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Effect of genistein supplementation on microenvironment regulation of breast tumors in obese mice

Breast Cancer Research volume 26, Article number: 147 (2024) Cite this article

Abstract

Obesity is an important risk factor for breast cancer in women before and after menopause. Adipocytes, key mediators in the tumor microenvironment, play a pivotal role in the relationship between obesity with cancer. However, the potential of dietary components in modulating this relationship remains underexplored. Genistein, a soy-derived isoflavone, has shown promise in reducing breast cancer risk, attenuating obesity-associated inflammation, and improving insulin resistance. However, there are no reports examining whether genistein has the ability to reduce the effects of obesity on breast tumor development. In this study, we constructed a mammary tumor model in ovariectomized obese mice and examined the effects of genistein on body condition and tumor growth. Moreover, the effects of genistein on the tumor microenvironment were examined via experimental observation of peritumoral adipocytes and macrophages. In addition, we further investigated the effect of genistein on adipocyte and breast cancer cell crosstalk via coculture experiments. Our findings indicate that dietary genistein significantly alleviates obesity, systemic inflammation, and metabolic disorders induced by a high-fat diet in ovariectomized mice. Notably, it also inhibits tumor growth in vivo. The impact of genistein extends to the tumor microenvironment, where it reduces the production of cancer-associated adipocytes (CAAs) and the recruitment of M2d-subtype macrophages. In vitro, genistein mitigates the transition of adipocytes into CAAs and inhibits the expression of inflammatory factors by activating PPAR-γ pathway and degrading nuclear NF-κB. Furthermore, it impedes the acquisition of invasive properties and epithelial‒mesenchymal transition in breast cancer cells under CAA-induced inflammation, disrupting the Wnt3a/β-catenin pathway. Intriguingly, the PPAR-γ inhibitor T0070907 counteracted the effects of genistein in the coculture system, underscoring the specificity of its action. Our study revealed that genistein can mitigate the adverse effects of obesity on breast cancer by modulating the tumor microenvironment. These findings provide new insights into how genistein intake and a soy-based diet can reduce breast cancer risk.

Introduction

The global obesity pandemic presents a growing public health challenge, with projections suggesting that up to 51% of the population could be obese by 2030 [1, 2]. This rise in obesity is particularly concerning in the context of breast cancer—the most prevalent cancer in women worldwide, with its risk increasing postmenopause [3]. Obese women face an increased risk of breast cancer invasion, distant metastases, and tumor recurrence and a reduced response to therapeutic interventions, cumulatively increasing mortality [4]. In obesity, increased and dysfunctional adipose cells fosters both local and systemic interactions that promote tumorigenic processes [5].

Adipose tissue is a dynamic endocrine organ responsible for energy homeostasis [6]. Uncontrolled excess adiposity often leads to metabolic disorders in the body, manifested as abnormal insulin, insulin-like growth factor 1 (IGF-1), leptin, and adiponectin secretion [7, 8]. In addition, obesity stimulates adipocytes to upregulate chemokines, inducing the production of proinflammatory proteins, proangiogenic factors and macrophages, which in turn cause systemic inflammation and immune hyporesponsiveness in obese individuals, thereby promoting the generation and progression of malignant tumors [4].

The tumor microenvironment (TME), which comprises cancer cells and surrounding nonmalignant cells, plays a crucial role in tumor cell survival and cancer progression [9]. Notably, cancer-associated adipocytes (CAAs) within the breast TME have emerged as key players in breast cancer progression [5, 10]. These CAAs, particularly those at the invasive front, can be transformed into adipocyte-derived fibroblasts (ADFs), which are characterized by reduced expression of adipocyte markers such as peroxisome proliferator-activated receptor gamma (PPAR-γ) and CCAAT enhancer-binding protein alpha (C/EBPα) [11, 12]. In addition, breast cancer cells can alter gene expression in CAAs and remodel their secretion profile in an MMTV integration site family member 3 A (Wnt3a)-dependent manner, resulting in sustained nuclear factor kappa B (NF-κB) pathway activation and increased secretion of inflammatory cytokines, which promote lipolysis and free fatty acid (FFA) release [13]. This altered adipocyte metabolism not only increases tumor-associated macrophage (TAM) production but also promotes epithelial‒mesenchymal transition (EMT), invasion, and metastasis in breast cancer cells [4, 14]. Therefore, targeting CAAs and the tumor microenvironment is currently an attractive approach for cancer therapy, but no safe and effective agents exist at present.

The long-term consumption of soy foods rich in isoflavones can reduce the risk of human breast cancer [15, 16]. Genistein (GEN) is the main active component of soy isoflavones and has a chemical structure similar to that of 17β-oestradiol [17], with beneficial antitumor and anti-inflammatory effects, alleviating obesity-related metabolic syndrome and preventing osteoporosis [18,19,20]. Notably, GEN is a natural agonist of PPAR-γ [21]. Studies have shown that nutrient-related concentrations of GEN can interfere with the production of proinflammatory cytokines by inhibiting the NF-κB signalling pathway and subsequently protect tissue cells from inflammatory mediators in neurodegenerative diseases, rheumatoid arthritis, and inflammatory disease models of the kidney and liver [22]. Moreover, GEN acts as a chemotherapeutic agent in different types of cancer, mainly by altering apoptosis, the cell cycle, and angiogenesis and inhibiting metastasis [23]. Clinical studies have shown that soy and isoflavone intake can reduce the risk of breast cancer in postmenopausal women [24]. Recent studies have shown that obesity increases the risk of postmenopausal, rather than premenopausal, breast cancer, which is mainly because oestradiol in premenopausal women alleviates low-grade inflammation in the breast caused by obesity [4, 25]. Therefore, GEN may play a role in inhibiting obesity-induced breast tumor development.

In this study, we confirmed that GEN supplementation attenuated the breast cancer-promoting effect caused by obesity via in vitro and in vivo methods. In addition, GEN affected inflammatory cell recruitment and CAA production in the tumor microenvironment. We also showed that GEN regulates the crosstalk between CAAs and tumor cells via the PPAR-γ/NF-κB signalling.

