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Published in final edited form as: Food Chem Toxicol. 2024 Feb 20;186:114540. doi: 10.1016/j.fct.2024.114540

Benzophenone-3 Alters Expression of Genes Encoding Vascularization and Epithelial-Mesenchymal Transition Functions During Trp53-null Mammary Tumorigenesis

Elena Morozova 1, Anastasia Kariagina 1, Calista Busch 1, Richard C Schwartz 1
PMCID: PMC10978255  NIHMSID: NIHMS1969818  PMID: 38387520

Abstract

Benzophenone-3 (also referred to as oxybenzone) is a putative endocrine disrupting chemical and common ingredient in sunscreens and other personal care products. We previously showed that benzophenone-3 was promotional for epithelial tumorigenesis in mice fed adult high-fat diet, while protective against the incidence of more aggressive spindle cell tumors in the same treatment group. In this study, we show that benzophenone-3 reduces epithelial to mesenchymal transition in the epithelial tumors of these mice. This reduction in epithelial to mesenchymal transition is associated with altered expression of several genes involved in regulation of angiogenesis and epithelial to mesenchymal transition. Among the genes altered in expression, Timp1 is of particular interest because benzophenone-3 suppressed both migration and Timp1 expression in a mammary tumor cell line that displays epithelial to mesenchymal transition characteristics. These alterations in gene expression plausibly stabilize the vasculature of epithelial carcinomas and contribute to benzophenone-3 promotion of epithelial tumors, while at the same time suppress epithelial to mesenchymal transition and suppress incidence of spindle cell tumors.

1. INTRODUCTION

Endocrine disrupting chemicals (EDCs), particularly estrogenic chemicals, are suspects in environmental promotion of breast cancer [1]. Environmental EDCs have the potential to act as agonists or antagonists in critical hormonally regulated processes, such as mammary gland development and mammary tumorigenesis. Benzophenone-3 (BP-3; also referred to as oxybenzone), a putative EDC and common ingredient in sunscreens and other personal care products, has been reported to be prevalent in human urine samples in the United States [2]. Several studies suggest an endocrine disrupting role for BP-3 as a weak estrogenic agonist, as well as an estrogenic, progestogenic, and androgenic antagonist [reviewed in 3]. Recent studies found plasma concentrations greater than 0.5 ng/mL among human cohorts using heavy topical applications of commercial sunscreens [4; 5]. This level, achieved after only one day exposure, exceeds Food and Drug Administration guidance for chemicals of “threshold of toxicological concern”, which was first adopted by the Food and Drug Administration in the regulation of food packaging substances that can migrate into food [6]. Beyond direct exposure by these products, BP-3 is present in household dust [7], in fish lipids [8], and, notably, in the aqueous environment [3]. BP-3 was demonstrated to have pathological effects on coral [9]. Several studies show that BP-3 can alter mouse mammary gland development [10; 11; 12]. In adult mice, BP-3 altered the proportion of stroma in the mammary gland [13]. BP-3 decreased stromal area with no change in epithelial area in parous mice, and decreased stromal area, while increasing adipose area, in mice fed a high-fat diet. Recently, BP-3 was found to promote mammary tumor metastasis in a 4T1 cell line metastasis model [14].

In work published in 2020 [15], we examined the interaction of BP-3 with high-fat diet (HFD) on mammary tumorigenesis in BALB/c mice, using the Trp53-null transplantation model [16]. In this study, we found that BP-3 elicited complex effects that were dependent upon dietary exposure to high fat. Further, BP-3 had differential effects on mammary tumors of differing histopathology. BP-3 was protective against epithelial carcinoma promotion in mice fed a lifelong low-fat diet (LFD), but promotional for epithelial tumorigenesis in mice fed HFD during adulthood (LFD-HFD). While BP-3 promoted epithelial tumorigenesis in mice fed LFD-HFD, BP-3 was protective in these same mice against poorly differentiated spindle cell carcinomas, which have an epithelial to mesenchymal transition (EMT) morphology [17; 18]. The only correlation to the specific promotion of epithelial carcinomas in mice fed LFD-HFD was a BP-3 induced increase in vascularization of these tumors [15].

2. Materials and Methods

2.1. Mice

BALB/c Trp53+/− breeding mice were obtained from Dr. D. Joseph Jerry (University of Massachusetts, Amherst MA), and Trp53-null mice were generated as described [19]. The female Trp53-null tissue donor mice were maintained on standard laboratory rodent diet (LabDiet 5001; LabDiet, St. Louis, MO) before mammary gland collection at eight weeks of age. Wild-type recipient female BALB/c mice were purchased from Charles River (Portage, MI) at 3 weeks of age.

