells

Our recent findings demonstrated that FA2H is a gene positively regulated by PPARα (Hirao-Suzuki et al., 2019b). As supported by the data in Fig. 1B, PFOA can activate PPARα transcription in MDA-MB-231 cells. We next examined the expression level of FA2H after exposure of the cells to PFOA. As shown in Fig. 2A and B, FA2H mRNA was significantly upregulated 3.19-fold by 100 μM PFOA; although FA2H protein levels tended to increase with 100 μM PFOA, the difference from untreated control cells (Ctl.) was not statistically significant. The expression of PPARα itself was not modulated by the same concentration of PFOA (Fig. 2C), suggesting that an engagement of PFOA with PPARα is important to induce FA2H.

Fig. 2

PFOA upregulates FA2H expression in MDA-MB-231 cells. (A and C) Real-time RT-PCR analyses of FA2H (A) and PPARα (C) in MDA-MB-231 cells after 48 hr of treatment with vehicle (indicated as Ctl.) or PFOA (50 and 100 μM). Data are presented as the mean ± S.E. (n = 6) of the fold induction relative to the vehicle-treated control. Significant differences (one-way ANOVA, followed by Dunnett’s post-hoc test) compared with the vehicle-treated control are marked with an asterisk (*P < 0.05). (B) Western blot analysis of FA2H in MDA-MB-231 cells after 48 hr of treatment with vehicle (indicated as Ctl.) or PFOA (50 and 100 μM). (B, lower panel) The band intensity of FA2H was quantified using ImageJ 1.46r software, and the intensities were normalized to those of β-tubulin. Data are presented as the mean ± S.E. (n = 3) of the fold induction relative to the vehicle-treated control.

PFOA stimulates migration of MDA-MB-231 cells without accompanying cell proliferation

Our recent study demonstrated that transfection of an FA2H or PPARα expression plasmid can enhance the migration of MDA-MB-231 cells (Hirao-Suzuki et al., 2020); thus, the PPARα-FA2H axis may be responsible for the aggressive nature of TNBC. Based on the results of Figs. 1B and 2, a PFOA concentration of 100 μM was selected for the following experiments. We first investigated the time-dependent effects of PFOA (12 and 24 hr after scratching; i.e., treatment with PFOA for 60 and 72 hr, respectively) on the migration of MDA-MB-231 cells. When compared to the vehicle-treated control cells (Ctl.), PFOA significantly stimulated cell migration during each time period (Fig. 3A, left and right panels). Increased cell proliferation could have contributed to the observed cell migration. Thus, we sought to determine the effects of PFOA on the proliferation of MDA-MB-231 cells. As shown in Fig. 3B, no stimulatory effects on cell proliferation were detected at PFOA concentrations from 1 to 100 μM. Furthermore, even after 24 hr in the scratched cells (i.e., treatment with 100 μM PFOA for 72 hr), FA2H mRNA and protein were significantly upregulated by PFOA (Fig. 3C and D). These results suggested that an increase in FA2H mediated by PFOA is involved in the enhanced cell migration.

Fig. 3

PFOA stimulates migration with no stimulatory effects on the proliferation of MDA-MB-231 cells. (A) Effect of PFOA on MDA-MB-231 cell migration. MDA-MB-231 cells were treated with vehicle (indicated as Ctl.) or PFOA (100 μM). (A, left panel) Representative images of the migrating cells were captured at 12 hr and 24 hr (i.e., treatment with PFOA for 60 and 72 hr, respectively). (A, right panel) Migration data presented in left panel were quantified on the basis of percent fill-in of the wounded area. Data are presented as the mean ± S.E. (n = 3). Significant differences (by Student’s t-test) compared with the vehicle-treated control are marked with asterisks (*P < 0.05). (B) Effects of PFOA on proliferation of MDA-MB-231 cells. MDA-MB-231 cells were treated with vehicle (indicated as Ctl.) or PFOA (1 to 100 μM) for 48 hr. Data are presented as the mean ± S.E. (n = 6) percentage of the vehicle-treated control. (C and D) Real-time RT-PCR analysis (C) and western blot analysis (D) of FA2H expression in MDA-MB-231 cells after 72 hr of treatment with vehicle (indicated as Ctl.) or PFOA (100 μM). (D, lower panel) The band intensity of FA2H was quantified using ImageJ 1.46r software, and the intensities were normalized to those of β-tubulin. Data are presented as the mean ± S.E. (n = 3) of the fold induction relative to the vehicle-treated control. Significant differences (Student’s t-test) compared with the vehicle-treated control are marked with an asterisk (*P < 0.05).