Materials and methods

Reagents

Genistein (C12H10O5, MW: 270.24, CAS: 446-72-0, HPLC purity ≥ 98%), was obtained from DESITE Biological Technology Co., Ltd. (Chengdu, China) and dissolved in DMSO (Sigma-Aldrich). Mouse research diets were purchased from Xietong Pharmaceutical Bioengineering Co., Ltd. (Jiangsu, China). Antibodies MHC II (M5/114, E-AB-F0990D, CD11b (M1/70, E-AB-F1081S), Gr-1 (RB6-8C5, E-AB-F1120UC) and F4/80 (CI: A3-1, E-AB-F0995E) used for flow cytometry were obtained from Elabscience (Wuhan, China). BODIPY 493/503 fluorochrome, Triton X-100, Oil Red O, 3-Isobutyl-1-methylxanthine (IBMX), bovine insulin and dexamethasone were provided by Sigma-Aldrich (Shanghai, China). Collagenase II, Matrigel and other reagents required for cell culture were purchased from Gibco (Shanghai, China). T0070907 (PPAR-γ inhibitors) was provided by Selleck (Shanghai, China), Reagents such as RIPA lysis buffer, phenylmethanesulfonyl fluoride and ECL Luminescent Reagent used in Western blotting were purchased from Beyotime Biotechnology (Shanghai, China). The primary antibodies of rabbit origin CD68, PPAR-γ, NF-κB, IL-1β, IL-6、IL-8, TNF-α, CCL2, E-cadherin, N-cadherin, Vimentin, Wnt3a, β-catenin, c-Myc, β-tubulin and Histone-H3 were purchased from Wanlei Biotechnology (Shenyang, China). The primary antibodies of mouse origin PPAR-γ, NFκB, β-actin and Pref1 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Goat anti-Rabbit, goat anti-Rabbit IgG horseradish peroxidase-conjugated secondary antibodies, FITC-labeled goat anti-mouse IgG H&L, Alexa Fluor 488-labeled goat anti-rabbit IgG H&L, DAPI solution and diaminobenzidine (DAB) substrate color liquid were all purchased from Bioss Biotechnology Co., Ltd.(Beijing, China).

Animal model

Female C57BL/6J mice were offered by CHANGSHENG Experimental Animals Co. Ltd (Changchun, Jilin province, China), and were housed at controlled temperature (25 ± 2 °C) and humidity (60 ± 10%) with 12 h light/dark cycle. They were ovariectomized at 6 weeks of age following isoflurane inhalation anesthesia. One week later, mice were divided into four groups (10 animals/group) and fed either Control (CON) diet [10% kcal from fat; ad libitum (Research Diets, D12450J)], GEN diet [Research Diets, D12450J containing 400 mg/kg GEN], Diet-Induced Obese (DIO) diet [60% kcal from fat; ad libitum (Research Diets, D12492)], D + G diet [Research Diets, D12492 containing 400 mg/kg GEN] for 105 days. Soybean oil was replaced with corn oil in both D12492 and 12,450 J diets. Additive dosages for GEN were cited from previously published literature [26]. On day 75 of feeding, venous blood was collected from the medial canthus of the eyes of mice.

E0771 breast cancer cells, derived from a C57BL6 mouse, were injected in the left fourth mammary fat pad (MFP) of mice at 250,000 cells in 100 µL of 40% Matrigel after 75 days of feeding with different diets. Tumor growth was monitored by measuring the length and width of the tumor using digital calipers. tumor Volume = (width)2 × (length)/2 [27]. One month after tumor implantation, the mice were sacrificed, the blood was collected and centrifuged, and serum was separated and stored in an ultra-low temperature freezer at -80˚C until testing. tumors, peritumoral adiposes, contralateral adiposes (inguinal subcutaneous white adipose tissue, iWAT), interscapular brown adipose tissue (BAT) and livers were collected for further investigation.

Flow cytometric analysis

Tumor blocks were digested and single cell suspension was obtained using the Miltenyi Biotec Mouse tumor Dissociation kit (Miltenyi Biotec, Auburn, CA). Peritumoral adiposes were washed with PBS and minced in FACS buffer (PBS containing 1% low endotoxin bovine serum albumin). The stromal vascular cells (SVCs) were prepared from Collagenase II digested fat pads. FACS analysis of SVCs and tumor cells for macrophage content and subtypes were performed as previously described [28]. All cell samples were stained with CD11b+, F4/80, Gr-1 and MHCII antibodies, and then washed with PBS containing 1%BSA. Finally, cell samples were detected using a FACS LSR Fortessa flow cytometer and data were analyzed with FlowJo software. Cells were gated as follows: macrophages (Macs) (CD11b+, F4/80+); M1-like Macs (CD11b+, F4/80+, Gr-1 low/MHCII high); M2-like Macs (CD11b + F4/80 + Gr-1 low / MHCII low).

Histological staining and imaging

Different tissues were fixed in 10% neutral formalin or adipose tissue fixative for 72 h, embedded, sectioned and subjected to hematoxylin and eosin (H&E) staining, Oil Red O staining, CD68 and PPAR-γ immunohistochemistry staining as previously described [29, 30]. Finally, we used Image-Pro Plus (IPP) image software to manually select positive regions and determine the mean value of the integrated optical density after completing the optical density calibration of the images.

Serum parameter test

Enzyme-linked immunosorbent assay (ELISA) kits (Jiangsu Jingmei Biotechnology Co., Ltd.) were used to detect the levels of insulin, leptin, adiponectin (ADP), free fatty acid (FFA), insulin-like growth factor 1 (IGF-1), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), chemokine ligand 2 (CCL2), chemokine ligand 5 (CCL5), transforming growth factor-β (TGF-β), and vascular endothelial growth factor (VEGF). Serum glucose levels were measured using assay kits (Nanjing Jiancheng Biotechnology).