Trp53-null transplanted mice were generated as follows. Fragments of donor mammary epithelium were collected from female BALB/c Trp53-null mice at 8 weeks of age and transplanted into the cleared inguinal mammary fat pads of 3-week-old female wild type BALB/c mice as previously described [20, 21]. To minimize donor bias from secondary genetic alterations, mammary duct fragments from 4 donor mice were transplanted to recipient mice in each diet group in equal distribution. Body weight and food consumption were monitored weekly. Mice were palpated for tumor development twice a week starting at 13 weeks of age. Tumors were harvested at 1 cm in diameter. The first tumor occurring in each mouse was removed by survival surgery for analysis and the tumorigenesis time course allowed to continue for the non-tumorous gland. Infrequent tumor recurrence in the first gland was not scored. Mammary glands that had no epithelium present were excluded from this analysis.

For experiments assessing the effects of BP-3 and diet, female Trp53-null transplanted mice were randomly assigned into diet groups. Food and water were provided ad libitum. Mice were housed in a standard laboratory housing environment with a 12:12 h light–dark cycle, at 20 to 24°C with 40 to 50% relative humidity. All animal experimentation was conducted in accord with accepted standards of humane animal care under guidelines approved by the All University Committee on Animal Use and Care at Michigan State University (AUF #07/17–128-00).

Low fat diet (LFD) (D11012202; 10% kcal fat) and high fat diet (HFD) (D11012204; 60% kcal fat) were purchased from Research Diets (New Brunswick, NJ). For the continuous LFD, the diet was initiated after transplantation at 3 weeks of age and maintained throughout the studies. For the LFD-HFD group, mice were initially fed LFD from 3 weeks until 10 weeks of age, and then switched to HFD thereafter. For the BP-3 treatments, BP-3 (Spectrum Chemical, New Brunswick, NJ) was compounded into the diet at 0.75 g/kg chow for pubertal animals (3 to 10 weeks of age) and 1.5 g/kg chow for adult animals; the difference in BP-3 between the diets for pubertal and adult animals was intended to compensate for the differences in food consumption and body weight with age and yielded BP-3 consumption of approximately 70 mg/kg body weight/d. BP-3 consumption was similar between LFD and HFD, and between pubertal and adult animals. BP-3 excretion in the urine voided at euthanasia for mice treated with BP-3 ranged between 1.0 and 6.1 mg/kg body weight with no significant differences between diet. We selected our dosage to yield levels of BP-3 urine excretion similar to values observed in a small human cohort that was exposed to heavy topical application of a BP-3 containing sunscreen [22], approximately 2.3 mg/kg body weight, assuming an average European adult body weight of 70.8 kg [23]. Considering the rapid excretion of BP-3 in mice [15], it is likely that our dosage generates urine excretion on the same order as that observed in heavily exposed humans. Determination of BP-3 levels in urine was performed by the Division of Laboratory Sciences of the National Center for Environmental Health, Center for Disease Control and Prevention (Atlanta, GA), as reported previously [24].

2.2. Histopathology

Portions of tumors were formalin-fixed, and paraffin embedded. 5 μm tumor sections were stained with hematoxylin and eosin (H&E). All tumors were reviewed and classified, as previously described [25]. Epithelial tumors show clear duct-like structures. While all malignant tumors show some invasion of surrounding stroma, spindle cell tumors are more pronounced in this regard and additionally acquire loss of differentiation, loss of cellular adhesion, loss of polarity, and the gain of a spindle shape. The acquisition of these features constitutes EMT. To score for epithelial tumors undergoing a transition to EMT, tumors with epithelial characteristics (e.g., duct-like structures) were examined for EMT features (e.g., loss of ductal structures, increased spindle cell morphology, signs of invasiveness, loss of epithelial cellular polarity, increased nuclear pleomorphism, increased mitotic figures) [reviewed in 26; 27]. Tumors arising in mammary glands with independent epithelial and spindle cell carcinomas were not included in the analysis. The entirety of tumor sections, including both central and border regions, were evaluated with a Nikon Eclipse E400 light microscope (Nikon, Inc., Melville, NY) at 40x and 100x magnification. The percentage of largely epithelial carcinomas with regions displaying EMT features is reported. Statistical significance was evaluated using Fisher’s exact test.

2.3. Immunofluorescence

5 μm tumor sections were deparaffinized and rehydrated. Antigen retrieval was then accomplished by boiling at 100°C for 10 min in citrate buffer (pH 6.0). Timp1 protein was detected using a mouse monoclonal antibody to Timp-1 (sc-21734; Santa Cruz, CA) diluted 1:50 with incubation at 4°C overnight, followed by AlexaFluor488–labeled goat anti-mouse secondary antibody (Invitrogen, Molecular Probes, Carlsbad, CA) diluted 1:100 with incubation at room temperature for 30 minutes. All immunofluorescent sections were counterstained with 4′,6-diamidino-2-phenylindole to visualize nuclei. Images were captured with a Nikon Eclipse TE2000-U fluorescence microscope (Nikon, Inc.) using a 40× objective lens. From 8 to 10 areas per tumor were analyzed. Integrated intensity of Timp1 fluorescence was measured using MetaMorph software (Molecular Devices, San Jose, CA).