PFOA stimulates migration of MDA-MB-231 cells in an FA2H-dependent manner

Given that the FA2H protein induced by PFOA exposure is critical to stimulate MDA-MB-231 cell migration, knockdown of FA2H by siRNA should be effective in abrogating PFOA-driven cell migration. We performed the same experiments as described in Fig. 3 in the presence of FA2H siRNA or Control siRNA (Fig. 4A and B); transfection with FA2H siRNA produced comparable migration between the control cells (Ctl.) and PFOA-treated cells (Fig. 4A). As shown in Fig. 4B, transfection with Control siRNA failed to abrogate the enhanced cell migration induced by PFOA at each time period (also see Fig. 3A). FA2H mRNA analysis verified that transfection of FA2H siRNA significantly reduced FA2H expression by 0.12-fold (P < 0.05) when compared to the expression in Control siRNA-treated cells; furthermore, addition of 100 μM PFOA resulted in upregulation of FA2H mRNA both in the Control siRNA (7.05-fold)- and FA2H siRNA (0.57-fold)-treated groups relative to the Control siRNA-treated group without PFOA incubation (Fig. 4C). Taken together, these results strongly suggest a critical involvement of FA2H in the PFOA-mediated migration of MDA-MB-231 cells.

Fig. 4

FA2H siRNA attenuates PFOA-stimulated cell migration, coupled with reduction in FA2H expression. (A and B) Effects of FA2H siRNA (A) or Control siRNA (B) on PFOA-stimulated MDA-MB-231 cell migration. MDA-MB-231 cells were treated with vehicle or PFOA (100 μM), followed by transfection with FA2H siRNA or Control siRNA. (A and B, left panel) Representative images of the migrating cells were captured at 12 hr and 24 hr (i.e., treatment with PFOA for 60 and 72 hr, respectively). (A and B, right panel) Migration data presented in left panel were quantified on the basis of percent fill-in of the wounded area. Data are presented as the mean ± S.E. (n = 3). Significant differences (by Student’s t-test) compared with the vehicle-treated control are marked with asterisks (*P < 0.05). (C) Effects of FA2H siRNA on PFOA-mediated FA2H upregulation in MDA-MB-231 cells. MDA-MB-231 cells were treated with vehicle or PFOA (100 μM), followed by transfection with Control siRNA or FA2H siRNA. FA2H mRNA expression was analyzed using real-time RT-PCR. Data are presented as the mean ± S.E. (n = 3) of the fold induction relative to the Control siRNA-transfected group in the absence of PFOA. Significant differences (by two-way ANOVA, followed by Tukey-Kramer post-hoc test) compared with the Control siRNA-transfected group in the absence of PFOA and the Control siRNA-transfected group in the presence of PFOA are marked with asterisks (*P < 0.05) and hashes (^#P < 0.05), respectively.

PFOA can release PPARβ/δ-mediated suppression of PPARα transcription

Shi et al. (2002) reported evidence demonstrating abolishment of PPARα transcriptional activity when it was co-expressed with the PPARβ/δ subtype in CV-1 and NIH/3T3 cells. MDA-MB-231 cells (i.e., human TNBC cells) also co-express functional PPARα and its “suppressive” subtype PPARβ/δ (Hirao-Suzuki et al., 2019b). Thus, how PFOA can activate PPARα-mediated transcription, which leads to FA2H upregulation, even in the restrictive environment mediated by PPARβ/δ is a question that remains unanswered.

To address this question, in this study, we utilized an ectopic expression strategy that enabled tight control of the expression levels of the PPARs α and β/δ. We first confirmed that ectopic expression of PPARα, which can be stimulated by fatty acids in the culture medium (ligands for PPARs) (Kane et al., 2006; Takeda et al., 2014), upregulated luciferase reporter activity 30-fold compared to that of the mock-transfected group (Ctl.), and this luciferase activity could be increased 1.75-fold more in the presence of PFOA. Co-transfection of PPARβ/δ expression plasmid (25 and 50 ng) with PPARα expression plasmid (100 ng) suppressed PPARα-dependent transcription in a concentration-dependent manner (Fig. 5). We next tested the idea that if PFOA could release the PPARβ/δ-mediated suppression of PPARα, exogenously added PFOA would still activate PPARα in the co-expression system (PPARα and PPARβ/δ). As anticipated, PPARα/PPRE transcription was significantly activated by PFOA even in the presence of 50 ng of transfected PPARβ/δ expression plasmid. We next studied whether the phenomenon demonstrated in the model system in Fig. 5 was relevant to PPARα-mediated cell physiology. Therefore, we tested whether PFOA could modify PPARα-mediated FA2H mRNA expression even in the presence of PPARβ/δ. As shown in Fig. 6, when compared to the mock-transfected control (arbitrarily set as 1.0), significant increases in FA2H mRNA were observed with 100 μM PFOA alone, PPARα alone, PFOA/PPARα, and PFOA/PPARα/PPARβ/δ. When focusing on the difference between PPARα and PPARα/PPARβ/δ, PPARα-driven transcription in the latter group was reduced to control levels. It should be noted that PFOA could induce FA2H mRNA levels even in the presence of PPARβ/δ. Furthermore, because i) the basal expression of PPARβ/δ was not downregulated by PFOA (Fig. 6, inset) and ii) PFOA could activate PPARα transcription coupled with FA2H induction (Figs. 5 and 6), PFOA might interfere with PPARβ/δ-mediated suppression of PPARα, possibly releasing transcriptional corepressors related to PPARβ/δ, such as NCoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) (Shi et al., 2002). Clearly, additional experiments are required to support this hypothesis.