Cell culture

E0771, 4T1, EMT-6 breast cancer cells and 3T3-L1 preadipocyte cells were purchased from the Chinese Biomedical Laboratory Cell Repository. E0771, 4T1, EMT-6 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium containing 10% fetal bovine serum (FBS), penicillin (50 U/ml) and streptomycin (50 U/ml). 3T3-L1 cells were maintained in Iscove’s modified Dulbecco’s medium (IMDM), containing 10% bovine calf serum (BCS), penicillin (50 U/ml), and streptomycin (50 U/ml). The cells were grown in a humidified atmosphere with 5% CO2 at 37 °C. The changes of cell proliferation ability were detected by CCK8 assay as previously described [31].

Adipocyte differentiation

3T3-L1 preadipocyte cells within 20 passages were seeded in 6-well plates for adipogenic induction and co-culture. After reaching approximately 100% confluency, 3T3-L1 cells were cultured with induction medium A, B, and C for 4 days each. Medium A: IMDM containing 10% BCS, 0.5 mM IBMX, 4 µg/mL insulin and 0.25 µM dexamethasone; Medium B: IMDM containing 10% BCS and 4 µg/mL insulin; Medium C: IMDM containing 10% FBS and 4 µg/mL insulin.

Co-culture conditions

A total of 3 × 105 E0771 cells (within 30 passages) were seeded in the top chamber of the Transwell culture system (0.4-µm pore size; Corning), and mature adipocytes were seeded in the bottom chamber for 4 days. There was no direct contact between adipocytes and E0771 cells. The top and bottom chamber culture media included DMEM containing 10% FBS, different concentrations of GEN and/or 1 µM T0070907 [32]. Adipocytes or tumor cells cultivated alone under similar conditions served as controls. After coculture, adipocytes and tumor cells were collected for further experiments.

Wound-healing assay

Conditioned medium (CM) generated from CAAs was collected and wound-healing assays were performed according to Liu et al. [33].

Transwell invasion assay

In a similar manner, tumor cells and adipocytes were co-cultured in 24-well plates. After 72 h, transwell inserts with 8 μm pores (spread Matrigel inside before the experiment) were replaced and inoculated with 1 × 105 tumor cells in the top chamber. After 36 h, the transwell inserts were removed and migrated cells were fixed with 4% paraformaldehyde and stained using 1% crystal violet. Membranes were cleaned and air-dried, and the number of migrated cells was counted under a microscope.

Cell lipid droplet observation

Oil Red O and BODIPY 493/503 staining were used to assess intracellular lipid droplet levels. Specific staining methods and parameter acquisition methods were described previously [29].

Cytofluorimetric analysis

E0771 cells co-cultured for 4 days were carefully digested with trypsin, seeded in new dishes and cultured with CM for 12 h for immunofluorescence staining. 3T3-L1 located in the lower chamber of the co-culture system can be directly fixed and stained. The specific staining steps are described by Liang. et al. [29].

RNA isolation, q-PCR, and Western blotting

RNA extraction and quantitative PCR (qPCR) were conducted as previously described [31]. Primers used to detect target mRNAs are listed in Table S1. In addition, the qRCR results of the samples were tested for Pearson correlation using Origin 2021.

The adipocytes were harvested, and the cytosolic and nuclear extracts were collected using the Nuclear/Cytosolic Extraction Kit (Active Motif, Carlsbad, CA, USA), according to the manufacturer’s instructions. The method for extracting total cellular protein was described previously [31]. Proteins were denatured by boiling and separated by SDS–PAGE followed by immunoblotting. Protein quantification was performed via ImageJ.

Molecular docking

The structure of PPAR-γ protein with ID P37231 was obtained from Uniprot Protein Data Bank. Structural files of GEN were acquired in Pubchem database (https://pubchem.ncbi.nlm.nih.gov/). PPAR-γ was subjected to simulated molecular docking with GEN using AutoDockTools 1.5.6 and AutoDock Vina software and the score of the combination was calculated. Force analysis and visualization were performed using Pymol and Discovery Studio 2019 software.

Statistical analysis

All the data were analysed via the SPSS 22.0 statistical package (SPSS Inc., Chicago, IL). Comparisons among different experimental groups for statistical significance were performed using a one-way analysis of variance (ANOVA) and the Tukey-Kramer test. Data were reported as mean ± standard deviation (M ± SD). The data was plotted using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, United States). p < 0.05 and p < 0.01 were considered statistically significant.

Results

GEN attenuated high-fat diet-induced obesity in mice and alleviated obesity-increased tumor burden

The experimental protocol, including grouped feeding and tumor implantation for ovariectomized mice, is outlined in Fig. 1A. Our results showed a significant increase in obesity in mice fed a High-Fat Diet (HFD) compared to those on the control (CON) diet, as depicted in Fig. 1B. Notably, GEN supplementation markedly alleviated the obesity induced by the HFD after 75 days of feeding. This anti-obesity effect of GEN was even more pronounced following tumor implantation, as shown towards the end of the experiment (Fig. 1C). In terms of tumor growth, mice on the HFD exhibited accelerated tumor development post-injection with E0771 cells, compared to those on a low-fat diet (Fig. 1D). While compared with mice in the DIO group, the tumor growth rate of mice in the D + G group was significantly slower, indicating that the addition of GEN alleviated the increase in tumor load caused by obesity (Fig. 1D, E). Regression analysis of tumor weight versus body weight revealed that the mammary tumor burden was positively correlated with body weight across all strains and diets (R = 0.87, P < 0.001, Fig. 1F).

Fig. 1
figure 1

Obesity-increased tumor burden was reduced by GEN. (A) Schematic diagram of the experimental design. (B) Representative photograph of mice. (C) Body weight curves of CON-fed and HFD-fed mice treated with GEN or not (n = 7/group). (D) Tumor size (Background 1 × 1 cm2). (E) E0771 tumor progression was measured by digital caliper over 1mouth after orthotopic injection (n = 7/group). (F) The weights of mammary tumors collected from all the mice were plotted against the body weights of corresponding mice. All values were expressed as mean ± S.D. * P < 0.05, ** P < 0.01 vs. CON group, # P < 0.05, ## P < 0.01 vs. DIO group

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GEN alleviated obesity-induced metabolic dysfunction and chronic systemic inflammation

The impact of diet and GEN supplementation on metabolic health was assessed through various parameters. Notably, lipid accumulation in the liver, a marker of metabolic decline, was significantly higher in DIO group mice compared to controls. However, GEN supplementation effectively alleviated this hepatic steatosis induced by a High-Fat Diet (HFD), as evidenced in Fig. 2A (top) and Fig. 2B. Additionally, GEN ameliorated mild vacuolation in the liver caused by estrogen deprivation in ovariectomized (OVX) mice (Fig. 2A, top). Histopathological examination of inguinal White Adipose Tissue (iWAT) revealed that adipocytes in both CON and GEN groups were small and densely packed (Fig. 2A, middle). In contrast, adipocyte expansion was markedly pronounced in the DIO group. GEN treatment significantly reduced adipocyte size, as shown in Fig. 2C. Brown Adipose Tissue (BAT), crucial for thermogenesis, exhibited more multilocular lipid droplets in DIO mice, indicative of BAT ‘whitening.’ Chronic GEN administration counteracted this effect of HFD on BAT (Fig. 2A, bottom).