2.4. RNA isolation and quantitative RT-PCR (qRT-PCR)

Total RNA was isolated using TRIzol reagent (ThermoFisher Scientific, Waltham, MA) according to the manufacturer’s protocol. RNA was reverse transcribed into cDNA using the RT2 First Strand Kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s protocol. cDNA reactions lacking reverse transcriptase were performed to confirm elimination of DNA. For qRT-PCR, each reaction (25 μl) included 12.5 μl of RT2 SYBR Green qPCR Mastermix (Qiagen), 1 μl of diluted first-stand cDNA synthesis reaction, 1 μl of primer, and 10.5 μl of deionized H2O. qRT-PCR was performed with the ABI 7500 Fast Real-Time PCR System (ThermoFisher Scientific) using the following program: step 1: 95 °C, 10 min; step 2: 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The abundance of all target RNA products was normalized to the abundance of beta-2 microglobulin RNA (B2m). Fold changes were calculated using the comparative Ct method (ΔΔCt). Amplification utilized QuantiTect (Qiagen) primers (Catalog number: 249900) angiopoietin-1 (Angpt1), GeneGlobe ID - PPM03054F; angiopoietin-2 (Angpt2), PPM03729F; angiopoietin-like protein 1 (Angptl1), PPM37158A; angiopoietin-like protein-4 (Angptl4), PPM05156B; beta-2 microglobulin (B2m), PPM03562A; fibronectin 1 (Fn1), PPM03786A; hypoxia-inducible factor 1 (Hif1a), PPM03799C, lymphoid enhancer-binding factor 1(Lef1), PPM05441F, transforming growth factor beta 1 (Tgfb1), PPM02991B, tissue inhibitor of metalloproteinases 1 (Timp1), PPM03693F; Twist-related protein 1 (Twist1), PPM05125C, vascular endothelial growth factor A (Vegfa), PPM03041F).

2.5. Cell Culture Migration Assay

The migration assay utilized MC7-L1, a mammary cell line of epithelial origin derived from a murine mammary ductal carcinoma [28; a gift from Dr. Claudia Lanari, Universidad de Buenos Aires, Buenos Aires, Argentina]. This cell line expresses both estrogen and progesterone receptors and is hormone responsive in cell culture, but displays EMT characteristics. Cells were maintained in DMEM (1:1) medium supplemented to 10% FCS, 100 units/ml penicillin and 100 mg/ml streptomycin. Cells were cultured at 37 °C with 5% CO2. Cells were cultured in 6-well plates and grown to confluency for 24 h. The growth medium was replaced with phenol red-free DMEM containing 10% charcoal-stripped fetal bovine serum for additional 24 h. Confluent plates were scratched by the plastic tip for a 10 μl pipette and the medium was replaced with phenol red-free DMEM supplemented with insulin, glutamine, and non-essential amino acids without or with 25, 50, and 100 μM BP-3 for additional 24 h. Cells were fixed with 70% ethanol and stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (ThermoFisher Scientific) at 12 and 24 h. Bright field images were captured for all wells at 0 h to delineate the scratch boundaries. Experiments were performed 3 times. Fluorescent images were captured with EVOS fluorescent microscope (ThermoFisher Scientific). The number of cells within the scratch area was counted for one representative experiment using Metamorph software (Molecular Devices Corporation, Downington, PA).

2.6. Statistics

The significance of morphological differences between tumor groups was assessed using Fisher’s Exact Test. The significance of differences in gene expression and cell migration assays were assessed using an unpaired two-tailed Student’s t-test. Immunofluorescence data were analyzed by a non-parametric two-tailed Mann-Whitney t-test from the GraphPad PRIZM 9.51 software package (San Diego, CA, USA). Outliers in individual treatment groups were determined using the ROUT method from the GraphPad PRIZM 9.51 software package. 2-way ANOVA was additionally used to assess effects in cell migration assays. Details are noted in figure legends.