Fig. 5

PFOA can stimulate PPARα-mediated transcriptional activity even in the presence of PPARβ/δ. MDA-MB-231 cells were transiently transfected with PPARα expression plasmid alone (100 ng) or in combination with PPARβ/δ expression plasmid (25 or 50 ng), in the presence of a PPRE-luciferase reporter plasmid. All plasmid concentrations were equalized using empty plasmid. Data are presented as the mean ± S.E. (n = 3) of the fold induction relative to the mock-transfected control in the absence of PFOA. Significant differences (by two-way ANOVA, followed by Tukey-Kramer post-hoc test) compared with the PPARα expression plasmid-transfected group and the PFOA-treated group are marked with asterisks (*P < 0.05) and hashes (#P < 0.05), respectively.

Fig. 6

PFOA can upregulate FA2H expression even in the presence PPARβ/δ. MDA-MB-231 cells were transiently transfected with PPARα expression plasmid alone or in combination with PPARβ/δ expression plasmid, followed by treatment with vehicle or PFOA (100 μM). Mock control cells (–/–/–) were transfected with a combination of empty plasmids in equal amounts. After 48 hr of treatment with vehicle or PFOA (100 μM), FA2H mRNA expression was analyzed using real-time RT-PCR. Data are presented as the mean ± S.E. (n = 3) of the fold induction relative to the mock-transfected control in the absence of PFOA (–/–/–). Significant differences (by two-way ANOVA, followed by Tukey-Kramer post-hoc test) compared with the mock-transfected control in the absence of PFOA (–/–/–) and the PPARα/PPARβ/δ expression plasmids-transfected group in the absence of PFOA (+/+/–) are marked with asterisks (*P < 0.05) and hashes (^#P < 0.05), respectively. Inset: Real-time RT-PCR analysis of PPARβ/δ mRNA after 48 hr of treatment with vehicle or PFOA (100 μM) without PPARα/PPARβ/δ expression plasmid transfection.

It has been reported that metastatic cells do not need to proliferate (Matus et al., 2015), which is consistent with our observation that PFOA-mediated increased migration of MDA-MB-231 cells (via upregulation of FA2H) did not require stimulation of cell proliferation (Fig. 3B). In the present study, we could not provide clear evidence of FA2H protein induction, only mRNA upregulation, with PFOA exposure (Fig. 2B). However, when combined with the results of siRNA treatments described in Fig. 4, these data suggest that FA2H mRNA upregulation (but very modest functional expression) can participate in PFOA stimulation of MDA-MB-231 cell migration.

The effects of PPARs on cancer progression or suppression are conflicting. Extensive activation of PPARα is related to cancer progression in some types of cancers, including TNBC (Suchanek et al., 2002; Chen et al., 2017; Fidoamore et al., 2017; Luo et al., 2019; Hirao-Suzuki et al., 2020a; Castelli et al., 2021). In addition, a recent study demonstrated that increased FA2H expression in TNBC patients is associated with decreased disease-free survival (Wang et al., 2018), and this epidemiological evidence may support the “PPARα-FA2H” axis as a key pathway for TNBC exacerbation.

In the present study, we utilized MDA-MB-231 cells as a model of human TNBC. We also detected PFOA-mediated FA2H upregulation and enhanced migration in 4T1 cells, a mouse TNBC model cell line (data not shown). This suggests that FA2H functions as a general molecule responsible for regulating the migration of TNBC cells.

In the present study, we showed that PFOA can stimulate PPARα transcription in human breast cancer MDA-MB-231 cells (a human TNBC model), coupled with FA2H upregulation at the same PFOA concentration. In addition, selective knockdown of FA2H with siRNA interfered with PFOA-stimulated cell migration. Mechanistically, PFOA could obviate the PPARβ/δ-mediated transcriptional repression of PPARα, leading to FA2H induction. Our findings indicate that FA2H, an inducible gene, might be a target for the prevention of TNBC metastasis, and that environmental chemicals able to “release” the suppression of PPARα by PPARβ/δ may act as migration stimulators.

ACKNOWLEDGEMENTS

This study was supported in part by a Grant-in-Aid for Scientific Research (C) [17K08402 and 21K12261 (to S.T.)] from the Japan Society for the Promotion of Science (JSPS) KAKENHI. This study was also supported in part by a Setouchi Satoyama-Satoumi Research Project Grant of FUKUYAMA UNIVERSITY (to S.T. and N.S.) and in part by the Cooperative Research Program of the Network Joint Research Center for Materials and Devices [Research No. 20211327 (to S.T.)].

Conflict of interest

The authors declare that there is no conflict of interest.

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