Serum analysis demonstrated that GEN treatment normalized hyperlipidemia and insulin resistance in the mice, as depicted in Fig. 2D and E. DIO mice exhibited elevated levels of Free Fatty Acids (FFA) and leptin, and reduced adiponectin levels, correlating with increased adipose tissue. Dietary GEN reversed these changes, decreasing serum FFA and leptin levels while increasing adiponectin (Fig. 2F-H). Moreover, GEN significantly lowered the serum levels of obesity-related adipokines, including TNF-α, IL1β, IL6, IL8, CCL-2, CCL-5, IGF-1, TGF-β, and VECF. This suggests that GEN can alleviate systemic inflammation caused by obesity and tumor implantation (Fig. 2I-Q). Collectively, these results indicate that GEN effectively improves body condition in HFD and OVX-induced obese mice.

Fig. 2
figure 2

GEN improved obesity-induced glucose metabolism and chronic systemic inflammation. (A) H&E or/and Oil-red O staining of liver, iWAT, and BAT. (B) The means integral optical density of Oil-red O postive area in liver (n = 3). (C) The relationship between the numbers and area of iWAT lipid droplets (n = 3). (D-Q) The serum levels of glucose, insulin, FFA, leptin, adiponectin, TNF-α, IL1β, IL6, IL8, CCL-2, CCL-5, IGF-1, TGF-β1 and VECF (n = 7/group) (“Pre” represents before tumor implantation, “Post” represents 30 days after tumor implantation); All values were expressed as mean ± S.D. * P < 0.05, ** P < 0.01 vs. CON group, # P < 0.05, ## P < 0.01 vs. DIO group

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Gen relieved adipose tissue inflammation and reduced CAAs and TAMs content in the tumor microenvironment

The transformation of cancer-associated adipocytes (CAAs) in the tumor microenvironment plays a critical role in the prognosis of breast cancer, particularly in obese patients [10]. Hematoxylin and Eosin (H&E) staining of tumor-surrounding adipose tissue revealed distinct morphological changes. Adipocytes in these areas appeared smaller and more irregularly arranged compared to normal mammary tissues, as depicted in Fig. 3A. This alteration was particularly pronounced in the Diet-Induced Obesity (DIO) group, where there was a noticeable increase in adipocyte-derived fibroblasts (ADFs, identified by black arrows) and immune cell recruitment (blue arrows) around tumor cells.

In contrast, the D + G group (DIO mice supplemented with GEN) exhibited minimal differences in adipocyte morphology between peritumoral and contralateral adiposes. Quantitative PCR analysis further supported these observations: Adipocyte differentiation markers such as APD, PPAR-γ, and C/EBPα showed higher expression in the peritumoral fat of D + G mice compared to the DIO group. Concurrently, there was a decrease in the expression of preadipocytokine-1 (Pref-1) and hormone-sensitive lipase (HSL). Similarly, adipokines like leptin, TNF-α, IL1β, IL6, CCL2, TGF-β1, and hypoxia-inducible factor-1 (HIF-1) were lower in the D + G group (Fig. 3B-D).

Immunohistochemistry staining highlighted a significant increase in CD68 + cells (a marker for macrophages) in the peritumoral adiposes of DIO mice. However, GEN feeding markedly reduced these CD68 + cells (Fig. 3E). Flow cytometry and immunofluorescent quantification demonstrated an increase in macrophage content in tumors and peritumoral adiposes of obese mice, which was significantly reduced by GEN treatment (Fig. 3F and H). Notably, obesity induced a rise in immunosuppressive M2-like macrophages in both tumors and peritumoral adiposes of obese mice, a trend that GEN effectively mitigated, particularly in tumor environments (Fig. 3G and I).

Fig. 3
figure 3

GEN reduced macrophage recruitment to tumor and pertumoral adiposes. (A) H&E staining of tumors and peritumoral adiposes. (B) Relative mRNA expression of PPAR-γ, C/EBPα, Pref-1 and HSL. (C) Relative mRNA expression of LEP, ADP, HIF-1 and TGF-β. (D) Relative mRNA expression of TNF-α, IL1β, IL6 and CCL-2. (E) Immunohistochemistry analysis and quantitative analysis of CD68 in peritumoral adiposes. (F) Analysis of infiltrating macrophages (F4/80+, CD11b+) in peritumoral adiposes by flow cytometry. (G) Analysis of M2-like macrophage subtypes (MHCII-, Gr-1-) of macrophages in peritumoral adiposes by flow cytometry. (H) Analysis of infiltrating macrophages (F4/80+, CD11b+) in tumors by flow cytometry. (I) Analysis of M2-like macrophage subtypes (MHCII-, Gr-1-) of macrophages in tumors by flow cytometry. All values were expressed as mean ± S.D.(n = 3 in each group). * P < 0.05, ** P < 0.01 vs. CON group, # P < 0.05 vs. DIO group

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GEN suppressed CAA-like phenotypic changes exhibited by adipocytes co-cultured with breast cancer cells

To elucidate how GEN influences the interaction between adipocytes and tumor cells, we utilized an in vitro co-culture model as illustrated in Fig. 4A. Results from the CCK-8 assay indicated that GEN concentrations up to 20 µM did not impact the growth of preadipocytes 3T3-L1 (Fig. 4B). BODIPY and Oil red O staining revealed that co-culturing with tumor cells prompted the release of lipid droplets from adipocytes. Notably, GEN partially restored this lipid accumulation in co-cultivated adipocytes in a dose-dependent manner (Fig. 4C and D). Under white light microscopy, mature adipocytes co-cultured with tumor cells for four days exhibited a highly elongated, fibroblastic shape (Fig. 4E, top). In these co-cultured adipocytes, Pref-1, a transmembrane protein of preadipocytes that hinders adipogenesis [11], was significantly elevated. However, the addition of GEN during co-culture effectively reduced Pref-1 expression (Fig. 4E, bottom).