3. Results

3.1. BP-3 diminished the proportion of epithelial tumors with regions of EMT.

In Kariagina et al. [15], we observed that BP-3 was protective against epithelial tumorigenesis in mice fed lifelong low-fat diet, while promotional for epithelial tumorigenesis in mice fed adult high-fat diet. The epithelial carcinomas occurring with BP-3 treatment in mice fed an adult high fat diet were more highly vascularized. At the same time, BP-3 reduced spindle cell tumorigenesis in these mice. Attempting to reconcile these opposing effects, we reexamined the histomorphology of epithelial carcinomas from our earlier study [15] to determine if BP-3 treatment impacted the number of epithelial carcinomas that exhibited regions spindle cell morphology indicative of EMT (Figure 1A). BP-3 treatment of mice fed LFD-HFD dramatically reduced the proportion of epithelial carcinomas showing regions of EMT from 20% to 4% (Figure 1B). The total number of epithelial tumors increased with BP-3 treatment on an LFD-HFD diet (Figure 1C), consistent with the tumor promotion observed with Kaplan-Meier analysis that we previously reported [15]. In contrast, BP-3 had no effect on EMT in epithelial carcinomas occurring in mice fed lifelong LFD.

Figure 1. In mice fed an adult-restricted HFD, BP-3 decreased the proportion of epithelial tumors showing regions with progression toward a spindle cell EMT-like morphology.

Figure 1.

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(A) Representative H&E-stained tissue sections of epithelial tumors. The left image shows a largely epithelial morphology, while the right image shows a region with a spindle cell EMT-like morphology. Magnification is 10×. Scale bar, 100 μm. (B) Proportion of epithelial tumors with regions of spindle cell EMT-like morphology across dietary and BP-3 treatments. LFD; LFD+BP-3; LFD-HFD; HFD-LFD; HFD-LFD+BP-3. Significance of differences between tumor groups was assessed using Fisher’s Exact Test. ****, p < 0.0001. (C) Total number of epithelial tumors with and without regions of spindle cell-EMT.

3.2. BP-3 reduced expression of several genes with roles in angiogenesis and EMT.

As tumor vascularization and EMT are linked in the process of metastatic progression [29], we examined expression of RNAs encoding proteins involved in angiogenesis, as well as RNAs encoding proteins critical to EMT in the epithelial carcinomas generated with and without BP-3 treatment in mice fed LFD and LFD-HFD dietary regimens. It should be noted that all these target genes have associated literature pertaining to both angiogenesis and metastasis/EMT. We examined RNA expression of angiopoietin-1(Angpt1) [reviewed in 30], angiopoietin-2 (Angpt2) [reviewed in 30], hypoxia-inducible factor 1 (Hif1a) [reviewed in 31], and vascular endothelial growth factor A (Vegfa) [reviewed in 32] to assess angiogenic processes, and RNA expression of angiopoietin-like protein 1 (Angptl1) [33], angiopoietin-like protein 4 (Angptl4) [34], lymphoid enhancer-binding factor 1 (Lef1) [35], transforming growth factor beta 1 (Tgfb1) [36], tissue inhibitor of metalloproteinases 1 (Timp1) [37], twist-related protein 1 (Twist1) [38], and fibronectin 1 (Fn1) [39] to assess EMT processes (Figure 2). Treatment with BP-3 reduced Angpt1 expression in LFD tumors, reduced Angpt2 expression in LFD-HFD tumors, reduced Hif1a expression in LFD-HFD tumors, and elicited a trend toward reduced Vegfa expression (p=0.06) in LFD-HFD tumors. BP-3 reduced Angptl4 expression in LFD-HFD tumors and induced a trend toward reduced expression in LFD tumors (p=0.1), reduced Lef1 expression in LFD-HFD tumors and induced a trend toward reduced expression in LFD tumors (p=0.08), reduced Tgfb1 expression in both LFD and LFD-HFD tumors, induced a trend toward reduced Timp1 expression in LFD-HFD tumors (0.06), reduced Twist1 expression in both LFD and LFD-HFD tumors, and reduced Fn1 expression in LFD-HFD tumors and induced a trend toward reduced expression in LFD tumors. BP-3 treatment did not elicit significant changes in Angptl1 expression in the tumors.

Figure 2. Epithelial tumor expression of a panel of genes involved in angiogenesis and EMT.

Figure 2.

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RNAs expressed from genes of interest were quantitated by qRT-PCR. Levels of expression are presented as the fold change compared to levels expressed in epithelial tumors arising in mice fed LFD and untreated with BP-3. Mean values are presented with standard error of the mean (SEM). Angpt1: LFD, n= 9; LFD+BP-3, n=7; LFD-HFD, n=7; LFD-HFD+BP-3, n=6. Angpt2: LFD, n=8; LFD+BP-3, n=8; LFD-HFD, n=8; LFD-HFD, n=7. Hif1a: LFD, n=8; LFD+BP-3, n=8; HFD-LFD, n=7; HFD-LFD+BP-3, n=5. Vegfa: LFD, n=11; LFD+BP-3, n=8; HFD-LFD, n=11; HFD-LFD+BP-3, n=9. Agptl1: LFD, n=6; LFD+BP-3, n=5; LFD-HFD, n=5; LFD-HFD+BP-3, n=5. Angptl4: LFD, n=9; LFD+BP-3, n=8; LFD-HFD, n=10; LFD-HFD+BP-3, n=9. Lef1: LFD, n=5; LFD+BP-3, n=7; LFD-HFD, n=4; LFD-HFD+BP-3, n=5. Tgfb1: LFD, n=8; LFD+BP-3, n=6; LFD-HFD, n=10; LFD-HFD+BP-3, n=8; Timp1: LFD, n=9; LFD+BP-3, n=10; LFD-HFD, n=8; LFD-HFD+BP-3, n=7. Twist1: LFD, n=6; LFD+BP-3, n=9; LFD-HFD, n=10; LFD-HFDS+BP-3, n=9. Outliers in individual treatment groups were determined using Grubbs’ test. Significance of differences from “LFD” was assessed using an unpaired two-tailed Student’s t-test. * p < 0.05; ** p < 0.01. Comparisons approaching significance are noted with p values.

3.3. Only Timp1 showed a trend toward downregulation in spindle cell tumors.

Since expression of Angpt2, Hif1a, Vegfa, and Timp1 were specifically reduced by BP-3 in epithelial carcinomas arising in LFD-HFD mice, we examined whether expression of these genes was also reduced by BP-3 in spindle cell carcinomas that exhibit EMT characteristics arising in LFD-HFD mice. Of these genes, only Timp1 showed a trend toward downregulation (p=0.09) (Figure 3).

Figure 3. BP-3 decreased expression of Timp1 in spindle cell tumors.

Figure 3.

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RNAs were quantitated by qRT-PCR. Levels of expression are presented as the fold change compared to levels expressed in spindle cell tumors arising in mice fed LFD and untreated with BP-3. Mean values are presented with standard error of the mean (SEM). Angpt2: LFD, n= 5; LFD+BP-3, n=5; LFD-HFD, n=5; LFD-HFD+BP-3, n=5. Vegfa: LFD, n= 5; LFD+BP-3, n=5; LFD-HFD, n=5; LFD-HFD+BP-3, n=4. Hif1a: LFD, n= 5; LFD+BP-3, n=5; LFD-HFD, n=5; LFD-HFD+BP-3, n=5. Timp1: LFD, n= 6; LFD+BP-3, n=6; LFD-HFD, n=7; LFD-HFD+BP-3, n=4. Outliers in individual treatment groups were determined using Grubbs’ test. Significance of differences from “LFD” was assessed using an unpaired two-tailed Student’s t-test. Comparison approaching significance is noted with p value.

3.4. BP-3 inhibited cell migration of a mouse mammary cancer cell line with EMT characteristics.

Since several genes associated with EMT were downregulated by BP-3 along with a decrease of EMT morphological features in tumors, we examined the effects of BP-3 on cellular migration, a functional feature of EMT. We utilized MC7-L1 cells, a mouse mammary cancer cell line of epithelial origin that expresses both estrogen and progesterone receptors and displays EMT characteristics [28], in an assay for cell migration (Figure 4). BP-3 inhibited cell migration of MC7-L1 cells in a dose-dependent manner over a 24-hour time course with no observable cell toxicity. We examined the expression of Timp1, which was downregulated by BP-3 in spindle cell carcinomas. Timp1 expression was reduced in MC7-L1 cells treated with BP-3 (Figure 5).

Figure 4. BP-3 suppresses migration of MC7-L1, a mouse mammary cancer cell line of epithelial origin that displays EMT characteristics.

Figure 4.

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(A) Representative photographs of cell migration scratch assays performed with MC7-L1 cells at 12 and 24 h after confluent plates were scratched. Concentrations of BP-3 in medium are noted. (B) Quantitation of cells filling the “scratch” area. Quantitation is presented as mean values (n=3) with SEM. 2-way ANOVA found significant effects of BP-3 concentration (p < 0.001) and time (p < 0.001), as well as an interaction between concentration and time (p < 0.0005). Significance of differences was assessed using an unpaired two-tailed Student’s t-test. *, p < 0.05 compared to control; #, p < 0.05 compared to 50 μM BP-3.

Figure 5. BP-3 suppresses Timp1 expression in MC7-L1 cells.

Figure 5.

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MC7-L1 cells were either untreated or treated with 100 μM BP-3 for 24 hours. RNA was isolated and quantitated by qRT-PCR. Levels of expression are presented as the fold change of BP-3 treated cells compared to untreated control cells. Mean values are presented with standard error of the mean (SEM). Control: n=6; BP-3, n=5. Outliers in individual treatment groups were determined using Grubbs’ test. Significance of difference from “Control” was assessed using an unpaired two-tailed Student’s t-test. ** p < 0.01.