Further qPCR analysis revealed that GEN alleviated the loss of adipogenic markers such as PPAR-γ, C/EBPα, APD, and FABP4 in 3T3-L1 cells post co-culture. Conversely, expression levels of Pref-1, HSL, and cytokines TNF-α, IL1β, IL6, and CCL-2 significantly decreased following GEN treatment (Fig. 4F). These findings suggest that GEN successfully maintains the adipogenic phenotype of co-cultivated adipocytes and prevents their transformation into CAAs, as shown in Fig. 4G and H. The experimental data collectively indicate that a concentration of 10 µM GEN is optimal and most effective in preventing the conversion of adipocytes into CAAs.

Fig. 4
figure 4

GEN suppressed extensive phenotypic changes in adipocytes co-cultured with E0771 in vitro. (A) Schematic diagram of 3T3-L1 differentiation and the coculture system. (B) Changes in viability of murine preadipocytes 3T3-L1 after treatment with gradient concentrations of GEN for 4 days (n = 6). (C) BODIPY 493/503 staining results and quantitative analysis of mature adipocytes cultured alone or in co-culture with E0771 cells (presence/absence of GEN) for 4 days (n = 3). (D) Oil-red O staining results of mature adipocytes cultured alone or in co-culture with E0771 cells (presence/absence of GEN) for 4 days. (E) Representative bright field and Pref-1 immunohistochemical staining photographs of 3T3-L1 cells. (F) Relative mRNA expression of PPAR-γ, C/EBPα, APD and FABP4 (n = 4). (G) Relative mRNA expression of Pref-1 and HSL (n = 4). (H) Relative mRNA expression of TNF-α, IL1β, IL6 and CCL-2 (n = 4). All values were expressed as mean ± S.D. ** P < 0.01 vs. Non-coculture group, # P < 0.05, ## P < 0.01 vs. Coculture group

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GEN inhibited adipocyte-stimulated migration and invasion potential of E0771 cells

To determine whether GEN could impede the metastatic potential of breast cancer cells induced by cancer-associated adipocytes (CAAs), we utilized wound-healing and Transwell invasion assays. The results showed that co-culture with CAAs significantly enhanced the metastatic ability of E0771 breast cancer cells, while GEN effectively slowed down the wound healing process and reduced cell invasion in a dose-dependent manner (Fig. 5A and B). Similarly, the addition of genistein at 10 µM inhibited the acquisition of invasive ability of 4T1 and EMT6 breast cancer cells after co-culture with adipocytes (Figure S1). We further assessed the expression of epithelial-mesenchymal transition (EMT)-associated regulators in E0771 cells from the upper chamber of the co-culture (Fig. 5C-G). Immunofluorescence staining of the cytoskeletal protein β-actin revealed that after 4 days of co-culture, E0771 cells lost their characteristic epithelial morphology, formed more dispersed colonies, and exhibited a scattered appearance. GEN addition ameliorated these changes (Fig. 5C). Moreover, co-culture notably increased N-cadherin and Vimentin expression while decreasing E-cadherin expression in E0771 cells, indicating a shift towards EMT characteristics. GEN treatment effectively counteracted these alterations (Fig. 5C-G). The Wnt3a/β-catenin signaling pathway is a critical driver in cancer progression and EMT, often regulated by inflammatory factors [34, 35]. Our investigation into this pathway showed that CAAs significantly upregulated the expression of Wnt3a, β-catenin, and c-Myc in E0771 cells. Conversely, GEN markedly reduced the activation of the Wnt3a/β-catenin pathway (Fig. 5H-K).

Fig. 5
figure 5

GEN inhibited adipocyte-stimulated migration and invasion potential of E0771 cells. (A) Wound healing of E0771 cells cultured in different CM (obtained after co-culture) was monitored under a phase-contrast microscope. (B) Transwell assays were performed to analyze the cell invasion ability of E0771 in the presence or absence of adipocytes, and four doses of GEN were added in the culture medium. (C) Immunofluorescence staining for E-cadherin/N-cadherin (red) and β-actin (green) in upper chamber E0771 cells. Nuclei were stained with DAPI (blue). (D-G) The protein expressions of E-cadherin, N-cadherin and Vimentin were measured by western blotting analysis, and β-actin protein level was used as a loading normal. (H-K) The protein expressions of Wnt3a, β-catenin and c-Myc were measured by western blotting analysis, and β-tubulin protein level was used as a loading norma. All values were expressed as mean ± S.D. (n = 3 in each group). * P < 0.05, ** P < 0.01 vs. Non-coculture group, # P < 0.05, ## P < 0.01 vs. Coculture group

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GEN activated PPAR-γ and negatively regulated the NF-κB pathway to inhibit inflammatory phenotypic changes in CAAs

Correlation analysis of 3T3-L1 mRNA detection results in adipocytes co-cultured with E0771 revealed a significant negative correlation between adipogenic differentiation-associated proteins and adipo-inflammatory factors (Fig. 6A), as did the same correlation analysis of peritumoral adipose mRNA in mice, especially the significance of the negative correlation between PPAR-γ and inflammatory factors (Fig. 6B). PPAR-γ is not only an important transcription factor for adipogenic differentiation, but also plays a pivotal role in the anti-inflammatory process, which is largely attributed to its role in inducing NFκB/p65 degradation [36, 37]. In addition, molecular docking simulation experiments showed that GEN bound to the PPAR-γ active site and obtained the lowest binding energy arrangement and the best 3D molecular docking structure (Fig. 6C). Under this structure, the lowest binding energy of PPAR-γ protein to GEN was − 7.9 kcal/mol. Solid blue lines indicate hydrogen bonds and dashed gray lines indicate hydrophobic forces (Fig. 6C). 2D presentation shows amino acid residues interacting with GEN in the binding site of PPAR-γ (Fig. 6D). And immunohistochemical staining highlighted that the protein expression of PPAR-γ was significantly decreased in peritumoral adiposes of DIO mice. However, GEN feeding significantly elevated PPAR-γ content in peritumoral adiposes. (Fig. 6E).