3.5. BP-3 inhibited Timp1 protein expression in LFD-HFD tumors.

Since several lines of experimentation suggested that BP-3 inhibition of Timp1 expression might be a key feature of BP-3 inhibition of EMT in epithelial tumors arising in LFD-HFD mice, we examined Timp1 protein expression in these tumors in the presence and absence of BP-3 treatment (Figure 6). Immunofluorescent staining with anti-Timp1 revealed that BP-3 treatment reduced Timp1 protein expression to 68% of that observed in tumors from untreated mice.

Figure 6. BP-3 suppresses Timp1 protein expression in epithelial tumors arising in mice fed LFD-HFD.

Figure 6.

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(A) Immunofluorescent detection of Timp1 (green) in representative tumor sections from mice fed LFD-HFD and LFD-HFD+BP-3, as labeled. Sections were counterstained with 4′,6-diamidino-2-phenylindole (blue) to visualize nuclei. A representative control stain with secondary antibody in the absence of primary antibody is presented. Yellow arrowheads indicate auto-fluorescent red blood cells. Scale bar, 100 μm. (B) Quantitation of Timp1 is presented as the fold change in LFD-HFD+BP-3 tumors (n=5) compared to levels expressed in LFD-HFD tumors (n=6). Mean values are presented with standard error of the mean (SEM). Significance of difference was assessed using an unpaired two-tailed Student’s t-test. ** p < 0.01.

4. Discussion

In our previous studies, we found that BP-3 treatment promoted mammary epithelial tumorigenesis and suppressed EMT-like spindle cell tumorigenesis in mice fed HFD through adulthood (LFD-HFD) [15]. In the current study, we observed that epithelial carcinomas occurring in mice fed LFD-HFD with BP-3 had fewer regions displaying an EMT morphology. Epithelial carcinomas in this treatment group in comparison to those arising without BP-3 treatment had lower levels of Angpt2, Hif1a, Vegfa, and Timp1 expression. Similar decreases were not observed in tumors arising in mice fed LFD+BP-3, which neither show BP-3 suppression of spindle cell tumorigenesis [15] nor suppression of EMT features in epithelial carcinomas. In our earlier report, we proposed that the suppression of spindle cell carcinomas in mice fed LFD-HFD was unlikely to be a consequence of BP-3 blocking progression of epithelial carcinomas to a spindle cell histopathology because one would then expect increased latency of spindle cell carcinomas with BP-3 treatment [15]. Our current findings suggest that a BP-3 block to tumor progression cannot be eliminated as a mechanism. Perhaps, the occurrence of spindle cell carcinomas was dependent upon EMT in epithelial carcinomas. The data may be complicated by some spindle cell carcinomas arising by progression from epithelial carcinomas and others by initiating events. Greater statistical power may have been needed to observe a predicted change in latency among spindle cell carcinomas.

Angpt2 expression is reported to stimulate breast cancer metastasis and its over-expression in cell culture promoted cell migration [40]. Angpt2 expression has also been closely associated with EMT in lung cancer [41]. This is consistent with our findings of reduced Angpt2 expression and reduced EMT for epithelial carcinomas arising in mice treated with BP-3 on the LFD-HFD regimen, as well as BP-3 inhibition of migration in MC7-L1 cells.

A decrease in Angpt2 expression is superficially at odds with our observation of increased vascularity in epithelial carcinomas arising in mice treated with BP-3 on the LFD-HFD regimen [15], since elevated Angpt2 expression has been broadly associated with tumor angiogenesis [reviewed in 42; reviewed in 43]. In Angpt2-deficient mice, the early stages of tumor growth are impaired and tumor vasculature is more mature without new growth [44]. However, Angpt2 expression is active in destabilizing existing vasculature [45] and requires Vegfa expression to drive angiogenesis [reviewed in 46]. Angpt-2 induces blood vessel regression in the absence of Vegf-A and promotes angiogenesis in the presence of Vegf-A. The elevated vascularity in the BP-3 treated LFD-HFD tumors may reflect maintenance of existing vasculature rather than angiogenesis, given reduced Vegfa expression in these tumors. Hif1a is reported to directly regulate Vegfa expression [47], and the reduction in expression of both genes fits well with a model where BP-3 spares existing vasculature but does not induce angiogenesis. This may, at least in part, explain BP-3 promotion of epithelial tumorigenesis in mice fed LFD-HFD, while BP-3 suppresses epithelial tumorigenesis in mice fed LFD.

The reduction in Angpt2 expression is also consistent with the other changes in gene expression observed in LFD-HFD tumors that predict inhibition of EMT. Notably, Angpt-2 acts in pathways that do not involve its role in angiogenesis as a Tie-2 ligand. These other Angpt-2 functions play a role in tumor invasion and metastasis across a variety of cancers, including breast cancer [reviewed in 48]. For example, Angpt-2 can upregulate Bcl2 expression, enhancing cell survival and suppressing apoptosis, through an α5β1 integrin-mediated pathway that can drive breast cancer cell migration and invasion [40; 49]. Furthermore, systemic expression of Angpt-2 is reported to promote metastasis, while antibody blockade of Angpt-2 inhibits metastasis [50]. The molecular mechanisms by which Angpt-2 modulates metastatic spread and outgrowth are yet to be fully elucidated. Generally, an increased ratio of Angpt-2 to Angpt-1 correlates with tumor angiogenesis and poor prognosis in many cancers [reviewed in 51]. A plausible underlying mechanism for converse Angpt1 and Angpt2 gene expression is regulation of Angpt1 by estrogen receptor α (ERα) and Angpt2 by ERβ [52].

Among the gene expressions more directly associated with EMT, the reduction in Timp1 RNA and protein expression in BP-3 treated epithelial LFD-HFD carcinomas is particularly interesting. Timp1 expression was shown to induce EMT-like changes at the levels of morphology, cell adhesion, and motility in human mammary epithelial cells [37]. A Timp1-mediated decrease in E-cadherin is dependent upon Twist1 expression, which we also found suppressed by BP-3 treatment in LFD-HFD and LFD tumors. Twist1 expression has also been closely associated with EMT, metastasis, and poor patient outcomes in breast cancer [53; 54; 55]. The spindle cell carcinomas that arise in Trp53-null mammary glands have been characterized as claudin-low-like and show high levels of Twist1 expression [17; 18; 56]. We note that BP-3 also suppresses Angptl4, Lef1 and Tgfb1 expression, also associated with EMT, in tumors arising in LFD-HFD as well as in LFD fed mice. This suggests that neither reduced Angptl4 nor Lef1 nor Tgfb1 nor Twist1 nor Fn1 expression is sufficient for the BP-3 suppression of EMT that we observe in tumors from LFD-HFD mice. Although not in the context of dietary fat exposure, the ability of BP-3 to inhibit migration of MC7-L1 cells, a cell line with EMT characteristics, is consistent with BP-3 suppression of gene expression associated with EMT. Furthermore, BP-3 induced inhibition of cellular migration in MC7-L1 cells was concomitant with reduced Timp1 expression, suggesting functional significance. BP-3 also reduced Timp1 expression in spindle cell carcinomas from LFD-HFD mice. When we explored the overall survival in human breast cancer patient samples using the Kaplan Meier Plotter [https://kmplot.com/analysis/; 57] for high versus low mRNA expression among the several gene expressions that were specifically reduced in epithelial tumors arising in mice fed LFD-HFD, we found that Timp1 expression was the only one with statistically significant reduced survival with high expression over a 5-year outcome window (Supplemental Figure 1).

Among the RNAs that we examined, BP-3 suppression of Angpt1 expression was unique to tumors from LFD mice. As the products of Angpt1 and Angpt2 act in antagonism to each other on the Tie2 receptor [reviewed in 58], one might expect that a lack of Angpt-1 would enhance the activity of Angpt-2. In this regard, it is interesting that tumors from BP-3 treated LFD mice do not exhibit any change in either vascularization or EMT characteristics, as do tumors from LFD-HFD mice that have reduced Angpt2 expression. It is likely that the antagonism between Angpt-1 and Angpt-2 is restricted to Tie-2 dependent pathways, and the alterations observed in tumors arising in LFD-HFD mice are elicited through other pathways, such as the α5β1 integrin-mediated pathway [40; 49].

Spindle cell carcinomas, which have EMT characteristics, are suppressed by BP-3 in mice fed LFD-HFD without a similar effect in mice fed LFD [15]. The current study found that BP-3 treatment also reduces the occurrence of EMT characteristics in epithelial tumors to a level similar to that observed in mice fed LFD. Comparing BP-3 modulation of angiogenesis and EMT related genes in epithelial tumors between mice fed LFD and LFD-HFD, BP-3 specifically reduced expression of Angpt2, Hif1a, Vegfa, and Timp1 in epithelial carcinomas arising in LFD-HFD mice. These genetic targets of BP-3 modulation present themselves as plausible candidates to play a role in both suppressing progression to EMT in epithelial carcinomas and suppressing promotion of spindle cell carcinomas. The mechanism by which dietary fat might alter the effects of BP-3 might include alteration of the target cell population, alteration of the immune milieu, and alteration of the spectrum of available growth factors. None of these are mutually exclusive. It is also possible that some effects are due to differential retention of BP-3 in mammary fat across the dietary regimens.