Immunofluorescence staining results demonstrated a marked decrease in PPAR-γ expression in adipocytes after 4 days of co-culture with tumor cells. Concurrently, changes in nuclear morphology were observed, alongside a significant increase in nuclear NF-kB expression. In stark contrast, GEN administration notably enhanced PPAR-γ content while inhibiting NF-κB expression in adipocytes (Fig. 6F). Western blot analysis further substantiated these findings. GEN treatment resulted in a significant increase in nuclear PPAR-γ content and a marked decrease in nuclear NF-kB levels (Fig. 6G). Additionally, GEN reduced the protein expression of intracellular pro-inflammatory cytokine TNF-α, IL1β, IL6 and CCL-2 after co-culture (Fig. 6H). These results collectively indicate that GEN attenuates the inflammatory state of adipocytes primarily through the PPAR-γ-induced degradation of nuclear NF-κB. This mechanism effectively disrupts the bidirectional communication between tumor cells and cancer-associated adipocytes (CAAs), underscoring the potential of GEN in modulating the tumor microenvironment.

Fig. 6
figure 6

GEN activated PPAR-γ and negatively regulate the NF-κB pathway to inhibit inflammatory phenotypic changes in CAAs. (A) Correlation analysis between adipogenic differentiation related proteins and adipose inflammatory factors in 3T3-L1 cells. “Red” represents positive correlation and “blue” represents negative correlation. (B) Correlation analysis between adipogenic differentiation related proteins and adipose inflammatory factors in mice peritumoral adiposes. “Red” represents positive correlation and “blue” represents negative correlation. (C) 3D presentation of interaction between genistein and PPAR-γ. (D) 2D presentation of interaction between genistein and PPAR-γ. (E) Immunohistochemistry analysis and quantitative analysis of PPAR-γ in peritumoral adiposes. (F) Immunofluorescence staining for PPAR-γ (green) and NF-κB (red) in bottom chamber 3T3-L1 cells. Nuclei were stained with DAPI (blue). (G) The protein expressions of nuclear PPAR-γ, NF-κB and cytosolic PPAR-γ, NF-κB. Histone-H3 was used as a loading control for the nuclear proteins, whereas β-actin and β-tubulin was used for the cytosolic proteins. (H) The protein expressions of TNF-α, IL1β, IL6 and CCL-2 were measured by western blotting analysis, and β-tubulin protein level was used as a loading norma. All values were expressed as mean ± S.D. (n = 3 in each group). * P < 0.05, ** P < 0.01 vs. Non-coculture group, # P < 0.05, ## P < 0.01 vs. Coculture group

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T0070907 abrogated the differentiation maintenance effect of GEN on adipocytes and reversed the anti-inflammatory activity of GEN on CAAs

To substantiate the pivotal role of PPAR-γ in GEN’s inhibition of adipocyte transformation into CAAs, we utilized the specific PPAR-γ inhibitor T0070907. Indeed, T0070907 treatment accelerated lipid loss in mature adipocytes after co-culture and interfered with the maintenance of adipogenic differentiation by GEN (Fig. 7A). Furthermore, T0070907 effectively reversed GEN’s maintenance of the adipocyte phenotype, accelerating the transformation of adipocytes into ADFs as depicted in Fig. 7B. The impact of T0070907 extended to the expression of adiposity markers. Compared to the group treated with GEN in co-culture, T0070907 led to a notable reduction in PPAR-γ-related adiposity markers and an increase in the expression of preadipocyte markers (Fig. 7C and D). To further validate the above observations, we examined the effect of T0070907 on PPAR-γ and nuclear NF-κB degradation. As expected, PPAR-γ inhibitor T0070907 suppressed PPAR-γ expression and weakened nuclear NF-κB degradation by GEN (Fig. 7E and F). Additionally, T0070907 effectively negated the anti-inflammatory impact of GEN during the transformation of adipocytes into CAAs. This was evidenced by the upregulated expression of inflammatory cytokines, including TNF-α, IL1β, IL6, and CCL-2, in the presence of T0070907 (Fig. 7G).

Fig. 7
figure 7

T0070907 reversed the inhibitory effect of GEN on adipocyte development to CAA and resulting inflammation in a co-culture system. (A) BODIPY 493/503 staining results and quantitative analysis of mature adipocytes cultured alone or in co-culture with E0771 cells (presence/absence of GEN and T0070907) for 4 days (n = 3). (B) Representative bright field and Pref-1 immunohistochemical staining photographs of 3T3-L1 cells. (C) Relative mRNA expression of PPAR-γ, C/EBPα, APD and FABP4 (n = 4). (D) Relative mRNA expression of Pref-1 and HSL (n = 4). (E) Immunofluorescence staining for PPAR-γ (green) and NF-κB (red) in bottom chamber 3T3-L1 cells. Nuclei were stained with DAPI (blue). (F) The protein expressions of nuclear PPAR-γ, NF-κB and cytosolic PPAR-γ, NF-κB. Histone-H3 was used as a loading control for the nuclear proteins, whereas β-actin and β-tubulin was used for the cytosolic proteins (n = 3). (G) The protein expressions of TNF-α, IL1β, IL6 and CCL-2 were measured by western blotting analysis, and β-tubulin protein level was used as a loading norma (n = 3). All values were expressed as mean ± S.D. * P < 0.05, ** P < 0.01 vs. Coculture group, # P < 0.05, ## P < 0.01 vs. Gen + Coculture group

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Targeting PPAR-γ by inhibitors T0070907 abrogated the tumor suppressor effect of GEN in co-culture systems

PPAR-γ, as a regulatory factor with a wide range of pro-differentiation and anti-proliferative effects, its low expression is also an important factor leading to the malignant development of cancer [38]. In this experiment, we also verified the interference of PPAR-γ deficiency with E0771 in the co-culture system by adding T0070907. Transwell assay showed that T0070907 interfered with the anticancer effect of GEN, allowing cells to regain invasive ability (Fig. 8A). Western blotting and immunofluorescence assay results also confirmed that T0070907 reversed the regulation of EMT-related protein E-cadherin, N-cadherin and Vimentin expression levels in upper chamber E0771 cells by GEN (Fig. 8B and C). The results also showed that addition of T0070907 to the co-culture further stimulated WNT/β-catenin signaling transduction in E0771 and similarly blocked the interference of GEN (Fig. 8D).