We are aware of only two studies that examined the effects of BP-3 on EMT and tumor metastasis. One study found that BP-3 enhanced metastatic seeding of mouse mammary carcinoma 4T1 cells in the lung [14]. Another study found that human lung carcinoma H460 cells acquired cell culture characteristics of metastatic cells along with altered gene expression characteristics of EMT [59]. This is at least superficially at odds with our findings. We note that the antagonism of EMT that we observe is dependent upon an adult HFD diet. Furthermore, we performed our experiments in an in vivo tumorigenesis model rather than a challenge with a pre-existing cell line of known metastatic potential [14] or a cell culture model of a tumor cell line [59]. The different outcomes may be attributed to dietary fat and the more diverse set of tumors obtained in the current study. It is clear from our previous studies that BP-3 effects are diet dependent [15].

The literature reports that many of the gene expressions that we examined are modulated directly or indirectly by estrogen, progesterone, or both. These include Angpt1 [57; 58; 61], Angpt2 [51; 62; 63], Hif1a [64; 65], Vegfa [66; 67], Angptl1 [68], Angptl4 [68], Lef1 [69; 70], Tgfb1 [71; 72], Timp1 [73], and Twist1 [74], and Fn1 [75; 76]. The ability of BP-3 to alter expression of these genes in this study is consistent with the notion that BP-3 acts in vivo as an EDC. Interestingly, the epithelial tumors arising in Trp53-null mammary glands are largely negative for estrogen and progesterone receptors [15]. This raises the possibility that endocrine signaling at a point earlier in the evolution of these tumors leads to epigenetic regulation of the genetic targets examined in this study. Irrespective of specific mechanism, the results reported here point to BP-3 effects in the expression of genes related to the regulation of angiogenesis and metastatic characteristics, as well as suggesting an interaction with dietary fat. This highlights angiogenesis and EMT as two pathways in which to further elucidate BP-3 effects on mammary gland development and tumorigenesis.

Supplementary Material

1

Supplemental Figure 1. Elevated Timp1 gene expression is associated with a statistically significant reduction in patient survival over a 5-year outcome window. Survival data for breast cancer patients with tumors carrying Trp53 mutations were queried over a 5-year window for elevated expression of Angpt2, Hif1a, Vegfa, and Timp1, as measured by RNA expression with gene chip technology. This was carried out using the Kaplan Meier Plotter [https://kmplot.com/analysis/; 56].

HIGHLIGHTS.

  • In mice fed an adult high-fat diet, BP-3 reduces the proportion of Trp53null epithelial tumors that show progression toward epithelial to mesenchymal transition (EMT).

  • BP-3 alters expression of several genes involved in regulation of angiogenesis and EMT.

  • Among the genes whose expression is altered, Timp1 appears to be a gene of particular interest with BP-3 decreasing expression at the RNA and protein levels in tumors arising in mice fed an adult high-fat diet, and decreasing both migration and Timp1 expression in a mammary tumor cell line that displays EMT characteristics.

  • These alterations in gene expression plausibly stabilize the vasculature of epithelial carcinomas and contribute to BP-3 promotion of epithelial tumors, while at the same time suppress epithelial to mesenchymal transition and suppress incidence of spindle cell tumors.

Funding

The work was supported by a Breast Cancer and the Environment Research Program Grant from the National Institute of Environment Health Sciences (NIEHS) and the National Cancer Institute (NCI), NIH, DHHS U01ES026119 to RCS.

Footnotes

CRediT authorship contribution statement

Elena Morozova: Methodology, Formal analysis, Investigation, Data curation, Writing - review & editing, Visualization. Anastasia Kariagina: Methodology, Formal analysis, Investigation, Data curation, Writing - review & editing, Visualization. Calista Busch: Formal analysis, Investigation, Data curation. Richard C. Schwartz: Conceptualization, Methodology, Formal analysis, Data curation, Writing - original draft, Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Declaration of interests

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Richard C. Schwartz reports financial support was provided by National Institute of Environmental Health Sciences.

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Associated Data

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Supplementary Materials

1

Supplemental Figure 1. Elevated Timp1 gene expression is associated with a statistically significant reduction in patient survival over a 5-year outcome window. Survival data for breast cancer patients with tumors carrying Trp53 mutations were queried over a 5-year window for elevated expression of Angpt2, Hif1a, Vegfa, and Timp1, as measured by RNA expression with gene chip technology. This was carried out using the Kaplan Meier Plotter [https://kmplot.com/analysis/; 56].

NIHMS1969818-supplement-1.docx (139.3KB, docx)

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