Fig. 8
figure 8

T0070907 abrogated the anti-tumor cell transfer effect exerted by GEN in the co-culture system. (A) Transwell assays were performed to analyze the cell invasion ability of E0771 in the presence or absence of adipocytes, GEN or/and T0070907 were added in the culture medium. (B) Immunofluorescence staining for E-cadherin/N-cadherin (red) in upper chamber E0771 cells. Nuclei were stained with DAPI (blue). (C) The protein expressions of E-cadherin, N-cadherin and Vimentin were measured by western blotting analysis, and β-actin protein level was used as a loading normal. (D) The protein expressions of Wnt3a, β-catenin and c-Myc were measured by western blotting analysis, and β-tubulin protein level was used as a loading norma. All values were expressed as mean ± S.D. (n = 3 in each group). * P < 0.05, ** P < 0.01 vs. Coculture group, # P < 0.05, ## P < 0.01 vs. Gen + Coculture group

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Discussion

Obesity is becoming one of the largest issues in public health, and its adverse effects on cancer have aroused concern. A survey revealed that obesity is a major risk factor for 11 types of cancer, a trend that is more prevalent in older adults [39]. Natural plant extracts have gained wide interest in the health care of the ageing population because of their few side effects and extraordinary health benefits [40]. GEN is an isoflavone found in almost all legumes [15]. Epidemiologic analyses indicate that among Asians, high soy intake is associated with an approximately one-third reduction in the risk of both pre- and postmenopausal breast cancer [41, 42]. GEN has therefore been considered a pharmacologically active natural compound with antitumor effects since its discovery, which is also supported by epidemiological evidence [43, 44]. This study is the first to demonstrate that dietary supplementation with GEN in ovariectomized mice attenuates the increased tumor burden associated with obesity, further enriching the application value of GEN in human and animal health.

In obese individuals, both local and systemic interactions between malignant tumor cells and dysfunctional adipose cells occur, thereby promoting tumorigenic processes [5]. Notably, adipose tissue expansion in obesity is associated with a metabolic syndrome characterized by critical metabolic changes, such as hyperinsulinaemia and hyperleptinaemia, which are related to increased cancer risk [6]. In our study, GEN administration effectively reduced the levels of insulin, leptin, and other adipokines, which aligns with existing evidence of the role of GEN in metabolic regulation and weight loss [45, 46].

Adipose tissue functions as an important endocrine organ, influencing the metabolism of distant organs and tumors through secreted hormones and cytokines [47]. Elevated levels of circulating lipids and adipokines also significantly alter the local microenvironment of tumors, promote angiogenesis, and affect cancer cell and CAA behaviour, thereby accelerating the occurrence and development of malignant tumors [48]. As a phytoestrogen, GEN exerts anti-inflammatory effects in the treatment of age-related diseases [49]. We first discovered that dietary GEN significantly inhibited adipokine efflux from the TME in obese mice after tumor implantation and one month of feeding, as reflected by decreases in the levels of circulating adipokines, especially CCL-2, leptin, and TNF-α, in the blood. These findings suggest that disruption of the inflammatory response by GEN plays a crucial role in interfering with the tumor microenvironment.

Furthermore, our study demonstrated that obese mice have an increased number of adipocytes with a CAA-like phenotype around the tumor, and some adipocytes even dedifferentiate into ADF-like cells, as described by Ludivine Bochet et al. [11]. However, dietary GEN allows most peritumoral adipocytes to maintain their lipid storage function, reduces adipokine release, and reduces leukocyte recruitment to the TME. As the primary leukocyte in the tumor stroma, TAMs play essential roles in tumor initiation and progression [50]. Adipocyte hypertrophy caused by obesity can increase the number of macrophages in the adipose tissue microenvironment several-fold [51]. After stimulation with chemokines, cytokines, and other factors secreted by tumor and immune cells, more monocytes are recruited to the TME, where they can differentiate into TAMs, whereas tissue-resident macrophages have the capacity to proliferate [52, 53]. TAMs affected by CAAs and local cytokines and metabolites generally exhibit functions similar to those of M2 macrophages and can be classified into the M2d subtype. M2ds or TAMs lose their antitumor function and even promote angiogenesis during tumor development [54, 55]. These phenomena are even more prominent in obese individuals [28, 50]. Our findings showed that GEN specifically reduced M2d macrophage recruitment in tumors and peritumoral adipose tissue, suggesting that the antitumor effects of GEN are closely tied to its impact on the peritumoral environment.

Isolated coculture systems using Transwell inserts have become popular in vitro models to study communication between tumor cells and other cells [56]. In this study, we differentiated 3T3-L1 fibroblast-like preadipocytes with directed differentiation potential into mature adipocytes. Afterwards, adipocytes cocultured with tumor cells presented phenotypic changes consistent with previous studies on CAAs [10, 11, 33]. However, there are no specific studies on the mechanism by which CAAs transition into an inflammatory state. In the present study, on the basis of the results of correlation analysis, we hypothesized that NF-κB activation and the transformation of inflammatory CAAs are modulated by the absence of PPAR-γ. Isoflavone phytoestrogens are similar to the PPAR-γ agonist rosiglitazone [21], and activation of PPAR-γ by GEN and inhibition of the NF-κB signalling pathway play pivotal roles in alleviating the transition from adipocytes to CAAs. In this study, we successfully confirmed this conclusion via the PPAR-γ inhibitor T0070907, which aggravated the phenotypic shift in CAAs, increase the secretion of inflammatory factors after coculture and reversed a series of protective effects of GEN. The present work not only further elucidates the mechanism of the conversion of adipocytes to CAAs but also confirms how GEN inhibits CAAs conversion during tumor development.

In many tissues or organs, PPAR-γ activation inhibits the β-catenin pathway, whereas activation of the canonical Wnt3a/β-catenin pathway inactivates PPAR-γ [35, 38]. The generation of CAAs and the ADF phenotype depends on reactivation of the Wnt/β-catenin pathway in tumor cells [11]. While we investigated CAAs in the coculture system, we also found that CAAs promoted EMT and the activation of the Wnt3a/β-catenin pathway in upper chamber E0771 cells. Moreover, GEN decreased the migration and invasion potential of E0771 cells seeded in the upper chamber of the Transwell insert. Phenotypic changes in cancer cells recovered from cocultures were found to be permanent [56]. This finding also indicates that GEN interferes with cancer cell‒adipocyte interactions, which can control tumor heterogeneity and decrease acquired malignancy in obese patients.

Although there is still controversy about the transduction of oestrogen signalling in breast cancer cells by GEN [57], . the ameliorative effect of GEN on obesity and insulin resistance is well known [58]. Obesity increases postmenopausal breast cancer risk rather than premenopausal risk [4]. After menopause, 17β-oestradiol production is low, and obesity causes uncontrolled systemic inflammation, increasing the risk of cancer and worsening patient outcomes [25]. In this study, we constructed a mammary tumor model in ovariectomized obese mice and first confirmed that GEN reduced obesity and controlled mammary tumor development by regulating the tumor microenvironment. GEN has also been shown to inhibit the conversion of adipocytes to CAAs via the activation of PPAR-γ in cell coculture experiments. This study provides new insights into how a soy-based diet and GEN intake reduces breast cancer risk (Fig. 9). This study also provides theoretical support for the use of GEN as a dietary component for adjuvant therapy in obese breast cancer patients. In the future, we will explore the interference effect of GEN on the intercellular metabolic crosstalk between adipose cells and tumor cells from the perspective of the acquisition of tumor cell metastatic properties, with the main aim of investigating the effect of GEN on fatty acid oxidation in tumor cells that uptake lipids.

Fig. 9
figure 9

A proposed schematic diagram illustrating the mechanism of genistein against obesity-associated breast cancer in mice

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Data availability

No datasets were generated or analysed during the current study.

Abbreviations

ADF:

Adipocyte-derived fibroblasts

ADP:

Adiponectin

BAT:

Brown adipose tissue

BC:

Breast cancer

CAA:

Cancer-associated adipocyte

CCL:

Chemokine ligand

C/EBPα:

CCAAT enhancer-binding protein alpha

DIO:

Diet-induced obese

DMEM:

Dulbecco’s Modified Eagle Medium

DMSO:

Dimethyl sulfoxide

ELISA:

Enzyme-linked immunosorbent assay

EMT:

Epithelial-mesenchymal transition

FABP4:

Fatty acid-binding protein 4

FACS:

Fluorescence-activated cell sorting

FFA:

Free fatty acid

HFD:

High fat diet

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GEN:

Genistein

H&E:

Hematoxylin and Eosin

HIF-1:

Hypoxia-inducible factor-1

HSL:

Hormone-sensitive lipase

IGF-1:

Insulin-like growth factor 1

IL:

Interleukin

iWAT:

Inguinal subcutaneous white adipose tissue

Macs:

Macrophage

MFP:

Fourth mammary fat pad

MHCII:

Major histocompatibility complex class II

NFκB:

Nuclear factor kappa B

OVX:

Ovariectomy

PBS:

Phosphate buffered saline

PPAR-γ:

Peroxisome proliferator-activated receptor gamma

Pref1:

Preadipocytokine-1

qRT-PCR:

Quantitative reverse transcriptase polymerase chain reaction

TAM:

Tumor-associated macrophage

TGF-β:

Transforming growth factor-β

TME:

Tumor microenvironment

TNF-α:

Tumor necrosis factor alpha

VECF:

Vascular endothelial growth factor

Wnt3A:

MMTV integration site family member 3 A

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Acknowledgements

None.

Funding

This research was funded by earmarked fund for CARS36 and National Natural Resources Foundation of China, Grant Nos. 31872527 and 32373083.

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Author notes

  1. Shengzi Jin and Yingce Zheng contributed to the work equally and should be regarded as co-first authors.

Authors and Affiliations

  1. College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, PR China

    Shengzi Jin, Ding Li, Xingyao Liu, Tingting Zhu & Yun Liu

  2. College of Life Sciences, Northeast Agricultural University, No. 600 Changjiang Road, Harbin, 150030, PR China

    Yingce Zheng & Zhonghua Liu

  3. College of Veterinary Medicine, Zhejiang A&F University, Hangzhou, 311300, PR China

    Shuang Wang

  4. Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine, Northeast Agricultural University, No. 600 Changjiang Road, Harbin, 150030, PR China

    Yun Liu

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  1. Shengzi Jin
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Contributions

S.J. and Y.L. conducted most of the experiments and collected the data. D.L., T.Z. and X.L. carried out the sample collection and data analysis. Y.Z. and S.W. conducted cells co-culture experiments. S.J. wrote the manuscript and Z.L. reviewed the manuscript. All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy.

Corresponding authors

Correspondence to Zhonghua Liu or Yun Liu.

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Ethical approval

Animal experiments were performed in accordance with the World Medical Association (WMA), the European Directive 2010/63/EU and the Laboratory Animal Management and Ethics Committee of Northeast Agricultural University (Approval Number: NEAUEC2023-03-38; approval date: March 6, 2023).

Competing interests

The authors declare no competing interests.

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Jin, S., Zheng, Y., Li, D. et al. Effect of genistein supplementation on microenvironment regulation of breast tumors in obese mice. Breast Cancer Res 26, 147 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13058-024-01904-8

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13058-024-01904-8

Keywords

Breast Cancer Research

ISSN: 1465-542X

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